Corneal Endothelial Transplant: (DSAEK, DMEK & DLEK)

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Corneal Endothelial Transplant (DSAEK, DMEK & DLEK)

System requirement: • Windows XP or above • Power DVD player (Software) • Windows Media Player version 10.0 or above • Quick time player version 6.5 or above Accompanying DVD ROMs are playable only in Computer and not in DVD player. Kindly wait for few seconds for DVD to autorun. If it does not autorun then please do the following: • Click on my computer • Click the drive labelled JAYPEE and after opening the drive, kindly double click the file Jaypee

DVD Contents.................................. DSAEK Surgical Techniques 1. Animation ........................................................................................ M Hodgkin MD 2. Surgery ............................................................................................. M Gorovoy MD 3. Surgery ............................................................................................. M Gorovoy MD 4. Surgery ............................................................................................. M Busin MD 5. Surgery ............................................................................................. F Price MD 6. Surgery ............................................................................................. T John MD 7. Donor Cornea ................................................................................. T John MD 8. Instruments ..................................................................................... T John MD 9. Instruments ..................................................................................... T John MD 10. Instruments ..................................................................................... T John MD 11. Instruments ..................................................................................... T John MD 12. Instruments ..................................................................................... T John MD 13. Instruments ..................................................................................... T John MD 14. Surgery ............................................................................................. M Terry MD

Corneal Endothelial Transplant (DSAEK, DMEK & DLEK) Editor Thomas John

MD

Clinical Associate Professor Loyola University at Chicago, Maywood, Illinois, USA Visiting Professor, Department of Defense Military Medical Academy, Belgrade, Serbia Thomas John Vision Institute, Tinley Park and Oak Lawn, Illinois, USA Chicago Cornea Research Center, Tinley Park, Illinois, USA

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Corneal Endothelial Transplant (DSAEK, DMEK & DLEK) © 2010, Jaypee Brothers Medical Publishers All rights reserved. No part of this publication and DVD ROM should be reproduced, stored in a retrieval system, or transmitted in any form or by any means: electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the editor and the publisher. This book has been published in good faith that the material provided by contributors is original. Every effort is made to ensure accuracy of material, but the publisher, printer and editor will not be held responsible for any inadvertent error(s). In case of any dispute, all legal matters to be settled under Delhi jurisdiction only.

First Edition: 2010 ISBN 978-81-8448-792-3

Typeset at JPBMP typesetting unit Printed at Nutech

To God of all faiths and religions. I give all credit to God, Jesus Christ, without whom I am nobody. To my wife, Annita and my kids Michelle, Andrea and Olivia for all their loving support. To my parents for their love and guidance. To all the teachers in this world for their immense contribution to society.

Contributors .................................... Richard L Lindstrom MD

Wisam A Shihadeh MD

Founder and Attending Surgeon Minnesota Eye Consultants Adjunct Professor Emeritus Department of Ophthalmology University of Minnesota, Associate Director, Minnesota Lions Eye Bank, USA Foreword

Assistant Professor/Medical School Jordan University of Science & Technology Consultant in Glaucoma, Cornea & Refractive Surgery King Abdullah University Hospital Irbid, Jordan Chapter 2

Roger F Steinert MD

Almamoun Abdelkader MD

Professor of Ophthalmology, Professor of Biomedical Engineering, Director of Cornea, Refractive & Cataract Surgery, Vice Chair of Clinical Ophthalmology, Department of Ophthalmology University of California, Irvine, CA, USA Introduction, Chapter 5

Assistant Lecturer of Ophthalmology Faculty of Medicine, Al-Azhar University Hospitals, Cairo, Egypt Chapter 2

Kenneth R Kenyon MD

Herbert E Kaufman MD

Founder, Cornea Consultants International, Boston, USA, & Munich, Germany, Associate Clinical Professor of Ophthalmology, Harvard Medical School, Boston, MA, USA, Senior Scientist, Schepens Eye Research Institute, Boston, MA, USA, Eye Health Vision Centers, North Dartmouth, MA, USA Chapter 39

Byod Professor of Ophthalmology; Pharmacology & Experimental Therapeutics; Microbiology, Immunology & Parasitology Louisiana State University Health Sciences Center in New Orleans, LSU Eye Center New Orleans, LA, USA Chapter 2

Thomas John MD

Senior Fellow, Cornea and Refractive Surgery, Massachusetts Eye & Ear Infirmary and Department of Ophthalmology Harvard Medical School Boston, MA, USA Chapter 3

Clinical Associate Professor, Loyola University at Chicago, Maywood, Illinois, USA, Thomas John Vision Institute, Tinley Park and Oak Lawn, Illinois, USA, Chicago Cornea Research Center, Tinley Park Illinois, USA Editor and Chapters 10, 11, 12, 13, 14, 23, 27, 28, 29, 31, 32, 39

Jay S Pepose MD, PhD

Pepose Vision Institute, St. Louis, MO, USA; Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA Chapter 1

Mujtaba A Qazi MD

Pepose Vision Institute, St. Louis, MO, USA; Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA Chapter 1

Pedram Hamrah MD

Eric C Amesbury MD, FACS

Corneal Fellow Department of Ophthalmology & Visual Sciences, University of Louisville, Louisville, KY, USA Chapter 3

Richard A Eiferman MD, FACS

Clinical Professor of Ophthalmology Department of Ophthalmology & Visual Sciences, University of Louisville, Louisville, KY, USA Chapter 3

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Corneal Endothelial Transplant George Baikoff MD

Clinique Monticelli, Marseille, France Chapter 4

Leejee H Suh MD

Assistant Professor of Clinical Ophthalmology Cornea and External Diseases Bascom Palmer Eye Institute Miami, FL, USA Chapter 6

William W Culbertson MD

Professor of Ophthalmology The Lou Higgins Distinguished Chair in Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Cornea and External Diseases, Miami, FL, USA Chapter 6

Harriet O Lloyd

Research Associate Department of Ophthalmology Weill Medical College of Cornell University, New York, NY, USA Chapter 7

D Dan Z Reinstein MD, MA(Cantab) FRCSC, FRCOphth

London Vision Clinic, London, UK; Department of Ophthalmology, St. Thomas’ Hospital - Kings College, London, UK; Department of Ophthalmology, Weill Medical College of Cornell University, NY, USA Chapter 7

D Jackson Coleman MD, FACS

The John Milton McLean Professor of Ophthalmology, Chairman Emeritus Department of Ophthalmology, Weill Cornell Medical College of Cornell University, NY, USA Chapter 7

Ronald H Silverman PhD

Professor of Computer Science in Ophthalmology Research Director, Bioacoustic Research Facility, Department of Ophthalmology Weill Medical College of Cornell University New York, NY, USA Member Research Staff Frederic L. Lizzi Center for Biomedical Engineering Riverside Research Institute New York, NY, USA Chapter 7

Monica Patel MD

Research Fellow, Bioacoustic Research Facility, Weill Medical College of Cornell University, New York, NY, USA Chapter 7

Jasmeet S Dhaliwal MD

Henry Ford Health System, Department of Ophthalmology, Cornea and Refractive Surgery, Troy, MI, USA Chapter 8

Auguste G-Y Chiou MD

Clinical Associate Professor LSU Eye Center, Av.de Montbenon 2 1003 Lausanne, Switzerland Chapter 8

Stephen C Kaufman MD, PhD

Omer Gal

Research Fellow, Bioacoustic Research Facility, Weill Medical College of Cornell University, New York, NY, USA Chapter 7

Professor and Lyon Endowed Chair of Ophthalmology Director of Cornea and Refractive Surgery University of Minnesota 420 Delaware St. SE, MMC-493 Minneapolis, MN 55455, USA Chapter 8

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Contributors Ramagopal Rao PhD

Executive Chairman 3D Vision Systems, LLC Irvine, CA, USA Chapter 9

David Miller MD

Associate Clinical Professor of Ophthalmology, Harvard Medical School Boston, MA, USA; Founder and Chief Medical Officer, 3D Vision Systems, LLC Irvine, CA, USA Chapter 9

Enrique S Malbran MD

Director of the Clínica Oftalmológica Malbran. President of the Fundación Oftalmológica Argentina Jorge Malbran. Chattered Member of the Academia Nacional de Medicina de Buenos Aires, Argentina Chapter 13

Mark A Terry MD

Director, Devers Eye Institute, Portland, OR, USA; Scientific Director, Lions Vision Research Laboratory of Oregon, Portland, OR, USA; Professor of Clinical Ophthalmology, Oregon Health Sciences University, Portland, OR, USA Chapters 14, 15, 16, 17, 20

Luiz F Regis-Pacheco MD Cornea and External Disease Service Department of Ophthalmology University of the State of Rio de Janeiro Rio de Janeiro, Brazil Chapter 14

Ashraf Amayem MD

Consultant Ophthalmologist Director of Cornea & Refractive Surgery Unit, Magrabi Eye Center, Jeddah Saudi Arabia Chapter 18

Magdi Helal MD

Consultant Ophthalmologist Director of Glaucoma Unit Magrabi Eye Center Jeddah, Saudi Arabia Chapter 18

Anastasios John Kanellopoulos MD

Associate Professor of Ophthalmology NYU Medical School, New York NY, USA, Director, Laser Vision Institute Athens, Greece Chapter 19

Massimo Busin MD

Head, Department of Ophthalmology Villa Serena Hospital Forli, Italy Professor of Ophthalmology, University of Bonn, Germany Clinical Professor of Ophthalmology University of Catanzaro, Italy Chapter 21, 37

Vincenzo Scorcia

MD

Villa Serena Hospital Department of Ophthalmology Forli, Italy Chapter 21

Marianne O Price PhD, MBA

José G Pecego MD

Executive Director, Cornea Research Foundation of America, Indianapolis, IN USA Chapter 22

Paula J Ousley MT

Francis W Price MD

Cornea Service Department of Ophthalmology Federal University of Rio de Janeiro Rio de Janeiro, Brazil Chapter 14

Past Research Director Lions Vision Research Laboratory of Oregon Portland, Oregon, USA Chapter 15

President, Price Vision Group Indianapolis, IN, USA Chapter 22

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Corneal Endothelial Transplant Mark S Gorovoy MD

Gorovoy MD Eye Specialists’ Office Fort Myers, FL, USA Chapters 24, 33

Anthony Kuo MD

Fellow, Cornea and Refractive Surgery Duke University Eye Center, Durham NC, USA Chapter 30

Keith A Walter MD

Associate Professor of Ophthalmology Wake Forest University Eye Center Winston-Salem, NC, USA Chapter 25

Marshall E Tyler

Terry Kim MD

Associate Professor of Ophthalmology Duke University School of Medicine Director of Fellowship Programs Associate Director Cornea and Refractive Surgery Duke University Eye Center Durham NC, USA Chapter 30

Wake Forest Univeristy Eye Center Winston-Salem NC, USA Chapter 25

Juan M Castro-Combs MD

Ciro Tamburrelli MD

Head, Ospedale Oftalmico di Roma, Rome, Italy Chapter 26

Agostino Salvatore Vaiano MD Ophthalmologist Ospedale Oftalmico di Roma Rome, Italy Chapter 26

Emilio Balestrazzi MD

Head of Ophthalmology Institute, Catholic University of Rome, Rome, Italy Chapter 26

Post-Doctoral Fellow, Cornea and Refractive Surgery Services, The Wilmer Ophthalmological Institute, The Johns Hopkins University School of Medicine The Johns Hopkins Hospital, Baltimore MD, USA Chapters 34, 35

Naima B Jacobs-El

The Wilmer Eye Institute The Johns Hopkins University School of Medicine Baltimore, MD, USA Chapters 34, 35

Ashley Behrens MD

Assistant Professor of Ophthalmology Cornea and Refractive Surgery Services The Wilmer Ophthalmological Institute The Johns Hopkins University School of Medicine, The Johns Hopkins Hospital Baltimore MD, USA Chapters 34, 35

xi

Contributors Panagiotis Georgoudis MRCOphth

Ophthalmology Resident, St Peter’s Hospital, Chertsey, Surrey, UK St Peter’s Hospital Guildford Road, Chertsey Surrey, KT16 0PZ, UK Chapter 36

Michael J Tappin FRCOphth

Ophthalmic Specialist, St Peter’s Hospital Chertsey, Surrey, UK Chapter 36

Jui-Yang Lai PhD

Assistant Professor, Institute of Biochemical and Biomedical Engineering Chang Gung University, Taoyuan Taiwan, Republic of China; Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan Republic of China Chapter 38

Ging-Ho Hsiue PhD

Department of Chemical Engineering National Tsing Hua University, Hsinchu Taiwan, Republic of China Chapter 38

Foreword .......................................... At the current moment, keratoplasty is undergoing an incredible paradigm shift in surgical technique. A field dominated by Penetrating Keratoplasty, where advances such as improved trephination systems, corneal preservation media, and suturing techniques while meaningful, have clearly been only incremental, is simultaneously going lamellar, minimally invasive, and sutureless. Lead by the extraordinary success of Deep Lamellar Endothelial Keratoplasty (DLEK), the concept of transplanting only the corneal layer which is diseased or damaged and needs replacing is gaining significant traction amongst corneal surgeons worldwide. Replacing the corneal endothelium only in a patient with Pseudophakic/ Aphakic Bullous Keratoplasty or Fuchs’ Dystrophy rather than replacing the entire cornea with a full-thickness Penetrating Keratoplasty has gone from the research interest of a few pioneering surgeons to mainstream in an amazingly short time. In addition to DLEK, we now have Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK) competing for our attention. At the same time, the Intralase femtosecond laser is being harnessed in an attempt to make Penetrating Keratoplasty more precise with the promise of more predictable refractive outcomes. It is truly not only an exciting time for the corneal surgeon, but also a demanding one requiring the rapid assimilation of new knowledge as well as the development of new surgical skills. Fortunately, Thomas John MD has produced for us a timely and outstanding educational offering in his new book “Corneal Endothelial Transplant (DSAEK, DMEK & DLEK)”. This very comprehensive book includes the history of lamellar transplantation; an in-depth discussion of the basic science of corneal structure, physiology, biomechanics and pathology; a primer on advanced corneal imaging; a review of the surgical instrumentation required; and of utmost importance, detailed instruction by the leaders in the field on the current best practices of surgical technique and complications management. A final section provides an enticing glimpse to the future. This book is complete enough to serve the corneal fellow well and advanced enough for even the most accomplished corneal surgeons to include in their personal library. Thank you, Dr John and colleagues, for providing we corneal surgeons with such an extraordinary educational resource. Richard L Lindstrom MD Founder and Attending Surgeon Minnesota Eye Consultants Adjunct Professor Emeritus Department of Ophthalmology, University of Minnesota Associate Director, Minnesota Lions Eye Bank, USA

Preface .............................................. This book entitled, Corneal Endothelial Transplant (DSAEK, DMEK & DLEK) is dedicated to the new way of performing corneal transplantation namely, without the use of corneal sutures and an absence of a full-thickness corneal wound. Such a move towards advanced corneal replacement surgery eliminates the induction of much disliked corneal astigmatism. Such a textbook provides the corneal surgeon with a variety of surgical techniques and instrumentation that will be a useful surgical resource for posterior lamellar keratoplasty procedures. For several decades, full-thickness penetrating keratoplasty (PKP) has dominated the field of corneal transplantation and has remained as the gold standard for corneal replacement surgery. However, the time has come when improved lamellar corneal techniques has re-appeared in the global horizon as a rapidly popular surgical technique and is beginning to challenge and possibly replace PKP as the gold standard in the times ahead. The editor has previously introduced a new term namely, Selective Tissue Corneal Transplantation (STCT) which may become the procedure of choice in many corneal disease processes. Why remove the whole cornea when the pathology may be limited regionally to either the front, middle or back part of the cornea? I have previously edited two books that covered both anterior and posterior lamellar keratoplasty, entitled, Surgical Techniques in Anterior and Posterior Lamellar Corneal Surgery, and Step by Step Anterior and Posterior Lamellar Keratoplasty. Due to the increasing popularity of posterior lamellar keratoplasty among corneal surgeons all over the world and the rapidly changing and evolving sutureless corneal transplantation techniques, this book collectively provides the reader with a wide assortment of surgical techniques from world leaders in the field of sutureless corneal transplantation as we know it today (at the time of writing this book). This book has numerous color photographs to assist in fully understanding the various surgical techniques described in the text. The editor and contributors have made it their priority to present the surgical techniques in a way that is easily understood by the readers of this textbook. This surgical text consists of 11 sections and 39 chapters. In Section 1, new areas of interest such as corneal hysteresis and biomechanical properties of the normal cornea are described. In addition, corneal physiology is covered. Also described in this section is the most important layer of the cornea that is responsible for corneal clarity, namely, corneal endothelium, both in health and in the disease state. In Section 2, new ways of imaging the human cornea are described. In vivo, real-time imaging of the cornea provides useful information both before and after surgery. This includes, Optical Coherence Tomography (OCT), Very High Frequency (VHF) ultrasound and confocal microscopy. The area of imaging covers both the cornea and the anterior segment. Section 3 presents the new generation operating microscope. This futuristic microscope provides a 3D perspective that is novel and may change the way we perform ophthalmic surgery in the future. Also included in this section is the use of intraoperative surgical slit-lamp microscope to assist in lamellar corneal surgery, namely, both anterior and posterior lamellar keratoplasty. The next section deals with the various new and useful surgical tools for the corneal surgeon to assist in performing sutureless corneal transplantation. Much like a paint brush is to an artist, so is the proper surgical instrument to the surgeon that will help in consistently performing high quality surgical work. It is not enough to have the best microscope and operating room setup. Equally or more important are the appropriate surgical instruments. This section describes the various surgical instruments that are commercially available to assist in performing posterior lamellar keratoplasty. Section 5 deals with an essential and important part of the instrumentation to perform posterior lamellar keratoplasty, namely, the artificial anterior chamber. A good understanding of the various types of artificial anterior chambers that are available will help the surgeon doing posterior lamellar keratoplasty. Both non-disposable and disposable types of artificial anterior chambers are described in this section of the book. This is especially important when the surgeon prefers to cut his or her own donor corneal tissue in the operating room rather than to use the corneal tissue that are pre-cut by eye bank technicians and supplied by the various eye banks in the United States for an additional fee. Newly introduced in the United States is a reimbursement code for surgeons preparing and cutting their own donor corneal disk for DSAEK, in addition to the DSAEK code for the DSAEK surgery.

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Corneal Endothelial Transplant

Section 6 deals with the definition, various terminologies, and classification of lamellar corneal surgery. This includes both the anterior and posterior lamellar keratoplasty. A good understanding of these terminologies that are currently in use will be beneficial to the lamellar surgeon and to all those interested in the field of lamellar corneal surgery. It is of great interest to go back in time and learn what the pioneers in the field of lamellar keratoplasty had to go through in order to arrive at the present-day surgical techniques that have simplified the posterior lamellar keratoplasty procedure. Such improved and simplified posterior lamellar techniques have fuelled the interest in lamellar surgery among corneal surgeons all over the world and their continued rapid conversion from the familiar, full-thickness penetrating keratoplasty to the not so familiar sutureless corneal transplantation. In this regard, Section 7 covers the history of lamellar and penetrating keratoplasty. Section 8 covers various aspects of Deep Lamellar Endothelial Keratoplasty (DLEK), including the large incision technique, small incision technique, combined phacoemulsification along with DLEK and there is a final chapter in this section that describes DLEK along with scleral-fixated posterior chamber intraocular lens implant. Section 9 is of great interest to all corneal surgeons looking at Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK). This section starts off with a chapter on eye banking issues and donor tissue preparation in DSAEK. This is an important chapter since there are many corneal surgeons who do not “cut” their own donor corneal tissue and instead elect to use eye bank technician cut donor corneal tissue for their patients. Also, in this section there is a chapter on the use of eye bank pre-cut donor tissue for DSAEK surgery. Also, in this section, various leading posterior lamellar corneal surgeons from both the United States and from other parts of the world describe their surgical techniques in performing posterior lamellar keratoplasty. Armed with this knowledge, the reader can elect to choose the surgical technique that appeals most to the individual surgeon. The techniques vary from folding the donor corneal disk into a taco-fold, to the burrito trifold, to no-fold (no taco fold). Varying amounts of donor corneal endothelial cell loss is inevitable at the present time with all exisiting surgical techniques, and such endothelial cell loss occurs during the handling of the donor corneal disk, insertion of the donor disk into the patient’s anterior chamber, and subsequent attachment of the disk to the inner surface of the patient’s cornea. There is a continued search for techniques that will allow for the least amount of endothelial cell loss in DSAEK surgery. Equally important, is the chapter on the visual and refractive outcomes following DSAEK. Also of great interest to the lamellar surgeon is the chapter on simplified technique and instrumentation in performing DSAEK. Also included in this section is the use of femtosecond laser (Intralase) for DSEK surgery. Once posterior lamellar keratoplasty is performed, the surgeon needs to learn various techniques to keep the donor disk attached to the inner surface of the patient’s cornea. In this regard, there is a chapter on surgical techniques to facilitate donor disk adherence to the patient’s cornea. The surgeon can use a single technique or a combination of techniques to decrease the disc detachment rate following DSAEK surgery. Also included in the same section is a chapter on the management of complications and a chapter on the unanswered questions in DSAEK. Section 10 compares the older form of sutureless corneal transplantation technique namely DLEK to the newer technique of DSAEK surgery. This section also describes the various staining techniques using the commercially available dyes that will allow better visualization of the donor corneal disk within the patient’s anterior chamber through a cloudy cornea. Staining in addition to providing better visualization of the donor disk also helps to identify the donor stromal surface from the donor endothelial surface. Included in the same section is a chapter on the comparative visual recovery in DSAEK, DLEK and PKP surgeries. The final section in this book, Section 11 gives a glimpse of what the future holds for posterior lamellar keratoplasty. In this section there are chapters on the use of tissue adhesive, to a novel approach for corneal endothelial cell transplantation using Descemet’s membrane as a carrier. There is also a chapter on true endothelial cell (Tencell) transplantation. Also included is a new technique of Descemet Membrane Endothelial Keratoplasty (DMEK). In addition, there is a chapter on corneal endothelial reconstruction with a bioengineered cell sheet. The final chapter in this book projects the possible future of posterior lamellar keratoplasty. This book is a comprehensive textbook in sutureless posterior lamellar corneal surgery that the reader would enjoy as he or she travels through this wide landscape of surgical techniques and instrumentation as it relates to the current status of posterior lamellar corneal surgery. Continued improvements and refinements of the surgical techniques by the ophthalmic surgeons can only incrementally benefit their patients all over the world. Thomas John MD

Acknowledgments ........................... I acknowledge all those who contributed to this book on “Corneal Endothelial Transplant,” by taking time from their busy schedules to write their chapter(s). A collective contribution and passion for their surgical pursuits makes this compilation valuable for readers all over the world. I wish to acknowledge all my teachers in the Cornea Service, Massachusetts Eye and Ear Infirmary (MEEI), Harvard Medical School, Boston, MA, USA, from whom I have learnt immensely both in the clinical and research aspects of “Cornea”. I am thankful to Drs Kenneth R Kenyon, Claes H Dohlman, C Stephen Foster, Roger F Steinert, Deberoah P Langston, Mark B Abelson, Michael D Wagoner, Jeffrey P Gilbard, Arthur S Boruchoff, and Ann M Bajart for all their dedication and effort in teaching surgical and medical skills relating to cornea and external diseases while I was a 2-year Clinical Cornea Fellow at Harvard. I wish to thank Dr Kenyon, under whose expert guidance I did my research work both at the Schepens Eye Research Institute and at the Massachusetts Institute of Technology (MIT), in Boston. I am fortunate to have worked with my colleagues, cornea fellows and residents at MEEI during my fellowship years, to name a few, Drs Mitchell C Gilbert, Eduardo C Alfonso, Kazuo Tsubota, Scheffer CG Tseng, Dimitri T Azar, John R Wittpenn, and Oliver D Schein. Special thanks to Drs James V Aquavella and Gullapalli N Rao for what they taught me in corneal surgery including epikeratoplasty, refractive surgical procedures, and keratoprosthesis. I am thankful to all my teachers in my formative years during my ophthalmology residency at the University of Pennsylvania. Although, not an all inclusive list, special thanks to Drs Ralph C Eagle, Jr, Myron Yanoff, John H Rockey, Irving M Raber, Alexander J Brucker, David M Kozart, William C Frayer, Harold Scheie, and Madeleine Q Ewing. I am especially thankful to Ralph C Eagle, Jr, MD, for all his support and professional inspiration, and for teaching me the various pathological basis of disease processes as it relates to the eye. I thank Myron Yanoff for accepting me into the ophthalmology residency program at the University of Pennsylvania. Teachers are one of the greatest assets of any society. I thank all my teachers from kindergarten to completion of my formal education both in the medical and pre-medical years. Without these teachers, I will be lacking in knowledge and I am indebted to each and every one of my teachers. I wish to acknowledge my wife, Annita, and the kids, Michelle, Andrea and Olivia for putting up with my late night academic work and for all their understanding and loving support. Thanks to Laura Phelps for the excellent medical illustrations in this book. To my office staff, for their patience and understanding. To all my patients, from whom I continue to learn everyday. Learning is a continuous and dynamic process that stimulates the mind and makes ophthalmology an even more interesting and challenging field in our life’s journey.

Introduction..................................... Sutures have been a necessary evil in most forms of corneal surgery. Sutures have historically been necessary to obtain a secure incision during the healing phase. The introduction of sutures in cataract surgery, utilizing large re-usable needles that the surgeon had to thread, in a manner like a tailor, represented a major breakthrough in rehabilitation after cataract surgery. As the needles and suture material became smaller and the number of possible sutures in a single incision increased, the patient rehabilitation time improved dramatically while the complication rate fell. No longer were cataract patients restricted to bed rest with their heads stabilized by sand bags, awaiting for healing of a limbal incision secured only by an overlying conjunctival flap. The problem with sutures in cataract surgery, of course, was the impossibility of precise control of the suture tension and placement. After prolonged healing, typically lasting several months, the patient might have high amounts of astigmatism representing either excessively tight or excessively loose sutures. These issues are now largely a historical footnote, as cataract surgery has come full circle. The drive for smaller incisions in cataract surgery allowed the creation of a self-sealing “valve” incision that, because of its inherent water tightness and structural stability, permits surgeons to use no sutures in many cases. In coming full circle and returning to sutureless cataract surgery, the evolution of the “valve” style incision represented a re-learning of the incision shape that helped cataract surgeons with sutureless incisions in the early 20th century, because the Graefe knife incisions of that era also created the same valve effect, unfortunately limited by the extreme width of the incision necessary to perform the whole lens surgery of that era. The lessons of sutures in cataract surgery apply even more to corneal surgery. In corneal transplantation, until recently, sutures have been mandatory to align and stabilize the junction of the donor and the recipient cornea. Because the cornea is slow to heal, those sutures must be retained much longer than in cataract surgery. In almost all cases, the sutures are also considerably closer to the optical center and the limbus. This proximity dramatically increases the negative impact of suture tension. Despite decades of improvement in suture materials, needles, and ingenious variations in suturing technique and suture patterns, the problem of distortion and slow healing of corneal incisions has remained as a powerful impediment to high quality vision after corneal transplantation. Indeed, another of the ironies in this story of sutures in ophthalmology came with a shift from silk sutures to fine nylon sutures for corneal transplantation. Silk sutures caused intense inflammation, vascularization, and higher rejection rates as well as patient discomfort. However, if the transplant survived, the patient benefited by the stimulation of more rapid incision healing and full suture removal much earlier than is possible with nylon sutures. The use of non-inflammatory material, therefore, caused a further shift in the direction of prolonged dependency on sutures and vulnerability to the negative impact of those sutures in corneal surgery. This outstanding text, conceived and edited by Thomas John, MD thoroughly explores the dramatic shift under way toward corneal transplantation without corneal sutures. The text thoroughly develops the background technologies that are the foundation of lamellar endothelial transplantation. Current endothelial transplant surgery typically still involves a few limbal sutures, but the future is clearly in the direction of transplantation of endothelial cells alone. That will complete the transformation to a completely sutureless corneal transplantation. Roger F Steinert MD Chair of Ophthalmology Director of the Gavin Herbert Eye Institute Professor of Ophthalmology and Biomedical Engineering Department of Ophthalmology University of California, Irvine, CA, USA

Perspective ....................................... Seems like just a year ago that we witnessed publication of Thomas John’s definitive magnum opus of lamellar keratoplasty, “Surgical Techniques in Anterior and Posterior Lamellar Corneal Surgery” (Jaypee Brothers, New Delhi, 2006). Can it really be time for yet another multifaceted, multi-authored work on posterior lamellar keratoplasty ?? Answer: Absolutely !! The Back to the Future of century old anterior lamellar keratoplasty techniques have now been extended and adapted for posterior corneal application plus technologically enhanced by current imaging and surgical instrumentation. This Paradigm Shift to Targeted TissueSpecific Keratoplasty is clearly the greatest advance in corneal surgery to occur within the last half century (Top 10 Ophthalmic Innovations of the Past 25 Years, Ocular Surgery News, Dec. 2007). Such rapid developments of the past decade, thanks to Dr. John and several of the nearly 50 international authors contributing to the current treatise, have propelled Endothelial Keratoplasty to have become the nearly standard approach for the surgical management of corneal endothelial disease. Little more than a year ago has passed since my own pilgrimage to Chicago (?or was it Lourdes or perhaps Mecca…?) to witness and learn from the Master K-Plaster, Tom John, himself. Thusly converted, I can now personally attest to the Miracle of Posterior Lamellar Keratoplasty. True, this surgery and its variations requires mastery of new surgical skills as well as problem solving and complication management, as does any novel operative technique. Yet having witnessed the surgery and its results (including the increasingly long-term published series, as are also included herein), the Bottom Line is all too obvious: Once you go DSEK, you never go back ! And so it is that Dr. John and friends rapidly push forward and relentlessly expand the frontiers through their current approaches to the state of the sutureless keratoplasty art. Including sections devoted to corneal basic science, tissue imaging, surgical instrumentation, basic themes plus multiple variations of endokeratoplasty technique, and future directions, this opus is a definitive work of equally magnum magnitude which should convince even more conservative corneal surgeons that the Back to the Future of lamellar keratoplasty is unquestionably now. Finally, I must also express my personal and professional appreciation to Thomas for both the honor of including me among his illustrious cast of coauthors but especially for the Epiphany of helping me to Perceive the Light. Kenneth R Kenyon MD Founder, Cornea Consultants International Boston, MA, USA & Munich, Germany Associate Clinical Professor of Ophthalmology Harvard Medical School, Boston, MA, USA Senior Scientist, Schepens Eye Research Institute Boston, MA, USA

Contents ........................................... Section 1: Cornea 1. Corneal Hysteresis and Biomechanics of the Normal Cornea ...................................................................................... 3 Mujtaba A Qazi, Jay S Pepose (USA) 2. Corneal Physiology ............................................................................................................................................................. 13 Wisam A Shihadeh (Jordan), Almamoun Abdelkader (Egypt), Herbert E Kaufman (USA) 3. Corneal Endothelium in Health and Disease ................................................................................................................ 23 Pedram Hamrah, Eric C Amesbury, Richard A Eiferman (USA)

Section 2: Corneal Imaging 4. Optical Coherence Tomography (OCT) of the Anterior Segment ............................................................................... 39 George Baikoff (France) 5. Optical Coherence Tomography in Corneal Implant Surgery ..................................................................................... 47 Roger F Steinert (USA) 6. Use of Optical Coherence Tomography (OCT) in Descemet’s Stripping with Endothelial Keratoplasty (DSEK) and Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK) ....................... 53 Leejee H Suh, William W Culbertson (USA) 7. Imaging of the Cornea and Anterior Segment with High-Frequency Ultrasound ................................................... 61 Ronald H Silverman, Monica Patel, Omer Gal, Harriet O Lloyd (USA) D Dan Z Reinstein (UK), D Jackson Coleman (USA) 8. Confocal Microscopy of the Cornea ......................................................................................................................................................71 Jasmeet S Dhaliwal (USA), Auguste G-Y Chiou (Switzerland), Stephen C Kaufman (USA)

Section 3: Next Generation Operating Microscope 9. Next Generation Operating Microscope: 3D Digital Microscope and Microsurgical Workstation ....................... 85 Ramagopal Rao, David Miller (USA) 10. Role of Surgical Slit-lamp in Endothelial Transplantation and Anterior Segment Surgery ................................... 95 Thomas John (USA)

Section 4: Surgical Instruments 11. New/Useful Surgical Instruments in DSAEK .............................................................................................................. 107 Thomas John (USA)

Section 5: Artificial Anterior Chambers 12. Artificial Anterior Chambers .......................................................................................................................................... 123 Thomas John (USA)

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Section 6: Classification of Lamellar Corneal Surgery 13. Definition, Terminology and Classification of Lamellar Corneal Surgery .............................................................. 133 Thomas John (USA), Enrique S Malbran (Argentina)

Section 7: History 14. History of Lamellar and Penetrating Keratoplasty ...................................................................................................... 143 Thomas John (USA), Luiz F Regis-Pacheco (Brazil), José G Pecego (Brazil), Mark A Terry (USA)

Section 8: Deep Lamellar Endothelial Keratoplasty (DLEK) 15. Deep Lamellar Endothelial Keratoplasty (DLEK): Large Incision Technique ....................................................... 157 Mark A Terry, Paula J Ousley (USA) 16. Deep Lamellar Endothelial Keratoplasty (DLEK): A Procedure for Special Cases of Endothelial Dysfunction .................................................................................................................................. 171 Mark A Terry (USA) 17. Deep Lamellar Endothelial Keratoplasty: Small Incision Technique Combined with Phacoemulsification and Posterior Chamber IOL .......................................................................... 183 Mark A Terry (USA) 18. Deep Lamellar Endothelial Keratoplasty (DLEK) Combined with Scleral-fixated Posterior Chamber Intraocular Lens Implantation ...................................................................................................................... 201 Ashraf Amayem, Magdi Helal (Saudi Arabia)

Section 9: Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK) 19. Eye Banking and Donor Corneal Tissue Preparation in DSAEK ............................................................................. 217 Anastasios John Kanellopoulos (Greece) 20. Endothelial Keratoplasty: A Step by Step Guide to DSEK and DSAEK Surgery ................................................... 225 Mark A Terry (USA) 21. Improved DSAEK Surgery for Enhanced Endothelial Survival ................................................................................ 237 Massimo Busin, Vincenzo Scorcia (Italy) 22. Endothelial Keratoplasty: Visual and Refractive Outcomes ...................................................................................... 245 Marianne O Price, Francis W Price (USA) 23. DSAEK Simplified Surgical Technique ....................................................................................................................... 253 Thomas John (USA) 24. Surgical Technique for Descemet Stripping Automated Endothelial Keratoplasty (DSAEK) ............................ 281 Mark S Gorovoy (USA) 25. Descemet’s Stripping Endothelial Keratoplasty (DSEK), Through a 3 mm Incision using the Tri-fold Technique ........................................................................................................................................................... 289 Keith A Walter, Marshall E Tyler (USA) 26. Femtosecond Laser (Intralase®) – Descemet’s Stripping Endothelial Keratoplasty (Femto-DSEK): Initial Studies of Surgical Technique in Human Eyes ............................................................................................... 293 Ciro Tamburrelli, Agostino Salvatore Vaiano, Emilio Balestrazzi (Italy)

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27. Techniques to Facilitate Disk Adherence to Recipient Cornea in DSAEK ............................................................. 303 Thomas John (USA) 28. Complication Management in DSAEK ......................................................................................................................... 311 Thomas John (USA) 29. Unanswered Questions in DSAEK ................................................................................................................................ 327 Thomas John (USA) 30. Use of Eye Bank Pre-cut Donor Tissue in DSAEK ...................................................................................................... 331 Anthony Kuo, Terry Kim (USA)

Seciton 10: DLEK Versus DSAEK 31. Comparison of Wound Architecture in DLEK Versus DSAEK ................................................................................. 343 Thomas John (USA) 32. Use of Dyes in DSAEK and DLEK ................................................................................................................................. 349 Thomas John (USA) 33. Comparative Visual Recovery in DSAEK, DLEK and PKP ........................................................................................ 361 Mark S Gorovoy (USA)

Section 11: DMEK and Future Directions in Posterior Lamellar Keratoplasty 34. Posterior Lamellar Keratoplasty Using Tissue Adhesive ........................................................................................... 367 Juan M Castro-Combs, Naima B Jacobs-El, Ashley Behrens (USA) 35. Novel Approach for Corneal Endothelial Cell Transplantation using Descemet Membrane as a Carrier ........ 377 Naima B Jacobs-El, Juan M Castro-Combs, Ashley Behrens (USA) 36. True Endothelial Cell (TEnCell) Transplantation ........................................................................................................ 389 Panagiotis Georgoudis, Michael J Tappin (UK) 37. Descemet Membrane Endothelial Keratoplasty (DMEK) ......................................................................................... 399 Massimo Busin (Italy) 38. Corneal Endothelial Reconstruction with a Bioengineered Cell Sheet .................................................................... 405 Jui-Yang Lai, Ging-Ho Hsiue (China) 39. Future of Posterior Lamellar Keratoplasty .................................................................................................................... 421 Thomas John, Kenneth R Kenyon (USA)

Index ..................................................................................................................................................................................... 425

Mujtaba A Qazi, Jay S Pepose

Corneal Hysteresis and Biomechanics of the Normal Cornea

1

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Corneal Endothelial Transplant

Introduction The structural characteristics of the cornea facilitate its essential functions, specifically to serve as both a transparent barrier and the predominant refractive element of the eye. Given the integral relationship between form and visual function, the biological and mechanical responses of the cornea to surgical interventions impact its optical performance. While major advances have occurred in the refinement and standardization of corneal surgical techniques, our ability to predict individual biological responses to surgery remains limited and can influence the predictability and stability of visual outcomes after corneal surgery. Some of these biomechanical responses are seen immediately, e.g., following lamellar keratectomy and/or laser ablation.1 Others may manifest in shape instability over time or with further surgery, or result in serious complications such as wound dehiscence, scarring, haze formation, and induction of irregular astigmatism. Corneal biomechanical characteristics often change with wound healing, and also may be better understood in the context of whole eye rigidity.2 In this chapter, we highlight the biochemical and ultrastructural features that most strongly contribute to the biomechanical properties of the normal cornea. Further understanding of these factors provide the basis for improving outcomes and reducing complications of corneal surgery, by identifying individual response outliers and developing strategies for regulating or compensating for these biomechanical features.3 While more familiar to engineers than to most ophthalmologists, we also review the definition of corneal hysteresis and other metrics that have been applied to studies of corneal biomechanics — a subject that encompasses the effects of corneal hydration, regional pachymetry, viscoelasticity and other inherent corneal characteristics that may not yet be fully defined.

Collagen Structure of the Cornea The tensile integrity and refractive curvature of the cornea is determined in large part by the stroma, which represent the bulk of the corneal thickness. On a weight basis, the stroma is approximately 78% water, 15% collagen and 7% non-collagenous proteins and proteoglycans.4 Approximately 300 collagen lamellae, spanning from limbus to limbus, comprise the center of the cornea.5 This number increases to about 500 as the cornea thickens toward the periphery.4,5 Presumably, this occurs from branching of the lamellae, with some lamellae branches merging with others.6 Branching is seen more extensively in the corneal periphery, where there is primarily a circumferential

orientation of the fibrils.7,8 Branching and interlacing of lamellae has been implicated to play an important role in corneal tensile strength.9 The orientation and spacing of the collagen fibrils appears to be controlled by stromal proteoglycans. Swelling studies have shown that the interlamellar adhesive strength of the central cornea depends upon proteoglycan bonding, whereas branching and interlacing of lamellae provides additional adhesive strength peripherally.7 Changes in the proteoglycan matrix may explain the increased pliability of the central cornea in keratoconus and may potentially impact the corneal response to keratorefractive surgery, contact lens wear and tonometric testing.10 The anterior-most stromal lamellae have oblique branching and interweaving fibers that insert into Bowman’s layer.11 Because of these features, the anterior stroma swells less and is about 25% stiffer than its posterior counterpart.12 Similarly, as there is greater interlacing of peripheral fibers, swelling of the peripheral cornea is usually less than in the central stroma.13 These findings suggest that peripheral and/or posterior incisional surgery may have less of a profound impact on corneal biomechanics than anterior, central surgery (Table 1-1). It appears that corneal shape is not determined on a random basis, but results from a steady state balance between the biomechanical properties of the cornea and intraocular pressure (IOP).14 The cornea assumes the shape for which its potential energy content is minimal and for which its stromal fibrils are in a relatively relaxed state, as a function of variables such as tissue elasticity, thickness, fibril length, rate of change of IOP, among others. External physiologic corneal stresses, such as from normal blinking or diurnal variation in IOP, and non-physiologic corneal stresses, caused by increases in IOP from forceful lid closure or rubbing, may potentially impact the corneal shape. However, normal corneas have been found to show low extensibility, measured by changes in anterior surface sagittal height, for a wide range of physiologic conditions and even with marked elevations in IOP in order to maintain refractive stability. 15 Conversely, when the corneal biomechanical properties are altered via incisional surgery TABLE 1-1: Local variation in corneal lamellar ultrastructure Collagen lamellae in the peripheral cornea: 1. Greater number 2. Greater branching and interlacing 3. Circumferential orientation 4. Greater resistance to swelling Collagen lamellae in the anterior cornea: 1. Anterior strands insert into Bowman’s layer 2. Greater proteoglycan bonding centrally 3. Greater stiffness than posterior cornea

Corneal Hysteresis and Biomechanics of the Normal Cornea

5

such as radial keratotomy, diurnal variation in IOP can lead to fluctuation in corneal refractive power by greater than one diopter (D).15

Metrics of Corneal Biomechanical Properties In the terminology of material science, the cornea is a complex composite (of collagen, other proteins, proteoglycans, water, and salts) with non-linear elastic and viscoelastic properties characterized by important local variation in organization in central versus peripheral and anterior versus posterior dimensions. Mathematical modeling of such a complex system is therefore quite difficult, but begins with identification of intrinsic properties of corneal tissue, as described below (Table 1-2). TABLE 1-2: Descriptors of corneal biomechanical properties 1. 2. 3. 4. 5. 6. 7. 8. 9.

Elasticity Viscoelasticity Hysteresis ORA corneal resistance factor Creep Stress relaxation Sheer strength Ocular rigidity PASCAL ocular pulse amplitude

Elasticity (Young’s modulus, E) is an indicator of material stiffness, with a higher modulus corresponding to a stiffer material. For example, a metallic rod would have a higher modulus than a wood rod (Figure 1-1). A perfectly elastic material returns to its original form, when an external stress is withdrawn, in a completely reversible and symmetric manner, i.e., along the same stress-strain pathway.16 Elasticity is traditionally measured ex vivo with an extensiometer that records the force generation required during steady axial elongation of a tissue sample. The slope of stress (force per unit area) over strain (the current length divided by the starting length) is calculated for a representative portion of the curve. A linear approximation can be obtained from the instantaneous slope of the stressstrain curve (tangent modulus) or as a chord between two points on the curve (secant modulus). A limitation of extensiometer measurement of elasticity is that the range of in vivo corneal elasticity modulus in healthy or diseased tissue is unknown,17 and reports on animal and human tissues can span orders of magnitude.15,18 Additionally, while most biological soft tissues approximate linear elastic behavior when a small range of stresses are introduced, their overall elastic behavior is highly non-linear. Nevertheless, understanding elastic

Figure1-1: The influence of structural and material properties upon the ability to deform the cornea. Bending a single chopstick is usually easy. However, bending three of the same type of chopsticks at once is much more difficult (top row). Hence, a larger deformation will be generated for thinner corneas given the same applied force. This partially explains the underestimation of IOP in eyes with thinner corneas. In contrast, it requires greater pressure to applanate or indent a thicker cornea, which contributes to overestimation of IOP in eyes with thicker corneas. Similarly, much more force is required to bend a steel rod than a wood rod of the same dimensions (middle row). The difference in this case is the elastic properties of the material, specifically Young’s modulus. Steel has a much higher Young’s modulus (w200 000 MPa) than wood (w10 000 MPa); therefore, if all other parameters are the same, it is much harder to deform a steel structure than a wood structure. Corneal curvature is another variable that can affect the accuracy of IOP measurement, possibly because of the difference in the volume of the displaced fluid after a given area is flattened (bottom row).17

properties is important for evaluating the instantaneous response of the cornea to surgery and can affect its subsequent viscous behavior. Some studies have demonstrated, for example, a decrease in stiffness and increase in extensibility of keratoconic tissue relative to normal tissue.19 The elastic modulus was identified by Guirao 20 in mathematical modeling as the most influential risk factor for posterior corneal steepening after keratorefractive surgery. Viscoelasticity extends the biomechanical response of biological tissues into complex mathematical descriptions of viscous fluids, where elastic responses are time and rate dependant.21 Viscoelastic materials return to their pre-stress shape via different stress-strain pathways that depend upon loading rates. Viscoelastic properties can be described through metrics of hysteresis, stress relaxation and creep. Viscoelastic creep is a time-dependent elongation of tissue (or increasing strain) that occurs under a sustained or constant stress (such as IOP).21 The effect of creep is a reduction in effective tissue stiffness, which can lead to a decrease in resistance to stretch. Creep may be a precursor to ectasia, where stressed collagen fibrils undergo a

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Figure 1-2: Experiments illustrating elastic and viscoelastic properties in a 7 mm, full-thickness horizontal corneal strip from a 63-year-old donor. Elliptical polarization allows visualization of non-homogeneous internal stresses. Progressive stretching of the sample (1, 2 and 3) and measurement of the induced load (stress) allows calculation of the elastic (Young’s) modulus from the slope of the stress-strain relationship. The relationship is non-linear. A second experiment in which a constant displacement is imposed in the same sample demonstrates time-dependent stress relaxation, a viscoelastic property of biological soft tissues (4 and 5). (Courtesy of W.J. Dupps, Jr., MD, PhD and T. Doehring, PhD.16)

pathologic weakening without an initial change in length. Once the collagen fibrils are weakened, a gradual stretching then occurs under constant stress or IOP. Viscoelastic stress relaxation refers to a situation where strain is increased then held constant (no more tissue elongation) while a slow but quantifiable time-dependent relaxation of the load is observed (Figure 1-2). Hysteresis, in general, is a property of physical and biological systems that do not instantly follow the forces applied to them but react slowly or do not return completely to their original state.21 Hysteresis describes a lag between making a change, such as increasing or decreasing power, and the response or effect of that change. Whereas a rubber band can be described as elastic because is springs back to its original shape at the same rate as when it is stretched, a putty exhibiting viscoelastic behavior quickly assumes a new shape when pushed upon but will not immediately return to its original shape when the mechanical pressure is released. Another example of hysteresis is a thermostat set at 80 degrees, which actually regulates the room temperature between 78 and 82 degrees. In broad terms, corneal hysteresis can be thought of as a metric of the ability of the cornea to absorb energy. Shear strength describes stromal resistance to lamellar sliding and bending. The shear resistance provided by collagen interweaving and other matrix forces has been

estimated from metrics such as the interlamellar cohesive strength.7 Corneal shear strength is low relative to its tensile strength, but provides a mechanism for tensile load transfer between lamellae.9 Volumetric distension experiments provide a measure of whole globe stiffness, or ocular rigidity. The slope of a pressure-volume curve can be recorded during such experiments. Ocular rigidity is non-linearly dependent upon IOP and has been shown to increase with age.2 The utility of metrics describing ocular rigidity may be limited with respect to their impact on the understanding of corneal surgery, given the contributory role of the scleral and uveal tissue to ocular rigidity.

New Techniques for in vivo Measurement of Corneal Biomechanical Properties While ex vivo diagnostic techniques, such as extensiometry, have provided valuable information on the biomechanical nature of normal and pathological corneas,22 a new era is dawning in biomechanical research with the development of techniques to measure structural and biomechanical properties in vivo. Imaging of the cornea can now be performed by confocal microscopy,23 very high frequency,24

Corneal Hysteresis and Biomechanics of the Normal Cornea and optical coherence technology (See Section 2: Corneal Imaging). Holographic interferometry uses optical comparison to evaluate corneal elasticity.25,26 Another method, dynamic corneal imaging, uses stepwise central indentation of the cornea and computer analysis of videokeratography images during indentation to assess corneal elastic properties in vivo. 27 A commercially available device that uses a dynamic, bidirectional, airpuff applanation to measure in vivo IOP and corneal viscoelastic properties is the Ocular Response Analyzer ([ORA], Reichert Ophthalmic Instruments, Depew, New York, USA).28

Biomechanics and Intraocular Pressure Increasing attention has focused on the impact of corneal parameters, particularly central corneal thickness (CCT), on the measurement of IOP.29 IOP measurements have been demonstrated to vary with CCT using the Goldmann applanation, 30 pneumotonometry,31 and non-contact tonometry. 32 The deformation of the cornea during applanation is determined by an interaction of the external applied force with the intrinsic properties of the cornea. With the same applied force, a larger deformation will be produced for less rigid corneas. This partially explains the underestimation of IOP in eyes with thinner corneas. In contrast, it requires greater force to applanate a more rigid cornea, partially explaining the overestimation of IOP in eyes with thicker corneas. Additionally, alteration of corneal biomechanics by LASIK flap creation and excimer laser ablation affects the postoperative measurement of intraocular pressure (IOP) using Goldmann applanation tonometry (GAT).33-35 The impact of pachymetry on intraocular pressure readings has been recently highlighted by the Ocular Hypertension Treatment Study,36 which demonstrated an inverse relationship between CCT and the risk of developing glaucoma. Goldmann tonometry is a static measurement, calculating IOP from the force applied during a steady state applanation of the cornea.37 Its design is based upon a number of assumptions, including that all corneas were of uniform thickness (i.e., 500 microns), that the eye’s volume was spherical and that the cornea behaved biomechanically as an infinitely thin and perfectly flexible membrane. Corneal biomechanics embody far more than central pachymetry alone, and include viscosity, elasticity, hydration, regional pachymetry and other factors.17 As an example, whereas corneas from patients with keratoconus are generally thinner than average and biomechanically

7

“floppy”, corneas from patients with Fuchs’ dystrophy are thicker than normal while they are also biomechanically similar to keratoconus.28 Other illustrative examples are the decrease in corneal rigidity following radial keratometry29 and hyperopic LASIK33 with subsequent drop in static applanation tonometry readings, yet little or no change in central corneal pachymetry. These examples highlight the complexity and potential flaws of simplistic attempts to linearly offset IOP based upon pachymetry alone. Alternative techniques that dynamically derive IOP from the corneal movement in response to a rapid air pulse simultaneously assess and compensate (to varying degrees) for the effect of the cornea’s viscous and elastic qualities on IOP measurement. Reichert’s ORA28 utilizes a metered collimated air pulse to applanate the cornea and an infrared electro-optical system to record inward and outward applanation events. The air-pulse deforms the cornea through an initial applanation event (peak 1), then beyond into concavity and then gradually subsides, allowing the cornea to rebound through a second applanation (peak 2) (Figure 1-3). Corneal hysteresis (CH) is defined as the difference between the applanation pressure at peak 1 (P1) and peak 2 (P2), so that CH = P1–P2. This dynamic assessment of corneal biomechanical properties yields metrics of both the cornea’s viscous and elastic qualities. Whereas corneal hysteresis may reflect mostly corneal viscosity, corneal resistance factor (CRF, defined by a linear function of P1 and P2) may predominantly quantify corneal rigidity. The equation used to determine CRF is: CRF = P1– 0.7 × P2. Pascal Dynamic Contour Tonometry (PDCT, Swiss Microtechnology AG; Port, Switzerland) employs a concave

Figure 1-3: The waveform generated from the ocular response analyzer identifies the pressure difference, or hysteresis, during inward (peak 1) and outward (peak 2) applanation events during non-contact tonometry.43

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Figure 1-4: Diurnal measurement of corneal hysteresis (left) and intraocular pressure (right) with the ocular response analyzer. While there was a statistically significant reduction in ORA IOP during the course of the day, diurnal measurements of hysteresis did not statistically differ.42

tip to “contour match,” rather than applanate, a convex segment of the central cornea and, thus, may be relatively independent of the effects of CCT or surgical intervention on IOP assessment.38-40 The instrument dynamically records over 100 IOP measurements per second, measuring IOP fluctuations throughout the cardiac cycle and digitally displaying the average diastolic IOP. Ocular Pulse Amplitude (OPA), the difference in IOP between systole and diastole (IOP systolic – IOP diastolic), is also reported and may be a marker for overall ocular rigidity,2 although it is also affected by ocular blood flow.

Clinical Applications of Hysteresis and Corneal Biomechanics Corneal hysteresis in normal eyes has been reported to range from 5.0 to 18.7, with a mean hysteresis of 9.6 to 12.7 mm Hg.28,41,42 Hysteresis values did not show a statistical difference in a cohort of 21 normal patients between right and left eyes, with a mean difference of 0.4 mm Hg (p>0.08). 42 Hysteresis values appear to be relatively insensitive to diurnal effects (Figure 1-4), although intrasubject variations have been observed. The correlation of CCT with CH and CRF was 0.59 and 0.62, respectively.41 Lower hysteresis was associated with visual field progression in a glaucomatous population.43 Corneas with keratoconus (Figure 1-5), Fuchs’ dystrophy and post-refractive surgery demonstrate a general decrease in corneal hysteresis compared to corneas in normal eyes.28 The low corneal hysteresis in the Fuchs’ eyes is seen despite unusually thick, but edematous, corneas. However, the large 99% confidence interval of corneal hysteresis seen in normal controls has considerable overlap with diseased

Figure 1-5: Distribution of hysteresis values in 339 normal (blue) and 60 keratoconic (red) eyes. Mean hysteresis for normal and keratoconic eyes was 9.6 mm Hg and 8.1 mm Hg, respectively.28

and post-surgical corneas, limiting its diagnostic value as a single metric in individual cases. What may turn out to allow better diagnostic differentiation are the significant changes seen in applanation waveform in diseased or postsurgical corneas. Keratoconic and post-LASIK corneas appear to have similar applanation signal morphology, indicating reduced or low corneal viscoelastic properties in both cases (Figure 1-6). Investigations to quantify morphologic characteristics of the ORA waveform are now underway that attempt to extract additional corneal biomechanical information. Corneal hysteresis may be useful as a qualification factor for LASIK in corneas that have similar CCT but display significantly different waveform properties. Thus, the ORA waveform, along with the derived biomechanical metrics of corneal hysteresis and resistance factor, may provide a more complete characterization of corneal biomechanical properties than corneal thickness alone and is perhaps a better tool for assessing refractive surgery qualification and outcomes.

Corneal Hysteresis and Biomechanics of the Normal Cornea

9

Figure 1-6: ORA applanation signal in keratoconic and post-LASIK eyes shows depressed applanation peak amplitudes and altered applanation peak widths. A: Case 1-4 are from keratoconic eyes. B: pre- and post-LASIK waveforms show a decrease in hysteresis postoperatively, along with reduced applanation peak amplitudes. C: waveform post penetrating keratoplasty shows marked alternation in the applanation signal, with increased noise during the applanation events.28

Modeling Based Upon Biomechanical Metrics: Implication for Surgical Planning Models of the cornea have taken many forms, including complex computational models that integrate structural, biomechanical and optical corneal properties.44 Dupps and

Wilson16 have proposed a strategy (Figure 1-7) for such modeling. By comparing these models to clinical experiments, useful models can be created, which can significantly improve our understanding of corneal biomechanics and allow us to better predict refractive effect after corneal transplant procedures.45 Results of finite element simulations indicate that significant changes in corneal refractive power could be introduced if refractive

Figure 1-7: An approach to biomechanical modeling of surgery and disease in the cornea. Disease is simulated by alteration of the substructural components or their material properties. Surgery is simulated by imposing an ablation profile or incisions. The model is optimized retrospectively by comparing model simulations to analogous experiments in tissue or clinical models. A model optimized with clinical data can then be used prospectively to design and evaluate patient-specific treatment algorithms.16

10

Corneal Endothelial Transplant wound healing and viscoelastic features of the normal and post-surgical cornea. As our understanding of these processes improves, so will our ability to offer rational interventions and strategies for further improving the predictability of keratorefractive surgery and minimizing its complications.

References

Figure 1-8: Major biomechanical loading forces in the cornea and a model of biomechanical central flattening associated with disruption of central lamellar segments. A reduction in lamellar tension in the peripheral stroma reduces resistance to swelling and an acute expansion of peripheral stromal volume results. Interlamellar cohesive forces and collagen interweaving, whose distribution is greater in the anterior and peripheral stroma and is indicated by grey shading, provide a means of transmitting centripetal forces to underlying lamellae. Because the central portions of these lamellae constitute the immediate postoperative surface, flattening of the optical surface occurs, resulting in hyperopic shift. The degree of flattening is associated with the amount of peripheral thickening. This phenomenon is exemplified clinically by PTK-induced hyperopic shift but is important in any central keratectomy, including PRK and LASIK. Simultaneous elastic weakening of the residual stromal bed may occur, and the threshold for inducing irreversible (plastic) or progressive (viscoelastic) steepening (or ectasia) is a matter of great clinical concern.16

procedures (Figure 1-8) are combined with corneal transplants. This requires high precision resections of corneal grafts, which may improve with the application of femtosecond laser technology.46 Treatment of the cornea with riboflavin and UVA to increase collagen crosslinking47 may also allow us to modulate the stiffness of the cornea before or after corneal transplant procedures.

Conclusion A review of the histology of corneal fibrils indicates evidence for inextensibility, under a wide range of physiologic conditions, which appears to be the basis for stability of refraction and corneal curvature. Given the major biomechanical loading forces of the normal cornea, surgical disruption of the central corneal lamellae may lead to central corneal flattening and peripheral thickening.1 Studying the dynamics of corneal shape changes that occur in response to collimated air pulses (via the Ocular Response Analyzer) may provide a basis for understanding the biomechanical effects of incisional and lamellar corneal surgery. The in vitro interactions of corneal fibroblasts and a fibrillar collagen substrate48 can be combined with advanced structural and functional in vivo diagnostic imaging techniques to develop mathematical models of the

1. Roberts C. The cornea is not a piece of plastic. J Refract Surg 2000;16:407-13. 2. Pallikaris IG, Kymionis GD, Ginis HS, Kounis GA, Tsilimbaris MK. Ocular rigidity in living human eyes. Invest Ophthalmol Vis Sci 2005;46:409-14. 3. Reinstein DZ, Roberts C. Biomechanics of corneal refractive surgery. J Refract Surg 2006;22:285. 4. Maurice DM. The cornea and sclera. In: Davson, H. (Ed.), The Eye. Academic Press, Orlando, FL, 1984;1-158. 5. Meek KM, Boote C. The organization of collagen in the corneal stroma. Exp Eye Res 2004;78:503-12. 6. Radner W, Zehetmayer M, Aufreiter R, Mallinger R. Interlacing and crossangle distribution of collagen lamellae in the human cornea. Cornea 1998;17:537-43. 7. Smolek MK, McCarey BE. Interlamellar adhesive strength in human eyebank corneas. Invest Ophthalmol Vis Sci 1990;31: 1087-95. 8. Meek KM, Newton RH. Organization of collagen fibrils in the corneal stroma in relation to mechanical properties and surgical practice. J Refract Surg 1999;15:695-9. 9. Dupps WJ, Roberts C. Effect of acute biomechanical changes on corneal curvature after photokeratectomy. J Refract Surg 2001;17:658-69. 10. McMonnies CW, Schief WK. Biomechanically coupled curvature transfer in normal and keratoconus corneal collagen. Eye Contact Lens 2006;32:51-62. 11. Komai Y, Ushiki T. The three-dimensional organization of collagen fibrils in the human cornea and sclera. Invest Ophthalmol Vis Sci 1991;32:2244-58. 12. Muller LJ, Pels E, Vrensen JM. The specific architecture of the anterior stroma accounts for maintenance of corneal curvature. Ophthalmology 2001;85:437-43. 13. Doughty MJ, Bergmanson JPG. Collagen fibril characteristics at the corneo-scleral boundary and rabbit corneal swelling. Clin Exp Optom 2004;87:81-92. 14. Sjontoft E, Edmund C. In vivo determination of Young’s modulus for the human cornea. Bull Math Biol 1987;49:217-32. 15. Jue B, Maurice DM. The mechanical properties of the rabbit and human cornea. J Biomech 1986;19:847-53. 16. Dupps WJ Jr, Wilson SE. Biomechanics and wound healing in the cornea. Exp Eye Res. 2006;83:709-20. 17. Liu J, Roberts CJ. Influence of corneal biomechanical properties on intraocular pressure measurement Quantitative analysis. J Cataract Refract Surg 2005;31:146–55. 18. Hoeltzel DA, Altman P, Buzard K, Choe K. Strip extensiometry for comparison of the mechanical response of bovine, rabbit, and human corneas. J Biomech Eng 2002;114:202–15. 19. Edmund C. Corneal topography and elasticity in normal and keratoconic eyes. A methodological study concerning the pathogenesis of keratoconus. Acta Ophthalmol Suppl 1989; 193:1-36. 20. Guirao A. Theoretical elastic response of the cornea to refractive surgery: risk factors for keratectasia. J Refract Surg 2005; 21:176-85. 21. Dupps WJ. Biomechanical modeling of corneal ectasia. J Refract Surg 2005;21:186-90.

Corneal Hysteresis and Biomechanics of the Normal Cornea 22. Andreassen TT, Simonsen AH, Oxlund H. Biomechanical properties of keratoconus and normal corneas. Exp Eye Res 1980;31:435-41. 23. Sherwin T, Brookes NH. Morphological changes in keratoconus: pathology or pathogenesis. Clin Exp Ophthalmol 2004;32: 211-7 24. Reinstein DZ, Silverman RH, Raevsky T, Simoni GJ, Lloyd HO, Najafi DJ, Rondeau MJ, Coleman DJ. Arc-scanning very high frequency digital ultrasound for 3D pachymetric mapping of the corneal epithelium and stroma in laser in situ keratomileusis. J Refract Surg. 2000;16:414-30 25. Smolek MK. Holographic interferometry of intact and radially incised human eye-bank corneas. J Cataract Refract Surg 1994; 20:277–86. 26. Jaycock PD, Lobo L, Ibrahim J, Tyrer J, Marshall J. Interferometric technique to measure biomechanical changes in the cornea induced by refractive surgery. J Cataract Refract Surg 2005;31:175-84. 27. Grabner G, Eilmsteiner R, Steindl C, Ruckhofer J, Mattioli R, Husinsky W. Dynamic corneal imaging. J Cataract Refract Surg 2005;31:163-74. 28. Luce DA. Determining in vivo biomechanical properties of the cornea with an ocular response analyzer. J Cataract Refract Surg 2005:31:156-62. 29. Doughty MJ, Zaman ML. Human corneal thickness and its impact on intraocular pressure measures: a review and metaanalysis approach. Surv Ophthalmol 2000;44:367-408. 30. Lleo A, Marcos A, Calatayud M, Alonso L, Rahhal SM, SanchisGimeno JA. The relationship between central corneal thickness and Goldmann applanation tonometry. Clin Exp Optom 2003;86:104-8. 31. Morgan AJ, Harper J, Hosking SL, Gilmartin B. The effect of corneal thickness and corneal curvature on pneumotonometer measurements. Curr Eye Res 2002;25:107-12. 32. Stabuc SM, Hawlina M. Influence of corneal thickness on comparative intra-ocular pressure measurements with Goldmann and non-contact tonometers in keratoconus. Klin Montasbl Augenheilkd 2003;220:843-7. 33. Jarade EF, Abi Nader FC, Tabbara KF. Intraocular pressure measurement after hyperopic and myopic LASIK. J Refract Surg 2005; 21:408-10. 34. Svedberg H, Chen E, Hamberg-Nystrom H. Changes in corneal thickness and curvature after different excimer laser photorefractive procedures and their impact on intraocular pressure measurements. Graefes Arch Clinic Exp Ophthalmol 2005; 243:1218-20.

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35. Chang DH, Stulting RD. Change in intraocular pressure measurement after LASIK: the effect of the refractive correction and the lamellar flap. Ophthalmology 2005:112:1009-16. 36. Brandt JD, Beiser JA, Kass MA, Gordon MO. Central corneal thickness in the Ocular Hypertension Treatment Study (OHTS). Ophthalmology 2001;108:1779-88. 37. Goldmann H, Schmidt T. On applanation tonography. Ophthalmologica 1965;150:65-75. 38. Siganos DS, Papastergiou GI, Moedas C. Assessment of the Pascal dynamic contour tonometer in monitoring intraocular pressure in unoperated eyes and operated eyes after LASIK. J Cataract Refract Surg 2005;31:458-9. 39. Duba I, Wirthlin AC. Dynamic contour tonometry for postLASIK intraocular pressure measurements. Klin Monatsbl Augenheilkd 2004:22:347-50. 40. Kaufman C, Bachmann LM, Thiel MA. Intraocular pressure measurements using dynamic contour tonometry after laser in situ keratomileusis. Invest Ophthalmol Vis Sci 2003;44: 3790-4. 41. Pepose JS, Feigenbaum SK, Qazi MA, Sanderson JP, Roberts CA. Changes in corneal biomechanics and in intraocular pressure pre- and post-LASIK using static, dynamic and non-contact tonometry. Am J Ophthalmol 2006; accepted. 42. Laiquzzaman M, Bhojwani R, Cunliffe I, Shah S. Diurnal variation of ocular hysteresis in normal subjects: relevance in clinical context. Clin Experiment Ophthalmol 2006;34:114-8. 43. Congdon NG, Broman AT, Bandeen-Roche K, Grover D, Quigley HA. Central corneal thickness and corneal hysteresis associated with glaucoma damage. Am J Ophthalmol 2006;141:868-75. 44. Buzard KA. Introduction to biomechanics of the cornea. Refract Corneal Surg 1992;8:127-38. 45. Cabrera Fernandez D, Niazy AM, Kurtz RM, Djotyan GP, Juhasz T. Biomechanical model of corneal transplantation. J Refract Surg 2006;22:293-302. 46. Sikder S, Snyder RW. Femtosecond laser preparation of donor tissue from the endothelial side. Cornea 2006;25:416-22. 47. Kohlhaas M, Spoerl E, Schilde T, Unger G, Wittig C, Pillunat LE. Biomechanical evidence of the distribution of cross-links in corneas treated with riboflavin and ultraviolet A light. J Cataract Refract Surg 2006;32:279-83. 48. Petroll WM, Cavanagh HD, Jester JV. Dynamic threedimensional visualization of collagen matrix remodeling and cytoskeletal organization in living corneal fibroblasts. Scanning 2004;26:1-10.

13

Corneal Physiology

Wisam A Shihadeh Almamoun Abdelkader Herbert E Kaufman

Corneal Physiology

2

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Corneal Endothelial Transplant

Introduction The cornea forms the transparent anterior part of the eye. It protects the contents of the eye and serves as the major refractive element in it. The principal layers of the cornea are the epithelium, Bowman’s layer, stroma, Descemet’s membrane, and the endothelium (Figure 2-1). The basis of understanding corneal physiology is to address the physiological importance of the corneal epithelial, endothelial barrier and metabolic pump functions. If either limiting layer is compromised, the cornea will increase in thickness, become edematous and show decrease in transparency. Loss of the corneal endothelial barrier will result in a much greater corneal edema than with loss of the epithelial barrier. This chapter addresses the fundamentals of corneal physiology to provide a foundation for understanding the normal as well as the pathological corneal conditions.

pachometers reported an average corneal thickness of 520 micrometers.4-7 Nowadays, because of practicality issues, automatic acoustic pachometers became the gold standard for measuring the corneal thickness despite calibration uncertainties. The corneal hysteresis phenomenon is a result of viscoelastic dampening in the cornea, i.e., the tissue’s ability to absorb and dissipate energy (See also Chapter 1, Corneal Hysteresis and Biomechanics of the Normal Cornea). Studies have shown that subjects whose corneas exhibit low corneal hysteresis, which can be thought of as having a “soft” cornea, are probable candidates for a variety of ocular diseases and complications. This parameter can be measured by the Ocular Response Analyzer from Reichert (Depew, NY),8 which utilizes a patented applanation process. Hysteresis, as an indicator of the viscoelastic properties of the cornea, can be used to enable a more accurate tonometry measurement. 9-12 Clinical research has shown that this measurement may be a valuable tool for identifying and classifying conditions such as corneal ectasia, Fuchs’ dystrophy as well as glaucoma.13

Physiology of the Corneal Epithelium The corneal epithelium serves as a barrier between the environment and the corneal stroma. Through its interaction with tear film, it forms a smooth refractive surface on the cornea.

Barrier Function

Figure 2-1: The cornea is a layered structure consisting of the epithelium (E), Bowman’s layer (Bw), stroma (St), Descemet’s membrane (De), and the endothelium (En).

Corneal Thickness and Hysteresis Corneal thickness is one of the important parameters of corneal health. Pachymetry is a useful tool in both cornea and glaucoma practices. Thin cornea can indicate keratoconus or other ectasic diseases, whereas a thickened cornea usually correlates with endothelial dysfunction and secondary corneal edema. Applanation tonometry underestimates the intraocular pressure in eyes with thin corneas and overestimates it in thick corneas.1-3 The early measures of corneal thickness using manual optical

The barrier is formed as a balance of cell shedding; basal cell division and the renewal of basal cells maintain epithelial cells by centripetal migration of new basal cells originating from limbal stem cells. The epithelial cells move from the basal layer to the surface of the cornea, progressively differentiating until the superficial cells form two layers of flattened cells. These are encircled by tight junction known as zonula occludens which serve as a semipermeable, highly resistant membrane. This barrier prevents the movement of fluid from tears into the stroma and also protects the cornea and intraocular structures from infection by pathogens.14

Refractive Function The microvilli on the surface of the most superficial epithelial cells are covered with a glycocalyx that interacts with the mucin layer of the tear film. This leads to smoothening of the surface of the cornea forming the smooth optical surface required for clear vision.15

Corneal Physiology Active Na+ Transport Na+

Transport of between tears and stroma has been demonstrated in the rabbit and frog.16-22 In humans,23 when factors such as corneal resting potential and other ion transport systems are taken into account, the net flow of Na + across the epithelium is from stroma to tears. Nevertheless, the corneal epithelium contains an active Na+ transport system directed from tear to stroma. The transepithelial transport of Na+ is most likely secondary to the Na+/K+ exchange activity of the deeper epithelial membranes, which accounts for the directionality of transepithelial Na+ transport.

Active Cl- Transport

15

coupling of the chloride secretion to sodium transport. In addition to the transport mechanisms above, the corneal epithelial cells also contain a sodium-hydrogen exchanger and a lactate-hydrogen co-transporter. These transport mechanisms serve to regulate intracellular pH by extrusion of lactate and hydrogen (H+) ions. Corneal epithelial cells have beta-adrenergic receptors that respond to stimulation by activation of adenylate cyclase and increase cyclic adenosine monophosphate (cAMP) levels in cells (Figure 2-2). cAMP increases the conductance of apical chloride channels, stimulating chloride transport. In vitro, the ion transport can osmotically move water from stroma to tears. However, in vivo epithelial ion transport probably has a minor role in corneal deturgescence as compared to the endothelium.29

Cl- transport across the epithelium is from the stroma to tears.19, 24, 25 Net Na+ absorptive transport and net Clsecretory transport can occur simultaneously only under special experimental conditions. In the living eye, the epithelium generates an electrical potential of about 30 mV, tear side negative, and in this situation the net movement of NaCl across the epithelium appears to be in the stromato-tear direction.21 The presence of active Cl- secretory transport in the epithelium raises the possibility that the epithelium might participate in the regulation of stromal hydration in addition to its role as a diffusion barrier. This has been demonstrated in frogs in which Cl- secretion by the epithelium produces significant stromal dehydration.26, 27

Electrophysiology of Corneal Epithelium and Ion Transport The mammalian cornea generates a transepithelial potential of 25-35 millivolts. This high voltage is consistent with the low ionic conductance of the optical epithelial cell membranes and high resistance of the tight junctions of the paracellular pathway. About 50% of the short circuit current across the corneal epithelium is carried by chloride ions moving through the apical membrane channels into the tears. This current is due to ionic gradients set up by epithelial transport of sodium and chloride ions. Ouabain sensitive Na+ - K+ ATPase present in the basolateral membrane of these cells, pumps sodium ions from the cells towards the stroma.28 A sodium-chloride co-transporter, also located in the basolateral membrane, facilitates the influx of sodium down its electrochemical gradient carrying with it chloride ions, which then diffuse through channels in the apical membrane. This chloride secretion is blocked when the Na+ - K+ ATPase is inhibited by Ouabain, demonstrating the

Figure 2-2: Scheme for the neuroregulation of Cl¯ transport in the corneal epithelium. It is proposed that serotonin and dopamine can evoke the release of norepinephrine from the sympathetic nerve fibers in the cornea. In turn, norepinephrine may activate adenocyclase via the β-adrenoreceptor to increase cell levels of cyclic AMP and, finally, to increase the chloride conductance of the apical epithelial membrane.

Physiology of the Corneal Stroma The corneal stroma is basically an extracellular compartment with keratocytes and nerves. It measures around 470 micrometers thick centrally in a human adult cornea. Collagen fibers approximately 22 to 32 nm30,31 in diameter appear to run uninterrupted from limbus to limbus in flat sheaths or lamellae. Although the concentration of Na+ and K+ may collectively be 35 mEq/L higher in the stroma than in the aqueous humor,32 the combined activity of these ions, and hence their effective osmolarity, is probably less in the stroma than in the aqueous humor. This fact is important for control of corneal hydration. When

16

Corneal Endothelial Transplant

the stroma swells, the diameter of collagen fibrils remains constant; swelling takes place in the ground substance, which is rich in glycosaminoglycans, and leads to an increased spatial separation of collagen fibrils.33 The stroma is maintained in a relatively dehydrated state, in comparison to its ability to swell. The stroma consists of 78% water, which is equivalent to a ratio of 3.45 parts water (by weight) to 1 part solid material. The corneal stroma scatters less than 10% of normal incident light. This is an unexpected property of the cornea given the disparity in refractive index between the collagen fibrils and the proteoglycan matrix. Maurice34 proposed that corneal transparency is a consequence of a crystalline lattice arrangement of collagen fibrils within the stroma. He also proposed that the light scattered by individual fibrils and uniform diameter is canceled by destructive interference with scattered light from adjacent fibers; therefore light is scattered only in the forward direction. Such an arrangement requires that all collagen fibrils be of equal diameter (275-350A) and that all fibrils be equidistant from each other. However, to maintain this transparency, it is required that the distance between the collagen fibrils be less than one half the wavelength of visible light. On the other hand, Goldman and Benedek35 and others36, 37 recognized that refractive elements in tissues whose dimensions are small (<200 nm) compared with the wavelength of light should not scatter as much as light as might be predicted by the stringent requirements of the crystalline lattice theory. Light scattering is wavelength dependent. This dependence of corneal transparency on the distribution and size of collagen fibrils is supported by observations of swollen corneas and by the structure of opaque sclera. When the epithelial or endothelial barrier of the cornea is damaged, the stroma imbibes water and swells, leading to loss of corneal transparency. This uptake of water causes formation of “lakes” devoid of collagen fibers within the stroma. This causes increased divergence of refractive index within the stroma as well as an increase in distance between collagen fibrils leading to a wavelength – dependent loss of light transmittance that increases with the amount of corneal swelling. It was found that fibril diameter is greater in the anterior cornea than in the posterior cornea and that the density of fibrils is lower in the anterior cornea than in the posterior cornea in both rabbits and humans. This leads to two-fold (in humans) and three-fold (in rabbits) increase in light scattered by the anterior cornea as compared with the posterior cornea. Proteoglycans are responsible for maintaining the regular spacing and packing of the collagen fibrils which is the basis of corneal transparency. The presence of functional epithelial and endothelial barriers and a metabolic pump

to maintain the corneal water content at 78% normally maintain the biochemical and physical properties of the corneal stroma and ultimately corneal transparency is maintained.34

Physiology of Corneal Endothelium The corneal endothelium is composed of a single layer of hexagonal cells which forms the posterior corneal surface. It is approximately 400,000 cells and 4-6 micrometers thick. The posterior cell membrane is thought by some to be coated with a viscous substance,38, 39 possibly of endothelial origin, which may reduce lipid membrane surface tension to promote wetting. A single primary cilium has been demonstrated in many endothelial cells, but its function is unclear.40, 41 Endothelial cells lack the ability to undergo mitosis. However, they do have the ability to enlarge and to maintain tight apposition with neighboring cells, preventing excessive diffusion of the aqueous into the stroma. The endothelium acts as a permeability barrier that restricts the movement of water and solutes into hydrophilic stroma. If the integrity of this layer is broken, corneal edema rapidly develops. Normally, the endothelium enjoys a privileged and protected environment in the anterior chamber, but it remains a fragile cell layer whose integrity and viability must be guarded to ensure the success of any intraocular procedure.42 Endothelial cell density is an important parameter to assess the condition of the cornea. The specular microscope has been a useful tool for noninvasive valuation of endothelial cell densities (Figure 2-3).43 Confocal microscopy is considered another important tool

Figure 2-3: (A) Specular photomicrograph of the corneal endothelium in a 28-year-old patient. The cell density is approximately 2,800 cell/ mm2. The dark bands are artifacts from applanation. (B) Endothelial cell density of approximately 400 cells/mm2 in a clinically successful corneal graft. Note the large cell size, pleomorphism and polymegathism.

Corneal Physiology to assess the integrity of the endothelium. Cell density varies with age. At birth, cell densities range from 35004000 cells/mm2, whereas the adult corneal normally has densities of 1400-2500 cells/mm2. Corneal transplants may have fewer than 1000 cells/mm2 and remain clear. A lower limit to this ability occurs at densities of 400-700 cells/ mm2, below which corneal edema and loss of vision ensue. When endothelial cells are subjected to stress, and especially when some cells are lost, the remaining cells may lose their regular hexagonal shape and become irregular in shape (pleomorphism) and size (polymegathism). These changes can occur with age, after trauma, and in long-term contact lens wear. The significance of these changes is unclear, but there is evidence that a cornea with these changes cannot withstand additional trauma as well as a normal cornea.

Endothelial Barrier and Pump Functions The corneal endothelium is leaky compared to the epithelium. In a physiologically normal and transparent cornea, aqueous humor crosses the endothelium and enters the stroma at a slow but constant rate. This constant leak of aqueous provides the principal source of glucose, amino acids and other nutrients for the cells of the avascular cornea. The permeability of the endothelial barrier results from the presence of low resistance intercellular junctions at the cells’ apical membrane. This leaky property made classic tracer experiments to demonstrate net ion transport mechanisms44 technically difficult. Nevertheless, the endothelium is clearly demonstrated to transport bicarbonate from the stroma to the aqueous humor in amounts sufficient to explain the simultaneous isotonic transport of fluid.45, 46 Subsequently, the transport of sodium ions in the same direction was inferred.47 Although the specific details of these transport mechanisms are unclear, several different schemes appear in the literature,48-50 indicating involvement of several alternate anion pathways.51 Since fluid and solutes are continuously entering the stroma, the maintenance of corneal thickness and transparency is dependent on the active removal of fluid that leaks into the stroma. A constant corneal thickness is maintained when the volume of fluid leaking into the stroma is equal to the volume of fluid actively removed from the stroma by the endothelium through the endothelial pump. Transport enzymes and ion channels in the endothelial cell membrane transport ions in a stroma-toaqueous direction.52 The osmotic gradient generated by this active transport mechanism draw water out of the stroma into the aqueous humor. The effect of endothelial ion transport results in net fluxes of sodium and bicarbonate

17

ions from stroma to aqueous. Fischbarg and coworkers53 promote the idea that endothelial fluid transport involves electroosmosis through the intercellular junctions as the primary process in a sequence of events secondary to active ion transport. Ruberti and Klyce54 report that transendothelial fluid transport may be rapidly self-modulating to control stromal hydration in response to small osmotic stresses, and this may assist in the regulation of corneal hydration. Corneal endothelial carbonic anhydrase, which catalyzes the conversion of carbon dioxide (CO2) and water (H2O) into bicarbonate (HCO3) and hydrogen (H+), is believed to provide an important source of bicarbonate for the endothelial pump. Carbonic anhydrase inhibitors have been shown to inhibit both corneal deturgescence and the electric potential across the endothelium. Na+ - K+ ATPase is an integral membrane protein localized to the lateral cell membrane in corneal endothelium. Ouabain, a specific inhibitor of this enzyme, has been shown to prevent temperature reversal of enucleated eyes and causes corneal swelling as it inhibits Na+ - K+ ATPase at doses comparable to those that cause corneal swelling. The endothelial cells in the human cornea decline constantly through out life. Despite this constant loss of cells, normal thickness and transparency are maintained. Other factors imposed on the endothelium markedly augment the normal aging process accelerating endothelial cell loss as in intraocular surgery, dystrophies, degenerations, glaucoma, and drug toxicity. When cell density declines to several hundred cells per square millimeter, corneal decompensation occurs. Corneal endothelium possesses a large reserve capacity in terms of the density of cells required for the maintenance of corneal transparency. If endothelial damage results in corneal edema, the restoration of the normal corneal hydration will be dependent on the extent to which the balance between barrier and pump functions can be reestablished.55

Corneal Metabolism and Nutrition Corneal metabolism is dependent on oxygen derived mainly from the atmosphere, the aqueous and the limbal vessels (Figure 2-4). The normal O2 in aqueous is low (40 mm Hg) compared to the tears (155 mm Hg). Most of the metabolic requirements for glucose, amino acids and vitamins are supplied through the aqueous humor and, to a lesser extent, via the tears and limbal vessels. Under both aerobic and anaerobic conditions glucose is diverted to the hexose monophasphate shunt (HMP) regulating levels of (NADPH) and converting hexoses to pentoses utilized in nucleic acid synthesis. Glucose derived from the aqueous

18

Corneal Endothelial Transplant of venular basement membrane followed by movement of the endothelial cells towards the stimulus. Endothelial cell proliferation occurs to form a solid sprout, which later develops a lumen. Two sprouts join each other to form a loop, the outer surface of which is lined by pericytes and blood flow begins. The process is repeated again by sprouting from apex of the loop. The angiogenic process consists of three basic steps: 1. Enzymatic degradation of basement membrane. 2. Endothelial cell movement. 3. Endothelial cell proliferation. The specific angiogenic factors include acidic fibroblast growth factor (FGF), basic FGF and transforming growth factor.56

Corneal Innervation Figure 2-4: Conceptual scheme of oxygen supply to the cornea. Although some oxygen enters the cornea from the limbus and aqueous humor, the anterior cornea derives essential amounts from the tears.

or from epithelial glycogen stores is converted to pyruvate by glycolysis under anaerobic condition yielding two molecules of ATP (adenosine triphosphate). Under aerobic conditions, pyruvate is oxidized in the Krebs or tricarboxylic acid cycle to yield water, carbon dioxide and 36 molecules of ATP per cycle. Under hypoxic condition as during contact lens use increasing amounts of pyruvate are converted by lactate dehydrogenase to lactate which diffuses from the epithelium into the stroma leading to epithelial and stromal edema.52

Corneal Avascularity The human cornea is normally avascular and transparent. Corneal neovascularization results from a variety of diverse conditions. The avascularity of the cornea is due to the compactness of the stromal cells, which acts as a mechanical obstacle. Also, the stroma is rich in mucopolysaccharides which is a barrier to neovascularization. Causes of corneal neovascularization include ocular surface diseases such as acne rosacea and staphylococcal hypersensitivity, immunological disorders such as cicatricial pemphigoid, corneal allograft rejection and collagen vascular disease with peripheral corneal ulceration and neovascularization. Corneal neovascularization is also found in keratitis caused by herpes zoster, a result of sprouting from perilimbal vessels. New capillaries arise from the perilimbal capillaries by focal degradation

The cornea is richly supplied with sensory nerves which are derived from the ciliary nerves originating from the ophthalmic division of trigeminal nerve. The cornea is surrounded by a perilimbal nerve ring from which fibers penetrate the middle and anterior layers of the stroma extending radially towards center of the cornea. The nerve fibrils divide dichotomously emerging from the deeper layers of cornea penetrating Bowman’s membrane to a subepithelial plexus. From the plexus, axon terminals spread among the epithelial cells innervating all layers of epithelium with sensory receptors. Adrenergic sympathetic nerves that originate in the superior ganglion also innervate the cornea. So it is one of the most sensitive tissues of the body which serves as a protective function. It contains A delta and C fibers, which respond to mechanical, thermal and chemical stimuli. They usually have the lowest threshold for mechanical stimulation. Neuropeptides including substance P and calcitonin gene-related peptide (CGRP) are present in corneal nerves and appear to have a direct but poorly understood trophic effect on the epithelium.28

Control of Corneal Hydration Several hypotheses have been proposed to explain the control of corneal hydration. Several components of corneal physiology are involved in this process (Figure 2-5). Underlying each of the hypotheses is the fact that stromal imbibition pressure (IP) must be counteracted to prevent swelling. In the past, it was suggested that although the cornea is surrounded by fluid, it would not swell if the cell membranes were impermeable to either water57 or salt.58, 59 Although both the epithelial and endothelial cell membranes have defined barrier properties, neither

Corneal Physiology

19

edema is usually irreversible and treatment varies depending on the nature of the underlying problem. Chronic corneal edema develops as a consequence of endothelial dysfunction, regardless of whether the original problem was dystrophy, inflammation or trauma. Endothelial dysfunction can be in the form of increased permeability, decreased ion transport function or both. In mild cases, increased stromal thickness occurs initially with minimal vision affection. In advanced cases, epithelial edema ensues, which affects vision dramatically. In late stages, painful bullous changes can develop. Thick subepithelial pannus eventually develops, leading to disappearance of the bullae.

Epithelial Edema Figure 2-5: Factors and forces involved in the control of corneal stromal hydration. Intraocular pressure and stromal imbibition pressure are forces that promote water accumulation in the stroma. Ion transport pumps in the corneal membranes reduce the osmotic pressure of the stroma such that the semipermeable membrane properties of the epithelium and endothelium balance the forces promoting edema.

membrane is impermeable to water or electrolytes.60 The effect of evaporation from the tear film has also been suggested as a major source for the control of corneal hydration61 and, in fact, the cornea is 5% thinner during waking hours than during sleep.62, 63 Disruption of the lipid layer of the tear film leads to evaporation from the epithelial surface, which can lead to dellen formation. High postoperative intraocular pressure can promote more rapid clearing of a corneal graft by accelerating the movement of fluid through the anterior corneal surface, thereby thinning the cornea. However, elevated intraocular pressure can also cause epithelial edema, as well as stromal swelling, in certain refractive surgical procedures.64 Maurice60 first proposed that, because they are not impermeable to solute or water, the corneal membranes could prevent corneal edema if ions were actively transported out of the stroma as fast as they leaked in by passive means (solvent drag, diffusion). Such a process would, in essence, lead to the sustaining of an osmotic gradient (2 to 3 mOsm) between the corneal stroma and the external solutions, which would balance the swelling pressure of the stroma. Davson 65 and Harris and Nordquist66 demonstrated that corneal hydration is loosely linked to the metabolic activity of corneal membranes.

Corneal Edema Corneal edema can be acute or chronic. Although acute corneal edema, as can be seen in contact lens wear and in angle-closure glaucoma, is often reversible, chronic corneal

Epithelial edema resulting from endothelial dysfunction or elevated intraocular pressure is predominantly extracellular.67 The underlying pathophysiologic mechanism seems to involve a forward movement of stromal fluid and aqueous, generated by the IOP. Thus if the endothelial functional reserve falls below a certain level, leading to edema and a reduction in stromal swelling pressure to below the value of the IOP, fluid from aqueous humor can collect.68 Because the otherwise healthy epithelium has such a high resistance to electrolytes and to the flow of water, the fluid can be trapped within the epithelium, resulting in the formation of cysts and bullae. The concept that the IOP is the driving force for the fluid movement is particularly supported by the fact that in phthisis bulbi with marked hypotony, epithelial edema does not occur, no matter how damaged the endothelium is and how thick the stroma is.42

Stromal Edema When the endothelial cell density falls below a critical level (200-400 cells/mm2), the ability of the endothelium to maintain stromal hydration begins to falter and the edema develops gradually.42 Because the stroma can swell only in the posterior direction, its thickness increases especially centrally. This flattening of the posterior surface can throw Descemet’s membrane into multiple folds.

Endothelial Changes Under stress, e.g. trauma, the endothelium changes in a way characterized by decreased cell number and enlargement and irregularity of the shape (polymegathism and pleomorphism).69 In chronic inflammation, the endothelium may undergo fibrous metaplasia.70 In Fuchs’ dystrophy the cells exhibit a change in form and show

20

Corneal Endothelial Transplant

vacuoles, phagocytized pigment, and irregular depositions (guttata). Even in advanced cases of Fuchs’ dystrophy, the endothelial surface appears intact.71, 72 Acknowledgements All figures were taken with permission from Kaufman HE, Barron BA, McDonald MB, Waltman SR, eds. The Cornea. Boston: Butterworth-Heinemann, 1988: 3-54.

References 1. Saleh TA, Adams M, McDermott B, Claridge KG, Ewings P. Effects of central corneal thickness and corneal curvature on the intraocular pressure measurement by Goldmann applanation tonometer and ocular blood flow pneumatonometer. Clin Experiment Ophthalmol 2006;34(6):516-20. 2. Kniestedt C, Lin S, Choe J, Nee M, Bostrom A, Sturmer J, Stamper RL. Correlation between intraocular pressure, central corneal thickness, stage of glaucoma, and demographic patient data: prospective analysis of biophysical parameters in tertiary glaucoma practice populations. J Glaucoma 2006;15(2):91-97. 3. Kohlhaas M, Boehm AG, Spoerl E, Pursten A, Grein HJ, Pillunat LE. Effect of central corneal thickness, corneal curvature, and axial length on applanation tonometry. Arch Ophthalmol 2006; 124(4):471-6. 4. Donaldson DD. A new instrument for the measurement of corneal thickness. Arch Ophthalmol 1966;76(1):25-31. 5. Mishima S, Hedbys BO. Measurement of corneal thickness with the Haag-Streit pachometer. Arch Ophthalmol 1968;80(6): 710-3. 6. Mishima S. Corneal thickness. Surv Ophthalmol 1968;13(2):5796. 7. McLaren JW, Bourne WM. A new video pachometer. Invest Ophthalmol Vis Sci.1999;40(7):1593-8. 8. Luce DA. Determining in vivo biomechanical properties of the cornea with an ocular response analyzer. J Cataract Refract Surg 2005;31(1):156-62. 9. Laiquzzaman M, Bhojwani R, Cunliffe I, Shah S. Diurnal variation of ocular hysteresis in normal subjects: relevance in clinical context. Clin Experiment Ophthalmol 2006;34(2): 114-8. 10. Kida T, Liu JH, Weinreb RN. Effect of 24-hour corneal biomechanical changes on intraocular pressure measurement. Invest Ophthalmol Vis Sci 2006;47(10):4422-6. 11. Randleman JB. Post-laser in-situ keratomileusis ectasia: current understanding and future directions. Curr Opin Ophthalmol 2006;17(4):406-12. 12. Medeiros FA, Weinreb RN. Evaluation of the influence of corneal biomechanical properties on intraocular pressure measurements using the ocular response analyzer. J Glaucoma 2006;15(5):36470. 13. Congdon NG, Broman AT, Bandeen-Roche K, Grover D, Quigley HA. Central corneal thickness and corneal hysteresis associated with glaucoma damage. Am J Ophthalmol 2006;141(5):868-75. 14. Thoft RA, Friend J. The X, Y, Z hypothesis of corneal epithelial maintenance. Invest Ophthalmol Vis Sci 1983;24(10):1442-3. 15. Gipson IK, Yankauckas M, Spurr-Michaud SJ, Tisdale AS, Rinehart W. Characteristics of a glycoprotein in the ocular surface glycocalyx. Invest Ophthalmol Vis Sci 1992;33(1):218-27. 16. Donn A, Maurice DM, Mills NL. Studies on the living cornea in vitro. I. Method and physiologic measurements. Arch Ophthalmol 1959;62:741-7. 17. Donn A, Maurice DM, Mills NL. Studies on the living cornea in vitro. I. Method and physiologic measurements. Arch Ophthalmol 1959;62:741-7.

18. Green K. Ion transport in isolated cornea of the rabbit. Am J Physiol 1965;209(6):1311-6. 19. Klyce SD, Neufeld AH, Zadunaisky JA. The activation of chloride transport by epinephrine and Db cyclic-AMP in the cornea of the rabbit. Invest Ophthalmol 1973;12(2):127-39. 20. Van der HC, Weekers JF, Schoffeniels E. Sodium and chloride transport across the isolated rabbit cornea. Exp Eye Res 1975; 20(1):89-96. 21. Klyce SD. Transport of Na, Cl, and water by the rabbit corneal epithelium at resting potential. Am J Physiol 1975;228(5): 1446-52. 22. Candia OA, Askew WA. Active sodium transport in the isolated bullfrog cornea. Biochim Biophys Acta 1968;163(2):262-5. 23. Fischer FH, Schmitz L, Hoff W, Schartl S, Liegl O, Wiederholt M. Sodium and chloride transport in the isolated human cornea. Pflugers Arch 1978;373(2):179-88. 24. Zadunaisky JA. Active transport of chloride in frog cornea. Am J Physiol 1966;211(2):506-12. 25. Wiederholt M. Physiology of epithelial transport in the human eye. Klin Wochenschr 1980;58(19):975-84. 26. Zadunaisky JA, Lande MA. Active chloride transport and control of corneal transparency. Am J Physiol 1971;221(6):1837-44. 27. Candia OA. Fluid and Cl- transport by the epithelium of the isolated frog cornea. Invest Ophthalmol Vis Sci 1976;15:12. 28. Klyce SD, Crosson CE. Transport processes across the rabbit corneal epithelium: a review. Curr Eye Res 1985;4(4):323-31. 29. Bonanno JA. Regulation of corneal epithelial intracellular pH. Optom Vis Sci 1991;68(9):682-6. 30. Giraud JP, Pouliquen Y, Offret G, Payrau P. Statistical morphometric studies in normal human and rabbit corneal stroma. Exp Eye Res 1975;21(3):221-9. 31. Hogan MJ, Alvarado JA, Weddell E. Histopathology of the Human Eye 1971. 32. Otori T. Electrolyte content of the rabbit corneal stroma. Exp Eye Res 1967;6(4):356-67. 33. Kanai A, Kaufman HE. Electron microscopic studies of swollen corneal stroma. Ann Ophthalmol 1973;5(2):178-90. 34. Maurice DM. The structure and transparency of the cornea. J Physiol 1957;136:263. 35. Goldman JN, Benedek GB. The relationship between morphology and transparency in the nonswelling corneal stroma of the shark. Invest Ophthalmol 1967;6(6):574-600. 36. Benedek GB. Theory of transparency of the eye. Appl Optics 1971;10:459. 37. Farrel RA, McCally RL, Tatham PER. Wavelength dependencies of light scattering in normal and cold swollen rabbit corneas and their structural implications. J Physiol 1973;233:589. 38. Wolf J. The secretory activity and the cuticle of the corneal endothelium. Doc Ophthalmol 1968;25(1):150-94. 39. Sperling S, Jacobsen SR. The surface coat on human corneal endothelium. Acta Ophthalmol (Copenh) 1980; 58(1):96-102. 40. Svedberg B, Bill A. Scanning electron microscopic studies of the corneal endothelium in man and monkeys. Acta Ophthalmol 1972;50(321). 41. Gallagher BC. Primary cilia of the corneal endothelium. Am J Anat 1980;159(4):475-84. 42. Klyce SD. Corneal Physiology 2005;4th:37-58. 43. Maurice DM. Cellular membrane activity in the corneal endothelium of the intact eye. Experientia 1968;24(11): 1094-5. 44. Ussing HH. The distinction by means of tracers between active transport and diffusion. Acta Physiol Scand 1949;19:43. 45. Hodson S, Miller F. The bicarbonate ion pump in the endothelium which regulates the hydration of rabbit cornea. J Physiol 1976;263(3):563-77. 46. Hull DS, Green K, Boyd M, Wynn HR. Corneal endothelium bicarbonate transport and the effect of carbonic anhydrase inhibitors on endothelial permeability and fluxes and corneal thickness. Invest Ophthalmol Vis Sci 1977;16(10):883-92.

Corneal Physiology 47. Lim JJ, Ussing HH. Analysis of presteady-state Na + fluxes across the rabbit corneal endothelium. J Membr Biol 1982;65(3):197-204. 48. Fischbarg J, Hernandez J, Liebovitch LS, Koniarek JP. The mechanism of fluid and electrolyte transport across corneal endothelium: critical revision and update of a model. Curr Eye Res 1985;4(4):351-60. 49. Widerholt M, Jentsch TJ, Keller SK. Electrical sodium bicarbonate symport in cultured corneal endothelial cells. Pflugers Arch 1985;405:S167. 50. Kuang KY, Xu M, Koniarek JP, Fischbarg J. Effects of ambient bicarbonate, phosphate and carbonic anhydrase inhibitors on fluid transport across rabbit corneal endothelium. Exp Eye Res 1990;50(5):487-93. 51. Bonanno JA. Identity and regulation of ion transport mechanisms in the corneal endothelium. Prog Retin Eye Res 2003;22(1):69-94. 52. Friend J. Biochemistry of ocular surface epithelium. Int Ophthalmol Clin 1979;19(2):73-91. 53. Sanchez JM, Li Y, Rubashkin A, Iserovich P, Wen Q, Ruberti JW, Smith RW, Rittenband D, Kuang K, Diecke FP, Fischbarg J. Evidence for a central role for electro-osmosis in fluid transport by corneal endothelium. J Membr Biol 2002;187(1):37-50. 54. Ruberti JW, Klyce SD. NaCl osmotic perturbation can modulate hydration control in rabbit cornea. Exp Eye Res 2003;76(3): 349-59. 55. Rae JL, Lewno AW, Cooper K, Gates P. Dye and electrical coupling between cells of the rabbit corneal endothelium. Curr Eye Res 1989;8(8):859-69. 56. Mishima S. Clinical investigations on the corneal endothelium. Ophthalmology 1982;89(6):525-30. 57. Leber T. Studies on fluid exchange in the eye. Graefes Arch Ophthalmol 2006;19:87. 58. Cogan DC, Kinsey VE. The cornea: V. Physiological aspects. Arch Ophthalmol 1942;28:661.

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59. von Bahr G. Measurements of the effect of solutions of different osmotic pressure on the thickness of the living cornea. Trans Ophthalmol Soc UK 1948;68:515. 60. Maurice DM. The permeability to sodium ions of the living rabbit’s cornea. J Physiol 1951;112(3-4):367-91. 61. Friedman MH. Unsteady transport and hydration dynamics in the in vivo cornea. Biophys J 1973;13(9):890-910. 62. Mishima S, Maurice DM. The effect of normal evaporation on the eye. Exp Eye Res 1961;1:46-52. 63. Mandell RB, Fatt I. Thinning of the human cornea on awakening. Nature 1965;208(7):292-3. 64. McPhee TJ, Bourne WM, Brubaker RF. Location of the stressbearing layers of the cornea. Invest Ophthalmol Vis Sci 1985 26(6):869-72. 65. Davson H. The hydration of the cornea. Biochem J 1955;59(1): 24-8. 66. Harris JE, Nordquist LT. The hydration of the cornea. I. The transport of water from the cornea. Am J Ophthalmol 1955;40(5 Part 2):100-10. 67. Iwamoto T, DeVoe AG. Electron microscopic studies on Fuchs’ combined dystrophy. II. Anterior portion of the cornea. Invest Ophthalmol 1971;10(1):29-40. 68. Ytteborg J, Dohlman CH. Corneal edema and intraocular pressure. II. Clinical results. Arch Ophthalmol 1965;74(4): 477-84. 69. Spencer WH. Ophthalmic pathology: An Atlas and Textbook 1985;(1):259. 70. Michels RG, Kenyon KR, Maumence AE. Retrocorneal fibrous membrane. Invest Ophthalmol 1972;11(10):822-31. 71. Iwamoto T, DeVoe AG. Electron microscopic studies on Fuchs’combined dystrophy. I. Posterior portion of the cornea. Invest Ophthalmol 1971; 10(1):9-28. 72. Waring GO, III, Bourne WM, Edelhauser HF, Kenyon KR. The corneal endothelium. Normal and pathologic structure and function. Ophthalmology 1982; 89(6):531-90.

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Corneal Endothelium in Health and Disease

Pedram Hamrah Eric C Amesbury Richard A Eiferman

Corneal Endothelium in Health and Disease

3

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Corneal Endothelial Transplant

Introduction The corneal endothelium separates the anterior chamber from the subjacent stroma. This unique monolayer is primarily responsible for control of corneal hydration (See also, Chapter 2, Corneal Physiology). It is essentially incapable of mitosis and, if damaged, causes corneal edema and loss of vision.

Embryology and Development Corneal development is induced by the separation of the lens vesicle from the surface ectoderm at around 33 days of gestation. The basal epithelial cells of the surface ectoderm secrete collagen fibrils and glycosaminoglycans that form the primary stroma. At five weeks gestation, the first of three waves of neural crest cells migrate along the posterior surface of the primary stroma to form the initially multilayered corneal endothelium.1-3 By the third month of gestation, the double-layered endothelium thins to a monolayer of endothelial cells that begin to deposit Descemet’s membrane. Descemet’s membrane initially consists of the anterior lamina densa and the posterior lamina lucida, which is adjacent to the endothelium. Between the third and fourth month of gestation, gap junctions and the apical band develop. Descemet’s membrane then goes on to form a complete layer of the fetal banded zone, which reaches a maximum thickness of 3 μm at birth. The endothelium shares its neural crest origin with stromal keratocytes, the sclera, the cells of the anterior iris, and the trabecular meshwork.4 This may explain the observation that congenital diseases of the endothelium often involve related structures in the anterior segment of the eye. Further evidence of this relationship is suggested by studies of genetic mutations affecting neural crest cell differentiation, providing insight into such conditions as Axenfeld-Rieger syndrome and Peters anomaly.5 The normal human fetal cornea appears to be primarily devoid of lymphatic and blood vessels and remains avascular into adulthood,6 an important factor contributing to the relative immune privilege of the cornea after penetrating keratoplasty and other procedures.

Morphological Characteristics, Aging and Wound Repair The corneal endothelium is composed of a monolayer of mostly hexagonal cells, covering the posterior surface of the Descemet’s membrane. Human corneal endothelial cells (HCECs) are 5 μm thick and 20 μm wide, with a surface

area of 250 μm 2 . In addition, they have numerous interdigitating cellular processes that increase the area of contact.7,8 In the early prenatal period, there is a rapid increase in total HCEC number through mitosis, while later, enlarging endothelial cells cover the rapidly growing surface of the cornea without a significant change in cell density.9 The HCEC density is highest at birth with about 500,000 cells and an average of about 4000 cells/mm2, although numbers as high as 7500 cells/mm2 have been reported.10,11 The endothelial cell density (ECD) decreases throughout life at variable rates. From birth to 14 years the rate of endothelial cell loss is approximately 3% per year. After age 14, endothelial cell loss slows to about 0.6% per year. Specular microscopy in normal young adult corneas reveals an ECD of about 3500 cells/mm2.12 The ECD declines to about 2000 cells/mm2 in older age.13 As ECD decreases, individual cells enlarge and lose their hexagonal shape. 2,14 The critical ECD below which the cornea decompensates is approximately 300-500 cells/mm2. 15 (See also Chapter 2, Corneal Physiology). The coefficient of variation of mean cell area (CV) is about 0.25 in the normal cornea, and is a clinically valuable marker. An increase of the CV is termed polymegathism which normally occurs with increasing age, or when the endothelium is stressed or traumatized. Another morphometric parameter is the hexagonality of the endothelial cells, which is 70-80% in the normal cornea. Deviation from hexagonality is termed pleomorphism, which is also seen with aging or in various endothelial disease states. Factors such as gender and ethnicity have also been noted to affect endothelial cell morphology and density in healthy subjects.16,17 There are important differences between the peripheral and central HCECs. The ECD is higher in the periphery as opposed to the central cornea. In addition, a higher replicative competence has been demonstrated in peripheral HCECs versus central HCECs.18 The endothelium forms the anterior border of the anterior chamber, and is therefore susceptible to blunt or penetrating trauma such as cataract extraction or anterior chamber intraocular lens implantation, as well as inflammatory and other conditions (Figures 3-1 to 3-4). As Edelhauser discussed in his 2006 Proctor Lecture, the peripheral HCEC population near Schwalbe’s line may represent an endothelial stem cell reservoir.19 With further investigation, findings such as this will improve our understanding, and avoidance, of critical areas of the endothelium during intraocular surgery. Recent studies revealed some factors that contribute to the observable age-related changes of HCECs. From in vitro and graft survival studies, the survival of endothelial cells

Corneal Endothelium in Health and Disease

Figure 3-1: Clinical photograph of an anterior chamber lens with corneal touch and corneal edema inferiorly (arrow).

Figure 3-2: Slit-lamp photograph displaying post-traumatic arcuate Descemet’s breaks (arrows).

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Figure 3-4: Low-power photomicrograph of posterior ulcer of von Hippel (arrow).

is better in younger donor grafts. 20-22 Injuries that accumulate with time, such as UV exposure23 and the effects of oxidative species, may play a role in the aging process. Animal studies suggest that intrinsic protective mechanisms, such as catalase, superoxide dismutase, and glutathione peroxidase, lose their activity in older corneas, while generation of reactive oxygen species remain unchecked.24-26 In previous studies, it has also been demonstrated that vitamin E, an antioxidant, is able to significantly increase the survival of treated endothelial cells.27 In humans, the corneal endothelium has a very limited capacity to mitose either in vivo or in vitro. Damage to this monolayer either by aging or disease heals by enlargement and sliding of remaining cells. Even when injured, HCECs appear to be “locked” in G-1 and cannot enter the S phase of mitosis. It is therefore critical to avoid damage to the endothelium during any intraoperative procedure.

Ultrastructural Characteristics

Figure 3-3: Photomicrograph of post-traumatic loss of corneal endothelium and iris pigment deposition (arrow).

Normal HCECs contain large numbers of mitochondria and a large nucleus, indicating their active metabolism. Further, they contain rough and smooth endoplasmic reticula, as well as a well-developed Golgi apparatus, reflecting their high level of protein synthesis. Pinocytotic vesicles and a terminal web of fine fibrils can be seen on cross-sections toward the apical surface of the cells. A circumferential band of actin-containing microfilaments, present at the cell periphery and beneath the plasma membrane, is probably involved in maintaining the endothelial cell shape.28 These filaments may also facilitate cell migration in response to injury. Apical F-actin, for example, dramatically reorganizes in response to transforming growth factor (TGF)-β1, which is known to

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Corneal Endothelial Transplant

enhance endothelial wound healing through migration and cell spreading.5 Endothelial cells interdigitate and contain several junctional complexes at the overlapping apicolateral boundaries that include zonula occludens, macula occludens, macula adherens, but no desmosomes, providing a leaky barrier to the aqueous humor.5,29 Further, gap junctions allow the transport of electrolytes and small molecules between these cells at all levels of the lateral plasma membrane, facilitating cell-to-cell communication.30 The anterior aspect of the endothelium is adjacent to Descemet’s membrane and has no known anchoring microstructures. The posterior aspect (apical) of the endothelium is covered by microvilli and free marginal folds. This creates a high surface area in contact with the aqueous humor, presumably in order to carry out the many metabolic functions of the HCECs, as discussed below.

Pump and Barrier Function The amount of corneal stromal hydration plays a role in maintaining corneal transparency, as described below.31-33 The mechanism of permitting the passage of nutrients into the cornea, while maintaining a barrier to the free flow of water into the stroma, is described by the pump-leak hypothesis of the endothelium32 (See also Chapter 2, Corneal Physiology). The leak rate of water and nutrients into the cornea is balanced by the pumping rate of endothelial cells. An imbalance would result in corneal swelling, clouding of the cornea and decreased vision. Normal HCECs maintain corneal transparency by regulating stromal water content. The precise arrangement of stromal collagen bundles, and therefore corneal transparency, is disrupted above or below 78% stromal hydration.31-33 Hydration homeostasis is, in part, achieved by HCEC ionic pumps that counteract the osmosis of water into the stroma. At least two active transport systems seem to contribute to the pumping mechanism of the endothelial cells: Na+/K+-dependent ATPase pumps,34 and functional Na+-HCO3– dependent cotransport.35,36 The Na+/K+dependent ATPase pumps are present in the basolateral membrane of HCECs. These pumps actively transport Na+ ions from the stroma into the aqueous. Additionally, carbon dioxide that diffuses into the endothelial cytoplasm is converted to HCO3– and water via carbonic anhydrase. HCO3– is then transported into the aqueous across the endothelium, along with Na+ by a cotransport pump, which apparently is the net effect of multiple subservient ionic mechanisms. The combined ionic

pumping creates an osmotic gradient, and therefore a flux of water, across endothelial cells into the aqueous humor. Corneal avascularity necessitates oxygen and nutrient delivery from the tear film and the aqueous humor. Vitamins, glucose and amino acids needed by the stroma and epithelium are mainly received from the aqueous humor through a paracellular route. This route requires endothelial cells to be permeable, or “leaky”, to these nutrients. Focal tight junctions, gap junctions, and the interdigitations of the lateral plasma membranes provide the relative permeability to nutrient molecules and water needed for the pump-leak mechanism to work.37,38

Descemet’s Membrane Descemet’s membrane, the basement membrane of the corneal endothelium, is a thick extracellular matrix that is secreted by endothelial cells. It increases in thickness from 3 μm at birth to around 10 μm in adults. Descemet’s membrane is laid down over time and is divided into a 0.3 μm thin anterior nonbanded zone adjacent to the stroma, an anterior 2-4 μm banded zone, and a posterior amorphous unbanded zone that is more than 4 μm thick.39 The positive correlation of age with the thickness of Descemet’s membrane allows the evaluation of endothelial cell function, which is particularly useful in studying endothelial dystrophies and diseases. An atypical striated collagen deposition in the posterior layer, for example, has been described in Fuchs’ endothelial dystrophy.40 Excessive extracellular matrix can be produced by individual endothelial cells, resulting in focal thickenings of the membrane, called Hassal-Henle bodies that are found in the corneal periphery. If similar structures are found in the central cornea, they are termed corneal guttae. These focal thickenings can increase with age, in Fuchs’ dystrophy, after trauma, and as a result of inflammation. Pigment phagocytosis by the endothelium at the level of guttata can be seen as brownish pigmentation. Further, pseudoguttata can be seen in conditions such as iritis, infections, and inflammation. However, these psudoguttata are usually reversible, as these conditions resolve. The adult Descemet’s membrane contains collagen type IV and VIII, fibronectin, laminin, dermatan sulfate and heparan sulfate proteoglycans. Descemet’s membrane is tightly adherent to the overlying stroma and reflects its changes. This leads to the observation of Descemet’s folds with swelling of the stroma on slitlamp biomicroscopy. The Descemet’s membrane usually remains intact in bacterial keratitis or corneal ulcers and protrudes as a descemetocele, but exposure to shearing stress tears it easily.

Corneal Endothelium in Health and Disease Tears in Descemet’s membrane allow penetration of the aqueous humor into the stroma, and corneal edema from inflammation and secondary loss of the endothelial pump. Although Descemet’s membrane does not regenerate, small defects in the Descemet’s membrane are healed by endothelial cells that migrate over the site of the tear; larger areas of Descemet’s membrane disruption require fibroblasts for the repair process.

Disease States Endothelial Dystrophies Congenital Hereditary Endothelial Dystrophy (CHED) Congenital hereditary endothelial dystrophy (CHED) is a rare disease that presents as a bilaterally symmetric, diffuse corneal edema with Descemet’s membrane thickening in children.41 Although cases consistent with this dystrophy were described under different names in the European literature as early as in 1893,42 it was Maumenee who initially described this dystrophy in the English literature in 1960, suggesting that the disease arises from an abnormality of the endothelium.43 CHED is thought to be caused by a primary dysfunction and degeneration of the corneal endothelial cells, leading to an increase in permeability, accelerated Descemet’s membrane secretion, and stromal edema.44 The corneal thickness is variable, but can reach two to three times the normal thickness, with epithelial microbullae and opacification extending to the limbus without any clear zones. Two inheritance forms of CHED are recognized. A more common autosomal recessive (AR) form, which is present at birth or within the first few weeks, is stationary, and it is associated with a nystagmus that is due to the severe visual loss. The cornea has a bluish-white ground-glass appearance and a uniform thickening of the Descemet’s membrane. The endothelium is typically difficult to observe, but if seen, it is atrophic, irregular or absent. Despite the severe corneal edema, symptoms of discomfort, epiphora or photophobia are absent in this form of CHED. In contrast, the autosomal dominant (AD) inheritance form presents later during the first or second year of life and it is slowly progressive without any nystagmus. The cornea has a diffuse, blue-gray, ground-glass appearance. Vision tends to be better than the AR form and the patients complain of photophobia and epiphora before the onset of corneal clouding.45 The primary abnormality in the AD form of CHED is thought to be a dysfunction of the corneal endothelium during or after the fifth month of gestation. Recent genetic studies have mapped the gene locus of CHED to chromosome 20 (AD CHED: 20p11.2-q11.2; AR

27

CHED: 20p13) with the autosomal recessive form being genetically distinct from the autosomal dominant form of CHED.46-48 Reports of similar pathology49 and the possible genetic allelism of AD CHED with PPMD suggest that AD CHED may represent an earlier and more severe spectrum of the posterior dystrophies.36,50 It is important to rule out congenital glaucoma, as with any other form of congenital corneal clouding, although congenial glaucoma has been reported in association with CHED.51 While congenital glaucoma results from abnormal neural crest cell migration, it is thought that CHED results from abnormal neural crest cell differentiation.52 Additional ocular abnormalities are usually absent, but corneal amyloidosis 53,54 and hearing deficiency 44 have been reported in association with CHED. Due to the early presentation of CHED in the amblyogenic period, penetrating keratoplasty is indicated in these patients. Despite the high rejection rates in this age group, good results have been reported.51,55 Schaumberg et al have reported in a recent multicenter retrospective study of penetrating keratoplasty for CHED that 69% of grafts remained clear after 70 months.56 The 2-year survival for first grafts was reported at 71%. It has been postulated that poor results are related to the early age of onset of the disease or the severity of the disease requiring penetrating keratoplasty.57 Posterior Polymorphous Dystrophy (PPMD) Posterior polymorphous dystrophy (PPMD) is a bilateral, asymptomatic, generally nonprogressive disease that has an autosomal dominant inheritance. PPMD, even within the same families, has a variable clinical spectrum and is easily overlooked.58 It occurs typically in the second and third decade of life, although presentation as a cloudy cornea in newborn infants has been reported.59 The clinical appearance that is characterized by grouped vesicular or annular opacities, geographic-shaped discrete gray lesions, or broad bands with scalloped edges, is produced by a thick collagenous layer, deposited by epithelioid cells that projects into the anterior chamber (Figure 3-5). Descemet’s membrane can be irregular or have a nodular appearance, and endothelial guttae can be seen. Further, peripheral anterior synechiae (PAS) have been reported in as high as 27% of PPMD patients.60 Although most patients are asymptomatic, stromal edema can occur. It is important to distinguish PPMD from CHED, since corneal edema in PPMD patients, unlike in CHED, may resolve. In case of persistent stromal edema, penetrating keratoplasty is indicated, but the presence of PAS, visible without gonioscopy in association with an increased intraocular pressure should be considered a relative contraindication.

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Corneal Endothelial Transplant

Figure 3-6: Iris atrophy in Iridocorneal Endothelial (ICE) syndrome. Figure 3-5: Slit-lamp photograph of posterior polymorphous dystrophy with vacuoles (arrows).

Following grafting, recurrence of PPMD has been reported.61,62 Histologically, PPMD is characterized by epithelioid cells that form multiple layers and overgrow normal cells. Unlike the normal endothelium, these cells have desmosomal junctions and microvilli. Immunohistochemical studies indicate the transformation of endothelial cells into epithelioid cells.63 However, recently Cockerham et al. reported that the epithelioid-cell like endothelium does represent a simple transformation from endothelium to surface epithelium. 49 They demonstrated the strong expression of the PPMD endothelium for cytokeratin 7, which is not expressed on the surface epithelium. As with CHED, the pathogenesis of PPMD is thought to be due to a dysfunction of the corneal endothelium and an aberrant differentiation of neural crest cells.46 PPMD is mapped to chromosome 20q11.64 In addition, recently a missense mutation was reported on chromosome 1q34.3p32.65 The mutated gene codes for the alpha 2 chain of collagen VIII that is the predominant collagen in the anterior banded zone of the Descemet’s membrane. Moreover, mutations in the TCF8 gene have recently been implicated in PPMD, causing ectopic expression of COL4A3 by corneal endothelial cells.66

Figure 3-7: Ectropion uveae (arrow) in Iridocorneal Endothelial (ICE) syndrome.

Iridocorneal Endothelial (ICE) Syndrome Iridocorneal endothelial (ICE) syndrome describes a spectrum of disorders that are distinguished by a spectrum of nonfamilial iris abnormalities, including pigmented iris nodules, full-thickness holes and stromal matting and effacement (Figures 3-6 to 3-8).67-70 These disorders are further characterized by varying degrees of unilateral secondary angle closure glaucoma, abnormal corneal

Figure 3-8: Early Iridocorneal Endothelial (ICE) syndrome with iridoschisis (arrow).

endothelium with corneal edema, and peripheral anterior synechiae (PAS), although rare bilateral cases have been published recently.71-73 The diagnosis of ICE syndrome should be considered when two of the three main clinical

Corneal Endothelium in Health and Disease features (endothelial abnormality, typical iris changes and PAS) are found. There are three subdivisions of ICE syndrome: Iris nevus 74 or Cogan-Reese 75 syndrome, Chandler syndrome, 76 and essential iris atrophy. Essential iris atrophy typically presents in young adults with a predelication for females. Patients present with blurry vision or coincidental findings of iris changes, which can extend from a slightly eccentric pupil, to severe corectopia, and partial or full thickness iris holes, caused by stretching or ischemia. In advanced cases that are associated with glaucoma and corneal edema, patients complain of pain. The pathophysiology of this disorder appears to involve an abnormal clone of endothelial cells that has the ultrastructural findings of epithelial cells, but retains its characteristics and lineage.77,78 Early in the disease process, these cells are interspersed with groups of normal endothelial cells, while replacing them slowly. The abnormal endothelial cells produce an abnormal basement membrane that grows along with endothelial cells on the trabecular meshwork and the iris, and is referred to as the “glass membrane”.79,80 The contraction of this membrane results in iris stretching, and results in atrophy and corectopia on the opposite side.67 Further, secondary angle closure glaucoma develops due to the progressive formation of PAS or the membrane overgrowth of the angle. Slit-lamp biomicroscopy does not always demonstrate a beaten-metal appearance that is typically seen in Chandler syndrome. On specular microscopy, however, typical ICEcells with an irregular shape, altered density, and loss of the hexagonal conformation are apparent in the affected eye.81 In cases with severe corneal edema, interfering with the use of specular microscopy, confocal microscopy has been used successfully.82,83 Chandler syndrome typically presents with severe corneal edema and ipsilateral glaucoma due to membrane formation and PAS in the angle.76 Although iris changes and nodules rarely occur in Chandler syndrome, no holes are formed. The corneal edema presents due to an abnormal endothelium that has a fine beaten-metal appearance and resembles Fuchs’ endothelial dystrophy, although looking finer than guttata in Fuchs’ dystrophy. Differentiating this disorder from PPMD is also important. In contrast to Chandler syndrome, PPMD is familial, bilateral and does not present with the typical iris findings. The progressive nature of this disorder eventually leads to corneal decompensation. As with the other ICE syndrome subtypes, specular and confocal microscopy are useful diagnostic tools that demonstrate endothelial changes. Patients with iris nevus74 or Cogan-Reese syndrome75 present with unilateral iris nodules, diffuse iris nevi, heterochromia, loss of iris surface architecture, PAS,

29

glaucoma and occasionally corneal edema. Patients essentially have a diffuse endothelialization of the iris with multiple pigmented nodules that are produced by the contracting endothelial basement membrane. The membrane extends from the posterior surface of the cornea, over the angle, and onto the iris surface. Endothelial changes, typical for Chandler syndrome, and iris holes are typically not seen in this disorder. Although the etiology of ICE syndrome is still unknown, the spectrum of disorders included in this syndrome has been attributed to an embryologic ectopia of the epithelium, delayed expression of the neural crest development, or an inflammatory disease. The presence of a normal Descemet’s membrane under the posterior collagenous layer indicates a postnatal onset of the etiology.79,80 Immunohistochemical studies indicating the coexpression of cytokeratin (epithelium) and vimentin (endothelium) in ICE cells suggests that metaplasia of endothelial cells is the more likely etiology. 78,84,85 Although the stimulus for this metaplasia is unknown, studies using PCR have detected the herpes simplex virus in a large number of patients with ICE syndrome, indicating a possible role for this virus.86 Medical treatment has been proven ineffective in ICE syndrome, although corneal edema in Chandler syndrome can initially be treated successfully with hypertonic saline solution. The initial mild form of glaucoma also responds to aqueous suppressants. However, once medical therapy fails and the cornea decompensates, penetrating keratoplasty is needed and, in contrast to essential iris atrophy,87 good results have especially been reported with Chandler syndrome.68,88-90 Eventually, filtering surgery is also required due to the progressive closure of the angle. The use of antifibrotic agents and drainage implants has lead to only variable success.91-94 Trabeculectomy with mitomycin-C is therefore the initial approach, followed by implantation of drainage devices. Fuchs’ Endothelial Dystrophy Fuchs’ endothelial dystrophy was initially described by Fuchs in 1910 as a combination of epithelial and stromal edema in older patients.95 Fuchs’ endothelial dystrophy manifests itself as bilateral, albeit asymmetric, central corneal guttae, corneal edema and reduced vision (Figures 3-9 to 3-11).96,97 It is the most common endothelial dystrophy and is usually seen beyond the fifth decade of life, although Biswas et al recently reported several families with early onset of this dystrophy in their third and forth decades of life.65 Fuchs’ endothelial dystrophy is, despite its dominant inheritance form, more common and progressive in women.98 It may also present in a sporadic form and is thought to be a primary disorder of the endothelium.

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Corneal Endothelial Transplant

Figure 3-9: Fuchs’ dystrophy with fixed Descemet’s folds and diffuse corneal stromal and epithelial edema.

Figure 3-10: Specular microscopic view of Fuchs’ corneal dystrophy with guttae adjacent to normal hexagonal endothelial cells.

Figure 3-11: Photomicrograph of endothelial guttae (arrows) in Fuchs’ corneal endothelial dystrophy.

However, according to recent findings, subtypes could be the result of abnormal basement membrane assembly rather than a primary defect.99 Late-onset Fuchs’ endothelial dystrophy has recently been linked to chromosome 13pTel13q12.13.100 It is thought that a gradual transformation of

the endothelium into fibroblast-like cells results in the deposition of collagen fibrils and basement membrane as well as thickening of the Descemet’s membrane. Recent reports investigated whether the loss of endothelial cells is due to apoptotic cells death.101,102 While apoptosis was detected in all corneal layers, it remains unclear whether apoptosis is the cause of endothelial cell death or is it secondary to corneal edema. Clinically, corneal stromal edema starts centrally and spreads peripherally. This is associated with spreading of corneal guttae towards the periphery, or a central coalescence of corneal guttae with a beaten-metal appearance. Initially, an increase in endothelial pump function delays the edema. Eventually, the pump function is, however, insufficient and with progression of stromal edema, epithelial edema develops. Epithelial edema, usually starting with the central corneal thickness exceeding 650 μm,103 presents with microcystic edema and continues to bullous keratopathy. Finally, rupture of bullae causes pain and foreign body sensation. This can initially be managed medically, but ultimately requires corneal transplantation. The primary histologic features of Fuchs’ corneal dystrophy are multiple excrescences and a diffuse thickening of the Descemet’s membrane, associated with decrease in endothelial cell density and an increase in cell size. The abnormal portions of the Descemet’s membrane consist of banded collagen and multiple layers of basement membrane material. In addition, the repeated epithelial edema causes abnormalities of the Bowman’s layer with occasional breaks and a fibrovascular pannus in the endstage of the disease. The course of this dystrophy can further be accelerated after intraocular surgery, specifically cataract extraction. A cell count of less than 1000 cells/mm2 or corneal thickness greater than 640 μm are considered major risk factors for corneal decompensation after cataract surgery.38,104,105 While specular microscopy is of use in diagnosis and follow-up in earlier stages, confocal microscopy can help in the diagnosis of Fuchs’ dystrophy in advanced cases with severe edema.106-108 Medically, corneal edema can initially be managed with hypertonic saline solution or ointment, dehydration of the cornea with a blow dryer, and reduction of intraocular pressure. Bandage contact lenses may be beneficial in the treatment of painful bullae or recurrent erosions. However, penetrating keratoplasty is the treatment of choice in patients with severe vision loss, although endothelial grafting [See also Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)] is being advocated with increasing frequency over the last years and might become the treatment of choice in the future.109,110

Corneal Endothelium in Health and Disease

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Mesenchymal Dysgenesis Mesenchymal dysgenesis, including endothelial dysgenesis, is due to an abnormal migration of neural crest cells. The conditions, previously called Axenfeld anomaly and syndrome or Rieger anomaly and syndrome, have now been grouped under the term Axenfeld-Rieger syndrome.111,112 This syndrome now represents a spectrum of disorders that is characterized by an anterior displacement of Schwalbe’s line, called posterior embryotoxon, iris strands, iris hypoplasia, limbal dermoids, and glaucoma in 50% of patients (Figures 3-12 to 3-14).111 In addition, patients have associated facial (maxillary hypoplasia, hypertelorism, telecanthus, prominent lower lip), cranial (mental retardation, empty sella syndrome) and dental abnormalities (hypodontia, microdontia). Corneal abnormalities associated with Axenfeld-Rieger syndrome

Figure 3-12: Slit-lamp photograph showing Axenfeld-Rieger syndrome with iridocorneal adhesion (arrow).

Figure 3-13: Axenfeld-Rieger syndrome with prominent anteriorly displaced Schwalbe’s line (arrow).

Figure 3-14: Photomicrograph of Axenfeld-Rieger syndrome with anteriorly displaced Schwalbe’s line (green arrow) and iris processes adherent to the cornea (white arrow).

present as pleomorphism of the endothelium, attenuated Descemet’s membrane, and posterior embryotoxon (present in 10-15% of normal eyes). The inheritance pattern is dominant in 75% of cases, while sporadic cases have been reported. The forkhead genes FOXC1, located in chromosome 6p25, are responsible for many of the congenital defects of the anterior segment.113 In addition, mutations in the PITX2 gene (4q25, RIEG1), and chromosome 13q14/RIEG2, can cause this syndrome.114-117 Further, Reneker et al have recently demonstrated the critical role of the endothelial formation on the anterior segment development in a murine transgenic model.118 Peters anomaly is a rare, central, corneal opacity that presents at birth, with sixty to eighty percent of cases being bilateral.119 In general, a central leukoma is present that is associated with defects of the endothelium, Descemet’s membrane, or stroma, and can be associated with iridocorneal adhesions (Figures 3-15 and 3-16). The peripheral cornea is usually unaffected. Most cases are sporadic, although both dominant and recessive inheritance patterns have been described. Peters anomaly can be caused by mutations in the PAX6 gene (11p13), PITX2 gene (4q25-26), CYP1B1 gene (2p22-21),120 and the FOXC1 gene (6p25). Additional ocular findings include, cataract, keratolenticular touch, microcornea, aniridia, buphthalmos, glaucoma, and persistent fetal vasculature. In sixty percent of cases, systemic malformations have been described that include heart defect, hearing loss, external ear abnormalities, CNS deficits, developmental delays, spinal defects, gastrointestinal and genitourinary defects, facial clefts, and skeletal abnormalities.121 The lenticular abnormalities demonstrate a stalk-like connection to the posterior cornea, suggesting an incomplete separation of

32

Corneal Endothelial Transplant polymegathism and polymorphism in diabetic patients.123,125-128 Moreover, the central corneal thickness in diabetics is increased in most studies.123,126,129 Abnormal aldose reductase activity in the endothelium has been implicated in the decrease in cell density and increased cell size seen in diabetics. 130 Patients with DM also demonstrate a decreased ability to maintain stromal hydration that is due to endothelial pump dysfunction. This may be related to polyhydroxy alcohols and lysine cross-linking, which could explain the slow recovery from post-operative corneal edema.131-134 Figure 3-15: Central leukoma of Peters anomaly (arrow).

Figure 3-16: Photomicrograph of Peters anomaly with iridocorneal adhesion; note the absence of corneal endothelium.

the lens vesicle. This could be due to incomplete migration of neural crest cells, resulting in endothelial and stromal defects. Alternatively, a subluxation of the lens in utero could interrupt the migration of the endothelium. The prognosis for keratoplasty is typically poor and is highest for the first graft, while decreasing significantly with subsequent surgery.122

Diabetes Corneal changes in diabetes mellitus (DM) present as basement membrane changes, punctate epithelial erosions, vertical Descemet’s folds (Waite-Beetham lines), and decreased corneal sensation. Further, patients have an altered endothelial cell morphology and function that differ from retinal vasculopathy.123 There is no thickening of Descemet’s membrane and the degree of endothelial abnormality does not correlate with the duration of disease or level of glycemic control.124,125 While some studies have shown similar cell areas in diabetics and nondiabetic patients, other studies have demonstrated an increased

Toxic Anterior Segment Syndrome (TASS) Toxic anterior segment syndrome (TASS) is a rare complication of intraocular surgery that has only recently been recognized.135 TASS is an acute sterile inflammation resulting from inadvertent introduction of noxious agents in the anterior segment, e.g., endotoxins, medications, residual viscoelastic agents, preservatives, or an altered osmolarity or pH.136-139 Permanent corneal endothelial damage can occur in severe cases of TASS. Furthermore, cystoid macular edema, glaucoma and iris atrophy can result. Clinical signs of TASS include immediate postoperative corneal edema that can extend from limbus-to-limbus, a fixed pupil, high intraocular pressure, and anterior segment inflammation. Patients usually complain of decreased visual acuity and pain. Mild to moderate forms of TASS can be treated successfully with hourly topical and oral steroids, together with nonsteroidal anti-inflammatory agents every 6 hours.136,138 In general, most cases of TASS resolve within weeks with aggressive treatment but more serious cases require endothelial replacement [See also Section 9, Descemet Stripping Automated Endothelial Keratoplasty (DSAEK)].

Future Directions The authors believe future advances will come from the field of cell biology. In rabbits, the endothelium is capable of robust, mitotic self-repair but this feature is lacking in human corneal endothelium. However, there is a recent report of BrdU activity in the periphery of human corneas, which is a characteristic marker of mitotic activity. In the future, it may be possible to “unlock” the potential of these cells to replicate by finding a biochemical key. Another possibility is the use of stem cells or genetic engineering (perhaps via a viral vector) to stimulate or replace diseased or damaged cells. Hopefully, not too far in the future, we may be able to decrease the need for corneal transplantation

Corneal Endothelium in Health and Disease by increasing our understanding of the basic science of the corneal endothelium.

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A, Bonsheck R, Ridgway A, McLeod D, Sheffield VC, Stone EM, Schorderet DF, Black GC. Missene mutation in COL8A2, the gene encoding the alpha2 chain of type VIII collagen, cause two forms of corneal endothelial dystrophy. Hum Mol Genet 2001;10:2415-23. Krafcheck CM, Pawar H, Moroi SE, Sugar A, Lichter PR, Mackey DA, Mian S, Nairus T, Elner V, Schteingart MT, Downs CA, Kijek TG, Johnson JM, Trager EH, Rozsa FW, Mandal MN, Epstein MP, Vollrath D, Ayyagari R, Boehnke M, Richards JE. Mutations in TCF8 cause posterior polymorphous corneal dystrophy and ectopic expression of COL4A3 by corneal endothelial cells. Am J Hum Genet 2005; 77:694-708. Campbell DG, Shields MB, Smith TR. The corneal endothelium and the spectrum of essential iris atrophy. Am J Ophthalmol 1978;86:317-24. Shields MB. Progressive essential iris atrophy, Chandler’s syndrome and the iris nevus (Cogan-Reese) syndrome: a spectrum of disease. Surv Ophthalmol 1979;24:3-20. Yanoff M. Iridocorneal endothelial syndrome: unification of a disease spectrum. Surv Ophthalmol 1979;24:1-2. Eagle RCJ, Front R, Yanoff M, Fine BS. Proliferative endotheliopathy with iris abnormalities: The iridocorneal endothelial syndrome. Arch Ophthalmol 1979;97:2104-11. Luca-Glass TC, Baratz KH, Nelson LR, Hodge DO, Bourne WM. The contralateral corneal endothelium in the iridocorneal endothelial syndrome. Arch Ophthalmol 1997;115:40-44. Hemady RK, Patel A, Blum S, Nirankari VS. Bilateral iridocorneal endothelial syndrome: case report and review of the literature. Cornea 1994;13:368-72. Huna R, Barak A, Melamed S. Bilateral iridocorneal endothelial syndrome presented as Cogan-Reese and Chandler’s syndrome. J Glaucoma 1996;5:60-62. Scheie HG, Yanoff M. Iris nevus (Cogan-Reese) syndrome. A cause of unilateral glaucoma. Arch Ophthalmol 1975;93: 963-70. Cogan DG, Reese AB. A syndrome of iris nodules, ectopic Descemet’s membrane and unilateral glaucoma. Doc Ophthalmol 1969;26:424-33. Chandler PA. Atrophy of the stroma of the iris; endothelial dystrophy, corneal edema, and glaucoma. Am J Ophthalmol 1956;41:607-15. Kramer TR, Grossniklaus HE, Vigneswaran N, Waring GO, Kozarsky A. Cytokeratin expression in the corneal endothelium in the iridocorneal endothelial syndrome. Am J Ophthalmol 1992;33:3581-5. Levy SG, McCartney AC, Baghai MH, Barrett MC, Moss J. Pathology of iridocorneal-endothelial syndrome. The ICE-cell. Invest Ophthalmol Vis Sci 1995;36:2592-2601. Alvarado JA, Murphy CG, Maglio M, Hetherington J. Pathogenesis of Chandler’s syndrome, essential iris atrophy and the Cogan-Reese syndrome. I. Alterations of the corneal endothelium. Invest Ophthalmol Vis Sci 1986;27:853-72. Alvarado JA, Murphy CG, Juster RP, Hetherington J. Pathogenesis of Chandler’s syndrome, essential iris atrophy and the Cogan-Reese syndrome. II. Estimated age at disease onset. Invest Ophthalmol Vis Sci 1986;27:873-82. Liu YK, Wang IJ, Hu FR, Hung PT, Chang HW. Clinical and specular microscopy manifestations of iridocorneal endothelial syndrome. Jpn J Ophthalmol 2001;45: 281-7. Chiou AG, Kaufman SC, Beuerman RW, Ohta T, Yaylali V, Kaufman HE. Confocal microscopy in the iridocorneal endothelial syndrome. Br J Ophthalmol 1999;83: 697-702. Garibaldi DC, Schein OD, Jun A. Features of the iridocorneal endothelial syndrome on confocal microscopy. Cornea 2005; 24:349-51. Howell DN, Damms T, Burchette JLJ, Green WR. Endothelial metaplasia in the iridocorneal endothelial syndrome. Invest Ophthalmol Vis Sci 1997;38:1896-1901.

Corneal Endothelium in Health and Disease 85. Hirst LW, Bancroft J, Yamauchi K, Green WR. Immunohistochemical pathology of the corneal endothelium in iridocorneal endothelial syndrome. Invest Ophthalmol Vis Sci 1995;36: 820-7. 86. Alvarado JA, Underwood JL, Green WR, Wu S, Murphy CG, Hwang DG, Moore TE, O’Day D. Detection of herpes simplex viral DNA in the iridocorneal endothelial syndrome. Arch Ophthalmol 1994;112:1601-9. 87. DeBroff BM, Thoft RA. Surgical results of penetrating keratoplasty in essential iris atrophy. J Refract Corneal Surg 1994;10:428-32. 88. Chang PC, Soong HK, Couto MF, Meyer RF, Sugar A. Prognosis for penetrating keratoplasty in iridocorneal endothelial syndrome. Refract Corneal Surg 1993;9:129-32. 89. Crawford GJ, Stulting RD, Cavanagh HD, Waring GOr. Penetrating keratoplasty in the management of iridocorneal endothelial syndrome. Cornea 1989; 8:34-40. 90. Buxton JN, Lash RS. Results of penetrating keratoplasty in the iridocorneal endothelial syndrome. Am J Ophthalmol 1984; 98:297-301. 91. Doe EA, Budenz DL, Gedde SJ, Imami NR. Long-term surgical outcome of patients with glaucoma secondary to the iridocorneal endothelial syndrome. Ophthalmology 2001; 108:1789-95. 92. Lanzl IM, Wilson RP, Dudley D, Augsburger JJ, Aslanides IM, Spaeth GL. Outcome of trabeculectomy with mitomycin-C in the iridocorneal endothelial syndrome. Ophthalmology 2000; 107:295-7. 93. Kim DK, Aslanides IM, Schmidt CMJ, Spaeth GL, Wilson RP, Augsburger JJ. Long-term outcome of aqueous shunt surgery in ten patients with iridocorneal endothelial syndrome. Ophthalmology 1999;106:1030-4. 94. Wright MM, Skuta GL, Drake MV, Chang LF, Rabbari R, Musch DC, Teikari J. 5-Fluorouracil after trabeculectomy and the iridocorneal endothelial syndrome. Ophthalmology 1991;98: 314-6. 95. Fuchs E. Dystrophia epithelialis corneae. Graefe’s Arch Clin Exp Ophthalmol 1910; 76:478-508. 96. Krachmer JH, Purcell JJj, Young CW, Bucher KD. Corneal endothelial dystrophy: A study of 64 families. Arch Ophthalmol 1978;96:2036-9. 97. Adamis AP, Filatov V, Tripathi BJ, Tripathi RC. Fuchs’ endothelial dystrophy of the cornea. Surv Ophthalmol 1993;38:149-68. 98. Cross HE, Maumenee AE, Cantolino SJ. Inheritance of Fuchs’ endothelial dystrophy. Arch Ophthalmol 1971; 85:268-72. 99. Gottsch JD, Zhang C, Sundin OH, Bell WR, Stark WJ, Green WR. Fuchs‘ corneal dystrophy: aberrant collagen distribution in an L450W mutant of the COL8A2 gene. Invest Ophthalmol Vis Sci 2005;46:4504-11. 100. Sundin OH, Jun AS, Broman KW, Liu SH, Sheehan SE, Vito EC, Stark WJ, Gottsch JD. Linkage of late-onset Fuchs’ corneal dystrophy to a novel locus at 13pTel-13q12.13. Invest Ophthalmol Vis Sci 2006;47:140-5. 101. Borderie VM, Baudrimont M, Vallee A, Ereau TL, Gray F, Laroche L. Corneal endothelial cell apoptosis in patients with Fuchs’ dystrophy. Invest Ophthalmol Vis Sci 2000;41: 2501-5. 102. Li QJ, Ashraf MF, Shen DF, Green WR, Stark WJ, Chan CC, O’Brien TP. The role of apoptosis in the pathogenesis of Fuchs endothelial dystrophy of the cornea. Arch Ophthalmol 2001;119:1597-1604. 103. Oh KT, Weil LJ, Oh DM, Mathers WD. Corneal thickness in Fuchs’ dystrophy with and without epithelial oedema. Eye 1998; 12:282-4. 104. Seitzman GD, Gottsch JD, Stark WJ. Cataract surgery in patients with Fuchs’ corneal dystrophy: expanding recommendations for cataract surgery without simultaneous keratoplasty. Ophthalmology 2005;112:441-6.

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105. Ophthalmic procedures assessment: Corneal endothelial photography. Ophthalmology 1991; 98:1464-8. 106. Chiou AG, Kaufman SC, Beuerman RW, Ohta T, Soliman H, Kaufman HE. Confocal microscopy in cornea guttatae and Fuchs’ endothelial dystrophy. Br J Ophthalmol 1999;83:185-9. 107. Kaufman SC, et al. Diagnosis of advanced Fuchs’ dystrophy with the confocal microscope. Am J Ophthalmol 1993;116: 652-3. 108. Mustonen RK, McDonald MB, Srivannaboon S, Tan AL, Doubrava MV, KIm CK. In vivo confocal microscopy of Fuchs’ endothelial dystrophy. Cornea 1998;17: 493-503. 109. Melles GR, Lander F, van Dooren BT, Pels E, Beekhuis WH. Preliminary clinical results of posterior lamellar keratoplasty through sclerocorneal pocket incision. Ophthalmology 2000;107:1850-6. 110. Terry MA, Ousley PJ. Replacing the endothelium without corneal surface incisions or sutures: the first United States clinical series using the deep lamellar endothelial keratoplasty procedure. Ophthalmology 2003;110:755-64. 111. Shields MB, Buckley E, Klintworth GK, Thresher R. AxenfeldRieger syndrome: A spectrum of developmental disorders. Surv Ophthalmol 1985;29:387-409. 112. Alward WL. Axenfeld-Rieger syndrome in the age of molecular genetics. Am J Ophthalmol 2000;130:107-15. 113. Nishimura DY, Searby CC, Alward WL, Walton D, Craig JE, Mackey DA, Kawas K, Kanis AB, Patil SR, Stone EM, Sheffield VC. A spectrum of FOXC1 mutations suggests gene dosage as a mechanism for developmental defects of the anterior chamber of the eye. Am J Hum Genet 2001;68:364-72. 114. Murray JC, Bennett SR, Kwitek AE, Small KW, Schinzel A, Alward WL, Weber JL, Bell GI, Buetow KH. Linkage of Rieger syndrome to the region of the epidermal growth factor gene on chromosome 4. Nat Genet 1992;2:46-49. 115. Semina EV, Reiter R, Leysens NJ, Alward WL, Small KW, Datson NA, Siegel-Bartelt J, Bierke-Nelson D, Bitoun P, Zabel BU, Carey JC, Murray JC. Cloning and characterization of a novel bicoidrelated homeobox transcription factor gene, RIEG, involved in Rieger syndrome. Nat Genet 1996;14:392-9. 116. Kulak SC, Kozlowski K, Semina EV, Pearce WG, Walter MA. Mutation in the RIEG1 gene in patients with iridogoniodysgenesis syndrome. Hum Mol Genet 1998; 7:1113-7. 117. Phillips JC, del Bono EA, Haines JL, Pralea AM, Cohen JS, Greff LJ, Wiggs JL. A second locus for Rieger syndrome maps to chromosome 13q14. Am J Hum Genet 1996;59:613-9. 118. Reneker LW, Silversides DW, Xu L Overbeek PA. Formation of corneal endothelium is essential for anterior segment development—a transgenic mouse model of anterior segment dysgenesis. Development, 2000;127. 119. Yang LI, Lambert SR. Peters’ anomaly. A synopsis of surgical management and visual outcome. Ophthalmic Clin North Am 2001;14:467-77. 120. Vincent A, Billingsley G, Priston M, Williams-Lyn D, Sutherland J, Glaser T, Oliver E, Walter MA, Heathcote G, Levin A, Heon E. Phenotypic heterogeneity of CYP1B1: mutations in a patient with Peters’ anomaly. J Med Genet 2001;38:324-6. 121. Traboulsi EI, Maumenee IH. Peters’ anomaly and associated congenital malformations. Arch Ophthalmol 1992; 110: 1739-42. 122. Yang LI, Lambert SR, Lynn MJ, Stulting RD. Long-term results of corneal graft survival in infants and children with peters anomaly. Ophthalmology 1999;l106:833-48. 123. Schultz RO, Matsuda M, Yee RW, Edelhauser HF, Schultz KJ. Corneal endothelial changes in type I and II diabetes mellitus. Am J Ophthalmol 1984;98:401-10. 124. Shetlar DJ, Bourne WM, Campbell RJ. Morphological evaluation of Descemet’s membrane and corneal epithelium in diabetes mellitus. Ophthalmology 1989;96:247-50.

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125. Matsuda M, Ohguro N, Ishimoto I, Fukata M. Relationship of corneal endothelial morphology to diabetic retinopathy, duration of disease and glycemic control. Jpn J Ophthalmol 1990;34: 53-56. 126. Keoleian GM, Pach JM, Hodge DO, Trocme SD, Bourne WM. Structural and functional studies of the corneal endothelium and diabetes mellitus. Am J Ophthalmol 1992; 113:64-70. 127. Pardos GJ, Krachmer JH. Comparison of endothelial cell density in diabetics and a control population. Am J Ophthalmol 1980;90:172-4. 128. Lass JH, Spurney RW, Dutt RM, Andersson H, Kochar H, Rodman HM, Stern RC, Doershuk CF. A morphological and fluorometric analysis of the corneal endothelium in type I diabetes mellitus and cystic fibrosis. Am J Ophthalmol 1985; 100:783-8. 129. Buettner H, Bourne WM. Effect of trans pars plana surgery on the corneal endothelium. Dev Ophthalmol 1981;2:28-34. 130. Datiles MB, Kador PF, Kashima K, Kinoshita JH, Sinha A. The effects of sorbinil, an aldose reductase inhibitor, on the corneal endothelium in galactosemic dogs. Invest Ophthalmol Vis Sci 1990;31:2201-4. 131. Whikehart DR. The inhibition of sodium potassium-stimulated ATPase and corneal swelling: the role played by polyols. J Am Optom Assoc 1995;66:331-3.

132. Sady C, Khosrof S, Nagaraj R. Advanced Maillard reaction and crosslinking of corneal collagen in diabetes. Biochem Biophys Res Commun 1995;214:793-7. 133. McNamara NA, Brand RJ, Polse KA, Bourne WM. Corneal function during normal and high serum glucose levels in diabetics. Invest Ophthalmol Vis Sci 1998;39:3-17. 134. Herse PR. Diurnal and long-term variations in the corneal thickness in the normal and alloxan-induced diabetic rabbit. Curr Eye Res 1990;9:451-7. 135. Mamalis N, Edelhauser HF, Dawson DG, et al. Toxic anterior segment syndrome. J Cataract Refractive Surg 2006;32:324-33. 136. Jehan FS, Mamalis N, Spencer TS, Fry LL, Kerstine RS, Olson RJ. Postoperative sterile endophthalmitis (TASS) associated with the memorylens. J Cataract Refract Surg 2000;26:1773-7. 137. Parihk CH, Edelhauser HF. Ocular surgical pharmacology: corneal endothelial safety and toxicity. Curr Opin Ophthalmol 2003; 14:178-85. 138. Moshirfar M, Whitehead G, Beutler BC, Mamalis N. Toxic anterior segment syndrome after Verisyse iris-supported phakic intraocular lens implantation. J Cataract Refract Surg 2006;32: 1233-7. 139. Monson MC, Mamalis N, Olson RJ. Toxic anterior segment inflammation following cataract surgery. J Cataract Refract Surg 1992;18:184-9.

George Baikoff

Optical Coherence Tomography (OCT) of the Anterior Segment

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Introduction In routine practice, the anterior segment is generally observed with the slit-lamp which gives frontal images with a subjective estimation of a few external measurements of the eye. An ultrasonic evaluation of corneal pachymetry and anterior chamber depth can be done if additional information is required. Today, with the development of sophisticated surgical techniques it has become essential to obtain elaborate static and dynamic measurements of the anterior segment in order to meet modern safety requirements. The choice now lies between optical and ultrasonic exploration of the anterior segment. Development of the Scheimpflug technique with oblique images resulted in a new capability to evaluate the distances in the eye’s anterior segment along different optical sections. The major drawback of this technology is a difficult mathematical reconstruction, as well as scleral overexposure when taking the photos. In particular, the entire angle area is masked by this overexposure and the fine structures are indiscernible (scleral spur, irido-corneal area and the angle). The idea of using infrared wavelengths in optical coherence tomography 1-2 is expanding rapidly (Visante™ OCT, Carl Zeiss Meditec, Jena, Germay) [See also Chapter 5, Optical Coherence Tomography in Corneal Implant Surgery, Chapter 6, Use of Optical Coherence Tomography in Descemet’s Stripping with Endothelial Keratoplasty (DSEK) and Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)]. About ten years ago, Izatt et al3 suggested using the OCT for anterior segment imaging. Reflection of the infrared light rays is captured and analysed by an optical sensor and appropriate software re-adjusts the dimensions of the images by erasing distortion errors due to different corneal optical transmission differences. Measuring software capable of evaluating the distance between two points, radius of curvature and angles is also integrated. Ultrasonic exploration of the anterior segment [See also Chapter 7, Imaging of the Cornea and Anterior Segment with High Frequency Ultrasound] appears to have reached its limits, whether in UBM or ultra high frequency ultrasound equipment (Artemis, ArcScan, Inc., Morrison, CO, USA) where resolution is identical to the anterior segment OCT’s 1310 nm wavelength (18µm for axial resolution, 60 µm for transverse resolution). Manipulation is fairly complex and even if some ultrasonic measurements are used as references to calibrate a certain number of instruments, there is no certainty concerning the exact in-vivo ultrasonic measurements. However, the error can be considered

relative, as long as the reference scale remains constant with each technology.

Anterior Chamber Exploration with the Visante™ OCT Anterior Chamber Measurements Using the Visante™ OCT (Figure 4-1), several studies were carried out on the static and dynamic4,5 anatomy of the anterior chamber. A large amount of data was obtained in the field of phakic implants and accommodation.6-8 Exploration and measurement of the anterior chamber’s internal dimensions, which was fairly imprecise until recently, is now possible where before only the external measurements of the anterior chamber were considered. Based on these internal measurements, considering that the device’s calibration assessment methods are reliable and that the error margin is minimal, our studies showed that in 75% of cases the internal diameter of the anterior chamber was an oval with a large vertical axis (Figure 4-2). On the basis of the external measurements of the cornea, which is the usual method of measuring, the shape of the anterior chamber is considered to be oval with a large horizontal axis in harmony with the palpebral cleft. These internal measurements are essential when considering angle-supported phakic implants.

Accommodation It was possible to study accommodation at different periods in life with the Visante™ OCT (Carl Zeiss Meditec, Jena,

Figure 4-1: Photograph of a Visante™ OCT device (Carl Zeiss Meditec, Jena, Germay).

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Figure 4-2: A cumulative study of 89 normal unoperated eyes showed that in 74% of cases the vertical diameter was larger than the horizontal diameter by at least 100 µm.

Figure 4-3: Visante™ OCT (Carl Zeiss Meditec, Jena, Germay) images showing the anterior displacement of the crystalline lens during accommodation in a 10 year-old child, accounting for the decrease in the anterior chamber depth during accommodation.

information that can play a role in the field of phakic IOLs.4-8 Each patient’s anterior chamber anatomy and the protrusion of the crystalline lens is of important consideration, as these individual variations can have an effect on phakic implant tolerance. For example, the study of a large series of phakic eyes with Artisan implants revealed that pigment dispersion syndrome appeared more frequently in eyes that had a significant protrusion of the crystalline lens.10 Figure 4-4 shows different aspects of the crystalline lens rise and Figure 4-5 represents a high myopic patient referred for refractive surgery. This patient suffered from microspherophakia. Although the anterior chamber was deep enough (over 3 mm), the forward protrusion of the crystalline lens was extremely significant, and because of this abnormality an Artisan type of lens implant would most certainly have been a contraindication in this case. Today, it is possible to study the shape of the crystalline lens with the OCT as long as this can be done within the

Germay) and the images revealed that some very important dynamic distortions occurred in the anterior chamber during accommodation. The anatomical relationship between the iris and the crystalline lens underwent modifications, and the decrease of the anterior chamber depth was inversely proportional to the degree of accommodation. During accommodation as shown in Figure 4-3 there was a forward thrust of the anterior pole of the crystalline lens and a decrease in the anterior chamber depth. Recent studies4,5,8 have confirmed von Helmholtz’s accommodation theory that was described more than 150 years ago.9

Crystalline Lens Observations of the anatomy of the crystalline lens both in its static and dynamic state has provided significant

Figure 4-4: Different aspects of crystalline lens rise.

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Figure 4-5: Visante ™ OCT (Carl Zeiss Meditec, Jena, Germay) images showing microspherophakia.

Figure 4-7: OCT image showing piggy-back intraocular lens implants. Notice the absence of any tissue in between the two intraocular lenses.

Figure 4-6: OCT image of a 2-year-old child with Peter’s anomaly and nystagmus.

Figure 4-8: OCT imaging provides a good view of the anterior chamber angles and the anterior segment of the eye. There is iris adhesion to the peripheral cornea resulting in angle closure in this case of angle-closure glaucoma.

pupillary area. Moreover, the OCT is a non-contact procedure and hence it is easy to use even in children. Studying congenital malformations of the anterior segment are fairly simple when using an OCT unit. Figure 4-6 is the OCT image of a 2-year-old child with Peters anomaly and nystagmus. The peripheral iris root is visible, as well as crystalline lens adhesion to the posterior surface of the cornea, leading to corneal edema. In this case, the different layers of the crystalline lens are also visible. However, studying the crystalline lens is not always as easy as in this extreme case and to date the technology at our disposal cannot give us a precise idea of the optical density of the crystalline lens. This limitation is due to the fact that along the optical axis there are significant reflection phenomena due to the laser beam, which is used as a fixation point. The OCT unit has a fairly limited depth of focus, and to study the thickness of the natural crystalline lens, focusing has to be done towards the back of the eye. On the other hand, studying intraocular lens implants in pseudophakic eyes is fairly simple. Figure 4-7 shows piggy-back intraocular lens implants, and it is quite clear that there is no tissue in between the two intraocular lenses. It is also possible to observe the development of inter-lenticular cellular proliferation when it exists. The relationship between the iris, crystalline lens and the cornea is easy to visualise and the OCT appears to be a useful tool for diagnosing angle-closure glaucoma (Figure

Figure 4-9: This is an example of a nanophthalmic patient, where there is a total absence of the anterior chamber.

4-8). Figure 4-9 shows the most extreme case, where there is a total absence of the anterior chamber in a nanophthalmic patient.

Cornea A profile study of the corneal morphology is relatively simple. Because of the non-contact technique that is used, it is easier to observe sensitive eyes that are otherwise difficult to image with ultrasonic techniques. Examinations are easy to repeat, image acquisition is rapid, and it can be performed several times on the same meridian, as alignment is along the visual axis. The three images in Figure 4-10 show patients at different stages of keratoconus, namely,

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Figure 4-10: Different stages of keratoconus from an early stage to an advanced stage with corneal hydrops as visualized using the Visante™ OCT (Carl Zeiss Meditec, Jena, Germay).

from the early stage, to corneal hydrops. The acute hydrops in keratoconus secondary to a break in the Descemet’s membrane (Figure 4-10) is particularly interesting as it shows the intrusion of aqueous humour into the corneal stroma resulting in corneal thickening and the formation of an aqueous bleb in front of the Descemet’s membrane, stretched across the deep, posterior corneal stroma. Corneal OCT is a remarkable clinical tool for the evaluation of both the preoperative diagnosis as well as postoperative follow-up in patients with varying disease states of the cornea. Figure 4-11 is an OCT image of a patient with iridocorneal-endothelial (ICE) syndrome taken shortly after a penetrating keratoplasty. The corneal graft has not yet recovered its normal shape and transparency following the surgery; however, it is possible with the OCT to visualize behind the graft, without any significant

Figure 4-11: OCT image of a patient with irido-corneal-endothelial (ICE) syndrome shortly after a penetrating keratoplsty. Gonio-synechiae are clearly visible in this case of ICE syndrome.

discomfort to the patient since it is a non-contact OCT. The development of pathological gonio-synechiae secondary to endothelial “metaplasia” in this case of ICE syndrome is clearly visible (Figure 4-11). As we mentioned in the introduction, the infrared light rays cannot penetrate pigments within the eye. It is therefore generally impossible to see just behind the iris pigments. However, this does not mean that one cannot see through opaque structures. The OCT penetrates milky-white corneas and the sclera as both these structures are without pigments. Figure 4-12 shows an OCT image of the preoperative aphakic corneal edema, where the cornea is completely white due to diffuse corneal stromal and epithelial edema. Even in this totally cloudy cornea, the iridocorneal synechiae and the pathological vitreous strands are clearly visible (Figure 4-12). In this case, there

Figure 4-12: Preoperative OCT image of an eye that has complete clouding of the cornea secondary to aphakic corneal edema. Even in this totally cloudy cornea, the iridocorneal synechiae and the pathological vitreous strands are clearly visible.

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Figure 4-13: OCT image of a patient with an inferior marginal keratoconus. The image information is helpful in planning a possible decentered PKP in this case.

is no artificial lens, namely, no pseudophakic IOL, and the surgeon can therefore decide on his surgical strategy. Preoperative sizing is also easy on the corneal cuts of the OCT imaging system. For example, Figure 4-13 displays an OCT image of a patient with an inferior marginal keratoconus. In this case, a large diameter deep lamellar graft can be surgically challenging, because, if there is an intra-operative corneal perforation during the lamellar dissection, the surgeon has to convert to a large diameter penetrating keratoplasty with a high risk of postoperative corneal graft rejection. In this case, it is possible to plan a graft with an 8.0 to 8.5 mm diameter, by not only including the ectatic zone of the patient’s cornea but also sufficiently covering the optical axis. The anterior segment OCT can be used routinely for the follow-up of penetrating and lamellar keratoplasties [See also Chapter 5, Optical Coherence Tomography in Corneal

Implant Surgery, Chapter 6, Use of Optical Coherence Tomography in Descemet’s Stripping with Endothelial Keratoplasty (DSEK) and Descemet’s Stripping with Automated Endothelial Keratoplasty (DSAEK)]. Figure 4-14 shows an OCT image of a patient with persistent corneal edema following corneal graft. OCT examination in this case shows that the edema was due to the non-adherence of the donor endothelial graft. As it was an old graft with longstanding corneal edema, a full-thickness penetrating keratoplasty was performed. A case of corneal stromal dystrophy that was treated by manual, deep anterior lamellar keratoplasty is shown in Figures 4-15 and 4-16. A small corneal perforation occurred during surgery which was closed with viscous substance. Postoperatively, there was a double anterior chamber and the recipient Descemetic-endothelial bed (Descemet’s membrane with its healthy endothelium) failed to adhere to the grafted donor corneal stroma that is devoid of its Descemet’s membrane and endothelium. Anterior chamber injection of C3F8 helped collapse the double anterior chamber, and facilitated donor-recipient tissue adherence with resolution of the corneal edema. Using standard software, focal areas of corneal stromal edema can be visualized as seen in Figure 4-17. Figure 4-17 shows a recent Artisan phakic implant dislocation, with focal area of corneal edema that was localized to the region of implant contact with the corneal endothelium. Once the implant was re-positioned, the focal corneal edema completely disappeared without any significant endothelial cell loss, due to the early (few days) surgical intervention following implant dislocation. Today, the Visante™ OCT

Figure 4-14: Non-adherent endothelial graft with corneal stromal edema and stromal thickening are clearly visible. Also, seen is a large fluid pocket in the region of greatest non-adherence between the donor corneal disk and the patient’s corneal stroma.

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Figure 4-15: Double anterior chamber after deep anterior lamellar keratoplasty in a case of corneal stromal dystrophy. Patient’s Descemet’s membrane that is not adherent to the donor corneal stroma is clearly visible.

Figure 4-18: Pachymetry mapping of an eye with keratoconus. Figure 4-16: Same eye as shown in Figure 4-15 after an anterior chamber injection of C3F8. Note the total collapse and disappearance of the double anterior chamber and good adherence of the patient’s Descemet’s membrane to the donor corneal stroma. Also seen is the resolution of the corneal edema.

Figure 4-19: LASIK flap measurement using OCT imaging.

Figure 4-17: Focal area of corneal stromal edema is visible, that was secondary to a dislocated Artisan phakic implant. The phakic implant was repositioned.

has a high resolution software giving an axial resolution of 8 µm. Images of the corneal stroma are more precise and with this new software a 10 mm diameter pachymetric mapping is available. Studying the thin or thick zones of the cornea is possible and this will probably be very helpful for an early diagnosis of keratoconus (Figure 4-18). However, great care is needed during the testing process, as the use of automatic software can lead to potential errors. In an ongoing study on postoperative LASIK flaps, the automatic method used to measure the corneal flaps (Figure 4-19), occasionally produced significant errors. The enveloping curve that reconstructs the anterior surface of the cornea has a serious defect leading to important errors, especially, if only taking into account the automated figures

produced by the device’s software. The advances in technology definitely make things simpler, but it is necessary to keep an open and critical mind, and personally check the results provided by the technicians. It is essential to compare the complimentary examinations with the clinical evaluations.

Conclusions and the Future of Anterior Segment OCT This general overview should give the reader an idea of the importance of this imaging technique in clinical practise. It is important to note that the equipment is fairly simple to use. Once the patient has fixated on the target, manipulation is as easy as with a corneal topography unit. It is a noncontact device, with quick image capture and the technician decides which axis he wishes to explore. Image resolution is similar to the ultra-high-frequency scanning devices. However, with the OCT the explored zones are easier to find because, the fixation point is on the optical axis. The

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irido-corneal angle is clearly visible in the OCT. When studying the measurements, or checking the evolution of the anterior segment, either the irido-corneal angle area or the scleral spur area, can be used as a reference point, since both these areas remain constant in the anterior chamber anatomy during various modes of testing using the OCT. Finally, in the laboratory, with a more appropriate wavelength, and/or a modification of the power of the light ray it has been possible to obtain images that are close in comparison to tissue histology. On pseudophakic cadaveric eyes, Linnola et al 10 was able to demonstrate cell proliferation on the posterior capsule. The images obtained with the high resolution OCT,10 were similar to histopathologic sections performed on the same cadaveric eyes. Future technological evolution of the Visante™ OCT for exploring the anterior segment is something to look forward to. Hopefully, in the near future, these improvements will be similar to those of the OCT for exploring the posterior segment of the eye, namely, more precise images, resolution to a few microns and a 3D image reconstruction of the anterior segment structures. All of these are under study. It is quite certain that this OCT imaging system will, in daily practice, replace ultrasound equipment for anterior segment exploration.

References 1. Huang D, Swanson EA, Lin CP, et al. Optical Coherence Tomography. Science 1991;254:1178-81. 2. Puliafito C, Hee MR, Schuman JS, et al. Optical Coherence Tomography of Ocular Diseases, Slack Inc, 1996. 3. Izatt JA, Hee MR, Swanson EA, et al. Micrometer-scale resolution imaging of the anterior eye in vivo with optical coherence tomography. Arch Ophthalmology 1994;112:1584-9. 4. Baikoff G, Lutun E, Ferraz C, et al. Analysis of the eye’s anterior segment with an optical coherence tomography: Static and dynamic study. J Cataract Refract Surg 2004;30:1843-50. 5. Baikoff G, Jitsuo Jodai H, Bourgeon G. Evaluation of the measurement of the anterior chamber’s internal diameter and depth: IOLMaster vs AC OCT. J Cataract Refract Surg 2005;31 (9):1722-8. 6. Baikoff G, Lutun E, Ferraz C, et al. Refractive Phakic Iols: Contact Of Three Different Models With The Crystalline Lens, An Ac Oct Study Case Reports. J Cataract Refract Surg 2004;30: 2007-12. 7. Baikoff G, Bourgeon G, Jitsuo Jodai H, et al. Pigment Dispersion and Artisan Implants. The crystalline lens rise as a safety criterion. J Cataract Refract Surg 2005;31:674-80. 8. Baikoff G, Lutun E, Ferraz C, Wie J. Anterior chamber optical coherence tomography study of human natural accommodation in a 19-year-old albino. J Cataract Refract Surg 2004;30:696-701. 9. von Helmholtz H. Uber die akkommodation des auges. Albrect von Graefes Arch Klein Exp Ophthalmol 1855;1(2):1-89. 10. Linnola R, Findl O, Hermann, B Sattmann H, Unterhuber A, Happonen RP, et al. Intraocular lens-capsular bag imaging with ultrahigh-resolution optical coherence tomography. Pseudophakic human autopsy eyes. J Cataract Refract Surg 2005;31: 818-23.

Roger F Steinert

Optical Coherence Tomography in Corneal Implant Surgery

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Principles of Optical Coherence Tomography Optical coherence tomography (OCT) utilizes multiple pulses of light to create a cross-sectional image [See also Chapter 4, Optical Coherence Tomography (OCT) of the Anterior Segment, and Chapter 6, Use of Optical Coherence Tomography in Descemet’s Stripping with Endothelial Keratoplasty (DSEK) and Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)], somewhat analogous to the manor in which a Bscan ultrasound cross-section image is created by the feedback signals of multiple pulses of sound waves. OCT performs cross-sectional imaging by measuring the light echo time delay and the intensity of the back reflected light from structures within the eye. Because OCT uses light, one of the essential requirements in developing an OCT image is that the structure be partially transparent to the wavelength of light. In ophthalmology, OCT is particularly well suited because of the optical properties of the eye. In addition, OCT can be performed without physical contact to the eye, unlike ultrasound. This improves patient comfort and ease of application. The light energy directed into the eye by OCT is reflected back from boundaries between different tissues. The light scatters differently in tissues with different optical properties. The distance and dimension of the different tissue structures are determined by the time delay of the “echo” of light that is back reflected or back scattered. Because the speed of light is almost one million times faster than the speed of sound, measurements involving light require sophisticated ultrafast time resolution. The ultrafast and high resolution measurements in OCT are possible because of an optical technique known as low-coherence interferometry. The physical technique of white-light interferometry was first described by Sir Isaac Newton.1 The fundamental principle is that of splitting the optical beam at its origin, and then comparing the downstream beam that is passed through the eye to a reference optical beam that is not passed through the eye structures. In the ophthalmic version of OCT, the light source is a laser with the ability to emit low energy short pulses of light. The optical beam from the laser is directed onto a partially reflecting mirror, commonly known as a beamsplitter. This mirror splits the light into two parts, the reflected beam and a transmitted beam. The beam that passes into the eye and then is reflected back is compared to the beam that is not passed through the eye. The disparity of these two beams creates the interferometry signal. Optical interference occurs when the pulses

coincide, and the interference is measured and quantitated by a photodetector.2 In order to measure the time delays of the light reflection (echoes) from the various structures within the eye, the reference mirror within in the OCT device can vary its position. The key principle is that the interferometer measures the time delays of the optical echoes by utilizing the comparison of the light reflected from the eye structure to the reference beam that has traveled the reference path within the device. Figure 5-1 shows the basic schematic principle of the ophthalmic OCT as developed by Zeiss, Inc. (Visante OCT, Zeiss-Meditec, Dublin, CA).

Figure 5-1: Schematic diagram of the optics of the Visante anterior segment OCT (Courtesy Zeiss-Meditec, Dublin, CA).

The images created by OCT can be displayed as a gray scale or a false-color scale. The gray scale image represents the intensity of the back reflected optical signal, where, a bright white color represents a strong return signal and black represents no return signal. The shades of gray are levels of back return signal in between. The gray scale is useful in anterior segment OCT, in particular, because it allows a finer definition than the representation of the falsecolor scale on a computer screen. In posterior segment OCT, the false-color scale is more commonly used because the colors can bring out subtle differences in the return signal, corresponding to different tissues. In anterior segment OCT, the color scale therefore has some utility in imaging iris structures, in particular. In retinal OCT imaging, the wavelength employed is typically 800 nm. In contrast, a wavelength of 1310 nm is used for the Visante OCT (Zeiss-Meditec, Dublin, CA). This is because longer wavelengths are scattered less and penetrate deeper, this is particularly important for penetrating the sclera in order to image the anterior angle and the anterior sclera. In addition, the safe level of light exposure is higher at a longer wavelength, and therefore more power can be employed. This results in the ability to

Optical Coherence Tomography in Corneal Implant Surgery increase the speed of capturing, with more frames per second. This speed helps avoid artifact from movement of the eye during the capture of an image. 3,4

OCT Imaging of Lamellar Corneal Dissection The appearance of a normal cornea is shown in Figure 5-2A. In addition to the frequently employed gray scale, the two choices for color scale are also shown. Figure 5-2B uses the same false-color scale that is familiar from posterior segment OCT, whereas Figure 5-2C uses a color scale that has better resolution of fine detail. Note that all of these images represent high resolution captures of the cornea. The vertical line and bright reflex in the center indicate that the image capture was well centered; this artifact occurs when the OCT is well centered on the vertex. In the upper right hand corner, one can see a circle with “OD” highlighted, indicating that the image is from the right eye.

A

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The triangle represents the nose. The direction of the arrow shows the orientation of the image plane. In this case, the image is a horizontal section. Just below that, on the right, is a marking for 0 degrees, and on the left, 180 degrees. These numbers change as the operator selects different orientations, either automatically or manually. The orientation of the section can be changed in steps as fine as one degree. HIPPA-compliant patient information is displayed at the top of the image. The operator can deselect the computer generated boundaries laterally and at the anterior and posterior corneal levels, if desired. A LASIK flap is shown in Figure 5-3A. Note that this particular section runs from inferotemporal to superonasal in the right eye at a 45 degree angle. The interface of the LASIK flap is best seen on the right side of the image as a slightly brighter line, representing increased light signal return from the interface. Figure 5-3B shows the addition of measuring devices to the image of a LASIK flap. Shown in orange, the “flap tool” allows the operator to measure both the overall thickness of the cornea and the thickness of the flap and underlying residual stromal bed. Up to seven measurements can be made simultaneously on the same cornea. Associated with each of these measurements are three numbers. At the top, the number anterior to the cornea is the distance from the corneal vertex. One can see that the central measurement is perfectly centered at the vertex, represented by 0.00 mm. The two numbers posterior to the cornea show the distance from the anterior surface to the horizontal mid-stromal line, and the second number

A

B

C Figures 5-2A to C: OCT images of a normal cornea. (A) Gray scale; (B) False color scale; (C) Rainbow scale.

B Figures 5-3A and B: (A) IntraLase LASIK flap; (B) IntraLase flap measured with the flap tool and calipers.

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represents the distance from the horizontal line to the posterior cornea. In this case, the central flap thickness is 155 microns and the residual bed is 477 microns. The operator selects the position of the interface marker, where the software automatically selects the anterior and posterior placements of the flap tool, based on the detection of the corneal boundaries. Therefore there is some element of subjectivity. In this particular example, on the left side at the -2.39 mm location, one can see that the flap location has not been accurately identified, with the horizontal line being slightly anterior to the interface. In addition to the flap tools in this example, in blue one can see a caliper set at 1.55 mm. This caliper is frequently used to measure structures within the eye. In this example, however, it has been placed to serve as a scale. Measuring the depth and placement of the lamellar dissection of the cornea is usually a critical element in corneal inlay surgery. It is critical for the surgeon to know the accuracy of the dissecting instrument used to create the pocket or flap. Moreover, the tolerance and optical performance of many inlays is dependent upon the anteroposterior location of the implant. Furthermore, the biocompatibility of a foreign element within the cornea usually requires placement of the element within a defined limited depth. The Visante OCT is uniquely capable of providing this information accurately and easily.

Refractive Inlays Studies are underway with refractive inlays under cornea flaps. Hydrogel corneal inlays were investigated in the past as a means to correct aphakia, hyperopia, and myopia. Early efforts in this area were optically unsatisfactory for a variety of reasons, particularly because of the inadequacies of early-generation microkeratomes as well as the attempt to create anterior corneal contours that were either optically unstable or associated with major aberrations.5 More recently, ReVision Optics (Lake Forest, CA) has been investigating a proprietary high water content material for correction of hyperopia and presbyopia. Figure 5-4A shows a 5 mm diameter positive power inlay. Note the thicker center and fine taper of the inlay in the periphery. In Figure 5-4B, the flap tool has been used to define the depth and thickness of the inlay, and a horizontal caliper has been used to measure the diameter. The diameter of 4.17 mm indicates that the cross-section, in this case vertical, is near but not exactly through the center of the inlay, which is 5.0 mm in diameter. The flap tools show that the flap has an anterior thickness between 114 and 122 microns, an important demonstration of uniformity with the flap created by the IntraLase laser (IntraLase Corporation, Irvine, CA).

A

B

C

D

E Figures 5-4A to E: (A) 5 mm ReVision Optics intracorneal inlay. (B) Inlay measured with flap tools and caliper. (C-E) Three different power ReVision Optics implants.

Optical Coherence Tomography in Corneal Implant Surgery However, one of the flap tools, positioned at 0.40 mm, has been placed with the horizontal bar at the posterior side of the inlay, compared to the flap tool at -0.08 mm which is positioned anteriorly. The difference between these two readings of 166 microns and 121 microns shows that the central thickness of the implant in this case is approximately 45 microns. This technology allows, for the first time, a direct measurement of the thickness of the high water content material once it has been placed and it has been stabilized. The thickness, of course, directly correlates to the optical power of the inlay. Figure 5-4C shows another case with a lower hyperopic correction. The flap tools show difference in thickness centrally of 25 microns. In yet another case, Figure 5-4D shows a center thickness of 32 microns, while Figure 5-4E shows a much higher powered

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implant, with a central thickness of 74 microns. The Visante OCT is demonstrated to be a critical tool in analyzing the postoperative position and characteristics of this type of corneal inlay. ReVision Optics (Lake Forest, CA) is currently investigating a 1.5 mm implant, designed to be placed either primarily or secondarily under a previous LASIK flap for the correction of presbyopia. The implant itself has positive power. It is centered on the pupil. A multifocal corneal optic is created as the corneal contour transitions from the area of the implant itself out to the original contour in the periphery. Figure 5-5A shows a slit lamp photograph of the implant on the left, and, on the right, the multifocal optic created in the transition zone labeled “2” in between the central power (“1”) and the more peripheral underlying corneal power (“3”). Figure 5-5B shows how thin and delicate this implant is, with the implant being seen as a thin separation under the LASIK flap centrally. In another case, shown in Figure 5-5C, the caliper shows the 1.5 mm implant at a depth of 159 microns.

Aperture Inlay for Presbyopia Correction A

B

C Figures 5-5A to C: (A) 1.5 mm presbyopic correcting ReVision Optics implant (left); illustration of resultant multifocal cornea (right). (B) 1.5 mm ReVision Optics presbyopic correcting inlay. (C) Flap tool and caliper measurement of the presbyopic inlay (images Courtesy of ReVision Optics).

A non-refractive intracorneal inlay is under investigation for unilateral implantation in the non-dominant eye to increase depth of focus and thereby correct presbyopia utilizing the well known pinhole effect. The technology is being developed by AcuFocus Corporation (Irvine, CA). The clinical appearance of this device is shown in two representative slit-lamp photographs (Figures 5-6A and B). The implanted material consists of a ring of a proprietary polymer. The polymer is extremely thin (on the order of 10 microns) and, as seen in Figure 5-6B, has multiple micropores to allow transmission of corneal nutrients while minimizing any light scattering that would degrade the optics. The manufacturer’s specifications are for an aperture of 1.60 mm centrally and an outer diameter of 3.80 mm. The central aperture diameter was selected as the optimum diameter to give maximum depth of focus while minimizing negative diffraction effects. The inlay is shown in Visante cross-section on a gray scale in Figure 5-6C. The section is not through the precise center, and therefore the caliper showing the aperture reads 1.39 mm and the overall diameter is read as 3.67 mm. The implant has been placed under a LASIK flap, as shown by the flap tool that is over the peripheral skirt. Note that the peripheral skirt blocks nearly all of the OCT light, and therefore the cornea appears dark under the skirt. However,

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A

B

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D

Figures 5-6A to D: (A) Low power slit-lamp photograph of the AcuFocus aperture inlay. Note shadow on iris in the upper left direction. (B) Light reflecting through the micropores of the peripheral skirt of the AcuFocus implant. (C) Gray scale OCT of the AcuFocus implant. (D) False color representation of an AcuFocus implant (Courtesy of Jack Holladay, MD).

a small amount of reflected signal can be seen as vertical lines. In Figure 5-6D, this affect is even more easy to see, with the colored signal against the dark background. The return signal under the skirt is because of the presence of the micropores, which are allowing a small amount of light to pass and be reflected back.

Conclusions The Visante OCT technology brings a critical new tool in anterior segment imaging. Knowledge of the performance of a microkeratome or the flap creation laser is critical, as the flap depth and uniformity will have a significant impact on the optical performance, and biological compatibility of intracorneal implants. The centration and optical characteristics of intercorneal implants are both quantitatively and qualitatively documented by the OCT technology. As shown in the

examples of the ReVision Optics (Lake Forest, CA) refractive implants and the AcuFocus (Irvine, CA) aperture inlay, the OCT technology plays an important role in optimizing intracorneal inlay procedures.

References 1. Born M, Wolf E, Bahatia AB. Principles of optics: Eectromagnetic theory of propagation, interference and diffraction of light. 7th ed. New York: Cambridge University Press, 1999. 2. Huang D, Swanson EA, Lin CP, et al. Optical coherence tomography. Science 1991; 254:1178-81. 3. Huang D, Wang J, Lin CP, Puliafito CA, Fujimoto JG. Micronresolution ranging of cornea an anterior chamber by optical reflectometry. Lasers Surge Med 1991; 11:419-25. 4. Radhakrishnan S, Rollins AM, Roth JE, et al. Real-time optical coherence tomography of the anterior segment at 1310 nm. Arch Ophthalmol 2001; 119:1179-85. 5. Steinert RF, Storie B, Smith P, McDonald MD, Van Rig G, Bores LD, et al. Hydrogel intracorneal lenses in aphakic eyes. Arch Ophthalmol 1996; 114:135-41.

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Use of OCT in DSEK and DSAEK

Leejee H Suh William W Culbertson

Use of Optical Coherence Tomography (OCT) in Descemet’s Stripping with Endothelial Keratoplasty (DSEK) and Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)

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Corneal Endothelial Transplant

Introduction Optical coherence tomography (OCT) is a high resolution imaging modality that is noninvasive and uses lowcoherence interferometry to provide in vivo cross-sectional images of tissue with a spatial resolution of 10 to 20 microns1 [See also Chapter 4, Optical Coherence Tomography (OCT) of the Anterior Segment, and Chapter 5, Optical Coherence Tomography in Corneal Implant Surgery]. Examination of the retina and posterior segment with OCT has been studied extensively. Recently, a high resolution anterior segment OCT (AC-OCT) has been made commercially available (VisanteTM OCT, Carl Zeiss Meditec, Dublin, CA). This instrument uses 1310 nm infrared light to visualize the anterior chamber to a resolution of 10 μm. Infrared-beam penetration at this wavelength is blocked by pigments, preventing examination behind the iris, but it can be used through an opaque cornea. AC-OCT provides easy, noncontact, and real-time images of the cornea, iris, lens, and iridocorneal angle.2 This instrument has been used to evaluate the static and dynamic properties of the anterior segment,2 the anterior chamber depth, corneal curvature and corneal thickness. 3 There are many potential applications of the AC-OCT. Changes in the anatomical configuration of the angle can be examined in such conditions as primary and secondary angle closure3 and analysis of the potential for occlusion of the anterior chamber angle. Real time imaging of the corneal layers is invaluable in corneal and refractive surgery. AC-OCT has been used to image corneal flap thickness after laser in situ keratomileusis (LASIK).4 Furthermore, the AC-OCT can be used in the preoperative and postoperative evaluation of patients undergoing phakic intraocular lens implantation.5 In planning lamellar keratoplasty, the depth of scarring can be assessed by OCT. Advances have occurred in the treatment of corneal endothelial disease, namely, deep lamellar endothelial keratoplasty (DLEK) and more recently Descemet’s stripping with endothelial keratoplasty (DSEK) and Descemet’s stripping with automated endothelial keratoplasty (DSAEK) (See also Chapter 13, Definition, Terminology and Classification of Lamellar Corneal Surgery). This chapter reviews the promising application of AC-OCT for DSEK and DSAEK.

Figure 6-1: AC-OCT image of recipient cornea. Note thickening of the stroma.

be imaged to assess preoperative corneal thickness across all radii and to assess details of the cornea and anterior chamber that may be obscured by corneal edema (Figure 6-1). For preparation of the donor lamellar button, the donor corneoscleral button is mounted in an artificial anterior chamber (See also Chapter 12, Artificial Anterior Chambers) which dovetails with a blade microkeratome (ALTK system, and CB microkeratome, Moria S.A., Antony, France). The donor corneoscleral button is mounted in the ALTK artificial anterior chamber unit with the lamellar plane created by the microkeratome (Figures 6-2A and B, arrows). The thickness parameters of the donor cornea are shown in Figure 6-2C. Although the ideal thickness of the donor lenticule has not been determined, we have found that donor corneal buttons measuring between 125 and 225 μm are

AC-OCT for DSEK and DSAEK The AC-OCT is useful in the preoperative and postoperative imaging of DSEK corneas. The Visante OCT (Carl Zeiss Meditec, Dublin, CA) has been used in all our patients undergoing DSEK. Before surgery, the recipient cornea can

Figures 6-2A to C: Donor cornea on anterior chamber after microkeratome application. Arrows show the lamellar plane of the dissection.

Use of OCT in DSEK and DSAEK easiest to handle and unfold in the anterior chamber.6 OCT corneal imaging may have a place in the pre- and post-cut analysis of the donor corneal dimensions. As more eye banks cut and prepare DSEK buttons for the surgeon’s use, this additional information relating to the donor disk may be helpful to the end-user surgeons (Editorial note: “Surgery by surgeons,” meaning, surgeon-cut tissue in the operating room possibly provide the best surgeon controlled environment, as it relates to the donor corneal tissue.) (See also Chapter 19, Eye Banking and Donor Corneal Tissue Preparation in DSAEK, and Chapter 30, Use of Eye Bank Pre-cut Donor Tissue in DSAEK). The corneoscleral button is then trephined with or without the anterior corneal portion to a desired diameter and placed aside in the preservation medium from the eye bank, namely, Optisol GS. (Editorial note: Trephination of the donor disk with the anterior, cut, corneal cap in place, prevents the introduction of potential debris into the donor-host interface). Then the recipient cornea is prepared for transplantation. Paracenteses are made in 4 oblique quadrants to provide access to the anterior chamber. Paracentral vents may be placed with a diamond knife for future removal of fluid or air in the interface (See also Chapter 27, Techniques to Facilitate Disk Adherence to Recipient Cornea in DSAEK). The epithelial surface is then marked with an 8.00 – 9.00 mm diameter circular mark to delineate the diameter of the descemetorhexis. Viscoelastic or continuous irrigation may be used to maintain the anterior chamber during recipient corneal surgery. Through a scleral tunnel or limbal incision, an instrument is then used to score the descemetorhexis and a scrapping instrument is used to strip off the Descemet’s membrane (See also Chapter 11, New/Useful Surgical Instruments in DSAEK). Alternatively a clinical femtosecond laser (Intralase, Irvine, CA) may be used to create the descemetorhexis at the desired position and diameter [See also Chapter 26, Femtosecond Laser (Intralase®)–Descemet’s Stripping Endothelial Keratoplasty (Femto-DSEK): Initial Studies of Surgical Technique in Human Eyes] prior to bringing the patient into the operating room. Attention is then redirected to the donor lamellar button, namely, the posterior lamellar button is carefully separated from the anterior cut-corneal cap and the viscoelastic is placed on the endothelial surface. (Editorial note: Avoid excess use of viscoelastic, as this can potentially interfere with donor disk attachment to the recipient cornea). The lamellar button is then folded endothelial side inward, grasped with forceps (See also Chapter 11, New/Useful Surgical Instruments in DSAEK) and then pulled or pushed into the anterior chamber. The button is unfolded endothelial side down (stromal side up) using air or balanced salt solution and attach to the bare stroma. Irrigation through the paracentesis may further position the disc. Residual viscoelastic is removed and air

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is injected into the anterior chamber to force the graft to appose to the stromal surface. Fluid or air in the graft interface may be expressed through the paracentral vents. The AC-OCT clearly demonstrates the postoperative image of the DSEK button and progression of stromal deturgescence. Figure 6-3 shows an image of the meniscusshaped DSEK button that has a tapered flange and is well apposed to the recipient stromal surface. Postoperative corneal deturgescence can be monitored with serial ACOCT (Figures 6-4A to F). Figures 6-4A and B is at postoperative month 5, while, Figures 6-4C and D is at postoperative month 8, and Figures 6-4E and F is at postoperative month 11. Note the progressive apical thinning of the cornea. Often, the host cornea becomes optically clear within about 3 to 4 weeks (See also Chapter 20, Endothelial Keratoplasty: A Step-by-Step Guide to DSEK and DSAEK Surgery, and Chapter 22, Endothelial Keratoplasty: Visual and Refractive Outcomes). At that point, refraction can be performed for spectacle correction. Unlike the high astigmatism encountered after PKP, there is minimal change in the refractive error after DSEK (See also Chapter 22, Endothelial Keratoplasty: Visual and Refractive Outcomes). Price et al has shown that postoperative corneal thickness does not necessarily correlate to optical clarity and as such, the thickness of the graft may not be as important for the overall success of the procedure.7

Figure 6-3: Graft of the lamellar button is nicely apposed to the recipient stroma. The lamellar button is meniscus-shaped with tapered flanges.

AC-OCT and DSEK Complications The most common complication encountered in DSEK is detachment of the lamellar button, which has been reported in 15-30% of cases in the early postoperative period.7 This complication, however, can be addressed by repositioning the button and/or injecting more air into the anterior chamber (called rebubbling). Figure 6-5A shows a detached DSEK button. The button was repositioned by manipulation through one of the paracenteses and rebubbled with air. Figure 6-5B shows the reattached button at postoperative month 1 that remained attached at postoperative month 3 (Figure 6-5C). Figures 6-5D to F shows the progressive deturgescence of the cornea. Another complication is the presence of air or fluid in the graft interface. Figures 6-6A and B shows a thickened

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Figures 6-4A to F: Progressive deturgescence of the corneas can be followed serially on AC-OCT. (A-B) is at postoperative month 5, (C-D) at postoperative month 8, and (E-F) at postoperative month 11. Note the progressive thinning of the corneas especially at the apex.

Figures 6-5A to F: Example of detached graft in immediate postoperative period and reattachment subsequent to repositioning and rebubbling. (A) Shows the detached button. After repositioning and rebubbling, the button reattached (B) and continued to remain attached (C) with progressive thinning (D-F).

Use of OCT in DSEK and DSAEK

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Figures 6-7A and B: (A) Epithelial ingrowth in the interface presumably from recipient at postoperative month 9. This layer is highly reflective on AC-OCT (B, arrows).

Figures 6-6A to C: (A-B) Example of thickened recipient cornea secondary to (C) fluid in interface.

cornea after DSEK. In this patient, fluid was noted in the graft interface, shown in some sections on AC-OCT (Figure 6-6C). Fluid or air can be evacuated postoperatively through the paracentral vents made during surgery. Epithelium or blood may also be present in the graft interface after DSEK. On AC-OCT the presence of such material is highly reflective. Figures 6-7A and B shows a highly reflective area in the graft interface. On examination, the patient was noted to have epithelial ingrowth in the nonvisual axis (Figure 6-7A), presumably from the recipient

cornea, introduced during surgery. Figure 6-8A shows blood at the donor-recipient interface border and on ACOCT (Figures 6-8B and C) there is a highly reflective area (arrow) correlating with the blood in the interface seen clinically (Figure 6-8A). Although the presence of epithelium or blood may not ultimately affect attachment of the donor button, other interface abnormalities can lead to persistent detachment. Figure 6-9A shows a detached button and upon closer examination, there is a thin highly-reflective membrane at the interface (Figures 6-9B and C). Despite repositioning and rebubbling, the button in this patient remained detached. Histopathology showed the presence of PASstaining Descemet’s membrane at the interface (Figure 6-9D, arrows). In another patient, the graft was detached and free-floating on the first postoperative day (Figure 6-10A) and on AC-OCT, the graft was noted to be thickened on one end (Figure 6-10B). Histopathology revealed that

Figures 6-8A to C: (A, arrows) Presence of blood in the interface; (B-C) AC-OCT of same patient (B-C, arrows) show highly reflective area at interface correlating to blood.

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Figures 6-9A to D: (A) Detached button secondary to presence of membrane in interface (B-C, arrows). Histopathology showed this membrane to be Descemet’s membrane (D, arrows).

Figures 6-10A to C: Free-floating button in anterior chamber post DSEK. One end of the button is thickened (B, arrow) and on histopathology the edge was full-thickness cornea (C, arrow), presumably from uneven lamellar dissection.

Use of OCT in DSEK and DSAEK the button had full-thickness epithelium, Bowman’s layer, stroma, and Descemet’s membrane at one margin with epithelial ingrowth into the interface (Figure 6-10C). This complication occurred most likely from uneven lamellar dissection with the microkeratome. Both these cases demonstrate the usefulness of AC-OCT images that are highly correlative to the histopathological findings.

Conclusion In summary, there are many applications for the AC-OCT in DSEK surgery. The preoperative thickness can be gauged by the AC-OCT and compared to postoperative thicknesses. The plane created by the microkeratome in the donor button preparation can be shown by AC-OCT. On AC-OCT the donor lamellar button is meniscus-shaped with tapered flanges. Progressive deturgescence of the recipient cornea can be followed by AC-OCT. Finally, complications of DSEK surgery, such as detachment and presence of interface material can be easily imaged with AC-OCT. Clearly, ACOCT is an invaluable tool in DSEK surgery.

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References 1. Huang D, Swanson EA, Lin CP, et al. Optical coherence tomography. Science 1991;254:1178-81. 2. Baikoff G, Lutun E, Ferraz C, Wei J. Static and dynamic analysis of the anterior segment with optical coherence tomography. J Cataract Refract Surg 2004;30:1843-50. 3. Dawczynski J, Koenigdoerffer, Augsten R, Strobel J. Anterior optical coherence tomography: a non-contact technique for anterior chamber evaluation. Graefe’s Arch Clin Exp Ophthalmol 2006; Epub ahead of print. 4. Maldonado MJ, Ruiz-Oblitas L, Munuera JM, Aliseda D, GarciaLayana A, Moreno-Montanes J. Optical coherence tomography evaluation of the corneal cap and stromal bed features after laser in situ keratomileusis for high myopia and astigmatism. Ophthalmology 2000;107:81-87. 5. Baikoff, G, Bourgeon G, Jodai HJ, et al. Pigment dispersion and Artisan phakic intraocular lenses: crystalline lens rise as a safety criterion. J Cataract Refract Surg 2005;31:674-80. 6. Culbertson WW. Descemet stripping endothelial keratoplasty. Int Ophthalmol Clin 2006;43:155-68. 7. Price FW, Price MO. Descemet’s stripping with endothelial keratoplasty in 200 eyes: early challenges and techniques to enhance donor adherence. J Cataract Refract Surg 2006;32:411-8.

Imaging of the Cornea and Anterior Segment with High-Frequency Ultrasound

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Ronald H Silverman, Monica Patel Omer Gal, Harriet O Lloyd D Dan Z Reinstein, D Jackson Coleman

Imaging of the Cornea and Anterior Segment with High-Frequency Ultrasound

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Introduction Ultrasound is a widely used technique for clinical diagnostic imaging. It is advantageous because it does not involve use of ionizing radiation, is relatively inexpensive, provides real-time imaging and is very portable. Ultrasonic imaging offers a view of tissue structures that are otherwise hidden by optically opaque overlying structures (Editorial Note: With optical coherence tomography (OCT) structures behind pigmented tissue are not visible.) [See also Chapter 4, Optical Coherence Tomography (OCT) of the Anterior Segment, Chapter 5, Optical Coherence Tomography in Corneal Implant Surgery, and Chapter 6, Use of Optical Coherence Tomography in Descemet’s Stripping with Endothelial Keratoplasty (DSEK) and Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)]. Its application to the eye can provide images with almost microscopic resolution. Sound waves are propagating disturbances in the density of a medium. These waves are characterized by their wavelength and frequency, the product of which is equal to the speed of sound. The speed of sound varies with the composition of the medium and other factors, such as temperature. Ultrasound is defined as any sound of frequency above the range of human hearing, i.e. approximately 25,000 cycles/second or more. In diagnostic ultrasound, frequencies in the megahertz (MHz = millions of cycles per second) range are used. Ultrasonic imaging systems require a piezoelectric transducer that converts a voltage transient into an acoustic pulse. When the propagating ultrasonic pulse encounters a density transition, reflections occur. When these reflections, or echoes, reach the piezoelectric material of the transducer, small voltages are generated that are then amplified and displayed by the ultrasound system. The time between pulse transmission and echo return is used to determine the range from the transducer to tissue interfaces. Echo amplitude is proportional to the change in acoustic impedance (the product of density and speed-of-sound) across a tissue interface. A plot of echo amplitude as a function of range along one line-of-sight is called an Ascan. Two-dimensional B-mode images are produced by scanning the transducer orthogonally to the beam axis. Since the transducer orientation and range to each echo are known, a two-dimensional image can then be formed in which pixel brightness is proportional to echo amplitude. In soft tissues, ultrasound travels at about 1540 meters/ second. Thus, an ultrasound pulse takes approximately 31 microseconds to go from the cornea to the retina and back (2 × 24 mm/1.54 × 106 mm/sec). The wavelength of an ultrasound pulse relates directly to obtainable resolution, and is defined as speed-of-sound/frequency. Thus, as

frequency increases, wavelength decreases and resolution improves. Most medical ultrasound systems operate at 1-10 MHz, equivalent to wavelength ranging from 1540 microns at 1 MHz to 154 microns at 10 MHz. While we always wish to obtain the best possible resolution in medical imaging, attenuation of ultrasound increases exponentially with frequency. Thus, abdominal exams would be performed at the lower end of the above range, while examination of superficial tissue structures, such as the eye, can be performed at 10 MHz or above. Ophthalmic ultrasonography was first described by Mundt and Hughes in 19561 with many other papers and books regarding this technique appearing in succeeding years.2-8 Almost from its beginnings, B-mode ophthalmic ultrasonography was performed at 10 MHz, a frequency that provides a good balance between resolution and sensitivity for examination of the vitreous, retina and orbit. However, the best-case 150 micron resolution obtainable at 10 MHz is inadequate for imaging of the structures of the anterior segment, including the ciliary body, iris and cornea. In the early 1990’s, instruments employing much higher frequencies (35-50 MHz) for imaging of the anterior segment became available.9,10 This frequency range is referred to as very high-frequency ultrasound (VHFU). While the eye can be scanned using coupling gel through a closed eyelid at 10 MHz, VHFU scans must be performed with open lids and a fluid coupling medium to prevent attenuation by the lids. Attenuation also limits scan depth to the anterior segment. However, VHFU can provide an axial resolution as fine as 30 microns. This has allowed VHFU systems to provide superbly detailed images of anterior segment structures, even in the presence of optical opacities such as hyphema or corneal scarring, and allows imaging of structures such as the ciliary body that are otherwise hidden by the sclera or iris. VHFU instruments are often called ultrasound biomicroscopes (UBM) after the first commercial system developed by Zeiss-Humphrey (Dublin, CA) and later Paradigm Medical Industries (Salt Lake City, UT). At the present time, handheld ophthalmic UBM systems are available from numerous companies. VHFU imaging of the cornea has long been an area of special interest. The short wavelength of VHFU systems allowed resolution of Bowman’s membrane, and hence provided a means for layered corneal biometry. VHFU ultrasound was also found capable of readily visualizing the interface between the flap and residual stroma following laser in situ keratomileusis (LASIK).11 In the mid-1990’s, our laboratory developed signal processing strategies suitable for imaging and automated detection of these interfaces.12-14 By scanning the cornea in a series of parallel

Imaging of the Cornea and Anterior Segment with High-Frequency Ultrasound planes, the depths of Bowman’s membrane, the flap and the posterior corneal surface could be measured and color maps representing the thickness of each layer produced. A shortcoming of that technology, however, arose from the specular nature of the corneal surface: outside the 3 mm central zone, echoes are deflected due to the curvature of the corneal surface and echo data not detected. To address this, we developed an arc-scan mechanism that maintained approximate normality and constant range between the ultrasound beam axis and the corneal surface.15 Using the arc-scan, the eye could be scanned in a sequence of meridians and layered pachymetry mapped over virtually the entire cornea. The first commercial system using the arc-scan for corneal imaging is the Artemis-2, developed by Ultralink, LLC (now merged with ArcScan Inc., Morrison, CO). A feature of this system is that unlike conventional Bscanners, the Artemis-2 incorporates optical means for eye fixation and visualization of eye position during scanning, a feature that is crucial for obtaining reproducible measurements.

Clinical Ultrasonic Imaging of the Anterior Segment In conventional 10 MHz B-mode imaging, the probe is placed in contact with the eyelid, or sometimes directly upon the globe after administration of a topical anesthetic. Because the transducer has a fixed focal length that is generally designed to fall near the retina, the anterior segment falls in the defocused near-field of the transducer. As demonstrated in Figure 7-1, the resulting image is nearly useless for evaluation of anterior segment structures. However, by standing off the transducer from the eye with a normal-saline waterbath, the focus can be placed on the anterior segment (Figure 7-2). This results in significant improvement, allowing depiction of anterior segment structures, although the resolution at 10 MHz limits the amount of detail that can be observed. Figure 7-3, a 20 MHz immersion scan image of the anterior segment, demonstrates a comparative improvement in resolution. The appearance of a normal anterior segment at 35 MHz is shown in Figure 7-4. Notice that while the anterior surface of the lens is sharply depicted, the equator is not seen. This is a consequence of the specularity of the lens surface and the oblique presentation of this surface at the equator. The anterior segment shown in Figure 7-5 has a somewhat shallow anterior chamber (1.71 mm from endothelium to lens surface) as well as an area of atrophic iris with narrowed angle temporally. While the lens equator is generally not visible, it can sometimes be seen if blood or

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Figure 7-1: Image of the normal eye acquired using a 10 MHz sector B-scan in contact mode. Note the relatively poor definition of the anterior segment.

Figure 7-2: Image of the whole eye of a normal subject acquired using a 10 MHz sector B-scan in immersion mode.

inflammatory material is present, as shown in Figure 7-6. The appearance of the anterior segment with post-traumatic hyphema is shown in Figure 7-7. Very high frequency ultrasound is of great value in evaluation of tumors of the iris and ciliary body. In many cases, a patient will present with a pigmented lesion in the angle (Figure 7-8), and the degree of involvement with the ciliary body is not apparent. Ultrasound can readily visualize the ciliary body and determine if melanoma is

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Figure 7-6: Imaging with a 35 MHz arc-scan axial (top) and temporal (bottom) of eye with endophthalmitis. Inflammatory material outlines the boundaries of the lens. The ciliary body is detached (arrow).

Figure 7-3: Image of the anterior segment of a normal subject acquired using a 20 MHz sector B-scan in immersion mode.

Figure 7-7: Anterior segment view with a 35 MHz arc-scan in a posttraumatic eye demonstrating hyphema and conjunctival thickening. Also note echoes just beneath anterior lens surface, suggesting cataractous change. Figure 7-4: Anterior segment view with a 35 MHz immersion arc-scan.

Figure 7-5: High resolution 35 MHz arc-scan of an anterior segment with shallow anterior chamber and area of atrophic iris (arrow).

Figure 7-8: Pigmented lesion seen in angle superiorly (arrow) is shown ultrasonically to involve both the iris and ciliary body.

Imaging of the Cornea and Anterior Segment with High-Frequency Ultrasound

Figure 7-9: Retroiridal cysts often cause changes in iris contour. Highresolution ultrasound penetrates through the iris and readily allows differentiation of cysts, an example of which is shown here, from solid lesions.

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Figure 7-12: Oversized phakic posterior chamber lens implant results in anterior displacement of iris and possible contact with crystalline lens.

Ultrasonic Imaging and Biometry of the Cornea

Figure 7-10: Displaced posterior chamber lens implant. Lens implants are readily visualized ultrasonically due to their difference in density from surrounding media.

Figure 7-11: Anterior chamber lens implant. Haptics are indicated by arrows.

present, as is the case in this figure. Because ciliary body involvement is associated with increased risk of metastases while iris melanomas are relatively indolent, this distinction impacts upon treatment. Differentiation of solid lesion from cysts (Figure 7-9) is readily accomplished ultrasonically. Very high frequency ultrasound allows visualization of lens implants in both the posterior (Figure 7-10) and anterior chambers (Figure 7-11) . Preoperative ultrasound biometry is of great importance for the measurement of ocular dimensions prior to phakic lens implant surgery (Figure 7-12), since white-to-white measurement is only weakly correlated with the angle and sulcus diameters.

With an approximate thickness of 0.5 mm, the need for high-resolution imaging systems is obvious. Resolution of the approximately 50 micron thick corneal epithelium requires the use of very high frequencies. A factor affecting corneal imaging and biometry is the cornea’s specularity. When imaging with a conventional sector scan probe, the corneal surface will present obliquely to the ultrasound axis, and echoes are deflected away from the probe, resulting in reduced or absent echoes outside the central cornea. This effect is exacerbated by the limited depth-offield of the focused transducer, because only a small region of the curved cornea will be within the focal zone during a scan. The Artemis system uses an arc-scan geometry to address these issues. By moving the transducer in an arc of appropriate radius, the cornea will present approximately normal to the beam axis and remain in the focal region across its entire diameter. The Artemis also incorporates an optical subsystem that allows the patient to gaze at a fixation target while the eye position is monitored with a video camera. This is crucial in obtaining measurement reproducibility. An image of a normal cornea obtained using the Artemis is shown in Figure 7-13. Following LASIK, the flap interface is evident, as shown in Figure 7-14. Because full-width arc-scan images are so much wider than deep, it is often advantageous to display them in geometrically uncorrected format so as not to loose the axial resolution to which we are entitled. Corneal pathologies such as edema (Figure 7-15) and scarring (Figure 7-16) can be readily visualized and their depth measured. Biometric analysis of digitized corneal ultrasound data involves detection of peaks associated with the anterior and posterior surfaces, Bowman’s membrane and the flap, where present. The Artemis stores raw echo data rather

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Figure 7-13: Artemis-2 image of normal cornea. The Artemis system, which is an immersion arc-scanner, also includes fixation lights and video monitoring of eye position during scanning. Figure 7-15: Example of corneal edema in high resolution arc-scan of the anterior segment.

Figure 7-14: Artemis images of post-LASIK cornea in proper geometry (top) and uncorrected rectilinear display (bottom). The corneal layers, epithelial surface (e), Bowman’s membrane (b), flap (f) and posterior surface (p) are indicated.

Figure 7-16: Corneal scar superiorly (arrow). Ultrasound measurement of scar depth can be of great value in clinical management.

Figure 7-17: Post-processing of digitized corneal scan data allows detection of corneal interfaces and determination of the thickness of each layer at any point in the scan. The example shows the thickness of the cornea (C) the epithelium (E) and the stroma (S).

than the images themselves, as the image data is of lower resolution. By applying digital signal processing techniques, the exact positions of each interface are determined. The Artemis automatically detects these interfaces and displays measurements as shown in Figure

7-17 for a normal cornea. Figure 7-18 shows an unusual thickness profile in a patient with a corneal graft. By acquiring a series of scans in a sequence of meridians, it is possible to map the thickness of each layer. Figure 7-19 shows representative B-mode images and pachymetric

Imaging of the Cornea and Anterior Segment with High-Frequency Ultrasound

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Figure 7-18: Corneal thickness profile eye with corneal graft.

Figure 7-19: Corneal pachymetric maps representing the thickness of each layer in a LASIK-treated cornea. Note the typical epithelial thickening over the ablated central zone. The residual stroma is shown to measure 282 microns, generally considered to be above the safety threshold of 250 microns.

maps of a post-LASIK cornea of a myope. Thickening of the cornea over the ablated central region is typical. In contrast, Figure 7-20 shows an annular thickening of the epithelium in a post-LASIK eye corrected for hyperopia. In this case as well, epithelial thickening corresponds to the ablation pattern. Images of a cornea that had received radial

keratotomy a decade previously are shown in Figure 7-21. While the incisions themselves are not evident in the B-mode images, their effect on the epithelial thickness is evident in the map. In a patient with keratoconus (Figure 7-22) , the epithelium is thinned to about 35 microns over the steepest part of the stroma with a surrounding halo of

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Figure 7-20: Corneal pachymetric maps following hyperopic LASIK. In this case, the epithelium thickens in an annulus consistent with the ablation pattern.

Figure 7-21: Pachymetric maps of a cornea treated over a decade previously with radial keratotomy. At several positions, epithelial defects are seen that are consistent with the prior surgery.

Imaging of the Cornea and Anterior Segment with High-Frequency Ultrasound

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Figure 7-22: Orbscan anterior and posterior difference-from-sphere (float) surface maps (top left) and corneal power maps (top right) and Artemis pachymetric maps (bottom) of cornea with keratoconus. Specific patterns of corneal thinning may develop in response to bulging forward of the underlying stroma in early stages of this disease. Because epithelial remodeling may mask this in conventional surface topography, ultrasound may offer a tool for early diagnosis.

epithelial thickening to as much as 75 microns. This remodeling of epithelium is a consequence of the bulging forward of the underlying stroma. Ultrasonic detection of this thinning pattern can be an early sign of this keratoconus that may be detectable before surface topographic changes are evident.

Conclusion New ultrasound technologies continue to be developed for improvement in sensitivity and resolution. High-frequency arrays may become available in the near future. Prototype 35 MHz annular arrays for ophthalmic imaging have been demonstrated.16 Dynamic focusing of such arrays offers a much improved depth of field, in fact, a sixfold improvement over current single-element probes. Prototype highfrequency linear array probes have also been developed.17-18 With such arrays, it would be possible to scan at high frame rates without mechanical motion, and it might be possible to replicate an arc scan, with its advantages for corneal imaging and biometry. Transducer technology is evolving to allow higher frequency

transducers in the range of 75 MHz providing improved definition and greater sensitivity to backscatter.19 These transducers might be particularly useful in screening for conditions involving stromal changes such as keratoconus and for imaging Schlemm’s canal. While optical methods such as OCT offer superb resolution, ultrasound will continue to serve a crucial role in anterior segment imaging, diagnosis and biometry due to its ability to penetrate optic opacities and probe the layered structure of the cornea.

References 1. Mundt G, Hughes W. Ultrasonics in ocular diagnosis. Am J Ophthalmol 1956;41:488-98. 2. Purnell E. Ultrasonic interpretation of orbital disease. In: al KG, (Ed). Ophthalmic Ultrasound. St. Louis: CV Mosby Co, 1969. 3. Coleman DJ, Konig WF, Katz L. A hand-operated, ultrasound scan system for ophthalmic evaluation. American Journal of Ophthalmology 1969;68(2):256-63. 4. Baum G. Aids in ultrasonic diagnosis. Journal of the Acoustical Society of America 1970;48(6):Suppl 2,1407. 5. Coleman DJ. Reliability of ocular and orbital diagnosis with Bscan ultrasound. Ocular diagnosis. American Journal of Ophthalmology 1972;73(4):501-16.

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6. Fisher YL, Bronson NR, Schutz JS, Llovera IN. Contact B-scan ultrasonography: clinicopathological correlations. Annals of Ophthalmology 1975;7(6):779-86. 7. Coleman DJ, Lizzi FL, Jack R. Ultrasonography of the Eye and Orbit. Philadelphia, PA: Lea & Febiger, 1977. 8. Coleman DJ, Silverman RH, Lizzi FL, Rondeau MJ. Ultrasonography of the Eye and Orbit. (2nd edn). Lippincott Williams & Wilkins, Philadelphia, 2005. 9. Pavlin CJ, Harasiewicz K, Sherar MD, Foster FS. Clinical use of ultrasound biomicroscopy. Ophthalmology 1991;98(3):287-95. 10. Pavlin CJ, Sherar MD, Foster FS. Subsurface ultrasound microscopic imaging of the intact eye. Ophthalmology 1990;97(2): 244-50. 11. Reinstein DZ, Silverman RH. Very high-frequency digital ultrasound: Artemis 2 scanning in LASIK. In: LASIK: Advances, Controversies, and Custom. Edited by Louis Probst, SLACK, 2004. 12. Reinstein DZ, Silverman RH, Trokel SL, Allemann N, Coleman DJ. High-frequency ultrasound digital signal processing for biometry of the cornea in planning phototherapeutic keratectomy. Arch Ophthalmol 1993;111:430-31. 13. Reinstein DZ, Silverman RH, Trokel SL, Coleman DJ. Corneal pachymetric topography. Ophthalmology 1994;101:432-8.

14. Reinstein DZ, Silverman RH, Rondeau MJ, Coleman DJ. Epithelial and corneal thickness measurements by highfrequency ultrasound digital signal processing. Ophthalmology 1994;101:140-6. 15. Reinstein DZ, Silverman RH, Raevsky T, Simoni GJ, Lloyd HO, Najafi DJ, Rondeau MJ, Coleman DJ. Arc-scanning very highfrequency ultrasound for 3-D pachymetric mapping of the corneal epithelium and stroma in laser in situ keratomileusis. J Refract Surg 2000;16:414-30. 16. Silverman RH, Ketterling JA, Coleman DJ. High-frequency ultrasonic imaging of the anterior segment using an annular array transducer. Ophthalmology In Press. 17. Lukacs M, Yin J, Pang G, Garcia RC, Cherin E, Williams R, Mehi J, Foster FS. Performance and characterization of new micromachined high-frequency linear arrays. IEEE Trans Ultrason Ferroelectr Freq Control 2006; 53(10):1719-29. 18. Ritter TA, Shrout TR, Tutwiler R, Shung KK. A 30-MHz piezocomposite ultrasound array for medical imaging applications. IEEE Trans Ultrason Ferroelectr Freq Control 2002; 49(2):21730. 19. Silverman RH, Cannata J, Shung KK, Gal O, Patel M, Lloyd HO, Feleppa EJ, Coleman DJ. 75 MHz ultrasound biomicroscopy of the anterior segment of the eye. Ultrasonic Imaging. In Press.

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Confocal Microscopy of the Cornea

Jasmeet S Dhaliwal Auguste G-Y Chiou Stephen C Kaufman

Confocal Microscopy of the Cornea

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History of Microscopy In the 17th century, a new invention revealed the microscopic world. The microscope marked the beginning of histology. Invented by Hooke, the Compound Microscope was used to describe pores in sections of ordinary cork.1 He coined the term “cell” to describe these pores, which was eventually applied to describe the “cells” of living systems. This early design eventually evolved into the modern microscope. The modern Light Microscope invented by Schleiden and Schwann in 1838 has significantly advanced our understanding of living structures.1 Although numerous optical design and manufacturing enhancements have improved microscopic resolution to an impressive half-micron, the inherent limitations of this technique have significantly hampered the study of “living” tissues. For example, this technique destroys “living” tissues during the fixation and sectioning process. Consequently, many natural associations between cells and microstructures that may have existed are lost. In addition, this process creates unwanted artifacts that further obscure microstructural details. More importantly, the optical performance of the light microscope is dreadful when used to examine thick tissue. The fundamental problem is that the image obtained not only contains light that is reflected from the focal plane, but also reflected light from structures both above and below the focal plane. Consequently, this unwanted reflected light blurs the fine structural details within the focused image; thus, decreasing contrast and both lateral and axial resolution.2 In an attempt to resolve these limitations, Maurice unveiled the Specular Microscope in 1968. 3 This new microscope provided a new ability to image tissues in vivo. Maurice’s design accomplished this task by exploiting the reflectivity of tissues. In the cornea, for example, the endothelial layer is highly reflective. By being more reflective than the structures below or above, the specular microscope is able to focus this reflective light unimpeded, producing sharp images with enhanced resolution and contrast.2 Soon thereafter, Bourne and Kaufman improved upon this novel design. Their design modifications introduced a practical version into ophthalmologic practice, allowing clinically useful corneal endothelial photography at high magnification (200X).4 Unfortunately, the specular microscope generally fails to satisfactorily image the less reflective structures within the cornea, which include the stromal keratocytes and the epithelial cells.1 In addition, corneal pathologic conditions, such as scars, may induce significant light scattering.2 Although the specular microscope advanced imaging of

in vivo tissues, its limitations required a different approach and eventually this lead to the development of the confocal microscope.

Development of Confocal Microscopy In 1955, Marvin Minsky, when he was a Junior Fellow at Harvard University, pioneered the first Confocal Microscope.5,7,18 Unlike earlier microscopic techniques that viewed sectioned tissue in a plane perpendicular to its surface, confocal microscopy views tissue images horizontal to its surface. His invention permitted tissue examination without the light scattering artifacts prevalent in prior microscope designs. He accomplished this feat by exploiting the pinhole effect. Minsky combined pinholes with microscope optics to produce remarkably clear images. The key was in the design. His microscope used two pinholes to produce a confocal effect. He placed the first pinhole before the condenser, which focuses the light rays onto the tissue and the second pinhole before the objective, which focuses the reflected light rays into an image. Minsky then calibrated this opticalpinhole system in a way that conjugated the tissue focal point to correspond precisely to both pinholes simultaneously. Thus, this effect permitted only light rays conjugate to the focal point within the tissue to pass, thereby blocking unwanted light rays reflected from other levels within the tissue.2,5-7 This early confocal microscopy concept is illustrated in Figure 8-1. This blocking phenomenon eliminated the lightscattering issue prevalent in earlier light microscopes. Furthermore, Minsky’s microscope could theoretically perform a point-by-point image reconstruction by sequentially shinning a pinpoint of light across a specimen instead of flooding the entire specimen with light. Minsky’s novel design set the focal length of the condenser and the objective lens as illustrated by the rectangle and oval respectively.18 As illustrated by the dotted line tracing in Figure 8-1, the focal point in the specimen is conjugate to the focal point at the pinhole. As one can appreciate, only the dotted light ray tracing can pass through the pinhole. If for example, reflected light from a point anterior to the focal point passes through the optical system, its light would focus before reaching the pinhole, and thus be blocked from passing through the pinhole (Figure 8-1). Likewise, reflected light from a point posterior to the focal point would focus behind the pinhole. Thus, the pinhole blocks unwanted light from passing through.

Confocal Microscopy of the Cornea

Figure 8-1: Pinhole concept. The focal lengths from the optical system to either the tissue or pinhole are identical. Therefore, only the dotted light ray tracing travels through the pinhole without being blocked. Thus, unwanted aberrant rays (red and blue) are filtered by the pinhole, creating a clear image of a single point within the tissue.

Hence, this arrangement not only illuminated, but also imaged a single point within a specimen simultaneously, blocking aberrant reflected light rays. He coined the term confocal to describe his design’s use of common focal points (Figure 8-1). His confocal technique produced sharp images with excellent contrast. Wilson and Sheppard further advanced this design that eventually lead to the modern confocal microscope.8 Modern confocal microscopy has replaced the condenser and objective eyepiece with the point illuminator and electronic detector, respectively. Thus, light from a point within the tissue can now be digitized and stored on a computer. In order to view structures below or above the point of interest, the optical focus is changed, creating a new focal plane that can be examined. If the focal length is continually changed, then numerous optical sections can be imaged (Figure 8-2).

Figure 8-2: In vivo confocal sectioning concept. The red and yellow planes illustrate this concept to dynamically image different planes within the block of tissue by changing the focal length of the optical system.

This ability to change focal length in real-time allowed a dynamic Z-axis scanning capability. Therefore, this technique enabled in vivo corneal scanning. In addition, digitizing the images allowed a computer to create threedimensional reconstructions. Thus, unlike earlier techniques, the natural association between cells and micro-

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structures can now be examined in vivo, without using stains or dyes. However, like earlier techniques, corneal edema or opacification degraded image quality.1 This new technology improved the lateral resolution to approximately 2 μm and axial resolution to 8 - 12 μm.9 Furthermore, this design improved magnification up to 600 times.9 This significant advance allowed impressive in vivo imaging of tissues, but there was a significant trade-off, namely, the field of view is small. To overcome this serious limitation, the concept of scanning was introduced. Just as this term implies, either the specimen must be moved past a fixed illuminated point or both the objective lens and condenser lens must be synchronously moved or scanned across the specimen within the X-Y plane.2,18 In Minsky’s device the specimen had to be moved using a precise registration system, in order to scan an entire field, which produced a multiple exposure photograph of the entire field.1 This process was tedious and time-consuming and hence never became a practical method to examine tissues.1

Tandem Scanning Confocal Microscope Approximately 10 years after Minsky’s original work, Petran and Hadravsky developed a novel technique that expedited the scanning process. They adapted the Nipkow Disk to improve confocal microscopy’s resolution and contrast. The Nipkow Disk was invented by Paul Nipkow in 1884 for encoding and decoding images for transmission over telegraph cables.1,5,10 This disk contains approximately 14,000 pinholes that are systematically placed 180° opposite each other, patterned in 40 identical Archimedean spirals.10,11 In other words, at any given time, there is a pinhole directly over the illuminator and detector, allowing only the light of interest to pass. The pinholes, which are 20 μm to 60 μm in diameter, are spaced at a set distance from each other to maximize the optics as explained in detail by Wilson and Sheppard.2,8 With separate light paths, or dual light path design, the illuminating light does not interfere with the light reflected from the specimen as it travels towards the detector.1 This diametric pinhole design was termed tandem and eliminated the primary cause of the decreased image contrast and the image degradation associated with white-light confocal microscopy (Figure 8-3).1 This design is critical, because, in order to scan a specimen within the X-Y plane, the disk must be rotated, while the specimen itself remains stationary. As the disk rotates, the spiraling set of pinholes act as camera shutters.

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Figure 8-3: Tandem scanning confocal microscope concept. The pinhole concept is used as described in Figure 8.1. To allow for scanning, a Nipkow disk is used. When a point in the tissue is imaged, the light ray essentially travels simultaneously through two pinholes. The light ray travels through the first pinhole, through the optical system and is focused onto a point within the tissue. The reflected light is then sent back through the optical system, redirected to the second pinhole. The second pinhole filters out aberrant light rays and allows the detector to image a single point within the tissue block.

Light can only pass through the system when a set of conjugate pinholes is present before the illuminator and detector. Since the pinholes are spaced apart, consecutive, corresponding points within the X-Y plane can be systematically imaged without moving the specimen. This spiral design, in effect, acts as thousands of confocal optical systems working in parallel to scan the specimen. At any given time, only a few hundred pinholes are simultaneously present over the specimen to scan.7 However, since the disk is rotated at speeds above 40 revolutions per second, the entire specimen can be scanned several times during a single rotation.7,12 This high scan rate permitted video cameras and monitors to be used, which possess inherently high capture rates. As a result, one could observe a continuous image on a video monitor. Furthermore, since the Z-axis can be changed quickly, this system can quickly scan through tissue, allowing in vivo dynamic optical sectioning of the scanned tissue.

Advantages of Tandem Scanning Design over Prior Designs2,6 1. Subtoxic white light levels are used to create the images without using stains or dyes. 2. Noninvasive. 3. Enhanced lateral and axial resolution. 4. Enhanced contrast. 5. Dynamic, real-time images can be obtained.

6. Optical sections allow imaging from different tissue depths. In the past, this design had a few significant disadvantages over other types of confocal microscopes.1 For a time, the calibration of the Nipkow disk itself was difficult. Since the outgoing light path and incoming light path utilize the same set of diametric pinholes in the Nipkow disk, any misalignment of the Nipkow disk resulted in severe image degradation.1 However, newer Tandem Scanning Confocal Microscopes (TSCM) contain a Nipkow disk system that is permanently aligned at the factory, eliminating this issue.1 Until recently, this microscope did not perform well during in vivo tissue examination secondary to an extremely low light-to-image capture rate. Because pinholes are used, only approximately 1% of the light sent into the system can be recovered for imaging.2 Additionally, any involuntary eye movements due to mircosaccades, pulse or respirations would result in large motion artifacts, making this novel design clinically useless. A rapid imaging capture system capable of at least 30 frames per second coupled with lowlux detectors was needed.1,9 Fortunately, technologic advances in video and electronic systems surpassed this threshold. These new capture imaging systems combined with low-lux cameras exploited the Nipkow disk’s potential. Combined with the development of a 20 X applanating cone objective lens, in vivo tandem scanning confocal microscopy of the cornea became a reality.12 Although, this design was the dominant confocal microscope used in ophthalmology, additional variations were developed.

Single-Sided Scanning Confocal Microscope Unlike the dual light path design of the tandem scanning confocal microscope, the single-sided confocal microscope uses a single light path design.1,2 This design utilizes the same pinhole in the rotating Nipkow disk for both the outgoing light destined for the specimen and the reflected light destined for the detector.1,2 A beam splitter mirror is required to separate the incoming and outgoing rays of light.1 The main disadvantage of this design is that the outgoing light rays, that do not pass through the Nipkow disk pinholes, reflect off the disk back toward the detector.1 These reflected light rays then interfere with specimen light rays of interest. Thus, this design potentially degrades image contrast and resolution.1 Kino and Xiao13 attempted to minimize this limitation by developing a tilted disk design. In their version, they tilted the Nipkow disk slightly.1 This tilt served to deflect

Confocal Microscopy of the Cornea the unwanted reflected light into a light sink, thus eliminating the reflected light interference prevalent in prior designs.1 However, this apparent simple solution required a complex arrangement of additional lenses and prisms within the optical system to bend the light rays to ensure that they correctly passed through the Nipkow disk’s pinholes.1 Overall, the end-result of these modifications was a moderate decrease in image quality secondary to induced astigmatism. In addition, a miscalculation in the location of the reflected ray’s focal point as small as 1 mm decreased confocality.1 A simplified light path using a single-sided confocal design was designed to eliminate the complications of Kino’s design, while maintaining the reliability and simplified alignment characteristics of the single-sided disk system. Although this new single-sided confocal microscopy system has been produced as a prototype, it is not commercially available.

Scanning-Slit Confocal Microscope Koester invented a different confocal technique that substituted a slit-beam for the Nipkow disk. He modified a specular microscope by adding a scanning-slit and mirror system1, 25 Maurice had also developed a similar system.1,14 Both designs used a novel approach without using the Nipkow pinhole disk. This design substituted a fine slitbeam of light. To allow for scanning, Koester and Maurice used movable mirrors to bend the light rays, allowing for X-Y plane scanning.1 They then combined these movable mirrors with lenses to create a mechano-optical scanning mirror apparatus.1 To eliminate aberrant rays, the reflected light rays passed through an additional slit before reaching the detector similar to the tandem confocal concepts.1 Like the pinhole effect, this scanning slit-beam design only imaged the focal plane of interest and allowed for dynamic, three-dimensional scanning. However, this microscope possessed a less precise Zaxis resolution.15 Early designs were plagued with slow scan times as compared to the tandem scanning technique. This limitation made it difficult to reach the 30 images per second scan speed threshold, which is required for full resolution video image capture.1 However, new low-lux detectors (cameras) and new, more powerful illuminators have helped to overcome these limitations.1

Laser Scanning Confocal Microscope Laser Scanning Confocal Microscopy (LSCM) is a relatively new confocal technique that has found wide applications

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in the biological sciences. Developed by the MRC Laboratory of Molecular Biology in Cambridge, UK in 1986, this design has evolved into a highly successful commercial product.12 The rapid utilization of this technique is easily explained by the wide use of fluorescent antibodies in biological research. Although the fluorescent-labeled antibodies were introduced in 1941, their true significance in identifying biological structures was not appreciated until the 1970’s when researchers realized that these antibodies could be used to tag biological proteins, such as actin and tublin.12,16 After this discovery, the florescent microscope quickly dominated biological research.12 However, researchers quickly grew frustrated with the aberrant light scattering inherent when examining thicker tissue specimens. To resolve this issue, they investigated and adapted Minsky’s concept of confocal microscopy. However, there were significant differences in design. The Nipkow disk design was abandoned since the pinholes did not allow enough light to pass through the system to make this system practical.12 After years of research, the modern laser scanning confocal microscope emerged. Currently, there are numerous design variations, but many of these designs encompass the same concept. Modern designs use a low power laser as the illuminator, which meets American National Standards Institute requirements for safe use in the eye.1 The detector digitizes the incoming photons for digital imaging. Similar to earlier confocal concepts, two pinholes are used, each placed before the laser and detector. Likewise, the focal points of both are identically calibrated. Optical lenses then focus the laser light to allow Z-axis scanning. However, instead of using the Nipkow disk to scan within the X-Y plane, the laser light reflects off a dichroic mirror, which directs it to an assembly of vertically and horizontally movable scanning mirrors. These motor-driven mirrors scan the laser across the specimen.7,12 Nevertheless, this design does possess some disadvantages. Unlike the tandem scanning design, the laser scanning design can only image one point at a time, decreasing its scan rate significantly. This limitation may decrease its significance in dynamic imaging. In addition, since this system utilizes movable mirrors, optical aberrations become more pronounced as larger angles are required to image a larger field of view.7 Despite these limitations, the laser scanning confocal microscope can produce high quality images. Furthermore, its powerful software can combine the two-dimensional optical sections into a three-dimensional reconstruction. The confocal microscope concept is a powerful tool that has been used for a long time in ophthalmology, for both

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research and clinically applications. Because of it transparency, the cornea is an ideal tissue to study in vivo, using this technique.

Confocal Microscopy Appearance of Corneal Anatomy The in vivo microscopic examination of the cornea’s avascular structure is more complex then its transparency would suggest. Therefore, a comprehensive knowledge of this structure is required to truly appreciate confocal microscopy’s power to view corneal anatomy in vivo.

Epithelium For review, the anterior surface of the cornea is covered with a layer of nonkeratinized, stratified squamous epithelium. This epithelium is comprised generally of three layers, namely, basal, wing and superficial cell layers. As compared to the entire cornea, the epithelium is relatively thin. The average corneal epithelium thickness as measured with a laser scanning confocal microscope (LSCM) was found to be 54 ± 7 μm centrally and 61 ± 5 μm peripherally.17 This difference can be explained by the back surface of the cornea having a smaller radius of curvature than the front surface. This curvature difference also explains why the central cornea is thinner than the periphery. The average central corneal thickness is 545 ± 25 μm and 652 ± 75 μm peripherally.17 The corneal epithelium is a highly metabolically active tissue that continually replenishes itself throughout life (See also Chapter 2, Corneal Physiology). The source of this proliferation emanates from the perilimbal stem cells. It is generally accepted that these stem cells go on to differentiate into basal cells that form the replicating layer that replenishes the epithelium. Overall, the epithelium is generally comprised of three layers. First, the basal epithelial layer, derived from the perilimbal stem cells, migrate onto the cornea and subsequently differentiate into polygonal wing cells. These wing cells comprise the intermediate cell layers that eventually differentiate into the superficial epithelial cells. Interestingly, the superficial and basal cell density are in a ratio of 10:1, respectively.17 The mean surface areas of superficial and basal cells as measured by one study are 624 ± 109 μm2 and 66 ± 5 μm2 respectively, with a ratio of 11.0 ± 4.5:1.18 Various studies have shown no correlation with cell density and age. 17,18

Basal Cells Basal cells measure approximately 10-15 μm in diameter and are generally uniform in size and reflection.18 These basal cells comprise a monolayer, situated at the base of the epithelium. Unfortunately, these basal cells reflect light poorly. For its power TSCM does not image this cell layer well.18 When imaged, the cell borders are visible as highly reflective outlines and highly reflective cell nuclei are visible.15,18 The cell cytoplasm is poorly imaged by the TSCM (Figure 8-4). The LSCM was no better at visualizing basal cell detail than the TSCM.17 Like the TSCM, the LSCM demonstrated morphologically well-defined cells with bright borders.17 The LSCM did show a central basal cell density of 8996 ± 1532 cells/mm2 compared to 5699 ± 604 cells/mm2 as measured with TSCM.15,17 Furthermore, the peripheral basal cell density was determined to be 10139 ± 1479 cells/mm2 using the LSCM.17 Thus far, various studies have not demonstrated a statistically significant relationship between gender or age and the basal cell density.15

Figure 8-4: Epithelial basal cells. The highly reflective cell borders with occasional nuclei can be seen in this confocal image. Note that the cell cytoplasm is poorly visible due to low reflectivity.

Wing Cells Overlying the basal cell layer are two to six layers of polygonal cells called wing cells. These differentiating cells appear uniform in shape and size with dark cytoplasm and bright borders similar to basal cell morphology as seen with confocal microscopy.15,17 Like the basal cells, wing cell details cannot be easily imaged due to poor

Confocal Microscopy of the Cornea reflectivity.15, 17 In general, wing cells tend to be larger than basal cells, but smaller than superficial cells. The average number of cells decrease to 5070 ± 1150 cells/mm2 centrally and 5582 ± 829 cells/mm2 peripherally as measured by the LSCM.17

Superficial Epithelial Cells The last step in differentiation creates the superficial epithelial cell layer. Generally, this cell layer is approximately one to two cells thick. The cells within this layer appear flat and polygonal on standard light microscopy. With white-light based confocal microscopy, these cells demonstrate light cell boundaries with bright nuclei (Figure 8-5).9,15,18 In addition, these cells tend to vary in size with the largest measuring 50 μm in one study.17 Conversely, one study used a LSCM that imaged epithelial cells with clear cell borders, bright cytoplasm and black nuclei.17 Both techniques showed cells that varied greatly in reflectivity, ranging from dark to bright cells.17 Generally, the bright cells represent metabolic active epithelial cells while the dark cells signify dead or desquamated epithelial cells.9,15,17,18

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example, the subepithelial nerve plexus lies just posterior to the Bowman’s layer. This plexus does image well on confocal microscopy and can be used indirectly to identify Bowman’s layer. This nerves plexus have a beaded appearance on confocal microscopy.15 (For additional details, see “Corneal Innervation” in this chapter).

Stroma The stroma comprises approximately 90% of corneal thickness. This layer is mostly comprised of keratocytes arranged in parallel lamella and ground substance. However, the stromal structure is not uniform throughout the cornea. For example, the orientation of the collagen fibrils differs in the anterior one-third and posterior twothirds of the corneal stroma and at the limbal regions of the cornea. These fibrils have an oblique arrangement in the anterior one-third of the corneal stroma, while they are parallel in the posterior two-thirds of the stroma and at the limbus they have a circumferential arrangement.19 Just posterior to the Bowman’s layer is the highest concentration of stromal keratoctyes. Mustonen et al15 used the scanning slit confocal microscopy to estimate a density of 1058 ± 217 cells/mm2. Furthermore, they found that these keratocytes progressively decreased in density to approximately 771 ± 135 cells/mm2 in the posterior stroma. Patel and co-workers20 verified this pattern. Additionally, they used the TSCM to calculate keratocyte volume-density in cubic millimeters. The results of this study are shown in Table 8-1.20 TABLE 8-1: Normal central human keratocyte density with respect to corneal depth. *

Figure 8-5: Epithelial superficial cells. These cells appear flat and polygonal with faint reflective cell boundaries and bright, prominent nuclei. Note that the cytoplasm is poorly reflective.

The average central cornea superficial cell density was measured to be 840 ± 295 cells/mm2 via LSCM and 1213 ± 370 cells/mm2 via TSCM.15,17 Peripherally, LSCM measured 833 ± 223 cells/mm2.

Bowman’s Layer Bowman’s layer is posterior to the basal cell lamina. It is an acellular layer comprised of randomly dispersed collagen fibrils approximately 12 μm thick. On confocal microscopy, Bowman’s layer appears as an amorphous membrane that is usually difficult to discern.15 This layer usually is identified with the aid of anatomical landmarks. For

Stromal depth (% Stromal thickness)

Keratocyte density (mm3 )

Full-thickness stromal

20522 ± 2981

Most anterior stromal apex

33050 ± 11506

0-10% (anterior)

28838 ± 8913

11-33%

20916 ± 4032

34-66% (mid)

19241 ± 2906

67-90%

19081 ± 2703

91-100% (posterior)

19947 ± 3254

* Table adapted from Patel SV et al who used the TSCM to generate this data20

Full-thickness central keratocyte density was negatively correlated with age that decreased 0.45% per year. Interestingly, this finding is similar to the estimated 0.6% per decrease in endothelial cells.21 Unfortunately, stromal keratocyte’s cytoplasm, cell boundaries and collagen substance absorb most of the

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incoming light.15 Despite this poor reflectivity, stromal keratocytes are imaged on confocal microscopy. They have bright, prominent nuclei suspended within a dark, amorphous ground substance.9,15 Sometimes, a small portion of the keratocyte cell body is also visible (Figures 8-6 and 8-7).9 Interestingly, the posterior keratocyte’s nuclei appear more elongated than the anterior keratocytes.15 However, neither the keratocyte processes nor the collagen network is visible.9 In addition to keratocytes, various nerve bundles can be seen weaving in between the collagen fibrils (Figure 8-8).

Figure 8-8: Corneal nerve. Numerous keratocytes can be seen with a large, reflective nerve bundle weaving in between the nonreflective collagen fibrils at the top of this image.

In general, the cornea is estimated to possess approximately 2.4 million keratocytes.22 Various studies, using fluorescent antibodies suggest that there may be at least three different types of keratocytes. These keratocytes’ product namely, collagen, is arranged in varying patterns, depending on the stromal depth. These varying arrangements are not likely to be seen in vivo as previously described. Figure 8-6: Anterior stromal keratocytes. The bright reflections in the image represent prominent keratocyte nuclei suspended within a dark, amorphous, non-reflective ground substance. Note, sometimes a small portion of keratocyte cell body can be seen as a reflective outline.

Descemet’s Membrane Descemet’s membrane represents the basal lamina of the corneal endothelium that thickens throughout life from 3.0– 4.0 μm at birth, to 10.0 – 12.0 μm during adulthood. More specifically, it has two layers, an anterior banded zone that develops in utero and a posterior nonbanded zone that is laid down by corneal endothelium throughout life.23 Unfortunately, the lack of cell nuclei makes Descemet’s membrane indistinguishable on confocal microscopy. The confocal microscopy can only image the surrounding keratocyte nuclei and endothelial cells, which can serve as anatomical landmarks, unless Descemet’s membrane thickens and develops fibrosis.15

Endothelium Figure 8-7: Tangential view through the basal epithelium and anterior stroma. The reflective epithelial basal cell outlines can be seen in the upper right. The highly reflective keratocyte cell nuclei with a small portion of the cell body can be seen suspended within a dark, amorphous ground substance in the lower left. Bowman’s layer appears as an amorphous membrane between the stromal keratocytes and epithelial basal cells.

The endothelium is a monolayer of hexagonal interdigitated cells. There are approximately 500,000 cells, giving an average cell count of 3055 ± 386 cells/mm2.15 However, since these cells cannot replicate, the absolute cell number decreases with age. Endothelial cell loss tends to be higher during childhood, but stabilizes after age 18.21

Confocal Microscopy of the Cornea Prior cross-sectional studies of adults have demonstrated an annual endothelial cell loss rate of 0.3% to 0.5%.21 However, a recent longitudinal study over 10 years demonstrated a slightly higher rate of cell loss, namely, 0.6% ± 0.5% per year in adults.21 Correlation between age and the rate of cell loss over the prior ten years in this longitudinal study was not statistically significant.21 In other words, the rate of cell loss appears to be constant throughout adulthood. Additional longitudinal studies over a shorter duration demonstrated between 0.3% and 1.0% cell loss per year.21Anterior segment surgery has been implicated in exacerbating the rate of cell loss. For example, three to five years after a penetrating keratoplasty, the measured endothelial rate of cell loss was 7.8%.24 However, this loss may likely represent the donor cells migrating onto the host, thus decreasing its absolute cell density. Morphologically, the apical surface of these cells faces the anterior chamber and their basal surface produce the posterior banded zone of the Descemet’s membrane. On confocal microscopy, the endothelial cells usually appear as bright cell bodies with dark, hexagonal cell boundaries (Figure 8-9).15 This appearance is similar to that of specular microscopy. Confocal microscopy is a useful clinical tool, in the diagnosis and follow-up of cases with endothelial decompensation as in Fuchs’ corneal endothelial dystrophy.

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Figure 8-10: Fuchs’ endothelial dystrophy. Multiple hyporeflective areas varying in size surrounded by hyperreflective structures can be seen, which are typical in Fuchs’ endothelial dystrophy. Many of these hyporeflective, round zones contain a central highlight. These dark, hyporeflective areas with occasional central highlights represent cornea guttata.

cells that is unrelated to an underlying disease state. On confocal microscopy, these cells display both polymegathism and pleomorphism. Usually, there are multiple hyporeflective areas varying in size surrounded by hyperreflective structures.25 These irregular bright bodies are typical in Fuchs’ endothelial dystrophy (Figure 8-10). Many of these hyporeflective, round zones have a central highlight. 25 In addition, other hyperreflective areas consistent with fibrous proliferation can also be imaged.25 These dark, hyporeflective areas with occasional central highlights represent guttata.25 Like the stroma, collagen reflects light poorly, and thus, appears dark on confocal microscopy; hence, the reason why guttata appear dark.

Corneal Innervation Figure 8-9: Endothelial cells. Normal endothelial cells characteristically appear as bright cell bodies with dark, hexagonal cell boundaries. Endothelial cell nuclei are not normally visible.

These diseased corneal endothelial cells have a characteristic appearance on confocal microscopy. For review, guttata, the clinical sign for Fuchs’ corneal endothelial dystrophy, likely represents focal collagen accumulation onto Descemet’s posterior surface by abnormal endothelial cells.25 However, this accumulation may also appear with normal aging corneal endothelial

The cornea, especially the epithelium, is one of the most densely innervated superficial tissues in the human body. For comparison, the corneal innervation is 20 to 40 times that of tooth pulp and 300 to 600 times that of skin.26 Overall, the central two-thirds of the corneal epithelium is equally, densely innervated that gradually decreases five to six times toward the periphery.26 For review, corneal innervation originates from the ophthalmic portion of the trigeminal nerve and eventually enters the eye along the long ciliary nerve within the perichoroidal space. Branches of this nerve bundle exits just posterior to the limbus and combine into an annular nerve plexus around the limbus. Approximately sixty to

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eighty branches radiate in a radial pattern into the substantia propria of the perilimbal conjunctiva and then pierce mostly into the anterior one-third of the corneal stroma.27 These nerve bundles lose their perineurium and myelin sheaths within approximately one millimeter of the limbus. These nerves then branch either di- or trichotomously or in a T-like division within the stroma.27 The details of this neural network had been elaborated by one study, using fluorescent antibodies. Most of these stromal nerve bundles were found to combine into a subepithelial plexus (SEP) just posterior to the Bowman’s layer.27 The corneal epithelium is thought to receive its innervation directly from this subepithelial plexus (Figure 8-11). This study found that terminal branches, measuring 10 to 12 μm in diameter, emanated from this plexus, penetrated Bowman’s membrane, weaved among basal cells and combined again to form another nerve plexus, named the basal epithelial plexus (BEP).26,27

From these intermediate bundles, smaller bundles bifurcate at right angles, traveling again in the nine-to-three direction.26 Confocal laser fluorescence microscopy with threedimensional reconstruction has been able to further elucidate corneal nerve architecture. Morphologically, A δfibers (1-5 μm) differs from C-fibers by possessing characteristic bulbous-like thickenings (~5-10 μm) that consistently remain below the basal epithelial layer.26,27 Many of these nerve fibers appear similar to a string of pearls.26,27 These fibers further divide di- and trichotomously, resulting in usually five to six fibers that are partly interconnected.27 C-fibers (0.2-2 μm), on the other hand, form short, branching clusters that run mostly perpendicular to the BEP, approaching the superficial epithelial cells.26,29 Furthermore, dichotomous dividing or bulbous-like thickenings associated with A δ-fibers were not found for C-fibers. Directly anterior to the Bowman’s layer, the Cfibers kink and travel within the BEP for only a short distance. Subsequent to traveling 8-10 μm after exiting the BEP, some of these fibers further divide dichotomously. These fibers then branch into fine fibers that weave their way to the superficial epithelial cell layer. They terminate blindly underneath the surface epithelial cell layer. It is estimated that there are approximately 16,000 nerve endings per one mm2 within the superficial epithelial layer.27

General Corneal Characteristics Figure 8-11: Subepithelial nerve plexus. The highly reflective lines in this confocal image represent the subepithelial nerve plexus. A recent study concluded that this plexus may lie just posterior to Bowman’s layer. 27 The corneal epithelium is thought to receive its innervation directly from this subepithelial plexus. The majority of nerve fibers in the subepithelial nerve plexus have been described as C-fibers26 that respond to mechanical, chemical, and thermal stimuli.

These corneal nerve bundles are composed of myelinated A δ-fibers and unmyelinated C-fibers.28 Both fiber types, known as nociceptors, can be activated by mechanical, chemical, and thermal stimuli.26,28 The majority of nerve fibers in the SEP of the human cornea have been described as C-fibers.26 Generally, from studies using electron microscopy, the architecture of the BEP has been elucidated. The large nerve bundles within the BEP were found to travel in the nine-tothree clock-hour direction. From these nerve bundles, smaller intermediate bundles bifurcated and traveled at almost right angles in the twelve-to-six clock direction.26

These confocal microscopes have also detailed the corneal thickness. For example, the average corneal thickness as measured with a LSCM is 545 ± 25 μm centrally and 652 ± 75 μm peripherally.17 Moreover, Patel and co-workers used TSCM to estimate each corneal layer’s thicknesses as shown in Table 8-2.20

Conclusion The confocal microscope has advanced our understanding of living systems, especially the cornea. This type of microscope has produced impressive photomicrographic images with excellent image resolution and contrast, and with dynamic in vivo scanning capabilities. In addition to research purposes, this microscope has the potential to make clinical diagnoses without performing corneal biopsies. For example, bacterial, fungal and acanthamoeba keratitis have all been detected in vivo without using stains or dyes.1 However, further studies still must be completed to validate this modality as a diagnostic tool. 1

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Confocal Microscopy of the Cornea TABLE 8-2: Corneal Thickness*

Corneal layer measured

Central thickness (μm)

Temporal thickness (μm)

Superficial epithelium to endothelium

563.0 ± 31.1

651.4 ± 37.3

Superficial epithelium to nerve plexus

48.6 ± 5.1

51.0 ± 8.7

16.7 ± 4.4

14.9 ± 6.1

498.5 ± 29.4

585.4 ± 36.0

Nerve plexus to most anterior keratocytes (Bowman’s layer thickness) Most anterior keratocytes to endothelium

* Table adapted from Patel SV et al who used the TSCM to generate this data. 20

The future role of confocal microscopy may likely become more significant with advances in computer, lighting and camera technology. However, the expense of this system has limited its use to mainly research purposes within the ophthalmologic community. As manufacturing costs decrease and new diagnostic roles are defined, the confocal microscope will become a more common tool in the ophthalmologist’s armamentarium.

References 1. Kaufman SC, Kaufman HE. How has confocal microscopy helped us in refractive surgery? Current Opinion in Ophthalmology 2006;17:380-8. 2. Cavanagh HD, et al. Confocal microscopy of the living eye. CLAO 1990; 16(1):65-73. 3. Maurice DM. Cellular membrane activity in the corneal endothelium of the intact eye. Experientia 1968; 24:1094–95. 4. Bourne WM, Kaufman HE. Specular microscopy of human corneal endothelium in vivo. American Journal of Ophthalmology 1976; 81:319–23. 5. Minsky M. Memoir on inventing the confocal scanning microscope. Scanning 1988; 10: 128-38. 6. Cavanagh HD, et al. Clinical and diagnostic use of in vivo confocal microscopy in patients with corneal disease. Ophthalmology 1993; 100(10): 1444-54. 7. Semwogerere D. Weeks ER. Confocal Microscopy Encyclopedia of Biomedical Engineering Academic Press, Taylor & Francis; 2005. 8. Wilson T, Sheppard C. Theoru and Practice of Scanning Optical Microscopy London: Academic Press, 1984. 9. Jalbert I, et al. In vivo confocal microscopy of the human cornea. British Journal of Ophthalmology 2003; 87: 225-36. 10. Petran MH, Hadravsky M, Egger MD, Galambos R. Tandem scanning reflected light microscope. J Optics Soc Am 1968; 58:661-4. 11. Lemp MA, et al. Tandem-Scanning (Confocal) microscopy of the full-thickness cornea. Cornea 1985/1986; 4:205-9. 12. Amos WB, White JG. How the confocal laser scanning microscope entered biological research. Biology of the Cell 2003;95: 335-42. 13. Kino GS, Corle TR, Xiao GQ. The scanning optical microscope: An Overview SPIE Proceedings: Scanning Microscopy Technologies and Applications 1988;897:32. 14. Maurice DM. A scanning slit optical microscope. Investigational Ophthalmology and Visual Science 1974;13:1033-7.

15. Mustonen RK, McDonald MB, et al. Normal human corneal cell populations evaluated by in vivo scanning slit confocal microscopy. Cornea 1998;17(5): 485-92. 16. Kasten FH. Chapter One: Cell Structure and Function by Microspectrofluorometry. The Origin of Modern Fluorescence Microscopy Academic Press, San Diego; 1989. 17. Eckard A, Stave J, Guthoff RF. In vivo investigations of the corneal epithelium with the confocal rostock laser scanning microscope. Cornea 2006;25(2):127-31. 18. Tomii S, Kinoshita S. Observations of human corneal epithelium by tandem scanning confocal microscope. Scanning 1994;16: 305-6. 19. Fundamental and Principles of Ophthalmology (section 2) & External Disease and Cornea (Section 8). Basic and Clinical Science Course Academic Press; American Academy of Ophthalmology: 2003. 20. Patel SV, McLaren JW, Hodge DO, Bourne WM. Normal human keratocytes density and corneal thickness measurement by using confocal microscopy in vivo. Investigational Ophthalmology & Visual Science 2001;42(2):333-9. 21. Bourne WM, Nelson LR, Hodge DO. Central Corneal Endothelial Cell Changes Over a Ten-Year Period. Investigational Ophthalmology & Visual Science 1997;38(3):779-82. 22. Muller LJ, Pels L, Vrensen GF. Novel aspects of the ultrastructural organization of human corneal keratocytes. Investigational Ophthalmology & Visual Science 1995;36(13): 2557-67. 23. Murphy C, Alvarado J, Juster R. Prenatal and postnatal growth of the human Descemet’s membrane. Investigational Ophthalmology & Visual Science 1984; 25:1402-15. 24. Bourne WM, Hodge DO, Nelson LR. Corneal endothelium five years after transplantation. American Journal of Ophthalmology 1994; 118:185-96. 25. Chiou A, Kaufman SC, Beuerman R, Ohta T, Soliman H, Kaufman HE. Confocal microscopy in cornea guttata and Fuchs’ endothelial dystrophy. British Journal of Ophthalmology 1999; 83: 185-89. 26. Muller LJ, Vrensen GF, Pels L, et al. Architecture of the human corneal nerves. Investigational Ophthalmology & Visual Science 1997; 38:985–94. 27. Guthoff RU, Wienss H, Hahnel C, Wree A. Epithelial Innervation of Human Cornea: A Three-Dimensional Study Using Confocal Laser Scanning Fluorescence Microscopy. Cornea 2005; 24(5): 608-13. 28. Muller LJ, Marfurt CF, Kruse F, et al. Corneal nerves: Structure, contents and function. Exp Eye Res 2003; 76:521–42. 29. MacIver MB, Tanelian DL. Free nerve ending terminal morphology is fiber type specific for A delta and C fibers innervating rabbit corneal epithelium. Journal of Neurophysiology 1993; 69:1779–83.

Ramagopal Rao David Miller

Next Generation Operating Microscope: 3D Digital Microscope and Microsurgical Workstation

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Introduction Direct visualization of a patient’s anatomy for diagnosis and surgery is an important component of medicine. A vast array of optical instrumentation that provide a high quality three-dimensional (or binocular) view are employed in medical subspecialties such as ophthalmology, ear, nose and throat (ENT) and neurosurgery, where anatomical microstructures are examined and operated upon using surgical microscopes that provide a highly magnified view of the tissues of interest. The current technology employed in devices such as the surgical microscopes, slit-lamps and endoscopes require binocular (two sets of) optics for obtaining three-dimensional views. These instruments provide magnification, illumination and mechanical manipulation so that a surgeon has an excellent visual access to the specific anatomy of the patient. Surgical microscopes have been in use for at least 80 years. Their use in ENT surgery and ophthalmic surgery enabled several innovative, less invasive surgical procedures. These instruments have seen several improvements since the early 1960’s such as a variable focus, ceiling mounted microscope systems, X-Y mechanism and fiber-optic illumination. Today, worldwide, it is estimated that there are about 50,000 surgical microscopes with different levels of functionality. Carl Friedrich Zeiss (1816-1888), an inventive optical designer from Jena in Germany, is credited with some of the early microscope designs. His company designed the first commercial compound microscope that incorporated the objective and eye piece into a single integrated instrument. Later Ernst Abbe (1840 -1905), Schott (18511935) and Horatio Greenough made significant contributions to advance the optical microscope technology. As early as 1887, the first stereo microscope was introduced. Stereo surgical microscopes have been in use for more than eighty years in microsurgery. Despite their wide acceptance, current optical microscope technologies have several limitations of functionality in terms of ergonomics, flexibility, image quality and archival storage. Most current instrument concepts do not derive any benefit from the recent advances in digital imaging technology, image processing software and related hardware. These newer digital technologies provide a significant potential for improvement in real time surgical imaging through image enhancements, interactive intelligence, storage and retrieval. Several recent advances in imaging technology such as high resolution sensors, high performance processors and image processing software allow acquisition of high resolution color images at frame rates that are very rapid. The digital display technologies can now display high resolution images in

brilliant colors at high frame rates. Three-dimensional images can now be displayed on two-dimensional display systems with advanced stereoscopic display systems. Several holographic three-dimensional display technologies are emerging for medical applications. Modern microsurgery places intense physical and mental demands on a surgeon and the surgical team. Besides the expected hand-eye coordination and manual dexterity, a surgeon is required to process vast amounts of visual and tactile information from the surgical field and environment, analyze this data and make timely decisions on the course of action. Ophthalmic and other microsurgeries require excellent coordination between all members of a surgical team. Conventional surgical microscope technology provides the surgeon with a good magnified view of the surgical field with limited options, namely, zoom and focus. Only the surgeon and assistant(s) can view through binocular eyepieces, placing the rest of the team at a disadvantage. These microscopes are “passive,” meaning, they can only present the image without any ability to provide further information or enhancements that could aid the surgeon. This is a serious limitation of the current state of the art equipment. There have been no significant technological innovations in the field of surgical microscopy for the past fifty years.

Digital 3D Microscope System Digital Microsurgical Workstation The authors are developing a digital microsurgical workstation (Figures 9-1A and B) based on various advances in digital imaging technology. Our concept may be parallel to the current revolution evident in the digital photography. Digital photography has garnered a large fraction of the photography market through its versatility, flexibility and superior image quality. Unlike the film where the acquired images can only be enhanced through chemical process in a laboratory, digital photography allows user to manipulate images through inexpensive software that is widely available. Post-processing allows users to obtain images with the desired attributes. This key concept can be carried over to the operating room where a surgeon can obtain a desired view with post-processing. Combination of real time view and processed images provides additional information to the surgeon, and such additional information could be critical for achieving better surgical outcomes. The major components of the system are illustrated in Figure 9-1B. The advent of digital (silicon-based) photography, novel display technologies and image processing software presents an excellent opportunity for

Next Generation Operating Microscope: 3D Digital Microscope and Microsurgical Workstation 87

Figures 9-1A and B: (A) Photograph of the digital microsurgical workstation and the 3D digital microscope; (B) Digital microsurgical workstation and the digital cameras.

the development of a new type of surgical imaging system that not only provides a high quality image but also much needed supporting information that aids the surgical process. The key elements of such technology are: 1. A three-dimensional image capture system provided through a stereo camera system. The camera system will have motor driven zoom and focus features. Magnification and stereo separation will be optimized to produce a high resolution three-dimensional image in true color.

2. A three-dimensional display system that is flexible and allows multiple users the same view as that of the surgeon. Such a system should also allow for different forms of display such as head mounted goggles, video monitors and large format screen for larger audiences. 3. An optional image processing system that incorporates hardware and software for image enhancement and other tasks provide additional information which may assist the surgeon. Such information could be presented as overlays over the real time image, measurements or

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navigation through a complex procedure. One could call such systems “smart” and applications as “intelligent”. Such “application” software could be procedure specific in that it provides guidance, metrology and overlay information as and when it is needed during the surgery. 4. Physical configuration that provides best ergonomic design features and flexibility.

Image Acquisition Stereoscopic image acquisition is accomplished through a dual camera system (Figures 9-2 and 9-3) with desired stereo separation for optimal depth perception. The sensor is typically a CMOS or CCD chip. The system has a single objective lens with a magnification of 8-12x for the chosen working distance and an optical zoom system that may provide 4x to 6x zoom. Any resolution over 1024 × 1024 is desirable in ophthalmology where the surgical field tends to be smaller than 60 mm. A frame rate of 30 or more frames per second per sensor is desirable for obtaining a flickerless image on the display device. The display frame rate is twice that of sensors as the dual frames are fused on to a single display screen.

Figure 9-2: Stereoscopic image acquisition through a dual camera system.

Display System A common display system for stereoscopic display is a conventional CRT monitor (Figure 9-3) with a polarizer screen (commonly known as a Z-Screen). The dual images

Figure 9-3: Dual camera system, “active” polarizing CRT monitor attached to a movable, on-wheels, floor-design stand.

from the stereo camera are displayed in alternate frames (a frame sequential mode) and the polarization of the Z-screen is altered (in orthogonal axes) in synchronization with the frame data. Such screens are known as “active” polarizing screens. The surgeon (observer) wears a spectacle with lenses polarized in orthogonal axes (same as the Z-screen) thus each eye is presented with the corresponding camera sensor view. As an alternative system may utilize a passive polarizing screen (over the monitor) with an active spectacle lenses (worn by the observer). High resolution and high brightness screen are required for stereo viewing, since polarizer screen can block almost 50% of the screen illumination. In other stereoscopic displays, a projection system (with dual projectors converging on to a single screen) is employed. LCD or DLP projectors are ideally suited for this. With conventional commercial projectors the resolution may be limited to 1200 ×1200 pixel range. Such systems require rigorous alignment of two projectors to achieve excellent pixel registration. Stereoscopic head mounted displays are yet another alternative display system where a 3D imaging mode is achieved without the need for convergence of dual images. The stereo image pair is projected or displayed on two small screens built into the head mounted displays. Some of the lower resolution displays can be fit into an oversized goggle configuration. The major disadvantage of these devices is that they are single observer displays and technologies for lower cost and higher resolution displays are still in the developmental stage.

Next Generation Operating Microscope: 3D Digital Microscope and Microsurgical Workstation 89 Displays with lenticular screens are yet another alternative for stereoscopic displays. These displays tend to be low resolution as lenticular films attached to the screens have the native resolution of the display. They also tend to be highly directional with a narrow angle of view. These displays require image information to be “interlaced” to conform to the screen configuration. This imposed some additional processing burden on the system. Newer display technologies may allow for flatter displays at higher resolutions and brightness in the future. Organic-LED, dual-LCD and compact high resolution DLP technologies hold great promise for future high performance 3D display systems. This will allow for configuration flexibility in the future 3D display systems.

Benefits of 3D Surgical Microscope There are several major benefits of such a digital microsurgical workstation, as stated below: 1. Ergonomics 2. Low light levels 3. Depth of focus 4. Form factor 5. Working distance 6. Intelligent microscopy a. Image enhancement tools b. Archival system c. Image framing d. Metrology e. Registration f. Diagnostics g. Computer aided surgery.

Ergonomics In an optical microscope the physical (or ergonomic) relationship between surgeon, patient and the system are fixed during the surgery. There is very little flexibility in positioning any one of these components thus requiring the surgeon to stay at a fixed position for optimum viewing. This may impose an inordinate amount of stress on the musculoskeletal system. The injury rate among surgeons who spend long hours in surgery is significantly high. Some surgeons have called this a “silent epidemic.” Several surgeons have curtailed their surgical time in operating room to minimize personal injury. A few surgeons have opted out of surgery. The injury rates reported by OSHA (that apply to all microscope users) support the fatigue factor of surgical microscope use (Table 9-1). With a digital microsurgical workstation a surgeon views the image on a monitor or a flat screen display monitor at a

TABLE 9-1: The injury rates among all microscope users, as reported by OSHA

Anatomical location

Employee percentage

Neck Shoulders Back (total) Lower back Lower arms Wrists Hands and fingers Legs and feet Eye strain Headaches

50-60 65-70 70-80 65-70 65-70 40-60 40-50 20-35 20-50 60-80

desired position and distance thus providing relief from a fixed position. This reduces pressure on the back and the neck, thus preventing injury. There is no need for adaptation between the view offered through the microscope and normal “room view”. This reduces potential errors. A surgeon and his team will have the same view. The digital workstation has twice the working distance as compared to an average optical microscope. This feature allows the surgeon to directly view the patient if necessary. This is often desirable for keeping track of the patient’s status.

Low Light Levels Conventional microscope systems require powerful light sources. Beam-splitters employed for assistant scopes and video camera feeds “steal” the light output, hence these microscopes require increased light to be delivered to the surgical field. In a typical system only 12 -25% of the light from the lamp actually is needed to view the field. Potential retinal light toxicity is a major concern with the conventional operating microscopes. A low-light budget reduces the total heat and noise (from fans). In a digital system assistant microscopes are not needed as the entire surgical team will have a full 3D view of the surgical filed on the screen or monitor. The light output on to the surgical field could be as low as about 30% of that needed for a typical optical microscope (Editorial Note: This is a major step forward in reducing potential light-induced retinal toxicity during ophthalmic surgery).

Depth of Focus Lower light levels needed in a digital system allow for smaller apertures for the delivery and collection of light delivered (and reflected from) to the surgical field. A smaller aperture results in higher depth of field. This allows surgeons to view most of the surgical field (up to 3 cm for eye surgery) without moving the microscope focusing optics.

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Form Factor

Archival System

In a digital workstation heavy optics are replaced by electronics thus reducing the size, weight and volume by a significant factor. This has a beneficial effect of reducing handling costs and installation costs.

Surgical procedures or components of a procedure can be stored in a central storage for later retrieval and review. Since the images are stored in 3D, a realistic view of the surgery is provided. Digitized images or procedures also provide a capability for random or partial retrieval unlike the analog video systems. The system provides a true documentation system for training and insurance purposes. Ability to recall prior surgical images during a surgery may aid in surgical decisions.

Working Distance In a conventional optical microscope, the ergonomics, namely, the relative position of eyepieces to the working surface imposes restrictions on the working distance. The normal working distance tends to be in the range of 1520 mm. In a digital system where the surgeon is “freed” from the eyepieces the distance is more determined by the available light budget and the sensitivity of the sensor element chosen. With CMOS or CCD sensors one can easily achieve working distances in the range of 300 to 400 mm.

Intelligent Microscopy The basic operative concept is that by introduction of an “image processor” between the image capture unit (camera) and the display system, the image is processed, analyzed, interpreted and then the enhanced image is displayed on the display system. Such image processing is computer bandwidth intensive as all the data contains highresolution, real time images at fairly high repetitive rates. We may have to incorporate unique pipeline processors that relieve the computer of certain routine processing chores. We call such image processing capability “image intelligence”. We plan to introduce various levels of functionality in gradual steps of sophistication and utility. We call this a “layering” methodology. It would be important for the surgeons to determine what kinds of information is essential and helps in the process and what would be either interfering or overwhelming in its content and presentation. This human (operator) interface will be decided in consultation with prominent surgeons. Some of the elements of such a system will incorporate training features to aid the learning process. The following is a partial list of functionality that can be incorporated as various layers of intelligence.

Image Framing With features such as “picture-in-picture” and “splitscreen” we will have the ability to present on the screen, during the surgery, the same procedure performed previously by the same surgeon, or another leading surgeon specializing in such procedures, in conjunction with the current surgery. This ability to view a “reference” source will be of significant benefit to the young surgeons. As newer procedures are developed the technique can be learned more easily if the comparative images are available during the surgery. We think this is an important training tool and will be adapted as standard tool in teaching programs. It will be possible to provide access to an entire library of surgical techniques and procedures.

Metrology With proper calibration of the microscope optics it would be practical to make actual measurement of tissue structures. This will save surgeons a significant amount of time during the surgery. It also minimizes the error rate. The software can calculate areas and volumes as necessary and display such information in real time. The range of measurements can extend to measurement of topography, refractive power and linear area of measurements. Some of these functions may require an additional accessory to be attached to the system. For example, the measurement of corneal topography under surgery may require an accessory that will project a simple Placido image on the cornea while the dual camera system will acquire the frame image for processing.

Image Enhancement Tools

Registration

This set of tools and processes will improve the image as seen by surgeons. Such controls can be activated by voice commands. Improvements in contrast, edge sharpness and color filtering can be provided. The display selection commands such as zoom and pan will also be made available.

This feature will allow a surgeon to place two (current real time view and a stored image) “images” of an anatomy, with one of the layers being a transparent layer, over the other with perfect registration. As the surgery progresses at different depths of tissue, the surgeon will have the ability to view on the screens, these sections, as layers. This

Next Generation Operating Microscope: 3D Digital Microscope and Microsurgical Workstation 91 positioning feature will enable surgeons to correctly locate appropriate sites of treatment. Any variations in anatomy specific to a patient can be viewed more vividly. As an example, such technology will immensely improve the accuracy of laser treatment for diabetic retinopathy. In a successful experiment that could help benefit the way surgery is performed, surgeons have begun viewing and manipulating 3D medical images of the very patients on whom they are operating while the operation is in progress. Early results indicate the potential for improving success rates on such procedures as the removal of cancerous tumors. In the first procedure of its kind, doctors at Britain’s Manchester Royal Infirmary used a standard laptop computer in the operating room to project and manipulate, in real time, a complex, three-dimensional image of a patient’s organ.

perspective volume rendering for clarity of view, full tissue texture and detail, volume sculpting for simulating incisions, and electronic contrasting by enhancing the volume display among regions of equivalence. The next step is to set up the system for the intervention. Then the intervention can begin; the monitoring system shows the surgical instruments in real time on an image chosen by the operator and utilizing alternative incisions. Surgical navigation, monitoring and training through virtual surgery are some of the applications that can be developed from the above technology.

Diagnostics

There are times when a corneal reflection is useful clinically. Although the size of the reflection, in most clinical settings is small, it can be magnified and the meticulously analyzed. For example the whole field of corneal topography and keratometry depend on such quantitative analysis. However, the reflection can also be an obstacle to the surgeon. For example, the size of the corneal reflection, as produced by the operating microscope light measures 1 to 2 mm. If the reflection is situated over a critical structure during the surgery, the reflection will obscure that structure. Thus, removal of the reflection is important in the operating room.

More sophisticated image processing techniques such as pattern recognition can be employed for image interpretation applications. In this methodology the surgical field image is interpreted for certain preset adverse states. Such alerts could relieve the surgeon from processing a multitude of images during surgery, and thus he can continue to stay focused on the surgical procedure. The processing algorithm can “spot” changes such as undesirable colors and dimensional changes and report them to the operating surgeon.

Computer-aided Surgery The advancements in computerized image processing enable surgeons to see the anatomy in 3D at full resolution. Data derived from recent medical imaging systems can be utilized for planning, simulating, and validating surgical procedures. The modern frameless stereotactic techniques enable surgeons to visualize the surgical site by providing interactive and intuitive access to surgical/anatomical images during the course of the surgical procedure. In this method the spatial relationship of moving surgical devices to the target lesion is viewed through the system. This technique allows surgeons to navigate through and around the site of surgery to determine the best surgical approach. The first step in computer-aided surgery is the acquisition of the medical images by conventional imaging technologies outside the operating room. Data is then downloaded to an appropriate computer allowing surgeons to view the images taken before the procedure. They can easily manipulate the volume and position views interactively and plan the surgical procedure by modeling multiple cuts of their choice. Software designed for computer-aided surgery offers surgeons interactive

Surgical Applications Removal of Unwanted Corneal Reflections and Artifacts

Artifact Removal Removal of the corneal reflection is not a new problem for ophthalmologists. For example, many ophthalmoscopes use polarizing filters to remove the reflected image, while some fundus cameras overlay a black dot on the corneal reflection. However, this latter approach also obscures the structure under the black dot. The 3D Vision System takes advantage of the advances in computer processing of digital images to remove the reflection in a new way. The present digital system described in this chapter can replace the reflection with an image of that area taken earlier, when the light source was moved, thus moving the reflection to another location. This is all possible because the surgeon does not view an optical image of the surgical field but a digital image of the field on a screen. A program can be built into the system to simply cancel any image that has the characteristics of the unwanted corneal reflection and substitute an earlier image of the area in question. Figure 9-4 shows an illustration of the way the surgical field appears when the 3D Digital Image System ‘erases’ the unwanted corneal reflection.

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Figure 9-4: Display of the 3D Vision System that has “erased” the unwanted corneal light reflection from the operating microscope light thus facilitating the surgical procedure.

Figure 9-5: 3D Vision System showing variable magnification of the operative field. The area of surgical interest namely, central magnified image is surrounded by a second image at a lower magnification of the same operative field.

Variable Magnification within the Surgical Field

interior of the lens in order to remove the cataractous nucleus and cortex by phacoemulsification followed by irrigation and aspiration. One of the unwanted result of the procedure is to produce a capsulorhexis of the wrong size. If the capsulorhexis is too small, then the implantation of the IOL becomes more difficult. If the capsulorhexis is too large, there is a greater chance of the IOL dislocation as well as a greater chance of a radial tear of the capsule with possible zonular disinsertion. To avoid these potential complications, a superimposed circular template of the correct capsulorhexis size would be a helpful guide to the surgeon. Using the 3D digital microscope, a capsulorhexis template (yellow circle) of the desired size can be superimposed on the digital image of the anterior lens capsule during the surgery (Figure 9-6), thus guiding the surgeon

In general, increasing the magnification of the surgical field reduces the field of view. A related analogy would be watching a football game with binoculars. If you focus on the quarterback with the magnification of most binoculars, this will result in loosing sight of the other important events taking place on the field. In other words, the observer is looking at reduced field of view. It would certainly be helpful to have a binocular that places a magnified image of the quarterback in the center of the field, and have this image surrounded by another image that shows the rest of the playing field at a lower magnification. The 3D system achieves this effect by limiting the magnification to only the center of the surgical field. This effect can only be achieved if the surgeon looks at a virtual digital image of the operative field on the screen instead of the real time optical image. Thus, the computer program that processes the image can be made to only zoom in on the central portion of the field and leave the rest of the field at a lower level of magnification. In this form of digital magnification (versus optical magnification) an image point that normally would cover one pixel is stretched out to cover many pixel areas (this accounts for a variable digital zoom effect). Figure 9-5 shows an operative field in which the center is at a higher magnification and the less important periphery is at a lower magnification.

Using a Template for Capsulorhexis An anterior capsulorhexis is the mechanical removal of the central portion of the capsule of the crystalline lens. The opening produced, allows the surgeon to enter the

Figure 9-6: Shows the capulorhexis template (yellow dotted circle), whose size can be controlled by the surgeon, is superimposed over the center of the anterior lens capsule of the cataractous lens. Also seen is the initial step of the surgical capsulorhexis path (blue dots).

Next Generation Operating Microscope: 3D Digital Microscope and Microsurgical Workstation 93 to perform the capsulorhexis of the desired opening. This ‘template’ for the correct size and shape of the capsulorhexis will always move so as to remain in the same location on the lens if the lens moves during the surgical procedure.

Metrology Presently, ocular entities such as pupil size, incision length, and lesion dimensions are measured by placing a caliper in the surgical field over the object to be measured. A less invasive, but also less accurate method, would involve superimposing a measuring reticule in the eyepiece of the microscope over the object of interest. The 3D system, by using a digital image of the surgical field, can employ a program which measures the dimensions of the object of interest by counting the pixels underlying the object of regard. Thus the diameter of the pupil is measured, in the computer program, by counting the pixels between the edges of the pupil and converting them into millimeters (Figure 9-7). The metrology of the 3D digital microscope system function can also be extended to more complex measurements which may involve range finding, triangulation, and topography. Intraoperative measurements of keratometry and refractive power are also possible with the digital technology.

Figure 9-8: The mentor system is demonstrated. The surgeon performs a phacoemulsification procedure, while a video of the same operation performed by the mentor is displayed in a split-screen mode, and it appears in another convenient part of the screen.

surgical procedures. These videos can be played in a splitscreen fashion (Figure 9-8). Thus, the young surgeon can view the real time surgical field on the central part of the screen while a 3D video of the mentor surgeon performing the same operation is seen on another part of the screen. This video of the experienced surgeon can be stopped or made to go fast or slow, forward or backward, to help review the steps taken to avoid or correct complications. The mentor system has broad implications in surgical training and education. It is possible to develop a training methodology that includes rating (or grading), interactive learning with either preceptor or computer aided interpretation. The system can be integrated with any surgical simulation systems.

Real Time Corneal Topography

Figure 9-7: Showing the pixel ruler of the 3D digital microscope system that is used to measure the pupillary diameter.

A Mentor System of Dealing with Complications Wouldn’t it be useful to have an experienced surgeon at your side when you are presented with a complication? The 3D system has developed a unique way to take advantage of the digital image presentation to simulate the help of an experienced surgeon (the mentor). It has in its memory, 3D videos of the mentors performing various

The design of an easy to use operating keratometer has been a legitimate goal for many years. The current methods of measuring the intraoperative corneal curvature includes placing a lighted circular target over the cornea and attempt to measure the dimensions of the corneal reflection to determine the real time corneal curvature. More recently, a portable corneal topography unit has been introduced, that is held over the cornea and a printed map created with the quantitative information. All of these systems require the surgical action to come to a halt while the circular lighted target is placed in position and a measurement of the reflection is taken. The 3D system takes advantage of the 3D image being continually created by the 2 digital cameras and uses a technique known as stereogrammetry to quantitate the corneal contour (Figure 9-9). Such a two camera

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Figure 9-9: Shows real-time intraoperative, corneal topography map that is superimposed on the cornea using the 3D digital microscope system.

photographic system has been used for many years to quantitate the contour of the 3D images of mountain ranges. Such a system can be useful in creating the proper tension in tying sutures or making relaxing corneal incisions of the proper length and depth in correcting corneal astigmatism.

Current Status and Future Directions The prototype systems have been used in actual surgery on humans (Figures 9-10A and B). The surgeon adaptation time was found to be negligible. Surgeons who used the systems in general had favorable impression of the system.

Bibliography 1. Barraquer JI. The history of the microscope in ocular surgery. J Microsurg. 1980;1:288-99. 2. Colvard DM, Kratz RP, Mazzocco TR, Davidson B. Clinical evaluation of the Terry surgical keratometer. J Am Intraocul Implant Soc 1980;6:249-51. 3. Cornic JC. The Zeiss surgical keratometer in cataract surgery. Dev Ophthalmol 1987;14:155-60. 4. Dhimitri KC, McGwin G, McNeal SF, Lee P, Morse PA, Patterson M, Wertz FD, Marx JL. Symptoms of musculoskeletal disorders in ophthalmologists. Am J Ophthalmol 2005;139:179-81. 5. Harmon RL. Microscope induced cervical spine disease ends surgical career. Eye World, June 2006. 6. Harms H. Evaluation of microsurgery. Trans Am Acad Ophthalmol Otolaryngol. 1969;73:439-40 7. Hollick EJ, Spalton DJ, Meacock WR. The effect of capsulorhexis

Figures 9-10A and B: (A) Actual use of the prototype systems of 3D digital microscope, and microsurgical workstation on humans; (B) Projection screen displaying the surgery in real-time.

8. 9. 10. 11. 12.

size on posterior capsule opacification: One year results of a randomized prospective study. Am J Ophthal 1999;128: 271-9. Oner FH, Durak I, Sovlev M, Ergin M. Long term results of various anterior capsulotomies and radial tears on intra ocular lens centration. Ophthalmic Surg Lasers 2001;32:118-23. Roach L. “The Silent Epidemic” ; AAO EyeNet Feature Two, October, 2002. Simon G, Parel JM, Nose I, Lee W. Modification, calibration and comparative testing of an automated surgical keratometer. Refract Corneal Surg 1991;7:151-60. Troutman RC. The operating microscope in ophthalmic surgery. Trans Am Ophthalmol Soc 1965;63:335-48. Troutman RC. The operating microscope. Past, present and future. Trans Am Ophthalmol Soc UK 1967;87:205-18.

Thomas John

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Introduction The earliest evidence of image magnification utilizing a magnifying glass dates back to the Book of Optics that was published by Ibn al-Haytham (Alhazen) in the year 1021. Since 1860 loupe magnification was used for surgical intervention. However, it was not until the year 1921, when Nylén from Sweeden transformed an ordinary laboratory microscope into a monocular operating microscope for ear surgery, that the first operating microscope was introduced.1,2 In the same year Holmgren first used the binocular operating microscope.1 These technological advances led to the development of true microsurgery. Ophthalmologists were the second group of physicians to use microscope in the operating theatre during the years 1940-1950.1 Since 1953, highly professional operating microscopes were made available and in the 1960s developments in the areas of microsurgical techniques, microinstruments and suture materials took place. The modern day operating microscope provides a wide range of magnifications and has the capability of zoom function, all of which are controlled by the foot pedals. This allows the surgeon’s hands to be free to perform surgery. This change in the way eye surgery was done forced the eye surgeons to use all four limbs during surgery. Thus, the introduction of the binocular operating microscope resulted in a paradigm shift in the way ophthalmic surgeons performed eye surgery. The operating microscope has served us well over several decades. During this time much of the surgical procedures in the anterior segment required only gross, full-field view with depth perception for both corneal and cataract surgeries. This scene is currently in a process of continual change as surgeons are slowly moving away from full-thickness, penetrating keratoplasty (PKP) to selective tissue corneal transplantation (STCT).3-5 STCT selectively replaces diseased parts of the cornea with similar donor tissue, while retaining the healthy portions of the patient’s cornea. With the increasing use of lamellar keratoplasty (LKP) techniques, the corneal surgeons require the ability to visualize the corneal thickness, and perceive the various corneal depths while doing surgery. In the absence of a cross-sectional view of the cornea, surgeons in the past have injected air into the anterior chamber (AC) and viewed the dark band between the reflective surface of the airbubble within the AC and the corneal surface blade edge, as the estimate of the remaining corneal thickness. This technique is laborious, and at best a rough estimate of the corneal thickness. At the present time we need the ability to directly view the corneal thickness in real-time with the aid of a slit-

view, much like the way we examine the anterior segment in a clinical setting. In order to meet this surgical need, the surgeon may consider the use of an intra-operative surgical slit-lamp. Over time, the use of an intra-operative surgical slit-lamp would possibly become the gold standard for ophthalmic microsurgery. Other areas of continued development in surgical viewing of the operative field include the ongoing experiments with video-assisted systems to further miniaturize the instruments for magnification and to gain a more comfortable working position for the surgeon. In this chapter, various surgical cases are presented to illustrate the use of an intra-operative surgical slit-lamp, demonstrating the usefulness of such a devise in corneal and anterior segment surgeries.

Surgical Cases Case 1 (Figure 10-1) In Descemet’s stripping automated endothelial keratoplasty (DSAEK), there is a surgical concern as to fluid being trapped within the donor-recipient interface that may contribute to post-operative donor disc detachment. In an attempt to eliminate potential fluid within this interface, various techniques have been described. One technique is to create “venting” incisions on the recipient cornea up to, and inclusive of, the interface without detaching the donor corneal disc. Four such incisions are usually made per eye. These incisions are routinely made on all cases undergoing DSAEK, since the interface is not visible with the conventional view provided by standard operating microscopes. With the use of a surgical slit-lamp the interface is clearly visible (Figure 10-1) and there is no fluid seen in the donorrecipient interface. Hence, no venting incisions are required. Surgical slit-lamp is beneficial in DSAEK surgery.

Case 2 (Figure 10-2) Patient was referred by his ophthalmologist with a large Descemet’s membrane (DM) detachment that occurred as a complication of clear cornea, phacoemulsification. This DM detachment resulted in significant corneal edema (Figure 10-2) and blurred vision following the cataract surgery. Large air bubble was used to attach the DM to the inner stromal surface of the patient’s cornea (Figure 10-2). Lindstrom roller is used to compress the corneal dome to facilitate a uniform attachment of the DM to the inner corneal stroma (Figure 10-2). Unlike the conventional, full-field view that does not allow adequate visualization of the DMstromal interface, the slit-view of the surgical slit-lamp

Role of Surgical Slit-lamp in Endothelial Transplantation and Anterior Segment Surgery

Figure 10-1: Top Row- 1. Descemetorhexis is performed using the John Dexatome (ASICO, Inc.), 2. Descemet’s membrane is removed as a single disc, 3. Donor corneal disc is folded over the patient’s cornea, 4. Double-ring sign of a well centered and uniformly adherent disc with smooth interface; Middle and Bottom Rows – Slit-beam scanning from the right to the left, showing the absence of any fluid collection and a smooth donor-recipient interface.

Figure 10-2: Top Row – 1. Full-field view showing significant corneal stromal and epithelial edema following Descemet’s membrane (DM) detachment during phacoemulsification, 2. Air is injected into the AC to attach the DM and the epithelium is removed to improve the surgical view, 3. Lindstrom roller is used to compress the corneal dome to facilitate a uniform attachment of the DM to the inner corneal stroma, 4. Well adherent DM; Middle Row – Broad and narrow slit-beam view of the edematous cornea; Bottom Row, Intraoperative clearing of the cornea following unifrom attachment of the DM as seen with the narrow slit-lamp view of the cornea and AC.

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Figure 10-3: Full-field and surgical slit-lamp views of the surgical steps in removing the retro-IOL, cloudy, cortical lens remnants using a bimanual technique.

Figure 10-4: Top and Middle Rows – Full-field view of the surgical steps in a combined penetrating keratoplasty with removal of a closed loop IOL and implantation of a scleral-fixated PC IOL; Bottom Row – Surgical slit-lamp view of the cornea and anterior segment at the completion of the surgical procedure. Notice the uniform corneal surface at the donor-recipient, circular margin, without any step configuration depicting appropriate suture tension and proper tissue orientation.

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Figure 10-5: Top Row – Intraoperative pre-surgical views of the cornea with keratoconus (KC) and apical, central corneal scar. Slit-view demonstrates the extreme corneal thinning and ectasia associated with the advanced stage of KC. Also seen are the partial-thickness, controlled corneal trephination using the Hanna vacuum trephine and lamellar dissection following intra-stromal air injection; Middle Row – Complete removal of the corneal stroma within the trephination circle, completely exposing the Descemet’s membrane within this circle. The surgical slit-view demonstrates the thin DM and the air that has entered the AC following intra-stromal air injection. Last figure – Removal of donor DM and endothelium; Bottom Row – Use of John ALK Compression Disc (ASICO, Inc.) is used to compress and facilitate a uniform attachment between the recipient DM and the inner stromal surface of the full-thickness donor corneal disc without its DM and endothelium, after application of fibrin glue in the donor-recipient interface. Middle Images – Notice the significant corneal flattening (compare it to the figures on the top row). Also seen is the uniform adherence between the patient’s DM and the inner stromal surface of the donor cornea without any fluid loculation or irregularities of the DM. The surgical slit-lamp view greately facilitates this intraoperative assessment by the surgeon. Last Image – Complete view of total anterior lamellar keratoplasty (TALK).

confirms good, uniform attachment of the DM in this case (Figure 10-2) and intra-operative clearance of the corneal stromal edema that is visible by the decreasing stromal thickness seen with the surgical slit-beam (Figure 10-2).

lens remnants without damaging the posterior lens capsule (Figure 10-3). The surgical slit-lamp view helps to assess this region during surgery (Figure 10-3).

Case 4 (Figure 10-4) Case 3 (Figure 10-3) Two weeks following an uneventful, clear cornea phacoemulsification, cortical lens remnants left behind in the superior, equatorial portion of the lens capsule migrated inferiorly and presented as cloudy lens material in the retrointraocular lens (IOL) potential space between the IOL and the posterior lens capsule, causing blurred vision. A bimanual surgical technique is used to clear the cortical

Figure 10-4 displays the surgical steps in managing pseudophakic bullous keratopathy caused by a closed-loop IOL. The procedure included a PKP, removal of the closedloop IOL, automated, anterior vitrectomy, and scleralfixated Posterior Chamber IOL (Figure 10-4). The junction between the donor corneal graft and the rim of the recipient cornea is important, since there may be a step, where the edge of the donor graft may be slightly higher as compared

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Figure 10-6: Top Row – Full-field, broad and narrow slit-lamp views of the failed DSAEK graft and edematous cornea. Both stromal and epithelial edema are seen; Middle Row – A reverse Sinskey hook is used within a Healon-filled AC to gently detach the failed donor corneal disc without damaging the inner stromal surface of the patient’s cornea. The failed donor disc is folded on its horizontal axis (temporal view) and the folded edge of the failed disc is brought out of the temporal entry wound. A 0.12 forceps is used to firmly hold the externalized portion of the failed donor corneal disc and it is gently pulled with a uniform force to peel-off the remaining portion of the donor disc from the patient’s corneal stroma. Last Image – Removed donor corneal disc; Bottom Row – Freshly prepared, surgeon-cut donor corneal disc with healthy donor endothelium is inserted into the recipient AC utilizing the “taco-fold” technique and delivered via the John DSAEK Insertion Forceps (ASICO, Inc.). Lindstrom roller is used to “iron” out any stromal wrinkles. Low-magnification (insert) and higher magnification full-field, frontal views of the well centered, and uniformly adherent donor corneal disc. Also seen is the double-ring sign. Right Images – Multiple surgical slit-lamp views demonstrating the absence of any interface fluid. Such an immediate real-time surgical confirmation, eliminates the need for any “venting” incisions on the recipient cornea.

to the edge of the recipient cornea causing surface irregularities. Additionally, very tight sutures may also contribute to irregularities at this donor-recipient circular junction. The slit-beam allows proper evaluation of this junction. In this case, a uniform, smooth junction is seen (Figure 10-4) confirming a good end-point for this surgery.

Case 5 (Figure 10-5) This is a case of advanced keratoconus with significant corneal thinning, apical cone scarring, and contact lens failure (per history) and the patient was referred for surgical management. The surgical options include full-thickness PKP, use of an intra-stromal Intacs (Addition Technology,

Inc.), and LKP. Of these three surgical options, LKP may be considered as the best option, since the patient’s endothelium is retained, and no prosthetic device is introduced into a very thin cornea. However, total lamellar keratoplasty (TALK) is a very challenging surgical procedure, where the surgeon needs to dissect the cornea and remove all the corneal stroma from a very thin cornea and not cause any tear or DM perforation during the TALK procedure. This challenging technique becomes even more difficult with the full-field view provided by the conventional, operating microscopes. The surgical slit-lamp provides direct visualization of the corneal thickness throughout the surgical procedure and helps prevent or decrease the chance of DM perforation during TALK procedure (Figure 10-5).

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Figure 10-7: Top Row - Slit-view of the stromal and epithelial microcystic edema associated with pseudophakic bullous keratopathy. Full-field views displaying John Dexatome-assisted descemetorhexis, and the use of the John DSAEK Scrubber (ASICO, Inc.) to roughen the peripheral, inner stromal surface within the epithelial circular mark. Middle Image – Surgical slit-lamp view of the fully detached, single-disc Descemet’s membrane (DM) suspended in the middle of Healon-filled AC. Slit-view confirms that the DM is fully detached as a single disc. Last Image – Tacofolded donor corneal disc, stromal stained with trypan blue (Vision Blue) within the fluid-filled recipient AC; Middle Row – Donor disc flipped in the wrong direction with the endothelium facing the recipient stroma and the donor stroma facing the iris surface. Last Image – Flipped, donor disc is removed out of the AC. Difficulty exists in confirming the stromal and endothelial side of the removed donor disc. Surgical slit-lamp helps in identifying the cut edge of the DM, the stromal surface (Bottom Row Middle) and the endothelial surface of the donor corneal disc thus assisting in proper orientation for the donor disc attachment to the recipient inner corneal surface; Bottom Row – Left - Full-field view of the properly oriented, and uniformly adherent donor corneal disc that is well centered. Bottom Row – Right – Surgical slit-lamp view of the donor disc and the donorrecipient interface.

Case 6 (Figure 10-6)

Case 7 (Figure 10-7)

DSAEK is soon becoming the preferred surgical procedure over a PKP in the surgical management of corneal endothelial decompensation. However, DSAEK can also result in endothelial graft rejection and graft failure. This is a case of DSAEK graft failure following graft rejection that did not fully resolve with standard medical management. Hence, a disc-exchange is required that is superior to a full-thickness PKP. Figure 10-6 demonstrates the surgical steps in this case of DSAEK disc-exchange. The surgical slit-lamp view aids in fully assessing the donor-recipient interface and no “venting” incisions on the recipient cornea is required (See also Figure 10-1).

This is an example of an intra-operative donor disc that “flipped” or unfolded in the wrong direction during a DSAEK procedure resulting in the donor endothelium facing the host, inner corneal stroma (Figure 10-7). Removal of the donor disc and reinsertion with the proper orientation is required. It may be difficult in such a case to clearly confirm the endothelial surface from the stromal surface using the full-field view afforded by conventional operating microscope. Here, the surgical slit-beam clearly identified the endothelial surface from the stromal surface and on evaluating the cut-edge of the donor disc the cut margin of the DM is clearly seen, that further confirms the endothelial side of the donor disc (Figure 10-7).

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Figure 10-8: Top Row, First Image – Patient had a previous keratoprosthesis OD. Patient presented without the keratoprosthesis and without a cornea. Iris and pupil are exposed to the room air. An iris surface membrane was seen. All Rows - Surgical slit-lamp views and full-field views display the surgical steps in removal of iris membrane, recreation of the AC angle, and a large, penetrating keratoplasty.

Case 8 (Figure 10-8)

Case 9 (Figure 10-9)

This is an extremely rare case presentation for surgical management. This patient had undergone an uneventful artificial cornea, namely, a keratoprosthesis. After about 6 months following the surgery the patient sees a cornea specialist with a history of redness and eye pain. The patient is then referred with a bandage soft contact lens. On clinical exam, the patient had no cornea and no keratoprosthesis. Removing the bandage contact lens provided direct visualization of the iris surface and the pupil with vitreous in the papillary space. Patient’s eye was immediately patched after antibiotic application and he received intravenous prophylactic antibiotics and underwent emergency eye surgery (Figure 10-8). The surgical slit-beam aided the surgical procedure especially during the iris membrane peeling and anterior segment reconstruction and a large diameter PKP (Figure 10-8).

Patient had a traumatic corneal laceration with lens rupture and secondary cataract. The primary corneal repair was performed by the referring ophthalmologist. There was a very large anterior lens capsular tear that did not extend to the posterior lens capsule. Surgical slitlamp view greatly facilitated the surgical management (Figure 10-9).

Surgical Pearls and Tips Take advantage of the surgical slit-lamp when available. Use both the broad-beam and narrow-beam as needed, along with the retro-illumination, by changing the angle of the beam to facilitate viewing various anterior segment tissues during surgery as demonstrated in the case examples provided above in this chapter.

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Figure 10-9: Top Row – Full-field and slit-views of the traumatic cataract and previously repaired corneal laceration by the referring ophthalmologist; Middle Row – Slit- and full-field views showing the use of trypan blue staining of the anterior capsule under air in the anterior chamber and needle capsulorhexis under Viscoat, of the previously torn anterior lens capsule; Bottom Row – Irrigation/aspiration tip is being used to remove the soft, fluffy, cloudy, lens material. Right side images show the slit-lamp views displaying an intact posterior lens capsule.

Conclusion The use of an intraoperative surgical slit-lamp provides the visualization that is needed for the present day newer, corneal dissection techniques and anterior segment surgery. STCT of the cornea requires meticulous lamellar dissections of the cornea without tearing or perforating the DM during such procedures. The surgical slit-lamp is helpful in these advanced corneal and anterior segment surgical procedures.

References 1. Haeseker B: [Microsurgery, a ‘small’ surgical revolution in the medical history of the 20th century]. Ned Tijdschr Geneeskd 1999;143:858-64. 2. Tamai S. History of microsurgery – from the beginning until the end of the 1970s. Microsurgery 1993; 14:6-13. 3. John T. Surgical Techniques in Anterior and Posterior Lamellar Keratoplasty. New Delhi, India: Jaypee Brothers Medical Publishers; 2006:1-687. 4. John T. Step by Step Anterior and Posterior Lamellar Keratoplasty. New Delhi, India: Jaypee Brothers Medical Publishers; 2006:1297. 5. John T. Selective tissue corneal transplantation: a great step forward in global visual restoration. Expert Rev Ophthalmol 2006;1:5-7.

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Thomas John

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Introduction Just like an artist needs good painting brushes to fully express his or her artistic talents, a corneal surgeon requires good surgical instruments to fully optimize his surgical skills. Having excellent machinery such as the operating microscope, phacoemulsification unit, best sutures, artificial anterior chamber and microkeratome, but, without the appropriate state-of-the-art surgical instruments, is like having a superb sound system without the best suited speakers. With this concept in mind this chapter is dedicated to introducing and familiarizing the reader to various surgical instruments that are specially designed for Descemet stripping automated endothelial keratoplasty (DSAEK) surgery [See Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)].

New Concepts in DSAEK Surgery1 In surgical ophthalmology, until now, we have been working on the “floor,” or doing “floor surgery,” meaning, inferiorly directed surgical maneuvers, whether working on the ocular surface, cornea, iris, lens, vitreous or retina that I call as “in-line-with-gravity” surgery (ILGS). In contrast, in DSAEK, for the first time in ophthalmology, we have changed the surgical direction to “against-the-lineof-gravity” surgery (ALGS), namely, we are working on the “ceiling” or “ceiling surgery,” ceiling meaning, the inner dome of the host cornea.1-21 This is a new direction in ophthalmic surgery. Hence, instruments that are designed with the usual “floor surgery” concept does not often work as well as one would like them to, when performing DSAEK surgery or “ceiling surgery.” With this concept in mind I designed several surgical instruments for DSAEK surgery (Table 11-1) and they are described in this chapter. Additionally, the surgeon has to be cognizant of the anterior chamber volume, namely, large, normal or small and the anterior chamber angle, all of which will have an influence in DSAEK surgery. Similarly, the choice of donor disk diameter, donor disk thickness, the type of fold that is used on the donor corneal disk [eg., taco-fold (bi-fold) [See also Chapter 20, Endothelial Keratoplasty: A Step by Step Guide to DSEK and DSAEK Surgery, Chapter 23, DSAEK Simplified Surgical Technique, Chapter 24, Surgical Technique for Descemet Stripping Automated Endothelial Keratoplasty (DSAEK)] 60/40, 70/30, 80/20, or a burrito fold (tri-fold)] (See also Chapter 25, Descemet Stripping Endothelial Keratoplasty (DSEK, Through a 3 mm Incision using the Tri-fold Technique), surgeon-cut versus eye-bank-technician pre-cut tissue, and the length of the entry wound into the anterior chamber (AC), will all have an influence on the overall outcome of

DSAEK procedure. Additionally, since we are doing ALGS in DSAEK, the donor corneal disk can detach from the patient’s inner corneal dome and fall and rest on the iris. Hence, the use of state-of-the-art DSAEK instruments and proper surgical techniques may be considered as essential ingredients to decreasing, if not, eliminating donor corneal disk detachment in DSAEK surgery.

Surgical Instruments for DSAEK A list of DSAEK surgical instruments are listed in Table 11-1 and shown in Figures 11-1 to 11-21. The Johninstruments were specifically designed to facilitate DSAEK surgery and increase surgeon-comfort in doing these newer corneal procedures. It appeared inappropriate to me, to try to work on the dome of the patient’s cornea with straight instruments from a fixed pivotal point (entry wound). The new instrument design that I developed, is reflected in the Dexatome and other similar instruments (Table 11-1) (Patent Pending). I coined the term “Dexatome” since it is used to score and detach the Descemet’s membrane (DM), a basement membrane, somewhat in keeping with the term cystatome that is also used to tear a basement membrane namely, the anterior lens capsule. The John Dexatome is curved, and has a sickle-like profile, so it can rotate to reach the entire inner surface of the patient’s cornea through a single entry wound, allowing for 360 degree scorring of the DM without removing the instrument from the anterior chamber. John Dexatome can then detach the DM as a single disk, leaving a pristine inner host stromal surface, and hence a superior donorrecipient interface. Such improved interface will contribute to better quality of vision and faster visual recovery.

John Dexatome DSAEK Spatula Identification of surgical step: Removal of DM and the corneal endothelium. Difficulty encountered: Reverse Sinskey Hook (RSH) is difficult to use, as it was not designed to work on the ceiling of the cornea. RSH requires multiple entries into the anterior chamber (AC). Main benefit: John DSAEK Dexatome Spatula (ASICO Inc., Westmont, IL, AE-2872) (Patent Pending) (Figures 11-1A to C) allows for DM and endothelium to be removed as a single disk almost every time (few exceptions) without exiting the AC (Figures 11-2 to 11-4). A DM tear that is 360 degrees is possible with a single AC entry. John Dexatome (Figures 11-1A to C) special design (Patent Pending) allows access to almost every point in the inner-corneal dome without exiting the AC. Currently, there is no other such instrument

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TABLE 11-1: Surgical instruments for DSAEK surgery Name Manufacturer Comments John Dexatome DSAEK Spatula (AE-2872) ASICO To score and detach DM as a single disk without exiting the anterior chamber; usually, no need for DM stripper John DSAEK Descemet’s Stripper (AE-2874)

ASICO

Used for stripping DM. Especially useful in failed corneal grafts with areas of scarring at the donor-host circular rim.

John DSAEK Inserting Forceps (AE-4227)

ASICO

For easy insertion of the folded donor disc into the recipient anterior chamber

John Retrocorneal Super-Micro Forceps (AE-4962)

ASICO

For holding tissues on the inner surface of the recipient cornea

John Super-Micro-Scissors (AE-5762)

ASICO

For cutting tissues on the inner surface of the recipient cornea

John Fixation-hook (AE-2182)

ASICO

To stabilize the donor corneal disk

John DSAEK Stromal Scrubber (AE-2878)

ASICO

Used to roughen the host inner corneal stroma

John DSAEK Corneal Glider (AE-2879)

ASICO

Used to smoothen the macro-folds in the donor disk

John DSAEK Marker (AE-2712)

ASICO

Used to make a circular mark on the corneal epithelium with a diameter of 8 or 9 mm

ALTK Artificial Anterior Chamber and CBm Microkeratome

Moria

Donor disk preparation

DSAEK Strippers (#19077/A) (#19077/B) DSAEK Irrigating Stripper (#19083/A) (#19083/B)

Moria

Stripping DM

Goosey Forceps (#19090)

Moria

DSAEK surgery

Price Hook (#19091)

Moria

To score DM

DSAEK Marker (#19095)

Moria

To mark host cornea

Moria

To introduce the donor disk into AC

Busin Glide Melles PLK Scraper

45o

&

90o

DORC

Stripping DM

Reverse Sinskey Manipulator (K3-5002)

Katena

For donor lamellar manipulations

Irrigating Endothelium Stripper (K7-5897)

Katena

Stripping and removal of endothelial layer

Optic Zone Marker 8 mm (K3-8150) 9 mm (K3-8154) Katena

Marking recipient cornea

Rosenwasser Forceps

Katena

For donor corneal disk

Steinert Forceps

Rhein

For donor corneal disk

Ambati Forceps

Rhein

For donor corneal disk

ASICO Inc., Westmont, IL, USA; Moria Inc., Antony Cedex, France; DORC Inc., The Netherlands; Katena Products Inc. (Denville, NJ); Rhein Medical Inc., Tampa, FL.

A

B

C

Figures 11-1A to C: (A) John Dexatome DSAEK Spatula (ASICO, Westmont, IL; AE-2872) (Patent Pending); (B) Higher magnification of the John Dexatome DXEK/DSAEK Spatula (ASICO, Westmont, IL; AE-2873) (Patent Pending); (C) Unique design of the John Dexatome DSAEK Spatula is displayed. The tip of this instrument is clearly seen in this magnified image.

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Figure 11-2: Intraoperative photographs (surgeon’s view, temporal approach) showing the clockwise direction of movement using the John Dexatome DSAEK Spatula (ASICO Inc., Westmont, IL) (Patent Pending) in initiating the Descemetorhexis (DX). The single anterior chamber (AC) entry wound is located in the lower right region of the photographs. This sequence completes one-half of the DX.

Figure 11-3: Intraoperative photographs (surgeon’s view, temporal approach) showing the counter-clockwise direction of movement using the John Dexatome DSAEK Spatula (ASICO Inc., Westmont, IL) (Patent Pending) in completing the Descemetorhexis (DX). The single anterior chamber (AC) entry wound is located in the lower right region of the photographs. This sequence completes second-half of the DX. Currently, John Dexatome is the only surgical instrument that allows for easy completion of the 360 degrees DX from a single AC entry wound using an instrument design that is in keeping with against-the-line-of gravity surgery (ALGS). It is also the only instrument currently available to easily remove the DM without touching the inner stromal surface of the central cornea, since the instrument is in contact only with the folded DM, thus leaving the central corneal surface pristine, contributing to a superior corneal interface and improved vision.

available commercially that will easily permit contact with all areas of the inner corneal dome with a single entry wound. John DSAEK Dexatome spatula is the only instrument that is currently available with which the DM can be removed as a single disk without scraping or touching the inner, central, stromal surface of the patient’s cornea. This is possible since the John Dexatome spatula is only in contact with the folded DM during the removal process (Figure 11-20), similar to rolling a carpet without touching the floor on which the carpet rests. Since there is no contact with the central stromal surface, it results in the best stromal interface and thus facilitates optimal postoperative vision for the patient.

John DSAEK Descemet’s Stripper Identification of surgical step: Complications with DM and endothelium removal. Difficulty encountered: Complications include, irregular tears in DM especially in failed cornea grafts, creation of unwanted stromal strands, inability to consistently remove DM as a single disk due to scarring from penetrating keratoplasty (PKP), and having to enter AC more than once to complete the 360 degree DM tear. Need three instruments to remove DM, namely, the RSH and two different strippers.

Main benefit: In these more difficult cases such as a failed corneal graft, the use of a John DSAEK Descemet’s Stripper (ASICO Inc., Westmont, IL, AE-2874) (Patent Pending) (Figures 11-5A and B) will facilitate the procedure and it requires only a single AC entry to remove the DM as a single disk from patient’s cornea. Usually need only one instrument to remove the DM disk, namely, John Dexatome (see above). However, in complicated cases like multiple failed PK with scarring, John Descemet’s stripper, John Super-microforceps and John Super-microscissors (Table 11-1) will facilitate smooth and sucessful completion of the procedure.

John Retrocorneal Super Micro Forceps Identification of surgical step: Complications related with DM and endothelium removal. Difficulty encountered: Very difficult to remove fragments of DM without damaging the stroma. Main benefit: Very “user-friendly” instrument, namely, John Super Micro Forceps (ASICO Inc., Westmont, IL, AE-4962) (Figures 11-6A and B) to complete the removal of the DM and endothelium especially in the regions of corneal stromal scarring after a PKP.

New/Useful Surgical Instruments in DSAEK

Figure 11-4: Intraoperative photographs showing complete detachment and removal of DM as a single disk using the John Dexatome (ASICO, Westmont, IL; AE-2872) (Patent Pending).

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A B

C Figures 11-7A to C: (A) John Retrocorneal Super Micro Scissors (ASICO, Westmont, IL; AE-5762) is displayed; (B&C) –Shows a higher magnification of the closed (B) and open (C) profile of the John Retrocorneal Super Micro Scissors.

John DSAEK Stromal Scrubber

B Figures 11-5A and B: (A) John DSAEK Descemet’s Stripper (ASICO, Westmont, IL; AE-2874) (Patent Pending); (B) Unique design of the John DSAEK Stripper is displayed. The tip of this instrument has a horizontal T-Bar that is clearly seen in this magnified image.

A

B Figures 11-6A and B: (A) John Retrocorneal Super Micro Forceps (ASICO, Westmont, IL; AE-4962) is displayed. Round, smooth handle allows for side-to-side rotation of the super-microforceps for holding, pulling and retracting tissues at different angles within the anterior chamber (AC) at the retrocorneal plane. Additionally, it permits the holding of tissues within the AC, in a closed system; (B) Magnified image of the tip of the John Retrocorneal Super Micro Forceps.

John Retrocorneal Super Micro Scissors

Identification of surgical step: To increase the adhesion of the donor corneal disk to the patient’s cornea and thus to decrease the rate of donor detachment. Difficulty encountered: RSH was used to roughen the peripheral exposed stroma within the area of the Descemetorhexis of the patient’s cornea. However, it was difficult to use, since it was not designed to work on the ceiling of the cornea. Main benefit: John DSAEK Stromal Scrubber (ASICO Inc., Westmont, IL, AE-2878) (Patent Pending) (Figures 11-8 to 11-10) is easy to use, and effectively roughens the inner corneal stroma within the circle of the Descemetorhexis in the inner surface of the patient’s cornea.

John DSAEK Inserting Forceps (AE-4227) Identification of surgical step: Insertion of taco-folded donor corneal disk into the recipient AC.

A

Identification of surgical step: Complications related with DM and endothelium removal. Difficulty encountered: Very difficult to remove fragments of DM without damaging the stroma. Main benefit: Very “user-friendly” instrument, namely, John Super Micro Scissors (ASICO Inc., Westmont, IL; AE-5762) (Figures 11-7A to C) to complete the removal of the DM and endothelium especially in the regions of corneal stromal scarring after a PKP. The round, smooth handle of this instrument allows for side-to-side rotation for cutting at different angles within the AC at the retrocorneal plane without damaging the adjacent tissues. It allows for cutting of the back surface of the cornea without making a large limbal opening in to the AC. Additionally, it works in a closed system within the AC.

B Figures 11-8A and B: (A) John DSAEK Stromal Scrubber (ASICO Inc., AE-2878) (Patent Pending); (B) Higher magnification image of John DSAEK Stromal Scrubber.

New/Useful Surgical Instruments in DSAEK

Figure 11-9: Intraoperative photographs displaying the profile of John DSAEK stromal scrubber (ASICO, Inc., Westmont, IL) (Patent Pending).

Figure 11-10: Photographs displaying the intraoperative use of John DSAEK Stromal Scrubber (ASICO, Inc., Westmont, IL) (Patent Pending).

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Corneal Endothelial Transplant Difficulty encountered: Kellman Forceps holds the folded disk in the horizontal plane and the hand must be rotated in or out for insertion and release of the taco-folded disk within the recipient AC. Other forceps with tissue clearance are also oriented to hold the disk in the horizontal plane. Also, the blades of these forceps release the disk less easily, especially when the AC is not deep enough for this maneuver.

A

B

C

Figures 11-11A to C: (A) John DSAEK Inserting Forceps (ASICO, Westmont, IL; AE-4227) is shown; (B) Higher magnification of John DSAEK Inserting Forceps (ASICO, Westmont, IL; AE-4227) with the tips open, displaying the holding platforms that are oriented 90o to the handle of this instrument to facilitate easy insertion of the folded donor taco-disk into the recipient anterior chamber; (C) Higher magnification of John DSAEK Inserting Forceps (ASICO, Westmont, IL; AE-4227) with the tips closed.

Main benefit: John DSAEK Insertion Forceps (ASICO Inc., Westmont, IL, AE-4227) (Figures 11-11 to 11-13) has vertically oriented platforms to hold the folded donor disk without any significant damage to the donor endothelium. No hand rotation is required. The special design of this forceps allows for more natural, smooth, “fluid-motion” of inserting the folded donor disk into the AC without causing any iris or pupillary damage.

John DSAEK Fixation Hook Identification of surgical step: Fixation of the donor corneal disk prior to unfolding the donor disk within the recipient AC.

Figure 11-12: (Top Left) Small amount of Healon being placed on the endothelial surface of the donor corneal disk before folding the disk; (Top Right and Bottom Left) The donor corneal disk is being folded into a 60/40 tacofold; (Bottom Right) The taco-folded donor corneal disk is held with the John DSAEK Inserting Forceps AE-4227, without any significant damage to the donor endothelium.

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Figure 11-13: Because of the vertically oriented handles and the horizontal platforms of the John DSAEK Insertion Forceps, the taco-folded donor corneal disk can be inserted into the recipient anterior chamber without the surgeon needing to change hand positions.

Difficulty encountered: RSH is difficult to use, as it was not designed to work on the ceiling of the recipient cornea. Main benefit: John DSAEK Fixation Hook (ASICO Inc., Westmont, IL, AE-2182) (Patent Pending) (Figures 11-14 to 1117) has a retractable central cable which allows for retracting the hook after use. It can also be used on the outer corneal surface to explore the slits “venting incisions”used to enhance donor disk attachment, and drain fluid from the donor-host interface.

A

John DSAEK Glider Identification of surgical step: Removal of wrinkles of folds in the donor corneal disk after attachment of the donor corneal disk to the inner surface of the recipient cornea. Difficulty encountered: Lindstrom Roller is designed for use with LASIK surgery. It has been used for DSAEK, however, it lacks curvature along the horizontal axis which creates the need for multiple rolls. Main benefit: John DSAEK Glider (ASICO Inc., Westmont, IL, AE-2879) (Patent Pending) (Figures 11-18 and 11-19) is specifically designed for use in DSAEK surgery. Its unique

B Figures 11-14A and B: (A) John DSAEK Fixation Hook (ASICO, Westmont, IL; AE-2182) (Patent Pending); (B) Close-up view of the tip of John DSAEK Fixation Hook (ASICO, Westmont, IL; AE-2182) (Patent Pending) showing the retractable central cable which allows for retracting the hook after use within the recipient anterior chamber.

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Figure 11-15: Use of John DSAEK Fixation Hook (ASICO, Westmont, IL; AE-2182) (Patent Pending) to fixate the donor disk against the inner surface of the recipient cornea and a 30-gauge cannula is used simultaneously to inject a steady stream of filtered-air to fully unfold the donor disk within the recipient anterior chamber.

Figure 11-16: Magnified view of the use of the John DSAEK Fixation Hook (ASICO, Westmont, IL; AE-2182) (Patent Pending) to fixate the donor disk against the inner surface of the recipient cornea and unfolding the fixated donor disk with an air-bubble.

Figure 11-17: After unfolding of the donor corneal disk, the John DSAEK Fixation Hook’s inner cable is retracted and withdrawn from the anterior chamber (ASICO, Westmont, IL; AE-2182) (Patent Pending); Insert Photo – Shows the fully unfolded donor corneal disk that is attached to the inner surface of the host cornea.

New/Useful Surgical Instruments in DSAEK A

117

design and curvatures allows for corneal contour-oriented contact between the outer corneal dome and the inner surface of the Glider. All regions of the outer corneal surface can be gently and smoothly “ironed” to diminish and/or eliminate donor disk wrinkles at the donor-host interface.

John DSAEK Marker (8 mm/9 mm) Identification of surgical step: Marking the corneal surface of the recipient cornea with a fixed diameter of either 8 mm or 9 mm diameter. Difficulty encountered: Using the disposable trephine to mark the recipient corneal surface can be difficult especially in a deep set eye and can result in an eccentric mark. B Figures 11-18A and B: (A) John DSAEK Glider (ASICO, Westmont, IL; AE-2879) (Patent Pending); (B) Higher magnification of the John DSAEK Glider.

Main benefit: John DSAEK Marker (ASICO Inc., Westmont, IL, AE-2712) (Figure 11-20) facilitates easy marking of the patient’s corneal surface with or without epithelium, using either end of the single instrument for a fixed diameter of

Figure 11-19: Intraoperative photographs of the John DSAEK Glider (ASICO, Westmont, IL; AE-2879) (Patent Pending) being used on the recipient outer corneal surface to remove the wrinkles in the donor corneal disk that is attached to the recipient cornea.

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Figure 11-20: Intraoperative photographs showing the John DSAEK Corneal Marker 8 mm / 9 mm diameter (ASICO, Westmont, IL) being used to mark the patient’s corneal epithelium. The descemetorhexis is performed within this circular mark using a single entry into the anterior chamber and complete detachment and removal of the Descemet’s membrane as a single disk is performed by using the John DSAEK Spatula (ASICO, Westmont, IL; AE-2872) (Patent Pending).

A

8 mm or 9 mm. This instrument helps in good centration and eliminates eccentric marks on the cornea. The Descemetorhexis is carried out within this circular mark (Figure 11-20).

Other DSAEK Instruments The other instruments for DSAEK made by Moria, Inc. (Antony Cedex, France), DORC, Inc. (The Netherlands), Katena Products Inc. (Denville, NJ); and Rhein Medical Inc. (Tampa, FL) are listed in Table 11-1 and Figure 11-21.

Conclusion B Figures 11-21A and B: (A) Busin Glide (Moria Inc., Antony Cedex, France) (Photo: Courtesy, Moria Inc.); (B) Intraoperative photograph of Busin Glide (Moria Inc., Antony Cedex, France), used to introduce the donor corneal disk into the recipient anterior chamber (Photo: Courtesy, Moria, Inc.).

In conclusion, this chapter has provided an in-depth listing and review of the types of surgical instruments for DSAEK surgery that are currently available commercially. In addition, intraoperative photographs display the use of some of these instruments. The surgeon has to choose his surgical instruments to facilitate the optimal DSAEK

New/Useful Surgical Instruments in DSAEK surgery thus providing a consistently good surgical outcome and best possible visual outcome for his patients.

References 1. John T. DXEK surgery, corneal disk detachment (Consultation Section). Ann Ophthalmol 2006;38:169-84. 2. John T. Selective tissue corneal transplantation: a great step forward in global visual restoration (Editorial) 2006;1:5-7. 3. John T (Ed.): Surgical Techniques in Anterior and Posterior Lamellar Corneal Surgery. Jaypee Brothers Medical Publishers (P) Ltd., New Delhi, India, 1-687. 4. John T (Ed.): Step by Step Anterior and Posterior Lamellar Keratoplasty, Jaypee Brothers Medical Publishers (P) Ltd., New Delhi, India, 1-297. 5. Melles GR, Eggink FA, Lander F, Pels E, Rietveld FJ, Beekhuis WH, Binder PS. A surgical technique for posterior lamellar keratoplasty. Cornea 1998;17:618-26. 6. Terry MA, Ousley PJ. Endothelial replacement without surface corneal incisions or sutures: topography of the deep lamellar endothelial keratoplasty procedure. Cornea 2001;20:14-18. 7. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty in the first United States patients: early clinical results. Cornea 2001;20:239-43. 8. Terry MA, Ousley PJ. Replacing the endothelium without corneal surface incisions or sutures: the first United States clinical series using the deep lamellar endothelial keratoplasty procedure. Ophthalmology 2003;110:755-64. 9. Terry MA, Ousley PJ. Rapid visual rehabilitation after endothelial transplants with deep lamellar endothelial keratoplasty (DLEK). Cornea 2004;23:143-53. 10. Terry MA, Ousley PJ. Corneal endothelial transplantation:

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21.

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advances in the surgical management of endothelial dysfunction. Contemporary Ophthalmology 2002;1(26):1-8. Terry MA, Ousley PJ. Small-incision deep lamellar endothelial keratoplasty (DLEK): six-month results in the first prospective clinical study. Cornea 2005;24:59-65. Ousley PJ, Terry MA. Stability of vision, topography, and endothelial cell density from 1 year to 2 years after deep lamellar endothelial keratoplasty surgery. Ophthalmology 2005; 112:50-57. John T. Stitchless corneal transplantation. Cataract and Refractive Surgery Today 2004;4(8):27-30. Melles GRJ, Wijdh RHJ, Nieuwendaal CP. A technique to excise the Descemet membrane from a recipient cornea (Descemetorhexis). Cornea 2004;23:286-8. Melles GRJ, Lander F, Rietveld FJR. Transplantation of Descemet’s membrane carrying viable endothelium through a small scleral incision. Cornea 2002;21:415-8. John T. Use of indocyanine green in deep lamellar endothelial keratoplasty. J Cataract Refract Surg 2003;29:437-43. Melles GR. Posterior lamellar keratoplasty: DLEK to DSEK to DMEK. Cornea. 2006;25:879-81. Price FW Jr, Price MO. Descemet’s stripping with endothelial keratoplasty in 200 eyes: Early challenges and techniques to enhance donor adherence. J Cataract Refract Surg. 2006;32: 411-8. Price MO, Price FW. Descemet’s stripping endothelial keratoplasty. Curr Opin Ophthalmol 2007;18:290-4. Price MO, Price FW. Descemet stripping with endothelial keratoplasty for treatment of iridocorneal endothelial syndrome. Cornea 2007;26:493-7. Mearza AA, Qureshi MA, Rostron CK. Experience and 12-month results of descemet-stripping endothelial keratoplasty (DSEK) with a small-incision technique. Cornea 2007;26:279-83.

Thomas John

Artificial Anterior Chambers

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Introduction The creation of an artificial anterior chamber (ACC)1 is a major milestone in the field of corneal surgery. This introduction of the ACC is changing the surgical landscape of corneal transplantation to a more favorable, and evolving field of lamellar corneal surgery. The pre-ACC era dealt with utilizing whole globes or corneoscleral button that was wrapped tightly around a glass orbital implant for the lamellar graft dissection.2 Issues during this era included difficulties in obtaining whole globes, stabilization of the whole globe for lamellar dissection, and less than optimal instrumentation. Artificial anterior chamber was first described by Ward and Nesburn in 1976.1 They described a way to trephine the donor cornea from the anterior surface when the donor corneal-scleral tissue was mounted on an instrument that formed a seal around the scleral rim of the excised donor cornea, allowing the endothelium to be supported physically by the liquid storage medium.1 Hence, the artificial anterior chamber protected the donor endothelial cells from damage as if they were still in the intact globe.1 Although it was thought to provide better donor-recipient match for full-thickness penetrating keratoplasty (PKP), since both the donor and the recipient corneas were trephined from the anterior corneal surface with the same diameter trephine, the use of such an ACC extended across boundaries from PKP to lamellar keratoplasty (LKP). Since the initial description, various modifications and improvements in the ACC has been made giving ophthalmic surgeons the ability to trephine to any desired donor corneal depth.2-16 Artificial anterior chambers may be utilized for both manual lamellar dissection, or automated lamellar dissection of the donor cornea using a microkeratome and the Moria ALTK system (Moria SA, Antony, France). This chapter provides an introduction to the various AAC that are currently available in the United States at the time of this writing.

Surgical Objective Surgical objective is to obtain a lamellar corneal disk of the desired thickness, diameter, an even surface with an uniplanar cut to augment the donor-host corneal interface, ease of operation, and avoidance of disk perforation.

Types of AAC 1. Reusable ACC 2. Disposable ACC

Reusable ACC Reusable ACC as the name suggests can be used repeatedly provided care is taken not to damage the ACC unit by improper handling.

Types a. Moria ACC (manual), and Moria ALTK system (automated with microkeratome) b Bausch and Lomb ACC (manual).

Moria ACC Moria ACC utilizes the Evolution3 console (Moria, Inc.) that is fully compatible with all Moria microkeratomes including the LSK, M2, and the CB units. Currently the CB microkeratome is the unit that is provided for the Moria ALTK system. The Evolution3 console has built-in safety features and provides ease of use for the surgeon and operating room staff. It runs on wall current and has a battery back-up for uninterrupted use. It has two high pumps that provide a quick and stable vacuum for the procedure. The Moria unit for lamellar surgical procedures is called the ALTK system. The Moria ALTK system can be used for anterior lamellar keratoplasty (ALK) (extra-ocular procedure) or for deep lamellar endothelial keratoplasty (DLEK) [See also Section 8, Deep Lamellar Endothelial Keratoplasty (DLEK)] or for Descemet Stripping Automated Endothelial Keratoplasty (DSAEK) [See also Section 9, Descemet Stripping Automated Endothelial Keratoplasty (DSAEK)]. The indications for these procedures are listed in the respective chapters. The donor corneoscleral tissue can be used for ALK using preferably, the automated technique for the best interface (with microkeratome use) or alternatively if the ALTK system is not available in the operating room, then, the Moria ACC can be used for manual dissection of the donor lamellar disc. When using the ACC with manual dissection, the partial thickness anterior lamellar dissection of the donor cornea can be performed after partial trephination to the desired depth using the Moria ACC and the Hanna trephine (Moria, Inc.) that is set to the required depth in microns. The Hanna trephine (Moria, Inc.) seats well on the Moria ACC and allows for stable partial thickness trephination of the donor cornea from the epithelial side. This is followed by manual lamellar dissection of the donor corneal tissue. The same trephine with the same blade can be used on the recipient cornea for the same diameter disk resection as that of the donor lamellar disk. Unlike the Moria ALTK system, with the

Artificial Anterior Chambers Moria ACC there is no control of the amount of corneal tissue that is exposed within the ring. Hence the diameter of tissue resection is determined by the diameter of the blade in the Hanna trephine. The Barron vacuum trephine does not seat optimally on the Moria ACC and hence proper trephination cannot be carried out with the Barron trephine in a Moria ACC. For automated donor corneal lamellar disk, the Moria ALTK system is used that utilizes both the Evolution3 console and the microkeratome described above. The Moria ALTK system utilizes a different design for its ACC (Figure 12-1). This ACC has the capability of raising or lowering the mounted donor corneo-scleral button (Figure 12-1), thus altering the final diameter of the resected donor tissue. The diameter can be approximated by using the applanating lenses. The diameter by measurement on the donor cornea, does not necessarily match exactly with the final resected donor lamellar disk. Hence, it may be better to resect the donor tissue first before using the microkeratome on the recipient cornea.

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Figure 12-2: The Moria ALTK system comprises of an artificial chamber that has a central post on which the donor corneoscleral button is placed with the endothelial side down after injecting Optisol solution from the donor corneal vial.

Figure 12-1: The Moria ALTK system utilizes an ACC that has the capability of raising or lowering the mounted donor corneo-scleral button, thus altering the final diameter of the resected donor tissue.

Figure 12-3: The Moria ACC encasing cylindrical unit with the central opening for the donor cornea.

The Moria ALTK system comprises of an artificial chamber that has a central post (Figure 12-2) on which the donor corneoscleral button is placed with the endothelial side down after injecting Optisol solution from the donor corneal vial. Next the encasing cylindrical unit with the central opening for the cornea (Figure 12-3) is moved into place such that the three posts (Figure 12-3) descend into the corresponding slots in the base of the ACC unit, taking care to maintain the central position of the donor cornea. Turning this encasing unit in a clockwise fashion locks it in place and the donor corneoscleral button is now firmly mounted on to this ACC, with uniform pressure being applied to 360° of the scleral rim. Titrate as needed, the intrachamber pressure by injecting additional fluid from

the attached syringe into the chamber, and locking the valve if the pressure is low, or alternatively aspirate fluid from the chamber if the intrachamber pressure is too high. The pressure can be checked using a Barraquer tonometer as needed. Next depending on whether a LSK or CB microkeratome is used with this unit, the appropriate ring is chosen (Figure 12-4) and screwed on to the unit. The amount of corneal tissue being exposed can now be adjusted by turning the rings and raising or lowering the central post (Figure 12-1). The applanation lenses provide an estimate of the diameter to be resected, ranging from 6 to 11 mm (Figures 12-5A and B). Figure 12-6 is a composite showing the set-up for the LSK microkeratome on the left and for a CB unit on the right. The LSK microkeratome

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Figure 12-4: The ring to the left is used with the Moria LSK microkeratome and the ring to the right is used with the Moria CB microkeratome.

Figure 12-6: Moria ALTK system showing sequential assembly for the Moria LSK microkeratome (left) and the Moria CB microkeratome (right).

A

(Figures 12-7A and B) or a CB microkeratome (Figure 12-8) can be used to complete the lamellar excision of the donor corneal disk. In summary, the 3 steps to the use of Moria ALTK system are as follows (Figure 12-9): 1. First, the donor cornea is sealed within the ACC, and the intrachamber pressure is adjusted to the required level. 2. Second, the surgeon selects the desired diameter of the cut. 3. Lastly, perform the donor corneal resection. The ALTK’s adjustability and versatility reduce the incidence of induced corneal astigmatism. In this system the high-speed, high-power turbine (30,000 cuts/minute) creates a smooth keratectomy for a seamless-edge margin. The single piece construction of the microkeratome heads are pre-calibrated for various depths of cut (130-400 µm).

Bausch and Lomb ACC

B Figures 12-5A and B: (A) Moria ALTK system with applanating lenses ranging from 6.0 to 9.0 mm in diameter. (B) Moria ALTK system showing sequential assembly prior to use.

Bausch and Lomb (B&L) ACC (Figure 12-10) can be used for manual dissection of the donor lamellar corneal disk. It is not designed for use with a microkeratome. The B&L ACC can be used for both DLEK and for anterior lamellar keratoplasty. It consists of a solid metallic rectangular base (Figure 12-10, labeled A), with a central post (Figure 12-10, labeled B) on which the donor corneoscleral rim is placed with the endothelial side down after applying viscoelastic material and fluid from the donor corneal vial. The central post has two openings for egress of fluid used to alter the intrachamber pressure. The metallic base has a circular space

Artificial Anterior Chambers

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A

Figure 12-8: Moria CB microkeratome being used with the Moria ALTK system.

B Figures 12-7A and B: Moria LSK microkeratome being used with the Moria ALTK system.

surrounding the central post (Figure 12-10, labeled C) for collecting the fluid that egresses out of the two openings in the central post. Two metallic channels (Figure 12-10, labeled D) are attached to the central post on one end and to metallic valves on the other end where a syringe filled with fluid can be attached. Once the donor corneoscleral rim is placed on the central post and the intrachamber space is primed with fluid, the circular fixation ring (Figure 12-10, labeled E) is placed over the donor corneoscleral rim and the C-arm (Figure 12-10, labeled F) is moved forwards such that it is above the flange of the fixation ring. Once the

C-arm is in its extended position the screw (Figure 12-10, labeled G) is tightened and this fixates the donor cornea within the B&L ACC. The intrachamber pressure is optimized by injecting fluid from the attached syringe and closing the valve to maintain the chamber pressure. The unit is now ready for lamellar dissection. Unlike the Moria ACC where the fixation pressure on the donor corneoscleral rim is applied 360° directly from the top (Figures 12-1 and 12-3), in the B&L ACC the C-arm applied pressure only on the proximal 180° of the flange of the fixation ring (Figure 12-10, labeled E) and the fixation ring in turn transmits the pressure to the scleral rim. A Barron radial vacuum trephine (Katena, Inc.) of the desired diameter is placed on the moistened, epithelial surface of the donor cornea and suction is applied to fixate the trephine. Partial thickness trephination to the desired corneal depth is followed by lamellar dissection of the donor cornea to obtain a lamellar donor corneal disk [See also Section VIII, Deep Lamellar Endothelial Keratoplasty (DLEK)].

Disposable ACC Barron Disposable ACC (Katena, Inc., Denville, NJ, USA) The Barron ACC (Figures 12-11 and 12-12) is sterile, disposable, and it comprises of three pieces, namely, base with tissue pedestal, tissue retainer, and a locking ring. The base has two parts with silicone tubing, in-line pinch

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A

Corneal Endothelial Transplant

B

C

Figures 12-9A to C: The 3 steps to the use of Moria ALTK system, (A) The donor cornea is sealed within the ACC, and the intrachamber pressure is adjusted to the required level. (B) Surgeon selects the desired diameter of the cut. (C) Perform the donor corneal resection.

Figure 12-10: Bausch and Lomb ACC, (A) Rectangular metallic base, (B) Central post, (C) Excess fluid collects in this space, (D) Two metallic channels with metallic valves for injection or aspiration of fluid to titrate the intrachamber pressure, (E) Fixation ring, (F) C-arm that can be extended or retracted, (G) Screw to tighten the C-arm in place.

Figure 12-12: Barron disposable ACC (Katena, Inc.).

Figure 12-11: Barron disposable ACC (Katena, Inc.).

clamps, and female luer-lock connectors. Either port may be used to inject or aspirate balanced salt solution, solution from the donor corneal vial, viscoelastic or air. This ACC is a companion to the disposable Barron radial vacuum trephine. This ACC allows for trephination from the epithelial side. It is designed to hold a donor cornea (14-18 mm) on a bed of viscoelastic. The bright blue color of the unit provides a high contrast background to view the cornea.

Artificial Anterior Chambers

Useful Surgical Suggestions and Avoiding Complications 1. The corneoscleral rim that is obtained from the eye bank should have adequate scleral rim 360° (see below) for proper housing and fixation within the ACC of any kind. If the scleral rim is short on any one side, this will lead to tissue slippage, loss of intrachamber pressure, fluid leakage from the unit and inability to perform an adequate lamellar dissection of the donor cornea. 2. A second back-up cornea from the local eye bank will be especially in the early learning stage of DSAEK surgery. However, currently most US eye banks charge-a-fee for the second cornea and hence this may not be an option for US surgeons. This will help complete the surgery if the first cornea is damaged during surgical dissection. 3. First dissect the donor cornea before operating on the recipient cornea. 4. If general anesthesia is used for surgery, the donor cornea can be prepared even before the patient is anesthetized. This will reduce the total time that the patient is under general anesthesia. Additionally, it will ascertain that a suitable lamellar corneal disk is ready for use even before the patient is anesthetized. 5. If there is tissue slippage and significant loss of intrachamber pressure, then it may be advisable to abandon the lamellar dissection and use the back-up cornea. Dissecting without proper stabilization of the donor cornea within the ACC, can result in multiplanar dissection, uneven interface, and more importantly, possible perforation of the lamellar disk. 6. If using the Moria ALTK system, check the blade and the microkeratome head before use and lubricate with BSS to limit any mechanical epithelial damage to the donor cornea. 7. If using the Moria ALTK system with the microkeratome, it is preferable that the surgeon has some prior experience with its use. 8. The Barron radial vacuum trephine usually does not fit on a Moria ACC, and hence use the appropriate matching units to facilitate and complete the procedure. A Hanna trephine will fit the Moria ACC and can be used routinely for this purpose. 9. The depth of cut on the Hanna trephine can be set in microns. With the Barron radial vacuum trephine, the depth is set by the number of quarter turns on its post. 10. Suitable lamellar dissection blades and knives should be available for the procedure. 11. In manual dissection, maintain a uniplanar lamellar dissection, by staying in the same plane of dissection

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until the disk is fully excised. This will help in attaining a better donor-host interface. 12. The intrachamber pressure should always be checked before any trephination or lamellar dissection of the donor cornea. Adjust the intrachamber pressure as needed by injecting or aspirating fluid via the attached syringe. 13. Maintain the intrachamber pressure by closing the valve or applying the clamps provided with the unit. 14. The intrachamber pressure may be checked digitally or with the Barraquer tonometer. Inadequate intrachamber pressure will result in potential complications with the donor tissue.

Results and Conclusions We compared three AACs to determine the optimal sizes of donor corneoscleral tissue (DCST).17 Twenty DCSTs were evaluated. The AACs tested were Bausch and Lomb, Moria ACC, and Moria ALTK. Twenty DCSTs were evaluated. The scleral skirt was measured from the limbus to the cut edge at 4 cardinal points (mean rim size, 3.14±0.61 mm; range, 2.07-4.19). DCSTs were mounted in the AACs, a target pressure of 65 mm Hg was set using Barraquer tonometer. Tissue slippage with seal rupture before reaching the target pressure was considered a failure. The mean scleral rim sizes that maintained a seal and failed to maintain a seal, respectively, were 3.4± 0.7 mm (range, 2.1-4.2) and 2.99 ± 0.51 mm (range, 2.13-3.5). Using Optimal Data Analysis (ODA) techniques, we found that the DCST should have a minimal scleral skirt of 3.6 mm for successful use in an AAC for lamellar or penetrating keratoplasty (epithelial approach).17 In summary, the introduction of these AAC into the field of corneal surgery has propelled further forward the field of lamellar corneal surgery and this momentum is expected to continue over time. Eye bank awareness and standardization of the required scleral rim size for lamellar corneal surgery are essential for successful outcome of the donor corneal tissue dissection and transplantation.

Acknowledgment The author thanks Moria, Inc., and Katena, Inc., for providing figures for this chapter.

References 1. Ward DE, Nesburn AB. An artificial anterior chamber. Am J Ophthalmol 1976;82:796-8. 2. Wong DW, Chan WK, Tan DT. Harvesting a lamellar graft from a corneoscleral button: A new technique. Am J Ophthalmol 1997;123:688-9.

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3. Ward DE, Nesburn AB. A modified artificial anterior chamber for use in refractive keratoplasty. Am J Ophthalmol 1980; 89: 742. 4. Beherens A, Dolorico AMT, Kara DT, et al. Precision and accuracy of an artificial anterior chamber system in obtaining corneal lenticules for lamellar keratoplasty. J Cataract Refract Surg 2002; 288:860-5. 5. Behrens A, Ellis K, Li L, Sweet PM, Chuck RS. Endothelial lamellar keratoplasty using an artificial anterior chamber and a microkeratome. Arch Ophthalmol 2003; 121:503-8. 6. Springs CL, Joseph MA, Odom JV, Wiley LA. Predictability of donor lamellar graft diameter and thickness in an artificial anterior chamber system. Cornea 2002; 21:696-9. 7. Behrens A, Dolorico AM, Kara DT, Novick LH, McDonnell PJ, Chao LC, Wellik SR, Chuck RS. Precision and accuracy of an artificial anterior chamber system in obtaining corneal lenticules for lamellar keratoplasty. J Cataract Refract Surg 2001; 27: 1679-87. 8. Ignacio TS, Nguyen TT, Sarayba MA, Sweet PM, Piovanetti O, Chuck RS, Behrens A. A technique to harvest Descemet’s membrane with viable endothelial cells for selective transplantation. Am J Ophthalmol 2005;139:325-30. 9. Wiley LA, Joseph MA, Springs CL. Tectonic lamellar keratoplasty utilizing a microkeratome and an artificial anterior chamber system. Cornea 2002; 21:661-3. 10. Azar DT, Jain S, Sambursky R, Staruss L. Microkeratome-

11. 12.

13. 14. 15. 16.

17.

assisted posterior keratoplasty. J Cataract Refract Surg 2001; 27:353-6. Buratto L. Globe holder and artificial anterior chamber. Refract Corneal Surg 1990; 6:205-6. Maguen E, Azen SP, Pinhas S, Villasenor RA, Nesburn AB. Evaluation of sources of variation on the accuracy and reproducibility of microkeratome sections with the modified artificial anterior chamber. Ophthalmic Surg 1982; 13:217-20. Maguen E, Villasenor RA, Ward DE, Nesburn AB. A modified artificial anterior chamber for use in refractive keratoplasty. Am J Ophthalmol 1980; 89:742-4. Springs CL, Joseph MA, Odom JV, Wiley LA. Predictability of donor lamellar graft diameter and thickness in an artificial anterior chamber system. Cornea 2002; 21:696-9. Wolter JR, Kunkel SL. Artificial anterior chamber made of rigid PMMA contact lenses. CLAO J 1985; 11:107-12. Li L, Behrens A, Osann KE, Sweet P, Chuck RS. Corneal lenticule harvest using a microkeratome and an artificial anterior system at high intrachamber pressure. Journal of Cataract and Refractive Surgery 2002; 28:860-5. John T, Selvadurai D, Ruszkowski E, Pivoney CJ, McCoy K: Evaluation of three artificial anterior chambers using donor human corneoscleral explants. Presented at the Annual Meeting of The Cornea Society and Eye Bank Association of America, Federated Scientific Session Meeting, New Orleans, LA, October 23, 2004.

Definition, Terminology and Classification of Lamellar Corneal Surgery

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Thomas John, Enrique S Malbran

Definition, Terminology and Classification of Lamellar Corneal Surgery

13

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Introduction A new terminology that I (TJ) introduced is called selective tissue corneal transplantation (STCT)1-4 that describes a new concept and, perhaps, the future direction of the field of corneal replacement surgery. STCT is defined as the selective removal of the diseased portion of the patient’s cornea and its replacement with anatomically similar healthy donor corneal tissue.2 Hence, in cases where the corneal opacities are confined to within the corneal stroma, with healthy corneal endothelium, then, only that portion of the cornea with the opacity is removed, leaving behind the remainder of the patient’s cornea. Such lamellar corneal surgery preserves the patient’s corneal endothelium and thus eliminates the possibility of corneal endothelial graft rejection. This is of great benefit to the patient of all ages. However, in cases of endothelial decompensation with corneal edema without associated corneal opacities or with only mild corneal opacity that does not involve the visual axis, then descemetorhexis (DX) with endokeratoplasty (EK) (DXEK) which is synonymous with Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)1-4 may be the preferred surgical choice, thus maintaining the patient’s corneal integrity without any corneal wound or corneal sutures. Lastly, if there is a full-thickness corneal scar that involves the endothelium, then the standard penetrating keratoplasty (PKP) will be required for patient’s visual rehabilitation. The term DX was first introduced by Sinha et al in 2003 to describe the excision of retained Descemet’s membrane (DM) following a PKP.5 They rationalized that both the lens capsule and DM are basement membranes, and since the term capsulorhexis is commonly used in cataract surgery, the term DX is appropriate for the DM and called it DX. Subsequently, others6,7 have used a similar technique to excise DM from a recipient cornea. The term EK was first used by Busin et al8 in flap-associated posterior lamellar keratoplasty (PLK) (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). EK refers to transplantation of a thin donor corneal disk comprising of donor stroma, DM, and a functional, healthy endothelium to the recipient’s cornea [See Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)].1 Based on the above descriptions, the term DXEK appears to be well suited for this type of corneal surgery. Other terms that describe the same surgical procedure include, Descemet’s stripping with endothelial keratoplasty (DSEK) and Descemet’s stripping automated endothelial keratoplasty (DSAEK).1 Since, the term DSAEK is well established, to avoid confusion, in this chapter the term DSAEK will be used.

Currently, full-thickness penetrating keratoplasty (PKP) is the gold standard worldwide for corneal replacement surgery. However, lamellar keratoplasty (LKP) is rapidly gaining popularity among corneal surgeons all over the world. Improved instrumentation (See also Chapter 11, New/ Useful Surgical Instruments in DSAEK), refinements in microsurgical techniques [See also Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)], availability of new artificial anterior chambers (See also Chapter 12, Artificial Anterior Chambers), and microkeratomes seem to have forward propelled this renewed interest in LKP to greater heights. Additionally, anterior LKP (ALK) for the most part is an extraocular, non-open-sky, less invasive procedure than a PKP and it avoids intraocular complications such as endophthalmitis, glaucoma, iatrogenic cataract formation, anterior synechiae, and iris prolapse.9-14 In contrast posterior lamellar keratoplasty (PLK) is an intraocular procedure. With the renewed interest in LKP there have been various terminologies that have been introduced into the medical literature that is often confusing to the ophthalmic surgeons. Some of these terminologies and abbreviations include, lamellar keratoplasty (LK), 15 deep lamellar anterior keratoplasty (DLAK), 15 deep lamellar endothelial keratoplasty (DLEK),15 deep stromal anterior lamellar keratoplasty (DSALK), 16 deep lamellar keratoplasty (DLKP),17 deep lamellar keratoplasty (DLK),18 deep anterior lamellar keratoplasty (DALK),19 and posterior lamellar keratoplasty (PLK).2° All of these varied terminologies refer to the recipient cornea.

Donor Cornea Another equally important area to be focused upon when dealing with LKP, is the thickness of the donor cornea. Most lamellar corneal surgeons are well aware that the donor corneal disk thickness can vary. The corneal thickness in: (a) anterior superficial lamellar resection is usually less than 160 µm (ALR), (b) mid-stromal lamellar resection (MSR) is 160 – 400 µm, and (c) almost fullthickness donor graft (FTDG), usually devoid of endothelium following Hallerman’s original idea,21-23 and later on adopted by many others.1,24-33 This chapter addresses these issues regarding LKP terminology and presents a unified, easy to use comprehensive classification for both anterior and posterior lamellar keratoplasty, for standardization purposes. These classifications for optical LKP, are named as “John-Malbran ALK Classification” (Figure 13-1) and “John PLK Classification”(Figure 13-2).

Definition, Terminology and Classification of Lamellar Corneal Surgery

Figure 13-1: Schematic representation of John-Malbran anterior lamellar keratoplasty classification of optical lamellar keratoplasty. SALK – superficial anterior lamellar keratoplasty, MALK – mid anterior lamellar keratoplasty, DALK – Deep anterior lamellar keratoplasty, TALK – Total anterior lamellar keratoplasty.

Figure 13-2: Schematic representation of John posterior lamellar keratoplasty classification of optical lamellar keratoplasty. DLEK - Deep lamellar endothelial keratoplasty, without flap – no surface wound or sutures, FDLEK - Flap-associated DLEK, with flap – surface wound and sutures present, DXEK - Descemetorhexis with endokeratoplasty (Synonymous term – DSAEK), DECT - Descemet’s membrane endothelial cell transplantation (Synonymous term – DMEK), ECT - Endothelial cell transplantation, ECA - Endothelial cell activation. FDLEK – Previous terminologies included endokeratoplasty, and endothelial replacement flap approach. ECT and ECA have not been established as a surgical procedure at the present time (at the time of writing this chapter).

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Outline • • • • • •

that are similar to the excised recipient corneal disk is used to complete the surgery.

Definition Classification of LKP Indications for LKP Differences between ALK and PLK Wound architecture in LKP General comments.

Posterior Lamellar Keratoplasty (PLK)

Definition Lamellar Keratoplasty LKP consists of partial thickness corneal tissue replacement.

Anterior Lamellar Keratoplasty (ALK) ALK is defined as varying amounts of anterior corneal tissue replacement with retention of the recipient Descemet’s membrane and endothelium. ALK violates the Bowman’s layer (Table 13-1). ALK in general would include superficial-ALK (SALK), mid-ALK (MALK), deep-ALK (DALK), and total-ALK (TALK) (Table 13-2). All of these procedures relate to the thickness of the excised recipient cornea. A matching donor corneal thickness with a matching donor corneal layers TABLE 13-1: Differences between anterior lamellar keratoplasty (ALK), posterior lamellar keratoplasty (PLK), and penetrating keratoplasty (PKP)

This Procedure Violates: Type of procedure Bowman’s layer

Descemet’s membrane

ALK

++

—

PLK

—

++

PKP

++

++

PLK is defined as any corneal lamellar procedure where the Descemet’s membrane and/or endothelium are excised with or without host corneal stroma. PLK usually violates the Descemet’s membrane (Table 13-3). PLK includes the following: • Deep lamellar endothelial keratoplasty (DLEK) • Flap-associated DLEK (FDLEK) • Descemet Stripping Automated Endothelial Keratoplasty (DSAEK)6,34 DSAEK [See Section 9, Descemet’s Stripping Automated Endothelial Keratoplqasty (DSAEK)] involves removal of the recipient Descemet’s membrane and endothelium and transplantation of a donor corneal disk that is about 150 µm thick and having a diameter of about 8.0 mm or 9.0 mm and rarely, 7.0 mm. This donor corneal disk comprises of deep donor corneal stroma with attached donor DM and endothelium.6,34 • Descemet membrane endothelial keratoplasty (DMEK) • Descemet membrane automated endothelial keratoplasty (DMAEK) • Endothelial cell transplant (ECT) (currently not possible) • Endothelial cell activation (ECA)(currently not possible)

Penetrating Keratoplasty (PKP) PKP is full-thickness corneal replacement procedure. PKP violates both the Bowman’s and Descemet’s membrane (Table 13-1). We define the difference between anterior and posterior LKP as the presence or absence of recipient Bowman’s layer. In ALK, Bowman’s layer is always violated and absent, while in PLK, Bowman’s layer is present (Table 13.1).

++: Yes; —: No TABLE 13-2: Types of procedures included under: (A) anterior lamellar keratoplasty (ALK) and posterior lamellar keratoplasty (PLK) ALK PLK • • • •

SALK – Superficial-ALK MALK – Mid-ALK DALK – Deep- ALK TALK – Total- ALK

• DLEK – Deep Lamellar Endothelial Keratoplasty • FDLEK – Flap-associated Deep Lamellar Endothelial Keratoplasty • Descemet Stripping Automated Endothelial Keratoplasty (DSAEK) (Synonymous term—DXEK) –Manual –Microkeratome assisted –Femtosecond laser assisted (Removal of recipient DM by descemetorhexis and transplantation of donor disk comprising of deep stroma, DM and endothelium) • EK • DMEK • DMAEK • ECT • ECA DSEK—Descemet stripping endothelial keratoplasty; DSAEK—Descemet stripping automated endothelial keratoplasty; EK—Endothelial keratoplasty; DMEK—Descemet membrane endothelial keratoplasty; DECT—Descemet’s membrane endothelial cell transplantation; ECT—Endothelial cell transplantation; DMEK (Synonymous term—DECT); DMAEK—Descemet membrane automated endothelial keratoplasty; ECA—Endothelial cell activation; ECT and ECA are not established surgical procedure at present.

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Table 13-3: Differences between anterior and posterior lamellar keratoplasty

Features

Anterior lamellar keratoplasty (ALK)

Posterior lamellar keratoplasty (PLK)

Host endothelial-DM-complex

++

Host Bowman’s layer

—

Usually –

Host Descemet’s membrane

++

Endothelial graft rejection/failure

—

++

Surface wound and sutures

++

— DSAEK, DMEK, and in flapless DLEK

Induced surgical astigmatism

++

— DSAEK, DMEK, and in flapless DLEK*

++ Usually –

Location of host-donor interface

Any level: • superficial in SALK • mid in MALK • deep pre-Descemetic in DALK • adjacent to DM in TALK

Deeper level: • Deeper corneal stroma in DLEK, FDLEK • At the level of Descemetorhexis in DSAEK, Femtosecond laser-assisted DSEK, and DMEK

Increased total corneal thickness

Sometimes

DLEK – sometimes FDLEK – sometimes DSAEK – always DMEK - No

Risk of wound dehiscence compared to PKP

Decreased

Decreased

Recipient manual corneal dissection

Yes

Yes for DLEK

Recipient automated dissection with microkeratome

Yes for SALK, MALK No for DALK, TALK

Yes for FDLEK No for flapless DLEK, DSAEK and DMEK

Can use donor cornea with defective endothelium that is rejected for PKP

Yes

No

Intraoperative complications

Rare in SALK, MALK, DALK; sometimes in TALK

More than in ALK More in FDLEK than DLEK

Risk of AC entry

No – SALK, MALK Yes – DALK, TALK

Yes, required for the procedure

Need for good to excellent quality donor corneas with healthy endothelium

No

Yes

Relatively simple surgical technique

Yes for SALK and MALK No for DALK and TALK

No

Long term topical immunosuppressive drops (Prednisolone acetate 1%)

No

Yes

Easy postoperative care

Yes

Yes

Superior wound strength compared to PKP

Yes

Yes

Surgical, technical difficulty

Less (except TALK)

More

DM: Descemet’s membrane; ++: Present; —: Absent; PKP: Penetrating keratoplasty; *: Absent or minimal astigmatism after flapless DLEK

Classification of LKP General Classification Based on Type 1. 2. 3. 4.

Optical Tectonic Therapeutic Cosmetic.

Based on Location 1. Central

2. Peripheral—Circular, oval, crescentic, annular, semilunar, rectangular or strip graft 3. Total—central and peripheral 4. Corneoscleral a. Total corneoscleral LKP b. Partial corneoscleral LKP—Circular, crescentic, annular, or strip graft. Central are usually round, but peripheral can adopt different configurations (see above)

Based on Stem-cell Transplantation 1. Non-stem cell LKP 2. LKP with stem cell transplantation (SCT)

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Corneal Endothelial Transplant

1. LKP only 2. LKP combined with PKP

Substitution LKP: In substitution-LKPs, the final recipient corneal thickness after surgery is in most cases, is expected to be closer to normal corneal thickness or slightly greater than normal.

Based on Age

Neutral LKP: Restores normal corneal thickness.

1. Pediatric LKP 2. Adult LKP

Addition LKP: In addition-LKP (DSAEK), the final recipient corneal thickness after surgery will “always” be greater than the preoperative, non-edematous, recipient corneal thickness (recipient cornea minus Descemet’s membrane and endothelium) plus the thickness of the donor disk (i.e. deep, donor stroma along with donor DM and endothelium). 1. Optical LKP: Lamellar keratoplasty that is performed for improving the eyesight is called optical LKP. 2. Tectonic LKP: Lamellar keratoplasty that is performed to restore structural integrity of the cornea is named as tectonic LKP. No emphasis is made here for optical outcome. Tissue addition is made in areas of corneal thinning and restoration to near normal surface contour of the cornea is carried out. 3. Therapeutic LKP: Lamellar keratoplasty that is performed to remove infected corneal tissue that has failed medical treatment. Medically failed corneal infections are usually treated with a therapeutic PKP. Therapeutic LKP may also be performed for treating Descemetocele with or without corneal perforation. Rarely, LKP is performed to remove corneal tumor that has not invaded the full-thickness of the cornea. Therapeutic LKP may also be utilized in corneal inflammatory processes, and in selected cases of fungal keratitis. Therapeutic LKP is not performed as a routine surgical procedure for fungal keratitis. 4. Cosmetic LKP: Lamellar keratoplasty that is performed to improve the cosmetic appearance of a blind or nearly blind eye.

Based on Number of Procedures

Based on the Use of Microkeratome 1. Manual LKP—Here no microkeratome is utilized for the procedure. 2. Automated LKP—Here a microkeratome is used for the dissection.

Based on Surgical Approach 1. Direct a. Cornea 2. Indirect a. Scleral-pocket.

Based on Diameter (donor and recipient) 1. 2. 3. 4.

Total ( up to 11-12 mm.) Subtotal (9-10 mm.) Partial (less than 9 mm) Corneoscleral.

Based on the Depth of the Recipient Corneal Resection (Figure 13-1) 1. Superficial ALK (SALK): <160 µm central cornea. 2. Mid ALK (MALK): Between 160-400 µm central cornea. 3. Deep ALK (DALK): Up to pre-Descemetic level, 400490 µm central cornea, 585-620 µm peripheral cornea. 4. Total ALK (TALK): Up to Descemetic level (almost 100%), >490 µm (490-520) central cornea, 630-650 µm peripheral cornea.

Based on Donor Corneal Thickness 1. 2. 3. 4.

Anterior lamellar dissection (ALD) upto 160 µm. Mid-stromal dissection (MSD) 160-400 µm. Full-thickness donor graft with endothelium (FTDGE). Full-thickness donor graft without endothelium (FTDG).

Based on Substitution or Addition of Donor Tissue 1. Substitution LKP—all ALKP procedures (see above), DLEK, and FDLEK. 2. Neutral LKP (Normal thickness)—DMEK/DECT (Synonymous). 3. Addition LKP —DSAEK/DSEK/DXEK (Synonymous).

John-Malbran ALK Classification of Optical LKP (Figure 13-1) I. Anterior Lamellar Keratoplasty (ALK) 1. Superficial ALK (SALK) (30%) (<160 µm) (Figure 13-1). 2. Mid ALK (MALK) (30-70%) (160-400 µm) (Figure 13-1). 3. Deep ALK (DALK) (90-95%) (400-490 µm) (Figure 13-1). 4. Total ALK (TALK) (almost 100% stromal, excludes Descemet’s membrane and endothelium) >490 µm (500-520 µm) (Figure 13-1).

Definition, Terminology and Classification of Lamellar Corneal Surgery John PLK Classification of Optical LKP (Figure 13-2) Posterior Lamellar Keratoplasty (PLK) (Figure 13-2). 1A. Deep lamellar endothelial keratoplasty (DLEK) (Figure 13-2) Without flap – no surface wound or sutures. 1B. Flap-associated DLEK (FDLEK) (Figure 13-2) With flap – surface wound and sutures present. 2. Descemet Stripping Automated Endothelial Keratoplasty (DSAEK)/ Synonymous term- Descemetorhexis with endokeratoplasty (DXEK) (Figure 13-2). 3. Descemet’s membrane endothelial cell transplantation (DECT)/Descemet membrane endothelial keratoplasty (DMEK) (Figure 13-2). 4. Endothelial cell transplantation (ECT) (Figure 13-2). 5. Endothelial cell activation (ECA) (Figure 13-2). FDLEK—Previous terminologies included endokeratoplasty, and endothelial replacement flap approach. ECT and ECA have not been established as a surgical procedure at the present time (at the time of writing this chapter).

Indications for LKP A. Optical 1. Scars a. Traumatic b. Herpetic (Herpes simplex & zoster) c. Surgical d. Chemical injury with healthy endothelium 2. Dystrophies a. Map-dot-fingerprint dystrophy with recurrent erosion and scars b. Epithelial, Bowman’s membrane, and stromal dystrophies 3. Degenerations a. Salzman’s nodular degeneration 4. Ectasia a. Keratoconus b. Keratoglobus c. Pellucid Marginal Degeneration 5. Refractive complications a. LASIK flap folds not responding to conventional treatment including mechanical, thermal, and hydration techniques b. Post-LASIK ectasia (iatrogenic ectasia) c. PRK complication with persistent haze B. Tectonic 1. Descemetocele 2. Pellucid marginal degeneration with Descemetocele 3. Terrienn’s marginal degeneration with Descemetocele

139

4. Sterile corneal melt and ulceration including Mooren’s ulcer. C. Therapeutic 1. Infective (usually PKP) 2. Tumors 3. Inflammatory D. Cosmetic 1. Opaque, cloudy cornea with healthy endothelium in a blind or near blind eye.

Differences between ALK and PLK There are several differences between anterior and posterior lamellar keratoplasty and these differences are listed in Tables 13-1 to 13-3. The interface in lamellar keratoplasty is important for the quality of visual outcome following such surgical procedures. In SALK, MALK, and DALK the host-donor corneal interface is stroma-to-stroma, and the location of the interface varies from superficial, mid to deep corneal stroma, while in TALK this interface is host Descemet’s membrane to the deep stroma of the donor cornea. Additionally, the interface in TALK is expected to be smoother since the DM is smooth as compared to the stroma-to-stroma interface described above. With an interface that is so deep in the cornea next to the Descemet’s membrane in TALK as compared to SALK, MALK, and DALK, the quality of vision may be expected to be better in TALK. However, recent refinements in DALK,35 can afford excellent visual results comparable to TALK with the advantage of reduced risk of Descemet’s membrane perforation, which in TALK without the use of dye [indocyanine green (ICG), trypan blue (Vision Blue)], has been reported to be between 8% - 36%. In 1972, Josè Barraquer outlined the favorable conditions to obtain better visual results with LKP including deepest possible interface to reduce corneal scarring.36 In general, PLK procedures are technically more difficult than ALK. Among ALK procedures TALK is the most difficult procedure to perform. The surgeon attempting these surgical procedures must have adequate experience in lamellar corneal surgery.

Wound Architecture in LKP Automated microkeratome-assisted ALK usually have a tapered lamellar disk margin with good adherence of the lamellar disk to the host corneal bed, especially in SALK. In contrast, the nearly perpendicular wound margins created by manual ALK procedures require corneal sutures to maintain the donor graft in secure position on the host

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corneal bed. Use of biologic tissue adhesive is better suited for SALK with the use of a microkeratome. When fibrin glue (Tisseel, Baxter Inc., Deerfield, IL) is used for deeper lamellar keratoplasty procedures, it needs to be supplemented with sutures.

12. 13. 14.

General Comments Automated microkeratome-assisted LKP usually should not be utilized in corneal disorders with considerable variations in corneal thickness, very deep stromal opacities and in conditions where there are extensive corneal and ocular surface irregularities. This limits the use of automated LKP procedures in pathologic corneas. The unified classification, namely, John-Malbran ALK classification for optical LKP, and the John PLK classification for optical LKP described in this chapter is expected to simplify and standardize the terminologies used in lamellar keratoplasty procedures worldwide.

References 1. John T. Descemetorhexis with endokeratoplasty. In: Surgical Techniques in Anterior and Posterior Lamellar Corneal Surgery. John T (Ed.). Jaypee Brothers Medical Publishers (P) Ltd., New Delhi, India, 2006;411-20. 2. John T (Editorial). Selective tissue corneal transplantation: a great step forward in global visual restoration. Expert Rev Ophthalmol 2006;1:5-7. 3. John T. Descemetorhexis with endokeratoplasty (DXEK). In: John T (Ed.). Step by Step Anterior and Posterior Lamellar Keratoplasty. Jaypee Brothers Medical Publishers (P) Ltd., New Delhi, India, 2006;177-96. 4. John T. Surgical management of diffuse corneal opacities (Fullthickness Grafting, Deep Lamellar Keratectomy). In: Becker MD, Davis J (Eds): Surgical Management of Inflammatory Eye Disease, Springer Publisher, ch II.A.1.2 (In Press). 5. Sinha R, Vajpayee RB, Sharma N, Tityal JS, Tandon R. Trypan blue assisted descemetorhexis for inadvertently retained Descemet’s membrane after penetrating keratoplasty. Br J Ophthalmol 2003;87:654-5. 6. Melles GR, Wijdh RH, Nieuwendaal CP. A technique to excise the descemet membrane from a recipient cornea (descemetorhexis). Cornea 2004; 23:286-8. 7. Zvi T, Nadav B, Itamar K, Tova L: Inadvertent descemetorhexis. J Cataract Refract Surg 2005; 31:234-5. 8. Busin M, Arffa RC, Sebastiani A. Endokeratoplasty as an alternative to penetrating keratoplasty for the surgical treatment of diseased endothelium: initial results. Ophthalmology 2000; 107:2077-82. 9. Vasco Posada J. Homoqueratoplastia interlaminar. Ann Inst Barraquer 1973;11:335. 10. Hamilton W, Wood TO. Inlay lamellar keratoplasty. In: Kaufman HE, McDonald MB, Barron BA, Waltman SR(Eds.): The Cornea, New York, Churchill Livingstone, 1988;683-95. 11. Panda A, Bageshwar LM, Ray M, et al. Deep lamellar

15. 16. 17. 18. 19. 20.

21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.

keratoplasty versus penetrating keratoplasty for corneal lesions. Cornea 1999;18:172-5. Wood TO. Lamellar transplants in keratoconus. Am J Ophthalmol 1977;83:543-5. Olson RJ. Corneal transplantation techniques. In Kaufman HE, McDonald MB, Barron BA, Waltman SR (Eds): The Cornea, New York, Churchill Livingstone, 1988;743-85. Rich LF. Expanding the scope of lamellar keratoplasty. Tr Am Ophth Soc 1999; vol XCVII: 771-814. Terry MA. The evolution of lamellar grafting techniques over twenty-five years. Cornea 2000;19:611-6. Melles GRJ, Lander F, Rietveld FJ, et al. A new surgical technique for deep stromal, anterior lamellar keratoplasty. Br J Ophthalmol 1999;83:327-33. Shimazaki J, Shimmura S, Ishioka M, Tsubota K. Randomized clinical trial of deep lamellar keratoplasty vs penetrating keratoplasty. Am J Ophthalmol 2002; 134:159-65. Sugita J, Kondo J. Deep lamellar keratoplasty with complete removal of pathological stroma for vision improvement. Br J Ophthalmol 1997; 81:184-8. Melles GR, Remeijer L, Geerards AJ, Beekhuis WH. A quick surgical technique for deep, anterior lamellar keratoplasty using visco-dissection. Cornea 2000; 19:427-32. Seitz B, Langenbucher A, Hofmann-Rummelt C, et al. Nonmechanical posterior lamellar keratoplasty using the femtosecond laser (femto-plak) for corneal endothelial decompensation. Am J Ophthalmol 2003; 136:769-72. Hallerman W. Verschiedenes uber keratoplastik. Klin Monatsbl, Augenh 1959;135:252-9. Hallerman W. Zur technik der lamellaren keratoplastik. Klin Monatsbl, Augenh 1963; 142:243-50. Hallerman W. Technik und ergebnisse einer operative behandlung des keratoglobus. Klin Monatsbl, Augenh 1975; 166:593-8. Stocker FW. In discussion of McCulloch C, Thompson GA, Basu PK. Lamellar grafts using full thickness donor material. Tr Am Ophthalmol Soc 1963;61:154-80. Stocker FW. Management of corneal dystrophies. Latest concepts. Highlights of Ophthalmology 1965;8:221. Malbran E, Stefani C. Lamellar keratoplasty in corneal ectasias. Ophthalmologica 1972;164:50-58. Malbran E, Stefani C. Lamellar keratoplasty in corneal ectasias. Ophthalmologica 1972;164:59-70. Anwar M. Technique in lamellar keratoplasty. Trans Ophthalmol Soc UK 1974;94:163-7. McCulloch C, Thompson GA, Basu PK. Lamellar grafts using full thickness donor material. Tr Am Ophthalmol Soc 1963;61:154-80. McPherson SD, Jr. In discussion of McCulloch C, Thompson GA, Basu PK. Lamellar grafts using full thickness donor material. Tr Am Ophthalmol Soc 1963;61:154-80. Vasco Posada, J Homoqueratoplastia interlaminar. Rev Soc Col Oftal 1973;4:99. Malbran E. Lamellar grafts may be reflourishing. Highligths of Ophthalmology 1989;27:5. Malbran E. Lamellar keratoplasty and keratoconus. International Ophthalmological Clinics 1966;1:99-109. Melles GR, Lander F, Rietveld FJ. Transplantation of Descemet’s membrane carrying viable endothelium through a small scleral incision. Cornea 2002;21:415-8. Malbran ES, Malbran E Jr, Malbran J. The actual scope of lamellar grafts. Highlights of Ophthalmol 2004;32:2-6. Barraquer J. Lamellar keratoplasty (special techniques). Ann Ophthalmol 1972;4:437-69.

History of Lamellar and Penetrating Keratoplasty

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Thomas John Luiz F Regis-Pacheco José G Pecego Mark A Terry

History of Lamellar and Penetrating Keratoplasty

14

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Corneal Endothelial Transplant

Introduction In this chapter, we shall “travel” on the “time-line” from the beginning of corneal transplantation surgery, through the turbulent times of development of techniques and instruments and complete our journey at the final stop, namely, the present time of modern day corneal transplantation. During this review, we shall try to recognize the major and minor contributors, in most, if not all aspects of corneal transplantation. Due to the long time periods, it is possible that we may unintentionally have overlooked some contributors. Also, it is possible that there exists yet unknown ophthalmologists who may have directly or indirectly contributed to further the field of corneal transplantation. We thank each and every ophthalmologist who has contributed to furthering our knowledge and improving the surgical techniques of corneal transplantation, so that our patients, all over the world can benefit from these improved surgical techniques and hopefully, improve their quality of vision. The history of lamellar keratoplasty is fascinating and is over 100 years old. The fathers of lamellar keratoplasty include Königshofer, von Walther, Muehlbauer, and von Hippel. It started in 1840 when Walther and Muehlbauer described the surgical principles of the technique (Table 14-1). The history of keratoplasty can be divided into four great periods as follows: 1. The period of the precursors and the first trials, before 1900. 2. The developmental period, between 1900 and 1945. 3. The period of constant improvement and changes, from 1945 until the middle of the nineties. 4. The current period, namely, the modern era.

lamellar keratoplasty (LKP). He was of the opinion that in many cases it was enough to perform partial removal of corneal tissue, leaving behind the intact deep layers of the cornea, including Descemet’s membrane and endothelium.3 In the same year, Muehlbauer, using von Walther’s idea, tried triangular lamellar grafts taken from the sheep and implanted it into human eyes, with poor results. The grafts corresponded to 2/3rds of the corneal thickness and corneal sutures were placed at the angles of the triangular graft. Although, this surgical trial was unsuccessful, Muehlbauer is credited as being one of the pioneers to use heterografts in humans. Additionally, he also described the surgical principles of LKP.3 During this early period, one of the most important event that occurred was in 1878, when Arthur von Hippel (1841–1916) invented the mechanical trephination of the cornea and described his technique for LKP. In 1888, using his mechanical trephine, Arthur von Hippel became the first surgeon to perform a successful keratoplasty in man (Figure 14-1). He achieved permanent improvement of visual acuity, after several years of experimental failures. This surgical procedure was a lamellar corneal graft.4 The mechanical trephine of von Hippel consisted of a circular cutting trephine with blades of different diameters, and a key for winding up the watch mechanism, which was a great evolutionary step at this time. Despite this improvement, the technique of lamellar graft continued to be rather a laborious procedure for most eye surgeons and it became an intermediate step towards penetrating keratoplasty.

The Period of the Precursors and the First Trials, Before 1900 The beginning of the nineteenth century was a period of great medical developments that included the idea of grafts, namely, keratoplasty.1 A very important author from this period was Resinger, who, in 1924, performed the first animal graft.2 In 1839, Königshofer, according to Ramon Castroviejo,2 pioneered the idea of lamellar homologous and heterologous keratoplasties in animals. To outline the graft and the host tissue, he used a double knife in order to obtain the same shape and size for both donor and host. The history of lamellar grafting began in 1840, when von Walther had a new concept of corneal surgery namely,

Figure 14-1: First corneal transplantation. LKP- Lamellar keratoplasty; PKP – Penetrating keratoplasty.

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History of Lamellar and Penetrating Keratoplasty TABLE 14-1: Historical landmarks in lamellar keratoplasty, full-thickness, penetrating keratoplasty, and anesthesia

Year

LKP

1824

Reisinger – First animal graft and coined the term “keratoplasty”

1831

Dieffenbach proposes partial-thickness keratoplasty (LKP, extraocular procedure)45

1834

Wilhelmus Thome – First use of the term “corneal transplantation

1839

Königshofer – Animal LKP (Homologous & heterologous)

1840

Muehlbauer (using von Walther’s idea) Triangular LKP, sheep to human eyes (2/3rds corneal thickness, sutures at angles of graft)

1844

Kissam – established guidelines for keratoplasty46

1846

First use of general anesthesia – ether, at the Massachusetts General Hospital, Boston, MA

1847

Use of chloroform

1878

Arthur von Hippel, invented circular cutting trephine, blades of different diameters, a key for winding up the watch mechanism

1884

Kohler – Use of topical cocaine

1888

Arthur von Hippel, first successful LKP in man

1905

PKP

Edward Zirm – First successful human PKP

1908

Plange – First human lamellar autograft (clear cornea from blind eye to opposite, scarred eye of the same patient). Graft remained clear for 5 years

1911

Magitot – Established homografts are superior to heterografts

1912

Magitot – Use of human cornea previously preserved in an antiseptic fluid for corneal transplantation.47

1914-1930

LKP abandoned for PKP

Interest in PKP for corneal transplant surgery

1921

Harry Gradle – First keratoplasty article in the US literature.48

1935

Filatov – Use of cadaveric corneas stored in ice49

1948

Paufique, Sordille & Offret – Published, Les Graffes De La Cornée

1940s

Charleus, Paufique, Sordille & Offret – Established French School of Transplants – emphasized importance of LKP

Paton – Established the first eye bank in the United States

1940s and 1950s

Ramon Castroviejo – Square corneal transplant and new surgical instruments

1947

First corneal transplant symposium by American Academy of Ophthalmology, Chicago – participants included, Castroviejo R, Paton RT, Maumenee E50

1949

José I. Barraquer – First manual microkeratome and keratomileusis procedure

1950s

José I. Barraquer – Concept of lamellar refractive keratomileusis. Steinway Instrument Co. with Professor Barraquer developed the first commercially available microkeratome and cryolathe to perform keratomileusis51

1950

José I. Barraquer* – Performed the first PLK, square anterior lamellar flap, resection & replacement of posterior lamella (with DM and endothelium), flap replaced and sutured (Flap-PLK).

*FO PLK

Contd...

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Corneal Endothelial Transplant TABLE 14-1: Contd...

Year

LKP

PKP keratoplasty52

1952

Stocker – Role of donor endothelium in

1956

Charles Tillett* – First flapless-PLK, sclera-corneal AC entry at 12 o’clock, posterior lamellar removal with right & left Katzin corneal scissors. Donor corneal lamella 300 µm. *FO PLK

1960s

Since 1960s, PKP became increasingly common

1961

Establishment of Eye Bank Association of America

1970 -1990

• • • • • • • •

Malbran – “peeling-off” technique 1970s McCarey and Kaufman – Donor corneal storage medium 197453 Ward and Nesburn – Artificial anterior chamber 1976 Werblin and Kaufman – Epikeratoplasty for aphakia correction 1980s; Epikeratophakia for keratoconus 198254 Late 1980s – First automated microkeratome by Luis A. Ruiz Arenas – Intrastromal air injection for deep lamellar keratoplasty 198455 Hanna – Variable depth controlled vacuum trephine 198556 Ruiz – First automated microkeratome in 1990, automated corneal shaper (ACS), called his procedure automated lamellar keratoplasty (ALK)51

1993

Ko et al – Animal PLK limbal approach (Tillett’s original approach 1956)

1998

Tsubota et al – Divide and conquer technique for ALK57

1998

W Culbertson – Flap endokeratoplasty

1999

Manche et al – Viscoelastic dissection for lamellar keratoplasty58

1999

G Melles – first sutureless PLK, with air bubble attachment of the donor cornea

2000

Jesus Vidauri – used Moria microkeratome with Moria ALTK system (Moria – personal communication)

2000

M Terry – publication of first PLK in the USA, and called it DLEK

2002

Anwar and Teichman – “Big-bubble” to bare Descemet’s membrane in ALK59

2002

T John – use of Moria LSK microkeratome with Moria ALTK system for DLEK surgery (Moria personal communication)

2002

G Melles – Small incision DLEK60

2002

E Balestrazzi – Trypan blue use within the cornea61

2003

Sinha – First use of the term Descemetorhexis62

2003

T John – First intracorneal use of ICG in DLEK surgery63

2003

B Seitz – use of femtosecond laser for PLK64

2003

T John – Upside-down phaco with DLEK65

2003

Steinert– Femtosecond laser corneal transplant, cadaver eye studies, Univ. of California, Irvine

2003

Kaufman – Use of fibrin glue for 200 µm lamellar graft66

2003

T John – Use of fibrin glue in total anterior lamellar keratoplasty (TALK), in the interface between host Descemet’s membrane and full thickness donor cornea devoid of endothelium, within the entire trephination area including visual axis67, 68

2004

T John – Use of trypan blue to stain donor corneal endothelium to facilitate visualization during endothelium and donor Descemet’s membrane removal for total anterior lamellar keratoplasty (TALK)67,68

2004

T John – Combined use of indocyanine green (ICG) and forced hydrodissection for TALK69

2004

G Melles – Excision of patient’s Descemet’s membrane and directly attaching donor disk to patient’s corneal stroma with an air bubble70

Contd...

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History of Lamellar and Penetrating Keratoplasty TABLE 14-1: Contd...

Year

LKP

PKP

2004

M Terry – Healon in AC for DSEK instead of fluid

2004

T John – New surgical instruments for DSEK. Dexatome – unique design to reach all points of the inner corneal dome of the recipient from a single entry wound

2005

Price called Melles’s procedure DSEK and popularized it in the USA71

2005

T John – Standardization of scleral rim size for donor corneas to be used in artificial anterior chamber73

2005

M Terry – Roughening of the peripheral recipient stroma within the area of Descemet membrane removal to enhance donor disk adherence

2005

T John – Use of trypan blue to stain stromal side of donor disk to facilitate visualization through a cloudy recipient cornea74

2006

Gorovoy – used microkeratome for DSEK and called it DSAEK75

2006

G Melles – first Descemet membrane endothelial keratoplasty (DMEK)76

2006

T John – first modern day textbook of lamellar keratoplasty (ALK & PLK)

2006

E Davis – Laser inferior peripheral iridotomy preoperatively prior to DSEK

2007

T John – Biomechanical properties of the cornea after DXEK/DSEK77

2008

Busin glide78

2008

T John – Use of surgical slit-lamp for lamellar corneal surgery (ALK and PLK)

Price – First femtosecond laser-enabled keratoplasty (FLEK), or IntraLaseenabled keratoplasty (IEK)72

LKP – Lamellar keratoplasty; PKP – Penetrating keratoplasty; PLK – Posterior lamellar keratoplasty; DM – Descemet’s membrane; FO – Father of; ALK – Anterior lamellar keratoplasty

The Developmental Period 1900 to 1945 During this period, keratoplasty passed, progressively, from a stage of isolated trials to one of permanent practice especially due to advances in anesthesiology and antisepsis. Seven years after the first LKP (Table 14-1) Edward Zirm on December 7, 1905, performed the first penetrating keratoplasty (PKP) in Olmutz near Prague in Slovakia, previously called Czechoslovakia. The patient Alois Golgar had sustained lime injury and was blind in both eyes. Zirm used von Hippel’s 5.0 mm trephine for the surgery. According to Tomas A. Casey, the first autograft was performed by Plange in 1908 and this was a lamellar transplant. He replaced a patient’s scarred cornea with a lamellar graft taken from the patient’s other eye, which, although blind, had a normal cornea. The graft remained transparent for five years.1 In 1911, the idea of homografts being superior to heterografts was established by the French ophthalmologist A. Magitot.5 Between 1914 and 1930 many surgeons contributed to the improvements in surgical techniques and instruments

for keratoplasty. However, during this period their efforts were primarily directed towards PKP rather than to lamellar corneal surgery. Penetrating keratoplasty was considered to be an easier surgical technique that required less surgical time and yielded better postoperative visual results. During the first thirty years of the last century many names were relevant in the development of lamellar transplants, such as, von Hippel and Lohlein in Germany; Magitot and Morax in France; Elschnig in Czechoslovakia (on January 1st, 1993 Czechoslovakia was split into the Czech Republic and Slovakia); Filatov in Russia; and Franceschetti in Switzerland. These authors further defined the indications and surgical techniques of lamellar corneal grafts. At that time PKP was not considered feasible.5 Between 1930 and the end of World War II, further surgical developments took place in PKP. In the mid 1930s, Ramon Castroviejo described rectangular PKP. Figure 14-2 displays a clear, square PKP. In contrast, the French school had consistently been emphasizing the value of lamellar corneal transplants throughout this same period.5

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Figure 14-3: Historic beginning of posterior lamellar keratoplasty (PLK).

Figure 14-2: Clear, square, full-thickness penetrating keratoplasty (Courtesy Kenneth R Kenyon, MD).

The Period of Constant Improvement and Changes, from 1945 until the Mid-Nineties Due to various limitations of LKP as compared to fullthickness PKP, lamellar surgical techniques were practically abandoned by most of the ophthalmic surgeons. However, in 1940 lamellar corneal surgery underwent resurgence, when the French surgeon from Lyon, Dr. Louis Paufique (1899–1981), improved the lamellar techniques and developed new surgical instruments for LKP. In 1948, Louis Paufique, G. Philippe Sourdille, and Guy Offret published an excellent book on keratoplasty, namely, Les Greffes de la Cornée. Paris; Masson & Cie, Editors. This was a rapport presented at the Société Française d’Ophtalmologie on May 23rd of that same year. It became a very important reference for LKP.5 Another French author of significant importance was Jacques Charleux of Lyon, who together with Paufique, Sourdille and Offret, played an important role in the development of the French School of Transplants that established the importance of LKP. They showed the usefulness of LKP not only as a tectonic or therapeutic procedure, but also as a surgical technique for improving vision. The first reference in the ophthalmic literature on the subject of posterior lamellar keratoplasty (PLK) can be found in a paper written in 1950, by José Ignácio Barraquer (1916– 1988), from Bogota, Colombia (Figure 14-3), who stated, that it is “for cases of incipient endothelial dystrophies.”

Barraquer’s surgical technique consisted of a square anterior lamellar flap, with resection and replacement of the posterior corneal lamella, including the dissected Descemet’s membrane and endothelium. The flap was then put in place and sutured.6 In 1956, Charles W. Tillett from Charlotte, North Carolina, published an elegant paper (“Posterior Lamellar Keratoplasty”, AJO 1956) (Figure 14-3) proposing a new technique for LKP, for the treatment of endothelial dystrophies and opacities of the posterior layer. Tillett’s technique consisted of a sclerocorneal anterior chamber entrance at the 12 o’clock meridian, combined with a 10 mm corneal, posterior lamellar excision using right and left Katzin corneal scissors. The donor material, with a thickness of 300 µm, was sutured at the 2, 4, 8, and 10 o’clock meridians. Unfortunately, the patient developed severe postoperative glaucoma without any visual improvement. However, it is important to note, that the posterior lamellar graft remained in good position even after one year following the surgery, and the iris details could be visualized through the graft. This paper was presented in part at the 14th Clinical Meeting of the Wilmer Residents Association, at the Wilmer Ophthalmological Institute on March 31, 1955.7 Hence, Tillett´s technique should truly be considered as the pioneer idea of modern day techniques for PLK. Also in 1955, yet another interesting historical paper was presented by Frederick Stocker, at a scientific section of the American Academy of Ophthalmology.8 The author presented in a motion picture a procedure, suggested by Louis Paufique, from Lyon, France, of scraping the endothelium off the posterior surface of the patient’s cornea, as a preliminary measure for the corneal grafts in patients with Fuchs’ endothelial dystrophy. This is the only

History of Lamellar and Penetrating Keratoplasty reference about this surgical technique in the literature. This surgical technique may be considered as the precursor for the newly established Descemet’s membrane stripping techniques in DSAEK surgery. During the period between 1970 and 1990s several ophthalmic surgeons contributed to the continued onward progress of LKP.

The Modern Era The year 1993 may be considered as the beginning of the modern era of PLK.9,10 In 1993, Ko et al,11 reported their animal study of PLK using a limbal approach. Five years later, in 1998, Gerrit Melles,12,13 from the Netherlands, pioneered the surgical principles for the modern day posterior endothelial keratoplasty with the first endothelial keratoplasty in humans where he used an air bubble in the anterior chamber to help in the adherence of the donor corneal disk to the patient’s cornea, eliminating the need for corneal suture attachment. The use of an air bubble to help attach the donor corneal disk to the patient’s cornea without the use of any corneal sutures may be considered a major milestone in the history of corneal transplantation. Melles et al called this technique PLK, the same term that Tillett7 used in 1956. In the United States, Mark A Terry worked in his laboratory in 1999 with PLK surgery testing the use of Healon viscoelastic (Bausch and Lomb Surgical, St. Louis, MO) rather than air to maintain the chamber, modifying the instrumentation, and adding an artificial anterior chamber for preparation of the donor tissue.14 In March 2000, Terry performed the first endothelial keratoplasty in the United States with this modified PLK technique that he renamed as deep lamellar endothelial keratoplasty (DLEK).15 Initially fluid was used to maintain the AC and air was used to unfold and attach the donor disk to the patient’s cornea. More recently, Terry M used Healon in the AC instead of fluid to maintain the AC in DSAEK. In 2002, Melles et al 16 modified his original PLK technique, to a smaller 5 mm incision technique by folding the donor corneal disk. Following Melles’ proposition of small incision and folded tissue insertion, Terry adopted this idea, moved the incision to the temporal side and advocated that the donor tissue be folded like a “taco,” in a 60/40 configuration to prevent “upside-down” unfolding.17 Terry went on to establish the legitimacy of DLEK surgery as an alternative to PKP with the largest prospective series in the world. 17 John T first used indocyanine green (ICG) to stain the donor corneal stroma to facilitate better visualization of the donor corneal disk

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through a cloudy cornea, to identify the stromal side from the endothelial side. For triple procedure of DLEK, phacoemulsification and PC IOL, John T. introduced a new technique which he called the upside-down phacoemulsification technique. Despite achieving good technical results with the DLEK surgery, irregularities in the surgical interface caused limitations in the Snellen visual acuity, and were deemed too difficult to perform by most surgeons. In an attempt to improve the donor-recipient interface in PLK/DLEK surgery, Melles et al in 2004,18 stripped the recipient Descemet’s membrane and placed the donor corneal tissue directly onto the posterior surface of the cornea and again used an air bubble to help assist in the donor disk adherence to the patient’s cornea. Most importantly, manual dissection of the recipient cornea was eliminated, making the surgery much easier. These two factors were the major stimuli for the revised surgical technique to become popular and accelerated the interest in this type of surgery among most corneal surgeons. However, the increased ease of stripping Descemet’s membrane compared to manual lamellar dissection came at the price of increased postoperative complications of donor tissue dislocation and iatrogenic graft failure. This was especially true among corneal surgeons in the early part of their learning curve. Price et al confirmed the high rate of tissue dislocation to be above 50% especially in the initial series of cases.19 Price19 re-named the surgery Descemet’s stripping endothelial keratoplasty (DSEK) [See also Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)]. The artificial anterior chamber (AAC) was first introduced in 1976 by Ward and Nesburn.20 The AAC was developed for PKP to facilitate trephination from the epithelial side on both the donor and recipient corneas. It is a natural progression to use the AAC in DLEK surgery with manual dissection of the donor cornea. Jesus Vidauri working with Moria (Moria SA, Antony, France) first combined the use of a microkeratome with the Moria AAC (personal communication, Moria SA). Subsequently, John T first used this combination of a Moria microkeratome and a Moria AAC for DLEK surgery (personal communication, Moria SA). DSEK, due to its relative simplicity over DLEK, soon became the dominant procedure for endothelial transplantation. Gorovoy21 used the microkeratome with the AAC in DSEK surgery and called it DSAEK, where the “A” represents automated. Presently, for the most part, donor corneal tissue for PLK procedures are prepared by the surgeon in the operating room or by a technician in the eye banks that distribute “pre-cut” tissues. The contribution of the AAC to the field of corneal transplantation further

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Figure 14-4: Left- First modern day Textbook of Lamellar Keratoplasty [John T (Ed.)]; Right – Subsequent book on Step by Step Lamellar Keratoplasty.

Figure 14-5: John surgical instruments (ASICO Inc, Westmont, IL, USA) for posterior lamellar keratoplasty (Patent pending).

facilitated the advancement of modern day LKP. In 2006, Thomas John from Chicago, edited the first modern day textbook on lamellar keratoplasty (ALK & PLK) (Figure 14-4). He also designed various surgical instruments to facilitate DSAEK surgery (Figure 14-5). The unique curvature of the John Dexatome Spatula and the John DSAEK Scrubber (ASICO, Inc., Westmont, IL) (patent pending) allows easy access to all regions on the inner aspect of the patient’s corneal dome (Figure 14-5) (See also Chapter 11, New/Useful Surgical Instruments in DSAEK).” DSAEK became the most popular procedure for endothelial keratoplasty (EK). The use of the microkeratome that was previously developed independently by Ramon Castroviejo and Jose I. Barraquer, improved the donorrecipient interface in both DLEK and DSAEK procedures. Since 2006, the primary complications of donor dislocation and iatrogenic primary graft failure (IPGF)

following DSAEK surgery have been reported by multiple surgeons. In their most recent published series, surgeons using different DSAEK techniques reported different complication rates. Gorovoy has reported a dislocation rate of 25% and an IPGF rate of 6% in his initial series of 16 eyes.21 Koenig et al have reported a dislocation rate of 35% and an IPGF rate of 12% in their initial series of 26 eyes.22 Melles’ group has reported a dislocation rate of 14% and an IPGF rate of 14% in their series of 22 eyes.23 Looking at larger series, Price et al have reported a total dislocation rate of 11% and an IPGF rate of 3% in their initial retrospective series of 200 DSEK eyes24 and Terry et al have reported a dislocation rate of 1.5% and an IPGF rate of 0% in their initial prospective series of 200 DSAEK eyes.25 While it is tempting to say that a “learning curve” influenced the complication rates in all of these series, these disparate rates of complications likely reflect the variation in DSAEK surgical techniques rather than individual surgeon skill level, as the large Price series and the large Terry series included their initial DSAEK cases as well. In addition, the low complication rate of the Terry series (1.5% dislocations, 0% IPGF) included 4 surgeons (1 experienced and 3 novice) using the same DSAEK technique.25 Various surgical techniques have been advocated to reduce the DSAEK complication of dislocation including recommendations by Price and Price,24 namely, the removal of interface fluid with the use of recipient surface massage and full thickness, recipient cornea “venting” incisions (See also Chapter 27, Techniques to Facilitate Disk Adherence to Recipient Cornea in DSAEK). Terry and colleagues25,26 have recommended the use of peripheral recipient bed scraping to promote exposure of recipient stromal fibrils and peripheral donor edge adhesion. John T has described the wound architectural differences between DLEK and DSEK and compared the donor disk to a “pizza on the ceiling” and recommended the use of a large air bubble in the AC, especially during the intraoperative waiting period to enhance donor-recipient adhesion, much like holding a pizza on the ceiling with two hands. The air bubble size is subsequently decreased. Extended full chamber air bubble support postoperatively, followed by partial air release one hour later has been advocated by Gorovoy,21 but pupillary block glaucoma remains a risk with this technique and the rate of dislocation remains high at 25%, with an unknown toxic effect to the donor endothelium from prolonged air exposure. John T. utilizes a preoperative, inferior, peripheral laser iridotomy (recommended by Elizabeth Davis), combined with an intraoperative use of a large air bubble that fills 100% of the AC during the waiting time to facilitate donor disk attachment to the recipient corneal stroma. Inferior peripheral iridotomy is preferred since in the sitting

History of Lamellar and Penetrating Keratoplasty up position the air bubble moves to the superior region of the AC. Following this intra-operative waiting period the air bubble size is slightly reduced and replaced with BSS. John T combines this technique of large air bubble with roughening of the peripheral recipient corneal roughening (personal communication Terry, M) using the John DSAEK Scrubber (ASICO Inc., Westmont, IL). Of greatest concern in DSAEK surgery is the acute and long term damage to the donor endothelium. The published 2 year prospective results reported by Terry et al27 on the endothelial survival in DLEK surgery on a large number of eyes (n=98) demonstrated that tissue inserted with folding through a 5 mm incision had much greater cell loss than tissue inserted through a 9.0 mm incision. In recent DSAEK surgery reports, surgeons are attempting to place donor tissue through an even smaller 3.0 mm clear corneal incision, using a variety of forceps, glides and “suture pull through” techniques.28-30 Mounting laboratory evidence indicates that tissue inserted through a 3.0 mm incision suffers extensively more overall acute damage to the donor endothelium than tissue inserted through a 5.0 mm incision, regardless of the method of insertion. 31,32 Advances in technology with “tissue injectors” which avoid wound compression damage to donor tissue may allow safe use of 3 mm incisions DSAEK in the future.33,34 However, continued monitoring of the donor endothelium pre- and postoperatively will shed light as to the overall success of these techniques. Long term donor endothelial cell death in DSAEK surgery is also of concern. Independently, Price35 and Terry36 have measured with central specular microscopy about a 35% loss of central endothelial cell density (ECD) 6 months after DSAEK surgery. While this cell loss may be stable from 6 to 12 months postoperatively,36 there appears to be a steady decline at 24 months and thereafter.35 In studies of EK, a plateau of this cell loss has not been documented (even out to 7 years) and the risk and rate of late endothelial graft failure in DSAEK surgery remains unknown. In this journey of the history of corneal transplantation, the most recent “stop” is called “DMEK” or Descemet membrane endothelial keratoplasty. Melles et al, and Tappin et al [See also Chapter 36, True Endothelial Cell (TEnCell) Transplantation and Chapter 37, Descemet Membrane Endothelial Keratoplasty (DMEK)] have described the use of donor Descemet’s membrane with healthy endothelium and directly transplanting it to the patient’s cornea that is devoid of Descemet’s membrane and endothelium and attaching the donor tissue with an air bubble. This advanced surgical technique restores the patient’s cornea closer to the preoperative status with regard to the corneal thickness. Thus,

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DLEK and DSAEK are additive procedures that result in an overall very thick cornea. In contrast, DMEK is a substitution procedure that restores the patient’s cornea back to its normal preoperative thickness. However, this surgical procedure is in its early developmental stage with various technical difficulties and hence it is not in the main stream of corneal transplant procedures. Busin recently simplified the technique of preparing the donor tissue [See also Chapter 37, Descemet Membrane Endothelial Keratoplasty (DMEK)] by using air injection to separate the donor Descemet’s membrane from the stroma, followed by trephination to obtain a donor disk of Descemet’s membrane and endothelium. Recently, preparation of the donor tissue for DSAEK surgery has moved from the operating room to the distributing eye bank, with many eye banks offering “precut” tissue to the surgeon. While there was some concern initially that pre-cut tissue would swell en-route to the operating room and increase the complication rate,37 a recent publication by Chen and colleagues on their first consecutive 100 cases of DSAEK using pre-cut tissue demonstrated a dislocation rate of only 1% (1 case) and a primary graft failure rate of 0% (0 case).38 In extensive data analysis of donor characteristics, they also found that surgical technique is the overriding factor determining complications in DSAEK surgery, and that postoperative visual acuity, topography and refractive outcome were not correlated with any specific donor characteristic. However, it has to be kept in mind that in EK, the donor endothelial cells are the most important. When we use pre-cut donor corneal tissue we are transferring this important surgical step of donor disk preparation from the surgeon to eye bank technicians, and somewhat compromising the concept of “surgery by surgeons” (John T). During this modern period of posterior lamellar keratoplasty transformation from DLEK to DMEK, it is of interest to mention that surgeons tried flap endokeratoplasty39,40 but it was short lived due to the limited clinical success of this surgical approach. Additionally, flap endokeratoplasty involved the creation of a surface corneal wound and surface corneal sutures. A relatively new concept with the changing landscape of corneal transplant surgery is the use of a single donor cornea for three recipients.41 The anterior donor corneal cap for ALK surgery, the posterior disk for PLK surgery and the rim tissue for stem cell transplantation.41 Thus, one donor cornea can be used for three separate surgeries and in doing so, one has immediately tripled the donor corneal pool. Although, this approach of one donor cornea for multiple recipients is practiced in some parts of the world, it is currently not permitted in the United States

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(personal communication with Illinois Eye Bank). Various surgical techniques are available for LKP42-44 and a review of these techniques would be beneficial for corneal surgeons who are interested in LKP. As the readers can see, this journey from the “beginning of time” for corneal transplantation surgery to the present selective tissue corneal transplantation (STCT) surgery, is truly fascinating. It may continue to evolve where pure donor endothelial cells may one day be transplanted to the patient’s cornea or even more exciting may be the possibility of “exciting” and “rejuvenating” the patient’s endothelial cells to “re-energize” and reactivate and clear the patient’s cornea. Such dreams may one day become reality. The various techniques in PLK have evolved due to the passion and hard work of corneal surgeons and the commitment of industry. Many other authors, who we did not have the opportunity to mention, were relevant in the development of our current knowledge on posterior lamellar techniques. To these colleagues and others, we extend our eternal gratefulness. “In the end, we will conserve only what we love, we will love only what we understand, we will understand only what we are taught” Baba Dioum

References 1. Casey TA, Mayer DJ. Corneal Grafting. Principles & Practice. Philadelphia: WB Saunders Company; 1984;11. 2. Castroviejo R. Atlas of Keratectomy and Keratoplasty. Philadelphia: WB Saunders Company; 1966;21. 3. Rezende CB. The use of cadaver cornea in keratoplasty. [thesis]. São Paulo: University of São Paulo; 1938. Portuguese. 4. Paufique L, Charleux J. Lamellar Keratoplasty. In: Casey T A. Corneal Grafting. London: Butterworths & Co; 1972; 121-76. 5. Paufique L, Sourdille GP, Offrett G. Les Greffes de la Cornée. Paris: Massom & Cie, Editors; 1948. 6. Barraquer JI. Keratoplasty. CVI Concilium Ophthalmologicum Britannia. 1950;(2):999. 7. Tillet CW. Posterior lamellar keratoplasty. Am J Ophthalmol 1956;41:530-3. 8. Stocker FW. Management of endothelial and epithelial corneal dystrophy (Fuchs) with corneal transplantation for endothelial and epithelial corneal dystrophy (Fuchs). (A Motion Picture). Tr Am Acad Opth 1955;59:577. 9. Terry M. The evolution of lamellar grafting techniques over twenty-five years. Cornea 2000;19(5):611-6. 10. Terry MA. Endothelial Keratoplasty (EK): History, current state, and future directions. Cornea 2006 (Editorial); 25: 873-8. 11. Ko W, Freuh B, Shield C, Costello M, Feldman S. Experimental posterior lamellar transplantation of the rabbit cornea. Invest Ophthalmol Vis Sci 1993;34:1102. 12. Melles GR, Eggink FA, Lander F, Pels E, Rietveld FJR, HoudijnBeekhuis W, Binder PS. A surgical technique for posterior lamellar keratoplasty. Cornea 1998;17(6):618-26.

13. Melles GRJ, Lander F, Beekhuis WH, Remeijer L, Binder PS. Posterior lamellar keratoplasty for a case of pseudophakic bullous keratoplasty. Am J Ophthalmol 1999;127:340-1. 14. Terry MA, Ousley PJ. Endothelial replacement without surface corneal incisions or sutures: topography of the deep lamellar endothelial keratoplasty procedure. Cornea 2001;20:14-18. 15. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty in the first United States patients: Early Clinical Results. Cornea 2001;20:239-43. 16. Melles GR, Lander F, Nieuwendall C. Sutureless posterior lamellar keratoplasty: a case report of a modified technique. Cornea 2002;21:325-7. 17. Terry MA, Ousley PJ. Small incision deep lamellar endothelial keratoplasty (DLEK): 6 months results in the first prospective clinical study. Cornea 2005;24:59-65. 18. Melles GR, Wijdh RH, Nieuwendaal CP. A technique to excise the Descemet’s membrane from a recipient cornea (descemetorhexis). Cornea 2004;23:286-8. 19. Price FW, Price MO. Descemet’s stripping with endothelial keratoplasty in 50 eyes: A refractive neutral corneal transplant. J of Refractive Surgery 2005;21:339-45. 20. Ward DE, Nesburn AB. An artificial anterior chamber. Am J Ophthalmol 1976;82:796-8. 21. Gorovoy MS. Descemet-stripping automated endothelial keratoplasty. Cornea 2006;25:886-9. 22. Koenig SB, Covert DJ. Early results of small-incision Descemet’s stripping and automated endothelial keratoplasty. Ophthalmology 2007;114:221-6. 23. Nieuwendaal CP, Lapid-Gortzak R, van der Meulen IJ, Melles GJR. Posterior lamellar keratoplasty using descemetorhexis and organ cultured donor corneal tissue (Melles technique). Cornea 2006;25:933-6. 24. Price FW Jr, Price MO. Descemet’s stripping with endothelial keratoplasty in 200 eyes: early challenges and techniques to enhance donor adherence. J Cataract Refract Surg 2006;32:411-8. 25. Terry MA, Shamie N, Chen ES, Hoar KL, Friend DF. Endothelial Keratoplasty: A simplified technique to minimize graft dislocation, iatrogenic graft failure and pupillary block. Ophthalmology 2008 (Epub ahead of print). 26. Terry MA, Hoar KL, Wall J, Ousley, PJ. The Histology of Dislocations in Endothelial Keratoplasty (DSEK and DLEK): Prevention of dislocation with a laboratory-based surgical solution in 100 consecutive DSEK cases. Cornea 2006;25:926-32. 27. Terry MA, Wall JM, Hoar KL, Ousley PJ. A prospective study of endothelial cell loss during the 2 years after deep lamellar endothelial keratoplasty. Ophthalmology 2007;114(4):631-9. 28. Mearza AA, Qureshi MA, Rostron CK. Experience and 12month results of descemet-stripping endothelial keratoplasty (DSEK) with a small-incision technique. Cornea 2007;26:27983. 29. Mehta JS, Por YM, Beuerman RW, Tan DT. Glide insertion technique for donor cornea lenticule during Descemet’s stripping automated endothelial keratoplasty. J Cataract Refract Surg 2007;33:1846-50. 30. Macsai MS, Kara-Jose AC. Suture technique for Descemet stripping and endothelial keratoplasty. Cornea 2007;26:112326. 31. Terry MA, Saad HA, Shamie N, Chen ES, Friend DJ, Holiman JD, Stoeger C. Endothelial Keratoplasty: The influence of insertion techniques and incision size on donor endothelial survival. Cornea 2008 (submitted). 32. Terry MA. Donor tissue damage in endothelial keratoplasty. Ophthalmology 2008;115:420-1. 33. Mehta JS, Thomas AS, Tan DT. Endothelial keratoplasty: Requirements of an insertion device. Ophthalmology 2008;115: 420. 34. Kuo AN, Harvey TM, Afshari NA. Novel delivery method to reduce endothelial injury in Descemet stripping automated endothelial keratoplasty. Am J Ophthalmol 2008;145:91-6.

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59. Anwar M, Teichmann KD. Big-bubble technique to bare Descemet’s membrane in anterior lamellar keratoplasty. J Cataract Refract Surg 2002;28:398-403. 60. Melles GR, Lander F, Nieuwendaal C. Sutureless, posterior lamellar keratoplasty: A case report of a modified technique. Cornea 2002;21:325-7. 61. Balestrazzi E, Balestrazzi An, Mosca L, Balestrazzi A. Deep lamellar keratoplasty with trypan blue intrastromal staining. J Cataract and Refract Surg 2002;28:929-31. 62. Sihna R, Vajpayee RB, Sharma N, Tityal JS, Tandon R. Trypan blue assisted descemetorhexis for inadvertently retained Descemet’s membrane after penetrating keratoplasty. Br J Ophthalmol 2003;87:654-5. 63. John T. Use of indocyanine green in deep lamellar endothelial keratoplasty. J Cataract Refract Surg 2003;29:437-43. 64. Seitz B, Langenbucher A, Hofmann-Rummelt C, SchötzerSchrehardt U, Naumann GOH. Nonmechanical posterior lamellar keratoplasty using the femtosecond laser (femtoPLAK) for corneal endothelial decompensation. Am J Ophthalmol 2003;136:769-72. 65. John T. Upside-down phaco with deep lamellar endothelial keratoplasty. Presented at the annual meeting of the American Society of Cataract and Refractive Surgery, San Francisco, CA, April 12-16, 2003. 66. Kaufman HE, Insler MS, Ibrahim-Elzembely HA, Kaufman SC. Human fibrin tissue adhesive for sutureless lamellar keratoplasty and scleral patch adhesion: A pilot study. Ophthalmology 2003;110:2168-72. 67. John T. New surgical technique: Use of indocyanine green and forced hydrodissection for total anterior lamellar keratoplasty. Presented at the Annual Meeting of the American Society of Cataract and Refractive Surgery (ASCRS), San Diego, CA, May 01-05, 2004. 68. John T. Use of fibrin glue in total anterior lamellar keratoplasty. Presented at the annual meeting of the American Academy of Ophthalmology, Chicago, Illinois, October 15–18, 2005. 69. John T. Use of indocyanine green (ICG) and forced hydrodissection in total anterior lamellar keratoplasty (TALK). Presented at the XXII Congress of the ESCRS, Paris, France, September 18-22, 2004. 70. Melles GR, Wijdh RH, Nieuwendaal CP. A technique to excise the Descemet membrane from a recipient cornea (descemetorhexis). Cornea 2004;23:286-8. 71. Price FW Jr, Price MO. Descemet’s stripping with endothelial keratoplasty in 50 eyes: A refractive neutral corneal transplant.. J Refract Surg 2005;21:339-45. 72. Culbertson W, Price FW, Steinert RF. Better PK results forseen with custom overlapping incision shapes. Ophthalmology Times Nov. 15, 2006, page 1. 73. John T, Selvadurai D, Ruskowski, Pivoney CJ, McCoy K. What is the ideal donor cornea scleral skirt size for successful use in artificial anterior chambers? Presented at the World Cornea Congress, Washington, DC, April 13-15, 2005. 74. John T. Advances in PLK surgery. Presented at the VII International Symposium on Ocular Trauma. June 29-July 1, 2006, Rome, Italy. 75. Gorovoy MS. Descemet-stripping automated endothelial keratoplasty. Cornea. 2006;25:886-9. 76. Melles GR, Ong TS, Ververs B, van der Wees J. Descemet membrane endothelial keratoplasty (DMEK). Cornea 2006; 25:987-90. 77. John T, Taylor DA, Shimmyo M, Siskowski BE. Corneal hysteresis following descemetorhexis with endokeratoplasty: early results. Ann Ophthalmol 2007;39:9-14. 78. Busin M, Bhatt PR, Scorcia V. A modified technique for Descemet membrane stripping automated endothelial keratoplasty to minimize endothelial cell loss. Arch Ophthalmol 2008;126:1133-7.

Mark A Terry Paula J Ousley

Deep Lamellar Endothelial Keratoplasty (DLEK): Large Incision Technique

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Introduction Surgical endothelial replacement for conditions such as Fuchs’ endothelial dystrophy and pseudophakic bullous keratopathy has been successfully accomplished with fullthickness penetrating keratoplasty (PKP) for nearly 100 years (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty).1 While the surgical technique of PKP is straightforward and relatively easy, the visual results and stability of the grafted corneal tissue are sometimes poor due to wound healing and suture-related problems.2-6 In 1993 Ko and Feldman presented an animal study at ARVO which described a new technique for endothelial replacement through a scleral limbal incision7 (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). In 1998, Melles et al described this technique in the first human patients and called it posterior lamellar keratoplasty8 (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). Terry and Ousley began laboratory work in 1999 on this technique, and after technical modifications and re-design of instrumentation, performed the first United States cases in 2000 and called the surgery Deep Lamellar Endothelial Keratoplasty (DLEK)9-20 (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). All of this work represents a radical departure from the PKP technique in that the DLEK surgery accomplished the goal of endothelial replacement without any surface incision on the recipient cornea. By eliminating surface corneal sutures and incisions, the advantages of preserving the preoperative corneal topography and faster wound healing were obtained, leading to faster visual rehabilitation and a more stable globe for the patient.10-14 While undoubtedly there will be further refinement of the technique and instrumentation in DLEK surgery, it is the purpose of this chapter to describe in detail a method of DLEK that has been proven (with prospective data) to work and to provide considerable advantages over standard PKP surgery.

Surgical Objective The purpose of DLEK surgery is to remove the diseased recipient endothelium and replace it with a healthy donor corneal endothelium. The advantage of DLEK surgery over PKP surgery is that it accomplishes this primary objective without violating the surface of the cornea with sutures or incisions. We have delineated the five ideal goals for endothelial replacement in previous papers as being (1) a smooth surface topography without significant change in astigmatism, (2) a highly predictable (and unchanged) corneal curvature, (3) a healthy donor corneal endothelium

which resolves all edema, (4) a tectonically stable globe, safe from injury and infection, and (5) an “optically pure” cornea.11,12,15-18 While standard PKP can consistently achieve good results for goals 3 and 5, the other goals have remained elusive.21-25 At the current time, DLEK surgery can accomplish the first 4 goals nearly perfectly, while the fifth goal of an optically pure cornea is good, but can still be improved. Instrumentation and technique modification to achieve a more perfect stromal interface between posterior donor tissue and anterior recipient tissue are the current directions of DLEK research.

Preferred Anesthesia DLEK surgery is usually done under general anesthesia, but retrobulbar block anesthesia has also been used. General anesthesia (either endotracheal or laryngeal mask airway technique) is preferred because it minimizes posterior pressure on the globe and this is important during the recipient resection and donor implantation phases of the surgery. Nonetheless, the surgery can be safely accomplished with good retrobulbar anesthesia combined with seventh nerve block (orbicularis block) local anesthesia as well. It is possible, in the severely medically frail patient, that this surgery could be accomplished with only topical anesthesia, similar to what has been done in PKP surgery under similar circumstances.26 However, this has not yet been done with DLEK surgery and likely would require a surgeon with the ability to accomplish this surgery in less than an hour.

Preoperative Preparation Like all intraocular procedures, the patient’s ocular health should be maximized prior to surgery and any blepharitis, dry eye, or lid abnormality should be treated before the DLEK surgery. Patients with some mild to moderate corneal surface haze or scarring from long-standing bullous keratopathy can still undergo DLEK surgery successfully. The surface scarring is simply scraped off at the time of surgery or weeks later in the clinic after the stromal edema has completely resolved. This eliminates the induced irregular astigmatism from the scars and restores normal corneal topography. In patients with pseudophakia, the pupil is constricted in order to stabilize the iris-lens diaphragm during the surgery. This is also done if the patient has a clear crystalline lens and concurrent cataract surgery is not planned. Preoperative medications include two sets of pilocarpine 1% drops applied one hour prior to surgery. One set of aproclonidine 0.5% drops is also given just prior

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to surgery to reduce pressure and minimize conjunctival injection. No preoperative antibiotics are necessary. The eye is prepped in the usual sterile ophthalmic fashion with the use of povidone-iodine solution. In the case of patients with cataract and endothelial failure, then cataract surgery is performed just prior to the DLEK endothelial transplant and the pupil is dilated preoperatively with the surgeon’s preferred dilating drops for cataract surgery. While pilocarpine is avoided, the rest of the preoperative medication regimen described above is utilized.

DLEK Surgical Procedure

Figure 15-2: Superior limbal peritomy.

Recipient Surgery For large incision DLEK surgery, the scleral access incision is 9.0 mm, and therefore the incision is usually placed at the superior limbal region (Figure 15-1) rather than temporally. The patient’s head is positioned with the chin up and the forehead back to minimize brow obstruction of the surgical field. In addition, the endotracheal tube is also positioned by the anesthesiologist to exit the mouth from the side opposite the surgical field to facilitate the surgeon’s field of movement. A superior and inferior rectus bridal suture can also be placed to aid in positioning and stabilizing the globe during DLEK surgery. Figure 15-3: Clear corneal stab incision.

Figure 15-1: Preoperative appearance

A superior limbal peritomy of the conjunctiva is performed with scissors (Figure 15-2) allowing exposure of the superior 10.0 mm arc length of limbal tissue. Prior to forming the DLEK scleral access incision, two clear corneal limbal stab incisions of about 1.0 mm in size are placed on either side of the peritomy area (Figure 15-3) , to be used as access points to the anterior chamber later in the operation. Through one of the stab incisions, the cohesive viscoelastic Healon (Pfizer, New York, NY) is placed into the anterior

Figure 15-4: Viscoelastic into the anterior chamber.

chamber (Figure 15-4) to replace the aqueous fully and to maintain normal pressure. It is important not to use Viscoat (Alcon, Fort Worth, TX) or other dispersive viscoelastic materials during any portion of DLEK surgery as the dispersive materials can cause stromal interface coating with subsequent non-adherence and dislocation of the donor tissue.

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Figure 15-5: A 9.0 mm diamond knife incision.

A trifaceted, guarded diamond knife is then set to a depth of 350 microns and a 9.0 mm incision is made approximately 1.0 mm posterior and concentric to the corneal limbus (Figure 15-5) . We have found that a deeper initial incision gives less of a beveled wound closure and also a greater chance of early perforation into the anterior chamber during

A

DLEK surgery. In lieu of a diamond knife, a sharp crescent blade or other steel scalpel can be used for the initial incision. A sharp crescent blade is then utilized to create a deep scleral-corneal lamellar pocket down to about 75 to 85% corneal depth along the entire length of the wound (Figures 15-6A and B). Perfect accuracy of the depth of the corneal stromal pocket does not appear to be critical for a good visual outcome.27 Pockets should be deeper than 50% in order to avoid interface scarring or haze, and should not exceed 95% depth in order to avoid a mismatch of the donorrecipient corneal thickness. Judgment of the initial depth of the pocket is based upon inspection of the anterior lip thickness and by the clarity of the underlying stromal bed. Obviously, experience with the DLEK procedure and with lamellar dissections in general aids in the surgeon confidence that the desired depth has been achieved. A specialized semi-sharp stromal dissector is then used to extend the pocket to the mid-pupillary region of the cornea (Figures 15-7A and B) and then a curved stromal dissector (Figures 15-8A and B) (Devers Dissector, Bausch and Lomb, St. Louis, MO) extends the pocket completely to

B Figures 15-6A and B: Crescent blade.

A

B Figures 15-7A and B: Straight Devers Dissector.

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A Figure 15-9: Release of Healon from paracentesis wound.

B Figures 15-8A and B: Curved Devers dissector. Figure 15-10: Terry trephine displayed.

the limbus for 360 degrees, creating a total area, deep lamellar corneal pocket. The Devers Dissectors are designed with a tip that is not as sharp as a crescent blade, but is sharper than a blunt dissector. The width of the dissecting heads are especially good for maintaining the stromal depth consistently throughout the dissection of the edematous deep stroma and the surgeon can actually feel the increased resistance to dissection if he/she deviates too anteriorly. The dissection is accomplished with a slow and methodical sweeping motion of the dissector heads, from central to peripheral tissue, and the surgeon can often see the reflections of Descemet’s membrane wrinkling during the sweeping motion, which is an assurance that the depth of the dissection is adequate. It is important that the pocket stromal dissection be carried out over the entire area of the cornea, extending it all the way to the limbus for 360 degrees, in order to allow adequate space for the intralamellar trephine. The resection of the posterior recipient tissue begins by first softening the globe with the release of Healon through the stab incisions (Figure 15-9) from the anterior chamber. A specialized intrastromal trephine (Figure 15-10) (Terry

Figure 15-11: Terry trephine in pocket.

trephine, Bausch and Lomb, St. Louis, MO) is then placed into the stromal pocket (Figure 15-11), taking care to position it centrally in the cornea. Once the blade is in position, additional Healon is placed into the anterior chamber to raise the pressure of the anterior chamber and provide a “back-pressure” for the trephination. The Terry

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trephine comes in diameters of between 7.0 mm and 8.5 mm in 0.5 mm increments, with the most common diameter of 8.0 mm used in our prospective clinical series.10-13 The circular blade has a profile height of 250 µm and is intended for one-time use. In reality, a well cared for blade can be used 3 or 4 times with good clinical results before it requires re-sharpening or replacement. Once the circular trephine blade has been inserted into the pocket, the surgeon grasps the knurled handle and rotates the trephine in the same manner as a standard surface hand-held free trephine blade. The globe may need to be stabilized with a separate forceps for the rotational cut to be made effective. The blade is rotated clock-wise and counter clock-wise along the arc length of the 9.0 mm scleral incision. It makes the surgery easier if the initial point of entry into the anterior chamber is at the distal position of the cornea (i.e. the 6 o’clock position), and so the surgeon pushes down on the blade slightly greater at 6 o’clock than at 12 o’clock position, during rotation of the Terry trephine. As soon as the intrastromal Terry trephine enters the anterior chamber, the Healon in the anterior chamber enters the interface, the pressure drops, and the trephination is stopped. If the entry point is distal as hoped, then another entry into the anterior chamber is made at the 12 o’clock position with a diamond or steel keratome. This is placed through the superior scleral wound, into the pocket and then perforating into the anterior chamber at the 12 o’clock trephination mark. It is through this entry point that the recipient posterior resection is completed utilizing special scissors designed for posterior lamellar tissue resection (Cindy I and Cindy II Scissors, Bausch and Lomb, St. Louis, MO). The Cindy I scissors are placed with one blade in the anterior chamber and one blade in the stromal pocket. The scissors complete the trephination cut, following the mark of the circular trephination made previously by the Terry trephine (Figures 15-12A and B). The Cindy I scissors have long, highly curved and low profile blades and are ideally suited for this procedure. Once the resection has progressed distally to about the 5 o’clock and 7 o’clock positions, then the Cindy II scissors are utilized for completion of the distal resection. The Cindy II scissors have long, low profile blades that are set at nearly a right angle to easily complete the more difficult distal resection. If the Terry trephine had entered the anterior chamber over 2 or 3 clock hours distally as earlier intended, then the Cindy II scissors are not needed and the resection is completed faster and more easily. Once the posterior recipient disk has been cut 360 degrees, then the tissue is removed from the eye (Figures 15-13A and B) and placed on the corneal surface for inspection. It is washed with balanced salt solution (BSS, Alcon, Fort Worth, TX), and dried with a sponge (Figure 15-14) . The stromal

A

B Figures 15-12A and B: Cindy scissors completion of resection.

surface is inspected for smoothness and the edges for regularity of cut, as well as the thickness of the resected tissue. With the removal of the recipient posterior edematous stromal tissue, the view into the anterior chamber through the central cornea clears significantly (Figure 15-15) , and often other intraocular surgery such as vitrectomy, IOL exchange, and iridoplasty can be performed at this stage of the DLEK procedure. After removal of the recipient posterior tissue, the superior scleral wound is temporarily closed with 3 interrupted 10-0 nylon sutures. An irrigation/aspiration tip is then introduced into the anterior chamber and extensive effort is expended to remove all of the viscoelastic material from the eye (Figure 15-16) . There should be no Healon left in the anterior chamber prior to insertion of the donor disk or the donor tissue will not “stick” in place. Therefore, care is taken to irrigate and aspirate the anterior chamber, pupillary area, angle, and even the peripheral pocket as necessary. Once the surgeon is confident that all Healon has been removed, then the eye pressure is left slightly soft and attention is turned to preparation of the donor.

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A Figure 15-15: Clearing of cornea after recipient disk removal.

B Figures 15-13A and B: Recipient disk removal. Figure 15-16: I/A removal of Healon.

Figure 15-14: The recipient disk is irrigated with balanced salt solution and dried with a sponge.

Donor Tissue Preparation The operating microscope is brought over to the separate donor table for preparation of the donor tissue. Because whole globes are rarely available in the United States, an artificial anterior chamber is necessary for preparation of the donor posterior disk. We utilize a Bausch and Lomb

(St. Louis, MO) artificial anterior chamber (AAC) (See also Chapter 12, Artificial Anterior Chambers) that is all stainless steel and has dual irrigation/aspiration ports. The OptisolGS preservation fluid (Bausch and Lomb, Rochester, NY) from the donor tissue container is aspirated into a syringe and is then used to fill the I/A ports of the AAC. The syringe is also attached to the port to be used to vary the pressure inside the chamber for the duration of the resection. The standard donor corneoscleral cap tissue is first coated with a thin layer of Healon on the endothelium (Figure 15-17). It is then placed endothelial side down onto the post of the AAC (Figure 15-18) and oriented with the largest diameter of the cornea in the horizontal meridian. This meridian is marked with a marking pen so that the horizontal meridian of the donor tissue can be identified later in the procedure. The donor tissue is capped into place and the chamber is filled with Optisol-GS and the pressure normalized. An 8.5 mm diameter Barron suction recipient trephine (Katena Products, Denville, NJ) is placed onto the surface of the donor tissue and suction is applied (Figure 15-19). Trephination is carried out to about 60% depth with the trephine. It is noteworthy that after the blade touches the

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Figure 15-17: Healon on endothelium.

Figure 15-18: Cornea placement onto artificial anterior chamber.

Figure 15-19: Barron recipient trephine.

epithelial surface of the donor, it only takes about 4 or 5 quarter turns of the Barron radial vacuum trephine to reach this depth. This is much sooner than when the same trephine is used on the recipient in standard PKP surgery. The trephine is then removed and the cut inspected for depth. Ideally, an 80% depth should be attained for the

Figure 15-20: Dissection of donor cornea.

plane of the pocket of the donor tissue. Any deeper than this, and the donor tissue is so thin that it spontaneously rolls up like a rug causing confusion as to which side is the endothelial side and undoubtedly causing endothelial damage. If the dissection depth of the donor is less than 60% depth, then the stromal surface may not be as smooth and the tissue may be much thicker than the depth of the recipient bed. However, whether disparity between donor and recipient disc thicknesses causes a later visual problem is unknown at this time. Similar to the recipient disc preparation, the crescent blade is used to cut down to the 80% depth (Figure 15-20) and this depth and pocket is then extended over the entire area of the cornea, all the way to the limbus 360 degrees, using the straight and curved Devers Dissectors (Bausch and Lomb). After completing the deep stromal pocket formation, the cap of the chamber is gently rotated, taking care not to collapse the chamber, and the cap is removed. The donor tissue is then left on the post with a formed chamber. The scleral edges of the donor are gently lifted to release the tissue and the tissue is removed from the post, once again taking care not to collapse the chamber and damage the endothelium. After the tissue is lifted off the post, the endothelial side is gently irrigated with BSS to remove excess Healon and prevent it from contaminating the stromal pocket during the next stage of the preparation. The donor tissue is then placed endothelial side up onto a standard punch trephine block (Figure 15-21). Barron donor punch (Katena, Denville, NJ) is used. The same size diameter punch is used as the diameter of the Terry Trephine used for the recipient resection. A diameter 0.25 mm larger for the donor has been used, but the incidence of donor folds and dislocations increased with this disparity (unpublished data). The tissue is punched out with the trephine (Figure 15-22) , and if the dissection has been done properly, the surgeon will not hear that

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A Figure 15-21: Cornea on punch block—before punch.

B Figures 15-23A and B: Tissue placement onto Ousley spatula. Figure 15-22: Cornea on punch block—after punch.

familiar “crunch” sound that is so common with full thickness PKP donor trephination. Instead the sound is much quieter or not present at all. While it is best to have good centration of the punch, if the stromal dissection of the donor has been carried out all the way to the limbus for 360 degrees, it will be fine. If the dissection has not been carried all the way out to the limbus, and the surgeon has an eccentric punch, then the posterior donor disc may have a 1 mm thick edge and an opposite 100 micron thick edge. This wedge of tissue will not adhere to the recipient bed, and so care in completion of the total donor stromal pocket is advised prior to punching out the tissue. The donor posterior disc is prepared for insertion into the eye by placing the donor disc on an Ousley insertion spatula (Bausch and Lomb) (Figures 15.23A and B). The Ousley spatula is a round, flat spatula with a diameter of 8.5 mm and a proximal anterior ledge that helps to prevent proximal movement of the donor tissue. The Ousley spatula is coated with a very thin layer of Healon to protect the endothelium, and then the donor disk is gently grasped at the stromal edge with fine forceps, separated from the

anterior stromal tissue and then placed endothelial side down onto the Ousley spatula (Figures 15-23A and B) . The donor tissue is then brought over to the operative field for insertion.

Transplantation of the Donor Tissue With the microscope in place, the temporary scleral sutures of the superior wound are cut. The anterior chamber of the patient is then filled completely with an air bubble (Figure 15-24). The Ousley spatula with the donor tissue is then brought into position by the limbal wound, the anterior lip of the wound is stabilized with a fine-toothed forceps, and then in one deft movement, the Ousley spatula is inserted into the anterior chamber while the anterior lip of the wound is slightly elevated (Figures 15-25 and 15-26). The spatula is inserted parallel to the iris and once in the anterior chamber it is lifted anteriorly until the stromal surface of the donor and recipient are coapted (Figure 15-27). The spatula is then gently removed from the eye (Figure 15-28), sliding on a layer of Healon, leaving the donor tissue behind, supported by a residual air bubble in the anterior chamber (Figure 15-29). A

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Figure 15-24: Air bubble in recipient anterior chamber.

Figures 15-27 and 15-28: Insertion of disk—end stage. Figure 15-25: Insertion of disk—beginning stage.

Figure 15-26: Insertion of disc—middle stage.

Figure 15-29: Air bubble behind donor corneal graft.

single suture of 10-0 nylon is then used to close the central scleral wound to secure the chamber and prevent escape of the donor tissue. An air bubble is then gently injected into the anterior chamber to further support the donor tissue. If the air is injected too rapidly and the superior wound does not have a suture in place, then the donor disk will be rapidly expulsed from the eye.

The donor disk may not be in perfect centration after insertion. If not, it can be positioned from either the endothelial side or the stromal side. If positioning is necessary, then the superior 9.0 mm wound must be completely closed first with 10-0 nylon suture (interrupted or running) to stabilize the chamber. Once secured, a large air bubble is injected into the anterior chamber to fill the

Deep Lamellar Endothelial Keratoplasty (DLEK): Large Incision Technique

Figure 15-30: Positioning of graft with a Sinskey hook.

chamber. A reverse Sinskey hook (Bausch and Lomb) is used for endothelial side positioning. The hook is placed through the stab incision, the peripheral endothelium is engaged, and the tissue moved over to whatever position is desired (Figure 15-30). Although this maneuver undoubtedly causes endothelial damage at that point of peripheral contact, we have not found that the central endothelial cell counts 6 months after surgery are any worse than after standard PKP.10,11,14 Care is taken, however to minimize this maneuver and also to avoid the central posterior striae that can occur and can compromise vision. An alternative technique for positioning can be done from the stromal interface side using a 30-gauge needle tip. A slight “barb” is placed on a standard short 30-gauge needle, and the tip is placed through the superior wound directly into the interface. The barb is rotated posteriorly to engage a few stromal fibers of the donor disc, and this grasp is used to move the tissue over into the proper centration. During both the endothelial and stromal positioning maneuvers, the anterior chamber is filled with air. Once the tissue is in proper centration, it is critical to make sure that all of the donor edges are anterior to all of the recipient bed edges for 360 degrees. Visual inspection is not enough, and manual verification is mandatory. If any portion of the donor tissue edge lies posterior to the recipient rim, then the donor tissue will likely be dislocated the next morning or present with a significant space in the interface (secondary anterior chamber). To accomplish proper donor edge position, the anterior chamber is filled completely with air and the reverse Sinskey hook is placed through a stab incision into the anterior chamber. The tip of the hook is then lifted anteriorly and placed between the edge of the donor and recipient rim. The hook is then rotated to engage the recipient rim posterior stromal edge, and then used to pull the edge posteriorly. With this maneuver, the air bubble in the anterior chamber immediately pushes the

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donor edge up anteriorly, into the recipient pocket, and upon release of the Sinskey hook, the recipient edge pops right up posterior to the donor edge. This “tire iron” maneuver is performed for 360 degrees, even when the donor tissue appears to be already in a good position. This is done because even small strands of recipient stromal edge tissue can get caught in the edge interface and prevent adherence of the graft or act as a wick for aqueous into the interface, causing late dislocation of the donor disc. Once satisfied that the donor disk is in final position with good edge position, the surgeon then removes the air in the anterior chamber and replaces it with BSS (Figure 15-31). Care is taken to avoid pupillary block by the air bubble in the anterior chamber, but if it occurs, simple suctioning of the air from the pupillary surface resolves the problem. Occasionally air can get trapped behind the iris, giving the impression of posterior pressure with the iris coming forward to the donor edges. Suctioning the air from the pupillary surface with a cannula will resolve this issue. The BSS placed into the anterior chamber creates a normal IOP and the chamber deepens. A small (3.0 mm wide) air bubble is usually left in place to help further stabilize the donor disc position over the first 24 hours postoperatively. All of the scleral wound sutures are tied slightly tight to induce about 2 diopters of vertical, steep, with-the-rule astigmatism. This is done because the 9.0 mm wound has a tendency to cause 1 or 2 diopters of against-the-rule astigmatism over time if the wound is left topographically neutral at the conclusion of surgery. The suture knots are cut short, and buried on the scleral side. The wound is checked to be watertight. The conjunctival peritomy is closed. We routinely place on the corneal surface a 24-hour collagen shield soaked in antibiotics and steroids at the end of the surgery in order to deliver medication until the patch is removed the next day. However, each surgeon’s usual routine for antibiotics (subconjunctival or otherwise) is certainly acceptable.

Figure 15-31: Appearance at end of surgery.

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An occlusive patch and shield are routinely placed over the operated eye, and the patient is brought to the recovery room. We usually instruct the nurses to have the patient lie in a supine position for the first few hours as much as possible to allow the retained air bubble to further stabilize the graft position, but this is not critical. The patient is discharged from this outpatient procedure when fully recovered from anesthesia.

Postoperative Course The patient is seen the next morning and the patch is removed. Most patients will remark that the eye was as comfortable as after standard cataract surgery and that they did not require any narcotic medication for relief of pain. Patients usually do not have eye pain after DLEK surgery. Once the patch is removed, the vision is usually about 20/ 400. The vision is unimportant on postoperative day one, and the only reason for the visit is to insure that the donor disc is attached and in good position. In our prospective series of over 115 patients (as of July 2004), we have experienced only 5 cases where the donor disc was dislocated on the first postoperative day. All five cases were easily treated by taking the patient back to surgery, and usually under topical anesthesia, another air bubble is placed in the anterior chamber and the disc repositioned as before. This is usually a 15-minute operation. We have been successful with all five re-positionings resulting in clear corneal grafts. If the graft is in good position on day one, it will heal in good position, and we have had no late graft dislocations. The edges of the graft seal down with solid healing sometime within the first 3 months. The overlying cornea has a variable rate of clearing, but some patients are able to see as well as 20/25 one week after DLEK surgery with a crystal clear central cornea. The usual visual progression postoperatively of patients with minimal or no macular disease, however, is as follows: One day, 20/400; one week, 20/100; one month, 20/60; three months, 20/50; six months, 20/40; one year, 20/30; and two years, 20/25. Of course, there is a high variability of vision in any series of elderly patients undergoing ocular surgery, but especially DLEK. The interface may clinically appear exceptionally clear, but it likely contributes about one line of visual loss to the macular potential.13,14 The postoperative medical therapy after DLEK surgery is identical at this time to what is done with PKP surgery patients. Topical prednisolone acetate 1% suspension (Pred Forte 1%, Allergan Inc., Irvine, CA) is used four times a day for 3 months, then three times a day until 6 months, then twice a day until 9 months, and then once a day until one

year postoperatively. The steroids are then tapered down further until discontinued entirely. We have experienced only a 3% rejection rate after DLEK surgery, and so steroid therapy may not be as critical as after PKP, but this remains as a speculation at this point. Fluoroquinolone antibiotics are used on a four times a day dosage for two weeks only and then discontinued. Outside of a scientific protocol, DLEK patients do not require the same degree of monitoring as standard PKP patients and therefore require less postoperative clinic time. With no sutures or corneal incisions, wound healing or corneal ulcerations are not an issue. Astigmatism management is also not an issue after DLEK surgery, much to the joy of patient and the surgeon alike! The only critical monitoring is for steroid-induced glaucoma as long as the patient is on topical steroids, and this is done according to the clinician’s standard routine. The DLEK surgical procedure is a difficult one and requires a commitment to exacting detail and thorough practice prior to incorporation of this procedure into the surgeon’s operative repertoire. However, with its superior topography, rapid wound healing and long-term safety, the DLEK procedure is well worth the effort.

References 1. Sugar A, Sugar J. Techniques in penetrating keratoplasty: A quarter century of development. Cornea 2000;19:603-10. 2. Abou-Jaoude ES, Brooks M, Katz DG, Van Meter WS. Spontaneous wound dehiscence after removal of single continuous penetrating keratoplasty suture. Ophthalmology 2002;109:1291-6. 3. Tseng SH, Lin SC, Chen FK. Traumatic wound dehiscence after penetrating keratoplasty: Clinical features and outcome in 21 cases. Cornea 1999;18:553-8. 4. Stechschulte SU, Azar DT. Complications after penetrating keratoplasty. Int Ophthalmol Clin 2000;40:27-43. 5. Akova YA, Onat M, Koc F, Nurozler A, Duman S. Microbial keratitis following penetrating keratoplasty. Ophthalmic Surg Lasers 1999;449-55. 6. Confino J, Brown SI. Bacterial endophthalmitis associated with exposed monofilament sutures following corneal transplantation. Am J Ophthalmol 1985;99:111-3. 7. Ko WW, Frueh BE, Shields CK, Costello ML, Feldman ST. Experimental posterior lamellar transplantation of the rabbit cornea [ARVO Abstract]. Invest Ophthalmol Vis Sci 1993;34(4):S1102. Abstract # 1967. 8. Melles GR, Eggink FA, Lander F, Pels E, Rietveld FJ, Beekhuis WH, Binder PS. A surgical technique for posterior lamellar keratoplasty. Cornea 1998;17:618-26. 9. Terry MA, Ousley PJ. Endothelial replacement without surface corneal incisions or sutures: Topography of the deep lamellar endothelial keratoplasty procedure. Cornea 2001;20:14-18. 10. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty in the first United States patients: Early clinical results. Cornea 2001;20:239-43. 11. Terry MA, Ousley PJ. Replacing the endothelium without corneal surface incisions or sutures: The first United States clinical series using the deep lamellar endothelial keratoplasty procedure. Ophthalmology 2003;110:755-64.

Deep Lamellar Endothelial Keratoplasty (DLEK): Large Incision Technique 12. Terry MA, Ousley PJ. In pursuit of emmetropia: Spherical equivalent refraction results with deep lamellar endothelial keratoplasty (DLEK). Cornea 2003;22:619-26. 13. Terry MA, Ousley PJ. Rapid visual rehabilitation after endothelial transplants with deep lamellar endothelial keratoplasty (DLEK). Cornea 2004;23:143-53. 14. Terry MA, Ousley PJ. Small incision deep lamellar endothelial keratoplasty (DLEK): Six-month results in the first prospective clinical study. Cornea 2005; 24:59-65. 15. Terry MA. Endothelial replacement: The limbal pocket approach. Ophthalmol Clin North Am 2003;16:103-12. 16. Terry MA. Deep lamellar endothelial keratoplasty (DLEK): Pursuing the ideal goals of endothelial replacement. Eye 2003;17:982-8. 17. Terry MA. A new approach for endothelial transplantation: Deep lamellar endothelial keratoplasty. Int Ophthalmol Clin 2003;43:183-93. 18. Terry MA, Ousley PJ. Corneal endothelial transplantation: Advances in the surgical management of endothelial dysfunction. Contemporary Ophthalmology 2002;1(26):1-8. 19. Terry MA. Endothelial replacement: New surgical strategies. In: Krachmer J, Mannis M, Holland E, eds. Cornea. Surgery of

20. 21. 22. 23. 24. 25. 26. 27.

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the cornea and conjunctiva. 2nd ed. St. Louis: Mosby-Year Book, Inc. 2004 (in press). Terry MA. The evolution of lamellar grafting techniques over twenty-five years. Cornea 2000;19:611-6. Duran JA, Malvar A, Diez E. Corneal dioptric power after penetrating keratoplasty. Br J Ophthalmol 1989;73:657-60. Binder PS. The effect of suture removal on postkeratoplasty astigmatism. Am J Ophthalmol 1988;105:637-45. Isager P, Hjortdal JO, Ehlers N. Stability of graft refractive power after penetrating keratoplasty. Acta Ophthalmol Scand 2000;78:623-6. Davis EA, Azar DT, Jakobs FM, Stark WJ. Refractive and keratometric results after the triple procedure: Experience with early and late suture removal. Ophthalmology 1998;105:624-30. Dursun D, Forster RK, Feuer WJ. Surgical technique for control of postkeratoplasty myopia, astigmatism, and anisometropia. Am J Ophthalmol 2003;135:807-15. Segev F, Voineskos AN, Hui G, Law MS, Paul R, Chung F, Slomovic AR. Combined topical and intracameral anesthesia in penetrating keratoplasty. Cornea 2004;23:372-6. Armour RL, Wilson DJ, Ousley PJ, Terry MA. Invest Ophthalmol Vis Sci 2004;45:ARVO E-Abstract 2898.

DLEK: A Procedure for Special Cases of Endothelial Dysfunction

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Mark A Terry

Deep Lamellar Endothelial Keratoplasty (DLEK): A Procedure for Special Cases of Endothelial Dysfunction

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Introduction Penetrating keratoplasty (PKP) has long been the standard treatment for patients with vision loss from advanced corneal edema due to endothelial dysfunction.1 However, the surface sutures and the vertical wounds which are produced by PKP present an inherent liability for long-term graft survival and visual function with most patients suffering some visual degradation from irregular astigmatism and a few suffering total visual loss from suture induced infections or wound induced traumatic globe rupture.2-6 A technique of selective endothelial keratoplasty (EK) which would avoid corneal sutures and vertical stromal wounds would eliminate many of the risks of transplant surgery and speed the visual recovery for the patient. In 1993 Drs Ko and Feldman from San Diego, California presented an animal study at the annual ARVO meeting which described a new technique for endothelial replacement through a scleral limbal incision7 (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). Dr Gerrit Melles, a Dutch ophthalmologist working as a fellow with Dr Perry Binder in San Diego developed this technique further in monkeys, utilizing a 9 mm scleral incision size. 8 In 1998 he described this technique in the first human patient and called it posterior lamellar keratoplasty (PLK)9 (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). Terry and Ousley began laboratory work in 1999 on this technique, and after their technical modifications, re-design of instrumentation, and safe introduction of Healon (Pfizer, New York, NY) to the procedure, this surgery was made considerably easier. Terry performed the first United States cases in 2000 and called the surgery Deep Lamellar Endothelial Keratoplasty (DLEK).10-19 All of this work represents a radical departure from the PKP technique in that the DLEK surgery accomplished the goal of endothelial replacement without surgically violating the surface of the recipient cornea. By eliminating surface corneal sutures and incisions, the advantages of normal corneal topography and faster wound healing were obtained, leading to faster visual rehabilitation and a more stable globe for the patient.20,21 In 2002, a small, 5 mm length scleral incision technique of PLK surgery was described in a case report by Melles et al.22 We have investigated this technique in the largest prospective series of small incision DLEK in the world and have found it to be valid for endothelial replacement surgery.15,21 Currently, further modifications have been made in the field of EK. A technique whereby only the posterior Descemet’s layer is stripped off was originally described by Melles and termed PLK with Descemetorhexis23 (See also

Chapter 14, History of Lamellar and Penetrating Keratoplasty). It was later popularized by Frank Price in the US as Descemet’s Stripping Endothelial Keratoplasty (DSEK),24 and most recently has been further popularized by Mark Gorovoy as Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK). Regardless of the name, the Descemet’s stripping technique of EK has been proven to be a technically easier alternative to DLEK surgery, and it has now been embraced by 50% or more of the corneal transplant surgeons in the US as the procedure of choice for EK surgery. Other developments in EK such as the use of the femtosecond laser for stromal dissections25-27 [See also Chapter 26, Femtosecond Laser (Intralase®)—Descemet’s Stripping Endothelial Keratoplasty (Femto-DSEK)]: Initial Studies of Surgical Technique in Human Eyes), the use of a microkeratome for donor preparation28,29 (See also Chapter 12, Artificial Anterior Chambers), and the use of donor tissue that has been “pre-cut” by the distributing eye bank (See also Chapter 19, Eye Banking and Donor Corneal Tissue Preparation in DSAEK, and Chapter 30, Use of Eye Bank Pre-cut Donor Tissue in DSAEK), are also contributing to the mainstream acceptance of EK surgery.

Indications and Rationale for DLEK Surgery Although DSAEK surgery is currently our procedure of choice for standard endothelial replacement surgery [See also Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)] we have found that DLEK surgery still has a place in selected complex cases which require EK surgery, and DLEK should remain part of the surgical armamentarium of the EK surgeon. Certain cases of endothelial dysfunction would entail too much risk of dislocation or graft failure with the standard DSAEK surgical treatment, and would be better off with a simple DLEK surgery. For example, in cases of pseudophakic bullous keratopathy (PBK) with the presence of an anterior chamber lens, we feel that DLEK is by far a safer procedure than DSAEK if the anterior chamber lens is to be retained postoperatively. This is due to the fact that maintenance of the air bubble placed at the end of DSAEK surgery is critical to the success of this surgery. If the recipient eye has an anterior chamber IOL in place, then an air bubble cannot be maintained in the anterior chamber long enough to support the tissue in DSAEK surgery because it migrates into the posterior chamber as soon as the patient sits up. Loss of the air bubble support in DSAEK with subsequent dislocation of the endothelial donor graft onto the plastic surface of the anterior chamber IOL can result in permanent

DLEK: A Procedure for Special Cases of Endothelial Dysfunction endothelial damage and possible graft failure. However, in DLEK surgery, an air bubble is not even necessary postoperatively for the support of a DLEK graft. In over 250 cases of DLEK, we routinely removed the entire air bubble at the conclusion of DLEK surgery without any significant graft dislocation (dislocation rate of 4%), and in all cases of DLEK with an anterior chamber IOL left in place, we have never had a dislocation. Therefore, in any instance in which an air bubble cannot be maintained for an extended period of time postoperatively for graft support due to an open communication between the anterior and posterior chambers (e.g. aphakic bullous keratopathy, PBK with an anterior chamber IOL in place, aniridia, or presence of a larger peripheral iridectomy), then, a small incision DLEK surgery is the procedure of choice. The purpose of this chapter is to describe the technique and results of standard small incision DLEK surgery in a large prospective series. The data from this DLEK prospective series21 should be used as a benchmark to determine the advantages and disadvantages of further modifications in EK.

The Small Incision DLEK Procedure We originally utilized a 9 mm length, scleral limbal incision for DLEK surgery [(See also Chapter 15, Deep Lamellar Endothelial Keratoplasty (DLEK): Large Incision Technique)] and this was utilized in our first 36 eyes in our series. It has been well described in our previous reports.10-12 With the advent of the smaller 5 mm incision technique, we then adopted this as our procedure of choice for the remainder of the 62 eyes in this study. The technique described here is the small incision technique of DLEK, with or without concurrent cataract extraction. We have previously described this surgical technique in both print and video,15-21,30 the step by step description given below is for more extensive instruction and explanation (See Figures 16-1A to L).

Anesthesia DLEK surgery is usually done under retrobulbar block anesthesia especially by the more experienced surgeon, but general anesthesia has also been used. General anesthesia (either endotracheal or laryngeal mask airway technique) is preferred for the novice EK surgeon because it minimizes posterior pressure on the globe and this is important during the recipient resection and donor implantation phases of the surgery. Nonetheless, the surgery is safely accomplished with good retrobulbar anesthesia combined with seventh nerve block (orbicularis block) local anesthesia as

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well. It is also quite possible, for the severely medically frail patient, that this surgery could be accomplished with only topical anesthesia, similar to what has been done in PKP surgery under similar circumstances. However, this has not yet been done with DLEK surgery and likely would require a surgeon with the ability to accomplish this surgery in less than an hour.

Preoperative Preparation Like all intraocular procedures, the patient’s ocular health should be maximized beforehand and any blepharitis, dry eye, or lid abnormality should be treated prior to surgery. Patients with some mild to moderate corneal surface haze or scarring from long-standing bullous keratopathy can still undergo DLEK surgery successfully. The surface scarring is simply scraped off at the time of surgery or weeks later in clinic after the stromal edema has completely resolved. This eliminates the induced irregular astigmatism from the scars and restores the normal topography. In the usual case of patients with pseudophakia (posterior chamber IOL) the pupil is constricted in order to stabilize the iris-lens diaphragm during the surgery. This is also done if the patient has a clear crystalline lens and concurrent cataract surgery is not planned. Preoperative medications include two sets of pilocarpine 1% drops applied one hour prior to surgery. One set of aproclonidine 0.5% drops is also given just prior to surgery to reduce pressure and minimize conjunctival injection. Preoperative antibiotics are used according to the surgeon’s preference. The eye is prepped in the usual sterile ophthalmic fashion with the use of povodone-iodine solution.

Recipient Surgery: Small Incision DLEK Portion The operating microscope is positioned for the surgeon to be seated at the temporal side of the patient. (For small incision DLEK surgery, the scleral access incision is 5 mm, and therefore the incision is usually placed at the temporal limbal region rather than superiorly). The patient’s head should be positioned facing the ceiling, parallel to the floor. In addition, if general anesthesia is employed, the endotracheal tube is also positioned by the anesthesiologist to exit the mouth from the side opposite the surgical field to facilitate the surgeon’s field of movement. A superior and inferior rectus bridal sutures can also be placed if necessary to aid in positioning and stabilizing the globe during DLEK surgery. A temporal limbal peritomy of the conjunctiva is performed with scissors allowing exposure of about 6 mm

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Figures 16.1A to L: (A) The surface of the recipient cornea is marked with a circular template of 8.0 to 9.0 mm diameter, depending on the size of the patient’s cornea; (B) A 5.0 mm scleral incision adjacent to the temporal limbus is made, and a scleral-corneal pocket incision is made into clear the cornea; (C) The anterior chamber is completely filled with Healon, and the straight Devers Dissector is used to begin the deep lamellar pocket dissection; (D) The curved Devers Dissector is used to create a total deep lamellar pocket, limbus to limbus, if possible; (E) The Cindy I Scissors are used to excise the posterior tissue, one blade in the pocket, one blade in the anterior chamber, with the anterior chamber filled with Healon. This is followed by the Cindy II Scissors for the distal resection edges; (F) All of the Healon is easily removed from the eye with an irrigationaspiration tip; (G) The donor tissue is mounted onto a Bausch and Lomb artificial anterior chamber. The tissue is prepared using manual dissection after reaching the 350 µm depth from a peripheral limbal corneal incision. Surface marks are present to help visualize and maintain the depth of the dissection; (H) The donor posterior disk is punched out using a same diameter trephine, and after a strip of Healon is placed on the central endothelium, the tissue is folded into a 60%/40% “taco” shape to prepare for insertion; (I) Special Charlie insertion forceps are used to grasp the donor tissue and insert it into the recipient anterior chamber; (J) The chamber is deepened with balanced salt solution to begin unfolding the tissue and then the tissue unfolding is completed with placement of an air bubble between the lips of the taco; (K) Once the donor tissue is unfolded and is in position, with the chamber filled with air, the Nick Pick is used to pull the recipient edges posterior to the donor tissue edges, locking the donor corneal tissue in place; (L) The air in the anterior chamber is replaced with balanced salt solution and a residual air bubble with a diameter of only 3 mm or less is left in place.

DLEK: A Procedure for Special Cases of Endothelial Dysfunction arc length of limbal tissue (about 3 clock hours). Prior to forming the DLEK scleral access incision, two clear corneal limbal stab incisions (about 1 mm diameter) are placed on either side of the peritomy area, to be used as access points to the anterior chamber later in the operation. Through one of the stab incisions, the cohesive viscoelastic Healon (Pfizer, New York, NY) is placed into the anterior chamber to replace the aqueous fully and to maintain normal pressure. We strongly oppose the use of Viscoat (Alcon, Fort Worth, TX) or other dispersive viscoelastic materials during any portion of DLEK surgery as the dispersive materials can cause stromal interface coating with subsequent non-adherence and dislocation of the donor tissue. In cases of corneal endothelial decompensation with cataract, a triple procedure can be performed, namely, DLEK combined with phacoemulsification and PC IOL.31, 32 Prior to creating the deep lamellar pocket of DLEK, a template mark is placed on the corneal epithelial surface. A circular marker with a diameter of 8.0 or 8.5 mm (depending upon recipient corneal diameter and surgeon preference) is used to make a circular impression on the central epithelial surface. If the position and centration of the mark is acceptable to the surgeon, then it is accentuated with ink marks. This circle on the cornea will later be used as a template for resection of the posterior recipient lamellar corneal tissue. A trifaceted, guarded, diamond knife is then set to a depth of 350 microns and a 5.0 mm length incision is made approximately 1 mm posterior to the corneal limbus and concentric with it. We have found that a deeper initial incision gives less of a beveled wound closure and also a greater chance of early perforation into the anterior chamber during DLEK surgery. In lieu of a diamond knife, a sharp crescent blade or other steel scalpel can be used for the initial incision. A sharp crescent blade is then utilized to create a deep scleral-corneal lamellar pocket down to about 75 to 85% corneal depth along the entire length of the wound. Perfect accuracy of the depth of the corneal stromal pocket does not appear to be critical for a good visual outcome. Pockets should be deeper than 50% in order to avoid interface scarring or haze, and conveniently should not be deeper than 95% depth in order to avoid donorrecipient thickness mismatch. Judgment of the initial depth of the pocket is based upon inspection of the anterior lip thickness and by the clarity of the underlying stromal bed. Experience with the procedure and with lamellar dissections in general aids in the confidence that the desired depth has been achieved. A specialized semi-sharp stromal dissector is then used to extend the pocket to the mid-pupillary region of the cornea and then a curved stromal dissector (Devers

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Dissector, Bausch and Lomb, St. Louis, MO) extends the pocket further. We prefer to have a pocket that extends at least 1 mm peripheral to the diameter of the surface template circular mark (i.e. 10.0 mm pocket diameter for an 8.0 mm mark). This creates a large area, deep lamellar corneal pocket. The Devers Dissectors are designed with a tip that is not as sharp as a crescent blade, but is sharper than a blunt dissector. The width of the dissecting heads are especially good for maintaining the stromal depth consistently throughout the dissection of the edematous deep stroma and the surgeon can actually feel the increased resistance to dissection if he/she deviates too anteriorly. The dissection is accomplished with a slow and methodical sweeping motion of the dissector heads, from central to peripheral tissue, and the surgeon can often see the reflections of Descemet’s membrane wrinkling during the sweeping motion, which is an assurance that the depth of the dissection is adequate. It is important that the pocket stromal dissection be carried out over the entire desired area of the cornea, in order to allow adequate edge space for the donor disk. The resection of the posterior recipient tissue begins by first entering the anterior chamber through the temporal scleral corneal pocket incision. We utilize a standard cataract surgery diamond blade with a 2.8 mm width, but any blade is acceptable. Entry into the anterior chamber is preferred at the exact corresponding position of the temporal edge of the surface template mark. It is through this entry point that the recipient posterior resection is started utilizing special scissors designed for posterior lamellar tissue resection (Cindy I and Cindy II Scissors, Bausch and Lomb, St. Louis, MO). The Cindy I scissors are placed with one blade in the anterior chamber and one blade in the stromal pocket. The scissors is used to perform a free-hand cut, following the marks of the circular template on the overlying epithelial surface. The Cindy I scissors have long, highly curved and low profile blades and are ideally suited for this procedure. Once the resection has progressed distally to about the 5 o’clock and 7 o’clock positions, then the Cindy II scissors are utilized for completion of the distal resection. The Cindy II scissors have long, low profile blades that are set at nearly a right angle to easily complete the more difficult distal resection. Once the posterior recipient disk has been cut for the full 360 degrees, then the tissue is removed from the eye and placed on the corneal surface for inspection. It is washed with balanced salt solution (BSS, Alcon, Fort Worth, TX), and dried with a sponge. The stromal surface is inspected for smoothness and the edges for regularity of cut, as well as the thickness of the resected tissue. With removal of the recipient posterior edematous stromal tissue, the view into

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the anterior chamber through the central cornea clears dramatically, and often other intraocular surgery such as cataract surgery, vitrectomy, IOL exchange, and iridoplasty can be performed at this stage of the DLEK procedure. After removal of the recipient posterior corneal tissue, the temporal scleral wound is temporarily closed with 1 interrupted 10-0 nylon suture. An irrigation/aspiration tip is then introduced into the anterior chamber and extensive effort is expended to remove all of the viscoelastic material from the eye. No residual Healon should remain in the anterior chamber prior to insertion of the donor disk or the donor tissue will not “stick” in place. Therefore, care is taken to irrigate and aspirate the anterior chamber, pupillary area, angle, and even the peripheral pocket as necessary. Once the surgeon is confident that all Healon has been removed, then the pressure is left slightly soft and attention is directed to the preparation of the donor corneal disk.

Donor Corneal Preparation This step of donor corneal preparation can be done just prior to the surgery on the patient’s eye, depending on surgeon preference. “Pre-cut” tissue is also available now through several EBAA certified Eye Banks (See also Chapter 30, Use of Eye Bank Pre-cut Donor Tissue in DSAEK) and its use is described below. The operating microscope is brought over to the separate donor table for preparation of the donor tissue. Because whole globes are rarely available here in the United States, an artificial anterior chamber is necessary for preparation of the donor posterior disc (See also Chapter 12, Artificial Anterior Chambers). The only automated microkeratome system available for DSAEK surgery is from Moria (Moria, Antony, France). The Optisol-GS preservation fluid (Bausch and Lomb, Rochester, NY) from the donor tissue container is aspirated into a syringe (which has a three way stopcock) and the syringe is then used to fill the I/A port of the artificial anterior chamber. The syringe is also attached to the port to be used to vary the pressure inside the chamber for the duration of the resection. The standard donor corneoscleral cap tissue is first coated with a thin layer of Healon on the endothelium. It is then placed with the endothelial side down onto the post/piston of the artificial anterior chamber (taking care not to include an air bubble in the chamber) and the tissue is oriented with the largest diameter of the cornea in the horizontal meridian. The post/ piston of the unit is then raised until the tissue is firmly locked into place (See also Chapter 12, Artificial Anterior Chambers). The Optisol filled syringe is then used to raise the pressure in the artificial chamber to over 65 mm Hg and

the stopcock is closed to stabilize the high pressure. The epithelial cells are then wiped from the surface of the cornea with a Miracel sponge. The horizontal meridian is marked at the peripheral cornea with a marking pen so that the horizontal meridian of the donor corneal tissue can be identified, and these marks are also used for the proper orientation of the anterior corneal cap following the microkeratome cut. For anterior automated lamellar keratoplasty, the guide ring for the microkeratome can be adjusted in height to yield the desired diameter for the tissue resection. For EK surgery, the greatest diameter possible of at least 10 mm is required, and therefore the guide ring should be placed at the lowest possible position. Usually, a 300 µm head is used with the microkeratome, and the diameter of resection is at least 9.5 mm or more. Intraoperative pachymetry is advised to determine if the tissue is thick enough to allow the 300 micron head resection and therefore avoid the occurrence of posterior perforation or “button holes” of the tissue [See also Section 9, Descemet Stripping Automated Endothelial Kerato-plasty (DSAEK)]. The ideal thickness for the posterior tissue to be transplanted is about 150 microns, but significant variance of greater or lesser depth can occur with the 300 micron head microkeratome. With the pressure in the artificial chamber elevated to at least 65 mm Hg and verified with a gravity tonometer or by finger touch, the microkeratome head is mounted on the guide ring, positioned for resection, and then passed over the donor cornea at a rate of about 4 to 5 seconds for the pass. A free cap of anterior tissue is resected and remains above the blade on the microkeratome head. After drying the residual stromal bed with Miracel sponges, checking the stromal bed for smoothness of cut and diameter of cut, several marks are placed at the peripheral edge of the resected bed to help with the centering of the subsequent trephination of the donor tissue. The anterior cap is placed back in position, using the previously placed peripheral reference surface marks. One additional mark is then placed at the exact central point of the anterior cap of tissue to further facilitate centration of the posterior trephination. A moment is given for the anterior cap to adhere to the bed. The donor tissue now must be carefully dismounted from the artificial anterior chamber without damaging the endothelial cells from chamber collapse. We have found that the easiest way to avoid chamber collapse is to leave the tissue attached to the post/piston. To achieve this, the stopcock on the syringe is turned to the position to allow Optisol flow from the syringe to the chamber. A tying forceps is then used to sweep along the inside ring of the metal cap which locks the tissue onto the post, pushing

DLEK: A Procedure for Special Cases of Endothelial Dysfunction slightly posterior on the scleral rim, and breaking the seal that binds the donor scleral tissue to the metal cap. Very gently the post/piston is lowered, and Optisol is gently infused, as needed, to keep the donor chamber from collapsing. The locking cap is then removed, with the donor tissue left on the post with a formed chamber. The scleral edges of the donor are gently lifted in each quadrant to release the seal of the tissue to the post, and the tissue is removed from the post very slowly, lifting the scleral edge up to allow air to enter the chamber, and as the tissue is further lifted off, the air slowly pushes the Healon along, and the Healon then simply flows off the opposite scleral edge in one cohesive unit from the endothelial layer onto the post. Once again care is taken not to collapse the chamber and damage the endothelium. We believe that minimal irrigation of the endothelium with Optisol during tissue removal from the Moria unit aids in the health of the endothelium and in the subsequent donor adherence to the recipient bed. Once the donor corneoscleral tissue has been removed from the post, we have been gently irrigating the sclera above and below the endothelial surface of the donor tissue with Optisol solution (taken from the same transport container for the donor tissue) in order to remove excess residual Healon from the donor. We then use a Miracel sponge to dry the excess fluid from the endothelium, placing the sponge tip at the scleral edge, away from the endothelium. The donor tissue is then placed endothelial side up onto a standard punch trephine block. The previously placed ink mark on the central point of the anterior resected cap is used as a guide to position the tissue for trephination, in order to make sure that the posterior punch trephination is centered on the bed of the previous keratome pass. We utilize a Barron donor punch (Katena, Denville, NJ). The same size diameter punch is used as the diameter of the Descemet’s membrane-stromal disk that was removed from the recipient. The tissue is punched out with the trephine. Because the 5 mm wound of small incision DLEK surgery is smaller than the 8.0 mm (or larger) diameter of the donor disk, the donor tissue must be folded prior to insertion. To accomplish this, a very thin strip of Healon is placed onto the endothelial surface along the previously identified and marked horizontal meridian of the donor button. Stabilizing the anterior edge of the donor button with a 0.12 mm forceps, the posterior stromal tissue edge is gently grasped with non-toothed delicate forceps such as Utrata forceps. The posterior tissue is then gently folded with the endothelium on the inside protected by the layer of Healon, and it is folded into an asymmetric “taco” shape, in a 60/40% ratio, the most anterior side of the taco being 60% and the posterior

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side 40%. We were the first to initiate the idea of an “overfolded, 60/40% ratio” over 6 years ago (in 2001) in order to avoid having the tissue unfold upside-down in the patients anterior chamber. The donor tissue is then brought over to the operative field on the trephine block.

Donor Tissue Preparation Using Pre-Cut Tissue When using a donor corneal tissue that was pre-cut by an eye bank using a microkeratome or a femtosecond laser and then shipped to the hospital or a surgery center, the surgeon must fully inspect the tissue prior to transplantation. The surgeon should be very careful to ensure that he knows the exact diameter of the resected bed and that he personally marks the edges of the resected bed of the donor tissue. In this way, the surgeon can avoid an eccentric cut that goes outside of the microkeratome cut bed, with the resultant 1mm thick donor edge. Using pre-cut tissue is very easy and fast, but to avoid complications, attention to details is paramount. The precut tissue is removed from the Optisol container and simply placed endothelial side down onto the lint-free plastic or metal surface of the donor table. Because the donor scleralcorneal tissue has a 3 mm rim of scleral “skirt” and tends to maintain its convex configuration, this manipulation of the tissue will not collapse the tissue or risk damage to the endothelium. Very gently, the surgeon dries the epithelial surface of the peripheral donor cornea with a Miracel sponge and reveals the edges of the microkeratome cut bed. Although the Eye Bank places marks on the donor corneal tissue prior to shipping, these are often smudged, difficult to see, and may not be accurate to the level of precision to avoid an eccentric cut. After drying, these edges are then measured by the surgeon for diameter and marked with an ink pen so that they are distinctly seen. The central free cap (which was left in place by the Eye Bank prior to shipment) can also be dried and marked centrally. The surgeon then takes the tissue and places it endothelial side up onto the trephine block, using the marks that he personally placed, to determine the best centration on the block, prior to trephination. The trephination and other steps that follow are well described above. One interesting feature of using pre-cut donor corneal tissue is that when folding the tissue into the 60/40% taco configuration, the adherence between the posterior stroma and the overlying free cap can sometimes seem a lot stronger than tissue that is cut “on site” by the surgeon, and so care should be taken to avoid causing stretching or striae when folding the graft prior to insertion.

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Donor Tissue Preparation Without a Microkeratome The operating microscope is brought over to the separate donor table for preparation of the donor tissue. Because whole globes are not usually available in the United States, an artificial anterior chamber is necessary for the preparation of the donor corneal disk. We utilized a Bausch and Lomb (St. Louis, MO) artificial anterior chamber that is all stainless steel and has dual irrigation/aspiration ports. The Optisol-GS preservation fluid (Bausch and Lomb, Rochester, NY) from the donor tissue container is aspirated into a syringe and is then used to fill the I/A ports of the artificial anterior chamber. The syringe is also attached to the port to be used to vary the pressure inside the chamber for the duration of the resection. The standard donor corneoscleral cap tissue is first coated with a layer of Healon on the endothelium. It is then placed endothelial side down onto the post of the artificial anterior chamber and oriented with the largest diameter of the cornea in the horizontal meridian. This meridian is marked with a marking pen so that the horizontal meridian of the donor tissue can be identified later in the procedure. The donor tissue is capped into place and the chamber is filled with Optisol-GS and the pressure normalized. There are several ways to create a deep dissection plane in the donor tissue in the absence of a microkeratome. Each method has advantages, and surgeon preference is the over-riding determinant as to which method is utilized. The peripheral incision and central manual trephine approaches are described below. In the peripheral incision technique, the preparation of the donor cornea is similar to the preparation of the recipient corneal bed at the deep lamellar plane. A diamond knife set to a depth of 350 µm is used to make a 3 or 4 clock hour-length incision in the peripheral donor limbal area, right next to the edge of the metal cap of the artificial anterior chamber. The crescent blade is then used to cut to the deeper stromal tissue and then once the desired plane has been reached, the Devers Dissectors are used to continue the dissection plane all the way to the limbus of the donor tissue for 360 degrees. We have found it helpful to place multiple ink marks onto the surface of the donor tissue. These marks accentuate the visualization of the surface of the donor tissue and therefore allow better depth perception (and estimation of the level of the dissection) when viewing the Devers Dissectors plane compared to the surface plane. It is important to make sure that the dissection plane is carried out all the way to the limbus in every quadrant in order to avoid problems later on that

may result from an eccentric trephination of the donor lenticule. In the manual trephine approach, an 8.5 mm diameter Barron suction recipient trephine (Katena Products, Denville, NJ) is placed onto the surface of the donor tissue and suction is applied. Trephination is carried out to about 60% depth with the trephine. It is noteworthy that after the blade touches the epithelial surface of the donor, it only takes about 4 or 5 quarter turns of the Barron trephine to reach this depth. This is much sooner than when the same trephine is used on the recipient in standard PKP surgery. The trephine is then removed and the cut inspected for the depth. Ideally, an 80% depth should be attained for the plane of the donor corneal pocket. Any cut deeper than this can result in the donor tissue being very thin and it can spontaneously roll up like a rug, causing confusion as to which side is the endothelial side and undoubtedly causing endothelial damage. If the dissection depth of the donor is less than 60% depth, then the stromal surface may not be as smooth and the tissue may be much thicker than the recipient bed is deep. However, whether disparity between donor and recipient disk thicknesses causes a later visual problem is unknown at this time, but separate reports by Terry and by Price on this issue at the recent ASCRS and ARVO annual meetings suggest that donor thickness is not a factor in final visual results.

Transplantation of the Donor Tissue With the microscope in place, the temporary scleral suture of the DLEK temporal wound is cut. The anterior chamber of the patient is then filled completely with BSS. The donor tissue is then brought into the field and the Charlie insertion forceps (Bausch and Lomb Surgical, St. Louis, MO) are used to grasp the stromal surface of the donor tissue along the horizontal meridian. The Charlie forceps are non-toothed fine forceps that only coapt at the distal tips and can be used for DLEK or DSEK surgery. The Charlie insertion forceps has a specially designed stop that permits a significant spacing, along the blade length to prevent crushing of the donor tissue. The folded donor tissue is placed into the anterior chamber in one deft movement, by inserting the donor tissue with the anterior 60% stromal side facing the recipient bed and the posterior 40% stromal side facing the iris. The endothelial layer remains protected on the inside by Healon. The tissue can be gently prodded with the forceps along the stromal sides if centration of the tissue within the recipient bed needs to be improved. As the anterior chamber is deepened with injection of BSS through the right paracentesis site, the tissue gently and

DLEK: A Procedure for Special Cases of Endothelial Dysfunction spontaneously opens up, with the opening of the taco shape to the surgeon’s left. The 60% stromal side gently adheres to the overlying recipient bed with the 40% stromal edge lying nearly perpendicular to the iris plane. Three sutures of 10-0 nylon is then used to close the scleral wound to secure the donor corneal disk within the anterior chamber. A cannula is then placed through the stab incision and the tip is placed onto the iris surface, between the donor sides, within the interior of the taco. BSS is then gently injected into the anterior chamber to deepen and fill the chamber. Irrigation with BSS also loosens the Healon from the endothelial surface and helps to gently unfold the tissue. Because the donor tissue was folded into an asymmetric shape, the tissue invariably will spontaneously unfold in the correct orientation (i.e. endothelium down), as long as the chamber is deep enough and there is no impediment to unfolding of the donor disk. Once the tissue has unfolded, then an air bubble is gently injected into the anterior chamber to stabilize the tissue. An alternative method of opening the tissue atraumatically involves deepening the chamber with BSS from the right side just enough to have the donor tissue unfold to a nearly perpendicular orientation to the iris surface. Then an air bubble is very slowly and gently injected from the left paracentesis site between the lips of the “taco” to complete the unfolding of the tissue and simultaneously lift it up into position into the recipient bed. (This technique is described in more detail in Chapter 20, Endothelial Keratoplasty: A Step by Step Guide to DSEK and DSAEK Surgery). The donor disk may not be in perfect centration after insertion. If not, it can be positioned from either the endothelial side or the stromal side. A reverse Sinskey hook (Bausch and Lomb, St. Louis, MO) is used for endothelial side positioning. The hook is placed through the stab incision, the peripheral endothelium is engaged, and the tissue moved over to whatever position is desired. Although this maneuver undoubtedly causes endothelial damage at that point of peripheral contact, we have not found that the central endothelial cell counts 6 months after surgery to be any worse than that seen after standard PKP procedure.15,21 Care is taken, however to minimize this maneuver and also to avoid the occurrence of a central posterior striae that can compromise vision. An alternative technique for positioning can be done from the stromal interface side using a 30-gauge needle tip. A slight “barb” is placed on a standard short 30-gauge needle, and the tip is placed through the superior wound directly into the interface. The barb is rotated posteriorly to engage a few stromal fibers of the donor disk, and this grasp is used to move the tissue over into proper centration. During both the endothelial

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and stromal positioning maneuvers, the anterior chamber is filled with an air bubble of about a 6 or 7 mm diameter. Once the tissue is in proper centration, it is critical to make sure that all of the donor edges are anterior to all of the recipient bed edges for 360 degrees. Visual inspection is not enough, and manual verification is mandatory. If any portion of the donor tissue edge lies posterior to the recipient rim, then the donor tissue will likely be dislocated the next morning or present with a significant space in the interface (secondary anterior chamber). To accomplish proper donor edge position, the anterior chamber is filled completely with air and the reverse Sinskey hook is placed through a stab incision into the anterior chamber. The tip of the hook is then lifted anteriorly and placed between the edge of the donor and recipient rim. The hook is then rotated to engage the recipient rim posterior stromal edge, and then used to pull the edge posteriorly. With this maneuver, the air bubble in the anterior chamber immediately pushes the donor edge up anteriorly, into the recipient pocket, and upon release of the Sinskey hook, the recipient edge pops right up posterior to the donor edge. This “tire iron” maneuver is performed for 360 degrees, even when the donor tissue appears to be in good position. This is done because even small strands of recipient stromal edge tissue can get caught in the edge interface and prevent adherence of the graft or act as a wick for aqueous into the interface, causing later disk dislocation. As an alternative to the reverse Sinskey hook, another specialized instrument for recipient edge positioning posterior to the donor tissue is available, and this is called the Nick Pick (Bausch and Lomb Surgical, St. Louis, MO). Once satisfied that the donor disk is in final position with good edge position, the surgeon then removes the air in the anterior chamber and replaces it with BSS. Care is taken to avoid pupillary block by the air bubble in the anterior chamber, but if it occurs, simple suctioning of the air from the pupillary area and surface of the posterior chamber IOL resolves the problem. Occasionally air can get trapped behind the iris, giving the impression of posterior pressure with the iris coming forward to the donor edges. Again, suctioning with a cannula from the pupillary area will resolve this issue. The BSS placed into the anterior chamber creates a normal IOP and the chamber deepens. A small (3 mm wide) air bubble is usually left in place to help further stabilize the donor disk position over the first 24 hours postoperatively, but this is not critical. The suture knots of the scleral incision are cut short, and buried on the scleral side. The wound is checked to be watertight. The conjunctival peritomy is closed. We routinely place on the corneal surface a 24 hours collagen shield soaked in antibiotics and steroids at the end of the surgery in order to deliver medication until the patch is

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removed the next day; however, each surgeon’s usual routine for antibiotics (subconjunctival or otherwise) is certainly acceptable. An occlusive patch and shield are routinely placed over the operated eye, and the patient is brought to the recovery room. We usually instruct the nurses to have the patient lie in a supine position for the first few hours as much as possible to allow the retained air bubble to further stabilize the graft position, but this is not critical. The patient is discharged from this outpatient procedure when fully recovered from anesthesia.

Postoperative Course The patient is seen the next morning when the patch is removed. Most patients will remark that the eye was no more uncomfortable than after a standard cataract surgery and that they did not require narcotic pain relief during the immediate postoperative period. Once the patch is removed, the vision is usually about 20/400. The vision is unimportant on postoperative day one, and the only reason for the visit is to insure that the donor disk is attached and that it is in good position. In our initial, prospective series of 100 cases, there were only 4 cases of donor disk dislocated on the first postoperative day.21 All four cases were easily treated by taking the patient back to surgery, and usually, under topical anesthesia, the disk is repositioned using another air bubble in the anterior chamber. This is usually a 15 minutes procedure. We have been successful with all the four cases of disc re-positioning, and all eyes resulted in a clear graft. If the graft is in good position on day one, it will heal in good position, and it is extremely rare to see a late disc dislocation. The edges of the graft seal down with solid healing sometime within the first 3 months, permitting any other necessary intraocular surgery (e.g. posterior vitrectomy, retinal detachment surgery, etc.) without the fear of dislocating the graft.33 We have also performed DLEK on eyes without any significant cataract, leaving the crystalline lens in place. However, 50% of these eyes developed cataracts in the first 2 years after DLEK surgery and required subsequent cataract surgery. The postoperative medical therapy after DLEK surgery is identical to what we do with our PKP surgery patients. Topical prednisolone acetate 1% is used four times a day for 3 months, then three times a day until 6 months, then twice a day until 9 months, and then once a day until one year postoperatively. The steroids are then tapered down further until discontinued entirely. Fluoroquinolone antibiotics are used four times a day for two weeks and then it is discontinued.

Clinical Results Following DLEK Surgery in a Large Prospective Study We have previously reported on the results from the largest prospective study in the world of DLEK surgery.21 The data from the study was from 100% follow-up of our initial 100 cases. These prospective results should serve as a benchmark for DLEK surgery.

Visual Results after DLEK There is, of course, a high variability of vision in any series of elderly patients undergoing ocular surgery, especially DLEK procedure. The interface may clinically appear exceptionally clear, but it likely contributes about one line of visual loss to the macular potential.14,21 The visual results at 6 months after small incision DLEK surgery averaged approximately 20/40, with 55% of the patients seeing 20/ 40 or better and no patients seeing worse than 20/200. In our prospective study, we did not exclude any patient that had known macular disease either before or after surgery. If we exclude those patients that have known macular disease, then approximately 80% will achieve vision of 20/ 40 or better. The vision after DLEK appears to get better from 6 to 12 months, and even from 12 to 24 months, as the stromal interface continues to remodel. 14,34 Visual rehabilitation can be very rapid, with some patients seeing as well as 20/25 only one week after surgery (Figure 16-2) but this is highly variable. Visual results are strongly and positively correlated with patient age (younger patients see better than older patients after DLEK) and with preoperative visual acuity (patients that have better vision preoperatively tend to have better vision postoperatively). Finally, in our series we have over 20 patients that have had a prior PKP in one eye and a DLEK in the fellow eye in

Figure 16.2: Five days after DLEK surgery combined with phacoemulsification in a 57-year-old patient with Fuchs’ dystrophy, the vision is 20/25 with a -0.75 + 0.50 x 130° refractive error.

DLEK: A Procedure for Special Cases of Endothelial Dysfunction treatment of their Fuchs’ dystrophy. All but one of the patients prefers the vision of the DLEK eye compared to the PKP eye, largely based upon the uncorrected visual acuity attained with each procedure.

Astigmatism after DLEK The advantage of DLEK surgery is that it usually retains the normal, presurgical corneal topography. While PKP surgery can result in an average of 4 to 6 diopters of astigmatism, 35 small incision DLEK surgery has no significant influence on the preoperative astigmatism. In our prospective study,21 we found that the average refractive astigmatism after small incision DLEK at 6 months was only 1.18 ± 0.74 diopters, representing only an insignificant 0.23 diopters of change from the preoperative level. The patients are able to wear spectacles after DLEK surgery, often the same pair of glasses that they wore before the onset of the corneal edema. No patients in this series required a contact lens or refractive surgery (e.g. LASIK, relaxing incisions, etc.) in order to achieve their best vision. Anisometropia was never an issue with DLEK surgery as it often is with PKP surgery.

Donor Endothelial Survival after DLEK The endothelial survival after small incision DLEK surgery is quite remarkable. Even with folding the tissue and other manipulations described above, the average endothelial cell count after small incision DLEK surgery is comparable to PKP surgery, and it is not significantly different at 6 months, from large incision DLEK surgery where the donor tissue is not folded. In our series, we found that the specular microscopy at 6 months after small incision DLEK surgery demonstrated an average central endothelial density of 2121 ± 420 cells/mm2. This represented an average 25% cell loss from preoperative donor eye measurements, and compared well to the 17 to 34% cell loss seen after standard PKP surgery.36,37 In addition, at one year of follow-up, we have not seen a significant decline in the endothelial cell counts from the 6-month measurements, for our large incision DLEK cases.34 It is important to note, however, that our most recent publication on DLEK surgery38 indicates that there is a delayed price to pay for folding the donor tissue in EK surgery. We have found that at the two year postoperative measurements, the large incision DLEK cases (where the tissue is not folded) had only a 27% loss of endothelial cells from the preoperative values, while in the small incision DLEK cases (where the tissue is folded, inserted, and unfolded) there was a 43% loss of endothelial cells compared to the preoperative measurements (p<.001).

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Current modifications of EK such as DSAEK [See also Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)] have shown a significantly higher cell loss, with an average of 40% cell loss at just 6 to 12 months after DSAEK surgery. This increase in acute, shortterm donor endothelial cell loss is of concern, and further data is crucial to determine the long-term outcome of eyes undergoing the DSAEK procedure.

Summary I believe that our prospective work with endothelial keratoplasty surgery over these past 7 years has established EK as a reasonable alternative to PKP for the patient that is suffering from visual loss due to endothelial dysfunction. Visual rehabilitation is faster with EK than with PKP, and the presence and magnitude of irregular astigmatism are largely eliminated. The globe is likely to be safer from longterm dangers of globe rupture secondary to minor trauma, and patients prefer the quality of vision from EK compared to PKP. Long-term study is necessary in order to determine if the endothelial survival rate and the graft rejection rate after EK surgery is comparable (or better) than after PKP surgery, and our experience over the past 7 years is encouraging. On a practical basis, outside of a scientific protocol, EK patients do not require the same degree of monitoring as standard PKP patients and therefore they require less postoperative clinic time. With no corneal sutures or incisions wound healing and corneal ulcerations are not an issue. Astigmatism management is also not an issue after EK surgery, much to the joy of patient and surgeon alike. The only critical monitoring is for steroid-induced glaucoma, as long as the patient is on topical steroids, and this is done according to the clinician’s standard routine. Surgeons learning the DSEK and DSAEK forms of EK surgery [See also Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)] should recognize that DSEK or DSAEK cannot be used safely for every form of endothelial dysfunction. Patients with an open communication between the anterior and posterior chambers (cases such as aphakia, anterior chamber IOL or sector iridectomy) will be unable to maintain an air bubble for support of the EK tissue, creating an extraordinarily highrisk of donor disk dislocation after DSEK/DSAEK surgery. In these specialized cases, DLEK is the EK procedure of choice, due to the adherence of donor tissue without the need of a postoperative air bubble. The DLEK surgical procedure is a more difficult one and requires a commitment to exacting detail and thorough practice prior to incorporation of this procedure into the surgeon’s operative repertoire. However, corneal transplant

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surgeons who wish to be able to safely treat all forms of endothelial dysfunction are well equipped when DLEK is included in their surgical armamentarium.

References 1. Sugar A, Sugar J. Techniques in penetrating keratoplasty: A quarter century of development. Cornea 2000;19:603-10. 2. Abou-Jaoude ES, Brooks M, Katz DG, Van Meter WS. Spontaneous wound dehiscence after removal of single continuous penetrating keratoplasty suture. Ophthalmology 2002;109:1291-6. 3. Tseng SH, Lin SC, Chen FK. Traumatic wound dehiscence after penetrating keratoplasty: Clinical features and outcome in 21 cases. Cornea 1999;18:553-8. 4. Stechschulte SU, Azar DT. Complications after penetrating keratoplasty. Int Ophthalmol Clin 2000;40:27-43. 5. Akova YA, Onat M, Koc F, Nurozler A, Duman S. Microbial keratitis following penetrating keratoplasty. Ophthalmic Surg Lasers 1999;449-55. 6. Confino J, Brown SI. Bacterial endophthalmitis associated with exposed monofilament sutures following corneal transplantation. Am J Ophthalmol 1985;99:111-3. 7. Ko WW, Frueh BE, Shields CK, Costello ML, Feldman ST. Experimental posterior lamellar transplantation of the rabbit cornea Invest Ophthalmol Vis Sci 1993;34(4):1102. 8. Melles GR, Eggink FA, Lander F, Pels E, Rietveld FJ, Beekhuis WH, Binder PS. A surgical technique for posterior lamellar keratoplasty. Cornea 1998;17:618-26. 9. Melles GR, Lander F, Beekhuis WH, et al. Posterior lamellar keratoplasty for a case of pseudophakic bullous keratopathy. Am J Ophthalmol 1999;127:340-1. 10. Terry MA, Ousley PJ. Endothelial replacement without surface corneal incisions or sutures: Topography of the deep lamellar endothelial keratoplasty procedure. Cornea 2001;20:14-18. 11. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty in the first United States patients: Early clinical results. Cornea 2001;20:239-43. 12. Terry MA, Ousley PJ. Replacing the endothelium without corneal surface incisions or sutures: The first United States clinical series using the deep lamellar endothelial keratoplasty procedure. Ophthalmology 2003;110:755-64. 13. Terry MA, Ousley PJ. In pursuit of emmetropia: Spherical equivalent refraction results with deep lamellar endothelial keratoplasty (DLEK). Cornea 2003;22:619-26. 14. Terry MA, Ousley PJ. Rapid visual rehabilitation after endothelial transplants with deep lamellar endothelial keratoplasty (DLEK). Cornea 2004;23:143-53. 15. Terry, MA, Ousley, PJ: Small Incision Deep Lamellar Endothelial Keratoplasty (DLEK): 6 months results in the first prospective clinical study. Cornea 2005;24:59-65. 16. Terry MA. Endothelial replacement: The limbal pocket approach. Ophthalmol Clin North Am 2003;16:103-12. 17. Terry MA. Deep lamellar endothelial keratoplasty (DLEK): Pursuing the ideal goals of endothelial replacement. Eye 2003;17:982-8. 18. Terry MA. A new approach for endothelial transplantation: Deep lamellar endothelial keratoplasty. Int Ophthalmol Clin 2003;43:183-93. 19. Terry MA, Ousley PJ. Corneal endothelial transplantation: Advances in the surgical management of endothelial dysfunction. Contemporary Ophthalmology 2002;1(26):1-8. 20. Terry MA. Endothelial Replacement. In: Krachmer J, Mannis M, Holland E, eds. Cornea: Surgery of the Cornea and Conjunctiva. St. Louis: Elsevier Mosby; 2005: Chapter 140:1707-18.

21. Terry MA, Ousley PJ: Deep Lamellar Endothelial Keratoplasty (DLEK): Visual acuity, astigmatism, and endothelial survival in a large prospective series. Ophthalmology 2005;112:154149. 22. Melles GR, Lander F, Nieuwendaal C. Sutureless, posterior lamellar keratoplasty: A case report of a modified technique. Cornea 2002 21:325-7. 23. Melles GR, Wijdh RH, Nieuwendaal, CP. A technique to excise the descemets’ membrane from a recipient cornea (descemetorhexis). Cornea 2004;23: 286-88. 24. Price FW, Price MO. Descemet’s stripping with endothelial keratoplasty in 50 eyes: A refractive neutral corneal transplant. J. of Refractive Surgery 2005; 21:339-45. 25. Terry MA, Ousley PJ, Wills B. A practical femtosecond laser procedure for DLEK endothelial transplantation: Cadaver eye histology and topography. Cornea 2005;24:453-9. 26. Soong HK, Mian S, Abbasi O, et al. Femtosecond laser-assisted posterior lamellar keratoplasty. Ophthalmology 2005;112: 44-49. 27. Sarayba MA, Juhasz T, Chuck RS, et al. Femtosecond laser posterior lamellar keratoplasty: A laboratory model. Cornea 2005;24:328-33. 28. Suwan-apichon O, Rizen M, Reyes JM, Herretes S, Behrens A, Stark WJ, Chuck RS. A new donor cornea harvesting technique for posterior lamellar keratoplasty. British Journal of Ophthalmology 2005; 89:1100-01. 29. Kang PC, McEntire MW, Thompson CJ, Moshirfar M. Preparation of donor tissue for deep lamellar endothelial keratoplasty (DLEK) using a microkeratome and artificial anterior chamber system: Endothelial cell loss and predictability of lamellar thickness. Ophthalmic Surgery, Lasers and Imaging 2005; 36:381-5. 30. Terry MA, Ousley PJ. Deep Lamellar Endothelial Keratoplasty: Small Incision Technique combined with Phacoemulsification and Posterior Chamber Intraocular Lens Implantation. In: John, T. editor. Surgical Techniques in Anterior and Posterior Lamellar Corneal Surgery. Jaypee Brothers Medical Publishers (P) LTD. 2005;345-64. 31. Terry MA, Ousley PJ. “The New Triple Procedure: First World Wide Patients with DLEK combined with Phaco/IOL surgery”. American Academy of Ophthalmology Video Library, Annual Meeting, “Best of Show”, October 2002. 32. John T. Upside-down Phacoemulsification in Deep Lamellar Endothelial Keratoplasty. In: John T (Eds). Surgical Techniques in Anterior and Posterior Lamellar Corneal Surgery. Jaypee Brothers Medical Publishers (P) LTD. 2005;372-9. 33. Amayem AF, Terry MA, Helal MH, Turki WA, El-Sabagh H, El-Gazayerli E, Ousley PJ. Deep Lamellar Endothelial Keratoplasty (DLEK): Surgery in complex cases with severe preoperative visual loss. Cornea 2005;24:587-92. 34. Ousley PJ, Terry MA. Stability of vision, topography, and endothelial cell density from one year to two years after deep lamellar endothelial keratoplasty (DLEK) surgery. Ophthalmology 2005;112:50-57. 35. Pineros OE, Cohen EJ, Rapuano CJ, et al. Triple vs nonsimultaneous procedures in Fuchs’ dystrophy and cataract. Arch Ophthalmol 1996;114:525-8. 36. Ing JJ, Ing HH, Nelson LR, et al. Ten-year postoperative results of penetrating keratoplasty. Ophthalmology 1998;105:1855-65. 37. Bourne WM. Cellular changes in transplanted human corneas. Cornea 2001;20:560-9. 38. Terry MA, Wall J, Hoar KL, Ousley PJ. Endothelial Keratoplasty: A prospective study of endothelial cell loss during the 2 years after deep lamellar endothelial keratoplasty. Ophthalmology 2007 (in press). 38. Gorovoy M. Descemet’s stripping automated endothelial keratoplasty (DSAEK).Cornea 2006;25:886-9.

DLEK: Small Incision Technique Combined with Phacoemulsification and Posterior Chamber IOL 183

Mark A Terry

Deep Lamellar Endothelial Keratoplasty: Small Incision Technique Combined with Phacoemulsification and Posterior Chamber IOL

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Introduction Surgical endothelial replacement for conditions such as Fuchs’ endothelial dystrophy and pseudophakic bullous keratopathy has been successfully accomplished with full thickness penetrating keratoplasty (PKP) for nearly 100 years.1 While the surgical technique of PKP is straightforward and relatively easy, the visual results and stability of the grafted tissue are sometimes poor due to wound healing and suture-related problems.2-6 In 1993 Ko and Feldman presented an animal study at the annual meeting of the Association for Research in Vision and Ophthalmology (ARVO) which described a new technique for endothelial replacement through a scleral limbal incision7 (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). In 1998, Melles et al described this technique in the first human patients and called it posterior lamellar keratoplasty (PLK)8 (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). Terry and Ousley began laboratory work in 1999 on this technique and after technical modifications and re-design of instrumentation, performed the first United States cases in 2000 and called the surgery Deep Lamellar Endothelial Keratoplasty (DLEK).9-20 All of this work represents a radical departure from the PKP technique in that the DLEK surgery accomplished the goal of endothelial replacement without altering the surface of the recipient cornea. By eliminating surface corneal sutures and incisions, the advantages of normal corneal topography and faster wound healing were obtained, leading to faster visual rehabilitation and a more stable globe for the patient.10-14 Recently, a small incision technique of PLK surgery was described in a case report by Melles et al.21 We have investigated this technique in the largest prospective series of small incision DLEK in the world and have found it to be valid for endothelial replacement surgery.14 While undoubtedly there will be further refinement of the technique and instrumentation in DLEK surgery, it is the purpose of this chapter to describe in detail this small incision method of DLEK, and to describe its combination with other intraocular procedures such as cataract extraction.

Surgical Objective The purpose of DLEK surgery is to remove the diseased recipient endothelium and replace it with healthy donor endothelium. The advantage of DLEK surgery over PKP surgery is that it accomplishes this primary objective without violating the surface of the cornea with sutures or incisions. We have delineated the five ideal goals for

endothelial replacement in previous papers as being (1) a smooth surface topography without significant change in astigmatism, (2) a highly predictable (and unchanged) corneal curvature, (3) a healthy donor endothelium which resolves all edema, (4) a tectonically stable globe, safe from injury and infection, and (5) an optically “pure” cornea.11,12,15-18 While standard PKP can consistently achieve good results for goals 3 and 5, the other goals have remained elusive.22-26 At the current time, DLEK surgery can accomplish the first 4 goals nearly perfectly, while the fifth goal of an optically “pure” cornea is good, but can still be improved. Instrumentation and technique modification to achieve a more perfect stromal interface between posterior donor tissue and anterior recipient tissue are the current, continued directions of DLEK research. The small incision technique of DLEK surgery described here reduces the scleral tunnel incision to 5 mm in length and moves it from the superior to the temporal side. It is felt that by making the scleral access incision even shorter than in standard DLEK, there will be less of a tendency for wound-induced corneal flattening over time and also provide more strength to the globe to withstand any future blunt trauma. Moving the position of the incision to the temporal side, allows easier visualization of the anterior segment during the surgery and spares the superior limbus, which may be utilized for cataract or glaucoma surgery at the same time as DLEK or at a later time.

Preferred Anesthesia DLEK surgery is usually done under general anesthesia, but retrobulbar block anesthesia has also been used. General anesthesia (either endotracheal or laryngeal mask airway technique) is preferred because it minimizes posterior pressure on the globe and this is important during the recipient resection and donor implantation phases of the surgery. Nonetheless, the surgery can be safely accomplished with good retrobulbar anesthesia combined with seventh nerve block (orbicularis block) local anesthesia as well. It is also quite possible, for the severely medically frail patient, that this surgery could be accomplished with only topical anesthesia, similar to what has been done in PKP surgery under similar circumstances.27 However, this has not yet been done with DLEK surgery and likely would require a surgeon with the ability to accomplish this surgery in less than an hour.

Preoperative Preparation Like all intraocular procedures, the patient’s ocular health should be maximized beforehand and any blepharitis, dry

DLEK: Small Incision Technique Combined with Phacoemulsification and Posterior Chamber IOL 185 eye, or lid abnormality should be treated prior to surgery. Patients with mild to moderate corneal surface haze or scarring from long-standing bullous keratopathy can still undergo DLEK surgery successfully. The surface scarring is scraped off at the time of surgery or weeks later in clinic after the stromal edema has completely resolved. This eliminates the induced irregular astigmatism from the scars and restores the normal topography. In patients with pseudophakia, the pupil is constricted in order to stabilize the iris-lens diaphragm during the surgery. This is also done if the patient has a clear crystalline lens and concurrent cataract surgery is not planned. Preoperative medications include two sets of pilocarpine 1% drops applied one hour prior to surgery. One set of aproclonidine 0.5% drops is also given just prior to surgery to reduce pressure and minimize conjunctival injection. No preoperative antibiotics are necessary. The eye is prepped in the usual sterile ophthalmic fashion with the use of povidone-iodine solution. In patients with cataract and endothelial failure, cataract surgery is performed just prior to the DLEK endothelial transplant and the pupil is dilated preoperatively (Figure 17-1) with the surgeon’s standard dilating drops for cataract surgery. While pilocarpine is avoided, the rest of the preoperative medication regimen described above is utilized.

Figure 17-1: Pre-operative appearance of the cornea.

Surgical Procedure: Combined DLEK and Phacoemulsification Recipient Surgery: Phacoemulsification Technique Small incision phacoemulsification cataract extraction, when combined with DLEK surgery, is usually performed utilizing a sclerocorneal tunnel access incision, rather than

a clear corneolimbal incision. The reasoning behind this is that the cataract wound needs to be water tight and as strong as possible to withstand the manipulations and pressures placed on the eye during DLEK recipient tissue dissections. In addition, it is preferable to completely eliminate any surface corneal incisions of any kind to maximize the topographic advantages of DLEK surgery. Although, phacoemulsification can be performed through the same incision of the DLEK surgery after the recipient posterior corneal tissue has cleared the visualization of the anterior chamber, it is our preference to perform the cataract extraction portion of the procedure from a separate site from the DLEK surgery whenever possible. Therefore, we currently begin “The New Triple Procedure” (Terry and Ousley, AAO instructional video, 2002) with the phacoemulsification performed from the superior 12 o’clock position. Most cases of Fuchs’ corneal endothelial dystrophy, corneal edema will allow adequate visualization of the anterior segment to safely complete the cataract surgery. However, high pressures within the eye during the phacoemulsification will force fluid into the already edematous cornea and create a foggy view. Therefore, the phacoemulsification wound is made long enough (usually 3.0 mm or more) to allow adequate egress of BSS during the phacoemulsification portion of the procedure and maintain an optimal corneal clarity. In some cases of advanced corneal endothelial decompensation, the preoperative corneal edema has created bullae on the central corneal surface, making visualization for cataract surgery difficult or impossible. In these cases, we have advocated one of the following remedies: (1) Apply sterile glycerin drops to the corneal surface to deterges the epithelial edema and improve the view of the anterior segment of the eye. This gives a very temporary effect of smoothing the surface and requires multiple applications; (2) Scrape the central 6 mm of the epithelium from the corneal surface to provide an immediate improvement in surface smoothness. This requires the use of a bandage contact lens or collagen shield at the end of the surgery and the application of a bandage contact lens during the 1st week following surgery. It does not seem to pose a risk to the later adhesion of the donor disk; or (3) Consider performing the cataract surgery through the same DLEK temporal wound, after the recipient tissue has been resected to clear the view. This may also need to be combined with surface scraping of epithelium when the bullae are severe. All of these techniques can be used and the choice of which one to use depends on the severity of the corneal surface edema and the comfort of the surgeon when working in situations with suboptimal visualization of the anterior segment.

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Once the sclerocorneal limbal phacoemulsification incision has been made (Figures 17-2 and 17-3) , the cataract surgery is performed according to the surgeon’s preferred technique. However, unlike standard cataract surgery, it is very important not to use a dispersive viscoelastic such as “Viscoat” (Alcon, Fort Worth, TX) during any portion of the procedure. Viscoat adheres significantly, not only to the endothelium, but also to the bare stroma, and any residual Viscoat that is left in the eye after cataract extraction threatens to coat the recipient stromal bed during the DLEK portion of the procedure. This will prevent the desired donor disc adherence later on and hence Viscoat (Alcon) and similar dispersive viscoelastics are contraindicated at any time during a surgery involving DLEK. Currently, we utilize Healon or Healon V (Pfizer, New York, NY) for all stages of the cataract surgery or the DLEK surgery. Additionally, during a combined cataract and DLEK surgery, we feel it is preferable to keep the diameter of the anterior capsulorhexis opening as small as possible (usually around 4 mm) (Figures 17-4A and B) in order to provide as much stability as possible to the IOL/ Iris diaphragm during the DLEK portion of the procedure.

A

B Figures 17-4A and B: Capsulorhexis is being performed.

Figure 17-2: Superior limbal peritomy being performed.

Figure 17-3: Scleral tunnel access incision is displayed.

We routinely perform a standard “phaco-chop” technique (Figures 17-5A to H) . One aspect that is unique to cataract surgery when combined with DLEK surgery is that the central endothelium is going to be replaced and so the surgeon can be indifferent to its care. The surgeon can bring the entire nucleus up into the anterior chamber for phacoemulsification, if necessary, and not worry about damage to the central endothelium, since the endothelium is going to be replaced by the DLEK portion of the combined procedure. Indeed, Dr. Thomas John has advocated a technique called “Upside-Down Phaco” when combining cataract surgery with DLEK in cases of severe corneal edema. Nonetheless, the surgeon should still respect the integrity and safety of the peripheral endothelium and should perform cataract surgery in the safest possible manner. We routinely place a foldable acrylic intraocular lens (Figure 17-6) , but the IOL type is based upon surgeon preference. Patients that have significant naturally occurring corneal astigmatism may even have a toric IOL placed for correction, since the small incision DLEK procedure does not seem to significantly induce large

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A

E

B

F

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H Figures 17-5A to H: Phaco-chop photos.

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Figure 17-6: Foldable posterior chamber IOL insertion.

Figure 17-7: Removal of Healon from capsular bag.

degrees of astigmatism.14 It is important to remove all of the Healon from the capsular bag (Figure 17-7), and to verify that no Healon is sequestered behind the IOL prior to closing the cataract wound. Any residual Healon left behind after the cataract surgery can have a detrimental effect on the adherence of the donor corneal stroma to the host corneal stroma during the following DLEK procedure. We place a pupil-constricting agent (Miochol acetylcholine chloride solution, Ciba Vision Ophthalmics, Duluth, GA) into the anterior segment to further stabilize the lens, and then close the wound at normal pressure. The cataract wound is selfsealing, but it is reinforced with one or two interrupted 100 nylon sutures. The conjunctiva is then closed with vicryl sutures (Figure 17-8), and attention is directed to the performance of the DLEK surgery from the temporal side.

Figure 17-8: Closing the conjunctiva with vicryl sutures.

limbal region rather than superiorly). The patient’s head should be positioned facing the ceiling, parallel to the floor. In addition, the endotracheal tube is also positioned by the anesthesiologist to exit the mouth from the side opposite the surgical field to facilitate the surgeon’s field of movement. A bridal suture can also be placed beneath the superior and inferior recti muscles, if necessary, to aid in the positioning and stabilization of the globe during the DLEK surgery. A temporal limbal peritomy of the conjunctiva is performed with scissors (Figure 17-9) allowing exposure of about 6 mm arc length (about 3 clock hours) of limbal tissue. Prior to forming the DLEK scleral access incision, two clear corneal limbal stab incisions (about 1 mm diameter) (Figure 17-10) are placed on either side of the peritomy area, to be used as access points to the anterior chamber later in the operation. Through one of the stab incisions, the cohesive viscoelastic Healon (Pfizer, New York, NY) is placed into the anterior chamber to replace the aqueous fully and to maintain normal pressure. We strongly oppose the use of Viscoat (Alcon) or other

Recipient Surgery: Small Incision DLEK The operating microscope is now positioned for the surgeon to be seated at the temporal side of the patient (for small incision DLEK surgery, the scleral access incision is 5 mm, and therefore the incision is usually placed at the temporal

Figure 17-9: Temporal limbal peritomy is being performed as the initial step for combined DLEK with phacoemulsification and posterior chamber IOL insertion.

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Figure 17-10: Stab incision being performed.

dispersive viscoelastic materials during any portion of DLEK surgery as the dispersive materials can cause stromal interface coating with subsequent non-adherence and dislocation of the donor tissue. Prior to creating the deep lamellar pocket of DLEK, a template mark is placed on the corneal epithelial surface (Figures 17-11A and B). A circular marker with a diameter of 8.0 or 8.5 mm (depending upon recipient corneal diameter

A

and surgeon preference) is used to make a circular impression on the central epithelial surface. If the position and centration of the mark is acceptable to the surgeon, then it is accentuated with gentian violet ink marks. This circle on the cornea will later be used as a template for resection of the posterior recipient lamellar tissue. A trifaceted, guarded diamond knife is then set to a depth of 350 µm and a 5.0 mm length incision is made approximately 1 mm posterior and concentric to the corneal limbus (Figure 17-12). We have found that a deeper initial incision gives less of a beveled wound closure and also a greater chance of early perforation into the anterior chamber during DLEK surgery. In lieu of a diamond knife, a sharp crescent blade or other steel scalpel can be used for the initial incision. A sharp crescent blade is then utilized (Figure 17-13) to create a deep sclerocorneal lamellar pocket down to about 75% to 85% corneal depth along the entire length of the wound. Perfect accuracy of the depth of the corneal stromal pocket does not appear to be critical for a good visual outcome.28 Pockets should be deeper than 50% in order to avoid interface scarring or haze, and should not be deeper than 95% depth in order to avoid donor-recipient

Figure 17-12: Diamond knife incision to a depth of 350 µm.

B Figures 17-11A and B: Template mark on recipient corneal surface.

Figure 17-13: Crescent blade being used.

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thickness mismatch. Judgment of the initial depth of the pocket is based upon inspection of the anterior lip thickness and by the clarity of the underlying stromal bed. Experience with the procedure and with lamellar dissections in general aids in the confidence that the desired depth has been achieved. A specialized semi-sharp stromal dissector is then used to extend the pocket to the mid-pupillary region of the cornea and then a curved stromal dissector (Devers Dissector, Bausch and Lomb, St. Louis, MO) (Figures 17-14 and 17-15) extends the pocket further. We prefer to have a pocket that extends at least 1 mm peripheral to the diameter of the surface template circular mark (i.e. 10.0 mm pocket diameter for an 8.0 mm mark) (Figure 17-16). This creates a large area of deep lamellar corneal pocket. The Devers Dissectors are designed with a tip that is not as sharp as a crescent blade, but is sharper than a blunt dissector. The width of the dissecting heads are especially good for maintaining the stromal depth consistently throughout the dissection of the edematous deep stroma and the surgeon

Figure 17-16: Peripheral Devers dissector being used in the DLEK portion of this triple procedure.

Figure 17-14: Straight Devers dissector is used to initiate the intrastromal corneal pocket.

can actually feel the increased resistance to dissection if he deviates too anteriorly. The dissection is accomplished with a slow and methodical sweeping motion of the dissector heads, from central to peripheral tissue, and the surgeon can often see the reflections of Descemet’s membrane wrinkling during the sweeping motion, which is an assurance that the depth of the dissection is adequate. It is important that the pocket stromal dissection be carried out over the entire desired area of the cornea, in order to allow adequate edge space for the donor disk. The resection of the posterior recipient tissue begins by first entering the anterior chamber through the temporal scleral corneal pocket incision (Figure 17-17). We utilize a standard cataract surgery diamond blade with a 2.8 mm width, but any blade is acceptable. Entry into the anterior chamber at the exact corresponding position of the temporal edge of the surface template mark is preferred. It is through this entry point that the recipient posterior resection is started utilizing special scissors designed for

Figure 17-15: Curved Devers dissector is used to complete the intrastromal corneal pocket.

Figure 17-17: Keratome entry into AC.

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Figure 17-18: Cindy I scissors is being used.

blades that are ideally suited for this procedure. Once the resection has progressed distally to about the 5 o’clock and 7 o’clock positions, then the Cindy II scissors are utilized for completion of the distal resection. The Cindy II scissors have long, low profile blades that are set at nearly a right angle to easily complete the more difficult distal resection. Once the posterior recipient disk has been cut for the full 360° then the tissue is removed from the eye (Figure 17-20) and placed on the corneal surface for inspection (Figure 17-21). It is washed with balanced salt solution (BSS) (Alcon Inc., Fort Worth, TX), and dried with a sponge. The stromal surface is inspected for smoothness and the edges for regularity of the cut, as well as the thickness of the resected tissue. Removal of the recipient posterior edematous stromal tissue, dramatically clears the view into the anterior chamber through the central cornea, and hence, other intraocular surgery such as cataract surgery, vitrectomy, IOL exchange and iridoplasty can be performed at this stage of the DLEK procedure. After removal of the recipient posterior tissue, the temporal scleral wound is temporarily closed with

A

Figure 17-20: Recipient disk removal.

B Figures 17-19A and B: Cindy II scissors is used to complete the circular cut.

posterior lamellar tissue resection (Cindy I and Cindy II Scissors, Bausch and Lomb) (Figures 17-18 and 17-19). The Cindy I scissors are placed with one blade in the anterior chamber and one blade in the stromal pocket. The scissors is used to do a free-hand cut, following the marks of the circular template on the overlying epithelial surface. The Cindy I scissors have long, highly curved and low profile

Figure 17-21: Recipient disk inspection.

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Figure 17-22: Suturing the wound.

Figure 17-23: I/A removal of Healon.

1 interrupted 10-0 nylon suture (Figure 17-22). An irrigation/aspiration (I/A) tip is then introduced into the anterior chamber and extensive effort is expended to remove all of the viscoelastic from the eye (Figure 17-23). No Healon should remain in the anterior chamber prior to insertion of the donor disk, otherwise, the donor tissue will not “stick” in place. Therefore, care is taken to irrigate and aspirate the anterior chamber, pupillary area, anterior chamber angle, and even the peripheral pocket as necessary. Once the surgeon is confident that all Healon has been removed, then the pressure of the eye is left slightly soft and attention is turned to preparation of the donor tissue.

Donor Tissue Preparation The operating microscope is brought over to the separate donor table for preparation of the donor tissue. Because whole globes are rarely available here in the United States, an artificial anterior chamber (See also Chapter 12, Artificial Anterior Chambers) is necessary for preparation of the donor posterior disk. We utilized a Bausch and Lomb (St. Louis,

Figure 17-24: Healon is being placed on the donor corneal endothelium.

MO) artificial anterior chamber that is all stainless steel and has dual irrigation/aspiration ports. The Optisol-GS preservation fluid (Bausch and Lomb, Rochester, NY) from the donor tissue container is aspirated into a syringe and is then used to fill the I/A ports of the artificial anterior chamber. The syringe is also attached to the port to be used to vary the intra-chamber pressure for the duration of the resection. The standard donor corneoscleral cap tissue is first coated with a thin layer of Healon on the endothelium (Figure 17-24). It is then placed endothelial side down onto the post of the artificial anterior chamber (Figures 17-25A and B) and oriented with the largest diameter of the cornea in the horizontal meridian. This meridian is marked with a marking pen so that the horizontal meridian of the donor tissue can be identified later in the procedure. The donor tissue is capped into place and the chamber is filled with Optisol-GS and the pressure normalized. An 8.5 mm diameter Barron suction recipient trephine (Katena Products, Denville, NJ) is placed onto the corneal surface of the donor tissue and suction is applied. Trephination is carried out to about 60% corneal depth with the trephine. It is noteworthy that after the blade touches the epithelial surface of the donor, it only takes about 4 or 5 quarter turns of the Barron trephine to reach this depth. This is much sooner than when the same trephine is used on the recipient in standard PKP surgery. The trephine is then removed and the cut inspected for depth. Ideally, an 80% corneal depth should be attained for the plane of the pocket of the donor tissue. Avoid going deeper than 80% corneal depth, since it will usually result in a thin donor corneal disc that will often spontaneously roll up like a rug causing confusion as to which side is the endothelial side and may result in endothelial damage. If the dissection depth of the donor is less than 60% depth, then the stromal surface of the donor corneal disk may not be as smooth and the donor

DLEK: Small Incision Technique Combined with Phacoemulsification and Posterior Chamber IOL 193

A Figure 17-26: Incision with diamond knife (Barron recipient trephine not used for this patient).

B Figures 17-25A and B: Donor cornea placement onto artificial anterior chamber.

corneal tissue may be much thicker than the recipient bed is deep. However, whether disparity between donor and recipient disk thicknesses causes a later visual problem is unknown at this time. Similar to the recipient disk preparation, the crescent blade is used to cut down to the 80% depth and it is then extended over the entire area of the cornea, all the way to the limbus, using the straight and curved Devers Dissectors. As an alternative to beginning the stromal pocket dissection with a measured trephine cut as described above, the surgeon may prefer to simply use a diamond knife (Figure 17-26) set to a depth of 350 µm and make a 3 or 4 clock hour-length incision in the peripheral donor limbal area. The crescent blade is then used to cut to the deeper stromal tissue and then once the desired plane has been reached, then the Devers Dissectors are used as described previously. After completing the deep stromal pocket formation (Figure 17-27), the cap of the chamber is gently rotated, taking care not to collapse the chamber, and the cap is removed. The donor tissue is then left on the post with a formed chamber. The scleral edges of the donor are gently

Figure 17-27: Dissection of donor cornea.

lifted to release the tissue and the tissue is removed from the post, once again taking care not to collapse the chamber and damage the endothelium. After the tissue is lifted off the post, the endothelial side is gently irrigated with BSS to remove excess Healon and prevent it from coating the stromal pocket during the next stage of the preparation. The donor tissue is then placed endothelial side up onto a standard punch trephine block (Figure 17-28). We utilize a Barron donor punch (Katena). The same size diameter punch is used as the diameter of the circular marker that was used to make the circular impression on the host central epithelial surface. A diameter 0.25 mm larger for the donor has been used, but the incidence of donor folds and dislocations increased with this disparity (unpublished data). The tissue is punched out with the trephine (Figure 17-29), and if the dissection has been done properly, the surgeon will not hear that familiar “crunch” sound that is so common with full thickness PKP donor trephination. Instead the sound is much quieter or not present at all. While it is best to have a good centration of

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Figure 17-28: Donor cornea is placed on the punch block (before punch).

Figure 17-30: Thin strip of Healon placed along center of the donor corneal disk.

The posterior tissue is then gently folded with the endothelium on the inside protected by the layer of Healon, and it is folded into an asymmetric “taco” shape, in a 60:40% ratio (Figure 17-31), the most anterior side of the taco being 60% and the posterior side 40%. The donor tissue is then brought over to the operative field still on the trephine block.

Figure 17-29: Donor cornea on punch block after it has been punched with a trephine blade.

the punch, if the stromal dissection of the donor has been carried out all the way to the limbus for 360°, it will be fine. If the dissection has not been carried all the way out to the limbus, and the surgeon has an eccentric punch, then the posterior donor disk may have a 1 mm thick edge and an opposite 100 µm thick edge. This wedge of tissue will not adhere to the recipient bed, and so care in completion of the total donor stromal pocket is advised prior to punching out the tissue. Because the 5 mm wound of small incision DLEK surgery is smaller than the 8.0 mm diameter of the donor disk, the donor tissue must be folded prior to insertion. To accomplish this, a very thin strip of Healon is placed onto the endothelial surface (Figure 17-30) along the previously identified and marked horizontal meridian of the donor button. Stabilizing the anterior edge of the donor button with a 0.12 mm forceps, the posterior stromal tissue edge is gently grasped with non-toothed specialized insertion forceps (Charlie forceps, Bausch and Lomb, St. Louis, MO).

A

B Figures 17-31A and B: Cornea folded over 60/40 taco fold.

DLEK: Small Incision Technique Combined with Phacoemulsification and Posterior Chamber IOL 195 Transplantation of the Donor Tissue With the microscope in place, the temporary scleral suture of the superior wound is cut (Figure 17-32). The anterior chamber of the patient is then filled completely with BSS (Figure 17-33). The donor tissue is then brought into the field and the Charlie insertion forceps are used to grasp the stromal surface of the donor tissue along the horizontal meridian (Figure 17-34). The Charlie forceps are non-

Figure 17-32: The temporary suture is cut.

toothed fine forceps that coapt only at the distal tips. The amount of space that is present along the long axis of the blades prevents crushing of the donor tissue. There is a specially designed stop for this purpose that enables the surgeon to transfer and hold the folded donor disk tissue without crushing it. The folded donor tissue is placed into the anterior chamber in one quick, smooth movement, by inserting the donor tissue with the anterior 60% stromal side facing the recipient bed and the posterior 40% stromal side facing the iris (Figures 17-35A to C). Again, the

A

B Figure 17-33: AC is filled with BSS.

C Figure 17-34: Tissue grasped with Charlie forceps.

Figures 17-35A to C: Insertion of the donor corneal tissue.

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endothelial layer remains protected on the inside by Healon. The tissue can be gently manipulated with the forceps along the stromal sides if centration of the tissue within the recipient bed needs to be improved. The tissue gently opens up on its own, with the opening of the taco shape to the surgeon’s left. The 60% stromal side gently adheres to the overlying recipient bed with the 40% stromal edge lying nearly perpendicular to the iris plane. Three 10-0 nylon sutures are then used to close the scleral wound to secure the anterior chamber (Figure 17-36). A cannula is then placed through the stab incision and the tip placed onto the iris surface, between the donor sides, within the interior of the taco. BSS is then gently injected into the anterior chamber to fill the chamber and deepen it (Figure 17-37). Irrigation with BSS also loosens the Healon from the endothelial surface and helps to gently unfold the tissue. Because the donor tissue was folded into an asymmetric shape, the tissue invariably will spontaneously unfold (Figures 17-38A to D) in the correct orientation (i.e. endothelium down), as long as the chamber is deep enough and there is no impediment. Once the tissue has unfolded,

Figure 17-36: The temporal incision is sutured.

Figure 17-37: BSS is added to deepen the anterior chamber.

then an air bubble is gently injected into the anterior chamber (Figure 17-39) to stabilize the tissue. The donor disk may not be in perfect centration after insertion. If not, it can be positioned from either the endothelial side or the stromal side. A reverse Sinskey hook (Bausch and Lomb, St. Louis, MO) is used for endothelial side positioning (Figure 17-40). The hook is placed through the stab incision, the peripheral endothelium is engaged, and the tissue moved over to whatever position is desired. Although this maneuver undoubtedly causes endothelial cell damage at that point of peripheral contact, we have not found that the central endothelial cell counts 6 months after surgery to be any worse than after a standard PKP.10,11,14 Care is taken, however, to minimize this maneuver and also to avoid the central posterior striae that can occur and can compromise vision. An alternative technique for positioning can be done from the stromal interface side using a 30-gauge needle tip. A slight “barb” is placed on a standard short 30-gauge needle, and the tip is placed through the superior wound directly into the interface. The barb is rotated posteriorly to engage a few stromal fibers of the donor disk, and this grasp is used to move the tissue over into the proper centration. During both the endothelial and stromal positioning maneuvers, the anterior chamber is filled with air. Once the tissue is in proper centration, it is critical to make sure that all of the donor edges are anterior to all of the recipient bed edges for 360 degrees (Figure 17-41). Visual inspection is not enough, and manual verification is mandatory. If any portion of the donor tissue edge lies posterior to the recipient rim, then the donor tissue will likely be dislocated the next morning or present with a significant space in the interface (secondary anterior chamber). To accomplish proper donor edge position, the anterior chamber is filled completely with air and a reverse Sinskey hook is placed through a stab incision into the anterior chamber. The tip of the hook is then lifted anteriorly and placed between the edge of the donor and recipient rim. The hook is then rotated to engage the recipient rim posterior stromal edge, and then used to pull the edge posteriorly. With this maneuver, the air bubble in the anterior chamber immediately pushes the donor edge up anteriorly, into the recipient pocket, and upon release of the Sinskey hook, the recipient edge “pops” right up posterior to the donor edge. This “tire iron” maneuver is performed for 360 degrees (Figure 17-41), even when the donor tissue appears already in good position. This is done because even small strands of recipient stromal edge tissue can get caught in the edge interface and prevent adherence of the graft or act as a wick for aqueous into the interface, causing later donor disk dislocation.

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A

C

D

B

Figures 17-38A to D: Donor corneal tissue is unfolded in the anterior chamber.

Figure 17-39: Air bubble injection into the anterior chamber.

Figure 17-40: Positioning of tissue with reverse Sinskey hook.

Once satisfied that the donor disk is in final position with good edge position, the surgeon then removes the air in the anterior chamber and replaces it with BSS (Figure 17-42). Care is taken to avoid pupillary block by the air bubble in the anterior chamber, but if it occurs, simple suctioning of the air from the pupillary surface resolves the problem. Occasionally air can get trapped behind the iris, giving the impression of

posterior pressure with the iris coming forward to the donor edges. Again, suctioning with a cannula from the pupillary surface will resolve this issue. The BSS placed into the anterior chamber creates a normal IOP and the chamber deepens. A small (3 mm wide) air bubble is usually left in place (Figure 17-43) to help further stabilize the donor disk position over the first 24 hours postoperatively.

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Corneal Endothelial Transplant steroids at the close of surgery in order to deliver medication until the patch is removed the next day; however, each surgeon’s usual routine for antibiotics (subconjunctival or otherwise) is acceptable. An occlusive patch and shield are routinely placed, and the patient is brought to the recovery room. We usually instruct the nurses to have the patient lie in a supine position for the first few hours as much as possible to allow the retained air bubble to further stabilize the graft position, but this is not critical. The patient is discharged from the recovery room when fully recovered from anesthesia.

Figure 17-41: “Running the rim” with reverse Sinskey hook.

Figure 17-42: Replacing the air with BSS.

Figure 17-43: Final view of eye after the triple procedure, namely, phacoemulsification, PC IOL and DLEK.

The suture knots of the scleral incision are cut short, and buried on the scleral side. The wound is checked to be watertight. The conjunctival peritomy is closed according to the surgeon preference. We routinely place on the corneal surface a 24-hour collagen shield soaked in antibiotics and

Postoperative Course The patient is seen the next morning and the patch is removed. Most patients will remark that the eye was as comfortable as after a standard cataract surgery and that they did not require any narcotic pain relief during the immediate postoperative period. Once the patch is removed, the vision is usually about 20/400. The vision is unimportant on postoperative day one and the only reason for the visit is to insure that the donor disk is attached and in good position. In our prospective series of over 115 patients (as of July 2004), we have experienced only 5 cases where the donor disk was dislocated on the first postoperative day. All these five cases were easily treated by taking the patient back to surgery and usually under topical anesthesia, another air bubble is placed in the anterior chamber and the disk repositioned as before. This procedure usually takes about 15 minutes. We have been successful with all five re-positionings, resulting in clear corneal grafts. If the graft is in good position on day one, it will heal in good position and we have had no late graft dislocations. The edges of the graft seal down with significant healing sometime within the first 3 months. The overlying cornea has a variable rate of clearing, but some patients are able to see as well as 20/25, only one week after DLEK surgery, with a crystal clear central cornea. The usual visual progression postoperatively of patients with minimal or no macular disease, however, is the following: One day: 20/400, one week: 20/100, one month: 20/60, three months: 20/50, six months: 20/40, one year: 20/30, two years: 20/25. There is, of course, a high variability of vision in any series of elderly patients undergoing ocular surgery, especially, DLEK. The interface may clinically appear exceptionally clear, but it likely contributes about one line of visual loss to the macular potential.13,14 The endothelial survival after small incision DLEK surgery is quite remarkable. Even with folding the tissue

DLEK: Small Incision Technique Combined with Phacoemulsification and Posterior Chamber IOL 199 and other manipulations described above, the average endothelial cell count after small incision DLEK surgery is comparable to PKP surgery and it is not significantly different from large incision DLEK where the tissue is not folded.14 The postoperative medical therapy after DLEK surgery is identical at this time to what is done with PKP surgery patients. Topical prednisolone acetate 1% is used four times a day for 3 months, then three times a day until 6 months, then twice a day until 9 months, and then once a day until one year postoperatively. The steroids are then tapered down further until discontinued. We have experienced only a 3% rejection rate after DLEK surgery, and so steroid therapy may not be as critical as after PKP, but this remains speculative at this time. Fluoroquinolone antibiotics are used on a four times a day dosage for two weeks and then discontinued. Outside of a scientific protocol, DLEK patients do not require the same degree of monitoring as standard PKP patients and therefore require less postoperative clinic time. With no corneal sutures or incisions, wound healing or ulcerations are not an issue. Astigmatism management is also not an issue after DLEK surgery. The only critical monitoring that is required is for steroid-induced glaucoma as long as the patient is on topical steroids, and this is done according to the clinician’s standard routine. The DLEK surgical procedure is a difficult one and requires a commitment to exacting detail and thorough practice prior to incorporation of this procedure into the surgeon’s operative repertoire. However, with its superior topography, rapid wound healing and long term safety, the DLEK procedure is well worth the effort.

References 1. Sugar A, Sugar J. Techniques in penetrating keratoplasty: A quarter century of development. Cornea 2000;19:603-10. 2. Abou-Jaoude ES, Brooks M, Katz DG, Van Meter WS. Spontaneous wound dehiscence after removal of single continuous penetrating keratoplasty suture. Ophthalmology 2002;109: 1291-96. 3. Tseng SH, Lin SC, Chen FK. Traumatic wound dehiscence after penetrating keratoplasty: Clinical features and outcome in 21 cases. Cornea 1999;18:553-8. 4. Stechschulte SU, Azar DT. Complications after penetrating keratoplasty. Int Ophthalmol Clin 2000;40:27-43. 5. Akova YA, Onat M, Koc F, Nurozler A, Duman S. Microbial keratitis following penetrating keratoplasty. Ophthalmic Surg Lasers 1999;449-55. 6. Confino J, Brown SI. Bacterial endophthalmitis associated with exposed monofilament sutures following corneal transplantation. Am J Ophthalmol 1985;99:111-3.

7. Ko WW, Frueh BE, Shields CK, Costello ML, Feldman ST. Experimental posterior lamellar transplantation of the rabbit cornea [ARVO Abstract]. Invest Ophthalmol Vis Sci 1993;34(4): S1102. Abstract nr 1967. 8. Melles GR, Eggink FA, Lander F, Pels E, Rietveld FJ, Beekhuis WH, Binder PS. A surgical technique for posterior lamellar keratoplasty. Cornea 1998;17:618-26. 9. Terry MA, Ousley PJ. Endothelial replacement without surface corneal incisions or sutures: Topography of the deep lamellar endothelial keratoplasty procedure. Cornea 2001;20:14-8. 10. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty in the first United States patients: Early clinical results. Cornea 2001;20:239-43. 11. Terry MA, Ousley PJ. Replacing the endothelium without corneal surface incisions or sutures: The first United States clinical series using the deep lamellar endothelial keratoplasty procedure. Ophthalmology 2003;110:755-64. 12. Terry MA, Ousley PJ. In pursuit of emmetropia: Spherical equivalent refraction results with deep lamellar endothelial keratoplasty (DLEK). Cornea 2003;22:619-26. 13. Terry MA, Ousley PJ. Rapid visual rehabilitation after endothelial transplants with deep lamellar endothelial keratoplasty (DLEK). Cornea 2004;23:143-53. 14. Terry MA, Ousley PJ. Small incision deep lamellar endothelial keratoplasty (DLEK): 6-month results in the first prospective clinical study. Cornea 2004 (in press). 15. Terry MA. Endothelial replacement: The limbal pocket approach. Ophthalmol Clin North Am 2003;16:103-12. 16. Terry MA. Deep lamellar endothelial keratoplasty (DLEK): Pursuing the ideal goals of endothelial replacement. Eye 2003;17:982-8. 17. Terry MA. A new approach for endothelial transplantation: Deep lamellar endothelial keratoplasty. Int Ophthalmol Clin 2003;43:183-93. 18. Terry MA, Ousley PJ. Corneal endothelial transplantation: Advances in the surgical management of endothelial dysfunction. Contemporary Ophthalmology 2002;1(26):1-8. 19. Terry MA. Endothelial replacement: New surgical strategies. In: Krachmer J, Mannis M, Holland E (Eds). Cornea. Surgery of the Cornea and Conjunctiva (2nd edn). St. Louis: Mosby-Year Book, Inc. 2004 (in press). 20. Terry MA. The evolution of lamellar grafting techniques over twenty-five years. Cornea 2000;19:611-6. 21. Melles GR, Lander F, Rietveld FJ. Transplantation of Descemet’s membrane carrying viable endothelium through a small scleral incision. Cornea 2002;21:415-8. 22. Duran JA, Malvar A, Diez E. Corneal dioptric power after penetrating keratoplasty. Br J Ophthalmol 1989;73:657-60. 23. Binder PS. The effect of suture removal on postkeratoplasty astigmatism. Am J Ophthalmol 1988;105:637-45. 24. Isager P, Hjortdal JO, Ehlers N. Stability of graft refractive power after penetrating keratoplasty. Acta Ophthalmol Scand 2000;78:623-26. 25. Davis EA, Azar DT, Jakobs FM, Stark WJ. Refractive and keratometric results after the triple procedure: Experience with early and late suture removal. Ophthalmology 1998;105:62430. 26. Dursun D, Forster RK, Feuer WJ. Surgical technique for control of postkeratoplasty myopia, astigmatism, and anisometropia. Am J Ophthalmol 2003;135:807-15. 27. Segev F, Voineskos AN, Hui G, Law MS, Paul R, Chung F, Slomovic AR. Combined topical and intracameral anesthesia in penetrating keratoplasty. Cornea 2004;23:372-6. 28. Armour RL, Wilson DJ, Ousley PJ, Terry MA. Invest Ophthalmol Vis Sci 2004;45:ARVO E-Abstract 2898.

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Introduction Endothelial dysfunction caused by pseudophakic bullous keratopathy (PBK) and Fuchs’ endothelial dystrophy is the leading cause of corneal visual loss in the United States.1,2 Penetrating keratoplasty (PKP) with implantation of an irisfixated or sclera-fixated posterior chamber intraocular lens (PC IOL) is the standard procedure for treating cases of PBK with an anterior chamber intraocular lens (AC IOL).³ Deep lamellar endothelial keratoplasty (DLEK) is a good alternative to PKP for endothelial replacement.4-13 In cases of aphakic or pseudophakic (AC IOL) bullous keratopathy, placement of a scleral-fixated PC IOL or replacement of the AC IOL with a scleral-fixated PC IOL may be performed by using the superior incision of DLEK together with a small superior and inferior scleral flaps.

Surgical Objective and Anesthesia The surgical objective is to exchange the posterior corneal lamella including the damaged endothelium for a healthy donor corneal disk of the same size combined with an automated anterior vitrectomy and implantation of a scleral-fixated PC IOL in cases of aphakia and in cases in which replacement of the anterior chamber IOL is necessary. It is preferable to place the IOL in the posterior chamber as shown by Holladay.14 The scleral-fixation of the PC IOL in cases with inadequate posterior capsular support has shown encouraging results as an alternative method of lens implantation.15-21 Dissection and excision of the posterior corneal lamellar disk allows a good surgical view of the anterior segment and facilitates performing anterior vitrectomy and scleral-fixated PC IOL even in severe cases of bullous keratopathy. Intraoperative epithelial scraping also can be performed in cases of severe epithelial edema, to improve visualization of the anterior segment. The use of general anesthesia is preferable. Monitored anesthesia care (MAC) can also be attempted if the patient has a high-risk of potential anesthesia-related complications with general anesthesia.

Surgical Procedure The basic surgical procedure of DLEK through a 9.0 mm superior limbal incision as described by Terry and Ousley8 is carried out [See also Section 8, Deep Lamellar Endothelial Keratoplasty (DLEK)]. This is combined with an automated anterior vitrectomy and scleral-fixated PC IOL as described by Helal et al.21 The DLEK instruments used for this procedure are shown in Figure 18-1.

Figure 18-1: Instrument tray showing Devers dissectors, scissors, Terry trephine, and artificial chamber.

The surgical steps are as follows: 1. A superior conjunctival periotomy of 11 mm is made with a Castroviejo corneal scissors (Katena, Denville, NJ) and the bleeding vessels are cauterized. 2. A 9.0 mm incision is made at the superior limbus (Figure 18-2) with a disposable super-blade (Alcon Surgical, Fort Worth, TX). Surgical tip: The incision should be 1 to 2 mm anterior to the original cataract incision so as not to perforate the sclera, if using the same incision, or posterior to it.

Figure 18-2: Limbal incision using a super-blade.

3. Sodium hyaluronate (Healon, Pharmacia, Peapack, NJ) is injected through a paracentesis into the anterior chamber at the 3 o’clock position. 4. A lamellar dissection is carried out (Figure 18-3) from the superior incision starting with a sclerotome blade (Katena). This lamellar dissection is performed about 350 µm from the corneal surface.

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Figure 18-3: Lamellar dissection with a sclerotome blade.

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Figure 18-5: Curved Dever dissector for the lower half of the cornea.

Figure 18-4: Straight Dever dissector for the upper half of the cornea.

5. A complete deep lamellar pocket is then created over the entire cornea, limbus to limbus, using the Devers dissectors (Bausch & Lomb, St. Louis, MO). The straight Devers dissector (Figure 18-4) is used for the lamellar dissection of the superior half of the cornea until the middle of the cornea (3 to 9 o’clock meridian) is reached and then a curved Devers dissector (Figure 18-5) is used for the lower half of the cornea. 6. A 7.5 mm, low-profile, Terry trephine (Bausch & Lomb) is introduced into the corneal pocket (Figure 18-6) and centered to the patient’s limbus. Trephination of the posterior corneal lamella including the Descemet’s membrane and the endothelium is then carried out after increasing the intraocular pressure by injecting

Figure 18-6: Terry trephine being introduced into the corneal pocket.

Healon 5 (Pharmacia) into the anterior chamber. Entry into the anterior chamber is marked by distortion of the pupil and iris entry into the corneal pocket. 7. Following entry into the anterior chamber and removal of the Terry trephine, superior entry into the anterior chamber is carried out using a 15-degree super-blade, if needed, and a right and left corneal micro-scissors (Katena) are used to cut the trephined disk on both sides (Figures 18-7 and 18-8). A highly curved Cindy I scissors (Bausch & Lomb) is used to cut the inferior part of the disk, which is then removed from the corneal pocket (Figure 18-9).

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Figure 18-7: Right corneal scissors being used.

Figure 18-9: A completely cut posterior lamellar disk is being removed through the superior limbal wound.

Figure 18-10: Anterior vitrectomy is performed using an automated ocutome unit. Figure 18-8: Left corneal scissors being used.

8. An anterior vitrectomy (Figure 18-10) is then carried out through the same superior incision by using an automated ocutome (Alcon Surgical). 9. An inferior 5.0 mm periotomy is made and the bleeders are cauterized. A 2.5 mm triangular scleral flap is then fashioned inferiorly at the 5:00 or 7:00 o’clock position. A second triangular flap is fashioned at the 11:00 or 1:00 o’clock position. Surgical tip: Avoiding the 6, 12, 3, and 9 o’clock positions decreases the risk of intraoperative bleeding, because the anterior and long posterior ciliary arteries are at the vertical and horizontal meridia.

10. A double-armed straight needle (Ethicon, Johnson & Johnson, Brussels, Belgium, W1713, 16 mm long, micropoint plus and 150 µm diameter) with 10-0 polypropylene (Prolene) is passed vertically about 0.75 mm from the posterior surgical limbus under the inferior flap (Figure 18-11). Surgical tip: This is the closest position to the ciliary sulcus as shown in cadaver eyes.22 11. A 27-gauge needle that is bent vertically (Figure 1812) is passed under the superior flap (Figures 18-13 and 18-14). 12. The straight needle is then directed tangentially to be fed into the lumen of the 27-gauge needle (Figure 18-15).

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Figure 18-11: Straight needle with Prolene suture is passed vertically under the inferior scleral flap.

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Figure 18-13: The 27-gauge needle is passed under the superior scleral flap.

Figure 18-14: The straight needle directed tangentially to meet the superior one. Figure 18-12: A 27-gauge needle is bent vertically.

13. The 27-gauge needle is then withdrawn, while guiding the straight 10-0 Prolene needle to exit under the superior flap, traversing the posterior chamber (Figure 18-16). 14. The Prolene suture is then pulled through the initial superior limbal incision using a Sinskey hook (Katena) and this suture is cut in the middle using microscissors (Figures 18-17 and 18-18). 15. The lower end of the Prolene suture is tied around the lower haptic of the PC IOL (AMO Duralens PS26TB, 1-piece polymethylmethacrylate, 7.0 mm optic and 14.0 mm overall diameter, Allergan Medical Optics,

Irvine, CA) and the upper end of the suture is tied around the superior haptic (Figure 18-19). Surgical tip: The tied suture knot, should be at the greatest haptic spread. In addition, the one-piece structure of the IOL provides torsional rigidity and stabilization for two-point fixation. 16. The IOL then is introduced through the original superior limbal incision into the anterior chamber and guided to the posterior chamber (Figures 18-20 and 18-21). Surgical tip: While introducing the IOL, the external lower Prolene suture should be pulled out slowly by the surgical assistant to avoid looping the suture around the IOL.

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Figure 18-15: The straight needle fed into the lumen of the 27 gauge needle. Figure 18-18: The Prolene suture (arrow) is pulled out using a Sinskey hook.

Figure 18-16: The 27 gauge needle is being withdrawn.

Figure 18-17: The Prolene suture (arrow) traversing the posterior chamber.

Figure 18-19: One piece polymethylmethacrylate PC IOL.

Figure 18-20: The IOL is introduced and the lower suture is pulled out simultaneously.

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Figure 18-21: The IOL optic is in position. Figure 18-23: The two sutures are pulled up to check lens position.

Figure 18-22: The superior haptic is being placed into the posterior chamber and into the ciliary sulcus.

17. The upper haptic is then placed in position and the superior suture is pulled out at the same time (Figure 18-22). 18. Final positioning of the PC IOL is done by pulling the two ends of the Prolene sutures tight (Figure 18-23) and then tying it to itself by passing the needle, after being bent vertically at the end, through the sclera, under the flap (Figures 18-24 and 18-25). This is done at the inferior (Figures 18-26 and 18-27) and the superior positions (Figures 18-28 and 18-29) followed by suturing of the flaps with 10-0 nylon sutures (Figure 18-30). This results in positioning the superior and inferior haptics of the PC IOL into the ciliary sulcus and the PC IOL is thus held in place by the 10-0 Prolene sutures (2-point fixation). The conjunctiva then is

Figure 18-24: The lower end of each needle is bent vertically by needle holder.

closed either by cautery or using 10-0 vicryl interrupted sutures. 19. The anterior chamber then is irrigated with a twoway canula (Simco canula, Model No. K7-4300, Katena) to remove the viscoelastic substance from the anterior chamber. The anterior chamber is then filled with filtered air. 20. The donor cornea is then mounted within the artificial anterior chamber (Bausch & Lomb) (Figure 18-31) (See also Chapter 12, Artificial Anterior Chambers). A 9.0 mm diameter disposable suction trephine (Barron radial vacuum trephine, Katena) is applied to the surface of the donor cornea and suction is engaged

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Figure 18-25: The bent needle is seen.

Figure 18-28: The upper needle is passed under the superior sclera flap.

Figure 18-29: The knot is tied under the superior sclera flap. Figure 18-26: The lower needle is passed through the sclera under the inferior flap.

Figure 18-27: The knot is tied under the flap.

Figure 18-30: The lens is in position and the scleral flaps covering the knots are seen.

DLEK Combined with Scleral-fixated Posterior Chamber IOL Implantation

Figure 18-31: The donor cornea is mounted within the artificial anterior chamber.

Figure 18-32: A 9.0 mm Barron trephine is positioned over the donor cornea.

(Figure 18-32). Trephination is carried out to approximately 60 to 75% of the corneal thickness. Surgical tip: This depth is achieved at eight quarter turns of the trephine wheel. The trephine is set at three quarter turns back after leveling with the suction barrel before applying the trephine to the donor cornea. 21. A lamellar dissection of the 9.0 mm partially trephined cornea is then performed with a crescent blade (Figure 18-33) until the lower end (Figure 18-34), leaving a small hinge. The lamellar cap is returned into position (Figure 18-35) to protect the posterior lamella during the use of the punch. 22. The donor cornea is then removed from the artificial anterior chamber (Figure 18-36) and mounted onto a 7.5 mm donor punch block (Barron vacuum donor corneal punch, Katena) with the endothelial side up (Figure 18-37) and trephined (Figure 18-38).

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Figure 18-33: Lamellar dissection of the partially trephined donor cornea with a crescent blade is carried out.

Figure 18-34: End of dissection of 9.0 mm partially trephined cornea.

Figure 18-35: The dissected lamellar cap is placed back into position.

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Figure 18-36: The donor cornea is removed from the artificial anterior chamber.

Figure 18-39: The donor lamellar disk is placed on the Ousley spatula with the endothelial side down.

23. The donor disk, which is comprised of the endothelium along with its attached deep corneal stroma, is separated from the anterior donor button and it is placed endothelial side down onto the Ousley spatula (Bauch & Lomb) coated with sodium hyaluronate (Figure 18-39). Surgical tip: A very fine film of viscoelastic substance (Healon 5) is spread over the spatula to protect the endothelium. Excessive viscoelastic material on the Ousley spatula can dislodge the disk from the host corneal stroma, because the viscoelastic material might enter the interface and prevent optimal adherence between the donor and host corneal stroma. Figure 18-37: The cornea is placed in a 7.5 mm punch block.

Figure 18-38: The donor cornea is punched with a 7.5 mm Barron trephine.

Figure 18-40: The lamellar disk is in position within the recipient anterior chamber with the donor disk approximated to the recipient cornea using the Ousley spatula.

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A

Figure 18-41: Suturing the wound with interrupted 10-0 nylon sutures.

B

Figure 18-42: The donor corneal graft is in position with an air bubble in the recipient anterior chamber.

24. The lamellar disk then is introduced by the Ousley spatula through the initial superior limbal incision and pressed gently upward (Figure 18-40) for the tissue to adhere to the recipient corneal lamella. The spatula is then guided out of the wound. Additional air is injected into the anterior chamber through the paracentesis port to stabilize the donor disk. 25. The superior limbal wound is closed with interrupted 10-0 nylon sutures (Figures 18-41 and 18-42). 26. The lamellar donor disk graft is positioned properly by the use of a Sinskey hook introduced through the wound and into the interface. Surgical tip: Any small air bubbles at the interface should be squeezed out, because they can interrupt the graft adhesion.

C Figures 18-43A to C: Preoperative and postoperative slit-lamp photographs. (A) Preoperative photograph showing severe aphakic bullous keratopathy with hand motion vision. (B) One week postoperatively with partial clearing of the corneal edema and a scleralfixated PC IOL in position. (C) Six months postoperatively with a clear cornea and a vision of 20/40.

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Corneal Endothelial Transplant 27. A collagen shield soaked in dexamethasone and levofloxacin is placed over the cornea and patched. The patient then remains in the supine positioned for 2 hours in the recovery room [See also Section 8, Deep Lamellar Endothelial Keratoplasty (DLEK)].

Postoperative Management

A

B

The position of the graft is evaluated every 4 hours postoperatively (Editorial Note: The timing of postoperative evaluations depend on the surgeon’s preference). Graft repositioning should be attempted as early as possible if the disk is dislodged. Topical prednisolone eye drops are given hourly during the first postoperative day with decreasing frequency according to the surgical inflammatory response [See also Section 9, Deep Lamellar Endothelial Keratoplasty (DLEK)]. Antibiotic drops also are administered four times daily for 2 weeks. The patient undergoes a follow-up examination on day 1, week 1, every month for 3 months, and then every 3 months. Examples of cases with severe aphakic bullous keratopathy pre- and postoperatively are shown in Figures 18-43 and 18-44. DLEK provides an adequate treatment for endothelial decompensation. This procedure facilitates obtaining a smooth corneal surface and for the most part maintains the patient’s own preoperative corneal topography and the degree of astigmatism. The combination of anterior vitrectomy and scleral-fixated PC IOL implantation performed through the same incision used for DLEK makes it a combined surgical approach in cases of aphakic or pseudophakic bullous keratopathy.

References

C Figures 18-44A to C: Preoperative and postoperative photographs of another case. (A) Severe aphakic bullous keratopathy and hand motion vision. (B) One month postoperatively with a clear cornea and a vision of 20/100. (C) One year postoperative with a clear cornea, 20/60 vision, and well-positioned scleral-fixated PC IOL.

1. Maeno A, Naor J, Lee HM, et al. Three decades of corneal transplantation: Indications and patient characteristics. Cornea 2000; 19:7-11. 2. Mamalis N, Anderson CW, Kreisler KR, Lundergan MK, and Olson RJ. Changing trends in the indications for penetrating keratoplasty. Arch Ophthalmol 1992;110:1409-11. 3. Muenzler WS, Hall JR. Lens replacement in pseudophakic bullous keratopathy. Posterior chamber intraocular lenses— iris fixated. In: Brightbill FS, ed. Corneal Surgery: Theory, Technique and Tissue, 2nd ed., St Louis, Mosby, 1993; 167-71. 4. Melles GR, Lander F, Beekhuis WH, Remejer L, and Binder PS. Posterior lamellar keratoplasty for a case of pseudophakic bullous keratopathy. Am J Ophthalmol 1999; 127:340-1. 5. Melles GR, Lander F, van Dooren BT, Pels E, and Beekhuis WH. Preliminary clinical results of posterior lamellar keratoplasty through a sclerocorneal pocket incision. Ophthalmology 2000; 107:1850-6. 6. Terry MA, Ousley PJ. Corneal endothelial transplantation: Advances in the surgical management of endothelial dysfunction. Contemporary Ophthalmology 2002;1:1-8. 7. Terry MA. Endothelial replacement: The limbal pocket approach. Ophthalmol Clin North Am 2003; 16:103-12.

DLEK Combined with Scleral-fixated Posterior Chamber IOL Implantation 8. Terry MA, Ousley PJ. Replacing the endothelium without corneal surface incisions or sutures: The first United States clinical series using the deep lamellar endothelial keratoplasty procedure. Ophthalmology 2003;110:755-64. 9. Terry MA. Deep lamellar endothelial keratoplasty (DLEK): Pursuing the ideal goals of endothelial replacement. Eye 2003; 17:982-8. 10. Terry MA. A new approach for endothelial transplantation: Deep lamellar endothelial keratoplasty. Int Ophthalmol Clin 2003;43:183-93. 11. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty in the first United States patients: Early clinical results. Cornea 2001;20:239-43. 12. Terry MA, Ousley PJ. In pursuit of emmetropia: Spherical equivalent refraction results with deep lamellar endothelial keratoplasty (DLEK). Cornea 2003;22:619-26. 13. Terry MA, Ousley PJ. Rapid visual rehabilitation after endothelial transplants with Deep Lamellar Endothelial Keratoplasty (DLEK). Cornea 2004;23:143-53. 14. Holladay JT. Evaluating the intraocular lens optic. Surv Ophthalmol 1986;30:385-90.

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15. Stark WJ, Goodman G, Goodman D, Gottsch J. Posterior chamber intraocular lens implantation in the absence of posterior capsular support. Ophthalmic Surg 1988;19:240-3. 16. Pannu JS. A new suturing technique for ciliary sulcus fixation in the absence of posterior capsule. Ophthalmic Surg 1988; 19:751-4. 17. Hu BV, Shin DH, Gibbs KA, Hong YJ. Implantation of posterior chamber lens in the absence of capsular and zonular support. Arch Ophthalmol 1988;106:416-20. 18. Malbran ES, Malbran E Jr, Aranguren AN. Scleral-fixated intraocular lenses. Arch Ophthalmol 1988;106:1347-8. 19. Girard LJ. PC-IOL implantation in the absence of posterior capsular support. Ophthalmic Surg 1988;19:680-2. 20. Gess LA. Scleral-fixation for intraocular lenses. Am Intra-Ocular Implant Soc J 1983;9:453-6. 21. Helal M, El Sayyad F, El Sherif Z, El Maghraby A, Dabees M. Transscleral fixation of posterior chamber intraocular lenses in the absence of capsular support. J Cataract Refractive Surg 1996;22:347-51. 22. Duffey RJ, Holland EJ, Agapitos PJ, Lindstrom RL. Anatomic study of transsclerally sutured intraocular lens implantation. Am J Ophthalmol 1989;108:300-9.

Anastasios John Kanellopoulos

Eye Banking and Donor Corneal Tissue Preparation in DSAEK

19

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Introduction The first experiments on keratoplasty took place in the early 19th century, with Franz Reisinger attempting to suture corneal grafts after excising host corneal tissue in rabbits (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). The experiments failed and the grafts were rejected; it would be decades until the first successful human keratoplasty procedure was performed. The procedure has evolved dramatically since the first successful keratoplasty, and today an expert corneal surgeon can perform a penetrating keratoplasty (PKP) in less than 30 minutes, significantly improve the preoperative visual acuity, and try to protect against corneal graft rejection with local steroid eyedrops. However, the procedure is far from being perfect; a host of autoimmune diseases and chemical injuries that cause corneal scarring, have a high rate of corneal graft rejection after a PKP. Furthermore, the PKP procedure requires corneal sutures, with the potential of poor wound healing, irregular astigmatism, and suture-induced corneal neovascularization leading to potential graft rejection and vision loss. Since the entire cornea is removed during a PKP procedure, there is a risk of intraoperative expulsive hemorrhage, with possible loss of the eye. Advances in keratoplasty research have focused on improving the surgical techniques to reduce these risks. The formation of eye banks for providing donor corneal tissues for keratoplasty procedures, has been a huge step forwards in the field of corneal transplantation. The first successful corneal transplant took place in 1906. The first US eye bank opened almost 40 years later, in the year 1944. Eye banks have an illustrious history and have been the stewards of the gift of sight throughout the world for over six decades. Eye banks are unique because they recover, evaluate, and distribute donor corneal tissue for transplantation, research and education. In 2005 alone, 31,947 corneas were transplanted in the United States. Additionally, 13,542 corneas were provided for research and 4,541 corneas were distributed for training purposes. The Eye Bank Association of America (EBAA) is a national association that represents 80 US eye banks and 13 international eye banks. Since 1961, the EBAA has been dedicated to the restoration of sight through the promotion of eye banking by promulgating medical standards, accrediting eye banks, and certifying eye bank technicians. The EBAA also helps to promote collegial relationships among the various eye banks in the US to assure an adequate supply of corneas for patients in the US. These relationships have introduced scheduled surgery for corneal transplants;

to date, there has been no need for a registry to list the availability of corneas. The EBAA was the first national transplant organization to establish medical standards (1980) and ethical codes of conduct. All EBAA member eye banks voluntarily submit for EBAA inspection; following inspection, an EBAA Accreditation Board meets to review the observations and to determine whether or not to accredit the eye bank. Accreditation of eye banks is awarded for up to three (3) years. The Association works closely with eye banks to provide resources for them to achieve the maximum 3-year accreditation status. In conjunction with the American Academy of Ophthalmology (AAO), the EBAA urges all patients, surgeons and health care facilities to work solely with organizations that are EBAA accredited. As there are marked differences in the medical, technical and practical aspects of organ, eye and tissue donation, the three categories of anatomical gifts have evolved as distinct specialty areas. Organ, eye and tissue donation differ significantly in their medical standards, federal regulatory requirements, timeliness of donor access, number of potential donors, costs structures and philanthropic community support. Anatomically, the cornea is thought to be uniquely protected from infection, significantly reducing the potential for transmission of systemic infectious diseases. Corneas are avascular and are bathed in clear fluids, namely, aqueous humor posteriorly and tears anteriorly on the corneal surface. Infectious agents that travel within the vascular tree, must exit the blood stream, and invade and permeate the clear aqueous humor and tears to reach the cornea. Viral adherence to the cornea may be inhibited by the proteins in these clear fluids. If there is significant gross bacterial contamination, it is often readily evident on slit-lamp microscope inspection of the clear cornea. This differs from bone, skin, and other tissues, which by their physical nature cannot be as readily inspected. Medical literature often refers to the cornea as “privileged” because of the absence of blood vessels, that in other anatomical sites, transport antigens and pathogens from the donor to the recipient. The relatively low rejection rate and the relative absence of systemic disease transmission through transplanted corneas are known to be attributable to the “privileged” nature of the cornea. When corneal avascularity is combined with strict donor screening criteria, the opportunity for systemic infectious disease transmission has proven to be virtually nonexistent, although it can occur. Timeliness is another critical issue that distinguishes eye recovery from organs and tissues. Delicate corneal cells remain viable for only a short period of time following death. Unlike many tissues that can be stored for extended period of time, the cornea can only be

Eye Banking and Donor Corneal Tissue Preparation in DSAEK preserved for a matter of days. Eye banks must attempt to gather all necessary medical data from multiple sources, interview the next of kin, and receive blood test results in as short a time as possible following the death of a donor. The information below outlines the thorough process that eye banks undertake to recover donor corneal tissue and ensure that it is safe for corneal transplantation. At each step, attention to detail and quality is of paramount importance.

Eye Banking: The Process from Donor to Recipient The Call An eye bank receives a call from a hospital or an organ procurement organization or another “federally designated” third party, that an individual has died, and has met preliminary criteria for donation. The eye bank has a very short time within which to contact the next of kin, obtain consent and recover the tissue. This generally needs to happen within 12 hours from the time of death.

The Contact The eye bank contacts the next of kin, as defined by the state law and the Uniform Anatomical Gift Act (UAGA), to obtain consent for the donation of the individual’s corneas.

The Consent If a consent is given, the next of kin is asked to complete a medical-social history. The medical-social history provides the eye bank with information to make a donor eligibility determination.

The Donor Medical Review After the consent is given, the eye bank obtains copies of relevant medical records for review from the hospital, a step in the process of creating a complete donor profile. Eye banks pay close attention to the cause of death, any medications that were administered to the individual and if there was any blood loss.

The Physicial Inspection In the absence of any medical “rule outs,” an eye bank technician performs a physical inspection of the donor. This physical inspection contributes to the donor profile, and screens for physical signs of infectious disease or behavior that may have put them at risk, such as

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intravenous drug use. The technician also draws a sample of blood from the donor to be tested for HIV I and II, hepatitis B & C, and syphilis.

The Recovery The donor’s eyes are then prepared for the procedure to recover the cornea. The technician wears a sterile gown and gloves, and drapes the donor eye to establish a sterile field. While the technician makes certain that the sterile field is not contaminated, the cornea itself is not considered sterile.

The Storgae After removing the cornea, the tissue is placed in a storage medium. This medium keeps the tissue viable and helps to reduce bacterial growth. The technician then transports the cornea to the eye bank’s laboratory for refrigeration.

The Evaluation Specially trained eye bank technicians evaluate the donor cornea through microscopes to ensure that it meets the eye bank’s strict criteria for corneal transplantation.

The Eligibility Determination The eye bank’s medical director or his/her designee reviews the records for the donor and makes a final eligibility determination.

The Release of Tissue If the medical director or his/her designee authorizes release of the tissue, the cornea is then sealed and packed in a container in wet ice (to ensure it remains between 2-8 degrees and does not freeze).

The Transport The cornea is labeled with a unique identification number to allow the eye bank to track the tissue from the donor to the recipient. It is then shipped to a surgeon or another eye bank for corneal transplant. Eye banks take their stewardship of the gift of sight very seriously. They train their staff to ensure that the recovered tissue is safe for corneal transplantation and is of the highest quality. The EBAA holds eye banks to a high level of professionalism through the promulgation of medical standards, a stringent accreditation program, examination and certification for technicians, research grants, and continuing education seminars and scientific sessions.1

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The demand for donor corneal tissue in the United States has increased dramatically over the last two decades. Cornea transplantation has become a major means of visual rehabilitation for several corneal disorders such as: 1. Pseudophakic bullous keratopathy. 2. Aphakic bullous keratopathy. 3. Advanced keratoconus. 4. Previous graft failure. 5. Significant corneal scarring, secondary to infection, trauma, etc. Currently, the number of corneal transplantations is under 40,000 per year, according to data from the EBAA. The demand of corneal tissue for transplantation is influenced by changes and the indications for PKP. These indications appear to change with the passing years. We have seen in the past literature, that the number of keratoplasties for pseudophakic and aphakic bullous keratopathies have changed according to the changes in the cataract surgery techniques. The criteria for corneal tissue required for PKP as set by the FDA were finalized in May 2004. In addition to screening and testing for HIV and hepatitis, as was done under an earlier regulation, the new regulation requires screening for diseases such as syphilis, West Nile virus, severe acute respiratory syndrome (SARS), and the neurological condition Creutzfeldt-Jakob disease (CJD). No lab tests are currently available to test for West Nile virus, SARS, and CJD. To identify a potential risk for these diseases, a tissue bank representative interviews the family of the deceased donor. Interviewers at Donor Alliance typically have social work or psychology backgrounds, says Mansfield, and they ask about 50 questions to gain extensive information about the donor’s medical and lifestyle history that may signal a risk, including sexual practices, injectable drug use, and travel history. Donors who lived in Britain, for example, at the height of the “mad cow” epidemic are excluded from donating because they are considered at risk for the human form of mad cow disease, a variant of CJD. To further protect transplant recipients, the donor eligibility rule builds in flexibility for the FDA to require screening for new disease threats as they emerge and reliable tests become available. Donor tissue cannot be released for use until all screening and testing processes are completed and medical experts review and evaluate the results. An exception may be made in the event of an urgent medical need where no suitable tissue is available and the recipient is likely to become gravely ill or die without the tissue (does not usually apply to corneas). Any donor found to have infected tissue or found to be at risk for infection is considered ineligible, and, with rare exceptions,

all tissue from that donor is destroyed or used only for research or educational purposes. http://www.fda.gov/ fdac/features/2005/305_tissue

Overview of Supply of Donor Corneal Tissue EBAA’s membership is comprised of 92 US member eye banks, a participation rate of 99% of the eye banks in this country. Our member banks provide approximately 97% of all corneal tissue for transplantation. All eye banks are 501(c)(3) organizations whose sole mission is to procure and provide donated human eye tissue for sight restoring transplantation procedures. The EBAA takes pride in ensuring the highest standards of safety for our member eye banks to practice and has established strict Medical Standards that are reviewed and revised annually. To be accredited, EBAA members are subject to an inspection and certification program to demonstrate adherence to such standards and other requirements. EBAA member banks provided 46,532 corneas for transplantation in 2001.2 A total of 83,075 were actually procured, with the difference deemed unsuitable for transplantation. These corneas did not meet strict eye bank standards, and based on exclusionary criteria, were not used for transplant, but were instead provided for research, education, or destroyed. Corneas are a gift of human eye tissue made by the donor prior to death, or by the donor’s family following the donor’s death. As a gift from a human donor, a supply cannot be ordered or assured. Further, a supply cannot be maintained, because a cornea loses its viability within several days of procurement. To meet the need of approximately 46,000 corneas each year, it is necessary to procure approximately twice the amount of donor corneal tissue. The future availability of corneas for transplant procedures is uncertain, given the increasing use of LASIK and other surgical procedures which modify the cornea. Currently, individuals who have undergone these procedures are not considered suitable donors for corneal transplantation. In the past five years alone, there has been a sharp increase in LASIK procedures, doubling from 1997 to 450,000 in 1998. The number of procedures in a five year period totals 5,415,000. This is cumulative and increasing and could well adversely affect the supply of corneas for transplantation. The future demand for transplantable corneas and ocular research tissue is likely to increase. The National Eye Institute and Prevent Blindness America released a report in March of this year, concluding that more Americans than ever are facing the threat of blindness from

Eye Banking and Donor Corneal Tissue Preparation in DSAEK age-related eye disease. “Over one million Americans aged 40 and over are currently blind and an additional 2.4 million are visually impaired. These numbers are expected to double over the next 30 years as the Baby Boomer generation ages.”3 In order to ensure a sufficient supply of corneal tissue for transplantation, necessary for the restoration of sight, eye banks must collect and distribute corneal tissue within strict time parameters and in sufficient volume to meet the need.4 At the present time there has been an increasing interest among corneal surgeons in lamellar corneal surgery [See also Section 8, Deep Lamellar Endothelial Keratoplasty (DLEK), and Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)], a gradual change in direction from full-thickness penetrating keratoplasty to partial thickness lamellar corneal surgery. Factors contributing to the increasing popularity of lamellar keratoplasty include the development and continued improvements in the microkeratome, the introduction of artificial anterior chambers (See also Chapter 12, Artificial Anterior Chambers), improved surgical instrumentation (See also Chapter 11, New/Useful Surgical Instruments in DSAEK), and simplified surgical techniques (See also Chapter 23, DSAEK Simplified Surgical Technique). Anterior lamellar keratoplasty (ALK) (Figure 19-1) for corneal stromal diseases and scars with a healthy recipient endothelium, and posterior lamellar keratoplasty (PLK) (Figure 19-2) such as deep lamellar endothelial keratoplasty (DLEK), Descemet’s stripping endothelial keratoplasty (DSEK), and Descemet’s stripping automated endothelial keratoplasty (DSAEK) are alternative surgical techniques to a fullthickness PKP for host endothelial cell failure.

Figure 19-1: Schematic representation of anterior lamellar keratoplasty.

Figure 19-2: An illustration of the donor corneal tissue in blue replacing the removed, host Descemet’s membrane and endothelium in DSAEK procedure.

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This relatively popular technique - DSAEK, relies on the observation that many causes of corneal decompensation occur as a result of corneal endothelial dysfunction, and that replacing the endothelium without transplanting the entire cornea is sufficient to restore vision. These techniques offer a clear cornea without any significant surgically induced corneal astigmatism, no corneal wound and the absence of corneal sutures. Furthermore, since the donor endothelium can be inserted through a small incision in DLEK and DSAEK surgery, instead of an open-sky approach, the relative chance for an expulsive hemorrhage is less, and since there are no corneal sutures, the risk for infection in these procedures are expected to be much less as compared with a PKP procedure. Endothelial replacement research dates back to 1993 when Ko et al performed posterior lamellar transplantation experiments in rabbits (See also Chapter 14, History of Lamellar and Penetrating Keratoplsty). Posterior lamellar keratoplasty (PLK) was then attempted in cadaver eyes and primates through the mid-1990s, and Melles et al reported the first successful human PLK in 1999 (See also Chapter 14, History of Lamellar and Penetrating Keratoplsty). Since then, the technique has been further refined by Terry et al, who termed the procedure deep lamellar endothelial keratoplasty (DLEK). Theoretically, DLEK or DSAEK can be performed for nearly half of the 38,000 transplants performed each year in the United States, since nearly half of these procedures are due to vision loss from endothelial dysfunction. DLEK is a technically challenging procedure [See also Section 8, Deep Lamellar Endothelial Keratoplasty (DLEK)] that relies on the surgeon’s ability to dissect into and through the corneal stroma, without perforating through the anterior surface of the cornea or into the anterior chamber. Both the corneal surface and the limbus are preserved in DLEK and DSAEK surgeries, and as such, it maintains the integrity of the globe. Keratoplasty has evolved over the last five years with the availability of elegant systems that offer artificial anterior chamber, microkeratomes that work in these artificial anterior chambers, and the ability for “sophisticated” preparation of the donor corneal tissue in vitro. We have presented in the past our experience with the artificial anterior chamber and a microkeratome in ALK (Figure 19-1). In this procedure a microkeratome is used to prepare a donor surface lenticule, to replace a complete lamellar flap removed from the host by the same microkeratome. More recently, the advent of DSAEK procedure has made it a clear indication for most endothelial cell failures. The actual parameters required for the donor corneal tissue with this procedure have changed. In DSAEK, the preparation of corneal tissue

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Figure 19-4: Schematic representation of the donor corneoscleral button within the Moria ALTK system.

A

Figure 19-5: A 350 µm LSK-microkeratome (Moria Inc., Antony, France) is assembled to perform a complete pass and remove the anterior 350 microns of donor tissue as a “free-cap.”

B

Figure 19-6: Schematic representation of the encased donor corneoscleral button within the Moria ALTK artificial anterior chamber with the LSK guide ring in place.

C Figures 19-3A to C: The placement of a large corneoscleral donor button endothelial-side down within the Moria ALTK system and the rings are attached and tightened (Moria Inc., Antony, France).

Figure 19-7: Diagrammatic display of the donor corneal tissue without the free-cap, after a complete pass with the LSK microkeratome.

requires a large corneoscleral button ideally over 14 mm in diameter. This will enable the successful donor tissue fixation within the artificial anterior chamber (Figures 19-3 and 19-4) and subsequent harvesting of the donor

deep stromal-endothelial button using an automated microkeratome and an artificial anterior chamber (Figures 19-5 to 19-7). The donor cornea is then trephined from the endothelial side using a disposable Hanna trephine with

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A Figure 19-8: The microkeratome cut donor corneal button is flipped over on itself such that the endothelial side is facing up on the cutting block and trephination is carried out using a Hanna trephine to create a corneal lenticule of a pre-determined diameter (8.75 mm in this example).

B Figures 19-11A and B: The prepared donor lenticule is folded and ready for insertion into the recipient anterior chamber. Figure 19-9: The Hanna trephined lenticule.

Figure 19-10: Schematic representation of trephination of the donor corneoscleral button using a Hanna trephine with a diameter of 8.75 mm.

a diameter that the surgeon chooses (Figures 19-8 to 1910). The donor lenticule is then folded as a 60%/40% fold with a strip of Healon within the “taco” fold (Figures 1911A and B) and it is now ready for insertion into the recipient anterior chamber.

DLEK and DSEAK procedures have been changing the offered tissue trends in several eye banks in the US. An increasing number of eye banks in the United States now offer pre-cut corneal tissue with an automated microkeratome and an artificial anterior chamber (See also Chapter 30, Use of Eye Bank Pre-cut Donor Tissue in DSAEK). The most popular device for the donor corneal preparation has been the one made by Moria (Moria Inc., Antony, France) (See also Chapter 12, Artificial Anterior Chambers). Donor corneal tissue that had previous LASIK surgery, is currently not offered by the eye banks as a donor cornea for transplantation. Since LASIK surgery has altered the cornea with regard to an intrastromal wound with altered biomechanics, it is not suitable for full-thickness PKP procedure. Since, only healthy donor corneal endothelium is required along with the deep corneal stroma for PLK procedures such as DLEK, DSAEK, it will be interesting to see whether in the future such corneas will be offered by

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eye banks for PLK procedures. Superficial corneal trauma as well as superficial corneal scar are another contraindication for donor corneal tissue for PKP. Again, this may not necessarily be a contraindication in the future for tissue use in DLEK and DSAEK surgery and eye banks may change their policy regarding such donor corneal tissues with healthy endothelium and deep corneal stroma. We have been using the 350 µm head with the LSK microkeratome (Moria Inc., Antony, France) in order to remove 320 to 380 µm of anterior cornea [See also Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)]. The corneal donor button is then trephined endothelial side up with a disposable 8.75 mm trephine blade and the Hanna donor block (Moria Inc., Athony, France). The reported results with the DSAEK procedure are very promising and there is a continued interest among corneal surgeons regarding the DSAEK procedure. Currently, many of the eye banks do provide pre-cut donor corneal tissue for those surgeons requesting pre-cut tissue for their DSAEK procedure. The appropriate donor tissue would be screened in the eye bank following necessary steps that are very similar to the current protocol in use. Use of pre-cut donor corneal tissue would significantly shorten the operative time. However, one has to keep in mind the tissue is cut by an eye bank technician and there is an additional charge by the eye bank for providing pre-cut donor corneal tissue (Editorial Note: “Surgery by surgeons” is somewhat modified in this setting. Healthy donor corneal endothelium is an important part of this transplant surgery. Currently, a new CPT code has been established in the US that will pay a surgeon fee for cutting

the donor corneal tissue by the surgeon in the operating room at the time of DSAEK surgery). The procedure would then entail the incision and removal of the host Descemet’s membrane, followed by the folding of the prepared endothelial donor corneal disk and insertion into the recipient anterior chamber. The use of the Femtosecond laser, has become popular both in the USA and internationally. This technology currently provides bladeless LASIK procedure. This technology has recently been used in the preparation of the donor corneal tissue for DSAEK surgery. Continuing technique refinements and studies will determine whether the overall graft survival in DSAEK is comparable to that of a PKP. DSAEK is currently a new but established alternative to PKP which is the golden standard treatment for most visually debilitating corneal diseases. Ongoing studies and further experience with the DSAEK procedure will determent the variability of this technique and the serious consideration from eye bank strategy in the USA and internationally, can facilitate the specific tissue needs that this new technique poses.

References 1. News from the EBAA, The uniqueness of eye banking: Eye Banks ensure the safe supply of corneal tissue for transplantation, research, and education – August 31, 2006. 2. Eye Bank Association of America. Statistical Report, 2002. 3. US. Department of Health and Human Services, Press Release: “More Americans Facing Blindness Than Ever Before.” March 20, 2002. 4. Keeping Human Tissue Transplants Safe. www.fda.gov/fdac/ features/2005/305_tissue.html

Mark A Terry

Endothelial Keratoplasty: A Step by Step Guide to DSEK and DSAEK Surgery

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Introduction Endothelial dysfunction from disease or trauma is one of the leading indications for corneal transplantation. Over the past 100 years, the only solution for endothelia’s replacement was through a full thickness corneal transplantation.1 While penetrating keratoplasty (PKP) has been shown to yield healthy donor tissue with good endothelial function, this procedure has been “plagued” by the inherent problems of unpredictable surface topography, retained surface sutures, and poor wound strength.2-6 In 1993 Ko and Feldman presented an animal study at ARVO which described a new technique for endothelial replacement through a scleral limbal incision7 (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). Their animal work allowed endothelial replacement, completely avoiding surface corneal incisions, but utilized sutures to attach the graft to the recipient. Gerrit Melles of the Netherlands had the insight to use an air bubble to hold the tissue in place, completely avoiding any sutures or incisions in the recipient cornea. He performed this selective endothelial transplantation in the first human patients in 1998 and called it posterior lamellar keratoplasty8 (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). Terry and Ousley began laboratory work in 1999 on this technique, and after technical modifications and re-design of instrumentation to make it faster and easier, performed the first United States cases in 2000 and called the surgery Deep Lamellar Endothelial Keratoplasty (DLEK).9-21 All of this work represents a radical departure from the standard PKP technique in that the DLEK surgery accomplished the goal of endothelial replacement without touching the surface of the recipient cornea. By eliminating surface corneal sutures and incisions, the advantages of normal corneal topography and faster wound healing were obtained, leading to faster visual rehabilitation and a more stable globe for the patient.10-14 We have investigated this technique in the largest prospective study of endothelial keratoplasty surgery in the world and have found it to be valid for endothelial replacement surgery.21 While undoubtedly there will be further refinements of the technique and instrumentation in DLEK surgery, it is the purpose of this chapter to describe in detail the recent modification in preparation of the recipient bed by stripping of the recipient Descemet’s membrane (See also Chapter 13, Definition, Terminology and Classification of Lamellar Corneal Surgery). This modification utilizing the stripping of Descemet’s membrane has been popularized as “Descemet’s Stripping Endothelial Keratoplasty”, or

“DSEK”, and has the advantage of being easier for the surgeon to perform and of providing a smoother interface on the recipient side for the visual axis [See also Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)]. Preparation of the donor tissue in endothelial keratoplasty has also been made easier with the utilization of an automated microkeratome, and the addition of this component to the surgical procedure has been popularized as “Descemet’s Stripping Automated Endothelial Keratoplasty”, or DSAEK. Currently, Moria has the only practical microkeratome for the preparation of donor material and other lamellar keratoplasty procedures (See also Chapter 12, Artificial Anterior Chambers). Recently, the use of the femtosecond laser for donor tissue preparation is also being explored [See also Chapter 26, Femtosecond Laser (Intralase®) – Descemet’s Stripping Endothelial Keratoplasty (Femto-DSEK): Initial Studies of Surgical Technique in Human Eyes]. These advantages of DSEK/DSAEK over DLEK have been mitigated in the past, however, by the reported increase in donor dislocation the day after surgery, requiring a further surgical intervention for re-attachment. In addition, the abnormally increased total corneal thickness following DSEK/DSAEK, has unknown visual consequences for the patient and is still being investigated. We describe in this chapter a method for reducing the incidence of donor dislocation in DSEK/DSAEK surgery, by specific roughening of the peripheral recipient bed to promote donor adhesion. Other surgical maneuvers to prevent dislocation are also described (See also Chapter 27, Techniques to Facilitate Disk Adherence to Recipient Cornea in DSAEK). The reader is cautioned, however, that DLEK or DSEK/ DSAEK surgery requires a separate skill set from standard full thickness PKP surgery, and the experienced PKP surgeon will find the maneuvers of DLEK or DSEK/DSAEK surgery initially unfamiliar. Therefore it is highly recommended that the new EK surgeon take a hands-on training course in EK surgery with an experienced EK surgeon first, and then follow this, with further cadaver eye practice sessions before embarking in the clinical and surgical treatment of patients.

Anesthesia DSAEK surgery is usually done under retrobulbar block anesthesia. General anesthesia (either endotracheal or laryngeal mask airway technique) is somewhat easier for DSAEK because it minimizes posterior pressure on the globe and this is important during the recipient resection and donor implantation phases of the surgery. In addition, general anesthesia minimizes the risk of patient movement

Endothelial Keratoplasty: A Step by Step Guide to DSEK and DSAEK Surgery during the more delicate portions of DSAEK surgery and may aid in the comfort level of the novice DSAEK surgeon. Nonetheless, DSAEK surgery can be safely accomplished with good retrobulbar anesthesia combined with seventh nerve block (orbicularis block) local anesthesia as well. It is even possible to perform DSAEK surgery on selected high risk patients under topical anesthesia, but this is not recommended for the initial cases of the novice DSAEK surgeon.

Preoperative Medications In the usual case of patients with pseudophakia, the pupil is constricted, in order to stabilize the iris-lens diaphragm during the surgery. This is also done if the patient has a clear crystalline lens and concurrent cataract surgery is not planned. We have stopped using Pilocarpine for this, and instead use Miochol (intraoperatively) in order to only have a short acting constriction of the pupil. The Miochol is placed just after the Healon has been removed from the anterior chamber, after the recipient stripped tissue has been removed. Preoperatively, one set of aproclonidine 0.5% drops is also given just prior to surgery to reduce pressure and minimize conjunctival injection. Preoperative antibiotics may be given according to the surgeon’s preference. The eye is prepped in the usual sterile ophthalmic fashion with the use of povidone-iodine solution. In the case of patients with cataract and endothelial failure, cataract surgery is performed concurrently with EK, just prior to the DSAEK endothelial transplant, and the pupil is dilated preoperatively with the surgeon’s standard dilating drops for cataract surgery. While pilocarpine is avoided, the rest of the preoperative medication regimen described above is utilized.

Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK): Recipient Peripheral Scraping Technique The essential steps of DSAEK surgery are shown in Figures 20-1A to L. A detailed account of DSAEK surgery maneuvers is presented as follows: The small incision DSAEK procedure is usually performed from the temporal side in order to provide the greatest manual access and visualization for the surgeon. Prior to forming the DSAEK scleral access incision, two clear corneal limbal stab incisions (about 1 mm diameter)

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are placed about 5 clock hours apart, to be used as access points to the anterior chamber later in the operation. We find that an inexpensive 1 mm diamond knife provides a better paracentesis wound than that made by a metal blade. The paracentesis wounds should be made relatively perpendicular to the corneal surface in order to allow quick access to the anterior chamber later in the surgery. Through one of the stab incisions, the cohesive viscoelastic Healon (Pfizer, New York, NY) is placed into the anterior chamber to replace the aqueous fully and to maintain normal pressure. We oppose the use of Viscoat (Alcon, Fort Worth, TX) or other dispersive viscoelastic materials during any portion of DSAEK surgery as the dispersive materials can cause interface coating with subsequent non-adherence and dislocation of the donor corneal tissue. Prior to the stripping of Descemet’s membrane, a template mark is placed on the corneal epithelial surface. A circular marker with a diameter of 8.0, 8.5, or 9.0 mm (depending upon recipient corneal diameter and surgeon preference) is used to make a circular impression on the central epithelial surface. If the position and centration of the mark is acceptable to the surgeon, then it is accentuated with gentian violet ink marks. This circle on the corneal surface will later be used as a template for stripping of the recipient Descemet’s membrane. A temporal limbal peritomy of the conjunctiva is performed with scissors allowing exposure of about 6 mm arc length (about 3 clock hours) of limbal tissue. After cautery of the scleral bed, a trifaceted, guarded, diamond knife is then set to a depth of 350 µm and a 5.0 mm length incision is made approximately 1 mm posterior to the corneal limbus and concentric with it. We have found that a deeper initial incision gives less of a beveled wound closure and also a greater chance of early perforation into the anterior chamber during DSAEK surgery. In lieu of a diamond knife, a sharp crescent blade or other steel scalpel can be used for the initial incision. A sharp crescent blade is then utilized to create a deep sclerocorneal lamellar pocket down to about 75 to 85% corneal depth along the entire length of the wound, extending centrally about 1 mm into clear cornea from the limbus. A simple, blunt tip, reverse Sinskey hook (Bausch and Lomb, St. Louis, MO) is used for the Descemet’s stripping portion of the procedure. The hook is introduced into the Healon-filled anterior chamber through one of the limbal paracentesis sites. The tip is passed across the chamber and then lifted anteriorly until it contacts the recipient endothelium at the distal location of the epithelial circular template mark. The diseased endothelium and thickened Descemet’s membrane of the recipient cornea is easily punctured by the blunt tip of the reverse Sinskey hook and

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Figures 20-1A to L: (A) The surface of the recipient cornea is marked with a circular template of 8.0 to 9.0 mm diameter, depending on the size of the patient’s cornea; (B) A 5.0 mm scleral incision adjacent to the temporal limbus is made, and a scleral-corneal pocket incision is made into the clear cornea; (C) The anterior chamber is completely filled with Healon, and a blunt tipped Sinskey hook is used to break through and score the recipient Descemet’s membrane, following the path of the overlying circular template; (D) The recipient Descemet’s membrane is stripped from the posterior corneal surface using the same blunt tipped Sinskey hook, while the chamber is maintained with Healon; (E) The Terry Scraper is used to scrape the peripheral recipient bed to create white stromal fibrils that will promote donor tissue edge adhesion; (F) All of the Healon is easily removed from the eye with an irrigation-aspiration tip; (G) The donor tissue is prepared using an automated microkeratome to cut a free anterior cap from the donor tissue, leaving a posterior residual tissue of between 125 µm and 175 µm; (H) The donor posterior disk is punched out using a same diameter trephine, and after a strip of Healon is placed on the central endothelium, the tissue is folded into a 60/40% “taco” shape, to prepare for insertion of this donor disk into the recipient anterior chamber; (I) Charlie insertion forceps are used to grasp the donor tissue and insert it into the recipient anterior chamber; (J) The chamber is deepened with balanced salt solution to begin unfolding the tissue and then the tissue unfolding is completed with placement of an air bubble between the lips of the taco; (K) Once the donor tissue is unfolded and in position, with the chamber filled with air, the Cindy Sweeper is used to remove interface fluid by compressing the surface and “milking” the fluid from the center to the periphery; (L) The air in the anterior chamber is replaced with balanced salt solution, dilating drops are placed on the surface, and a residual air bubble with a diameter that just covers the donor tissue is left in place.

Endothelial Keratoplasty: A Step by Step Guide to DSEK and DSAEK Surgery the hook then is used to score the recipient Descemet’s membrane, following the path of the overlying circular template mark. This maneuver easily cuts a nearly perfect circle in the Descemet’s membrane, outlining the edge limits of the recipient bed for endothelial transplantation. A blunt tip hook is far preferable to a sharp point or needle point in that it selectively punctures the Descemet’s membrane, but does not penetrate the stronger overlying stromal fibers (See also Chapter 11, New/Useful Surgical Instruments in DSAEK). This allows true Descemet’s stripping (and not stromal dissection), yielding the easiest stripping and the smoothest possible surface for the visual axis. Fuchs’ dystrophy causes the recipient Descemet’s membrane to be significantly thicker than what is normally encountered in other conditions of endothelial failure. This fact, in addition to posterior separation of posterior stromal lamellae by edema, works to the advantage of the surgeon in allowing very easy and smooth stripping of the diseased Descemet’s membrane to occur. In addition, the stripped Descemet’s membrane is easily visualized, and does not require special staining to help with resection. Once the diameter of recipient bed has been scored, the same tip is used to gently peel the Descemet’s membrane away from the overlying stromal tissue, pulling the edges 360 degrees into the anterior chamber. The tissue can then be completely stripped off from the central posterior cornea using either the same reverse Sinskey hook tip or by using simple forceps or an irrigation/aspiration tip during the step of viscoelastic removal. Once Descemet’s membrane has been completely stripped with the reverse Sinskey hook, the diseased tissue can be removed from the anterior chamber and sent to pathology. We utilize a standard cataract surgery diamond blade with a 2.8 mm width to enter the anterior chamber, but any suitable blade is acceptable. Entry into the anterior chamber is done by passing the blade through the corneoscleral tunnel, directing the blade posteriorly, and then entering the anterior chamber. We prefer to enter the anterior chamber at a site at least 1 mm peripheral to the most temporal portion of the circular template mark. Capsular or other gentle forceps are used to grasp the recipient Descemet’s membrane, and remove it from the eye and send it to pathology. It is at this point of the surgery that we feel it is critical to scrape the periphery of the recipient bed in order to aid in later donor adherence. When viewed by scanning electron microscopy, we have found that the recipient bed after stripping of Descemet’s membrane to be exquisitely, “glassy” smooth.25 Compared to DLEK, there is complete absence of cut stromal fibrils. We believe that these cut fibrils

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of the recipient bed significantly contribute to donor adhesion, and their absence after stripping may partially explain why donor tissue after DSAEK has more of a propensity to dislocate than after DLEK. We therefore advocate a hybrid of the two techniques whereby the advantages of both are retained. In this hybrid modification, the periphery of the recipient bed is scraped to roughen the surface and expose stromal fibrils, while the central recipient bed is left untouched to yield the smoothest interface possible for the visual axis. Peripheral scraping of the recipient bed is accomplished with the use of a specialized instrument, the Terry Scraper (Bausch and Lomb, St. Louis, MO) (See also Chapter 11, New/ Useful Surgical Instruments in DSAEK). This instrument is similar to a reverse Sinskey hook or Descemet’s stripper, but instead has a broad (1 mm) roughened tip like an ice scraper. The tip is introduced into the anterior chamber through the scleral-tunnel incision, with Healon still filling the anterior chamber. The tip of the Terry Scraper is then used to scrape the peripheral 1.5 mm of the recipient bed, and the creation of stromal fibrils is visually verified. The periphery of the recipient bed is scraped for 360 degrees, taking care to leave the central bed of 5 to 6 mm diameter untouched and smooth. After scraping of the recipient bed, the temporal scleral wound is temporarily closed with 1 interrupted 10-0 nylon suture. An irrigation/aspiration (I/A) tip is then introduced into the anterior chamber and extensive effort is expended to remove all of the viscoelastic from the eye. Absolutely no Healon should remain in the anterior chamber prior to insertion of the donor disc or the donor tissue will not “stick” in place. Therefore, care is taken to irrigate and aspirate the anterior chamber, pupillary area, angle, and even the peripheral recipient bed as necessary. Once the surgeon is confident that all Healon has been removed, then Miochol is injected into the anterior chamber to constrict the pupil. The intraocular pressure of the eye is left slightly soft and attention is turned to preparation of the donor corneal disk.

Automated Donor Tissue Preparation (DSAEK) This step of donor preparation can be done just prior to the surgery on the patient’s eye, depending on surgeon preference. “Pre-cut” tissue is also available now through several EBAA certified Eye Banks (See also Chapter 19, Eye Banking and Donor Corneal Tissue Preparation in DSAEK). The operating microscope is brought over to the separate donor table for preparation of the donor tissue. Because

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whole globes are rarely available in the United States, an artificial anterior chamber (See also Chapter 12, Artificial Anterior Chambers) is necessary for preparation of the donor corneal disk. The only automated microkeratome system available for DSAEK surgery is from Moria (Moria, Antony, France). The Optisol-GS preservation fluid (Bausch and Lomb, Rochester, NY) from the donor tissue container is aspirated into a syringe (which has a three way stop-cock) and then the syringe is used to fill the I/A port of the artificial anterior chamber with the Optisol-GS fluid. The syringe is also attached to the port to be used to vary the pressure inside the artificial anterior chamber for the duration of the resection. The standard donor corneoscleral cap tissue is first coated with a thin layer of Healon on the endothelium. It is then placed endothelial side down onto the post/piston of the artificial anterior chamber (taking care not to include an air bubble in the chamber) and the tissue is oriented with the largest diameter of the cornea in the horizontal meridian. With the donor corneal tissue within the artificial anterior chamber, the piston/post of the unit is then raised until the tissue is firmly locked into place. The Optisol filled syringe is then used to raise the pressure within the artificial anterior chamber to over 65 mmHg and the stopcock is closed to stabilize the high pressure. The epithelial cells are then wiped from the surface of the cornea with a Miracel sponge. The horizontal meridian is marked at the peripheral cornea with a marking pen so that the horizontal meridian of the donor tissue can be identified and for proper anterior cap orientation later in the procedure. For anterior automated lamellar keratoplasty, the guide ring for the microkeratome can be adjusted in height to yield the desired diameter for the tissue resection. For DSAEK surgery, the greatest diameter possible of at least 10 mm is required, and therefore the guide ring should be placed at the lowest possible position without the use of a spacer. A 300 µm microkeratome head is used and the diameter of resection is at least 9.5 mm or more. For corneas that are thicker than 580 microns, a 350 µm microkeratome head can be utilized to obtain an even thinner posterior donor tissue. Caution should be utilized, however, when using a 350 µm head in order to avoid the occurrence of posterior perforation or “button holes” of the tissue (Editorial Note: When using a 350 µm head, pachymetry of the donor cornea is recommended). With the pressure in the artificial anterior chamber elevated to at least 65 mmHg and verified with a gravity tonometer or by digital palpation, the microkeratome head is mounted on the guide ring, positioned for resection, and then passed over the donor cornea at a rate of about 4 to 5 seconds for the pass.

A free cap of anterior tissue is resected and remains above the blade on the microkeratome head. After drying the residual stromal bed with Miracel sponge, checking the stromal bed for smoothness of cut and diameter of cut, several marks are placed at the peripheral edge of the resected bed to help with the centering of the subsequent trephination of the donor tissue. The anterior cap is placed back in position, using the previously placed peripheral reference surface marks. One additional mark is then placed at the exact central point of the anterior cap of tissue to further facilitate centration of the posterior trephination. A moment is given for the anterior cap to adhere to the bed. The donor tissue should then be carefully dismounted from the artificial anterior chamber without damaging the endothelial cells from chamber collapse. We have found that the easiest way to avoid chamber collapse is to leave the tissue attached to the post/piston. To achieve this, the stopcock on the syringe is turned to the position to allow Optisol flow from the syringe to the chamber. A tying forceps is then used to sweep along the inside ring of the metal cap which locks the tissue onto the post, pushing slightly posterior on the scleral rim, and breaking the seal that binds the donor scleral tissue to the metal cap. Very gently the post/piston is lowered, and Optisol is gently infused, as needed, to keep the donor chamber from collapsing. The locking cap is then removed, with the donor tissue left on the post with a formed chamber. The scleral edges of the donor corneal button are gently lifted in each quadrant to release the seal of the tissue to the post, and the tissue is removed from the post very slowly, lifting the scleral edge up to allow air to enter the chamber, and as the tissue is further lifted off, the air slowly pushes the Healon along, and the Healon then simply flows off the opposite scleral edge as one single sheet, from the endothelial layer onto the post. Once again, care is taken not to collapse the chamber and damage the endothelium. We believe that minimal irrigation of the endothelium with Optisol during tissue removal from the Moria unit aids in the health of the endothelium and in the subsequent donor adherence to the recipient bed. Once the donor corneoscleral tissue has been removed from the post, we have been gently irrigating the sclera above and below the endothelial surface of the donor tissue with Optisol solution (taken from the same transport container for the donor tissue) in order to remove any excess residual Healon from the donor. We then use a Miracel sponge to wick the excess fluid from the endothelium, placing the sponge tip at the scleral edge, away from the endothelium.

Endothelial Keratoplasty: A Step by Step Guide to DSEK and DSAEK Surgery The donor tissue is then placed endothelial side up onto a standard punch trephine block. The previously placed ink mark on the central point of the anterior resected cap is used as a guide to position the tissue for trephination, in order to make sure that the posterior punch trephination is centered on the bed of the previous keratome pass. We utilized a Barron donor punch (Katena, Denville, NJ). The same size diameter punch is used as the diameter of the Descemet’s membrane disc that was removed from the recipient. The tissue is punched out with the trephine. Because the 5 mm wound of small incision DSEK surgery is smaller than the 8.0 mm (or larger) diameter of the donor disc, the donor tissue must be folded prior to insertion into the recipient anterior chamber. To accomplish this, a very thin strip of Healon is placed onto the endothelial surface along the previously identified and marked horizontal meridian of the donor button. Stabilizing the anterior edge of the donor button with a 0.12 mm forceps, the posterior stromal tissue edge is gently grasped with non-toothed delicate forceps such as Utrata forceps. The posterior tissue is then gently folded with the endothelium on the inside protected by the layer of Healon, and it is folded into an asymmetric “taco” shape, in a 60%/40% ratio, the most anterior side of the taco being 60% and the posterior side 40%. We were the first to initiate the idea of an “overfolded, 60%/40% ratio” in order to avoid having the tissue unfold upside-down in the patients anterior chamber. The donor tissue is then brought over to the operative field still on the trephine block.

Donor Tissue Preparation Using Pre-Cut Tissue When utilizing tissue that has been pre-cut by the eye bank (See also Chapter 30, Use of Eye Bank Pre-cut Donor Tissue in DSAEK) with a microkeratome or femtosecond laser and then shipped to the surgery center, the surgeon must be very careful to insure that he knows the exact diameter of the resected bed and that he personally marks the edges of the resected bed of the donor tissue. In this way, the surgeon can avoid an eccentric cut that goes outside of the microkeratome cut bed, with the attendant 1 mm thick donor edge. Using pre-cut tissue is very easy and fast, but to avoid complications, attention to details is paramount. The precut tissue is removed from the Optisol container and placed endothelial side down onto the lint-free plastic or metal surface of the donor table. Because the donor sclerocorneal tissue has a 3 mm rim of scleral “skirt” and tends to maintain its convex configuration, this manipulation of the tissue

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will not collapse the tissue or risk damage to the endothelium. Very gently, the surgeon dries the epithelial surface of the peripheral donor cornea with a Miracel sponge and reveals the edges of the microkeratome cut bed. Although the Eye Bank places marks on the donor cornea, these are often smudged, difficult to see, and may not be accurate to the level of precision to avoid an eccentric cut. After drying, these edges are then measured by the surgeon for the diameter and marked with an ink pen so that they are distinctly seen. The central free cap (which was left in place by the Eye Bank prior to shipment) can also be dried and marked centrally. The surgeon then takes the tissue and places it endothelial side up onto the trephine block, using the marks that he personally placed, to determine the best centration on the block, prior to trephination. The trephination and other steps that follow are well described above. One interesting feature of using pre-cut tissue is that when folding the tissue into the 60/40 taco configuration, the adherence between the posterior stroma and the overlying free cap can sometimes seem a lot stronger than the tissue that is cut “on site” and so care should be taken to avoid causing stretching or striae when folding the graft prior to insertion into the patient’s anterior chamber.

Donor Tissue Preparation Without a Microkeratome: The “DSEK” Procedure The operating microscope is brought over to the separate donor table for preparation of the donor tissue. Because whole globes are rarely available here in the United States, an artificial anterior chamber (See also Chapter 12, Artificial Anterior Chambers) is necessary for preparation of the donor posterior disc. We utilized a Bausch and Lomb (St. Louis, MO) artificial anterior chamber that is all stainless steel and has dual I/A ports. The Optisol-GS preservation fluid (Bausch and Lomb, Rochester, NY) from the donor tissue container is aspirated in a syringe and is then used to fill the I/A ports of the artificial anterior chamber. The syringe is also attached to the port to be used to vary the pressure inside the chamber for the duration of the resection. The standard donor corneoscleral cap tissue is first coated with a layer of Healon on the endothelium. It is then placed endothelial side down onto the post of the artificial anterior chamber and oriented with the largest diameter of the cornea in the horizontal meridian. This meridian is marked with a marking pen so that the horizontal meridian of the donor tissue can be identified later in the procedure. The donor tissue is capped into place and the chamber is filled

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with Optisol-GS and the pressure normalized. There are several ways to create a deep dissection plane in the donor corneal tissue in the absence of a microkeratome. Each surgical approach has advantages and surgeon preference is the over-riding determinant as to which method is to be utilized. The peripheral incision and central manual trephine approachs are described below. In the peripheral incision technique, the preparation of the donor is similar to the preparation of the recipient deep lamellar plane. A diamond knife set to a depth of 350 µm is used to make a 3 or 4 clock hour-length incision in the peripheral donor limbal area, right next to the edge of the metal cap of the artificial anterior chamber. The crescent blade is then used to cut to the deeper stromal tissue and then once the desired plane has been reached, the Devers Dissectors (Bausch and Lomb, St. Louis, MO) are used to continue the dissection plane all the way to the limbus of the donor tissue for 360°. We have found it helpful to place multiple ink marks onto the surface of the donor tissue. These marks accentuate the visualization of the surface of the donor tissue and therefore allow better depth perception (and estimation of the level of the dissection) when viewing the Devers Dissectors plane compared to the surface plane. It is important to make sure that the dissection plane is carried out all the way to the limbus in every quadrant in order to avoid problems that may result from an eccentric trephination of the donor lenticule later on. In the manual trephine approach, an 8.5 mm diameter Barron suction recipient trephine (Katena Products, Denville, NJ) is placed onto the surface of the donor tissue and suction is applied. Trephination is carried out to about 60% depth with the trephine. It is noteworthy that after the blade touches the epithelial surface of the donor, it only takes about 4 or 5 quarter turns of the Barron trephine to reach this depth. This is much sooner than when the same trephine is used on the recipient in standard PKP surgery. The trephine is then removed and the cut inspected for depth. Ideally, an 80% depth should be attained for the plane of the pocket of the donor tissue. Any deeper than this, and the donor tissue is so thin that it spontaneously rolls up like a rug causing confusion as to which side is the endothelial side and undoubtedly causing endothelial damage. If the dissection depth of the donor is less than 60%, then the stromal surface may not be as smooth and the tissue may be much thicker. Whether increased disk thickness causes a later visual problem is unknown at this time, but separate reports by Terry and by Price on this issue at recent ASCRS and ARVO annual meetings suggest that donor thickness is not a factor in final visual results.

Transplantation of the Donor Tissue With the microscope in place, the temporary scleral suture of the superior wound is cut. The anterior chamber of the patient is then filled completely with BSS. The donor tissue is then brought into the field and grasped along the horizontal meridian of the stromal surface of the donor tissue with specialized insertion forceps (Charlie Forceps, Bausch and Lomb, St. Louis, MO). The Charlie forceps are non-toothed fine forceps that coapt only at the distal tips. The forceps have a block that allows a significant spacing along the blade length to prevent crushing of the donor corneal tissue. This specially designed stop in the Charlie forceps enables the surgeon to transfer and hold the folded donor disc tissue without crushing it. The folded donor tissue is placed into the anterior chamber in one deft movement, by inserting the donor tissue with the anterior 60% stromal side facing the recipient bed and the posterior 40% stromal side facing the iris. Again, the endothelial layer remains protected on the inside by Healon. The tissue can be gently manipulated with the forceps along the stromal sides if centration of the tissue within the recipient bed needs to be improved. Additionally, the surface of the cornea can be massaged, and this too often allows the donor taco tissue to be gently adjusted into proper centration. The opening of the taco is placed to the surgeon’s left. The 60% stromal side gently adheres to the overlying recipient bed with the 40% stromal edge lying nearly perpendicular to the iris plane. Three sutures of 10-0 nylon are then used to close the scleral wound to secure the chamber. A cannula is then placed through the right hand stab incision and the tip placed onto the iris surface. BSS is then gently injected into the anterior chamber to fill the chamber and deepen it. With deepening of the anterior chamber, the “taco” will usually begin to open. The ideal position is to open the taco just enough so that the posterior 40% edge lies at an 80 degree angle, just short of being perpendicular to the iris plane. If the tissue does not open at all, then the irrigating cannula can be moved to the left paracentesis site, and irrigation with BSS can be used to loosen the Healon from the endothelial surface and gently unfold the tissue to the 80 degree angle noted above. Because the donor tissue was folded into an asymmetric shape, the tissue invariably will spontaneously unfold in the correct orientation (i.e. endothelium down), as long as the chamber is deep enough and there is no impediment to the unfolding of the donor corneal disc. Once the tissue has unfolded to the 80 degree angle, then an air bubble is gently injected into the anterior

Endothelial Keratoplasty: A Step by Step Guide to DSEK and DSAEK Surgery chamber from the left paracentesis site, very gently into the interior of the 80 degree folded taco. This gentle injection will then fully open the taco and push it up into position onto the recipient bed. Once the tissue is unfolded fully, then air is forcibly and quickly injected to fill the anterior chamber and stabilize the donor tissue, locking it into position. If at any time the surgeon has trouble opening or positioning the donor taco, then the first thing to do is to close the wound with three sutures to stabilize the anterior chamber and then repeat the unfolding and chamber deepening maneuvers mentioned above. The donor disc may not have perfect centration after insertion. If not, it can be positioned from either the endothelial side or the stromal side. A reverse Sinskey hook (Bausch and Lomb, St. Louis, MO) is used for endothelialside positioning. The hook is placed through the stab incision, the peripheral endothelium is engaged, and the tissue moved over to whatever position is desired to achieve optimal centration of the donor corneal disc. This should only be done with a partial air bubble in the anterior chamber. If the chamber is completely filled with air, then such a maneuver will cause striae in the donor tissue. Although this maneuver undoubtedly causes endothelial damage at that point of peripheral contact, we have not found that the central endothelial cell counts 6 months after surgery are any worse than that after a standard PKP procedure.10,11,14,21 Care is taken, however, to minimize this maneuver and also to avoid the central posterior striae that can occur and can compromise vision. An alternative technique for positioning can be done from the stromal interface side using a 30-gauge needle tip. A slight “barb” is placed on a standard short 30-gauge needle, and the tip is placed through the temporal wound directly into the donor-recipient interface. The barb is rotated posteriorly to engage a few stromal fibers of the donor disc, and this grasp is used to move the tissue over into the proper position of centration. During both the endothelial and stromal positioning maneuvers, the anterior chamber is only partially filled with air, namely, an air bubble with a diameter of 9 mm or less. Once the tissue is properly centered, the anterior chamber is completely filled with air and the wound is sutured. If the two paracentesis sites are not air tight, then these too can be temporarily sutured shut for the next step of the procedure. Once the tissue is in proper centration, it is critical to make sure that there is no residual fluid in the central or peripheral interface which can interfere or delay adhesion of the donor disc to the recipient cornea. One technique described by Price et al involves the “milking” of interface fluid from the central interface to the periphery and into

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the anterior chamber (See also Chapter 27, Techniques to Facilitate Disk Adherence to Recipient Cornea in DSAEK). In this technique, the anterior chamber must be completely filled with air, past the edges of the donor tissue. The epithelial surface of the cornea is kept wet with BSS, and then the smooth surface of the shaft of an irrigating cannula is used to deeply compress the central surface of the cornea. The cannula is then swept across the surface from central to peripheral, stroking the corneal surface, attempting to move central interface fluid to the periphery and then into the anterior chamber. Significant compression of the surface must be employed. This maneuver is done from center to periphery in every quadrant, until the surgeon is satisfied that there is no residual fluid in the interface. An instrument called the Cindy Sweeper (Bausch and Lomb Surgical, St. Louis, Missouri) can be for this maneuver (See also Chapter 11, New/Useful Surgical Instruments in DSAEK). Another maneuver for interface fluid removal involves the placement of stab incisions from the surface to the interface, which are placed in the mid-peripheral cornea in each quadrant. Price has advocated these stab incisions (See also Chapter 27, Techniques to Facilitate Disk Adherence to Recipient Cornea in DSAEK) as a means of further releasing interface fluid and reducing donor dislocations. We do not advocate this technique at this time as it violates the primary philosophy of endothelial keratoplasty, which is to avoid surface corneal incisions or sutures of any kind. Furthermore, we do not find that this additional manipulation offers any further protection against dislocation than the recipient preparation steps we have outlined above. With the tissue in good position, the interface fluid removed, and the anterior chamber completely filled with air, the microscope light is turned off and the tissue is left undisturbed for a full ten minutes intraoperatively. We believe that this time allows for the donor corneal tissue to fully warm-up to the patient’s body temperature and for the donor corneal endothelial pump function to begin. This also allows for the cohesive interaction of the peripheral recipient corneal stroma with the donor corneostromal fibrils. Admittedly, the decision for 10 minutes of intraoperative waiting time to facilitate donor disc adhesion to the patient’s cornea is arbitrary, and less or more time may be recommended in the future as multiple surgeons experience dictate. It is also at the beginning of this 10 minute waiting time that we place several drops of dilating solution (Cyclogyle 1% and Phenylephrine 2.5%) onto the corneal surface to allow for the pupil to dilate postoperatively. Because we leave about an 8 or 9 mm air bubble in place at the end of

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surgery, the dilated pupil will prevent any pupillary block. In nearly 500 cases of endothelial keratoplasty, we have not had a single case of pupillary block. Once satisfied that the donor disc is in final position with no interface fluid, the surgeon then removes the air in the anterior chamber and replaces it with BSS. Care is taken to avoid pupillary block by the air bubble in the anterior chamber, but if it occurs, simple suctioning of the air from the pupillary area resolves the problem. Occasionally air can get trapped behind the iris, giving the impression of posterior pressure with the iris coming forward to the donor edges. Again, suctioning with a cannula from the pupillary area will resolve this issue. The BSS placed into the anterior chamber creates a normal IOP and the chamber deepens. An air bubble of approximately 8 to 9 mm is usually left in place to help further stabilize the donor disc position over the first 24 hours postoperatively. The suture knots of the scleral incision are cut short, and buried on the scleral side. The wound is checked to be watertight. The conjunctival peritomy is closed with either sutures or cautery. We routinely place on the corneal surface a 24-hour collagen shield soaked in antibiotics (moxifloxacin) and steroids (dexamethazone) at the close of surgery in order to deliver medication until the patch is removed the next day; however, each surgeon’s usual routine for antibiotics (subconjunctival or otherwise) is certainly acceptable. A gentle occlusive patch and shield are routinely taped into place, and the patient is brought to the recovery room. We usually instruct the nurses to have the patient lie in a supine position, flat, facing the ceiling, for the first hour after surgery and then as much as possible to allow the retained air bubble to further stabilize the graft position, but this is not critical. The patient is discharged following this outpatient procedure when fully recovered from the anesthesia.

Combined Surgery with DSAEK The most common procedure done in combination with DSAEK surgery is cataract extraction with intraocular lens placement. This is usually accomplished with phacoemulsification, but other nucleus manipulative techniques can be employed. The cataract extraction phase of the combined procedure is usually done first and from the same surgical sclerocorneal tunnel incision site. If there is a problem with visualization of the procedure due to surface bullae, nodular scarring or epithelial edema, we simply scrape the surface of the cornea over a 7 mm diameter area or larger and remove any irregularities. Even with poor visualization, the nucleus can be easily removed by bringing the lens up into

the anterior chamber and performing the emulsification there. Damage to the central endothelium is not an issue, because the endothelium will be removed during the DSEK procedure which follows. The viscoelastic material used must be the most cohesive type available, therefore we prefer Healon or Healon V (Pfizer, New York, NY) for use during any surgery that is combined with DSEK. By no means should a dispersive viscoelastic like Viscoat (Alcon, Fort Worth, TX) be used at any time of a combined procedure, and a concentrated effort must be made to ensure that absolutely no viscoelastic of any kind remains in the eye prior to insertion of the donor tissue lenticule. After the cataract extraction with IOL placement is completed, the surgeon then strips the Descemet’s membrane as described above using either the paracentesis sites or the scleral tunnel incision for access (Editorial Note: Removal of the recipient Descemet’s membrane can be performed either before or after the phacoemulsification depending upon surgeon preference. Also, phacoemulsification through a cloudy cornea can also be performed by a technique described by Dr. Thomas John namely, “upside-down phacoemulsification”). Other surgeries such as IOL exchange, vitrectomy, iridoplasty, or suturing of an IOL to the ciliary sulcus can also be accomplished at the same time as DSEK surgery. Because many of these surgeries require a larger incision, namely, larger than a 3 mm cataract wound, they can be carried out through the DSAEK wound, and the DSAEK wound fashioned for whatever length that is necessary to accomplish the task (Editorial Note: When using a much larger incision, the surgeon may consider the superior limbus area rather than the temporal location of the surgical wound). All surgical maneuvers must be completed however, before the donor tissue is inserted, to insure the secure adhesion and safety of the donor endothelium. Interestingly, many intraocular surgeries can safely be accomplished only 3 or 4 months after endothelial keratoplasty surgery without concern of dislodging the grafted corneal endothelial tissue.22

Postoperative Course The patient is seen the next morning and the patch is removed. Most patients will remark that the eye was as comfortable as after standard cataract surgery and that they did not require any narcotic pain relief following the surgery. Once the patch is removed, the vision is usually about 20/200. The vision is unimportant on postoperative day one, and the only reason for the visit is to insure that the donor corneal disc is attached to the recipient cornea, and that it is in good position. In our prospective series of our first 100 DLEK patients, we experienced only 4 cases

Endothelial Keratoplasty: A Step by Step Guide to DSEK and DSAEK Surgery where the donor disc was dislocated on the first postoperative day.21, 23 All four cases were easily treated by taking the patient back to surgery, and usually under topical anesthesia, with a 15 minute operation, another air bubble is placed in the anterior chamber and the disc is repositioned. All repositioned grafts resulted in clear corneas, but the endothelial cell counts at 6 months postop are significantly lower than grafts that have not had to undergo repositioning.23 In our first 4 cases of DSEK without the benefit of scraping and roughening of the peripheral recipient bed, we experienced a 50% rate (2 of 4 eyes) of dislocation. Utilizing the modification of peripheral scraping, however, we have experienced only 4 cases (4%) of dislocation in our initial 100 DSEK cases using peripheral scraping.24 As of January 2007, we have experienced no cases of donor dislocation into the anterior chamber in the past consecutive 100 DSAEK cases, reducing our complication rate of dislocation to 2% or less (unpublished data). If the graft is in good position on day one, it generally will heal in good position. Although we had no late dislocations in DLEK surgery, of the 4 cases of dislocation in DSEK/DSAEK surgery, 3 of those cases which were attached at day one following surgery, became dislocated on days 2, 3 and 4 after the surgery date. This has led us to recommend to patients that they lie in the supine position for one more day after DSEK (to utilize the residual air bubble in the anterior chamber) and to not rub their eye for 2 weeks after DSEK surgery. The edges of the graft seal down with solid healing some time within the first 3 months. The overlying cornea has a variable rate of clearing, but some patients are able to see as well as 20/20 only one week after DSAEK surgery with a crystal clear central cornea (Figure 20-2).

Figure 20-2: One week following DSAEK surgery in a 43 year old phakic eye with Fuchs’ corneal dystrophy, the vision is 20/20 with a +0.50 spherical refraction.

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Only prospective data with control of the variables of patient age and preoperative vision will sort out the differences in DLEK and DSEK/DSAEK surgical outcome. As a guide for DSAEK, however, we have found that the usual visual progression postoperatively of patients with minimal or no macular disease, is as follows: One day: 20/200 One week: 20/70 One month: 20/20 to 20/40 Three months: 20/20 to 20/40 Six months: 20/20 to 20/40 One year: 20/20 to 20/30 Two years: 20/20 to 20/30 There is, of course, high variability of vision in any series of elderly patients undergoing ocular surgery, but especially, DLEK or DSEK/DSAEK surgery. The interface may clinically appear exceptionally clear following surgery. However, it is the stromal interface of the donor tissue that most likely contributes about one line of visual loss to the macular potential.13,14 Extensive work continues to be done to improve the interface in DSAEK surgery. Investigators are working in the areas of femtosecond laser preparation of the donor tissue, but currently, the interface after femtosecond resections in the deep stroma are inferior to that created by a microkeratome [See also Chapter 26, Femtosecond Laser (Intralase®) – Descemet’s Stripping Endothelial Keratoplasty (Femto-DSEK): Initial Studies of Surgical Technique in Human Eyes]. While all of these high and low technology approaches have individual appeal, the worth of any modification will have to be determined by comparing the safety level of the modified technique to the safety of the current DLEK and DSEK/DSAEK techniques. The ultimate visual results must also be compared in like fashion. If any of these modifications cause a higher dislocation rate or other complication for the grafted tissue, then any apparent advantage for the ease of surgery is “fatally” mitigated. Patient safety and improved patient results should always take precedence over the comfort level in surgery for the surgeon. The endothelial survival after small incision DLEK/ DSEK/DSAEK surgery is quite remarkable. Even with folding the tissue and other donor manipulations described above, the average endothelial cell count after small incision DLEK surgery at 6 months is comparable to PKP surgery and is not significantly different from large incision DLEK where the tissue is not folded.14,21 However, our most recent 2 year analysis indicates that folded tissue has a more accelerated loss of endothelial cells from one to two years postoperatively than that found in eyes with large incision DLEK surgery where the tissue is inserted without folding it.

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The postoperative medical therapy after DSAEK surgery is identical at this time to what is done with PKP surgery patients and in our DLEK surgery patients. Topical prednisolone acetate 1% is used four times a day for 3 months, then three times a day until 6 months, then twice a day until 9 months, and then once a day until one year postoperatively. The steroids are then tapered down further until discontinued entirely. In our first 100 cases of DLEK with follow-up between 6 months and 5 years, we have experienced only 4 episodes of graft rejection, inflammation, and only one case where the graft function was lost.24 This is a lower rate of rejection and graft loss due to rejection than that seen in a similar cohort of PKP patients. Steroid therapy after DLEK surgery, therefore, may not be as critical as after PKP surgery, but this remains speculative at this time. Fluoroquinolone antibiotics are used on a four times a day dosage for the first two weeks after DSAEK surgery and then discontinued. Outside of a scientific protocol, DLEK/DSEK/DSAEK patients do not require the same degree of monitoring as standard PKP patients and therefore require less postoperative clinic time. With no sutures or corneal incisions to worry about, wound healing or ulcerations are not an issue. Astigmatism management is also not an issue after DLEK/DSEK/DSAEK surgery. The only critical monitoring that is required, is for steroid-induced glaucoma as long as the patient is on topical steroids, and this is done according to the clinician’s standard routine. The DLEK/DSEK/DSAEK surgical procedure is different than PKP, and requires a commitment to exacting detail and thorough practice prior to incorporation of this procedure into the surgeon’s operative repertoire. However, with its superior topography, rapid wound healing and long term safety, the endothelial keratoplasty by DSAEK procedure is well worth the effort.

References 1. Sugar A, Sugar J. Techniques in penetrating keratoplasty: a quarter century of development. Cornea 2000;19:603-10. 2. Abou-Jaoude ES, Brooks M, Katz DG, Van Meter WS. Spontaneous wound dehiscence after removal of single continuous penetrating keratoplasty suture. Ophthalmology 2002;109:1291-6. 3. Tseng SH, Lin SC, Chen FK. Traumatic wound dehiscence after penetrating keratoplasty: Clinical features and outcome in 21 cases. Cornea 1999;18:553-8. 4. Stechschulte SU, Azar DT. Complications after penetrating keratoplasty. Int Ophthalmol Clin 2000;40:27-43. 5. Akova YA, Onat M, Koc F, Nurozler A, Duman S. Microbial keratitis following penetrating keratoplasty. Ophthalmic Surg Lasers 1999;449-55.

6. Confino J, Brown SI. Bacterial endophthalmitis associated with exposed monofilament sutures following corneal transplantation. Am J Ophthalmol 1985;99:111-3. 7. Ko WW, Frueh BE, Shields CK, Costello ML, Feldman ST. Experimental posterior lamellar transplantation of the rabbit cornea. Invest Ophthalmol Vis Sci 1993;34(4):1102. 8. Melles GR, Eggink FA, Lander F, Pels E, Rietveld FJ, Beekhuis WH, Binder PS. A surgical technique for posterior lamellar keratoplasty. Cornea 1998;17:618-26. 9. Terry MA, Ousley PJ. Endothelial replacement without surface corneal incisions or sutures: Topography of the deep lamellar endothelial keratoplasty procedure. Cornea 2001;20:14-8. 10. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty in the first United States patients: Early clinical results. Cornea 2001;20:239-43. 11. Terry MA, Ousley PJ. Replacing the endothelium without corneal surface incisions or sutures: The first United States clinical series using the deep lamellar endothelial keratoplasty procedure. Ophthalmology 2003;110:755-64. 12. Terry MA, Ousley PJ. In pursuit of emmetropia: Spherical equivalent refraction results with deep lamellar endothelial keratoplasty (DLEK). Cornea 2003;22:619-26. 13. Terry MA, Ousley PJ. Rapid visual rehabilitation after endothelial transplants with deep lamellar endothelial keratoplasty (DLEK). Cornea 2004;23:143-53. 14. Terry MA, Ousley PJ. Small incision deep lamellar endothelial keratoplasty (DLEK): 6 months results in the first prospective clinical study. Cornea 2005;24:59-65. 15. Terry MA. Endothelial replacement: The limbal pocket approach. Ophthalmol Clin North Am 2003;16:103-12. 16. Terry MA. Deep lamellar endothelial keratoplasty (DLEK): Pursuing the ideal goals of endothelial replacement. Eye 2003;17:982-8. 17. Terry MA. A new approach for endothelial transplantation: Deep lamellar endothelial keratoplasty. Int Ophthalmol Clin 2003;43:183-93. 18. Terry MA, Ousley PJ. Corneal endothelial transplantation: Advances in the surgical management of endothelial dysfunction. Contemporary Ophthalmology 2002;1(26):1-8. 19. Terry MA. “Endothelial Replacement. In: Krachmer J, Mannis M, Holland E (Eds). Cornea: Surgery of the Cornea and Conjunctiva. St. Louis: Elsevier Mosby; 2005;140:1707-18. 20. Terry MA. The evolution of lamellar grafting techniques over twenty-five years. Cornea 2000;19:611-6. 21. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty (DLEK): Visual acuity, astigmatism, and endothelial survival in a large prospective series. Ophthalmology 2005;112:1541-9. 22. Amayem AF, Terry MA, Helal MH, Turki WA, El-Sabagh H, El-Gazayerli E, Ousley PJ. Deep Lamellar Endothelial Keratoplasty (DLEK): surgery in complex cases with severe preoperative visual loss. Cornea 2005;24:587-92. 23. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty (DLEK): Early complications and their management. Cornea 2006;25:37-43. 24. Terry MA, Hoar KL, Wall J, Ousley PJ. The histology of dislocations in endothelial keratoplasty (DSEK and DLEK): Prevention of dislocation with a laboratory-based surgical solution in 100 consecutive DSEK cases. Cornea 2006;25:92632. 25. Terry MA, Wall JM, Hoar KL, Ousley PJ. A prospective study of endothelial cell loss during the 2 years after deep lamellar endothelial keratoplasty. Ophthalmology 2007;114:631-9.

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Introduction Descemet stripping automated endothelial keratoplasty (DSAEK) [synonymous with Descemetorhexis with endokeratoplasty (DXEK)], [See also Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)], has been developed over the past decade and has recently established itself as the most successful alternative to conventional penetrating keratoplasty (PKP) for the surgical treatment of decompensated corneal endothelium. 1-5 DSAEK derives from the continuous improvement of the initial endokeratoplasty procedure, which we introduced in 1996.6 This technique included peeling of the recipient Descemet’s membrane, preparation of a donor lamella of deep stroma, Descemet’s membrane and endothelium, insertion of the graft into the anterior chamber through a clear-cornea tunnel, and fixation of the graft onto the bare posterior corneal surface by means of trans-corneal sutures (Figure 21-1). Results in the rabbit model (Figure 21-1) were not encouraging, as the postoperative course was complicated by frequent misplacement of the donor lenticule and high endothelial cell loss, thus making the use of this technique in humans unreasonable. In 1999 Melles demonstrated that corneal stromal layers can adhere to each other permanently without the need for sutures and contributed substantially to the development of the new posterior lamellar keratoplasty techniques.7 However, Melles and other surgeons used hand dissection for the preparation of both the donor corneal tissue and for the recipient corneal bed, and the corneal surfaces obtained with this type of dissection create an interface with an optical quality that is very rarely compatible with 20/20 vision.

More recently, Price4,5 and others8 have substantially improved the optical quality of the DSAEK-interface by introducing the microkeratome-assisted dissection of the donor corneal tissue (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). Excellent visual results, comparable, if not superior to those of conventional PKP, have been reported by both authors. Nonetheless, the same complications encountered with the original endokeratoplasty procedure (graft displacement and, especially, endothelial cell loss up to 40-50% one year after surgery) still seem to complicate the postoperative course of DSAEK in a relatively high number of patients. The modified surgical technique presented below was developed with the purpose of eliminating the major drawbacks of the present DSAEK technique, while retaining the major advantages of a relatively simple, reproducible and safe surgical procedure [See also Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)].

Surgical Technique A marker, usually 9.0 mm in diameter, is used at the beginning of the procedure to outline the limit of the internal surface, from which the endothelium will be peeled off during this surgery (Figure 21-2). The tip of a 25-gauge needle mounted on a 2.5 cc empty syringe is bent upwards (Figure 21-3) before it is introduced into the anterior chamber (AC) at the 12 o’clock position. About 0.2 to 0.4 cc of aqueous humor is aspirated and air is injected, filling-up the AC. The tip of the needle is then used to cut through endothelium and Descemet’s membrane following the contour of the superficial mark (Figure 21-4). Then the needle is retracted and a 25-gauge cannula is mounted on a syringe and

Figure 21-1: Schematic representation of endokeratoplasty as described by Busin et al. (Ophthalmology, 1996). (1) Peeling of recipient Descemet’s membrane and endothelium; (2) Preparation of a clear-cornea tunnel; (3) Insertion of the donor lamella into the anterior chamber; (4) Fixation of the graft with transcorneal sutures; (a-d) Intraoperative photographs of the rabbit cornea showing the endokeratoplasty procedure; (A,B) Postoperative slit-lamp view of the rabbit cornea following the endokeratoplasty procedure.

Improved DSAEK Surgery for Enhanced Endothelial Survival

Figure 21-2: A circular mark, 9.0 mm in diameter is made on the corneal epithelium outlining the extent of Descemet’s membrane stripping.

Figure 21-3: A 25-gauge needle bent both proximally and at its tip, is used to perform Descemetorhexis.

re-inserted through the same entry site. The cannula is employed to sweep away the Descemet’s membrane and the endothelium, usually in a single piece (Figure 21-5). The blunt cannula avoids mechanical damage to the posterior bare stromal surface of the recipient cornea. Whenever air is lost through the entrance site of the needle/ cannula, the AC is reformed by injecting additional air with the syringe. Performing the whole maneuver under air allows perfect visualization of the Descemet’s membrane and endo-

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Figure 21-4: Descemetorhexis is performed under an air-bubble, using a 25 gauge needle.

Figure 21-5: A blunt 25-gauge cannula is used to remove in a single piece both the Descemet’s membrane and the endothelium under an air-bubble.

thelium without the need for any type of dye or much more importantly, for any viscoelastic substance in the AC (Figure 21-5). In fact, if viscoelastic substance is used in the AC and the viscoelastic substance is removed incompletely before insertion of the graft, it may reach the donor-recipient interface, preventing adhesion of the donor tissue to the stromal surface and causing the procedure to fail. A clearcornea tunnel, 1.0 mm in length and 5.0 mm in width is

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Figure 21-6: Clear-cornea nasal tunnel is prepared with a keratome blade.

prepared nasally (Figure 21-6) [Editorial Note: For alternative techniques and instrumentation, See also Chapter 11, New/Useful Surgical Instruments in DSAEK, Chapter 32, Use of Dyes in DSAEK and DLEK, and Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK)]. The dimensions of the tunnel are critical for the uneventful insertion of the graft into the AC. If the tunnel is too long the donor tissue may remain partly trapped during insertion, while a too narrow tunnel may cause an increase in endothelial cell loss secondary to pronounced deformation of the donor corneal graft. After coating the endothelial side of the donor cornea with viscoelastic substance, it is mounted on an artificial anterior chamber of the automated lamellar therapeutic

A

Figure 21-7: Microkeratome-assisted removal of the anterior stroma from the donor cornea mounted on the artificial anterior chamber (Moria S.A., Antony, France).

keratoplasty (ALTK) system (Moria S.A., Antony, France) (See also Chapter 12, Artificial Anterior Chambers) and most of the anterior stroma is removed by means of the microkeratome with a 300 µm head, which usually cuts lamellae with a thickness of about 350 µm (Figure 21-7). The same marker that was used to mark the corneal surface is employed to mark the stromal side of the remaining tissue (usually with a thickness between 100 and 200 µm) (Figure 21-8A), which is then punched to the desired size (8.0 to 9.0 mm), as shown in Figure 21-8B. As opposed to what is being done by most corneal surgeons, the donor corneal tissue is not folded and inserted into the AC using the socalled “taco” technique. Instead, a specially designed glide is loaded with the donor corneal disk, endothelial side up

B

Figure 21-8: (A) Donor cornea is seen in the artificial anterior chamber after microkeratome-assisted removal of the anterior stroma using the 300 µm microkeratome head. The area to be punched using a trephine is marked within the area of stromal removal and an additional mark is made peripherally to identify the stromal and endothelial sides of the donor cornea. (B) Donor lamella punched to size from the endothelial side. Notice reversal of the identification mark as the cornea lies upside down (endothelium upwards) on the trephination block [ Editorial Note: Stromal marking may be associated with endothelial cell loss (Terry M., personal communication)].

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Figure 21-9: The donor tissue is pulled into the glide opening by means of a crocodile vitreous forceps. A

and crocodile vitreous forceps are used to grasp the donor button and pull it into the opening of the glide (Figure 21-9). A side entry into the AC is created temporally. The glide is then inverted and positioned at the entrance of the nasal clear-cornea tunnel. The crocodile forceps is then inserted through the side entry wound and passed across the AC, exiting through the clear-cornea tunnel, to grab the donor corneal graft and drag it into the AC (Figures 21-10A and B). The donor lamella is allowed to unfold spontaneously under continuous irrigation. Gentle tapping at the inferior limbus may facilitate centring of the donor graft. The whole maneuver is performed with the aid of continuous irrigation with an AC maintainer inserted at the limbus at the 12:00 o’clock position. An iridectomy is performed, if not already present, in order to prevent pupillary block and Urrets-Zavalia syndrome, secondary to filling of the anterior chamber with air at the end of the surgery. Both the clear-cornea tunnel and the side entry are sutured water-tight with interrupted 10-0 nylon stitches (Figure 21-11). The graft is finally attached to the posterior stromal surface by means of air (Figure 21-12). Triamcinolone acetonide and gentamicin are injected subconjunctivally at the end of the procedure. The eye is patched and the patient is required to lie on his back for 68 hours following the surgery. When DSAEK is combined with other procedures (e.g. cataract surgery, secondary IOL implantation or exchange, anterior vitrectomy, etc.), these must be usually performed first, as both the donor endothelium itself and the attachment of the graft to the posterior surface of the recipient cornea may be compromised by any additional surgical maneuver (Editorial Note: In combined procedures, the editor first removes the Descemet’s membrane as a single disk, then

B Figures 21-10A and B: Insertion of the graft into the anterior chamber with the “pull through” technique, using vitreous forceps and tissue glide.

performs the combined procedures such as phacoemulsification with PC IOL, etc. and finally the donor corneal disk is attached to the recipient, bare corneal stroma).

Visual and Refractive Results The visual and refractive results obtained in 20 consecutive cases operated on with the technique described above do not differ substantially from those reported in the past.4,5,8 At a follow-up examination 3 months postoperatively, best spectacle corrected visual acuity (BSCVA) equal to or better than 20/200 was obtained in 19 of 20 cases (95%), while BSCVA better than or equal to 20/40 was recorded

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Figures 21-13A and B: Slit-lamp photographs broad-beam (A) and narrow-beam (B), showing a clear central cornea with a well centered 9.0 mm posterior donor graft, 1 month after combined DSAEK, phacoemulsification and PC IOL implanted in the capsular bag. The uncorrected visual acuity was 20/20. The slit-lamp detail (b) outlines the excellent adherence of the graft to the posterior surface of the recipient cornea as well as the clarity of both recipient and donor corneal tissues. Figure 21-11: The clear-cornea tunnel and the side entry are sutured with interrupted 10-0 nylon stitches, obtaining water-tight closure of both surgical wounds.

Figure 21-12: Graft perfectly centered and adherent to the posterior surface of the recipient cornea. The anterior chamber is left full with air and the patient instructed to lie on his back for 6-8 hours.

in 14 of 20 cases (70%). The outcome was not negatively affected when catarct surgery was combined with DSAEK: BSCVA of at least 20/40 was measured in all 9 patients with advanced Fuchs’ endothelial dystrophy undergoing combined DSAEK and phacoemulsification with implantation of an IOL in the capsular bag. In particular, patient in Figure 21-13 represents the most outstanding result in this series, with an uncorrected visual acuity of 20/20 as early as 1 month postoperatively.

On the other hand, 5 of 6 patients with BSCVA worse than 20/40 had undergone a phacoemulsification procedure complicated by rupture of the posterior capsule and had developed cystoid macular edema. BSCVA of 20/400 in the remaining patient was secondary to advanced age-related macular degeneration. It must be noticed, that in general, younger patients with primary endothelial failure (Fuchs’ corneal endothelial dystrophy), with or without cataract, tend to have a better visual prognosis. On the other hand, patients with longstanding corneal edema require a longer period of time for visual rehabilitation and are prone to have a more limited visual improvement. Decreasing the postoperative astigmatism represents one of the most distinctive advantages of DSAEK over conventional PKP surgery. Also in our patients, final astigmatism is low (within 2 diopters in practically all cases) and of the regular type (Figure 21-14). A limited amount of corneal distortion may be present initially in some cases, but it disappears when all corneal sutures are removed (usually 4 to 6 weeks after surgery), similar to what is seen after cataract surgery. We have observed a slight hyperopic shift (less than 1 diopter) in most of our patients in the early postoperative period. However, this hyperopic shift has regressed completely in the majority of the patients within a few months, corresponding to the reduction of the initial amount of edema at the graft edge. As a consequence of this observation, we believe that transient hyperopic shift may be secondary to an increase in posterior corneal curvature secondary to peripheral edema at the bare edge of the

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Endothelial Cell Density and Corneal Transparency

Figure 21-14: Corneal topographic map obtained from a post-DSAEK patient 1 week following surgery, showing the presence of low-degree regular astigmatism. As opposed to conventional PKP, topographic analysisi is possible as early as few days after DSAEK, confirming the negligible effect of this procedure on the corneal curvature.

grafted donor lamella. Sealing of the wound with time, leads to reduction of the posterior corneal curvature, and normalization of the refraction. In those patients undergoing combined surgery, DSAEK was found to be particularly effective in targetting the optimal postoperative refraction. In fact, as keratometry of the recipient cornea is not affected substantially by DSAEK, the power of the IOL to be eventually implanted secondarily or exchanged may be calculated more precisely.

To date, the relatively high postoperative endothelial cell loss (up to almost twice as much as after a conventional PKP) remains the main drawback of DSAEK surgery. Preparation of the donor lamella by means of microkeratome-assisted dissection has shown a negligible effect on donor corneal endothelial cells. Instead, intraoperative manipulation of the donor graft (i.e. folding, grabbing with forceps, inserting into the AC and finally unfolding) has shown to closely correlate with donor endothelial survival rate. Thin donor grafts (below 100 µm thickness) are especially difficult to manipulate and are therefore exposed to the risk of higher cell loss. For this reason, a too deep dissection of the donor cornea may result in a disappointing final result. Before resorting to the technique described in detail in this chapter, we operated eight patients using the “taco” technique, and we experienced early decompensation of the graft in two cases, while in the remaining six, an endothelial cell loss up to 50% was recorded (Figure 21-15A). Instead, “dragging” the graft into the AC, as illustrated in Figures 21-10A and B, has proven to minimize the loss of endothelial cells to a level comparable to that of conventional PKP. The endothelial cell counts obtained 6 months postoperatively from the first 9 patients operated on with this technique showed an endothelial cell loss of 18% to 26% of the preoperative counts (Figure 21-15B).

Figures 21-15A and B: Confocal microscopic images of endothelial cells after DSAEK: Cell loss is about double with the use of the “taco” technique (A) than it is when the graft is dragged into the anterior chamber (B), employing the technique described in detail in this chapter (the magnification used for both images is the same).

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Postoperative transparency and the optical quality of the cornea is an “unsolved problem” of DSAEK. Although corneal clarity seems to recover to normal levels early after surgery, the clinical assessment does not always correlate with vision. Some patients continue to improve their vision, even if refraction has stabilized long before and no change in corneal transparency is clinically evident. On the other hand, some patients with perfectly transparent corneas at slit-lamp examination, still complain of “hazy” vision, with visual acuity failing to reach the expected 20/20 value. Various parameters have been investigated, albeit unsuccessfully, with the purpose of explaining these findings. Interestingly, it has been proven that visual acuity is not related to post-DSAEK corneal thickness and even an increase of up to 30% is still compatible with 20/20 vision, confirming data recorded after epikeratophakia that was published in the past. Understanding the optical properties of corneas undergoing various types of lamellar keratoplasty probably represents the key issue and the most challenging task that corneal researchers and surgeons are presently facing in an attempt to further improve the results of these lamellar procedures.

References 1. Terry MA, Ousley PJ. Replacing the endothelium without corneal surface incisions or sutures: The first United States clinical series using the deep lamellar endothelial keratoplasty procedure. Ophthalmology 2003;110:755-64. 2. Melles GR, Kamminga N. Techniques for posterior lamellar keratoplasty through a scleral incision. Ophthalmologe 2003; 100:689-95. 3. Terry MA, Ousley PJ. Small-incision deep lamellar endothelial keratoplasty (DLEK): Six-month results in the first prospective clinical study. Cornea 2005;24:59-65. 4. Price FW Jr, Price MO. Descemet’s stripping with endothelial keratoplasty in 200 eyes: Early challenges and techniques to enhance donor adherence. J Cataract Refract Surg 2006;32: 411-8. 5. Price MO, Price FW Jr. Descemet’s stripping with endothelial keratoplasty comparative outcomes with microkeratomedissected and manually dissected donor tissue. Ophthalmology 2006;113:1936-42. 6. Busin M, Monks T, Arffa RC. Endokeratoplasty in the rabbit Model: A new surgical technique for endothelial transplantation. Ophthalmology 1996;103:167. 7. Melles GR, Lander F, Beekhuis WH, Remeijer L, Binder PS. Posterior lamellar keratoplasty for a case of pseudophakic bullous keratopathy. Am J Ophthalmol 1999;127:340-1. 8. Van Rij G, Bartels M. Descemet’s stripping with endothelial keratoplasty in 50 eyes: A refractive neutral corneal transplant (Comment on). J Refract Surg 2006;22:529-30.

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Marianne O Price Francis W Price

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Introduction By selectively replacing dysfunctional endothelium while retaining the patient’s overlying stroma, endothelial keratoplasty (EK) causes little to no change in corneal surface topography or refractive status.1-3 This allows rapid recovery of useful vision and provides significant benefits for patients compared with traditional penetrating keratoplasty (PKP).2,4-6 Although PKP has been performed for over 100 years, penetrating grafts still suffer from serious drawbacks, including the potential induction of large and unpredictable refractive changes.7-9 Furthermore, after PKP, the eye is permanently weakened and remains forever at increased risk of traumatic rupture, because the full-thickness incision never heals back to the strength of the “virgin” cornea.10,11 After PKP, there is also significant risk that ocular surface problems will interfere with recovery, because the fullthickness incision serves all corneal nerves, reducing the tendency to blink and produce tears, and loose or broken sutures can serve as a conduit for infection.12,13 With EK these concerns are minimized, or in many cases eliminated. EK can be performed through a small (3.0 – 5.0 mm) incision so that most of the structural integrity of the eye is maintained. 5,14,15 Corneal innervation is retained,16 and with a properly constructed scleral tunnel incision, EK can be performed as a completely sutureless procedure, so postoperative ocular surface problems are minimal.14 Finally, EK is essentially refractive-neutral.5

Evolution of EK Techniques and Manifest Cylinder Outcomes A series of advances in EK techniques has allowed the use of progressively smaller incision sizes and essentially eliminated any concerns about induced astigmatism. Early methods of endothelial keratoplasty involved creation of an anterior flap and replacement of posterior stroma and endothelium, with use of sutures to secure the donor graft 17-21 (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). However, these early methods suffered from many of the same drawbacks as PKP, including unpredictable corneal topography and induced irregular astigmatism, and hence, they were never widely adopted. The current popularity of EK can be attributed to a series of key breakthroughs pioneered by Melles, along with contributions from many other surgeons (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). In 1997, Melles reported a posterior lamellar keratoplasty (PLK) technique whereby he replaced a 7.5 mm diameter area of

posterior stroma and endothelium through a 9 mm incision and used air rather than sutures to secure the donor graft to the recipient cornea.22,23 In 2000, Terry initiated a prospective clinical study of this fundamental technique, which he called deep lamellar endothelial keratoplasty (DLEK).24 In early PLK and DLEK series utilizing a 9.0 mm incision, mean postoperative manifest cylinder was 1.5 to 1.6 D.2,3 In the DLEK series, this represented a small, but statistically significant, increase of 0.4 D compared with the preoperative mean manifest cylinder (P=0.035, Table 22-1).2 In 2002, Melles demonstrated that a 9.0 mm posterior donor graft could be folded in half and inserted through a 5 mm incision in a completely sutureless PLK procedure.14 Terry showed that with small incision DLEK, postoperative mean manifest cylinder was statistically comparable to the preoperative value (Table 22-1).2,25 The next major EK advance occurred in 2003, when Melles reported that dysfunctional endothelium could be successfully removed from the recipient cornea by descemetorhexus, or stripping Descemet’s membrane, in lieu of the more difficult lamellar dissection and excision of posterior recipient stroma used in his original PLK technique.26 With some procedural refinements, this EK procedure became known as Descemet’s stripping with endothelial keratoplasty (DSEK).5 We found that DSEK produces no change in the mean manifest cylinder (Table 22-1).5 Gorovoy subsequently reported excellent clinical outcomes using a microkeratome to dissect the donor tissue, in an EK iteration known as Descemet’s stripping automated endothelial keratoplasty (DSAEK).27 We have found that DSAEK likewise does not induce any statistically significant change in mean refractive cylinder (Table 22-1).1 In summary, while the initial large-incision PLK/DLEK technique caused a small but statistically significant increase in mean manifest cylinder, subsequent smallincision EK iterations do not cause any significant change (Table 22-1).1,2 However, a transient increase in mean manifest cylinder may be noted during the first 1-3 months after surgery, if sutures are used to close the small incision (Figure 22-1).5 Also, even though the average manifest cylinder for the patient population does not change after small incision EK, individual patients may notice a mild shift in their refraction (Figure 22-2).5 Nevertheless, for most individuals, post-EK cylinder is likely to be within 1 D of the preoperative value, which is a major improvement over the prognosis with a PKP. After PKP, 4.0 – 5.0 D of mean refractive cylinder is common, and 10-15% of PK patients typically require hard contact lenses to help normalize the corneal surface so that they can obtain best visual acuity

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Endothelial Keratoplasty: Visual and Refractive Outcomes TABLE 22-1: Comparison of preoperative and postoperative refractive cylinder for various keratoplasty techniques

Keratoplasty technique Small incision EK DSAEK DSEK DLEK Large incision EK DLEK PLK

Preoperative refractive Postoperative refractive Number of eyes cylinder (mean ± SD) cylinder (mean ± SD)

Reference

1.5 ± 1.2 0.96 ± .79 1.5 ± 1.3 0.97 ± 0.71

1.5 ± 1.2 1.5 ± 1.2 1.5 ± 1.0 1.2 ± 0.74 1.5 ± 1.2

100 16 100 62 15

Price1 Gorovoy 27 Price1 Terry 2 Fogla25

1.2 ± 0.92

1.6 ± 0.97 1.5 ± 0.81

36 7

Terry 2 Melles3

4.2 ± 2.9 3.9 ± 1.9

54 64

Claesson31 Pineros29

PK for Fuchs’

Figure 22-1: Transient increase in mean refractive cylinder that may occur when an EK incision is closed with sutures.

(Table 22-1).28-30 In addition, a small but significant percentage of PKP patients require subsequent refractive procedures, such as limbal relaxing incisions or laser in situ keratomileusis, to help reduce their high astigmatic errors.29,31 In a preliminary report of outcomes from the first prospective, randomized study directly comparing EK with PK, the mean postoperative refractive cylinder was 5.0 ± 3.1 D in 6 PK eyes, compared with 1.0 ± 0.9 D in 4 large incision DLEK eyes.32 These results certainly confirm the findings from the larger, non-randomized series cited in Table 22-1.

Spherical Equivalent Outcomes EK also causes minimal change in mean spherical equivalent refraction, with some surgeons reporting no change and others reporting only a mild hyperopic shift.1,5,27 Spherical equivalent outcomes are probably

Figure 22-2: Scatterplot comparing preoperative and postoperative refractive cylinder in 50 consecutive DSEK patients with 6-month followup.

influenced by donor dissection technique. For example, during manual dissection of a donor corneal/scleral rim mounted on an artificial anterior chamber (See also Chapter 12, Artificial Anterior Chambers) there is a tendency for the dissection plane to be shallower in the periphery, resulting in a meniscus-shaped donor lenticule, which can cause a mild hyperopic shift. Many microkeratomes, including the Moria CB microkeratome (Moria S.A., Antony, France) used for DSAEK, tend to cut deeper in the periphery, which helps to compensate for the increased thickness in the peripheral cornea compared with the central cornea, and therefore a microkeratome can produce a posterior donor button that has a relatively planar contour across the visual axis (Figure 22-3). However, using a slower translational speed with the microkeratome tends to produce deeper cuts, and if the translational speed varies during the microkeratome pass, the posterior donor button may have a tapered contour. Extensive experience with cutting LASIK flaps

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Figure 22-3: Visante optical coherence tomography cross sectional image of a DSAEK eye. The 9 mm diameter donor graft has a uniform thickness across the visual axis and becomes slightly thicker at each edge.

shows that microkeratome dissection depths can vary significantly from the nominal depth plate dimension and have a large standard deviation.33,34 Despite these variables, in our experience, microkeratome-dissection of the donor cornea results in no change in the mean spherical equivalent refraction after DSAEK.1 Some other physicians report a mild hyperopic shift with DSAEK.27 The relatively predictable refractive outcomes achieved with EK allow surgeons to perform cataract extraction and intraocular lens (IOL) implantation prior to corneal transplantation, and thus avoid endothelial cell loss from subsequent surgical trauma. By tracking their post-EK refractive outcomes, individual surgeons can develop a nomogram to use in their IOL calculations, to compensate for any shift in spherical equivalent refraction they are likely to get after subsequent EK surgery. It should be noted that performing cataract extraction prior to corneal transplantation represents a real paradigm shift. With PKP, it was usually preferable to postpone cataract extraction until after transplantation so that the selection of IOL power could be used to help address some of the unpredictable post-PKP refractive outcomes. With EK the cataract surgery can be performed first to help protect the donor endothelium.

Visual Outcomes Current EK techniques provide rapid visual recovery and an easy postoperative course compared with PKP. Full recovery of best vision could be a year with the original PLK/DLEK technique. 35 The apposition of 2 handdissected surfaces in PLK/DLEK probably resulted in interface irregularities that may have caused some visual degradation in the early postoperative period. Over a period of time, stromal remodeling by the keratocytes probably helped normalize the interface and improve best-corrected vision.

Figure 22-4: Visual acuity vs. time for consecutive series of patients with endothelial dysfunction treated with DSEK and DSAEK. All eyes are included regardless of whether pre-existing retinal problems limit visual potential.

Visual recovery seems to be faster with DSEK and particularly with DSAEK compared with the earlier EK techniques ((Figure 22-4). Removal of Descemet’s membrane leaves an extremely smooth recipient interface, and microkeratome dissection of the donor tissue typically produces a smoother donor interface compared with hand dissection. This combination can produce faster visual recovery. However, an important caveat when comparing the outcomes with different EK techniques is that the patients in the various reported series were not randomized, so there may have been demographic differences or differences in mean visual potential in different treatment groups. Fuchs’ corneal dystrophy is the most prevalent indication and bullous keratopathy the second most common indication for EK. We have also found that excellent visual outcomes can be obtained when EK is used to treat iridocorneal endothelial (ICE) syndrome. So far we have treated 3 ICE syndrome patients and all have achieved visual acuities of 20/20 to 20/30 after DSAEK.36 Achieving 20/40 vision is a key goal for many patients because that is often the benchmark required to obtain a driver’s license.5 The percentage of patients who achieve 20/40 visual acuity after EK compares quite favorably with the percentage after PKP.5 Since PK substantially alters corneal topography, it is not uncommon for 10-15% of the treated eyes may require a hard contact lens to normalize the anterior surface of the cornea in order to achieve best visual acuity.29,30 In contrast, EK does not significantly alter corneal topography, so hard lenses are not required for best vision. This is an important advantage with EK because some patients cannot tolerate hard lenses and older patients in particular may find them difficult to manage.

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Factors Affecting Visual Outcomes What preoperative or surgical factors correlate with visual acuity after EK? Multivariate analysis indicates that preexisting retinal disease or amblyopia in the treated eye is the most statistically significant factor (P<0.0001) limiting visual acuity after EK.1 Interestingly, post-EK visual outcomes are also significantly correlated with recipient age even after eyes with known pre-existing retinal disease are excluded (Figure 22-5),1 indicating that younger patients have a better visual prognosis. Figure 22-6: Scatterplot showing no significant correlation between central corneal thickness measured by ultrasonic pachymetry and best spectacle-corrected visual acuity after DSEK/DSAEK.

Figure 22-5: Scatterplot showing correlation between patient age and best spectacle-corrected visual acuity (BSCVA) after DSEK or DSAEK. Eyes with pre-existing retinal problems or amblyopia were excluded.

Whereas DLEK and flap-associated DLEK (FDLEK) do not significantly increase total corneal thickness, DSEK and DSAEK do, because they add donor stroma without removing any recipient stroma (See also Chapter 13, Definition, Terminology and Classification of Lamellar Corneal Surgery). Initially there was concern that the increased corneal thickness might degrade visual acuity. However, our analysis of several hundred eyes has shown that postEK central corneal thickness is not significantly correlated with Snellen acuity (Figure 22-6, P= 0.25). Retinal problems could be a confounding variable in this type of analysis, so the analysis was restricted to those eyes with no known retinal problems.1 Anterior stromal scarring also can cause significant light scattering and degrade visual acuity after EK (Figure 22-7). Therefore, patients are likely to recover better vision if the EK is performed before long-standing edema causes anterior stromal scarring, because EK does not replace anterior stromal tissue, and it is not yet known, whether stromal remodeling can ultimately reverse long-standing changes. The most frequent post-EK complication is detachment of the donor tissue in the first week after surgery.16,37 The donor tissue can usually be re-attached by simply reinjecting an air bubble to again press the donor graft up

A

B Figure 22-7: Confocal microscopy images of the anterior stroma in 2 DSAEK patients. The eye shown in (A) has 20/25 visual acuity and normal stromal appearance. This image was taken just beneath the epithelium as evidenced by the columnar basal epithelial cells near the bottom of the image and the sub-epithelial nerve crossing the center of the image. The stroma appears dark except for bright bean-shaped keratocyte nuclei. This indicates that most of the incident light is passing through to the retina and not being reflected back into the microscope. The eye shown in (B) has 20/60 visual acuity; the scarring in the anterior stroma is causing significant light scattering and is probably responsible for the sub-optimal best corrected vision.

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against the recipient cornea. We have not detected a statistically significant difference in visual acuity between eyes that had a second air injection and those that did not.1 Sometimes full-thickness folds can occur in an EK donor graft and degrade visual acuity.38 In some cases, folds may occur if an oversized PLK/DLEK donor button is squeezed into an undersized excised area of the recipient cornea. With DSEK or DSAEK, the donor is simply laid on the flat recipient stromal surface, but folds can still develop and be difficult to remove especially with very thin donor grafts.

Implications The excellent visual and refractive outcomes achieved with EK are producing a paradigm shift in determining the best time for a patient to have a corneal transplant. The risks and long recovery period after a traditional PKP caused many patients to postpone transplantation for as long as possible. In fact, patients often tried to postpone having a PKP until after retirement so that the prolonged recovery period would not jeopardize their job. In contrast, postoperative recovery after EK is quite rapid. In fact, some patients recover 20/25 vision within a week of their EK procedure. Thus, EK significantly improves the risk/benefit ratio of having a corneal transplant. Often patients with Fuchs’ corneal dystrophy have already begun to experience significant impairment of their daily activities, like driving on sunny days or reading in an office with overhead fluorescent lighting, although their Snellen acuity measured in a darkened room still measures 20/30 or 20/40. It has been our experience that when patients with Fuchs’ corneal dystrophy with Snellen acuity of 20/30 in both eyes has an EK performed in one eye, they usually report at their one-month exam that the treated eye has much better quality of vision, even if the Snellen acuity is 20/30 in both the treated and untreated eyes. They frequently mention that colors appear more brilliant with the treated eye. This suggests that Fuchs’ guttata cause significant light scattering and visual disturbances that are not detected with Snellen acuity testing in a darkened room. By removing the guttata, EK can significantly reduce visual disturbances and improve the quality of life for many patients with endothelial dysfunction.5 Another nice feature of the EK technique is that visual outcomes are relatively predictable. Fewer eyes may achieve 20/20 visual acuity after EK compared with PKP. On the other hand, anisometropia after PKP can limit some eyes to a final visual acuity no better than 20/200, whereas after EK visual acuity of 20/70 or worse is extremely rare in eyes that do not have pre-existing retinal problems.1

What’s Next? Melles and Tappin are currently perfecting methods of reliably implanting only Descemet’s membrane and healthy endothelium from donor corneas, without including any donor stromal tissue39-41 [See also Chapter 36, True Endothelial Cell (Tencell) Transplantation, and Chapter 37, Descemet Membrane Endothelial Keratoplasty (DMEK)]. Their techniques, respectively called Descemet’s membrane endothelial keratoplasty (DMEK) and true endothelial cell transplantation (Tencell), do not increase the total corneal thickness nor introduce dissected surfaces. Early results suggest these techniques may yield more 20/20 outcomes. A current challenge is that without attached stroma for support, bare Descemet’s membrane is quite fragile, so a significant percentage of donor corneas are rendered unusable during the harvesting and implantation procedures.39-42 The excellent outcomes achieved with EK are increasing the demand for cornea transplants, and the demand is outstripping the worldwide supply of donor corneas. A number of groups are working to culture endothelial cell sheets for use with EK (See also Chapter 38, Corneal Endothelial Reconstruction with a Bioengineered Cell Sheet, and Chapter 39, Future of Posterior Lamellar Keratoplasty). Success in such an endeavor may ultimately help alleviate the current worldwide shortage of suitable donor corneas.43-46

References 1. Price MO, Price FW, Jr. Descemet’s stripping with endothelial keratoplasty comparative outcomes with microkeratomedissected and manually dissected donor tissue. Ophthalmology 2006;113:1936-42. 2. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty visual acuity, astigmatism, and endothelial survival in a large prospective series. Ophthalmology 2005;112:1541-8. 3. Melles GR, Lander F, van Dooren BT, Pels E, et al. Preliminary clinical results of posterior lamellar keratoplasty through a sclerocorneal pocket incision. Ophthalmology 2000;107:18506; discussion 1857. 4. Price FW, Jr. Corneal transplantation as a refractive surgical procedure. Journal of Refractive Surgery 2005;21:216-7. 5. Price FW, Jr., Price MO. Descemet’s stripping with endothelial keratoplasty in 50 eyes: A refractive neutral corneal transplant. J Refract Surg 2005; 21:339-45. 6. Melles GR. Posterior lamellar keratoplasty: DLEK to DSEK to DMEK. Cornea 2006;25:879-81. 7. Riddle HK, Jr., Parker DA, Price FW, Jr. Management of postkeratoplasty astigmatism. Curr Opin Ophthalmol 1998; 9:15-28. 8. Binder PS, Waring GO. Refractive Keratotomy for Myopia and Astigmatism (Ed. Waring GO) 1157-1186 (Mosby Year Book, St. Louis, MO), 1992. 9. Touzeau O, Borderie VM, Allouch C, Scheer S, et al. Effects of penetrating keratoplasty suture removal on corneal topography and refraction. Cornea 1999;18:638-44.

Endothelial Keratoplasty: Visual and Refractive Outcomes 10. Elder MJ, Stack RR. Globe rupture following penetrating keratoplasty: how often, why, and what can we do to prevent it? Cornea 2004;23:776-80. 11. Renucci AM, Marangon FB, Culbertson WW. Wound dehiscence after penetrating keratoplasty: Clinical characteristics of 51 cases treated at Bascom Palmer Eye Institute. Cornea 2006;25:524-9. 12. Thompson RW, Jr., Price MO, Bowers PJ, Price FW, Jr. Longterm graft survival after penetrating keratoplasty. Ophthalmology 2003;110:1396-402. 13. Price FW, Jr., Whitson WE, Johns S, Gonzales JS. Risk factors for corneal graft failure. J Refract Surg 1996;12:134-43; discussion 143-7. 14. Melles GR, Lander F, Nieuwendaal C. Sutureless, posterior lamellar keratoplasty: A case report of a modified technique. Cornea 2002;21:325-7. 15. Terry MA, Ousley PJ. Small-incision deep lamellar endothelial keratoplasty (DLEK): Six-month results in the first prospective clinical study. Cornea 2005;24:59-65. 16. Price FW, Jr., Price MO. Descemet’s stripping with endothelial keratoplasty in 200 eyes: Early challenges and techniques to enhance donor adherence. J Cataract Refract Surg 2006;32:4118. 17. Barraquer J. In The Comea World Congress (Eds. King H Jr MJ) 586-604 (Rutterworths, Washington, DC), 1965. 18. Busin M, Arffa RC, Sebastiani A. Endokeratoplasty as an alternative to penetrating keratoplasty for the surgical treatment of diseased endothelium: Initial results. Ophthalmology 2000;107:2077-82. 19. Ehlers N, Ehlers H, Hjortdal J, Moller-Pedersen T. Grafting of the posterior cornea. Description of a new technique with 12month clinical results. Acta Ophthalmol Scand 2000;78:543-6. 20. Guell JL, Velasco F, Guerrero E, Gris O, et al. Preliminary results with posterior lamellar keratoplasty for endothelial failure. Br J Ophthalmol 2003;87:241-3. 21. Azar DT, Jain S. Microkeratome-assisted posterior keratoplasty. J Cataract Refract Surg 2002;28:732-3. 22. Melles GR, Eggink FA, Lander F, Pels E, et al. A surgical technique for posterior lamellar keratoplasty. Cornea 1998;17:618-26. 23. Melles GR, Lander F, Beekhuis WH, Remeijer L, et al. Posterior lamellar keratoplasty for a case of pseudophakic bullous keratopathy. Am J Ophthalmol. 1999;127:340-1. 24. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty in the first United States patients: Early clinical results. Cornea 2001;20:239-43. 25. Fogla R, Padmanabhan P. Initial results of small incision deep lamellar endothelial keratoplasty (DLEK). Am J Ophthalmol 2006;141:346-51. 26. Melles GR, Wijdh RH, Nieuwendaal CP. A technique to excise the descemet membrane from a recipient cornea (descemetorhexis). Cornea 2004;23:286-8. 27. Gorovoy MS. Descemet-stripping automated endothelial keratoplasty. Cornea 2006;25:886-9. 28. Davis EA, Azar DT, Jakobs FM, Stark WJ. Refractive and keratometric results after the triple procedure: Experience with early and late suture removal. Ophthalmology 1998;105:62430.

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29. Pineros O, Cohen EJ, Rapuano CJ, Laibson PR. Long-term results after penetrating keratoplasty for Fuchs’ endothelial dystrophy. Arch Ophthalmol 1996;114:15-8. 30. Price FW, Jr., Whitson WE, Marks RG. Progression of visual acuity after penetrating keratoplasty. Ophthalmology 1991;98:1177-85. 31. Claesson M, Armitage WJ, Fagerholm P, Stenevi U. Visual outcome in corneal grafts: A preliminary analysis of the Swedish Corneal Transplant Register. Br J Ophthalmol 2002;86:174-80. 32. Baratz KH NC, Hodge DO, Bourne WM. A prospective, randomized study of deep lamellar endothelial keratoplasty versus penetrating keratoplasty: Early results. Invest Ophthalmol Vis Sci 2005;46:E-Abstract 2703. 33. Thompson RW, Jr., Choi DM, Price MO, Potrezbowski L, et al. Noncontact optical coherence tomography for measurement of corneal flap and residual stromal bed thickness after laser in situ keratomileusis. J Refract Surg 2003;19:507-15. 34. Reinstein DZ, Srivannaboon S, Archer TJ, Silverman RH, et al. Probability model of the inaccuracy of residual stromal thickness prediction to reduce the risk of ectasia after LASIK part II: quantifying population risk. J Refract Surg 2006;22:861-70. 35. Terry MA, Ousley PJ. Rapid visual rehabilitation after endothelial transplants with deep lamellar endothelial keratoplasty (DLEK). Cornea 2004;23:143-53. 36. Price MO, Price FW, Jr. Descemet’s stripping endothelial keratoplasty for treatment of iridocorneal endothelial syndrome. Cornea 2006;in press. 37. Terry MA, Hoar KL, Wall J, Ousley P. Histology of Dislocations in Endothelial Keratoplasty (DSEK and DLEK): A LaboratoryBased, Surgical Solution to Dislocation in 100 Consecutive DSEK Cases. Cornea 2006;25:926-32. 38. Faia LJ, Baratz KH, Bourne WM. Corneal graft folds: A complication of deep lamellar endothelial keratoplasty. Arch Ophthalmol 2006;124:593-5. 39. Melles GR, Ong TS, Ververs B, van der Wees J. Descemet membrane endothelial keratoplasty (DMEK). Cornea 2006;25:987-90. 40. Melles GR, Lander F, Rietveld FJ. Transplantation of Descemet’s membrane carrying viable endothelium through a small scleral incision. Cornea 2002;21:415-8. 41. Tappin M. A method for true endothelial cell (Tencell) transplantation using a custom made cannula for the treatment of endothelial cell failure. Eye 2007;21:775-9. 42. Zhu Z, Rife L, Yiu S, Trousdale MD, et al. Technique for preparation of the corneal endothelium-Descemet membrane complex for transplantation. Cornea 2006;25:705-8. 43. Sumide T, Nishida K, Yamato M, Ide T, et al. Functional human corneal endothelial cell sheets harvested from temperatureresponsive culture surfaces. FASEB J 2006;20:392-4. 44. Shimmura S, Miyashita H, Konomi K, Shinozaki N, et al. Transplantation of corneal endothelium with Descemet’s membrane using a hyroxyethyl methacrylate polymer as a carrier. Br J Ophthalmol 2005;89:134-7. 45. Mimura T, Yamagami S, Yokoo S, Usui T, et al. Cultured human corneal endothelial cell transplantation with a collagen sheet in a rabbit model. Invest Ophthalmol Vis Sci 2004;45:2992-7. 46. Chen KH, Azar D, Joyce NC. Transplantation of adult human corneal endothelium ex vivo: A morphologic study. Cornea 2001;20:731-7.

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Introduction Descemet membrane stripping automated endothelial keratoplasty (DSAEK) is currently the most popular surgical technique among corneal surgeons all over the world for selective tissue corneal transplantation (STCT),1 namely, where there is patient corneal endothelial decompensation and it needs to be replaced with healthy donor corneal endothelium in order to clear the corneal edema and visually rehabilitate the patient and return him back to his normal activities, in a relatively short time as compared to full-thickness penetrating keratoplasty (PKP). In this chapter, every effort is taken to introduce the reader to a step-wise fashion of DSAEK procedure and thus increase the comfort level of the surgeons in doing this procedure2,3 (See also Chapter 20, Endothelial Keratoplasty: A Step by Step Guide to DSEK and DSAEK Surgery).

Surgical Technique DSAEK may be considered as a 3-step procedure (Figure 23-1): 1. Choosing the trephine diameter 2. Donor disk 3. Host cornea.

the most suitable eye in which to use a 9.0 mm diameter donor corneal disk. In this eye a smaller diameter disk, e.g. 7.5 mm or occasionally even a 7.0 mm diameter donor disk may be the most desirable diameter. On the other hand, in a large diameter cornea, with a deep anterior chamber, a 9.0 mm diameter donor disk may be the best choice. The basic premise is to introduce as much healthy donor endothelium as possible, without subjecting the patient to any additional postoperative complications. A large diameter donor corneal disk in a small diameter, crowded anterior segment, will be prone to potential peripheral anterior synechiae (PAS), angle closure glaucoma, peripheral donor disk deformation due to progressive PAS, chronic anterior uveitis due to peripheral iris rubbing on the donor disk, inflammation-related macular edema, and even graft failure. A simple approach to choosing the donor disk diameter is to open a surgical caliper that is set at 9.0 mm and if there is at least 1.0 mm clearance, 360 degrees, then the donor disk diameter of choice is 9.0 mm. If this caliper (9.0 mm setting) reaches the limbus or beyond the limbus, then decrease the setting to 8.0 mm, or 7.5 mm, or occasionally 7.0 mm, until there is a 1.0 mm or more peripheral room (Figure 23-2) for introduction of various surgical instruments as needed during the DSAEK surgery and postoperatively to prevent PAS.

Step 2: Donor Disk

Figure 23-1: DSAEK may be considered as a 3-step procedure.

Step 1: Choosing the Trephine Diameter This is one of the most important steps in doing DSAEK surgery. Not all eyes are “made” the same. Hence, in my opinion it may not be a good idea to go with a fixed-diameter donor corneal disk, e.g. 9.0 mm diameter for all eyes undergoing DSAEK surgery. A small diameter cornea in a hyperopic eye with a crowded anterior segment may not be

The second step is the preparation of the donor corneal disk. I prefer to cut my own tissue in the operating room rather than to use pre-cut tissue that is prepared in an eye bank by an eye bank technician (See also Chapter 19, Eye Banking and Donor Corneal Tissue Preparation in DSAEK, and Chapter 30, Use of Eye Bank Pre-cut Donor Tissue in DSAEK). This entails the use of a Moria automated lamellar therapeutic keratectomy (ALTK) system and a Moria CB microkeratome to perform DSAEK procedure (Moria, Antony, France). There are many ways to set-up the Moria ALTK system, and Figure 23-3 shows a relatively simple way to set-up the artificial anterior chamber (AAC) for DSAEK procedure. Make sure all tubing connections are tight to prevent leakage of fluid. The donor cornea is mounted on to the central post within the AAC that is primed with the Optisol GS fluid such that during the mounting process there is no contact between the healthy donor corneal endothelium and the metal surface. While mounting the donor cornea if the surgeon simultaneously injects Optisol GS fluid, it will prevent capture of any air bubble within the AAC. The mounting rings are tightened to encase the donor cornea

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Figure 23-2: Upper Row—Intraoperative photograph of an eye with pseudophakic bullous keratopathy to undergo DSAEK procedure. A Castroviejo surgical caliper is set at 9.0 mm (Bottom figure), and there is at least 1.0 mm clearance in the periphery. Hence, the trephine diameter of choice in this case is 9.0 mm.

Figure 23-3: Moria automated lamellar therapeutic keratoplasty unit (Moria, Antony, France) with a donor cornea that is mounted within the artificial anterior chamber (AAC). The AAC is filled with Optisol GS, and a CB microkeratome is mounted on the post and ready for use. The ALTK unit is attached to a 10.0 ml sterile syringe that is partially filled with the Optisol GS solution and it is connected to the ALTK unit by a short sterile tubing via a valve unit.

(Figure 23-3). The syringe plunger is depressed to increase the intrachamber pressure that is confirmed by finger palpation and the valve is turned to the locked position. I routinely use finger palpation at this stage, however, if one prefers a Barraquer tonometer it may be used. Remove the epithelium with a Meracel sponge (Figure 23-4) and mark the center of the cornea with a dot and a radial mark at the corneal periphery with a sterile surgical marker for reference points. Removal of the corneal epithelium (Figure 23-4) gives an additional 50 micron deeper cut with the microkeratome. Moria makes different microkeratome heads. I prefer to use a 300-micron head on all my donor corneas for DSAEK procedures. When using a 300-micron head (Figure 23-5), I am of the opinion there is no need to take pachymetry readings on any of the donor corneas (personal experience). However, if a surgeon uses 350 micron head then pachymetry is usually a must to prevent inadvertent corneal perforation in some cases. Check the microkeratome after inserting a new blade into the blade slot, and also make sure there are no blade edge defects. Use sterile balanced salt solution to moisten both

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Figure 23-4: Donor corneal epithelium is removed after the corneal button is encased within the Moria ALTK system.

the donor corneal button and prevent any tissue slippage during the cutting process. The Moria CB microkeratome is then mounted on the post on the ALTK system and it is moved in a curvilinear fashion to create a free cap (Figure 23-6). Following the removal of the free cap, the anterior cut surface of the donor corneal stroma is exposed wthin the circular opening of the ALTK system. Vision Blue (Trypan blue 0.06%, Dutch

Figure 23-5: Photograph of a 300 micron Moria microkeratome head for the DSAEK procedure.

the microkeratome head in the region of the blade and on the donor corneal surface prior to cutting the cornea. Surgical Pearl: Just before cutting the donor cornea re-tighten the ring in the Moria ALTK unit, as this will ensure good holding of

Figure 23-6: Upper left – Donor cornea being cut using a Moria ALTK system; Main Figure – A free-cap of the donor cornea is seen within a Moria 300 micron head.

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Figure 23-7: Following the removal of the free cap, the exposed anterior corneal stroma is bathed, and stained with trypan blue 0.06% (Vision Blue), and the excess dye is removed.

Figure 23-8: Following trypan blue staining of the donor corneal stroma, the free cap is replaced to completely cover the cut corneal stroma.

Ophthalmics, USA) is then placed on the cut corneal surface to bathe the surface, thus staining only the cut stromal surface (Figure 23-7) (See also Chapter 32, Use of Dyes in DSAEK and DLEK). The adjacent uncut cornea with its intact epithelium does not stain. The excess Vision Blue is removed using a sterile Merocel® eye spears (Figure 23-7) (Medtronic Ophthalmics, Jacksonville, FL). It is essential not to introduce any debris on the corneal surface at this stage, since it can subsequently get trapped within the donor-host interface and be present postoperatively. The free cap is replaced (Figure 23-8) and aligned using the

pre-placed markings (Figure 23-9). The outer ring in the Moria ALTK unit is rotated to the unlocked position. Surgical Pearl: Do not pull the outer sleeve immediately from the unlocked position, since the cornea can collapse inwards and the endothelium can contact the metal surface of the central post and result in endothelial cell loss. I recommend that the outer sleeve and cornea be raised hydraulically by pressing on the plunger after almost completely filling the syringe with Optisol GS solution. If the cornea collapses inwards during his step it will not hit the metal surface. Once the outer sleeve is raised it can be removed from the ALTK system and the cornea is

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Figure 23-9: The free cap is replaced using the pre-placed markings for proper alignment. Trypan blue stained (top images) and unstained (bottom images) corneas.

removed using a 0.12 forceps and placed on a corneal punch with the epithelial side down (Figure 23-10). Using a Moria Hanna Punch trephination is carried out from the endothelial side after suction keeps the cornea in place (Figure 23-10). This is a “2 unit” cornea with a free anterior cap and the posterior stroma and endothelium. Avoid lateral displacement of the “2 units” of the cornea. Use the area of trypan blue staining as a guide to trephination such that the circular blade cuts within the blue stained region to prevent eccentric trephination (Figure 23-10). Figure 23-11 shows an example of trypan blue stained and unstained corneas. The blue staining helps in DSAEK procedure both in the donor corneal preparation and within the anterior chamber of the recipient.

Step 3: Host Cornea The preferred anesthesia is topical anesthesia using 2% lidocaine jelly with monitored anesthesia care (MAC), whether it is a primary procedure, or a secondary disk exchange procedure (Figure 23-12). Complete hemostasis is essential before entering the anterior chamber via the temporal wound (Figure 23-13). Surgical Pearl: Take the

additional time for complete hemostasis to prevent donorrecipient interface entrapment of blood. Any blood in the interface will decrease vision postoperatively and result in potential interface inflammation, depending on the location and amount of the entrapped blood. The preferred wound size is 5.0 mm (Figure 23-14) to minimize endothelial cell damage. The limbal wound depth is 350 microns (Figure 23-15). An intra corneal pocket is constructed via the temporal wound (Figure 23-16). The anterior chamber is not entered at this time. The anterior chamber is entered both to the right and left of the temporal wound using a 15-degree super blade (Alcon Inc., Fort Worth, TX). Surgical Pearl: Removal of the Descemet’s membrane under fluid (Figures 23-17 and 23-18) is much more difficult than under viscoelastic such as Healon. Always use a cohesive viscoelastic such as Healon (Figure 2319) at this stage of the surgery and it will simplify DSAEK surgery significantly as compared to working under fluid. The use of Healon does not jeopardize the donor disk adherence to the recipient cornea since this Healon will be removed (see below). With Healon filling the anterior chamber, Descemetorhexis and removal of the Desecemet’s membrane as a single disk is carried out using the John Dexatome (ASICO Inc.,

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Figure 23-10: The donor cornea is removed from the Moria ALTK system and placed on a Moria Donor Punch with the epithelial side down. Vacuum holds the cornea in place while trephination is carried out using a Moria Hanna Punch. The circular trephine blade lands within the blue stained area to prevent eccentric trephination of the donor cornea.

Figure 23-11: Donor cornea following trephination. Trypan blue stained (bottom) and unstained (top) donor cornea.

Westmont, IL) (Figures 23-20 and 23-21). Surgical Pearl: Always touch the folded Descemet’s membrane and not the stromal surface. This will result in the best possible recipient side of the interface and thus may contribute to improved vision postoperatively. John Dexatome (ASICO Inc., Westmont, IL) will permit removal of the Descemet’s membrane as a single disk every time without the use of a second instrument. Remain in the same plane when removing the Descemet’s membrane without entering into the stroma. The unique curvature design of the

John Dexatome permits easy access to any part of the inner dome of the recipient cornea. Removing the Descemet’s membrane as a single disk (Figure 23-22) is essential so that re-entry into the anterior chamber is not necessary to remove Descemet membrane tags and remnants. Descemetorhexis is begun in the distal point from the anterior chamber entry site and it is continued first in a clockwise direction (Figure 23-23) and then starting again from the distal starting point it is continued in a counter-

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Figure 23-12: Topical anesthesia with monitored anesthesia care (MAC) is used for DSAEK procedure. This is an example of a failed penetrating keratoplasty with subsequent DSAEK procedure that later resulted in endothelial graft failure. A disc exchange is planned in this case.

Figure 23-13: Complete hemostasis is essential before entering the anterior chamber via the temporal wound.

Figure 23-14: Preparation of the temporal region of the patient’s eye. A circular mark is placed on the corneal epithelium of the chosen diameter. A Castroviejo caliper is set at 5.0 mm to mark the limit of the limbal wound.

DSAEK Simplified Surgical Technique

Figure 23-15: The limbal wound depth is 350 microns.

clockwise direction to complete the 360-degrees (Figure 23-24). While detaching the Descemet’s membrane both the leading and the trailing edges need to be watched so that the Descemet’s membrane is removed as a single disk every time (Figures 23-25 to 23-28). The Descemetorhexis is performed about 0.5 mm within the epithelial mark. Since the donor disk has the same diameter as the epithelial mark, the disk overlaps the peripheral 0.5 mm area and prevents any break through corneal edema. When performing this

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step in a failed PKP cornea, the PKP wound is used as the circular guide and no epithelial mark is made in these cases. The fully detached Descemet’s membrane is removed from the anterior chamber after entering the anterior chamber through the previously made limbal wound in the temporal region (Figures 23-14 to 23-16) using a 3.2 mm keratome blade. The peripheral stroma within the epithelial circular mark is made rough by using the John DSAEK scrubber (ASICO Inc., Westmont, IL) to enhance donor disk attachment to the recipient cornea (First described by Terry, M). This is completed 360-degrees, first in a clockwise direction, followed by a counter-clockwise direction (Figure 23-29). Complete removal of Healon from the anterior chamber using an irrigation/aspiration unit is essential for donor corneal disk adherence to the recipient corneal stroma (Figure 23-30). Peripheral iridectomy is not performed during DSAEK surgery since this can result in intraoperative bleeding. The donor corneal disk is folded into a “taco-fold” after placing a small amount of Healon on the endothelial surface (Figure 23-31). Donor corneal disk is placed in the recipient anterior chamber after enlarging the entry wound to 5.0 mm. The wound is then closed with three interrupted

Figure 23-16: Temporal wound construction using a crescent blade.

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Figure 23-17: Descemet membrane stripping under fluid. Do not use fluid, instead use Healon.

Figure 23-18: Descemet membrane stripping under fluid. Do not use fluid, instead use Healon.

Figure 23-19: Anterior chamber is filled with Healon before performing Descemetorhexis.

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Figure 23-20: Schematic representation of Descemetorhexis and removal of Descemet’s membrane.

10-0 nylon sutures (Figure 23-32). The donor disk is unfolded using filtered-air (Figure 23-33). The air is injected in a steady, controlled fashion. Donor disk is uniformly adherent to the patient’s cornea and it is well centered. Also seen is the double-ring sign (Figure 23-34). “Fork-lift” taco helps in initial stroma to stroma adherence and facilitates unfolding of the taco. The cannula is placed between the folded taco and the anterior iris surface and sterile balanced salt solution is injected to gently lift the taco (Figure 23-35). The taco fold is usually a 60/40 fold. This may be done as an over-fold or more recently an under fold in which case the hand is rotated for proper orientation while inserting the taco into the anterior chamber. In some cases the taco fold may be 70/30 or occasionally an 80/20 fold. However, as one moves away from the 60/40 towards 80/20 there is more endothelial cell exposure and hence potentially increased cell damage. A large air bubble facilitates adherence. Wait 8 to 10 minutes with an air-filled anterior chamber to facilitate initial disk adherence. The air bubble size is then decreased by airfluid exchange.

In some patients with a deep anterior chamber, fluid unfolding of the donor disk may be possible (Figure 23-36). If the donor disk begins to unfold in the wrong direction then the John Fixation Hook (ASICO Inc., Westmont, IL) may be used to pin the donor disk against the recipient cornea, followed by air-unfolding of the donor disk (Figures 23-37 to 23-39). Following air attachment of the donor corneal disk, if there are macrofolds in the donor disk, then the Lindstrom roller or the John DSAEK Glider (ASICO Inc., Westmont, IL) may be used to decrease or eliminate the folds (Figure 23-40). Figure 23-41 shows the presence and absence of macrofolds in the donor corneal disk. There are different insertion techniques to place the donor corneal disk within the recipient anterior chamber (Figures 23-42 to 23-46). These are either “push” or “pull” –through techniques (Figure 23-42). Residual air bubble is usually seen 1 day following DSAEK procedure (Figure 23-47). Howeevr, the air bubble partially or fully clears the pupillary diameter and hence there usually is no risk of acute pupillary block glaucoma attack on or after day 1 following DSAEK surgery. There often is relatively rapid clearing of the corneal edema

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Figure 23-21: The corneal surface is marked with the John DSAEK marker of the chosen diameter (ASICO Inc., Westmont, IL). Descemetorhexis is carried out within the circular mark using the John Dexatome spatula and the same instrument is used to detach the Descemet’s membrane as a single disk and it is suspended in the Healon and then removed through the temporal wound.

Figure 23-22: Removal of the patient’s Descemet’s membrane as a single disk.

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Figure 23-23: John Dexatome spatula (ASICO Inc., Westmont, IL) is introduced into the anterior chamber from the right temporal wound and Descemetorhexis is performed 180-degrees in a clockwise direction with Healon filling the anterior chamber.

Figure 23-24: John Dexatome spatula (ASICO Inc., Westmont, IL) is then taken back to the distal staring point and Descemetorhexis is continued in a counter-clockwise direction to complete the 360-degrees of Descemetorhexis from a single wound entry site.

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Figure 23-25: Following Descemetorhexis, initial controlled, surgical detachment of the Descemet’s membrane (arrow) using the John Dexatome spatula (ASICO Inc., Westmont, IL).

Figure 23-27: Progressive detachment of the Descemet’s membrane (arrows) using the John Dexatome spatula (ASICO Inc., Westmont, IL).

Figure 23-26: Progressive detachment of the Descemet’s membrane (arrows) using the John Dexatome spatula (ASICO Inc., Westmont, IL).

Figure 23-28: Total detachment of the Descemet’s membrane (arrows) using the John Dexatome spatula (ASICO Inc., Westmont, IL).

following DSAEK surgery as compared to a PKP procedure (Figures 23-48 and 23-49). Following DSAEK, the cornea can be studied using corneal OCT and Confoscan units (Figure 23-50). Both DLEK and DSAEK usually look similar clinically after donor-recipient interface has cleared (Figure 23-51). Cornea may also be evaluated using wavefront analysis techniques that gives information as to the quality of vision and also helps in removal of the limbal wound sutures (Figure 23-52). Fuchs’ corneal dystrophy with endothelial decompensation is a common diagnosis for

DSAEK (Figure 23-53). Following DSAEK there usually is good clearance of the corneal edema (Figure 23-54) and with postoperative healing process there is good uniform adherence of the donor corneal disk to the patient’s cornea (Figure 23-55). Corneal epithelium may be removed to increase intraoperative visualization of the donor corneal disk and the anterior chamber (Figure 23-56). When a triple procedure is planned, namely, phacoemulsification along with DSAEK surgery, the cataract may be removed using the upside-down phacoemulsification (John technique)

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Figure 23-29: The peripheral stroma within the epithelial circular mark is made rough by using the John DSAEK scrubber (ASICO Inc., Westmont, IL) to enhance donor disk attachment to the recipient cornea. This is completed 360-degrees, first in a clockwise direction, followed by a counterclockwise direction.

Figure 23-30: Complete removal of Healon from the anterior chamber using an irrigation/aspiration unit. The peripheral iridectomy is from the previous surgery performed elsewhere.

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Figure 23-31: The donor corneal disk is folded into a “taco-fold” after placing a small amount of Healon on the endothelial surface (upperleft).

Figure 23-32: Donor corneal disk is introduced into the recipient anterior chamber after enlarging the entry wound to 5.0 mm and the wound is closed with three interrupted 10-0 nylon sutures.

DSAEK Simplified Surgical Technique

Figure 23-33: The donor disk is unfolded using filtered-air. The air is injected in a steady, controlled fashion.

Figure 23-34: Donor disk is uniformly adherent to the patient’s cornea and it is well centered. Also seen is the double-ring sign.

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Figure 23-35: “Fork-lift” taco helps in intial stroma-to-stroma adherence and facilitates unfolding of the taco. The cannula is placed between the folded taco and the anterior iris surface and sterile balanced salt solution is injected to gently lift the taco.

Figure 23-36: Fluid unfolding of the donor disk in a patient with a deep anterior chamber.

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Figure 23-37

Figure 23-38

Figure 23-39

Figures 23-37 to 23-39: John Fixation Hook (ASICO Inc., Westmont, IL) pins the donor disk against the recipient cornea, followed by air-unfolding of the donor disk.

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Figure 23-40: Use of John DSAEK Glider to remove macrofolds from the donor corneal disk following air attachment of the donor corneal disk to the recipient cornea.

Figures 23-41A and B: A– The donor corneal disk is uniformly adherent to the patient’s cornea without any macrofolds; B – Macrofolds are seen in the donor corneal disk and requires the use of Lindstrom Roller (insert) or John DSAEK Glider.

DSAEK Simplified Surgical Technique

Figure 23-42: Various donor corneal disk insertion techniques.

Figure 23-43: Suture-drag technique to pull the folded donor corneal disk into the recipient anterior chamber.

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Figure 23-44: Use of Busin Glide and vitreous forceps to pull the donor corneal disk into the anterior chamber.

Figure 23-45: Various features associated with the suture-drag technique.

Figure 23-46: Various features associated with the pull-through Busin Glide technique.

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Figure 23-47: Residual air bubble in the anterior chamber 1 day following DSAEK procedure.

Figure 23-48: Preoperative and day 1 postoperative slit-lamp photos following DSAEK surgery, showing rapid clearing of the recipient corneal edema. PBK – Pseudophakic bullous keratopathy.

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Figure 23-49: Pre- and postoperative slit-lamp photographs showing rapid clearing of the corneal edema.

Figure 23-50: Following DSAEK, the cornea can be studied using corneal OCT and Confoscan units.

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Figure 23-51: Same patient with DLEK in one eye and DSAEK in the opposite eye with clear corneas OU. PTPatient

Figure 23-52: Following DSAEK, wavefront analysis may be performed.

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Figure 23-53: Fuchs’ corneal dystrophy with cloudy cornea.

Figure 23-55: Profile view of the cornea following DSAEK for pseudophakic bullous keratopathy (PBK) showing good uniform adherence of the donor corneal disk to the patient’s cornea.

Figure 23-54: : Good corneal clearance of edema following DSAEK.

Figure 23-56: Epithelial removal to increase intraoperative visualization of the donor corneal disk and the anterior chamber.

Figure 23-57: Schematic representation of the upside-down phacoemulsification (John technique).

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Figure 23-58: Intraoperative photographs of the upside-down phacoemulsification (John technique).

Figure 23-59: Descemetorhexis can be difficult due to scarring following penetrating keratoplasty (PKP) and a failed corneal graft.

Figure 23-60: DSAEK surgery is often more difficult in eyes following a filtering bleb.

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(Figures 23-57 and 23-58). Descemetorhexis can be difficult due to scarring following penetrating keratoplasty (PKP) and a failed corneal graft (Figure 23-59). DSAEK surgery is often more difficult in eyes following a filtering bleb (Figure 23-60). It may be advisable to avoid doing DSAEK surgery in aphakic eyes depending on the level of surgeon experience in DSAEK surgery, since there is a higher risk of the donor corneal disk falling into the vitreous cavity and on to the retinal surface. The same applies to eyes with a very large complete peripheral iridectomy without any PC IOL partially covering the iris opening. DSAEK surgery has been simplified with the use of Healon in the anterior chamber and not using fluid for the initial steps in this surgical procedure. Additionally,

improved surgical instruments and surgical textbooks have all helped the corneal surgeon to transition from fullthickness penetrating keratoplasty to STCT, namely DSAEK.

References 1. John T. Selective tissue corneal transplantation: A great step forward in global visual restoration. Expert Rev Ophthalmol 2006;1:5-7. 2. John T. Surgical Techniques in Anterior and Posterior Lamellar Keratoplasty. New Delhi, India: Jaypee Brothers Medical Publishers; 2006;1-687. 3. John T. Step by Step Anterior and Posterior Lamellar Keratoplasty. New Delhi, India: Jaypee Brothers Medical Publishers; 2006;1-297.

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Surgical Technique for DSAEK

Mark S Gorovoy

Surgical Technique for Descemet Stripping Automated Endothelial Keratoplasty (DSAEK)

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Introduction The surgical technique of Descemet stripping automated endothelial keratoplasty (DSAEK) for corneal endothelial failure and secondary corneal edema is explained with clinical pearls to facilitate the use of this technique. DSAEK is a novel procedure that replaces full-thickness penetrating keratoplasty (PKP) for corneal endothelial disease. The visual results of DSAEK exceed those of PKP.

Historic Perspective Penetrating keratoplasty has been the standard of care for corneal transplantation for over 50 years (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). Modern microsurgical techniques and improved eye banking has resulted in a very highly successful operation, especially if the measures of outcome is a clear cornea. PKP is a fullthickness corneal procedure and applicable for a large range of diseases including corneal decompensation from endothelial failure, stromal scars and corneal ectatic diseases. However, visual results with PKP are highly variable, unpredictable and often very delayed. Unacceptable astigmatism both regular and irregular limits spectacle correction following PKP. Full-thickness trephinations and long term sutures contribute to the refractive dilemmas and surface disease. The susceptibility to traumatic wound dehiscence and loss of vision remains permanent. Clearly, a procedure that not only improves the anatomy (i.e. corneal clarity), but also consistently and reproducibility improves the function (i.e. visual acuity as measured by best corrected spectacle visual acuity (BCSVA) is highly desirable. DSAEK, by avoiding full-thickness trephinations and long term corneal sutures, avoids the functional short-comings of PKP and results in a rapid high quality visual recovery. DSAEK is a “focused” surgery for endothelial disease, such as Fuchs’ corneal endothelial dystrophy, pseudophakic or aphakic bullous keratopathy, prior endothelial graft failure following a PKP or the irido-corneal-endothelial (ICE) syndrome. Is it not applicable for stromal scars or ectatic disease? The origins of DSAEK stems from the work of Dr. Gerritt Melles (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). His original technique of posterior lamellar keratoplasty (PLK) involved a large limbal incision and deep manual lamellar corneal dissections with excision and transplantation of a similarly dissected donor corneal disk. Next evolved a smaller (5 mm) clear cornea temporal incision and a “taco-fold” donor corneal insertion into the recipient anterior chamber (AC). Dr Mark Terry renamed the procedure Deep Lamellar Endothelial Keratoplasty (DLEK) and has been instrumental in promoting the superior outcomes of DLEK. PLK (DLEK) requires extensive surgical skill to master the arduous lamellar dissections

on both the patient and donor corneas [performed using an artificial anterior chamber (AAC)]. The next evolution by Dr Melles was the substitution of the patient’s corneal stromal dissection with Descemet stripping. This pivotal change gave birth to Descemet’s Stripping Endo Keratoplasty (DSEK). Descemet stripping eliminated the manual dissection of the patient’s cornea and my adaptation of the Moria anterior lamellar therapeutic keratoplasty (ALTK) system eliminated the manual dissection of the donor and gave rise to DSAEK (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). The elimination of all manual lamellar dissections has resulted in a more consistent and reproducible surgical outcome while simplifying the surgical procedure. Visual recovery is hastened by the two smooth lamellar surfaces.

Surgical Technique DSAEK is my procedure of choice for all patients requiring corneal transplantation due to corneal endothelial disease. DSAEK surgery is totally dissimilar to PKP surgery and because it is a new and novel procedure, I continue to modify my technique to improve patient outcomes. The “Achilles heel” of DSAEK is donor corneal dislocation and most changes to the procedure are in an effort to reduce this complication. I will describe my present surgical technique (Figures 24-1 to 24-18) and postoperative maneuvers in detail.

Patient Selection DSAEK is indicated for corneal endothelial disease. Fuchs’ corneal dystrophy comprises the largest cohort followed

Figure 24-1: Cutting the corenal cap using a Moria ALTK system and a 300 micron microkeratome head.

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Figure 24-2: Intraoperative photograph showing the surface circular mark of 9.0 mm diameter and temporal 5.0 mm limbus marks with surgical calipers.

Figure 24-5: Irrigating Descemet stripper used for scoring the inside surface of the cornea, using the surface circular mark as a guide.

Figure 24-3: Diamond blade is used to make the 1.0 mm limbal entry wounds to the anterior chamber.

Figure 24-6: Descemet stripping is performed using an I and A unit.

Figure 24-4: A 2.75 mm keratome blade is used to make a clear corneal incision.

Figure 24-7: Stab incisions are made with 1.0 mm diamond blade from the corneal surface to the donor-recipient interface.

by PBK and prior corneal endothelial graft failure. The technique is essentially the same for all diagnoses. All patients must be pseudophakic with a posterior chamber intraocular lens implant (PC IOL). I prefer to do DSAEK as a stand alone procedure, not combined with other surgical

procedures. All phakic patients are rendered pseudophakic even if it requires a clear lensectomy (Editorial Note: Clear lens extraction if performed, should be discussed in detail with the patient prior to the procedure and documented in the patient’s chart). The only exception is a cornea too opaque to perform

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Figure 24-8: The clear corneal wound is enlarged to 5.0 mm.

Figure 24-11: The donor corneal disk is folded as a 60/40 “taco” fold.

Figure 24-9: Donor endothelium is coated with HealonTM.

Figure 24-12: Donor corneal disk is inserted into the recipient anterior chamber with the Goosey forceps.

Figure 24-10: Intraoperative photograph showing the cornea after stripping the Descemet’s membrane.

Figure 24-13: Corneal wound is closed with a 10-0 nylon suture.

phacoemulsification. This deepens the anterior chamber (AC) for donor corneal disk unfolding and avoids future cataract surgery, that is almost inevitable, thereby avoiding phacoemulsification induced endothelial cell loss. All AC IOL is replaced with sutured scleral-fixated PC IOL. Glaucoma must be well controlled and if necessary filtering or shunt surgery is performed pre-DSAEK. The staged procedures are performed approximately 4-6 weeks apart.

Preoperative Treatment A fourth generation fluoroquinolone is started qid the day prior to surgery [Editorial Note: Alternative medication to consider include, levofloxacin 1.5% (Iquix 1.5%, Vistakon Pharmaceuticals, Jacksonville, FL) used qid for 3 days prior to surgery and continued postoperatively]. Topical anesthesia using Lidocaine hydrochloride jelly 2% (Akorn Inc., Buffalo

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Figure 24-14: “Taco-folded” donor corneal disk is positioned nasally.

Figure 24-17: Air is injected under the donor corneal disk in the anterior chamber.

Figure 24-15: I and A unit is used to unfold the donor corneal disk.

Figure 24-18: Drainage of fluid through the stab incisions if any donor-recipient interface fluid.

Grave, IL) and intravenous sedation as needed are utilized. The pupil is left neutral, i.e. no miosis or mydriasis.

A lid speculum is placed after the lid margin and lashes are draped with tegaderm (3M, St. Paul, MN). The surgeon is seated temporally, with the operating microscope and the corneal surface is marked with a 9.0 mm trephine marker inked with a sterile marking pen. This allows the surgeon to judge the trephine size for donor tissue trephination. The surgeon almost always uses a 9.0 mm donor corneal disk unless this corneal marking approaches 2.0 mm to the limbus, in which case, a smaller diameter trephine is preferable such as an 8.5 or 8.75 mm trephine. Avoid the donor lenticule from impinging too close to the AC angle. The patient’s eye is left open with the eyelid speculum and attention is then directed to the donor corneal tissue preparation. This air drying allows a swollen cornea to deturgesce to some extent and improve operative visibility.

Surgical Steps

Donor Corneal Preparation

In the operating room, Betadine 5% ophthalmic solution (Alcon Laboratories, Fort Worth, TX) to the skin and lids is performed with direct instillation onto the ocular surface.

The donor cornea is placed within the ALTK system (Moria, Doyleston, PA) and the donor cornea is cut with a 300 µm microkeratome head (Moria, Doyleston, PA). Air is used to

Figure 24-16: A second corneal suture is applied and the donor corneal disk is centered.

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inflate the donor cap within the Moria ALTK system. A very firm corneal dome is verified with finger palpation. The donor corneal rim must be a consistent 16 mm rim size, without any edge irregularities or defects. The eye bank must be made aware that a large donor scleral rim size is required. The anterior cap is then removed from the keratome head and the remaining posterior lamellar donor tissue is removed from the ALTK system and placed on a cutting block and trephined endothelial side up. When lifting up the outer ring, simultaneous forced air pressure prevents the donor tissue from collapsing on the ALTK central post. Centration of the donor tissue on the cutting block is essential to avoid a decentered cut and a fullthickness donor disk edge. If that occurs, recenter the tissue and recut it to remove the full-thickness section. A perfectly round donor tissue is not required and causes no optical problems. A drop of Optisol GS (Bausch and Lomb Surgical, Irvine, CA) is placed on the donor cornea and attention is directed to the patient’s cornea. Using a 1.0 mm diamond blade vertical limbal paracentesis are made superiorly, inferiorly and nasally. A clear cornea 2.75 mm keratome incision is made temporally. Through the right handed paracentesis, the Gorovoy irrigating Descemet stripper (Harvey Instruments, Rotonda West, FL) is introduced into the AC and Descemet’s membrane is scored along the surface trephination mark for close to 360°. Descemet’s membrane is then stripped for 2-3 mm for several clock hours. The I and A 4.0 mm port handpiece (B&L millennium) is introduced through the keratome incision and the loose Descemet’s membrane edge is aspirated and removed through the wound. This may take several passes with the I and A unit to remove all of the Descemet’s membrane within the scored area. Three full-thickness stabs using the same diamond paracentesis blade are made equal distance in the mid-periphery for future interface fluid drainage as described by Dr. Frances Price. The temporal incision is enlarged to 5.0 mm.

Donor Corneal Disk Insertion The donor corneal tissue within the cutting block is placed under the microscope and excessive fluid is removed with a Weck-cel™ spear (Medtronic Xomed Inc., Jacksonville, FL). Several drops of Healon (Advanced Medical Optics, Santa Ana, CA) are placed on the donor corneal tissue which is then folded over like a taco in a 60%/40% fold. It is then held with Goosey insertion forceps (Moria, Doyleston, PA) and the donor corneal disk is then inserted into the AC. A single 10-0 nylon suture (Ethicon # 9061) is applied to partially close the incision and sterile balanced

salt solution (BSS) is used to deepen the AC. The donor corneal disk may unfold spontaneously. If it does not unfold, the I and A handpiece is used to deepen the AC and hold the underside of the “taco” and unfold it. A final 10-0 nylon suture is placed on the wound and the donor disk is centered to the limbus. This is accomplished by reforming the AC about 50% depth and applying gentle pressure indentations at the limbus with any device, even your finger. The donor corneal disk will move away from the spot at the limbus where focal pressure is applied. Once the donor corneal disk is well centered, a full air bubble fills the AC outlining the donor disk edge. If the donor disk decenters during this step, the air is removed and the donor corneal disk is re-centered. The corneal stab incisions are then probed with a spatula or cannula and any interface fluid is expressed out. Dilating drops are applied and the patient is left in a supine position for 1 hour in the holding area. After 1 hour, a slit-lamp exam verifies the position of the donor and air is expressed out thru the paracentesis or the main wound site, until the lower air bubble meniscus is above the inferior pupil edge. This prevents potential pupillary block. A shield is applied. Topical antibiotics and corticosteroid eyedrops qid are begun and the patient is examined the next day in the office. Patient leaves the operating room with a patch and a shield taped over the eye.

Postoperative Day 1 Biomicroscopic examination confirms the position of the donor corneal disk. Is the cornea grossly edematous? Has the donor disk slipped inferiorly? If the answer is “yes” to those two questions, then donor dislocation is likely. Careful slit-lamp examination can identify very tiny adhesion gaps, even with 4-plus corneal edema (scale of 1-4). Repeat air injection into the AC is done in the minor operating room using the surgical microscope. Removal of excess air bubble is done as needed in the operating room. I have had to place three bubbles in several patients. There is no harm to the “loose” donor disk in the AC as the aqueous humor provides endothelial nourishment independent of stromal attachment. One patient had a delayed donor disk adhesion at 7 weeks, and has a perfect result with an excellent endothelial cell count. The goal is a donor disk dislocation rate of less that 5%. Postoperative topical medication regimen is 1 week of antibiotic drops, and corticosteroid drops qid for 3 months. After 3 months, the corticosteroid medication is tapered to once a day for six months. Steroid responders with increased IOP may need accelerated tapering and even a switch to loteprednol

Surgical Technique for DSAEK etabonate ophthalmic suspension 0.5% (Lotemax, Bausch & Lomb, Rochester, NY) bid plus glaucoma drops. Cyclosporine ophthalmic emulsion 0.05% (Restasis, Allergan Inc., Irvine, CA) are used for dry eye and to decrease local inflammation. BSCVA is obtained at 6 and 12 weeks at which time a new eye glass prescription can be given. Limbal sutures are cut after 6 weeks for astigmatism control.

Discussion DSAEK has revolutionized the surgical management of corneal endothelial failure. This is a significant paradigm shift in corneal replacement surgery worldwide. It allows the surgeon to explain the improved benefits of this new surgical procedure while minimizing some of the potential complications associated with an open-sky full-thickness procedure like the penetrating keratoplasty. In the absence of concomitant retinal and/or optic nerve disease, one can expect a BSCVA of 20/40 by 6 weeks in over 80% of the patients. This improves to over 90% by 12 weeks. BSCVA of 20/20 is reached in 16% by 6-12 months following surgery. There are no significant refractive surprises. Patients with bilateral disease are requesting second eye surgery by 3 months. However, appropriate patient counseling must never ignore the risks of any corneal transplant surgery; namely, infection, primary graft failure, graft rejection and secondary glaucoma. I no longer recommend DSAEK as an alternative to penetrating keratoplasty. DSAEK has replaced

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PKP in my surgical practice and such a paradigm shift is expected to continue globally.

Bibliography 1. Gorovoy MS. Descemet stripping automated endothelial keratoplasty (DSAEK). Cornea 2008;27:632-3. 2. Melles GRJ, Eggink FAGJ, Lander F, et al. A surgical technique for posterior lamellar keratoplasty. Cornea 1998;17:618-26. 3. Melles GRJ, Lander F, Beekhuis WH, et al. Preliminary clinical results of posterior lamellar keratoplasty for a case of pseudophakic bullous keratopathy. Am J Ophthalmology 1999; 127:340-1. 4. Melles GRJ, Lander F, Dooren BTH, et al. Preliminary clinical results of posterior lamellar keratoplasty through a sclerocorneal pocket incision. Ophthalmology 2000;107:1850-7. 5. Melles GRJ, Landerd F, Nieuwendaal C. Sutureless, posterior lamellar keratoplasty. Cornea 2002;21:325-7. 6. Melles GRJ, Wijdh, RHJ, Nieuwendaal CP. A technique to excise the Descemet membrane from a recipient cornea (Descemetorhexis). Cornea 2004;23:286-8. 7. Price F, Price M. Descemet stripping with endothelial keratoplasty in 200 eyes. Cataract and Refractive Surgery 2006;32:411-8. 8. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty in the first United States patients: Early clinical results. Cornea 2001;20:239-43. 9. Terry MA, Ousley PJ. Endothelial replacement without surface corneal incisions or sutures: Topography of the deep lamellar endothelial keratoplasty procedure. Cornea 2001;20:14-18. 10. Terry MA, Ousley PJ. Rapid visual rehabilitation after endothelial transplants with deep lamellar endothelial keratoplasty (DLEK). Cornea 2004;23:143-53. 11. Terry MA, Ousley PJ. Replacing the endothelium without corneal surface incisions or sutures: The first United States clinical series using the deep lamellar endothelial keratoplasty procedure. Ophthalmology 2003;110:755-64.

DSEK, Through a 3 mm Incision using the Tri-fold Technique

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Keith A Walter Marshall E Tyler

Descemet’s Stripping Endothelial Keratoplasty (DSEK), Through a 3 mm Incision using the Tri-fold Technique

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Background A sutureless small incision surgery is most desirable in the modern era of micro-incisional cataract surgery. There are several advantages of such a technique when considering DSEK. Less pain, less patient anxiety, less induced astigmatism and lower incidence of complications such as iris prolapse or wound leak. However, the concern has been that placing an 8.0 mm or larger donor corneal graft through a small entry-wound can result in crush injury to the donor endothelial cells and endothelial cell loss can occur. Perhaps, the benefits of small incisional DSEK would likely be vanquished in the potential high graft failure rate. As it turns out, the techniques of folding the endothelial graft in half and unfolding it in the limited confines of the anterior chamber under low pressure requires a bit of skill and patience. Often times, the additional manipulations in “unsticking” the two halves, can further damage the endothelial cells or even cause an inversion of the tissue with the donor endothelium facing the patient’s corneal stroma, an undesired incident. Considering that most anterior chamber depths are only a 2.0 to 3.0 mm deep, the iris or lens may obstruct the proper unfolding of an 8.09.0 mm diameter endothelial graft. Conversely, a tri-folding or “burrito” configured endothelial graft, is quite easily unfolded with plenty of space for the edges to unfold. Thus a trifolded donor corneal disc requires less room within the AC to unfold as compared to a bifolded donor corneal disk. The author is of the opinion that tri-folding the endothelial graft is likely to be a distinct advantage for both the patient and the surgeon.

Description of Technique The donor corneal disk that is “freshly” trephined and a “S” mark is made on the stromal surface of the donor corneal disk using a surgical marking pen (Figure 25-1) (Editorial Note: Recent study seem to indicate that stromal

Figure 25-1: Schematic representation of the tri-fold technique.

Figure 25-2: Healon being placed on the endothelial cells of the donor corneal disk.

markings with a marking pen may be associated with increased endothelial cell loss). It is then placed on a stable surface with the endothelial side up. A small amount of viscoelastic, namely, Healon (American Medical Optics, Inc., Santa Ana, CA) is placed over the endothelial cells (Figure 25-2). Using a toothed forcep, the edge of the anterior lamellar cap is secured, while peeling the edge of the endothelial graft up and across with a pair of folding forceps (Goosey or Phakic IOL insertion Forceps) (Figure 25-3). Once one-third of the tissue is folded over, the stromal side of the donor graft is grasped across the length of the graft, parallel to the initial fold (Figure 25-4). Using the right hand the tissue is then supinated and rolled over so that the tissue now is in a “stroma out/endothelium in” configuration much like a cinnamon roll (Figures 25-5A and B). Prior to insertion, the anterior chamber is filled with air to prevent collapse during insertion. The tissue roll is then inserted through the entry wound, taking care not to allow the loose edge to unfold exposing endothelial cells (Figure 25-6). Once this is

Figure 25-3: Forceps peeling 1/3rd of the donor graft back as the initial step in the tri-fold technique.

DSEK, Through a 3 mm Incision using the Tri-fold Technique

Figure 25-4: Forceps being applied across the stromal side of the donor corneal disk.

Figure 25-5A: Tissue rolled over forceps into a “burrito” fold prior to insertion into patient’s anterior chamber.

Figure 25-5B: Tissue rolled around forceps in a “burrito” fold.

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Figure 25-6: Insertion of rolled endothelial graft through a 3.0 mm clear corneal wound.

Figure 25-7: Forceps grasping entry wound and graft to prevent tissue from slipping.

accomplished, the tissue is released within the anterior chamber without irrigation. The edge of the endothelial graft and wound should be grasped as the insertion forceps are retracted out of the eye (Figure 25-7). The endothelial graft typically remains in its tri-folded configuration within the anterior chamber (Figure 25-8). Evacuation of any remaining air, followed by gently deepening the anterior chamber with irrigating balanced salt solution will typically automatically unfold the “wings” of the endothelial graft. Occasionally, a small amount of air is required to be placed in the middle of the endothelial graft which further facilitates the unfolding of the donor corneal disk. No instruments are required in the anterior chamber at this stage of the procedure, and in some cases an externo pressure can be used to complete unfolding of the donor cornea, prior to injection of an air bubble.

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Corneal Endothelial Transplant pressure should not be used when inserting. Also, a small enlargement to 3.5 mm or 4 mm may be necessary if the wound tunnel is long or the graft is especially large (greater than 8.5 mm) or the donor graft is thick. The tissue should slide easily through the entry wound and not force the tissue which can result in crush injury to the donor endothelial cells.

Combining with Phaco

Figure 25-8: Endothelial graft in anterior chamber in tri-fold configuration.

Advantages Over the Bi-fold Method In most cases, the tri-fold technique allows one-third or two-third of the tissue to be unfolded and in the correct position immediately after the release of the donor disk within the anterior chamber. The portion of the corneal graft that is rolled and not grasped typically unfolds at the 3 o’clock position with the stroma against stroma. The remaining one-third requires very little manipulation. Less tissue manipulation and negligible risk of inversion affords a great benefit to endothelial survival. With a smaller 3 mm incision, the procedure can be performed through a clear corneal incision. Such a sutureless surgical technique results in less corneal astigmatism and possibly less postoperative pain for the patient. Additionally, such a small 3.0 mm incision can also decrease or prevent potential iris prolapsed during DSEK surgery.

Learning Curve and Precautions The surgeon acquiring endothelial transplant skills must be cautious when manipulating the graft tissue. Specifically, using the tri-fold technique requires that the surgeon take special care not to damage the endothelium when folding or grasping the tissue. The use of Healon probably “cushions” the endothelial cells and provides some additional safety in this procedure. The folding should be gentle and avoid any direct contact with the endothelial cells. Care should be taken when grasping the tissue with the forceps and only apply minimal pressure to hold the tissue. The tissue should not be crushed between the holding forceps. Additionally, one tong of the forceps will touch the endothelial side (Figure 25-1), so downward

Using the Tri-fold technique through a small cataract incision is an excellent way to combine phacoemulsification with endothelial cell replacement. After routine clear corneal cataract surgery, and immediately following IOL insertion, the viscoelastic (Healon, AMO) can be retained during Descemet’s stripping. After this, the viscoelastic can be removed with irrigation/aspiration and the corneal graft is prepared in the usual fashion using the tri-fold technique. The pupil should remain dilated and no miotics should be used to prevent the air bubble from getting behind the iris (Editorial Note: Alternative techniques include removing the patient’s Descemet’s membrane prior to phacoemulsification and IOL insertion, and constricting the pupil and removing the Healon prior to insertion of the donor corneal disk. Also, upsidedown phacoemulsification technique (John technique) may be considered for cataract surgery with DSEK). The tissue is inserted through the same cataract incision into the anterior chamber and then unfolded in the usual fashion described above. Care must be taken so as to not inject the air into the capsular bag but only in the anterior chamber. Only a small amount of additional time is required to perform this triple procedure of combined cataract and corneal surgery. Intraocular lens selection should be targeted towards myopia which will compensate for the typical hyperopic shift seen after DSEK. Using the Tri-fold technique prevents the surgeon from having to enlarge the wound and later suture it.

Future Considerations The tri-fold technique will likely be precursory to the evolution of an even better technique for small incisional sutureless endothelial transplantation. An insertion tool that would limit any crushing forces and deploy the tissue in the correct orientation would be readily accepted by the corneal surgeons worldwide. Such an instrument would likely bear resemblance to the current IOL injectors (Editorial Note: Currently, various donor corneal insertion cartridges are in various stages of development). The tissue could be rolled or tri-folded for loading and then inserted through a protective sheath.

Femto-DSEK: Initial Studies of Surgical Technique in Human Eyes

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Ciro Tamburrelli Agostino Salvatore Vaiano Emilio Balestrazzi

Femtosecond Laser ® (Intralase )–Descemet’s Stripping Endothelial Keratoplasty (Femto-DSEK): Initial Studies of Surgical Technique in Human Eyes

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Corneal Endothelial Transplant

Introduction Among the most frequent indications (30-40%) for penetrating keratoplasty in developed countries are pseudophakic or aphakic bullous keratopathy and Fuchs’ corneal dystrophy. For these corneal endothelial diseases, Melles and associates1 in Europe and Terry2 in the United States introduced the technique of posterior lamellar keratoplasty (PLK), namely, deep lamellar endothelial keratoplasty (DLEK), to selectively exchange patient’s corneal endothelium, Descemet’s membrane (DM), and adjacent deep corneal stroma with a similar donor corneal disk with healthy endothelium. A limbus-to-limbus intrastromal lamellar dissection was carried out within the host cornea through a 9.0 mm scleral incision. The posterior lamellar disk, consisting of posterior corneal stroma, DM, and endothelium, was excised by using an intralamellar trephine, and a disk of similar donor posterior lamellar tissue was placed in the recipient opening without suture fixation. Donor disk adhesion was made possible by using an air bubble in the anterior chamber (AC). Melles has subsequently described two important modifications in this procedure. The first was reducing the size of the scleral incision to 5 mm,3 which mandates removal of the posterior lamella with corneal microscissors. More important, with a 5 mm incision, donor posterior lamellar tissue, which is typically 8.0 to 9.0 mm in diameter, is folded into a “taco” configuration with the endothelium on the inside and coated with viscoelastic material such as Healon (American Medical Optics, Santa Ana, CA). The folded graft is then inserted into the AC and unfolded and positioned against the host corneal stroma. The second modification was to prepare the host cornea by stripping DM and endothelium without removing the posterior corneal stroma,4 a technique known as Descemet stripping with endothelial keratoplasty (DSEK). With preparation of lamellar donor tissue evolving from manual to microkeratome-assisted dissection, the descriptive terminology is Descemet stripping with automated endothelial keratoplasty (DSAEK). This is an important improvement in the surgical preparation of the posterior corneal lamella from the donor cornea. The donor cornea with its scleral rim is mounted on an artificial anterior chamber (AAC), namely, the Moria ALTK system (Moria SA, Antony Cedex, France) and a Moria microkeratome with a 300 μm head is used to excise the anterior corneal lamella. The anterior corneal cap is discarded, and the posterior corneal lamellar tissue is placed in the corneal storage medium. At the beginning of the surgery, the posterior corneal lamellar tissue is punched from the endothelial side using a corneal trephine of chosen

diameter. The donor corneal lenticule is then placed into the recipient AC.

Femtosecond Laser The femtosecond laser (IntraLase® Corp.) is a solid-state laser used to create flaps in laser in-situ keratomileusis (LASIK) and recently to perform penetrating keratoplasty with different shapes of stromal cuts. The laser uses an infrared wavelength (1053 nm) to deliver closely spaced, 3 μ spots that can be focused to a preset depth to photodisrupt tissue within the corneal stroma.5-10 The laser bursts are short (1 quadrillionth of a second). The resultant plasma produces a cavitation bubble, consisting of water and carbon dioxide primarily. We used the femtosecond laser to create a dissection plane on the donor cornea mounted on an AAC. After laser dissection of the donor cornea, the donor tissue was then transferred to a punching system and cut with an 8.0 mm diameter trephine. Forceps separation of the posterior lamella from the anterior stroma is then carried out before placement into the AC. To study the feasibility of the femtosecond laser to prepare posterior corneal lamellae, three eye bank corneoscleral buttons, not suitable for corneal transplantation, underwent a femtosecond laser lamellar dissection of 400 μm stromal depth, with subsequent side cuts completed by manual trephine. In all the corneoscleral buttons, the posterior disk was peeled off the cornea easily with forceps after laser treatment, and all posterior lamellae presented with smooth stromal surface and no gaps or breaks occurred during manual separation from the anterior stroma. Good results encouraged us to apply the same technique in 4 patients requiring endothelial replacement due to endothelial failure by pseudophakic bullous keratopathy and Fuchs’ corneal dystrophy.

Surgical Technique For DSEK, the donor cornea was prepared first in the femtolaser room. This was followed by surgery on the recipient cornea. Time interval between donor corneal preparation and recipient surgery ranged between 1.0 to 26 hours. An AAC seated firmly under the femtolaser and the corneoscleral rim preserved in organ culture storage media was accurately placed within the AAC (Figures 26-1A to D). To reduce the number of air bubbles beneath the cornea, rims were placed on the chamber base after the infusion was released. Once the cornea was stabilized and centered and the absence of air bubbles was confirmed, the infusion was closed, the superior metal support was placed

Femto-DSEK: Initial Studies of Surgical Technique in Human Eyes

Figures 26-1A to D: Intralase applanation phases. (A) Centering, (B) Initial contact, (C) Incomplete applanation, (D) Applanation completed. Note in B and C the formation of air/fluid meniscus between the applanation lens of the Intralase applanating cone and the side portion of the corneal surface not yet applanated. In D full applanation with almost complete disappearance of air/fluid meniscus.

and locked by turning it clockwise. High internal pressure was constantly obtained by marked elevation of the infusion bottle (not less than 1.5 m above the AAC). Corneal thickness was measured using an ultrasound pachymeter (model 850, Bausch and Lomb) in the center of the cornea and in 4 quadrants on the vertical and horizontal meridians. As the Intralase applanation lens makes a full contact with the corneoscleral button a green LED is illuminated and is visible in the operating microscope and on the display panel (Figure 26-2). As the beam delivery device is lowered further after initial contact, the applanation force increases. At a preset position of the objective lens deflection corresponding to the maximum allowed eye pressure, a sensor is actuated and a red LED, which is visible in the operating microscope as well as on the video display, is illuminated. Further downward movement of the beam delivery device is disabled, but upward movement is still enabled. We considered a good applanation a slight further downward movement of the beam delivery device after the green light is illuminated, at this level a homogeneous applanation is obtained and concentric folds visible in the posterior stroma are minimal. We think that edematous stroma in donor tissue causes circular folds as the cornea is flattened by the intralase applanation lens and they tend to increase as the applanation force increases. Proper setting of the femtolaser was then carried out. Laser parameters were: stromal depth 400 μm, bed energy 1.1 μJ (lamellar cut) and no side cut, line/spot separation 9/9 μm in a raster pattern, firing rate 60 kHz, and 9.0 mm diameter. Intralase software enabled 400 μm as maximum stromal depth. The

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Figures 26-2A to D: Intralase applanation phases as observed through the operating microscope or the video display. (A) Initial contact: large air-fluid meniscus is present between the applanating surface of intralase cone and the area of the non-applanated cornea. (B) Further lowering of the cone, minimum decentering and almost complete disappearance of the air meniscus. (C) Full contact with cornea surface. Absence of air-fluid meniscus. Counter-pressure has not reached the right level to ensure good quality laser dissection. Note circular folds appearance in the deeper layer of the stroma (D) Green LED illumination indicates the pressure has reached a safe level to allow for a good laser dissection. Note that the circular folds in the midperipheral cornea have increased in number.

pre-laser corneal pachymetry was used to calculate the expected posterior lamellar thickness subtracting 400 μm from the central corneal pachymetry. Energy setting was input as a compromise between the high level required to obtain a good stromal dissection with smooth surface and minimal residual stromal bridges and the low energy to prevent possible endothelial damage. Forceps separation of the posterior lamella required more tissue stress with bed energy setting below 1.1 μJ. Line/spot separation 9/9 μm is the shortest distance allowed between single spots and lines of spots. Larger distances can be used, but in such a case bridges of uncut stroma are more easily seen. The raster pattern was chosen instead of the double raster to reduce the amount of bed energy released. The 9.0 mm diameter was chosen to ensure inclusion of all the dissected plane in the 8.00 mm diameter of the corneal punch allowing for inclusion of all the dissected plane even with a 1 mm decentered punch trephination (Figure 26-3). At the end of the procedure the treated corneoscleral button is again placed in the storage medium and sent to the surgical theatre. Few minutes after laser treatment, both visible air bubbles and dissection plane cannot be visualized under the operating microscope. The corneoscleral button is punched from the endothelial side using an 8.0 mm Hanna trephine (Moria, S.A., Doylestown, PA).

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Figure 26-3: Gas bubble formation are the byproducts of the femtolaser stromal photodisruption. Laser parameters: stromal depth 400 μm, bed energy 1.1 μJ (lamellar cut) no side cut, line/spot separation 9/9 μm in a raster pattern, firing rate 60 kHz, and 9.0 mm diameter.

Proper centration of the donor tissue is essential (Figure 26-4). Both anterior and posterior cut corneal lamellae remain resting on the donor punching block adherent to each other and covered by the organ culture storage media until use. Two Uttrata forceps are then used to gently separate anterior from posterior lenticule. The cleavage plane is usually found in the posterior fourth of the stromal tissue and the two lamellae are separated taking care to avoid excessive stress and folding of the posterior lamella (Figure 26-5). Once separation has occurred, three small marks are placed gently touching the stromal side cut by a

Figure 26-4: Donor corneal trephination is carried out from the endothelial side using the Hanna trephine.

marker pen: a single one on the distal part and pair of two close marks 90 degree apart clockwise after the single mark [Editorial Note: Alternative staining technique (See also Chapter 32, Use of Dyes in DSAEK and DLEK) may be considered, since recent studies (Terry et al personal communication) seem to

Figure 26-5: (Top and bottom rows)—Posterior lenticule is gently separated from the anterior stroma. Care is taken to find the cleavage plane created by the femtolaser dissection and to exert gentle traction with minimal folds on the thin posterior lamella. Note that the two lamellae are attached by residual microscopic stromal bridges easily broken with forceps separation.

Femto-DSEK: Initial Studies of Surgical Technique in Human Eyes

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Figure 26-6: Three small marks are placed by gently touching the stromal side-cut of the separated posterior lenticule, with a sterile marking pen: a single mark on the distal part of the donor lenticule and a pair of two additional marks 90 degrees apart and clockwise from the single mark. Once placed in the recipient anterior chamber, these marks on the donor lenticule will help ensure that the stromal side of the lenticule comes in contact with the stromal side of the recipient cornea. The paired marks are positioned in a clockwise direction from the single mark.

indicate endothelial cell loss associated with the use of marking pen on the stromal surface of the donor corneal disk]. Once placed in the recipient anterior chamber, to ensure that the stromal side of the lenticule comes in contact with the stromal side of the recipient cornea, the pairs of marks have to be positioned clockwise proximal to the single mark (Figure 26-6). After preparation of the corneal lenticule, attention is directed to the host cornea and a limbal temporal incision is made with a 3.2 mm keratome blade. In patients undergoing a triple procedure (keratoplasty, cataract extraction, and intraocular lens [IOL] implantation), surgery is performed through the same incision using phacoemulsification and a phaco-chop technique. The host corneal epithelium is marked with an 8.0 mm Weck trephine (Solan Medtronics, Jacksonville, FL) stained with gentian violet dye. A paracentesis is made 2 hours clockwise from the limbal incision to allow manipulation of a second instrument. The AC is maintained with an irrigating cannula, and a reverse-bent 30 gauge needle is used to incise the host endothelium and Descemet’s membrane corresponding to the 8.0 mm epithelial trephine mark. A John DSAEK Descemet’s Stripper (ASICO, Westmont, IL, AE-2874, Patent Pending) is then used to carefully remove the diseased host endothelium and Descemet’s membrane within the circumference of the Descemet’s incision (Figures 26-7A to D) (See also, Chapter 11, New/Useful Surgical Instruments in DSAEK). The limbal incision then is widened to approximately 5.2 mm using the keratome. The endothelial surface of the donor lenticule is coated with a small amount of viscoelastic (Healon), and the donor disk is gently folded into a “taco-shape” using the Uttrata forceps. The folded donor corneal lenticule is then inserted

into the anterior chamber choosing between two different techniques. To minimize risk of crush injury to the donor endothelium an IOL holder with curved platforms that do not oppose centrally, is used to grasp and insert gently the folded lenticule (Figure 26-8). In a second technique, a 5.2 mm keratome is used as a glide. The folded lenticule resting on the keratome is gently pushed with a spatula in the AC through the limbal incision that can be widened to 6.2 mm to allow a smoother insertion (Figure 26-9). A double 10-0 nylon suture is used to close the limbal wound before filling the AC with balanced salt solution (BSS) (Alcon, Fort Worth, TX). To properly unfold the donor corneal lenticule having the endothelial side facing the AC and stromal surface in contact with the recipient stroma, the balanced salt solution is inserted directing the fluid in the virtual space between the touching endothelial surfaces. Proper contact is checked noting that the double marks are located clockwise before the single one (Figure 26-10). The AC is then filled with air and 8 minutes is allowed for the air to help in the donor-recipient corneal adherence. After 8 minutes, part of the air bubble is removed and replaced with BSS (Figure 26-11). Patient receives 1 drop each of ciprofloxacin (Alcon, Fort Worth, TX) [Editorial Note: Newer antibiotics such as levofloxacin 1.5% (Iquix 1.5%, Vistakon Pharmaceuticals, Jacksonville, FL) may be used one drop QID], cyclopentolate 1% (Bausch and Lomb, Tampa FL), and prednisolone acetate 1% (Pred Forte 1%, Allergan, Inc., Irvine, CA) at the end of the procedure. The operated eye is patched and a shield is applied over the patch and they are taped in place. Postoperatively, patients receive topical antibiotics and cycloplegia for 1 week and topical prednisolone acetate 1% ophthalmic suspension (Pred Forte 1%, Allergan, Inc., Irvine, CA) 3 times per day for 1

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Figures 26-7A to D: Stripping of the patient’s Descemet’s membrane under fluid using an irrigating cannula. (A) Incision of the Descemet’s membrane with a reversed anterior chamber needle. (B-C) Stripping and detachment of the Descemet’s membrane along the edges of the external circular template mark that was made initially on the patient’s corneal epithelial surface. (D) Removal of the Descemet’s membrane. Also seen are the guttata on the Descemet’s membrane.

Figure 26-8: After folding the donor posterior lenticule into a “taco-fold”, It is grasped with an IOL-holder and the donor lenticule is inserted into the recipient anterior chamber through a 5.2 mm limbal incision.

month. After 1 month, the topical prednisolone is tapered gradually over a 3-month period.

Results The group of patients included 4 consecutive cases with corneal edema from (1) Fuchs’ endothelial dystrophy, (3) pseudophakic bullous keratopathy, without significant corneal stromal scarring who underwent Femto-DSEK.

Preoperative and postoperative pachymetry of the donor cornea and the calculated and actual thickness of the endothelial lenticule are reported in Table 26-1. In vivo measurements of the endothelial lenticule were carried out with the Heidelberg Cornea Tomograph (Heidelberg Engineering, Inc., Heidelberg, Germany). Cell count density in each donor cornea and in the endothelial lenticule 6 months after surgery, as well as postoperative visual acuity are reported in Table 26-2.

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Femto-DSEK: Initial Studies of Surgical Technique in Human Eyes

Figure 26-9: The folded lenticule resting on a blunt keratome and it is gently pushed with a spatula into the recipient AC through the limbal incision that can be widened to 6.2 mm to allow a smoother insertion. Note that tissue folding occurred on the axis of the single mark.

Figure 26-10: (Top left)—The posterior lenticule is seen within the recipient anterior chamber. (Top right and Bottom left)—An irrigating cannula is placed within the space created by the edges of the donor lenticule that are in contact. The donor endothelium is on the inside surface while the donor stroma is on the outer surface. The fluid from the irrigating cannula allows for a smooth separation and complete unfolding of the donor lenticule. (Bottom right)—Proper lenticular orientation within the anterior chamber is confirmed using the pre-placed single and double marks. TABLE 26-1: Pre- and postoperative pachymetry and endothelial lenticule thickness

Donor cornea pachymetry

Intralase horizontal cut

Endothelial lenticule calculated thickness

Endothelial lenticule measured thickness

Difference

590 μm

400 μm

190 μm

170 μm

–20 μm

610 μm

400 μm

210 μm

220 μm

+10 μm

605 μm

400 μm

205 μm

210 μm

+5 μm

590 μm

400 μm

190 μm

175 μm

–15 μm

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Figure 26-11: The anterior chamber is filled with air and allowed to remain in the anterior chamber for 8 minutes. Following the 8 minute waiting period, the air bubble size is decreased and replaced with sterile balanced salt solution.

Figure 26-12: In vivo measurements of the endothelial lenticule thickness obtained with the Heidelberg Cornea Tomograph (Heidelberg Engineering, Inc.). TABLE 26-2: Endothelial cell density and visual acuities following femtosecond laser (Intralase®) - Descemet’s stripping endothelial keratoplasty (Femto-DSEK)

Donor cornea cell density

Cell density 6 mo postop

Postop visual acuity

2150/mm2

1350/mm2

20/30

2500/mm2

985/mm2

20/40

2450/mm2

1550/mm2

20/30

2350/mm2

787/mm2

20/100

Conclusions Despite excellent postoperative results, visual acuity (VA) after deep lamellar endothelial keratoplasty (DLEK) rarely exceeded 20/30 due to presumed optical aberrations at the graft-host interface.11 Unlike DLEK, Descemet’s stripping automated endothelial keratoplasty (DSAEK), employs mechanical stripping of the diseased host endothelium along with the patient’s Descemet’s membrane and replacement with a healthy homograft of endothelium,

Descemet’s membrane, and a thin layer of donor stromal tissue harvested with an automated microkeratome.12 These technical refinements have resulted in improvements in smoothness of both recipient and donor stromal surfaces thus minimizing interface aberrations. We used femtosecond laser to create posterior lenticules of variable thickness from donor corneas. Previous studies have demonstrated by scanning electron microscopy that in the posterior lamellar cuts, there is a slightly more irregular texture of the interface than in the standard anterior femtosecond laser assisted in-situ keratomileusis (LASIK) cuts. Increased scatter and attenuation of the laser energy that accompanies deep treatment in edematous corneas8 and the less compactly organized lamellar layers of the posterior stroma as compared with the superficial stroma13 are the likely causes for the increased roughness noted in the posterior cut surface. Nevertheless, one major advantage of the femtolaser cut over the mechanical microkeratome cut is the ability to set deeper cuts, thus obtaining thinner lenticules with less donor stroma included in the donor corneal disk. Replacement of the diseased endothelial cells along with the Descemet’s membrane with only the donor Descemet’s membrane along with healthy donor endothelial cells represents the ideal treatment for patients with diseased corneal endothelium. Femtosecond laser theoretically may achieve these results provided that deeper than 400 µm cuts can be obtained and no damage to the endothelial cells is induced by the laser energy. Further studies are needed mainly to evaluate the endothelial cell damage by laser energy. Our small series disclosed that marked cell loss occurred after 6 months and two patients required lenticule replacement. Femtosecond laser (Intralase, Advanced Medical Optics, Santa Ana, CA) damage to endothelial cells during posterior laser dissection to cut a 7 mm diameter, 100 µm lamellar disk from the endothelial side was recently investigated.14 The average endothelial cell loss in human eye bank donor buttons ranged between 6% and 14% according to different types of viscoelastics used as a “cushion” to protect the endothelium during applanation and laser delivery. Applanation alone without laser dissection resulted in cell loss of 9% therefore, the laser application although not marked, laser dissection causes endothelial damage in addition to the applanation process. Many other factors affect endothelial cell counts in DLEK surgery. Recently, it has been reported that folding of the donor corneal disk and added manipulations of the donor disk in small-incision DLEK surgery, results in deleterious effect on the endothelial cell survival. Techniques that involve even more donor disk manipulations, such as triple folding and rolling of the donor tissue, combined with

Femto-DSEK: Initial Studies of Surgical Technique in Human Eyes squeezing of the tissue through an even smaller 3 mm incision15 may have an even greater endothelial cell damage and death of these cells as compared to other surgical techniques described above. In conclusion, the femto-DSEK offers potential advantages over the automated microkeratome with regard to a better sizing of the posterior donor lenticule with reduced lenticule thickness. Additionally, the femtosecond laser has the unique capability of obtaining a smooth surface and precise stromal cuts for laser-assisted donor corneal preparation. Further work needs to be done to explore the possibilty of removing only the host Descemet’s membrane and endothelium and replacing it with a similar donor disk. Such an approach, if possible, would result in the postoperative thickness of the recipient cornea to be almost comparable to that of the normal corneal thickness. At the present time DSEK remains as an additive procedure with an increased overall corneal thickness.

References 1. Melles GR, Eggink FA, Lander F, et al. A surgical technique for posterior lamellar keratoplasty. Cornea 1998;17:618-26. 2. Terry AM, Ousley PJ. Endothelial replacement without surface corneal incisions or sutures: Topography of the deep lamellar endothelial keratoplasty procedure. Cornea 2001;20:14–18. 3. Melles GR, Lander F, Nieuwendaal C. Sutureless, posterior lamellar keratoplasty: A case report of a modified technique. Cornea 2002;31:325–7. 4. Melles GR, Lander F, Rietveld FJ. Transplantation of Descemet’s membrane carrying viable endothelium through a small scleral incision. Cornea 2002;21:415–8.

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5. Kurtz RM, Liu X, Elner VM, et al. Photodisruption in the human cornea as a function of laser pulse width. J Refract Surg 1997; 13:653–8. 6. Nordan LT, Slade SG, Baker RN, et al. Femtosecond laser flap creation for laser in situ keratomileusis: Six-month follow-up of initial U.S. clinical series. J Refract Surg 2003;19:8–14. 7. Sugar A. Ultrafast (femtosecond) laser refractive surgery. Curr Opin Ophthalmol 2002;13:246–9. 8. Soong K, Mian S, Abbasi O, Juhasz T. Femtosecond Laser– Assisted Posterior Lamellar Keratoplasty Initial Studies of Surgical Technique in Eye Bank Eyes. Ophthalmology 2005; 112:44–49. 9. Seitz B, Langenbucher A, Hofmann-Rummelt C, SchlotzerSchrehardt U, Naumann GOH. Nonmechanical Posterior Lamellar Keratoplasty Using the Femtosecond Laser (femtoPLAK) for Corneal Endothelial Decompensation. Am J Ophthalmol 2003;136:769–72. 10. Tamburrelli C, Mosca L, Fasciani R, Balestrazzi E. Femtosecond Laser Descemet’s Stripping Endothelial Keratoplasty: Initial Studies of Surgical Technique in Human Eyes ASCRS Session: 2-C San Diego 2007 April 28-May 01. 11. Terry MA, Ousley PJ. Replacing the endothelium without corneal surface incisions or sutures. The first United States clinical series using the deep lamellar endothelial keratoplasty procedure. Ophthalmology 2003;110:755–64. 12. Price FW Jr, Price MO. Descemet’s stripping with endothelial keratoplasty in 200 eyes: Early challenges and techniques to enhance donor adherence. J Cataract Refract Surg 2006;32: 411–8. 13. Fine BS, Yanoff M. Ocular Histology: A Text and Atlas (2nd edn). Hagerstown, MD: Harper and Row; 1979:163–92. 14. Sikder S, Snyder RW. Femtosecond laser preparation of donor tissue from the endothelial side. Cornea 2006;25(4):416-22. 15. Terry MA, Wall JM, Hoar KL, Ousley PJ. A Prospective Study of Endothelial Cell Loss during the 2 Years after Deep Lamellar Endothelial Keratoplasty. Ophthalmology 2007;114: 631–39.

Techniques to Facilitate Disk Adherence to Recipient Cornea in DSAEK

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Thomas John

Techniques to Facilitate Disk Adherence to Recipient Cornea in DSAEK

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Corneal Endothelial Transplant

Introduction Descemet stripping automated endothelial keratoplasty [(DSAEK), synonymous with Descemetorhexis with endokeratoplasty (DXEK)]1-14 is an “against-gravity-line” surgery (AGLS) (Author’s terminology) (See also Chapter 13, Definition, Terminology, and Classification of Lamellar Corneal Surgery). Excluding DSAEK surgery, all other ophthalmic surgical procedures such as surgeries on the cornea, iris, lens, vitreous and retina, are all “in-gravityline” surgery (IGLS) (Author’s terminology). Since, DSAEK is an AGLS, if the donor disk is not well attached to the inner surface of the patient’s cornea and remain attached, then the disk will detach and rest in the inferior part of the anterior chamber (AC) and rest on the iris surface.1 This is a potentially unwanted postoperative result and every measure should be taken by the surgeon to decrease his donor disk detachment rate.

Surgical Techniques to Increase Donor Disk Adherence to Recipient Cornea Currently, there are at least 3 different techniques to increase and augment donor disk attachment to the recipient cornea (Figure 27-1) as listed below: 1. Corneal slits (Price FW) 2. Roughening the peripheral circular area of recipient corneal stroma (Terry MA)10 3. Use of large air bubble (John T).

Corneal Slits Price FW described the use of corneal slits to increase donor disk attachment. Conceptually, this is a good procedure to enhance donor disk adherence. In Dr. Price’s experience, this surgical technique has significantly decreased the donor disk detachment rate. Four corneal slits are made, one in each quadrant within the circle of Descemetorhexis (DX) (Figures 27-2A to D). Each of these recipient corneal slits are about 2.0 mm in length and they vary in depth to enter the donor-recipient interface. These corneal slits are of no use if the interface is not reached. Once the slit incision reaches the donor-recipient interface, it will often result in draining any aqueous humor that may be trapped in the interface (Figure 27-3). Drainage of such trapped fluid will collapse the fluid layer in the interface, and help in the adherence of the donor corneal disk to the recipient cornea. The same type of incision is then repeated in the remaining 3 quadrants. One or more of these slit-incisions may drain

Figure 27-1: Schematic representation of 3 different surgical techniques to increase donor corneal disk attachment to the recipient cornea. One or two inferior peripheral iridotomies are performed pre-operatively when the large air bubble technique is used.

any trapped fluid from the interface. It may be difficult to ascertain that the depth of these incisions is adequate. One such indication is the mild movement of the donor disk, when the incising blade has actually reached the donorrecipient interface. These slit-incisions should only be performed with an air bubble inside the anterior chamber (AC) that pushes the donor disk against the inner corneal surface of the recipient cornea (Figure 27-4). There is significant resistance from the recipient cornea when making these slits with a steel blade (Figures 27-2 and 27-3). Such an incision should only be made gently, by a “to-and-fro” motion without any significant corneal deformation. The amount of fluid that may be drained from the slit-incision is usually small (Figure 27-3). If a diamond blade is used for these slit incisions, there is very little or no resistance from the recipient cornea and it makes the procedure much easier as compared to using a steel blade. There is little or no recipient corneal deformation when using a diamond blade. Postoperatively, these four quadrant slits are visible at the slit-lamp and remain visible over time. These corneal slits do not disappear. Also, one has to keep in mind that these slits although small, are made on the host corneal surface. The author does not routinely perform slit-incisions

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Figures 27-2A to D: (A) Intraoperative photograph (Surgeon’s view, temporal approach) showing the use of a steel blade in creating a corneal slit incision in the temporal quadrant of the cornea at 3:00 o’clock position; (B) Corneal slit incision in the superior quadrant of the cornea at 12:00 o’clock position; (C) Corneal slit incision in the nasal quadrant of the cornea at 9:00 o’clock position; (D) Corneal slit incision in the inferior quadrant of the cornea at 6:00 o’clock position.

on the recipient cornea. The author reserves the slit-incision to those occasional cases, where there is entrapped fluid in one quadrant, with 360 degrees of peripheral attachment of the donor disk to the recipient cornea. In such a case, the author performs one-slit that corresponds to the quadrant of “loculated” interface fluid.

Roughening the Peripheral Circular Area of Recipient Corneal Stroma

Figure 27-3: Arrow shows fluid drainage from the donor-recipient corneal interface associated with the slitincision in the temporal quadrant of the cornea.

Terry M first described this technique of roughening the peripheral circular area of the recipient exposed corneal stroma, after DX to increase the donor disk attachment to the recipient cornea. We know from the deep lamellar endothelial keratoplasty (DLEK) experience that there is good adherence between the roughened host corneal stromal surface to the roughened manually dissected donor

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Figure 27-4: Intraoperative photograph showing 4-slits (asterisks), one in each quadrant to drain any interface fluid and increase adherence of the disk to the recipient corneal stroma. Also seen is the double-ring sign (arrows) of a well centered donor corneal disk. The outer ring represents the border of the air bubble inside the anterior chamber and the inner ring represents the margin of the attached donor corneal disk. The donorrecipient interface is very smooth and uniform without any interface debris or donor corneal folds in DSAEK surgery.

corneal disk, during the time prior to the use of a microkeratome in posterior lamellar keratoplasty (PLK). During that period of DLEK surgery, the roughened stroma also included the central area of visual axis. Postoperatively, the interface was quite pronounced at the slit-lamp, but such interface haze and opacity fully cleared over time, often 1 year or less after a DLEK procedure. This concept of increased stromal adherence when the stromal surface is rough, is carried over from DLEK surgery to DSAEK surgery, except the central region of the host cornea including the visual axis is not roughened. Only a narrow peripheral band of the recipient corneal stroma is

roughened inside the circle of the host DX. Such a surgical step, is thought to further augment the adherence of the peripheral area of the donor disk 360 degrees. The author has designed a special instrument called the John DSAEK Stromal Scrubber (ASICO Inc., Westmont, IL, AE-2878, Patent Pending) (Figures 27-5A and B) (See also Chapter 11, New/Useful Surgical Instruments in DSAEK) to facilitate this procedure. This instrument takes advantage of the special design of the John DSAEK Dexatome Spatula (ASICO Inc., Westmont, IL, AE-2872, Patent Pending) (See also Chapter 11, New/Useful Surgical Instruments in DSAEK) that permits the John DSAEK Stromal Scrubber to complete the circular scrubbing process with a single entry into the AC. The John DSAEK Stromal Scrubber has a hemispherelike tip which is roughened by sand-blasting technique, makes a narrow band of roughened area within the DX circle. The curvilinear design of this instrument allows for easy access to the inner corneal dome. The author used this surgical technique to further augment the adhesion of the donor disk in combination with a large air bubble (see below).

Use of Large Air Bubble The author consistently uses a large air bubble within the recipient AC (Figures 27-6 and 27-7) to enhance disk attachment. This is usually combined with a preoperative laser peripheral iridotomy (PI) procedure in the inferior aspect of the iris (Recommended by Elizabeth Davis, MD, personal communication). This is performed in an attempt to prevent postoperative pupillary block glaucoma attack. He combines such a procedure often with peripheral scrubbing of the recipient peripheral corneal stroma (see

Figures 27-5A and B: (A) Showing the profile of the John DSAEK Stromal Scrubber (ASICO Inc., Westmont, IL, AE2878, Patent Pending); (B) John DSAEK Scrubber being used to roughen the outer region of the exposed donor cornea within the circle of Descemetorhexis to increase the adhesion between the donor corneal disk and the patient corneal stroma. This instrument is used to create the peripheral roughening of the recipient cornea.

Techniques to Facilitate Disk Adherence to Recipient Cornea in DSAEK

Figure 27-6: The use of a large air bubble in the recipient anterior chamber is displayed in DSAEK surgery.

above). These techniques have significantly decreased the disk detachment rate to about 2%. One may consider an acceptable disk detachment rate to be about 5% or less. In all DSAEK procedures, the surgeons use an air bubble to attach the donor disk to the recipient cornea. The attachment of the donor disk may be compared to “slapping” a pizza on to the ceiling and holding it in place with two hands (See also Chapter 31, Comparison of Wound

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Architecture in DLEK versus DSAEK). The author is of the opinion that the diameter of the air bubble within the recipient AC should be greater than the diameter of the donor corneal disk to prevent disk detachment (Figures 27-6 and 27-7). If the air bubble has a smaller diameter than the disk diameter there is increased risk of disk detachment (Figure 27-8). The author usually leaves a large air bubble in the AC at the end of the procedure. The globe is palpated intraoperatively to assure that the intraocular pressure (IOP) is within the acceptable range. The IOP with the air bubble in the AC will also depend on the scleral rigidity, the configuration of the AC, etc. The potential downside to a large air bubble includes pupillary block glaucoma attack on the evening of surgery. The preoperative laser PI as described above should prevent pupillary block glaucoma. There is usually no such risk of a pupillary block glaucoma attack the next day, since the air bubble consistently is at or above the horizontal meridian bisecting the central pupil through the visual axis (Figure 27-9). Thus, the disk detachment rate is correlated to the relative size of the air bubble within the AC to the donor disk diameter (Figure 27-10). However, when a peripheral scrapping is performed, this surgical step helps in the disk attachment to the recipient cornea and the size of the air bubble may be decreased in such cases. There is increased risk of donor

Figure 27-7: A large air bubble is used to attach the donor corneal disk to the inner stromal surface of the recipient cornea. The air is injected in a gradual and steady manner to prevent donor disk dislocation or inversion of the disk (flipped disk).

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Figures 27-8A and B: (A) Surgical photograph showing an air bubble in the anterior chamber. The diameter of the air bubble is smaller than the diameter of the donor corneal disk and this increases the risk of donor corneal disk detachment. (B) Cartoon showing that when only one hand is used o hold a pizza against a ceiling, the unsupported side of the pizza comes off from the ceiling and when the hand is removed the entire pizza falls to the ground.

Figure 27-9: The day after surgery, showing partial absorption of air and the residual size of the air bubble in the patient’s anterior chamber. Notice that the air bubble does not fully cover the pupil and hence there is no risk of pupillary block glaucoma attack on day 1 following DSAEK surgery.

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large air bubble within the AC. Although air may be considered to have a deleterious effect on the host corneal endothelium, in the author’s experience there has not been any endothelial decompensation that has been attributed to the air in the AC. In some cases, where there may a slight increase in the intraoperative tactile IOP check, oral Diamox sequels may be given following the DSAEK surgery if the patient has no medical contraindications to its use and has no allergy to sulfa medications. It is essential to dilate the pupil intraoperatively in all cases of DSAEK. In conclusion, disk detachment is a true concern following DSAEK surgery. Hence, any surgical technique that possibly reduces the disk detachment rate should be entertained by the surgeon. Any of the above mentioned surgical techniques by itself or in combination, should help reduce the rate of disk detachment following DSAEK surgery.

References

Figure 27-10: Schematic representation of the relative size of the air bubble in the anterior chamber to the size of the donor corneal disk. Top – there is increased risk of donor corneal detachment when the air bubble diameter (ABD) is smaller than the donor corneal disk diameter (DKD). Middle – When ABD=DKD there is still increased risk of disk detachment. Bottom – When ABD>DKD there is decreased risk of disk detachment.

disk detachment when the air bubble diameter is equal to or smaller than the donor disk diameter (Figure 27-10), while, there is decreased risk of disk detachment when the air bubble diameter is greater than the donor disk diameter (Figure 27-10). A double-ring sign with equal spacing between the two rings suggests that the donor corneal disk is well centered on the recipient cornea (Figure 27-4). In high risk cases of potential acute glaucoma attack with air in the AC, such as those patients with a small corneal diameter, with hyperopia and convexity to the iris surface and narrow angles, the author performs two NdYAG laser peripheral iridotomies (PIs) preoperatively. The site of the laser PIs are located inferiorly as suggested by Dr. E. Davis (personal communication). The inferior peripheral iridotomy is preferred since the air bubble rises in the patient upright position, and this will not usually block the inferior iris openings made with the laser. In patients with pre-existing PIs, there usually is no risk of an acute pupillary block glaucoma attack associated with a

1. John T. Corneal disk detachment. Annals of Ophthalmol 2006; 38:169-84. 2. John T. Selective tissue corneal transplantation: A great step forward in global visual restoration. Expert Rev Ophthalmol 2006;1:5-7. 3. John T. Descemetorhexis with endokeratoplasty. In: Surgical Techniques in Anterior and Posterior Lamellar Corneal Surgery. John T (Ed.). Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India, 2006; 411-20. 4. John T. Descemetorhexis with endokeratoplasty (DXEK). In: Step by Step Anterior and Posterior Lamellar Keratoplasty. John T (Ed.). Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India, 2006;177-96. 5. Melles GR. Posterior lamellar keratoplasty: DLEK to DSEK to DMEK. Cornea 2006;25:879-81. 6. Price FW Jr, Price MO. Descemet’s stripping with endothelial keratoplasty in 200 eyes: Early challenges and techniques to enhance donor adherence. J Cataract Refract Surg 2006; 32:411-8. 7. Price MO, Price FW. Descemet’s stripping endothelial keratoplasty. Curr Opin Ophthalmol 2007;18:290-4. 8. Terry MA. Endothelial keratoplasty: History, current state, and future directions. Cornea 2006;25:873-8. 9. Kymionis GD, Suh LH, Dubovy SR, Yoo SH. Diagnosis of residual Descemet’s membrane after Descemet’s stripping endothelial keratoplasty with anterior segment optical coherence tomography. J Cataract Refract Surg 2007;33:1322-4. 10. Terry MA, Hoar KL, Wall J, Ousley P. Histology of dislocations in endothelial keratoplasty (DSEK and DLEK): A laboratorybased, surgical solution to dislocation in 100 consecutive DSEK cases. Cornea 2006;25:926-32. 11. Price MO, Price FW Jr. Descemet stripping with endothelial keratoplasty for treatment of iridocorneal endothelial syndrome. Cornea 2007; 26:493-7. 12. Mearza AA, Qureshi MA, Rostron CK. Experience and 12-month results of descemet-stripping endothelial keratoplasty (DSEK) with a small-incision technique. Cornea 2007; 26:279-83. 13. Price MO, Price FW Jr. Descemet stripping with endothelial keratoplasty for treatment of iridocorneal endothelial syndrome. Cornea 2007; 26:493-7. 14. Cheng YY, Pels E, Nuijts RM. Femtosecond-laser-assisted Descemet’s stripping endothelial keratoplasty. J Cataract Refract Surg 2007;33:152-5.

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Introduction Descemet membrane stripping automated endothelial keratoplasty (DSAEK)1-3 may be associated with complications that may be intraoperative or postoperative and may manifest early or late in the post-surgical period. This chapter will review some of these complications associated with DSAEK. Some of these complications can also occur with other types of sutureless corneal transplant, namely, deep lamellar endothelial keratoplasty (DLEK) and Descemet membrane endothelial keratoplasty (DMEK).

Complications Intraoperative Complications 1. 2. 3. 4. 5. 6. 7.

Blood in the anterior chamber Iris prolapse Fluid in the donor-recipient interface Macro-folds Flipped-disk Disk detachment during unfolding Dropped disk into vitreous cavity

Blood in the Anterior Chamber It is best to prevent this intraoperative complication by making sure that complete hemostasis is achieved before entering the anterior chamber through the temporal wound. If the anterior chamber is entered with the donor corneal disk while there is active bleeding or if there is blood on the ocular surface while entering the anterior chamber, this blood will easily enter into the anterior chamber and decrease visualization of the donor disk, and more importantly, it can get into the donor-recipient interface and cause postoperative decrease in vision and intracorneal inflammation and possible subsequent scarring. If the donor disk is within the anterior chamber and a small amount of blood is in the anterior chamber this blood should be aspirated using a sterile syringe and a blunt 30gauge or a 27-gauge cannula.

wound and make another entry wound further away from the iris surface and use this new site for completion of the surgical procedure. The further the entry wound is away from the iris surface, the less chance of iris prolapse. However, the wound should not go past the surface epithelial circular mark. Alternatively, the iris prolapse may be due to a positive pressure and this can be determined easily by gentle palpation of the globe. If the globe is hard to palpation, then there is a positive pressure within the eye that contributes to the iris prolapse. If that is the case, then first priority is to decrease the intraocular pressure (e.g. by using intravenous Mannitol). Preoperative intravenous Mannitol along with globe compression with a Honan balloon can often prevent intraoperative positive pressure. Alternatively, the intraocular pressure can be decreased by doing a limited anterior vitrectomy. If the positive pressure is excessive, it is also important to rule out a choroidal hemorrhage. If the intra-globe pressure continues to be excessive, it may be safer to close the wound and bring the patient back later in the day or even the next day to complete the surgical procedure.

Fluid in the Donor-Recipient Interface During DSAEK surgery, following the donor disk adherence to the recipient cornea, there may be fluid within the potential space between the donor and recipient corneas. Price has described the use of slit-incisions passing from the corneal surface to the interface and thus drain the interface fluid and promote adherence of the donor disk to the inner surface of the recipient cornea (Figure 28-1). These incisions are

Iris Prolapse If there is iris prolapse through the anterior chamber, temporal entry wound, it can be due to “premature” entry into the anterior chamber. A premature entry into the anterior chamber through a temporal wound will result in the wound being very close to the iris surface, and hence there can be an increased tendency for the iris to prolapse out of the anterior chamber during surgery. If the iris keeps prolapsing out during surgery, it may be best to close the

Figure 28-1: Selective drainage of donor-recipient interface fluid, namely a single slit incision in the temporal region. Fluid draining from the slit incision is seen (arrow).

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Figure 28-2: Four-quadrant slit-incisions to drain interface fluid during DSAEK surgery.

Figure 28-3: Good adherence of the donor corneal disk following interface fluid drainage in 4-quadrants. Also seen is the double-ring sign of good uniform donor disk adherence to the recipient cornea.

sometimes referred to as “vent” incisions. Price advocated the use of four-quadrant slit-incisions to drain interface fluid during DSAEK surgery (Figure 28-2). Good adherence of the donor corneal disk is usually seen following interface fluid drainage in 4-quadrants (Figure 28-3). Double-ring sign of good uniform donor disk adherence to the recipient cornea may also be seen intra-operatively (Figure 28-3). These slit-

incisions are almost always visible by slit-lamp examination, regardless of the postoperative duration after the initial surgical procedure. Occasionally, there has also been incidence of epithelial ingrowth through these “vent” incisions (Figures 28-4 and 28-5). I do not perform routine slit-incisions in all DSAEK procedures. Total air-filled anterior chamber during the 8 to 10 minutes of intraoperative waiting period often expels most of the interface fluid. Additionally, the corneal dome may be gently massaged with a Lindstrom roller (Figure 28-6) or a John Glider (Figure 287) (ASICO Inc., Westmont, IL) to further drain any interface fluid. Intraoperative surgical slit-lamp can also assist in evaluating the interface for any fluid collections (See also Chapter 10, Role of Surgical Slit-lamp in Endothelial Transplantation and Anterior Segment Surgery). If there is only an area of localized fluid collection, then a single slitincision overlying this area may be used to drain the fluid (Figure 28-8).

Macro-folds If there is any macro-folds in the donor cornea after airassisted donor corneal adherence to the recipient cornea, these folds may be diminished or eliminated using the John Glider (Figure 28-7). The amount of relief will depend on the number and severity of the macro-folds.

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Figure 28-4: Slit-lamp views, broad and narrow beam, of epithelial ingrowth through corneal vent incision extending from the corneal epithelial surface to the donor-recipient interface (Photos Courtesy: Dr. Arun K. Jain).

Figure 28-5: Left—Light photomicrographs of DSAEK cornea with epithelial ingrowth through a venting incision following DSAEK. Corneal specimen was studied following a successful, subsequent penetrating keratoplasty; Right—Specimen of epithelial ingrowth within the anterior chamber (Photos Courtesy: Dr. Cathy Newton).

Figure 28-6: Use of Lindstrom roller to remove any donor-recipient interface fluid.

Flipped-Disk Every effort should be made to prevent the donor corneal disk from flipping upside down, resulting in the endothelial

surface of the donor corneal disk facing the inner corneal surface of the recipient cornea. If not recognized, the disk may be attached by the wrong surface to the recipient cornea, namely, the donor endothelium at the donorrecipient interface. Flipped disk attachment to the recipient cornea will result in postoperative graft failure. This event when unrecognized at the time of surgery, can only be confirmed when a penetrating keratoplasty is performed and the tissue is available for histopathology showing the donor endothelial cells at the donor-recipient interface. Options to consider in preventing the wrong surface facing the inner recipient corneal surface include the following: • Promoting optimal unfolding of the “taco-folded” donor corneal disk • 60/40 or greater under- or over-fold • Steady controlled air injection to unfold the donor corneal disk • Correct immediately any tendency to unfold the donor corneal disk in the wrong direction

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Figure 28-7: Use of John DSAEK Glider to remove any donor-recipient interface fluid and macro-folds from the donor corneal disk following air attachment of the donor corneal disk to the recipient cornea.

• Staining the donor surface with trypan blue (Vision Blue) to identify the stromal surface from the donor endothelial surface (See also Chapter 32, Use of Dyes in DSAEK and DLEK) • Use of surgical slit-lamp (See also Chapter 10, Role of Surgical Slit-lamp in Endothelial Transplantation and Anterior Segment Surgery) • Introducing donor corneal disk with the endothelial side facing the anterior iris surface (e.g. Busin glide, sheetsglide, suture-pulling techniques, injecting device delivering donor disk with the endothelial side down). Do take into consideration the amount of endothelial cell loss during endothelium-down delivery of donor corneal disk into the recipient anterior chamber.

Disk Detachment during Unfolding Figure 28-8: Single slit-incision to drain localized interface fluid collection.

• Recognizing and correcting any impediments to unfolding of the donor corneal disk within the recipient anterior chamber (e.g. vitreous band through pupil to cornea, vitreous in the anterior chamber, very shallow, crowded anterior chamber, extensive anterior synechiae)

Re-attach the disk with additional air.

Dropped Disk into Vitreous Cavity Dropped disk in most instances can be prevented if proper preoperative assessment is made prior to surgery. Increased risk for dropping the donor disk into the vitreous cavity includes the following:

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• Aphakic eyes • Large complete iridectomy without any posterior support such as a PC IOL blocking most of the iris opening • Aniridia. If the donor corneal disk is accidentally dropped into the vitreous cavity, avoid deep vitreous cavity “fishing.” Trying to grab a dropped disk can cause additional ocular tissue damage and possible permanent visual loss. Close the eye and refer the patient to a vitreoretinal surgeon to surgically remove the dropped donor corneal disk.

Postoperative Complications 1. 2. 3. 4. 5. 6. 7.

Disk detachment Macro-folds Interface blood Epithelial ingrowth Graft rejection Failed graft Infection.

Figure 28-9: Temporal approach—30-gauge cannula is inserted through a stab incision and the tip of the cannula is placed between the detached donor corneal disk and the anterior iris surface.

Disk Detachment Disk detachment in the immediate postoperative period may occur following DSAEK surgery. This is usually detected on the day following surgery. However, late disk detachment can also occur. Disk detachment may be a result of the patient rubbing the eye following DSAEK, or the surgical techniques used during surgery may not have been sufficient for proper donor disk adherence to the inner surface of the recipient cornea. When the donor disk is detached it usually rests inferiorly with the edge of the disk being captured within the inferior aspect of the anterior chamber angle. Disk reattachment is a relatively simple surgical procedure (Figures 28-9 to 28-12) and may be done on the next day or within about 7 to 10 days following the initial surgery. Any delay past 10 days may result in anterior synechiae formation and risk of disk damage and difficulty with the removal and re-attachment surgery. The re-attachment (Figures 28-9 to 28-12) is done under topical anesthesia, namely, 2% lidocaine jelly and monitored anesthesia care (MAC). A 30-gauge cannula is placed in the region between the detached disk and the anterior iris surface and filtered-air is injected to raise and attach the donor disk to the recipient corneal stroma. The disk is centered using a reverse Sinskey hook. Selective slit incision may be made to drain any localized fluid collection. The epithelium may be removed to enhance the view of the donor disk, the interface and the anterior chamber.

Figure 28-10: Air bubble in the anterior chamber raises the detached disk and brings it into contact with the recipient inner corneal stroma.

Figure 28-11: The donor corneal disk is moved to proper centration using a reverse Sinskey hook. The epithelium is removed to increase the visualization of the donor corneal disk and the donor-recipient interface.

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Figure 28-12: A single slit-incision is made to drain an area of localized interface fluid.

Macro-folds Macro-folds may rarely be seen following DSAEK surgery and may occasionally persist (Figures 28-13 to 28-15). If postoperative follow-up shows continued wound remodeling and decrease in the degree of the donor disk folds then one can continue to watch without any surgical intervention. If the patient is very symptomatic from these folds and if there is no improvement over time, then a disk exchange may be considered.

Interface Blood Interface blood is to be avoided if possible. Meticulous hemostasis before entering the anterior chamber via the temporal wound is essential. If there is surface blood this can enter into the eye and get trapped in the donor-recipient interface. Interface blood can last for a long time and may contribute to visual degradation, visual symptoms, and interface inflammation.

Epithelial Ingrowth This is a rare complication of the “vent” incisions that is made to drain any interface fluid and enhance donor corneal adherence to the recipient cornea. Management is similar

to any epithelial ingrowth within the cornea. Watch for progression of the epithelial island, any overlying corneal melt. Surgical intervention will be necessary if the above mentioned complications occur.

Graft Rejection Since DSAEK involves endothelial transplantation from a donor cornea, endothelial graft rejection can occur (Figure 28-16). The treatment of graft rejection is similar to endothelial graft rejection following a penetrating keratoplasty, namely, frequent topical corticosteroid drops and cycloplegia.

Failed Graft Graft rejection not resolved with medical treatment can result in a failed graft following DSAEK, DMEK, and DLEK procedures. Figure 28-17 displays the ultra-structural findings in a case of failed DLEK graft. In DSAEK, a disk exchange may be the preferred choice over a full-thickness penetrating keratoplasty. Figures 28-18 to 28-27 display the surgical steps in disk exchange following a failed DSAEK graft, after a failed penetrating keratoplasty (PKP).

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Figure 28-13: Bilateral DSAEK procedures, with postoperative macro-folds in the right eye.

Figure 28-14: Confocal microscopy showing donor disk folds.

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Figure 28-15: Corneal OCT showing donor disk folds.

Figure 28-16: Top Row—Slit-photographs showing endothelial graft rejection following DSAEK; Bottom—Confocal microscopic image showing keratic precipitates adherent to the endothelium, following DSAEK procedure.

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Figure 28-17: Transmission and scanning electron microscopy showing lymphocytes adherent to donor endothelium in a failed DLEK graft.

Figure 28-18

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Figure 28-27 Figures 28-18 to 28-27: Surgical steps in disk exchange following a failed DSAEK graft after a failed penetrating keratoplasty (PKP).

Infection John T first described a new clinical entity namely, “sandwich keratitis” following DLEK in 2003.4 In selective tissue corneal transplantation1-3 only the diseased portion of the patient’s cornea is surgically removed and replaced with a similar, healthy donor corneal tissue, and in so doing, it results in an interface between the donor and the recipient corneal surfaces. Any infectious agent(s) inadvertently introduced into this potential space at the interface during surgery gets “trapped” between the two tissue layers and be localized in that region. Since the infection occurs between two layers of the cornea much like a sandwich, the term “sandwich keratitis was given to this clinical entity (Figures 28-28 and 28-29).4 In sandwich keratitis, the patient may not be symptomatic in the early stages and the eye may not be significantly red, except for the redness usually noted after surgery. This often can result in a delay in diagnosis and management. These findings both symptoms and signs are quite different from the usual corneal ulcer that begins from the corneal surface and progresses into the corneal stroma. In the corneal ulcer that starts from the corneal surface, the

Figure 28-28: Sandwich keratitis 6 weeks after a DLEK procedure. The infectious organisms are located in the donor-recipient interface.

patient presents early while in sandwich keratitis the patient may present much later. Unlike a corneal ulcer where the topical antibiotics will achieve high concentration in the region of the ulcer, in a sandwich keratitis the antibiotics have to reach the interface

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Corneal Endothelial Transplant not to open the sandwich during a therapeutic keratoplasty, since the organisms from the interface can spill into the anterior chamber, converting a sandwich keratitis to a potential endophthalmitis and even loss of the eye. This new clinical entity, namely sandwich keratitis should be recognized by clinicians and treated appropriately, as there is a continued trend in the direction away from a full-thickness penetrating keratoplasty to a selective tissue corneal transplant technique such as DSAEK, DLEK, DMEK, and all types of ALKs, all of which can result in a donor-recipient interface infection called, sandwich keratitis.

References Figure 28-29: Transmission electron micrograph of the donor-recipient interface in a case of sandwich keratitis following DLEK, showing Candida (Torulopsis) glabrata (arrow) surrounded by Staphylococcus sp.

and hence the concentration will be lower than that will be achieved when treating a surface corneal ulcer. In a surface corneal ulcer that fails medical treatment and goes onto a therapeutic keratoplasty, one of the main concerns is usually infection free margins. In a sandwich keratitis the same applies, but in addition it is important

1. John T. Selective tissue corneal transplantation: a great step forward in global visual restoration. Expert Rev Ophthalmol 2006;1:5-7. 2. John T. Surgical Techniques in Anterior and Posterior Lamellar Keratoplasty. New Delhi, India: Jaypee Brothers Medical Publishers; 2006:1-687. 3. John T. Step by Step Anterior and Posterior Lamellar Keratoplasty. New Delhi, India: Jaypee Brothers Medical Publishers; 2006:1-297. 4. John T, Delany C, John ME. Sandwich keratitis: New clinical entity following deep lamellar endothelial keratoplasty. Presented at the Annual Meeting of the American Society of Cataract and Refractive Surgery, San Francisco, CA, April 1216, 2003.

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Terminology Descemetorhexis with endokeratoplasty (DXEK) is synonymous with Descemet’s stripping with endothelial keratoplasty (DSEK), and Descemet’s stripping automated endothelial keratoplasty (DSAEK) (See also Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty). The term DSAEK will be used in this chapter, since it is a more commonly used term.

Unanswered Questions in DSAEK In DSAEK, first a centrally placed circular area of the inner corneal surface of the patient’s cornea is operated upon to expose the recipient corneal stroma.1-9 Unlike deep lamellar endothelial keratoplasty (DLEK)10-16 [See also Section 8, Deep Lamellar Endothelial Keratoplasty (DLEK)], there is no posterior corneal recess in DSAEK. The recipient cornea is intact except for the absence of a circular area of Descemet’s membrane (DM) with its attached endothelium. Next, the donor corneal disk is attached to this area of exposed corneal stroma in the inner surface of the patient’s cornea (See also Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty). The surgical approach of adding a disk of donor circular tissue to the surface of another tissue/organ namely, the patient’s cornea, is a very rare approach, if not the only one of its kind in the whole field of transplant surgery. Additionally, the attachment is done without any sutures or tissue adhesives of any kind. In any segmental tissue transplantation, the edges of the transplanted tissue are usually approximated to the edges of the host tissues to which the transplantation is performed. Such tissue-totissue contact results in contact inhibition and prevents any tissue expansion. Since there is no edge-to-edge approximation in DSAEK surgery, this may be considered as a “step-up-stage” type of tissue addition procedure. In this case, the “stage” is the circular disk of tissue and the “ground level’ would be the patient’s cornea. The edge of this “stage” is the cut-edge of the donor corneal stroma, DM, and endothelium. Thus, the cut-edge of the donor corneal disk is fully exposed and bathed in the aqueous humor within the anterior chamber. Since, the cut-edge of the donor corneal disk is exposed; does this translate to expansion of these tissues onto the inner surface of the patient’s cornea? Does this mean that the stromal keratocytes will extend onto the surrounding patient’s inner cornea? Although the endothelial cells for the most part do not multiply, will the donor endothelial cells slide outward and extend onto and over the patient’s endothelium? If so, will the expanded endothelial cell layer

lay down new DM? Will such potential cellular expansion, if it occurs over time, extend onto the anterior chamber angle, iris surface, and progress further, resulting in peripheral anterior synechiae, secondary angle closure glaucoma, and pupillary abnormalities? The answers to these questions are currently not available since the DSAEK procedure has a short time track from its inception to the present time (at the time of writing this chapter). In the future, as some of the DSAEK corneas undergo graft rejection and graft failure, and go on to possible full-thickness penetrating keratoplasty (PKP), these corneas will provide the answers to some of the above questions, as these tissues are then studied in the ophthalmic pathology laboratories. The interface relates to the area of contact between the donor and the recipient corneal stroma. This interface moves from the mid-stromal region of the recipient cornea in DLEK to a more posterior region of the recipient cornea namely, the posterior corneal stroma close to the level of the excised DM of the recipient cornea in DSAEK. This posterior shift in the interface is believed to have a positive effect on the overall quality of vision following DSAEK. This interface is more noticeable in the immediate postoperative period following DSAEK and it becomes less prominent, to almost non-visible in some cases, over time. The question arises as to what extent does the donor-host tissue integration take place? We do know that initially this tissue integration is not pronounced and the donor disk can be removed with relative ease. However, it becomes more difficult to remove a disk that has been in place for several months following DSAEK. Does this imply that there is extensive tissue remodeling and integration over time? These questions will be answered as we study these tissues when they become available over time. The author’s experience has shown keratocyte activation, wound healing, and tissue integration at the level of the donorrecipient interface.17 In DSAEK, the edge of the donor corneal disk overlaps the cut-edge of the host endothelium and DM. Since the cut-edge of the host endothelium is “free” without any cellular contact inhibition, will these endothelial cells expand into the donor-host interface to interfere with the interface clarity? The answer to this question is possibly a “no.” The reason being, there is direct contact between the host corneal stroma and the donor corneal stroma, and continued adhesion and remodeling that takes place between these tissues will allow for no free space for the host endothelium to slide into. Also, the host endothelium is usually not healthy and usually comprises of decompensated cells at the time of DSAEK surgery. DSAEK surgery is an additive procedure. Donor corneal disk is added on to the host cornea, resulting in the final

Unanswered Questions in DSAEK thickness of the host cornea always being greater than the normal human corneal thickness. If a donor corneal disk of 150 µm thickness is added to a host central corneal thickness of about 520 µm, this will result in an increase in the host corneal thickness by about 29%. In the above example, the host corneal thickness to begin with may be greater than 600 µm, which then gradually decreases in thickness as the healthy donor corneal endothelium clears the host cloudy cornea. The healthy donor corneal endothelium has to now function to clear a cornea with an increased thickness of about 29% or even greater depending on the donor corneal disk thickness and the final host corneal thickness. The question then arises what is the upper limit of total corneal thickness that a monolayer of healthy endothelium can keep clear? Is there an upper limit of total corneal thickness beyond which the endothelium will fail to function? Currently, these questions remain unanswered. To date, clinically the increased corneal thickness does not appear to have any known deleterious effects on the patient’s cornea or on the quality of vision.

References 1. John T. Corneal disk detachment. Annals of Ophthalmol 2006; 38:169-84. 2. John T. Selective tissue corneal transplantation: A great step forward in global visual restoration. Expert Rev Ophthalmol 2006;1:5-7. 3. John T. Descemetorhexis with endokeratoplasty. In: John T (Ed): Surgical Techniques in Anterior and Posterior Lamellar Corneal Surgery. Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India, 2006;411-20. 4. John T. Descemetorhexis with endokeratoplasty (DXEK). In:

5. 6.

7.

8. 9. 10. 11. 12. 13.

14. 15. 16. 17.

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John T (Ed): Step by Step Anterior and Posterior Lamellar Keratoplasty. Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India, 2006;177-96. Melles GR. Posterior lamellar keratoplasty: DLEK to DSEK to DMEK. Cornea 2006;25:879-81. Price FW Jr, Price MO. Descemet’s stripping with endothelial keratoplasty in 200 eyes: Early challenges and techniques to enhance donor adherence. J Cataract Refract Surg 2006;32: 411-8. Mearza AA, Quershi MA, Rostron CK. Experience and 12month results of descemet-stripping endothelial keratoplasty (DSEK) with a small-incision technique. Cornea 2007;26: 279-83. Price MO, Price FW. Descemet’s stripping endothelial keratoplasty. Curr Opin Ophthalmol 2007;18:290-4. Terry MA, Ousley PJ. Corneal endothelial transplantation: Advances in the surgical management of endothelial dysfunction. Contemporary Ophthalmology 2002;1(26):1-8. Melles GR, Eggink FA, Lander F, Pels E, Rietveld FJ, Beekhuis WH, Binder PS. A surgical technique for posterior lamellar keratoplasty. Cornea 1998;17:618-26. Terry MA, Ousley PJ. Endothelial replacement without surface corneal incisions or sutures: Topography of the deep lamellar endothelial keratoplasty procedure. Cornea 2001;20:14-8. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty in the first United States patients: Early clinical results. Cornea 2001;20:239-43. Terry MA, Ousley PJ. Replacing the endothelium without corneal surface incisions or sutures: The first United States clinical series using the deep lamellar endothelial keratoplasty procedure. Ophthalmology 2003;110:755-64. Terry MA, Ousley PJ. In pursuit of emmetropia: Spherical equivalent refraction results with deep lamellar endothelial keratoplasty (DLEK). Cornea 2003;22:619-26. Marcon AS, Terry MA, Kara-José N, Wall J, Ousley PJ, Hoar K. Influence of final corneal thickness in visual acuity after deep lamellar endothelial keratoplasty. Cornea 2007;26:543-5. Yepes N, Segev F, Hyams M, McAllum P, Slomovic AR, Rootman DS. Five-millimeter-incision deep lamellar endothelial keratoplasty: One-year results. Cornea 2007;26:530-3. John T, Fraenkel GG, John ME. Keratocyte densities and interface changes in failed deep lamellar endothelial keratoplasty. Presented at the ARVO Annual Meeting, 2004.

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Use of Eye Bank Pre-cut Donor Tissue in DSAEK

Anthony Kuo Terry Kim

Use of Eye Bank Pre-cut Donor Tissue in DSAEK

30

332

Corneal Endothelial Transplant

Introduction Over the last 250 years, corneal transplant surgery has progressed from a mere idea to a successfully practiced means of restoring corneal clarity and integrity for a multitude of patients. Similarly, within the last decade, posterior lamellar keratoplasty has been refined to provide a viable alternative to full-thickness transplantation for those with endothelial disease.1-4 As patients with endothelial disease (Fuchs’ dystrophy, aphakic/pseudophakic bullous keratopathy, and graft failure) comprise up to 40% of the recent full-thickness transplant population, 5 Descemet’s stripping with endothelial keratoplasty (DSEK) represents a less invasive surgical advance that offers faster recovery than penetrating keratoplasty (PKP) for these patients. Dissection of the donor corneoscleral tissue for DASEK is described elsewhere in Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK) and in the literature.1-4 Recently, pre-cut allograft tissue from eye banks has become available for DSEK and can eliminate tedious and time-consuming intraoperative dissection of donor tissue for the DSAEK surgeon. In this chapter, we will discuss the third party’s preparation of the cornea for DSEK, our surgical technique for handling pre-cut DSEK tissue, and finally, our outcomes with pre-cut DSEK tissue.

Third Party Preparation of Corneal Tissue for DSAEK Pre-cut DSEK tissue can currently be obtained from select eye banks. Our center utilizes pre-cut DSEK tissue from both our local eye bank – The North Carolina Eye Bank (NCEB), as well as a private company, Ocular Systems Inc. (OSI; Winston-Salem, NC). While this chapter discusses the practices of NCEB and OSI due to our familiarity with them, there are other third party preparers of DSEK tissue nationwide. Regardless of the source of pre-cut DSEK tissue, the surgeon should personally ensure that the institution he or she chooses to provide pre-cut DSEK tissue meets federal regulations as well as the surgeon’s standards. When a request for tissue is received, there are two models for the acquisition of tissue. For an eye bank, this may simply mean redirecting appropriate corneal tissue on hand for DSEK cutting, while a private company would obtain corneal tissue from a partnering eye bank. Once the tissue is acquired, both models will subject the tissue to administrative and medical verification for appropriateness of use. The donor tissue is screened for concerning medical history, positive or reactive infectious testing, and poor endothelial cell counts or tissue quality. An eye bank may perform the testing and

specular microscopy in house, while a private company will rely on the information supplied by their partnering eye bank. In addition, a private company will also examine details specific to the transit of the tissue from their partner, such as shipping information, intact packaging and carrier, and storage/refrigeration conditions. For both models, the carrier media will be inspected for color and clarity, and the donor tissue itself examined under high magnification for defects or unusual endothelial morphology. This is akin to the inspection process any ophthalmologist would undertake prior to using any donor tissue. Once the tissue passes inspection, it is ready to be prepared. The tissue carrier is brought into the clean room, and the tissue is prepared under sterile technique with sterile instrumentation. NCEB uses a NSF Type II laminar flow biosafety hood, while OSI utilizes a dedicated clean room with HEPA filtered, laminar airflow that is graded Class 100 (no more than 100 particles greater than 0.50 μm per cubic square foot of the work area). To create the DSEK graft, both NCEB and OSI use of an automated lamellar therapeutic keratoplasty (ALTK) system (Moria SA, Antony, France) (See also Chapter 12, Artificial Anterior Chambers). The components and detailed use of this system are described in Chapter 12, Artificial Anterior Chambers. The corneoscleral donor of at least 16 mm is centered, mounted on the Artificial Chamber, and brought to appropriate pressure (Figure 30-1). An ultrasound pachymetry is then used to measure

Figure 30-1: NCEB tissue markings for pre-cut DSEK tissue. This diagram represents the donor tissue, epithelial side up. The cornea is light blue, while the sclera is cream colored. The inner-most circle represents the anterior cap cut by the ALTK system. NCEB places an alignment mark at the 12 o’clock position crossing both the anterior cap and the uncut tissue. In addition to a centering dot, NCEB places 3 additional marks just outside of the anterior cap to give a visual approximation of the bed diameter (anterior cap). NCEB places all of its marks on the epithelium only.

Use of Eye Bank Pre-cut Donor Tissue in DSAEK the full-thickness of the graft. A microkeratome head is selected and then used to cut the graft. OSI typically uses a 300 μm microkeratome head, reserving the 350 μm head for tissue thicker than 575 μm. NCEB uses the same heads but will use a 250 μm head for tissue thinner than 500 μm. The resulting bed diameter (anterior cap) is then measured. This is typically intended to be 9.0 mm to 10.0 mm. At this point, the tissue is marked by the third party preparer with Gentian Violet [Editorial Note: Use of marking pen on the corneal stroma has been associated with endothelial cell loss (Terry M, personal communication)]. Each third party preparer may have its own unique method of marking the tissue and documentation forms (Figures 30-1 to 30-4), and the surgeon will need to familiarize himself or herself to the markings of their tissue provider. For instance, NCEB indicates the anterior cap with the dots shown in figure 1. On the other hand, OSI uses arcs to indicate the margins of the anterior cap and to clearly delineate its extent (diagram on lower half of Figure 30-3). Both preparers also create an anterior cap alignment mark. The anterior cap is now temporarily removed. With the anterior cap removed, the stromal side of the endothelial graft is exposed. A pachymetry is taken on the exposed stroma to provide the endothelial graft thickness. This is generally between 100 to 150 μm depending on the original pachymetry of the donor tissue. OSI places additional graft polarity marks at this point: both a grossly visualized centering mark and an orientation mark (the letter “S”) are placed [Editorial Note: Stromal markings appears to be associated with endothelial cell loss (Terry M, personal communication) and hence, it may be advisable not to perform such markings]. The stromal surfaces are dried, and the anterior cap is replaced and repositioned based on the earlier alignment mark. The complete corneoscleral donor is then removed from the ALTK system and replaced into a new transport container with fresh Optisol-GS. The transport container is sealed and finally brought out of the sterile clean room, ready to be packaged for delivery to the surgeon. Most third party preparers will attempt to acquire tissue that can be implanted within five days postmortem and within 24 hours of DSEK preparation. The pre-cut DSEK tissue from OSI comes packaged with three sheets of documentation. The first (Figure 30-2) is a cover sheet that reiterates that the pre-cut tissue be used only for endothelial keratoplasty and also contains the OSI tissue ID number, full-thickness and endothelial graft pachymetry, and legal information. Another sheet is labeled “Recipient Information Form” and is to be completed by the surgeon to be returned to OSI for patient tracking and quality control purposes. The sheet titled “Donor Summary

333

and Allograft Preparation Form” is the most important one for the operating surgeon (Figure 30-3). This sheet contains the donor information, the endothelial cell count, the pachymetry, the bed diameter, and the OSI processing times. It should be noted that the listed endothelial cell count is that provided by the original eye bank. OSI does not perform another endothelial cell count after preparing the tissue. NCEB provides similar information in their documentation titled “EK Processing and Tissue Evaluation Form” as seen in Figure 30-4. NCEB does perform its own endothelial cell count and tissue examination post ALTK dissection, and this is reported on the form. NCEB also includes copies of the original paperwork from the tissue source. Practical use of the information provided in the tissue documentation is described below under Surgical Technique. If you use a different pre-cut DSEK tissue provider, the documentation may differ, but it is important to locate the donor information, the endothelial cell count, the pachymetry, and especially the bed diameter within the documentation. As described, the preparation of tissue for DSEK by either an eye bank or private tissue processor requires only a brief detour in the tissue’s transit from the donor to the surgery center. During this detour, the tissue preparer assumes the task of cutting the endothelial graft, thus making it ready for DSEK before it arrives in the surgeon’s hands in the operating suite.

Surgical Technique for Pre-Cut Corneal Tissue for DSEK The use of pre-cut corneal tissue for DSEK does not change the overall techniques of the procedure. The surgeon must only be aware of how the pre-cut tissue has been cut and marked to help in centering the trephination and to help in confirming stromal and endothelial side of the disk. We use the tissue markings of OSI as an example in this section. There may be differences among the third-party preparers with regard to tissue markings.

Examination of the Tissue As in all corneal transplant surgeries, the surgeon should examine the donor tissue prior to its use to confirm that the tissue is suitable for transplantation. This includes visual examination of the donor cornea, preferably under magnification, for signs of disease (opacities, vascularization, etc.). The included documentation provides information regarding the donor’s demographics, endothelial cell

334

Corneal Endothelial Transplant

Figure 30-2: Cover sheet of documentation from OSI. The tissue identification number should be confirmed with the tissue transport container. The donor pachymetry and the thickness of the prepared endothelial graft are recorded at the top of this sheet (Courtesy of Ocular Systems, Inc.).

Use of Eye Bank Pre-cut Donor Tissue in DSAEK

335

Figure 30-3: Donor summary and Allograft Preparation Form from OSI documentation. The usual demographic information including endothelial cell count is in the top section. The lower section titled “OSI Preparation Information” includes important information. In addition to the pachymetry, the bed diameter is shown. This diameter is the maximum size endothelial graft this pre-cut tissue can support. It is visually indicated by the three arcs labeled “Bed Diameter Marks (3)” on the lower right diagram. The trephine punch should not exceed the bed diameter and should be centered within the arcs. Note the letter “S” which will help with graft orientation in the anterior chamber (Courtesy of Ocular Systems, Inc.).

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Corneal Endothelial Transplant

Figure 30-4: Documentation from NCEB. The tissue number should be confirmed with the tissue transport container. Basic donor, tissue, and dissection information as well as a specular microscope image are contained on this sheet. Note the Graft Bed Size in the right column. This is the absolute maximum size endothelial graft diameter that can be punched from this tissue (Courtesy of the North Carolina Eye Bank).

Use of Eye Bank Pre-cut Donor Tissue in DSAEK

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Cutting the Pre-cut Tissue

Figure 30-5: Pre-cut donor tissue with markings in trephine punch stage. In this instance, a Weck system is used as its Teflon stage is white, allowing the markings to be clearly visualized. The three bed diameter marks are seen close to the limbus indicating the predissected area. The trephination should be carried out within the arcs. A backwards “S” can also be seen inferiorly, indicating that the endothelial surface is facing the viewer. The centering mark can be seen overlying the center of the stage. Compare this to the diagram in Figure 30-3.

counts, and characteristics. It is also important to note the bed diameter as this is the maximum diameter of the prepared DSEK graft. The surgeon will use all of this information to confirm the health of the tissue and to determine whether the donor tissue is of appropriate size for the desired graft (Figures 30-3 and 30-4). Removing the donor corneoscleral tissue from the transport container should reveal the markings in Figure 30-5. In the paracentral area, there are three arcs which indicate the extent of the anterior cap generated by the ALTK system (Moria S.A., Antony, France) (See also Chapter 12, Artificial Anterior Chambers). These arcs can be used to help guide centration during donor punching. There is also a centering dot for the same purpose. The surgeon should however, visually confirm that the dot is centered in the donor cornea. Finally, there is an “S” mark which helps with confirming the stromal side of the donor corneal disk (“S” is seen when viewed with the stromal side proximal to the viewer; “Z” (Editorial Note: “reversed –S”) is seen when viewed with the endothelial side proximal to the viewer). [Editorial Note: In this method Gentian Violet is introduced into the anterior chamber (See also Chapter 32, Use of Dyes in DSAEK and DLEK). Recent evidence suggests that such use of markings with a marking pen can result in endothelial cell loss].

When determining the graft size to transplant, the surgeon must not only take into account the host dimensions, but he must also take into account the bed diameter of the precut tissue. This is listed in the documentation and is indicated visually on the tissue by the three arcs. The donor punching should occur within these arcs, as they describe the maximum extent of the pre-cut area. Cutting the tissue outside of the bed diameter and arcs will result in a fullthickness rim in addition to the endothelial graft once the free anterior cap is removed (Figure 30-6). We recommend selecting a trephine punch diameter at least half a millimeter less than the bed diameter to prevent this from occurring (i.e. with a donor bed diameter of 9.0 mm, we recommend using a 8.5 mm or smaller trephine) (Editorial Note: An 8.5 mm trephine on a donor bed of 9.0 mm allows for a surgeon error of 0.25 mm). The pre-cut tissue may be cut with the surgeon’s preference of trephine punch system. We recommend the

Figure 30-6: Choosing a punch diameter smaller than the bed diameter. (A) Indicates the bed diameter, which is the extent of the anterior cap. (B) Shows a well-centered punch using a punch diameter smaller than the bed diameter. This yields the desired partial-thickness endothelial graft after the anterior cap is removed. (C) Shows a punch diameter larger than the bed diameter. This yields undesired full-thickness edges once the anterior cap is removed. This may also occur if the punch is decentered and cuts outside of the anterior cap. We recommend using a trephine punch size at least 0.5 mm less than the bed diameter.

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Corneal Endothelial Transplant

Figures 30-7A to C: Punching the donor button. (A) Shows the importance of confirming a full-thickness punch by lifting the rim to ensure that the button has been cut free. (B) The center image displays the cut button with the donor rim removed. (C) Illustrates the examination of the donor rim to ensure that the punch is well-centered within the three bed diameter marks.

use of a trephine system that contains a white or lightcolored Teflon block for holding the donor cornea (e.g. Troutman trephine system) so that the centration mark and peripheral arc marks are more readily visible. The Troutman system’s block has a central hole that can be used to align the centration mark of the donor tissue. External operating room lighting directed on the donor corneal tissue can also aid in visualization of these marks. Regardless of the trephination system, care must be taken to handle the tissue without contacting and damaging the endothelial cells as in any corneal transplant surgery. We use a separate sterile table to perform the donor punching. The tissue is placed on the punch block, endothelial side up, and then centration visually confirmed. The tissue can then be cut with the individual system’s trephine punch per the manufacturer’s instructions. This will yield the donor rim and the donor button (Figures 30-7A to C). The donor rim should be examined to ensure that the punch was centered within the three arcs. If the trephination occurred outside of the three arcs, then the excess full-thickness rim tissue must be carefully trimmed with Vannas scissors from the donor button before it can be used as a partial-thickness graft. Otherwise, the excess tissue can potentially interfere with full apposition of the donor graft against the posterior stroma of the host. The donor rim can then be sent for the usual testing similar to a PKP. The endothelium of the donor

button should then be protected with either Optisol-GS or a small amount of cohesive viscoelastic (i.e. Healon) and kept covered until transplantation into the recipient cornea.

Transplanting the Tissue At this point, the donor button is the same as if the surgeon had cut the tissue himself or herself (see Table 30-1). It consists of an anterior stromal layer (cap) and the posterior endothelial graft. The only addition is the inked letter “S” on the stromal side of the endothelial graft. Once Descemet’s membrane has been stripped and the host corneal bed prepared, the donor button is brought into the surgical field. The endothelial graft is carefully separated from the anterior stromal cap and then delivered into the anterior chamber by the surgeon’s preferred method (Figures 30-8A to D). Once these graft is unfolded and tamponaded against the host stroma by either balanced salt solution or air, the surgeon must verify the stromal side from the endothelial side of the graft. If the letter “S” of the graft is seen through the cornea, then the stromal side is up, and the endothelial side is appropriately oriented (Figure 30-9). If the reverse is seen (“Z”) (Editorial Note: “reversed-S”), then the endothelial side is incorrectly contacting the host stroma, and the graft must be carefully reoriented (Editorial Note: If there is excessive

TABLE 30-1: Differences between pre-cut and surgeon-cut donor corneal tissue Pre-Cut

Number

Description

1.

Donor tissue cut by

Technician

Surgeon

2.

Upto 24 hours delay from tissue cutting to transplantation

Yes

No

3.

Surgeon in control of all parameters relating to cutting of donor tissue

No

Yes

4.

Additional cost

Yes

No

5.

Require ALTK system (Moria Inc.)

No

Yes

Unknown*

Unknown*

6. Superior surgical outcome *at the time of composing this table

Surgeon-Cut

Use of Eye Bank Pre-cut Donor Tissue in DSAEK

339

Figures 30-8A to D: Cut donor button and insertion. (A) The pre-cut tissue is being prepared for folding. Note the ideal position of the centration mark and backwards “S” mark on the donor tissue. (B) Shows the separation (and folding) of the pre-cut posterior lamellar tissue from the underlying anterior cap. (C) Shows the tri-folding technique of the lamellar graft onto a pair of angled McPherson forceps. (D) Shows insertion of the tri-folded graft through a clear corneal incision.

endothelial damage due to improper disk orientation and tissue handling, a disk exchange may become necessary). Once the disk is properly oriented, centered, and tamponaded, then the case is concluded in the surgeon’s usual fashion.

Discussion and Outcomes

Figure 30-9: Successful placement of pre-cut endothelial graft into host. Here the letter “S” is seen, which helps confirm the correct orientation of the posterior lamellar graft (stromal side up and endothelial side down). If a backwards “S” or “Z” is seen, then the graft is incorrectly positioned upside down and must be inverted into the correct orientation.

As with any technique, there are benefits and drawbacks of using pre-cut tissue for DSEK. The surgeon has advance information regarding the specific characteristics of the precut tissue that can help anticipate various issues during surgery (i.e. handling of a very thin or thick posterior graft, and the limits of the DSEK graft diameter). Secondly, the tissue-processor assumes the responsibility of cutting the tissue for DSEK. If a surgeon buttonholes a graft or generally performs a poor lamellar dissection intraoperatively, this will impede, if not potentially end, the progress of the case. A trusted tissue-processor, though, would be responsible for delivering only suitably cut tissue for DSEK

340

Corneal Endothelial Transplant

transplantation [Editorial Note: This may be considered as a partial modification from “surgery by surgeons” (see also Table 30-1)]. Thirdly, a reliable DSEK tissue-processor will have extensive experience cutting grafts specifically for DSEK. As an example, OSI has prepared over 500 corneas in a year for their client surgeons. Freeing the surgeon from performing the lamellar dissection also reduces case time in the operating room, decreasing the patient’s time under anesthesia and the costs associated with increased operating room time (Editorial Note: There is an added cost for pre-cut donor corneal tissue see Table 30-1). The costs of acquiring and maintaining an ALTK or similar dissection system are also assumed by the tissue-processor and not the surgeon – in the pre-cut tissue model. The ability to order the desired number of precut DSEK grafts also allows the surgeon to successively schedule the appropriate number of DSEK cases on the same operative day without having to wait on the sterilization and cooling of a microkeratome system which can delay the operating room schedule. However, using pre-cut DSEK tissue has its disadvantages as well. The tissue-processor or eye bank will typically charge a fee for performing the lamellar dissection. This fee currently ranges from $ 500 to $ 1000 (US dollars) per graft depending on the supplier. Aside from financial considerations, not every surgeon may feel comfortable having a third party perform the donor tissue dissection beforehand. Another question is whether using pre-cut tissue instead of intraoperatively-cut tissue affects the outcome for the patient. As DSEK is a recently popularized procedure and the availability of pre-cut tissue is even more recent, few studies regarding the use of pre-cut DSEK tissue have been published. Carlson and colleagues examined the endothelial cell density after the preparation of ten research corneas for DSEK. There was a 2.5% and 11.3% decrease in endothelial cell density at 48 and 72 hours post-section. 6 As a comparison, whole human corneas stored in Optisol-GS have been shown to have an average decrease in endothelial viability of 9.5% after 96 hours.7 Functionally, the presumed result of this endothelial cell loss would be an increase in graft thickness. Griffin has shown that in 12 pre-cut DSEK tissues, the increase in thickness of the endothelial graft from the time of third-party microkeratome dissection to surgeon’s insertion the following day averaged only 7 μm (5%).8 The clinical implications of these results are still not known. At our institution, we have used only pre-cut tissue since we began performing DSEK. A review of our first 50 cases shows a graft dislocation rate of 12%.9 As a comparison, Price reported an overall dislocation rate of 16% in his first 200 cases.4 However, it should be noted

that both Price’s and our data are heterogeneous, and thus do not lend themselves to easy direct comparison. With more time, we expect that more basic science studies and larger, case-controlled comparisons of pre-cut versus intraoperatively-cut DSEK tissue will provide more information regarding the outcomes of using pre-cut tissue.

Conclusion The recent availability of pre-cut donor tissue in DSEK frees the surgeon from the cost of investing in specialized corneal dissection instrumentation, minimizes the time-consuming dissection of the donor graft in the operating room, and eliminates the potential complications associated with intraoperative cutting of the donor tissue (i.e. button-holing, perforation, decentration). Using pre-cut tissue does not alter the overall DSEK procedure; the surgeon need only be aware of the cut bed diameter and the marks indicating the endothelial graft orientation. Not all surgeons, however, may be comfortable with having a third party prepare the graft, and there is currently a lack of peer-reviewed literature comparing the outcomes of DSEK using pre-cut versus surgeon-cut grafts. We recommend that the interested surgeon research the tissue-processor or eye bank of choice to inquire about the availability of pre-cut DSEK tissue and to develop a trusted, working relationship with the facility in providing quality endothelial grafts for this exciting advance in corneal transplantation surgery.

References 1. Melles GR, Egglink FA, Lander F, Pels E, Rietveld FJ, Beekhuis WH, Binder PS. A surgical technique for posterior lamellar keratoplasty. Cornea 1998;17(6):618-26. 2. Melles GR, Wijdh RH, Nieuwendaal CP. A technique to excise the descemet membrane from a recipient cornea (descemetorhexis). Cornea 2004;23(3):286-8. 3. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty visual acuity, astigmatism and endothelial survival in a large prospective series. Ophthalmology 2005;112(9):1541-8. 4. Price FW, Price MO. Descemet’s stripping with endothelial keratoplasty in 200 eyes. J Cataract Refract Surg 32:411-8. 5. Kang PC, Klintworth GK, Kim T, Carlson AN, Adelman R, Stinnett S, Afshari NA. Trends in the indications for penetrating keratoplasty, 1980-2001. Cornea 2005;24(7):801-3. 6. Carlson AN, Lee AC, Afshari NA. Endothelial cell viability of pre-cut corneal donor tissue for endothelial keratoplasty. Invest Ophthal Vis Sci 2006; 47: E-Abstract 2355. 7. Means TL, Geroski DH, Hadley A, Lynn MJ, Edelhauser HF. Viability of human corneal endothelium following Optisol-GS storage. Arch Ophthalmol 1995;113(6):805-9. 8. Griffin NB. Thickness changes in donor tissue prepared and stored for DSEK. Paper presentation at the American Academy of Ophthalmology Annual Meeting 2006. 9. Chau F, Kim T, Carlson AN, Stinnett S, Afshari NA. Risk Factors for Donor Tissue Dislocation after Descemet Stripping Endothelial Keratoplasty (DSEK). Poster presentation at the American Academy of Ophthalmology Annual Meeting 2006.

Comparison of Wound Architecture in DLEK Versus DSAEK

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Introduction “No-stitch” (no corneal sutures) corneal transplant is an increasingly popular surgical procedure, both in the United States and globally that threatens to take the place of fullthickness penetrating keratoplasty (PKP) over time. Such surgical procedures like deep lamellar endothelial keratoplasty (DLEK)1-13 and Descemet stripping automated endothelial keratoplasty (DSAEK)14-21 requires working on the inner corneal surface of the patient’s cornea while removing a host corneal disk in DLEK or Descemet’s membrane (DM) with endothelium in DSAEK and replacing it with a donor corneal disk with healthy endothelium in the process of surgical, visual rehabilitation of the patient. I introduced a new term for these types of surgeries namely, Selective Tissue Corneal Transplant (STCT).20 STCT may be viewed as a superior surgical procedure as compared to full-thickness corneal replacement surgery such as a PKP. STCT does not violate the patients corneal surface and thus avoids any induced surgical astigmatism, has no corneal sutures and hence it eliminates any suturerelated complications including breakage of corneal sutures, suture induced corneal neovascularization and possible infection including keratitis and corneal ulceration. Unlike a PKP, there is no full-thickness corneal wound in either DLEK or DSAEK and hence, the patient’s

cornea maintains the globe integrity and will be less prone to corneal rupture following traumatic injury to the eye. The wound architecture is very interesting in DLEK and DSAEK. In DLEK, a pocket is surgically created within the stroma of the patient’s cornea without perforating either to the surface or into the anterior chamber (AC) (See chapters on DLEK). This requires some amount of expertise in lamellar keratoplasty. The intrastromal pocket extends to almost the full diameter of the patient’s cornea up to the limbus. Following the creation of this intrastromal pocket, a Terry trephine (Bausch & Lomb, Inc., Rochester, NY) is gently inserted into this intrastromal pocket and a posterior corneal trephination is carried out. This surgical step is performed after injecting a viscoelastic, such as Healon 5 (Advanced Medical Optics, Santa Ana, CA) into the AC prior to the posterior corneal trephination (see above). Next, a specially designed surgical scissors, called Cindy scissors (Bausch & Lomb, Inc., Rochester, NY) is used to excise the trephined posterior host corneal disk and it is removed using a 0.12 forceps. Removal of such a host corneal disk results in the creation of a circular recess in the posterior, inner corneal surface of the patient’s cornea (Figure 31-1A). Such a recess may be compared to a manhole (Figure 31-1B). This corneal recess is then closed with a donor corneal disk (Figure 31-2A) comprising of posterior corneal stroma, DM and healthy donor corneal endothelium. This

Figure 31-1: (A) Schematic representation of DLEK surgery, showing the posterior corneal recess within the recipient cornea and the donor disk. (B) Cartoon depiction of the corneal wound being compared to a “manhole” and the donor corneal disk is compared to the manhole cover.

Figure 31-2: (A) The donor disk is attached to the recipient cornea in DLEK. The donor corneal disk comprising of stroma, Descemet’s membrane, and healthy endothelium has to be placed precisely within the recipient corneal wound. A reverse Sinskey hook is used along the margins of the donor disk 360 degrees (running-the-rim) to prevent any “edgeoverride.” This donor corneal disk is held in place primarily by the surrounding tissue forces and to a lesser extent by a large air bubble within the anterior chamber. (B) The attachment of the donor corneal disk to the recipient cornea is compared to a manhole being covered with the manhole cover.

Comparison of Wound Architecture in DLEK Versus DSAEK

Figure 31-3: At the end of DLEK surgery, the air bubble size is decreased without the risk of disk detachment. This is considered to be due to the tissue forces assisting in this attachment process.

may be compared to closing a manhole with a lid (Figure 31-2B). Once the donor corneal disk is placed in position, the AC is filled with an air-bubble (Figure 31-2A) which initially holds the donor corneal disk in place. Next, a reverse Sinskey hook is used to “run-the rim” of the donor corneal disk to ensure proper placement of the donor corneal disk within the surgically created recess within the host cornea. Next, the size of the air-bubble is decreased (Figure 31-3) such that the diameter of this air-bubble is often smaller than the diameter of the donor corneal disk. The donor corneal disk usually stays in place without being detached from the host cornea (Figure 31-3). Thus, the wound architecture in DLEK is much like a manhole that is covered with a lid (Figures 31-1A to 31-2B). Since the donor disk is pushed gently into the host corneal recess, the stroma-to-stroma corneal rim contributes to holding the donor disk in place without being detached even when the size of the air-bubble is decreased (Figure 31-3). In contrast to DLEK, in DSAEK there is no such recess that is created in the host cornea (Figures 31-4A and B). Only the host DM and the attached monolayer of host corneal endothelium is detached and removed as a single

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disk (See also Section 9, Descemet’s Stripping Automated Endothelial Keratoplasty). The removal of this circular area of patient’s DM and endothelium, results in the exposure of the host corneal stroma in this central area. Following this, a donor corneal disk comprising of posterior donor corneal stroma, DM, and healthy donor endothelium, of about 150 µm thickness and often about 8.0 or 9.0 mm diameter (occasionally 7.0 mm disk) is created using a Moria mickrokeratome and an artificial anterior chamber (Moria Inc., Antony Cedex, France). This donor corneal disk is usually folded as a taco-fold, introduced into the AC, unfolded within the AC and attached to the patient’s inner corneal surface corresponding to the circular area of exposed host corneal stroma. An air-bubble is then used within the AC to hold the donor corneal disk in place (Figures 31-4A and B). This is very much like placing a pizza on a ceiling and holding it in place with two hands (Figures 31-4A and B). If one hand is removed, the pizza edge corresponding to the side where the hand is removed will come-off the ceiling (Figures 315A and B and 31-6A and B). If both hands are removed, obviously, the pizza will drop from the ceiling (Figure 317). Similarly, when the air bubble diameter is greater than the diameter of the donor disk, the donor disk will remain attached to the recipient cornea. But, when the air bubble size is decreased, making it smaller than the donor disk diameter, then both sides of the disk will begin to detach from the host cornea (Figures 31-5A and B) in primary position of gaze, and when the eye moves to the left, the air bubble will move to the right and keep the right margin of the disk attached while the left edge of the disk will begin to detach (Figures 31-6A and B), with aqueous humor entering the detached space (Figures 31-6A and B). When the eye moves to the right, a similar sequence of events will result in the detachment of the right side of the donor corneal disk. With aqueous entering the interface from both sides of the disk periphery, it will often result in total disk detachment, with the disk landing in the inferior aspect of

Figures 31-4A and B: (A) Schematic representation of the attachment of a donor corneal disk of about 150 µm thickness in DSAEK surgery. This donor disk is attached to the posterior, exposed stromal surface of the recipient cornea using a large air bubble within the anterior chamber. (B) The disk attachment to the inner dome of the recipient cornea in DSAEK surgery, is compared to a “pizza slapped onto the ceiling” (cartoon). To prevent the “pizzadrop” it has to be held in place with two hands.

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Figures 31-5A and B: (A) In DSAEK surgery, in primary eye position, if the air bubble is smaller than the donor corneal disk diameter, then the edges of this disk will usually detach and aqueous humor gets into the interface. (B) Cartoon showing, the pizza edge drop from the ceiling when only one hand is used to hold the pizza in the center.

Figures 31-6A and B: (A) When the eye is turned to the left, there is a shift in the air bubble position towards the opposite side and this results in pushing the right edge of the donor disk closer to the recipient cornea and further detachment of the left side of the donor disk with more aqueous humor entering the left side of the donor-recipient interface. (B) Cartoon showing when the pizza is held with one hand on the right edge, there is a left-sided “pizza-drop”.

Figures 31-7A and B: (A) When aqueous humor fully enters the donor-recipient interface this results in donor disk detachment in DSAEK surgery. (B) Cartoon showing a “pizza-drop” when it is not supported onto the ceiling.

the anterior chamber. Thus, in the initial period, immediately after attaching the donor corneal disk to the host cornea, it is only the air-bubble that holds the disk in place. Hence, the diameter of the air-bubble must be greater than the diameter of the donor corneal disk in order for the donor corneal disk to remain attached to the host cornea (Figure 31-4). Unlike DLEK, in DSAEK there is no circumferential tissue “adhesive forces” to hold the donor disk in place. Thus, the wound architecture is quite different in DSAEK as compared to DLEK (Figures 31-8 to 31-11). Postoperatively, both DLEK and DSAEK look somewhat similar in the frontal view, but differ in the profile view, namely, the

“broad-band-wound” in DLEK (Figure 31-9) and the “narrow-band-wound” in DSAEK (Figure 31-11) are clinically evident under the biomicroscope. These differences in the wound architectures between DLEK and DSAEK are important factors to keep in mind when trying to reduce the donor disk detachment rate following such STCT procedures. A recent modification is the use of a scraping instrument [Terry Scraper (Bausch & Lomb, Inc., Rochester, NY), John DSAEK Stromal Scrubber (ASICO Inc., Westmont, IL)] to scrape a band of corneal stroma on the inner corneal surface (Described by Terry, M, personal communication) just within

Comparison of Wound Architecture in DLEK Versus DSAEK

Figure 31-8: Broad beam slit-lamp photograph of a cornea of a patient, 2 years and 2 months following a deep lamellar endothelial keratoplasty (DLEK) procedure showing a clear and compact cornea, and the wellcentered and fully integrated donor corneal disk to the inner stromal surface of the host cornea.

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Figure 31-9: Side profile view of a slit-lamp photograph of a patient’s cornea, 2 years and 2 months after a DLEK procedure (see also Figure 31-8), showing a circumferential broad-band-wound configuration (arrow).

Figure 31-10: Broad beam (A) and narrow beam (B) slit-lamp photographs of a cornea of a patient, 9.5 weeks following a Descemet stripping automated endothelial keratoplasty (DSAEK) procedure showing a clear and compact cornea, and the well-centered and fully integrated donor corneal disk to the inner surface of the host cornea.

the corneal epithelial circular mark. Such a technique is expected to increase the adhesion of the donor corneal disk to the recipient inner cornea and decrease postoperative disk dislocation rate.

References

Figure 31-11: Side profile view of a slit-lamp photograph of a patient’s cornea, 9.5 weeks after a DSAEK (see also Figure 31-10), showing a circumferential narrow-band-wound configuration (arrow).

1. Melles GR, Eggink FA, Lander F, Pels E, Rietveld FJ, Beekhuis WH, Binder PS. A surgical technique for posterior lamellar keratoplasty. Cornea 1998;17:618-26. 2. Terry MA, Ousley PJ. Endothelial replacement without surface corneal incisions or sutures: Topography of the deep lamellar endothelial keratoplasty procedure. Cornea 2001;20:14-18. 3. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty in the first United States patients: Early clinical results. Cornea 2001;20:239-43. 4. Terry MA, Ousley PJ. Replacing the endothelium without corneal surface incisions or sutures: The first United States clinical series using the deep lamellar endothelial keratoplasty procedure. Ophthalmology 2003;110:755-64.

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5. Terry MA, Ousley PJ. In pursuit of emmetropia: Spherical equivalent refraction results with deep lamellar endothelial keratoplasty (DLEK). Cornea 2003;22:619-26. 6. Terry MA, Ousley PJ. Rapid visual rehabilitation after endothelial transplants with deep lamellar endothelial keratoplasty (DLEK). Cornea 2004;23:143-53. 7. Terry MA. Endothelial replacement: The limbal pocket approach. Ophthalmol Clin North Am 2003;16:103-12. 8. Terry MA. Deep lamellar endothelial keratoplasty (DLEK): Pursuing the ideal goals of endothelial replacement. Eye 2003;17:982-8. 9. Terry MA. A new approach for endothelial transplantation: Deep lamellar endothelial keratoplasty. Int Ophthalmol Clin 2003;43:183-93. 10. Terry MA, Ousley PJ. Corneal endothelial transplantation: Advances in the surgical management of endothelial dysfunction. Contemporary Ophthalmology 2002;1(26):1-8. 11. Hyams M, Segev F, Yepes N, Slomovic AR, Rootman DS. Early postoperative complications of deep lamellar endothelial keratoplasty. Cornea 2007;26:650-3. 12. Marcon AS, Terry MA, Kara-José N, Wall J, Ousley PJ, Hoar K. Influence of final corneal thickness in visual acuity after deep lamellar endothelial keratoplasty. Cornea 2007;26:543-5. 13. Yepes N, Segev F, Hyams M, McAllum P, Slomovic AR, Rootman DS. Five-millimeter-incision deep lamellar endothelial keratoplasty: One-year results. Cornea 2007;26:530-3.

14. Price MO, Price FW. Descemet’s stripping endothelial keratoplasty. Curr Opin Ophthalmol 2007;18:290-4. 15. Mearza AA, Quershi MA, Rostron CK. Experience and 12-month results of descemet-stripping endothelial keratoplasty (DSEK) with a small-incision technique. Cornea 2007;26:279-83. 16. Melles GR. Posterior lamellar keratoplasty: DLEK to DSEK to DMEK. Cornea 2006;25:879-81. 17. Price FW Jr, Price MO. Descemet’s stripping with endothelial keratoplasty in 200 eyes: Early challenges and techniques to enhance donor adherence. J Cataract Refract Surg 2006;32:4118. 18. Price FW Jr, Price MO. Descemet’s stripping with endothelial keratoplasty in 50 eyes: A refractive neutral corneal transplant. J Refract Surg 2005;21:339-45. 19. Price MO, Price FW Jr. Descemet’s stripping with endothelial keratoplasty: Comparative outcomes with microkeratomedissected and manually dissected donor tissue. Ophthalmology 2006;113:1936-42. 20. John T. Descemetorhexis with endokeratoplasty. In: Surgical Techniques in Anterior and Posterior Lamellar Corneal Surgery. John T (Ed) Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India, 2006;411-20. 21. John T. Descemetorhexis with endokeratoplasty (DXEK). In: Step by Step Anterior and Posterior Lamellar Keratoplasty. John T (Ed). Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India, 2006;177-96.

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Use of Dyes in DSAEK and DLEK

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Introduction Endothelial transplant (ET), also known as endothelial keratoplasty (EK) encompasses various surgical techniques,1-17 namely, deep lamellar endothelial keratoplasty (DLEK) (Figures 32-1 and 32-2), Descemet stripping automated endothelial keratoplasty (DSAEK), and Descemet’s membrane endothelial keratoplasty (DMEK). All of these surgical techniques, although, vary in the degree of surgical complexity, attains a common end goal of transplanting healthy donor corneal endothelial cells to the inner surface of a decompensated, recipient cornea, thus bringing corneal clarity, improved visual acuity without inducing any significant corneal astigmatism following surgery. These newer corneal surgical procedures may replace conventional penetrating keratoplasty (PKP) over time. DLEK can be surgically challenging regarding the surgical dissection within the host cornea to create an intracorneal pocket that can temporarily house the Terry trephine for subsequent posterior stromal trephination. It is essential for the intrastromal dissection to be carried out without creating perforations either anteriorly onto the

Figure 32-1: Schematic representation of “no-stitch” corneal transplant (no corneal sutures) in deep lamellar endothelial keratoplasty (DLEK).

Figure 32-2: Upper left – Donor cornea is held in place within the artificial anterior chamber (ACC) and lamellar dissection of the donor cornea is carried out. The donor endothelium is pre-coated with viscoelastic, and the ACC is pressurized with Optisol. Upper right – Moria Teflon block with the indocyanine green (ICG) stained donor cornea with the stromal side down is held in place with vacuum. Trephination is carried out with a Moria disposable trephine in a guillotine manner. Lower left – Introduction of the ICG stained donor disc into the recipient anterior chamber under air. The donor disk is positioned into the prepared host corneal bed with the assistance of the green color of the ICG stained disk. Lower right – Intraoperative view of the ICG stained donor lamellar disc that is well centered within the recipient cornea. Also seen is the residual air bubble.

Use of Dyes in DSAEK and DLEK corneal surface or posteriorly into the anterior chamber. Further challenges include proper placement of the donor corneal disk within the host cornea, from the anterior chamber, under air. It is also essential that the endothelium on the donor corneal disk is not iatrogenically damaged before placement in the host cornea. The newer surgical technique namely, DSAEK has become easier to perform as compared to DLEK, since there is no intrastromal dissection in DSAEK, and thus it is continuing to gain rapid popularity among corneal surgeons worldwide. DMEK, however, is in its earlier stages of development and requires further improvement in the surgical techniques before it will become widely accepted. All these surgical techniques do have one common factor, namely, the surgeon looks through a cloudy cornea while doing surgery. This compromised view of the anterior chamber makes it difficult to clearly view the donor corneal disk within the recipient anterior chamber. Staining the donor corneal stroma with dyes such as indocyanine green (ICG) or trypan blue, enhances the visibility of the donor disk within the recipient anterior chamber. The author first reported the use of indocyanine green (ICG) within the human cornea, namely, in DLEK.5 This chapter describes the technique of using ICG on the stromal side of the donor corneal disk in DLEK (Figure 32-2) and

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the use of trypan blue staining of the donor disk in DSAEK procedures (Figures 32-3 to 32-6). These quick staining methods effectively localizes the donor disk in the host anterior chamber at all times during surgery and increases the accuracy of proper placement of the donor disk on to the inner surface of the recipient cornea.

Surgical Technique of Using ICG in DLEK ICG Preparation ICG (IC-Green, Akorn, Inc., Buffalo Grove, IL) is packaged in a 25-mg vial and a 10-ml ampule of aqueous solvent (Akorn, Inc.). The aqueous solvent (pH 5.5-6.5) is a prepared sterile water for injection used to dissolve the ICG. Aqueous solvent, 0.5 ml, is added to the ICG bottle and 4.5 ml of sterile balanced salt solution (BSS) is added to the bottle containing the ICG. The bottle is shaken until the contents of the bottle are well dissolved. Two ml of the ICG solution is aspirated into a 5 ml sterile syringe and an additional 2 ml of sterile BSS is aspirated into the same syringe for a total volume of 4 cc (1:1 dilution). A sterile, blunt, 30-gauge cannula is attached to the syringe and the solution is ready for corneal use as described below.

Figure 32-3: Donor corneal disk is folded like a “taco” prior to its introduction into the recipient anterior chamber.

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Figure 32-4: Trypan blue staining enhances the visibility of the donor corneal stroma within the recipient anterior chamber.

ICG Staining of the Donor Corneal Stroma The freshly prepared ICG is used to stain the anterior corneal stroma of the donor cornea. Following excision of the anterior lamellar corneal stroma from the donor cornea, the anterior bare stroma is exposed to room air. The freshly prepared ICG from the 5-cc syringe is dropped onto the exposed donor corneal stroma using the 30-gauge cannula to completely cover the donor corneal stroma. The excess ICG is removed using a Meracel sponge (Medtronic, Jacksonville, FL). ICG only stains the bare corneal stroma, and the surrounding full-thickness donor corneal rim with the intact corneal epithelium is not stained.

Completion of Donor Corneal Surgery Following staining of the anterior corneal stroma of the donor cornea, the donor corneoscleral tissue is removed from the ACC and placed on a Moria Teflon block (Moria

Inc., Doylestown, PA) with the exposed, ICG stained, stromal side down and centered on the Teflon block using the ICG stained area as an indicator of the outer demarcation of the corneal stroma with a total diameter of 9 mm. The outer full-thickness donor cornea with the intact epithelium is devoid of staining. Suction is applied to this donor disk to hold it in place and trephination is carried out using an 8.0 mm diameter Moria (Moria Inc.) trephine in a guillotine fashion (Figure 32-2 upper right). The ICGstained donor disk is seen as a green disk on the white Teflon block following the removal of the remainder of the donor corneoscleral rim. The thickness of the donor disk is not measured directly to avoid distortion to the tissue before transfer into the host corneal opening. The estimated thickness of this disk is between 200 and 300 µm. This deep stromal-endothelial donor disk is carefully transferred onto a Healon-coated, Ousley spatula (Bausch & Lomb) with the endothelial side down.

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Figure 32-5: Filtered-air is being injected in a controlled manner to unfold the taco-folded donor corneal disk. Trypan blue staining helps in good visualization of the donor cornea through a cloudy cornea.

Donor Disk Transplantation Additional air is injected into the anterior chamber of the host eye via the side port incision. The scleral sutures were cut and removed. The Ousley spatula with the donor disk is introduced into the pre-dissected corneal pocket wound, dropped into the anterior chamber, and then elevated placing the donor disk into the host bed of the resected central area (Figure 32-2 lower left). The donor corneal stroma is approximated to the host corneal stroma, creating a new corneal interface for the host. The donor corneal disk that is stained green with the ICG dye is well visualized through the host anterior corneal stroma. The donor disk margins are well approximated to the cut edges of the host corneal rim without any overriding of the donor disk edge to the cut edges of the inner host corneal stroma. The Ousley spatula is then gently removed from the eye. The donor corneal disk adheres to the central posterior recipient bed (Figure 32-2 lower right), the interface green dye is visible, and the donor disk is held in place initially by the air bubble in the anterior chamber. Minor adjustments to the

coaptation of the edges of the donor disk and the recipient bed can be made if needed using a Sinskey hook (Stephens Instruments, Lexington, KY). The superior scleral wound is then closed with interrupted 10-0 nylon sutures without adding any additional tension to prevent induced astigmatism. The air bubble is decreased in size and additional BSS is injected into the anterior chamber as needed through the side port incision. A collagen shield soaked in antibiotics and steroid is placed on the ocular surface at the end of the procedure. Patients are examined the following day in the office.

Trypan Blue Staining of the Donor Corneal Stroma in DSAEK Procedure The selective staining properties of trypan blue (Vision Blue, Dutch Ophthalmic International, Exeter, NH, USA) were used to visualize and manipulate the donor corneal disk during EK surgery for proper centration and attachment of the donor corneal disk to the recipient cornea (Figures 32-3 to 32-6).

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Figure 32-6: Trypan blue stromal stain helps to better visualize the donor-recipient interface. Lower Row—Well-centered donor corneal disk is seen. Also seen is the double ring sign (bottom row) showing uniform adherence of the donor corneal disk to the recipient cornea.

Surgical Technique Moria anterior lamellar therapeutic keratoplasty (ALTK) system (Moria Inc., Antony, France) was used as the artificial anterior camber (AAC) and a microkeratome with a 300 µm CB head was utilized for the procedure (Moria Inc., Antony, France). The set-up consisted of a sterile syringe filled with the Optisol-GS solution from the eye bank vial (Illinois Eye Bank, Chicago, IL) containing the donor corneal button. This syringe was attached to one end of a short single-use sterile tubing with a stop-valve, and the other end of the tubing was attached to the ALTK system. Optisol-GS solution was gently injected from the syringe such that the solution filled the well in the central post. The donor corneal button with a large scleral rim was placed with the endothelial side down on the central post of the AAC filled with the Optisol-GS solution. The OptisolGS solution bathed the donor corneal endothelium. The cylindrical fixation ring was then locked in place, encasing the donor corneal button within the AAC.

The corneal epithelium was removed using dry Merocel spear (Medtronic, Jacksonville, FL). A central dot was placed on the dome of the donor cornea with a marking pen and a linear mark was made on the periphery of the cornea. The microkeratome head was moistened with sterile balanced salt solution (BSS) (Meditech Inc, Manassas, VA, USA) and the head was mounted on to the post, and a corneal cap was cut with the CB-microkeratome head in a steady curvilinear fashion. A free-cap was then obtained on the microkeratome head and the donor corneal stroma was exposed in a circular manner in the central opening of the ALTK system.

Stromal Staining Trypan blue ophthalmic solution (Vision Blue, Dutch Ophthalmic USA, Exeter, NH) was then applied on to the exposed corneal stroma using a sterile syringe with a blunt cannula. The excess trypan blue was dried with Merocel spears (Medtronic, Jacksonville, FL). This step stained the

Use of Dyes in DSAEK and DLEK exposed donor corneal stroma blue and can be seen well even through a cloudy cornea, upon placement of the donor disk into the recipient AC (Figures 32-4 and 32-5). The freecap was then gently removed from the microkeratome head and replaced on to the exposed donor corneal stroma, such that the pre-placed marks were aligned. The outer cylinder in the AAC was then rotated into the unlocked position. The corneal button with the outer corneal cap was then removed from the AAC and it was placed on a Hanna corneal punch with the epithelial side down (Moria Inc., Antony, France). Looking through the central opening in the Hanna trephine the surgeon could see the area of blue staining. Trypan blue only stained the cut stromal surface and the surrounding epithelium remained unstained. The blue coloration helped in the centration of the trephine and prevented any eccentric trephination of the donor corneal disk. Centration was more critical with a larger diameter trephine, such as a 9.0 mm diameter trephine, as compared to a smaller trephine, such as a 7.5 mm trephine blade. With a 9.0 mm trephine, there is a greater potential for eccentric trephination as compared to a smaller 7.5 mm trephine. The circular blade should land within the area of blue coloration to obtain a properly cut donor corneal disk and avoid eccentric trephination of the donor corneal disk. A temporal approach was used in all cases. Following trephination of the donor cornea from the endothelial side, the trephine blade edge was ink-marked, and the recipient corneal surface on the epithelium was marked using the same diameter trephine as used on the donor cornea. Alternatively, the low-profile, John DSAEK corneal marker (ASICO Inc., Westmont, IL) was used to mark the recipient cornea. The subsequent descemetorhexis was performed using the John Dexatome spatula in all cases (ASICO Inc., Westmont, IL), 0.5 mm within this circular mark, such that the host Descemet’s membrane (DM) skirt covered the edge of the donor disk 360 degrees without any area devoid of DM, and thus prevented any postoperative peripheral corneal edema. Conjunctiva and Tenon’s membrane were cut in the temporal region to expose the bare sclera. Complete hemostasis was achieved using a disposable cautery. A Castroviejo caliper was ink-marked at the tips with a marking pen, and the marks were placed at the perilimbal sclera, with a chord length of 5.0 mm. A curvilinear incision was then made at the limbus using a 350 µm fixed-depth diamond blade. A peripheral intracorneal pocket was created in the temporal region using an angled crescent blade (Alcon Inc., Fort Worth, TX). The AC was not entered at this time. Two stab-incisions were made at the limbus, one to the right (about 2:00 o’clock position) and a second-

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stab incision was made on the left side of the temporal incision (about 5:00 o’clock position) using a 15-degree superblade (Alcon Inc., Fort Worth, TX). The 2:00 o’clock incision was used to inject the Healon and the 5:00 o’clock incision was used to inject air to unfold the “taco-folded” donor disk within the AC. These incisions were also used to introduce other instruments such as a John Dexatome spatula (ASICO Inc., Westmont, IL), reverse Sinskey hook, John super micro-scissors (ASICO Inc., Westmont, IL), John super-micro forceps (ASICO Inc., Westmont, IL), etc. as needed. Descemetorhexis was performed under a Healon-filled AC. Descemet’s membrane was scored using the John Dexatome Spatula (ASICO Inc., Westmont, IL) first in a clockwise direction, followed by a counterclockwise scoring to complete a 360-degree scoring of the DM that was about 0.5 mm inner to the circular mark on the corneal surface. John Dexatome Spatula allowed easy Descemetorhexis, 360-degrees from a single wound entry into the AC, without exiting the AC, and without using a second instrument such as a Descemet’s stripper. Following 360-degrees scoring of the DM, the DM was then detached as a single disk and it was removed from the AC using the John EK/DSAEK insertion forceps (ASICO Inc., Westmont, IL). The John Dexatome Spatula allowed for the Descemet’s membrane to be removed as a single, complete disk, almost 100% of the time, except, in occasional cases with severe focal DM adhesions due to scarring from previous penetrating keratoplasty (PKP), such as in cases of failed corneal graft. In such cases, the John supermicro forceps (ASICO, Westmont, IL) and the John supermicro scissors (ASICO, Westmont, IL) helped to release the Descemet’s membrane in these areas of focal scarring and allowed the removal of the Descemet’s membrane as a single disk. Next, the AC was entered through the temporal pre-made groove, using a 3.2 mm keratome blade (Alcon Surgical, Fort Worth, TX). The detached Descemet’s membrane disk was then removed from the AC using a John insertion forceps (ASICO, Westmont, IL) and quickly examined under the microscope to confirm the removal of the Descemet’s membrane as a single disk. Peripheral stromal scrubbing was performed within the epithelial circular mark to increase adhesion of the donor corneal disk to the exposed host corneal stroma. The author (TJ) performed peripheral scrubbing of the host corneal stroma routinely using a John scrubber (ASICO, Westmont, IL). This technique of peripheral stromal scrubbing was first described by Terry.48 During the scrubbing of the peripheral stroma, Healon remained in the AC to prevent AC collapse. Following stromal scrubbing, Healon was completely removed from the AC using an irrigation/

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aspiration unit attached to the Alcon Legacy phacoemulsifier (Alcon Surgical, Fort Worth, TX). Healon was also removed from the inner corneal surface. The temporal peri-limbal wound was then enlarged to its full length of 5.0 mm using the angled crescent blade, bevel-up (Alcon Surgical, Inc., Fort Worth, TX). The donor corneal disk was brought to the surgical field and placed on the host corneal dome, such that the epithelial surface of the donor corneal disk was in contact with the host corneal epithelium. A small amount of Healon was placed on the endothelial surface in the center of the donor disk. One edge of the donor corneal disk was held with the John insertion forceps (ASICO Inc., Westmont, IL) and the donor disk was folded on itself in the shape of a “taco” (Figure 32-3). The donor disk was folded into a 60%/40% overfold such that the trypan blue stained stromal surface was facing up. The folded donor taco corneal disk was then introduced into the recipient AC in one continuous, smooth motion, and it was deposited on the iris surface (Figure 32-4), past the pupillary margins and the John insertion forceps was withdrawn out of the AC. The peri-limbal wound was closed with three interrupted, 10-0 nylon sutures. A 30-gauge sterile cannula was introduced from the previously placed stab incision at the 5:00 o’clock position, and the tip of the cannula was placed between the leaflets of the folded taco. Filtered-air was gently injected in a gentle, steady stream to unfold the taco completely (Figure 32-5) and attach the donor corneal disk to the host corneal stroma. If the donor corneal disk was decentered (Figure 32-5), centration of the donor disk was achieved (Figure 32-6) using a reverse Sinskey hook. Following proper attachment and centration of the donor corneal disk a waiting period of 8 to 10 minutes was allowed to further facilitate donor disk adhesion.

Results ICG helped in better visualization of the donor disk within the recipient anterior chamber during DLEK, due to the green color of the stained donor stromal surface (Figure 32-2). Further ICG stained disk was easier to position properly within the host cornea as compared to those cases where ICG was not used. The day after surgery there was no ICG visible in any of the cases by slit-lamp examination. ICG disappears within 24 hours after surgery. There was no increased anterior chamber inflammation in any of the cases as compared to DLEK without ICG. The donor corneal disk edges and the interface were better approximated in the ICG cases as compared to the non-ICG cases. There was no posterior segment toxicity noted in any of the cases

from the ICG use. Similarly, the use of trypan blue during DSAEK surgery helps to easily visualize the donor cornea even through a cloudy cornea (Figures 32-3 to 32- 6).

Discussion ICG has been widely used in various medical fields, including cardiovascular, hepatic and ophthalmic specialties.18 ICG has been used both in animal19-26 and human studies.27-31 In ophthalmology, it has been used initially for ophthalmic angiographic studies27 and more recently to stain the anterior lens capsule32 under air before capsulorhexis during phacoemulsification. When this solution of ICG is used in the anterior chamber under air, the aqueous humor that is in the anterior chamber immediately dilutes the injected dye within the anterior chamber. Since dilution does not take place when the dye is directly added to the exposed corneal stroma of the donor corneal disk attached to the ACC, the dye is further diluted 1:1 with sterile BSS before its direct use on to the corneal stroma of the donor cornea. Following donor corneal exposure to this dye, it stains only the exposed donor corneal stroma and it does not stain the uncut, full-thickness, donor corneal rim with its intact epithelium. Such selective staining of the donor corneal stroma provides an added advantage to the corneal surgeon in proper placement and centration of this donor disk on the Teflon block before trephination. The trephine that is used to trephine the donor disk is 8.0 mm in diameter. The exposed corneal stroma is 9 mm in diameter. This allows only a 0.5 mm margin on all sides of the donor disk to trephine within the exposed corneal stroma. Any eccentric trephination will carry with it the uncut corneal rim at the eccentric margin that will then have to be manually trimmed with scissors. Manual trimming can cause irregularities and result in improper approximation of the donor disk to the host cornea creating irregularities in the newly created corneal interface. Since the disk is stained with the dye in a circular area 9.0 mm in diameter, this allows for good centration of this disk on the Teflon block to match the ring mark on the Teflon block and allows for the 8.0 mm trephination to be carried out well within the 9.0 mm corneal stained area and prevents any eccentric cut. This eliminates any inadvertent cutting of the peripheral full-thickness donor cornea. Staining of the corneal donor disk with the ICG allows good visualization of the donor disk in the host anterior chamber through the host anterior corneal stroma. Before this staining technique, the visualization of the donor disk within the host anterior chamber was suboptimal and made it very difficult for the corneal surgeon to properly

Use of Dyes in DSAEK and DLEK approximate the donor disk to the host corneal opening. This staining technique greatly facilitates this surgical technique and helps in proper positioning of the donor disk within the recipient cornea. ICG is a sterile, water soluble, tricarbocyanine dye with a peak spectral absorption of 800-810 nm in blood plasma or blood.33 ICG is taken up from the plasma almost exclusively by the hepatic parenchymal cells and is secreted entirely into the bile. 27 ICG contains not more than 5.0% of sodium iodide. 33 ICG is marketed for intravenous administration. Anaphylactic or urticarial reactions have been reported in patients with or without a history of allergy to iodides. 33 If reactions occur, treatment with appropriate agents, e.g. epinephrine, antihistamines, and corticosteroids, should be initiated. It is unknown whether this drug is excreted in human milk,33 and caution should be exercised when ICG is administered to a nursing woman.33 However, in the present method of ICG usage described in this chapter a dilute solution is used to stain the donor disk, and the excess dye is removed before placement of the stained disk into the anterior chamber of the host. This is a negligible amount of the dye that is already adhering to the cornea as compared to the larger dose that is used for ophthalmic angiography. DLEK is a relatively new anterior segment surgical procedure that may selectively replace PKP in a subset of patients to visually rehabilitate those individuals with corneal endothelial decompensation and corneal edema. There are several advantages to the intraoperative use of ICG dye to stain the donor corneal stroma (Table 32-1). ICG greatly facilitates performing this new surgical procedure and contributes to a good postoperative result. TABLE 32-1: Advantages of intraoperative use of ICG in DLEK Advantages 1. Instant staining of donor corneal stromal tissue from clear to green. 2. The staining step takes less than 60 seconds and with no significant surgical delay. 3. ICG disappears from the host cornea within 24 hours following surgery. 4. Helps confirm that the donor corneal disk is in the host anterior chamber. 5. Helps in good approximation of donor stromal tissue to the recipient corneal stroma. 6 Facilitates good coaptation of donor-host graft margins 360 degrees. 7 Helps to visualize any interface fluid pocket, air, or debris.

Trypan Blue In 2004, Food and Drug Administration approved the use of Vision Blue (Trypan Blue ophthalmic solution) (Dutch

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Ophthalmic International, Exeter, NH, USA) for staining of the anterior lens capsule during cataract surgery. Trypan blue has various surgical and laboratory applications.34-47 Georgiadis et al34 used trypan blue to stain donor cornea and evaluate their suitability for human corneal transplantation. Subsequently in 2002, Balestrazzi et al35 first used trypan blue to stain the human corneal stroma during anterior lamellar keratoplasty. In 2004, Food and Drug Administration approved the use of Vision Blue (Trypan Blue ophthalmic solution) (Dutch Ophthalmic International, Exeter, NH, USA) for staining of the anterior lens capsule during cataract surgery. Trypan blue has various surgical and laboratory applications.34-47 Trypan blue, also known as diamine blue and Niagara blue, is a stain used to selectively color dead tissues or cells.40 It cannot transverse the membrane of live cells but does through membranes of dead cells and therefore, only the dead cells are shown with a distinctive blue color.40 In the present study the donor corneal cells are viable as assessed by the distributing eye banks. Therefore, the selective staining of the cut stromal surface may be due to the presence of glycosaminoglycans and other cellular matrix substances present in the corneal stroma. Trypan blue does not stain the surrounding intact corneal epithelium. This selective staining of the cut stromal surface is beneficial to the surgeon throughout the ET procedure. However, this is an off-label use of trypan blue. In addition to laboratory uses of trypan blue, it is used in various anterior and posterior segment ophthalmic procedures including capsulorhexis prior to phacoemulsification (especially in the absence of a red reflex), perioperative staining of the corneal endothelium, staining of internal limiting membrane and epiretinal membrane in vitrectomy, and evaluation of corneal graft tissue. 34-47 Ocular toxicity due to the use of trypan blue has been studied.41 In vitro studies have shown that rat neurosensory retina (R28) cells are more sensitive than human retinal pigment epithelial cells (RPE) (ARPE-19) to trypan blue. 41 Human RPE cells showed no evidence of toxicity to four different concentrations of trypan blue (0.1%, 0.05%, 0.025%, and 0.0125%) in vitro. 41 Trypan blue has also been evaluated in vitreoretinal surgery.39 The application of trypan blue onto the internal limiting membrane (ILM) and epiretinal membranes (ERM) in vitreoretinal surgery resulted in a useful bluish staining, facilitating the identification, delineation, and removal of the membranes in all surgeries.39 However, no residual staining or adverse effects related to the dye were observed.39 No adverse effects were noted when trypan blue 0.02% was used in vivo, inside the eye, or in postmortem, histological studies.42 However,

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when trypan blue was used at concentrations of 0.15% and 0.25% there was disorganization of the inner retinal layers and the ILM was absent in the postmortem studies.42 Vision blue (Trypan blue, Dutch Ophthalmic, USA) is commercially available at a concentration of 0.06%. The risks include potential staining of high water content hydrophilic acryclic intraocular lens, and inadvertent staining of the posterior lens capsule and vitreous face. The staining of the posterior lens capsule and vitreous face is usually self-limited and can last up to 1 week. The use of Vision Blue in the donor corneal stromal staining in DSAEK procedures has not been associated with any known permanent intraocular damage. Unlike in cataract surgery where it is used directly in the anterior chamber to stain the anterior lens capsule under filtered-air, in DSAEK the staining of the donor corneal stroma is done outside the eye and the area of staining then comes into apposition with the recipient inner corneal stroma at the donorrecipient interface. Such staining of the donor corneal stroma has not been associated with any interface adverse effects that is seen clinically. Selective staining of the donor corneal stroma aids in proper alignment and centration of the disposable tryphine during donor corneal tryphination. This further helps to prevent eccentric tryphination of the donor corneal disk. Further, if the donor disk unfolds to the wrong side, i.e. the donor endothelium facing up towards the inner surface of the recipient cornea, the trypan blue staining will help identify the stained stromal side from the unstained donor endothelium. Trypan blue can be used for staining of patient’s Descemet’s membrane as well. However, we do not recommend staining of both the donor corneal disk and Descemet’s membrane of patient’s cornea, as it would be difficult to distinguish the two and could lead to error and potential complications. We recommend the use of trypan blue ophthalmic solution to corneal surgeons who perform EK surgery to facilitate viewing the donor corneal disk through the patient’s cloudy cornea. Trypan blue appears to be safe in the staining of donor corneal stroma during EK surgery.

Summary and Conclusions The use of dye, namely, trypan blue (Vision Blue, Dutch Ophthalmic USA), or ICG to stain the donor corneal stroma is recommended for ET surgery. The use of these dyes for corneal staining is an “off-label” use of the drug in the United States. Staining the donor corneal stroma greatly improves the visualization of the donor corneal disk

through a cloudy cornea. The use of such dyes as described, appears to be clinically safe and there have been no clinically observable adverse side-effects from its use.

References 1. Ousley PJ, Terry MA. Stability of vision, topography, and endothelial density from 1 year to 2 years after deep lamellar endothelial keratoplasty surgery. Ophthalmology 2005; 112:5057. 2. Terry MA, Ousley PJ. Small-incision deep lamellar endothelial keratoplasty (DLEK): Six month results in the first prospective clinical study. Cornea 2005;24:59-65. 3. Van Dooren B, Mulder PG, Nieuwendaal CP, Beekhuis WH, Melles GR. Endothelial cell density after posterior lamellar keratoplasty (Melles techniques): 3 years follow-up. Am J Ophthalmol 2004;138:211-7. 4. Sano Y. Corneal endothelial transplantation: Results of a clinical series using deep lamellar endothelial keratoplasty (DLEK). Cornea 2004; 23:S55-S58. 5. John T. Use of indocyanine green in deep lamellar endothelial keratoplasty. J Cataract Refract Surg 2003;29:437-43. 6. Guell JL, Velasco F, Guerrero E, Gris O, Calatayud M. Preliminary results with posterior lamellar keratoplasty for endothelial failure. Br J Ophthalmol 2003;87:241-3. 7. Melles G, Eggink F, Lander F, et al. A surgical technique for posterior lamellar keratoplasty. Cornea 1998;17:618-26. 8. Melles G, Rietveld F, Beekhuis W, et al. A technique to visualize corneal incisions and lamellar dissection depth during surgery. Cornea 1999;18:80-86. 9. Melles G, Lander F, vonDooren B, et al. Posterior lamellar keratoplasty for a case of pseudophakic bullous keratopathy. Am J Ophthalmol 1999;120:346-8. 10. Melles GRJ, Lander F, vanDooren BTH, et al. Preliminary clinical results of posterior lamellar keratoplasty through a sclerocorneal pocket incision. Ophthalmology 2000;107:1850-7. 11. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty in the first united states patients: Early clinical results. Cornea 2001;20:239-43. 12. Terry MA, Ousley PJ. Endothelial replacement without surface corneal incisions or sutures: Topography of the deep lamellar endothelial keratoplasty procedure. Cornea 2001;20:14-18. 13. Prasher P, Muftuoglu O. Herpetic keratitis after Descemet stripping automated endothelial keratoplasty for failed graft. Eye Contact Lens 2009;35:41-42. 14. Phillips PM, Terry MA, Kaufman SC, Chen ES. Epithelial downgrowth after Descemet-stripping automated endothelial keratoplasty. J Cataract Refract Surg 2009;35:193-6. 15. Jun B, Kuo AN, Afshari NA, Carlson AN, Kim T. Refractive change after descemet stripping automated endothelial keratoplasty surgery and its correlation with graft thickness and diameter. Cornea 2009;28:19-23. 16. Terry MA, Shamie N, Chen ES, Hoar KL, Phillips PM, Friend DJ. Endothelial keratoplasty: The influence of preoperative donor endothelial cell densities on dislocation, primary graft failure, and 1-year cell counts. Cornea 2008;27:1131-7. 17. Ham L, van der Wees J, Melles GR. Causes of primary donor failure in descemet membrane endothelial keratoplasty. Am J Ophthalmol 2008;145:639-44. 18. Maarek JM, Holschneider DP, Harimoto J. Fluorescence of indocyanine green in blood: Intensity dependence on concentration and stabilization with sodium polyasparate. J Photochem Photobiol B 2001;65:157-64. 19. Springett R, Sakata Y, Delpy DT. Precise measurements of cerebral blood flow in newborn piglets from the bolus passage of indocyanine green. Phys Med Biol 2001;46:2209-25.

Use of Dyes in DSAEK and DLEK 20. Mordon S, Devoisselle JM, Soulie-Begu S, Desmettre T. Indocyanine green: Physiochemical factors affecting its fluorescence in vivo. Microvasc Res 1998;55:146-52. 21. Center SA, Bunch SE, Baldwin BH, Hornbuckle WE, Tennant BC. Comparison of sulfobromophthalein and indocyanine green clearances in the cat. Am J Vet Res 1983;44:727-30. 22. Tsao SC, Sawada Y, Iga T, Hanano M. Effect of chlorpromazine on hepatic transport of indocyanine green in rats. Biochem Pharmacol 1983;32:1105-12. 23. Chan PK, Hayes AW. Effect of penicillic acid on biliary excretion of indocyanine green in mouse and rat. J Toxicol Environ Health 1981;7:169-79. 24. Chong LP, Ozler SA, de Queiroz JM Jr, Liggett PE. Indocyanine green-enhanced diode laser treatment of melanoma in a rabbit model. Retina 1993;13:251-9. 25. Klaassen CD, Plaa GL. Plasma disappearance and biliary excretion of indocyanine green in rats, rabbits, and dogs. Toxicol Appl Pharmacol 1969;15:374-84. 26. Hansen DA, Spence AM, Carski T, Berger MS. Indocyanine green (ICG) staining and demarcation of tumor margins in a rat glioma model. Surg Neurol 1993;40:451-6. 27. Yannuzzi LA, Sorenson JA, Guyer DR, Slakter JS, Chang B, Orlock D. Indocyanine green videoangiography: Current status. Eur J Ophthalmol 1994;4:69-81. 28. Valada M, Lodato G, Cillino S. Mutifocal choroiditis: Indocyanine green angiographic features. Ophthalmologica 2001;215:16-21. 29. Avram MJ, Krejcie TC, Henthorn TK. The relationship of age to the pharmacokinetics of early drug distribution: The concurrent disposition of thiopental and indocyanine green. Anesthesiology 1990;72:403-11. 30. Burk SE, Mata AP, Snyder ME, Rosa RH, Foster RE. Indocyanine green-assisted peeling of the retinal limiting membrane. Ophthalmology 2000;107:2010-14. 31. Haglund MM, Berger MS, Hochman DW. Enhanced optical imaging of human gliomas and tumor margins. Neurosurgery 1996;38:308-17. 32. Pandey SK, Werner L, Escobar-Gomez M, Roig-Melo EA, Apple DJ. Dye-enhanced cataract surgery. Part 1: Anterior capsule staining for capsulorhexis in advanced/white cataract. J Cataract Refract Surg 2000;26(7):1052-9. 33. Physicians Desk Reference (PDR) for Ophthalmic Medicines, Edition 30, Medical Economics Company, Inc., Montvale, NJ. 2002;203-4.

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34. Georgiadis N, Kardasopoulos A, Bufidis T. The evaluation of corneal graft tissue by the use of trypan blue. Ophthalmologica 1999;213:8-11. 35. Balestrazzi E. Deep lamellaer keratoplasty with trypan blue intrastromal staining. Journal of Cataract and Refractive Surgery 2002;28:929-31. 36. Bhartiya P, Sharma N, Ray M, et al. Trypan blue assisted phacoemulsification in corneal opacities. Brit Jour Ophthal 2002;86:857-9. 37. Perrier M. Epiretinal membrane surgery assisted by trypan blue. Am J Ophthalmol 2002;135:909-11. 38. Perrier M, Sebag M. Trypan blue-assisted peeling of the internal limiting membrane during macular hole surgery. Am J Ophthalmol 2004;137:207-08. 39. Teba FA, Mohr A, Eckardt C, Wong D, Kusaka S, Joondeph BC, Feron EJ, Stalmans P, Van Overdam K, Melles GR. Trypan blue staining in vitreoretinal surgery. Ophthalmology 2003;110:2409-12. 40. Van Dooren B. Corneal endothelial cell density after trypan blue capsule staining in cataract surgery. Journal of Cataract & Refractive Surgery 2002;28:574-5. 41. Narayanan R, Kenney C, Kamjoo S, et al. Trypan blue: Effect on retinal pigment epithelial and neurosensory retinal cells. Investigative Ophthalmology and Visual Science 2005;46:304-9. 42. Haritoglou C, Gandorfer A, Schaumberger M, et al. Trypan blue in macular pucker surgery: An Evaluation of Histology and Functional Outcome. Retina 2004;24:582-90. 43. Jacob S, Agarwal Am, Agarwal At, et al. Trypan blue as an adjunct for safe phacoemulsification in eyes with white cataract. Journal of Cataract and Refractive Surgery 2002;28:1819-25. 44. Sharma N, Gupta V, Vajpayee R. Trypan-blue-assisted posterior capsule plaque removal. Journal of Cataract and Refractive Surgery 2002;28:916-7. 45. Gouws P, Merriman M, Goethals S, et al. Cystoid macular edema with trypan blue use. Br J Ophthalmology:2004; 88:1348-49. 46. Jackson T, Kwan A, Laldlaw A, et al. Identification of retinal breaks using subretinal trypan blue injection. Ophthalmology 2007;114:587-90. 47. Grisanti S, Szurman P, Tatar O, et al. Histopathological analysis in experimental macular surgery with trypan blue. Br J Ophthalmol 2004;88:1206-8. 48. John T, Shah AA. Use of trypan blue stain in endothelial keratoplasty. Annals of Ophthalmology 2009;41:10-15.

Comparative Visual Recovery in DSAEK, DLEK and PKP

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Mark S Gorovoy

Comparative Visual Recovery in DSAEK, DLEK and PKP

33

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Introduction Improved vision is the goal in the vast majority of patients undergoing corneal transplantation. Any of the cornea’s 5 layers can be the underlying source of decreased corneal transparency. Endothelial dysfunction is the most common indication for penetrating keratoplasty (PKP). Loss of the non-regenerating, single-layered endothelium results in stromal edema. This deterioration of endothelial-pump function may be gradual at first, with only morning edema with minimal loss of vision, but over time, classically worsens to gross bullous keratopathy, with severe loss of vision and the patient may also have intermittent pain due to rupture of bullae. In Fuchs’ dystrophy, edema is preceded typically with central guttata, which by themselves contribute to loss of visual function. Acute loss of endothelium can also result from a variety of surgical mishaps and rarely viral infections. Regardless of the etiology, loss of endothelial function always precipitates stromal edema and subsequent loss of corneal clarity and visual acuity. The only therapeutic procedure is replacement of the endothelium with donor tissue, i.e. corneal transplantation. In my practice, which typifies the western world, the most common indications for corneal transplantation secondary to endothelial failure are the following in numerative order; 1) Fuchs’ endothelial dystrophy with endothelial decompensation, 2) pseudophakic bullous keratopathy (PBK)/aphakic bullous keratopathy (ABK), 3) failed graft (PKP or DSAEK), 4) other (ICE syndrome). Most patient’s symptoms, unless acutely caused by an intraocular surgery such as phacoemulsification, typically experience gradual loss of vision from worsening corneal edema. The decision to undergo corneal transplantation is therefore elective, depending on the patient’s symptoms and informed consent as to the risk-benefit ratio of surgery. This ratio is key in determining the timing of transplantation. Historically, surgical success has defined the indications for surgical intervention. As an example, cataract couching was delayed until the cataract was ripe (almost gross blindness). In the early 1980s, with intracapsular cataract extraction and no IOL surgery, the surgical bar was raised to visual acuity less than than 20/70. Presently, 20/20 clear lensectomy for refractive indications with modern phacoemulsification techniques and multifocal IOLs are accepted by surgeons and patients alike. Similarly, modern microsurgery and eye banking advancements have elevated the surgical bar for present day penetrating keratoplasty (PKP) for endothelial disease. If success is defined as just obtaining an anatomically clear donor button, then PKP, the standard of care for the last 50

years, is highly successful. However, if success is defined as the recovery of excellent functional vision, then the success rate plummets. Functional visual recovery requires not only a clear corneal donor button, but predictable and regular surface keratometry. The full-thickness trephinations of PKP commonly induce irregular astigmatism, only correctable with a rigid contact lens. The resultant corneal power is unpredictable, often inducing unacceptable anisometropia. Long-term sutures and the slow healing rate of the avascular cornea delay final refractive outcome. Suture breakage or removal results in a large refractive shift, sometimes deleterious for the patient. Surgically induced neurotrophic keratitis can diminish vision from surface disease. Late traumatic wound rupture can have devastating results, often with loss of the globe. Avoiding the visual risks of PKP has led to the surgical development of the posterior lamellar keratoplasty (PLK) techniques, such as deep lamellar endothelial keratoplasty (DLEK), and Descemet stripping automated endothelial keratoplasty (DSAEK). They are small limbal incision techniques that eliminate full-thickness trephinations and transplant only a donor endothelial layer with some accompanying posterior stroma. By and large, DSAEK has supplanted DLEK as the preferred technique because of its surgical consistency and improved visual results. DSAEK incision size has been reduced to just over 3 mm, similar to clear corneal phacoemulsification incisions (Editorial Note: Unlike a phacoemulsification incision, with a DSAEK incision, with decreasing size from a 5.0 mm wound to a smaller wound size, there appears to be an increase in the endothelial cell loss due to mechanical tissue compression). Without surface penetrating trephinations, the refractive irregularities and unpredictabilities of the procedure have been eliminated. Corneal clarity is excellent within weeks and continues to improve over the first 3 months in most patients. The surgical bar has been lowered and less severe visual disability is not a barrier to surgical intervention. The visual outcomes following fullthickness and partial-thickness corneal transplantation are shown in Figures 33-1 and 33-2.

PKP Visual Recovery The surgical bar in my practice for PKP surgery is 20/70 BCVA. Of course, this is just a guideline and patients always make the final decision. In the state of Florida, this decision is often tied to the minimum visual acuity that is required for a driver’s license, which is 20/70 in both eyes if the best eye is less than 20/40. I counsel patients that final visual outcome is typically delayed to over 1 year or longer. Depending on their BSCVA, the running suture is left in place indefinitely and a GPCL is often required,

Comparative Visual Recovery in DSAEK, DLEK and PKP

Figure 33-1: Relative visual acuities following penetrating keratoplasty (PKP), deep lamellar endothelial keratoplasty (DLEK), Descemet stripping endothelial keratoplasty (DSEK), and Descemet stripping automated endothelial keratoplasty (DSAEK).

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the patient and donor corneas in DLEK; 2) the visual recovery of DSAEK is more rapid. The originally higher donor dislocation rate of DSAEK compared to DLEK, has been eliminated with further surgical modifications and surgeon experience. However, DLEK greatly improved the quality and rate of visual recovery over PKP by eliminating full thickness trephinations. Based on the results of only one experienced DLEK surgeon, BSCVA recovered to the 20/40 -20/60 range by 6 months. Surface topography was regular and predictable and the limiting factor for faster recovery was the interface wound, which continued to improve for over 1 year postoperative. This was a vast improvement over the results obtained with PKP, except that no patients achieved 20/20 vision. Other advantages of DLEK over a PKP include a relatively small 5 mm limbal incision, without induced neurotrophic surface problems or suture-related complications.

DSAEK Visual Recovery

Figure 33-2: At one year following surgery, percentage of best corrected visual acuity (BCVA) of 20/40 or better following penetrating keratoplasty (PKP), deep lamellar endothelial keratoplasty (DLEK), and Descemet stripping automated endothelial keratoplasty (DSAEK).

usually not for at least 3 months, but this may be delayed until the suture is removed at 1 year the earliest. Additional refractive procedures are often required to improve the visual quality, such as relaxing incisions, compression sutures or LASIK. This causes further delay and adds uncertainty as to the final visual outcome. If the condition is bilateral, I do not recommend the second eye for surgery until the first eye is the better eye. This unpredictable quality and rapidity of their visual recovery is the driving force behind these conservative recommendations.

DLEK/PLK Visual Recovery DLEK is the forerunner of DSAEK, and has largely been replaced by it. The reasons are twofold; 1) DSAEK eliminates the difficult manual lamellar dissections of both

DSAEK has quickly become the dominant procedure for endothelial keratoplasty. Despite a significant learning curve and capital equipment outlay ( unless donor cutting done by eye bank), it provides the best visual quality at the fastest pace of all keratoplasty techniques. Built upon the surgical ideas of DLEK, it also eliminates full-thickness trephinations of PKP, thereby leaving undisturbed the normal surface anatomy. It improves upon DLEK by eliminating all manual lamellar dissections. This increases the reproducibility of the procedure. Visual quality is enhanced by the regularity of the interface tissues. Descemet stripping leaves a pristine smooth recipient surface. The keratome cuts an extremely regular surface on the donor, equal to the quality of LASIK flaps. Incision size has been reduced to 3.2 mm, close to the typical phacoemulsification incision size (Editorial Note: Compared to 3.2 mm incision in DSAEK, a 5.0 mm incision causes less donor corneal endothelial cell loss). In my patients, when macula or optic nerve pathology is eliminated, the rate of visual recovery is extremely rapid. Over 80% of patients usually achieve a BSCVA of 20/40 by 6 weeks and this increases to over 90% by 3 months. While 20/20 is obtained by only 16% of patients, over one-third usually reach 20/25 in the first 6 months. This compares favorably to DLEK, with only 28% reaching 20/40 at 6 months and none at 20/20. However, the one year DLEK data continues to improve, narrowing this gap. The visual recovery of DSAEK is so predictable, the interface wound so imperceptible, that a patient who fails to reach a

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postoperative Snellen milestone should be evaluated for undetected macular disease. The improved visual recovery of DSAEK patients, compared to those of PKP patients has caused a paradigm shift in the recommendations to prospective surgical candidates. The surgical bar has been lowered, not dissimilar to the evolutionary rise seen with improved cataract surgery techniques. Patient complaints, consistent with their corneal disease, now drive the decision toward surgery. Rather than a conservative 20/70 minimum visual acuity used for PKP counseling, in Fuchs’ dystrophy, dense guttata, decrease visual quality which is not accurately depicted by Snellen acuity may be part of the surgical considerations. My “second eye rule”, refers to deferring surgery on the second eye until the first eye is the better eye. With DSAEK, patients are routinely requesting surgery for their second eye as early as 2-3 months following surgery, even if the Snellen acuity in the operated eye is less than the

unoperated eye. The postoperative visual quality is now superior with DSAEK surgery as compared to PKP. This is not too dissimilar to patients with posterior sub-capsular cataracts who are significantly disabled by their vision in spite of near normal Snellen measurements. Glare and contrast are important clinical indicators of patient satisfaction. Like DLEK, refractive surprises are usually non-existent. There appears to be a small early hyperopic shift of 1.0-2.0 D (Editorial Note: Usually 1.0 diopter or less) that partially fades over the first 3 months. Rigid contact lenses are unnecessary as there is no induced irregular astigmatism. Spectacle correction can be given confidently by 3 months postoperative. The functional recovery is consistent, with less than 10% not reaching 20/40 BSCVA. This compares very favorably to PKP for Fuchs’ disease with 36% failing to achieve 20/40 BCVA (15% required contacts). Long-term results only extend to 2 and ½ years and NEI sponsored endothelial cell count study is in progress.

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Juan M Castro-Combs Naima B Jacobs-El Ashley Behrens

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Introduction Before the 1930s, lamellar keratoplasty was the favored transplantation procedure for corneal opacification (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty). Magitot in 1913, successfully performed lamellar autografts for small, central corneal scars and in 1916 treated recurrent pterygia with lamellar transplan-tation.1-3 After the 1930s, penetrating keratoplasty (PK) has become the gold standard to manage surgical corneal disorders. Despite the advantages of lamellar procedures, such as less risks of intraocular complications and allograft rejection, PK increased in popularity while lamellar keratoplasty steadily declined.1-3 The main drawbacks of a lamellar procedure may be that the procedures are surgically demanding and carry the risk of interface haze development reducing the best corrected visual acuity. However, in the 1960s, Malbran described the “peelingoff” technique, in which the recipient cornea of patients with keratoconus was removed by applying traction to the partially dissected cornea overlying the cone.4-6 Other authors describe a surgical technique for lamellar keratoplasty that consists of a partial trephination of the anterior corneal stroma, and a single plane dissection of the recipient stroma. The depth of resection is judged by the corneal structure and the location of the Descemet’s membrane, which is recognized by its “glossy” appearance. It is often demanding to meticulously dissect the recipient stroma, especially when variable corneal thicknesses are present in the diseased stroma, which increases the likelihood of corneal perforation. In other words, by performing a PK, one is usually able to skip the challenges of a lamellar dissection.7 In 1972, Barraquer was the first to describe the use of the microkeratome in lamellar keratoplasty8 (See also Chapter 14, History of Lamellar and Penetrating Keratoplasty) Previous to the introduction of the microkeratome, lamellar keratoplasty was a tedious and time-consuming manual process used for reconstructive or tectonic surgery, rather than for optical purposes. The approach then consisted in performing an intralamellar dissection of the anterior corneal stroma creating a cap using a piriform spatula. The flap was lifted, and the posterior recipient stroma was trephined out. The tissue was replaced with a donor posterior lamellar button and the overlying flap was then sutured into place. With the application of the microkeratome technique there was an increase in the speed of the operation, but not necessarily in the final quality of vision. Barraquer also outlined the conditions to achieve good visual results with lamellar keratoplasty: (1) achieve the deepest possible interface to reduce scarring, (2) create a posterior layer of uniform

thickness, (3) perform smooth surface sectioning of both the graft and bed, (4) make the graft tissue of appropriate thickness, (5) obtain the highest quality donor material, (6) ensure good coaptation of the edges and uniform traction of the sutures, and (7) make sure there is perfect cleanliness of the interface.8 These conditions have served as a directive for the evolution of lamellar grafting techniques over the subsequent 30 years.

Penetrating Keratoplasty The total number of cases of corneal transplants with reported recipient diagnoses in the United States in the year 2000 was 31,532, which represents 67% of the corneas distributed by the 80 US eye banks. The most common recipient diagnosis among these cases was pseudophakic bullous keratopathy accounting for 19.6% of the cases.9 Other major indications for PK include Fuchs’ dystrophy, keratoconus, aphakic bullous keratopathy, and regraft. Corneal endothelial failure due to dystrophies or trauma ultimately accounts for most of the cases requiring corneal transplantation. Up to 53% of the US grand total of corneal transplants comprises some form of endothelial cell dysfunction.10 PK is a safe and effective method for restoring corneal transparency and obtaining improved visual acuity in posterior corneal disorders, but it can be complicated by high and/or irregular astigmatism,11-13 insufficient wound healing, 14,15 prolonged recovery time, and tissue rejection.16,17 Wound dehiscence after PK may also occur from days to decades after transplantation. Although some patients retain clear grafts and excellent vision following wound dehiscence repair, others suffer significant visual loss, most of whom have associated ocular complications. Mechanisms of injury producing wound dehiscence tend to be mild to moderate blunt trauma (i.e., poked with finger, hit with an object, or punched in the eye), which would be unlikely to rupture a globe in an intact cornea.14,15 Graft failure after PK can be defined as an irreversible loss of graft clarity due to endothelial cell failure.18 Repeated corneal graft rejection reactions may lead to graft failure. Previous studies have highlighted risk factors for graft rejection reactions after PK.19,20 The Collaborative Corneal Transplantation Studies Research Group identified a recipient age less than 40 years, a history of corneal graft, a combined surgery, a graft diameter more than 8.0 mm, and recipient corneal neovascularization as significant risk factors for rejection reactions. 19 Rates of graft rejection reaction depend on the series and range from 3.5 to 65%, according to the extent of the recipient corneal neovascularization.20

Posterior Lamellar Keratoplasty Using Tissue Adhesive Corneal transplantation is characterized by an overall high first year graft survival rate that reaches 90%, of cases with only local immunosuppression.16 Some long-term studies reflect that a graft survival in low risk conditions (keratoconus, corneal dystrophies) remains over 90% after 10 years of follow-up. However, the 10-year success rate in high risk recipients (with a history of anterior segment inflammation, corneal neovascularization, etc.) is much lower, achieving less than 35%.16,17 There are other studies in which cases were not divided in low and high risk conditions. These studies reported that the graft survival rate at 5-year follow-up for Fuchs’ dystrophy was 85%, pseudophakic bullous keratopathy 84%, and re-grafts 55% with an overall graft survival of 66%.15 Primary graft failure is a gradual corneal decompensation, unresponsive to corticosteroids, and with no history of a rejection episode. It is the most common cause of late graft failure, causing more than 90% of failures beyond 5 years post-keratoplasty.21 Endothelial cell loss rate is a good indicator of the PK outcome. Endothelial cells are indispensable to retain clear grafts and excellent vision after corneal transplantation. An overall endothelial cell loss of approximately 33% has been reported 1 year after PK,22 and the cell density has been found to continue to decrease at an accelerated rate up to 20 years after surgery.23,24 This finding suggests that after the initial surgical trauma, donor endothelial cell survival is compromised in the host ocular environment.25 Another factor that affects quality of vision after PK is surgery-related astigmatism. Many patients with successful corneal grafts have poor vision postoperatively associated with disabling astigmatism. A full spectacle correction of the postoperative refractive error is often poorly tolerated because of anisometropia. Contact lenses are effective in restoring good vision and binocularity in some but not all patients. 26 Studies report that early postoperative astigmatism levels vary between 3.0 and 7.0 diopters and are rarely less than the baseline preoperative astigmatism in patients without keratoconus.11-13 Other long-term study suggests that the best corrected visual acuity at 5 years is 6/18 in 53% of cases while the mean keratometric astigmatism is 3.4 diopters.15

Posterior Lamellar Keratoplasty Newer techniques of lamellar corneal surgery aim at selective replacement of the diseased endothelium in posterior corneal disorders such as Fuchs’ dystrophy, pseudophakic bullous keratopathy and aphakic bullous keratopathy. These diseases have been the subject of study in the last few years.27-30

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Currently, there are two main approaches for corneal endothelium and posterior stromal transplantation in Posterior Lamellar Keratoplasty. In 1998, Busin and coworkers, inspired by previous work of Barraquer, accessed the posterior stroma by creating an anterior flap with the use of a microkeratome.29 They named this lamellar flap approach as endokeratoplasty (EKP). The other approach is through a sclerocorneal pocket incision that was first described by Melles and co-workers in 199831 (See also Chapter 14, History of Lamellar and Penetrating Keratoploasty).

Microkeratome-assisted Posterior Lamellar Keratoplasty Lamellar corneal surgery has become more popular in the last decade with the development of the laser-assisted in situ keratomileusis (LASIK) and microkeratome instrumentation, now capable of achieving an excellent cut quality.32 The smoothness of the cut surface may lead to a better surgicalt outcome with a clearer interface, which is essential to obtain a good optical result.33 In parallel, posterior lamellar keratoplasty is re-emerging as an alternative to PK in patients with endothelial cell dysfunction. Busin et al reported the use of EKP in seven patients with aphakic bullous keratopathy (n = 2), pseudophakic bullous keratopathy (n = 4), and Fuchs’ endothelial corneal dystrophy (n = 1). To perform the procedure they placed a microkeratome on the cornea. The suction ring and its 160 μm base plate was used to create a central, hinged flap of approximately 9.5 mm in diameter and 160 μm in thickness. The flap was lifted and the central posterior recipient bed excised using a 6.5 mm trephine and corneal scissors. On the donor side, the corneoscleral rims are prepared using two modalities. The first is simple trephination through the endothelial side of a corneoscleral rim by means of a 7.0 mm hand-held trephine. The entire thickness of the donor cornea is transplanted to the recipient. A cellulose sponge is used to gently apply 70° alcohol for few seconds to the button surface. After washing out the alcohol, extreme care is taken to completely remove the donor epithelium from the button surface by means of a blunt spatula. The cardinal sutures are placed, and the flap is replaced and properly aligned. The flap is then sutured in position using a running 8-bite antitorque 10-0 nylon suture. In the second approach the donor corneoscleral rim is placed in an artificial anterior chamber, the chamber is filled with methylcellulose-based viscoelastic material, and an anterior corneal disk is created with a 160 μm thick plate of the microkeratome. The corneoscleral rim is then inverted on a Kaufman trephine block and a 7.0-mm button is obtained by

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trephination from the endothelial side. The obtained partial thickness donor button is placed in the recipient bed, and sutured into position using four cardinal 10-0 nylon sutures. A running 8-bite antitorque 8-0 polyglactin suture is then placed around the circumference of the graft.29 Busin and co-workers concluded that EKP appeared to have the potential to restore sight to eyes with endothelial decompensation and to significantly reduce the time it takes to achieve useful vision. With the use of this technique all patients can be refracted as early as 1 month after surgery, a time when this is usually not possible for PK patients. Although the number of patients included in the study was small and follow-up was short, the technique appeared to hold great promise.

Scleral-pocket Incision Approach The posterior lamellar keratoplasty technique described by Melles et al31 begins by filling the anterior chamber of the donor eye with air through a paracentesis. A 4.0 mm peripheral corneal incision is made, and with a custommade spatula,34 a stromal pocket is dissected across the cornea at 60% stromal depth, using the air-to-endothelial interface as a reference plane for dissection depth.35-37 A plastic strip is inserted into the pocket, and a corneoscleral rim is gently excised from the globe. The rim is mounted endothelial side up onto a punch block, and with a 7.0 or 7.5 mm punch trephine a full-thickness corneal button is excised. The button is placed endothelial side down onto a custom-made, spoon-shaped glide covered with a viscoelastic substance. The anterior lamella and the plastic strip at the lamellar interface are removed, so that a posterior lamellar disk is in situ on the glide.31 In the recipient eye, the anterior chamber is also completely filled with air through a paracentesis. The superior conjunctiva is opened, and a 9.0-mm partial thickness scleral incision is made. With the spatula, a stromal pocket is dissected across the cornea at 80 % stromal depth, using the air-to-endothelium interface as a reference plane for dissection depth. A custom-made, 7.0 or 7.5 mm diameter flat trephine is inserted into the pocket to excise a posterior lamellar disk. After perforation, remaining posterior corneal tissue is cut with custom-made microscissors, and the excised, recipient posterior disk is removed from the eye with fine forceps. The spoon-shaped glide carrying the posterior donor disk is introduced into the recipient stromal pocket, and the ‘same-size’ disk is slid into the recipient posterior opening. The scleral incision is closed with 10-0 monofilament nylon sutures.31 Terry and Ousley slightly modified this 9.0 mm incision stromal pocket technique and renamed it as deep lamellar

endothelial keratoplasty (DLEK).28 Melles and associates further described a modified technique (small-incision DLEK) in a clinical report in which a 5.0 mm scleral tunnel incision is performed in the recipient eye and a stromal pocket is dissected across the cornea, just above Descemet’s membrane, at a visually controlled depth. Then, trypan blue 0.06% is diluted 1:6 with balanced salt solution and injected into the stromal pocket to stain the stromal interface. An 8.5 mm punch trephine is used to make an indentation in the corneal surface epithelium to outline the size of the posterior lamellar disk that is to be excised. Custom-made curved microscissors are used to excise an 8.5-mm diameter recipient posterior lamellar disk. In a whole donor globe a corneal pocket is dissected at 80% stromal depth. Then the corneal disk is excised from the globe, and an 8.5-mm diameter posterior lamellar disk is trephined (endothelium to epithelium). After covering the donor endothelial surface with a viscoelastic material, the posterior lamellar disk is folded with the endothelium at the internal side, using a custom made inserter. After removing all air from the eye, the donor posterior disk is positioned into the recipient anterior chamber. After unfolding the posterior lamellar disk, it is positioned in the recipient posterior opening without suture fixation. The anterior chamber is then completely filled with air, and 5 minutes later all air is exchanged by balanced salt solution. No sutures are used to close the scleral incision.27 A midterm endothelial cell density was evaluated after posterior lamellar keratoplasty. Fourteen consecutive eyes with at least 3 years of follow-up were measured to determine the rate and the pattern of cell loss after this new approach for corneal surgery.38 The comparison was made using two different surgical approaches: in the disks that were implanted into the recipient eye by using a spoonshaped glide covered with viscoelastic or by folding the donor for implantation through a small tunnel incision. In the first group of patients, the endothelial cell counts averaged 2,062 cells/mm 2 (27.6% cell loss) at 1 year postoperatively. Except for two, all eyes had an endothelial cell density of 2,000 cells/mm2 or more.38 A similar result was found by Terry and associates, who performed this procedure with some minor modifications in eight patients.39,40 These findings suggest that these techniques may cause considerable endothelial cell loss, but still within acceptable ranges. In addition, the first group showed a rapid decline of the cell density of endothelial cell density measurements in the following years to 1,126 cells/mm2 at 3 years (cumulative cell loss, 61%). However, this rate of cell loss appears to be similar to that after penetrating keratoplasty (53% SD 19).23,39 In the second group of patients, in which the donor tissue was implanted

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after being folded, the average endothelial cell counts was 1,215 cells/mm2 at 1 year postoperatively.22 Authors finally conclude that donor endothelial cell density after posterior lamellar keratoplasty may be similar to that after conventional full-thickness penetrating keratoplasty.38

Last Advances in Posterior Lamellar Keratoplasty Compared to full-thickness keratoplasty performed for corneal endothelial cell disorders, the 5 mm incision for posterior lamellar keratoplasty may represent an advantage because of: (1) a fast visual recovery with a relatively stable refractive error, (2) no sutures are used and the surgeryinduced astigmatism may be minimized, (3) suture-related complications are eliminated, and the risk of wound dehiscence is reduced. (4) suture removal is not required to monitor the astigmatism, and less frequent follow-up visits may be possible.27 However, this approach is laborious requiring a highly skilled surgeon and the endothelial cells may be damaged during the insertion of the donor tissue through the scleral tunnel.38 The creation of a flap by means of a microkeratome to perform a posterior lamellar keratoplasty (PLK) is easier and faster than the manually dissected slerocorneal approach, and the risk of damaging the endothelial cells may be lower. However, in the corneal flap technique, a minimal number of sutures are required to secure the corneal flap and the transplanted corneal disk, which still may be a disadvantage. Use of corneal sutures has been associated to several drawbacks following corneal surgery. A 5-year retrospective study of 361 grafts between 1993 and 1994 reported erosions over the nylon sutures in 10.8% of cases, infiltrates at suture entrance site in 9.4% and infectious keratitis in 3.3%.41 Similarly, the use of sutures in microkeratome-assisted PLK may induce some astigmatism, although significantly lower and for a shorter period than in classic PK.42 Behrens et al and Li et al previously reported 2 different techniques of PLK using an artificial anterior chamber and microkeratome.42,43 In the first report, they used 8 interrupted 10-0 nylon sutures in the stromal bed to secure the graft.42 In the second study, they used a running graft suture to secure the graft.43 The mean astigmatism in the first group was 3.3 D,42 and 1.47 D in the group in which running graft suture was used.43 During the execution of these two experiments, they observed that the transplanted corneal disk tended to remain attached to the flap stroma without sutures, when the pressure was within physiologic limits and the flap was secured (Behrens, unpublished data). The stromal surface tension at the donor-recipient interface

A

B Figure 34-1: Miyake view (posterior approach) of the cornea with lens and uvea removed. A. White arrows show the line that demarcates the anterior flap edge. Blue arrows show the edge of the transplanted posterior stromal disk, smaller than the flap size. B. Stress exerted to the flap/disk adhesion by partially lifting the flap (blue arrow) with forceps (asterisk). Note that the donor disk maintains its location and adhesion to the anterior stromal interface without sliding.

together with a physiologic intraocular pressure, contribute to keep the disk in place without sliding. With the additional pump of endothelial cells, these adherent forces may be stronger. We have performed the sutureless approach of the donor disk in a Miyake approach to demonstrate the adherence of the donor disk to the flap (Figure 34-1). 44 We therefore started performing microkeratome-assisted posterior lamellar keratoplasty in patients in year 2002, without using sutures to the grafted corneal disk, and only to the corneal flap. Transplant slippage after the surgery was not observed in these cases, and patients showed significant improvement in best corrected visual acuity after surgery, similar to what has been reported previously in the literature (Behrens, unpublished data).

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Modified Microkeratome-assisted Posterior Lamellar Keratectomy (MMAPLK) Piroushmanesh et al44 based on these previous observations developed a novel approach to solve the problem of flap suture-induced astigmatism after microkeratome-assisted posterior lamellar keratoplasty. A 300 µm-thick partial flap keratectomy was performed in 8 human donor corneoscleral rims using an artificial anterior chamber and a manual microkeratome. Differing from previously published techniques, this approach attempts to obtain a wide flap hinge to provide more stability to the whole cornea by reducing the total corneal opening.39,42,45 After lifting the flap in the recipient cornea, a 6.25 mm trephination was performed to remove a disk of posterior stroma, Descemet’s membrane, and endothelium. The donor disk was obtained in a similar fashion from a corneoscleral rim using an artificial anterior chamber with the same trephination size, and positioned in a sutureless fashion in the recipient stromal bed. Only the flaps were secured either with a novel chondroitin-sulfatealdehyde–based adhesive or standard nylon 10-0 sutures. The mean astigmatic change after surgery was 1.13 (SD 0.55)D for the adhesive group vs. 3.08 (SD 0.84) D for the suture group (p=0.008), and the mean resisted bursting pressure in both groups was close to 100 mmHg, without significant difference between groups.44 The use of tissue adhesive without any corneal sutures in this approach simplified the surgical technique, considerably decreased surgical time, and produced less corneal astigmatism. The major concerns of this technique are the stability of the graft because of the reduced support of the sutureless disk in the posterior stroma and the wide anterior opening with the flap. In the first case, the donor disk tends to be more stable compared to other similar approaches [especially that observed in the Descemet’s stripping endothelial keratoplasty (DSEK)], because in this approach we have a recipient bed with perpendicular cuts to the stromal lamellae. This wound configuration may induce a stronger healing than a simple apposition of collagen fibers, which may result in a disk more stable over time. Actual wound healing activity can be observed in perpendicular cuts at histology over time contrasted to the weak adhesion “LASIK style” of the donor disk in the DSEK technique. In the second case, the wider opening is certainly of concern. However, from the experience we have gathered from the two-step LASIK procedure in PK, there might be a stronger and faster wound healing response in these patients, possibly because of the particular wound configuration. During the two-step LASIK after PK, a corneal flap covering the trephination is performed in the first step, and the laser treatment is performed in the second step after stabilization of the surface corneal topography (at least

a month apart). It is common to observe significant adhesion of the flap in the area where the donor-recipient interface is located at the time of the flap lift, only after a few weeks. We believe that a similar event may occur in the microkeratomeassisted posterior lamellar keratoplasty, adding more strength to the wound and therefore, to the whole cornea.

Modified Microkeratome-assisted Posterior Lamellar Keratoplasty Surgical Technique The modified microkeratome-assisted posterior lamellar keratoplasty (MMAPLK) is essentially similar to previously described techniques by Barraquer and others.8,29,42,43 In an attempt to reproduce a more stable postoperative cornea and reduce the dependence on sutures, a wider flap hinge is created in the recipient cornea as a result of a partial flap cut up to the pupillary margin (Figure 34-2). Since the flap hinge will obstruct some of the available stromal area for trephination, a dissection with a blunt spatula underneath the hinge is required to expose the stromal bed (Figure 34-3). After the space is created, a hand trephine smaller than the flap diameter

Figure 34-2: In the recipient cornea, a partial flap of 300 microns is created with a large hinge (arrows) that is just past the pupil but close to the pupillary margin.

Figure 34-3: The area underneath the hinge is dissected with a blunt spatula (arrow) in order to have sufficient space for the trephination of the posterior stroma and diseased endothelium.

Posterior Lamellar Keratoplasty Using Tissue Adhesive

Figure 34-4: A trephine is centered according to the flap margin (black arrow) to allow for some space for the flap to be secured with the adhesive. The hinge area is slightly pushed away with the hand trephine (white arrow) to cut the area that was previously undermined with the spatula.

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Figure 34-6: The donor button of the same size consisting of posterior stroma and healthy endothelium is placed in the recipient bed.

Figure 34-5: The diseased posterior stroma is cut with scissors in the area underneath the flap hinge.

is placed on the stromal bed and a trephination is performed to discard the posterior stroma and diseased endothelial layer (Figures 34-4 and 34-5). A similar disk is obtained from a corneoscleral rim using an artificial anterior chamber, and placed in the recipient bed (Figure 34-6). The flap is then repositioned and a two component adhesive is applied to the flap edge to seal the corneal wound (Figure 34-7). We have observed in the lab that the adhesive tends to efficiently close the wound with comparable sealing capacity to standard sutures. Moreover, the advantage of a complete seal of the wound (flap) and less postoperative astigmatism may be factors of improvement over suture closure. We observed no donor button dislocations in any of the corneas we tested with this approach.

Corneal Adhesive Sutures are the gold standard for corneal incisions and wound repair because of the efficiency and strength of the

Figure 34-7: The flap is repositioned prior confirmation that the bubbles in the interface are removed. Air in the anterior chamber should be left in place, as it helps promoting adherence of the disk (arrows) to the flap. Corneal adhesive (blue) should be applied to the flap edges.

closure. However, sutures may not be the ideal method for wound closure, especially in the cornea. Many corneal surgeons recognize that sutures can be a source of potential problems. Suturing is usually labor intensive and may lead to infection,46 induced astigmatism,47 erosion,48 foreign body sensation, and corneal vascularization,49 among others. In addition, suture removal is required when a nonreabsorbable material is used, which increases the number of patient visits, extends the follow-up period and results in higher costs and inconveniences to the patient. Alternative methods to the conventional closure methods have been investigated for decades.50-54 Particularly in the past few years, we have been testing two novel adhesives for corneal surgery, and both of these compounds are

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biodegradable, which represents an excellent alternative for patient comfort. One of these adhesives is based on chondroitin sulfate, and the other compound is based on collagen, both natural constituents of the cornea.55,56 The chondroitin sulfate aldehyde adhesive was originally developed as an adhesive for cartilage. In initial trials in corneal wounds, it was demonstrated that its adhesive capacity was comparable to nylon 10-0 sutures for clear corneal cataract wounds and for MMAPLK.56 It requires two components to be activated and provide collagen crosslinking. The collagen-based adhesive requires activation by a laser in the 1.45 µm wavelength to induce crosslinking. It showed similar adhesive capabilities when compared to nylon 10-0 sutures for similar wounds. We expect that these adhesives will be available soon for corneal clinical applications, which may be a major advancement for these microkeratome-based corneal transplantation techniques.

Conclusions The various techniques previously described for posterior lamellar keratoplasty are being currently used in clinical applications. One of the major disadvantages of the microkeratome-based approaches is the requirement of suturing for the flap component. However, the use of novel corneal adhesives may improve the results and safety of the procedure, thereby facilitating the adoption of this technique that appears easier to perform. Further in vivo studies are underway to evaluate the biocompatibility of these adhesives and the feasibility of these techniques in the near future.

References 1. Fine M. Lamellar corneal transplants In: Dabezies OH, Citter KA, Saimson CLM (Eds). Symposium on the Cornea. Transactions of New Orleans Academy of Ophthalmology. St Louis: Mosby;1972;204-13. 2. Arentsen JJ. Lamellar grafting. In: Brightbill FS (Ed). Corneal Surgery Theory, Technique and Tissue. St Louis: Mosby; 1993;360-8. 3. Eye Bank Association of America 1997 Eye Banking Statistical Report. Washington DC: Eye Bank Association of America; 1997. 4. Malbran E. La queratoplastia laminar total en el queratocono. Arch Soc Oftalmol Hisp-Am 1964;286-93. 5. Malbran E. Lamellar keratoplasty in keratoconus. In King JH, McTigue JW (Eds): Cornea World Congress 1965. Washington, DC: Butterworth & Co; 1965;511-518. 6. Polack FM. Lamellar keratoplasty: Malbran’s “peeling off” technique. Arch Ophthalmol 1971; 86:293-5. 7. Melles GR. Posterior lamellar keratoplasty. Arch Soc Esp Oftalmol 2002;77:175-6. 8. Barraquer JI. Lamellar keratoplasty. (Special techniques). Ann Ophthalmol 1972;4:437-69.

9. Aiken-O’Neill P, Mannis MJ. Summary of corneal transplant activity, Eye Bank Association of America. Cornea 2002;21:13. 10. Brady SE, Rapuano CJ, Arentsen JJ, Cohen EJ, Laibson PR. Clinical indications for and procedures associated with penetrating keratoplasty, 1983–1988. Am J Ophthalmol 1989; 108:118–22. 11. Maeno A, Naor J, Lee HM, Hunter WS, Rootman DS. Three decades of corneal transplantation: indications and patient characteristics. Cornea 2000; 19:7–11. 12. Williams KA, Ash J, Pararajasegaram P, Harris S, Coster DJ. Long term outcome after corneal transplantation. Ophthalmology 1991; 98:651–7. 13. Riddle H, Parker D, Price F. Management of post-keratoplasty astigmatism. Curr Opin Ophthalmol 1998; 9:15–28. 14. Nagra PK, Hammersmith KM, Rapuano CJ, Laibson PR, Cohen EJ. Wound dehiscence after penetrating keratoplasty. Cornea 2006;25:132-5. 15. Beckingsale P, Mavrikakis I, Al-Yousuf N, et al. Penetrating keratoplasty: Outcomes from a corneal unit compared to national data. Br J Ophthalmol 2006;90:728-31. 16. Williams KA, Muehlberg SM, Lewis RF, et al. The Australian corneal graft registry 1996 report. Adelaide: Mercury Press, 1997;11:47–50. 17. Williams KA, Muehlberg SM, Lewis RF, Coster DF. Long-term outcome in corneal allotransplantation. The Australian Corneal Graft Registry. Transplant Proc 1997;29:983. 18. Bourne WM, Hodge DO, Nelson LR. Corneal endothelium five years after transplantation. Am J Ophthalmol 1994;118:18596. 19. Maguire MG, Stark WJ, Gottsch JD, Stulting RD, Sugar A, Fink NE, Schwartz A. Risk factors for corneal graft failure and rejection in the Collaborative Corneal Transplantation Studies. Ophthalmology 1994;101:1536–47. 20. Khodadoust AA. The allograft rejection reaction: the leading cause of late graft failure of clinical corneal grafts. In: Porter R, Knight J (Ed.): Corneal Graft Failure. Amsterdam: Elsevier, 1973:151–64. 21. Ing JJ, Ing HH, Nelson LR, Hodge DO, Bourne WM. Ten-year postoperative results of penetrating keratoplasty. Ophthalmology 1998;105:1855-65. 22. Culbertson WW, Abbott RL, Forster RK. Endothelial cell loss in penetrating keratoplasty. Ophthalmology 1982;89:600-604. 23. Bourne WM. Cellular changes in transplanted human corneas. Castroviejo lecture. Cornea 2001;20:560-9. 24. Böhringer D, Reinhard T, Spelsberg H, Sundmacher R. Influencing factors on chronic endothelial cell loss characterized in a homogeneous group of patients. Br J Ophthalmol 2002;86:35-38. 25. Nishimura JK, Hodge DO, Bourne WM. Initial endothelial cell density and chronic endothelial cell loss rate in corneal transplants with late endothelial failure. Ophthalmology 1999;106:1962-5. 26. Malecha MA, Holland EJ. Correction of myopia and astigmatism after penetrating Keratoplasty with laser in situ keratomileusis. Cornea 2002;21:564-9. 27. Melles GR, Lander F, Nieuwendaal C. Sutureless posterior lamellar keratoplasty: A case report of a modified technique. Cornea 2002;21:325-7. 28. Terry M, Ousley PJ. Endothelial replacement without surface corneal incisions or sutures: Topography of the deep lamellar endothelial keratoplasty procedure, Cornea 2001; 20:14-18. 29. Busin M, Arffa RC, Sebastiani A. Endokeratoplasty as an alternative to penetrating keratoplasty for the surgical treatment of diseased endothelium. Ophthalmology 2000;107:2077-82. 30. Melles GR, Lander F, van Dooren BT, Pels E, Beekhuis WH. Preliminary clinical results of posterior lamellar keratoplasty through a sclerocorneal pocket incision. Ophthalmology 2000; 107:1850-7.

Posterior Lamellar Keratoplasty Using Tissue Adhesive 31. Melles GR, Eggink FA, Lander F, Pels E, Rietveld FJ, Beekhuis WH, Binder PS. A surgical technique for posterior lamellar keratoplasty, Cornea 1998;17:618–26. 32. Behrens A, Langenbucher A, Kus MM, Rummelt C, Seitz B. Experimental evaluation of two current-generation automated microkeratomes: The Hansatome and the Supratome. Am J Ophthalmol 2000;129:59-67. 33. Haimovici R, Culbertson WW. Optical lamellar keratoplasty using the Barraquer microkeratome. Refract Corneal Surg 1991;7:42-5. 34. Melles GR, ten Hoope GW, Rietveld FJ, Beekhuis WH, Binder PS. Depth predictability of stromal pockets in the posterior cornea. Cornea 1998;17:174–9. 35. Melles GR, Rietveld FJ, Beekhuis WH, Binder PS. A technique to visualize corneal incision and lamellar dissection depth during surgery. Cornea 1999;18:80–6. 36. Melles GR, Lander F, Rietveld FJ, Remeijer L, Beekhuis WH, Binder PS. A new surgical technique for deep stromal, anterior lamellar keratoplasty. Br J Ophthalmol 1999;83:327–33. 37. Melles GR, Remeijer L, Geehards AJ, Beekhuis WH. The future of lamellar keratoplasty. Curr Opin Ophthalmol 1999;10: 253-9. 38. Van Dooren B, Mulder PG, Nieuwendaal CP, Beekhuis WH, Melles GR. Endothelial cell density after posterior lamellar keratoplasty (Melles technique): 3 years follow-up. Am J Ophthalmol 2004;138:211-7. 39. Terry MA, Ousley J. Replacing the endothelium without corneal surface incisions or sutures. Ophthalmology 2003;110:755-64. 40. Melles GR, A disagreement. Ophthalmology 2004;111:193. 41. Christo C, van Rooij J, Geerards AJ, Remeijer L, Beekhuis WH. Suture-related complications following keratoplasty. A 5-Year retrospective study. Cornea 2001;20:816-9. 42. Behrens A, Ellis K, Li L, Sweet PM, Chuck RS. Endothelial lamellar keratoplasty using an artificial anterior chamber and a microkeratome. Arch Ophthalmol 2003;121:503-8. 43. Li L, Ellis KR, Behrens A, et al. Laboratory model for microkeratome-assisted posterior lamellar keratoplasty utilizing a

44.

45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56.

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running graft suture and a sutureless hinged flap. Cornea 2002;21:192-5. Pirouzmanesh A, Herretes S, Reyes J, Suwan-apichon O, Chuck RS, Wang DA, Elisseeff JH, Stark WJ, Behrens A. Modified microkeratome-assisted posterior lamellar keratoplasty using a tissue adhesive. Arch Ophthalmol 2006;124:210-4. Azar DT, Jain S, Sambursky R, Strauss L. Microkeratomeassisted posterior keratoplasty. J Cataract Refract Surg 2001; 27:353-6. Heaven CJ, Davison CR, Cockcroft PM. Bacterial contamination of nylon corneal sutures. Eye 1995;9:116-8. Azar DT, Stark WJ, Dodick J, et al. Prospective, randomized vector analysis of astigmatism after three-, one-, and no-suture phacoemulsification. J Cataract Refract Surg 1997;23:1164-73. Dana MR, Goren MB, Gomes JA, Laibson PR, Rapuano CJ, Cohen EJ. Suture erosion after penetrating keratoplasty. Cornea 1995;14:243-8. Nirankari VS, Karesh JW, Richards RD. Complications of exposed monofilament sutures. Am J Ophthalmol 1983;95:515-9. White RA, Kopchok G, Donayre C, et al. Comparison of laser welded and sutured aortomies. Arch Surg 1986;121:1133-5. White JV. Laser tissue repair with the CO 2 laser. Proc SPIE 1989;1066:35-40. Oz MC, Bass LS, Popp HW, et al. In vitro comparison of THC:YAG and argon ion lasers for welding of biliary tissue. Lasers Surg Med 1989;9:248-53. White RA, Kopchok G, Donayre C. Argon laser welded arteriovenous anastomoses. J Vasc Surg 1987;6:447-53. Stanley CM, Boisjoly H. Advances in the use of adhesive in ophthalmology. Curr Opin Ophthalmol 2004;15:305-10. Noguera GE, Lee WS, Castro-Combs J, Chuck RS, Soltz B, Soltz R, Behrens A. A novel laser-activated solder for sealing corneal wounds. Invest Ophthalmol Vis Sci (in press). Reyes JM, Herretes S, Pirouzmanesh A, Wang DA, Elisseeff JH, Jun A, McDonnell PJ, Chuck RS, Behrens A. A modified chondroitin sulfate aldehyde adhesive for sealing corneal incisions. Invest Ophthalmol Vis Sci 2005;46:1247-50.

Novel Approach for Corneal Endothelial Cell Transplantation

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Naima B Jacobs-El Juan M Castro-Combs Ashley Behrens

Novel Approach for Corneal Endothelial Cell Transplantation using Descemet Membrane as a Carrier

35

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Corneal Endothelial Transplant

Introduction Penetrating Keratoplasty For over one hundred years, penetrating keratoplasty (PK) has been the standard of care for corneal diseases.1,2 PK is commonly indicated for failed corneal grafts, pseudophakic bullous keratopathy (PBK), phakic keratopathy, Fuchs’ dystrophy, keratoconus, and corneal scars.3 Being the most commonly performed transplant, the procedure offers patients with these diseases improved vision by way of clearer corneas.4 PK is also practical because it is an “open sky” procedure which allows for concurrent intraocular procedures (i.e. cataract extraction and IOL placement, vitrectomy, iridoplasty, etc.) to be carried out. PK involves replacing a full thickness, circular portion of recipient cornea with that of a donor cornea secured into place with 32, 16 or even 8 sutures. The procedure is widely accepted by corneal surgeons, as it requires the use of familiar surgical skills and instrumentation; these qualities afford the procedure technical simplicity with excellent chance of success. The development of topical corticosteroids, antibiotics, surgical microscopes, improved trephines, viscoelastics and fine suture materials enable this delicate procedure to be routinely performed with the prospect of success.1 Suture technique has also been modified several times to help improve the outcomes of this surgery. One of the major advancements of corneal transplantation lies in the changes in management of donor tissue by eye banks, which are now able to screen and handle donor tissue in such a fashion that recipient wait time has been significantly reduced. The average maximum Snellen visual acuity after PK is an important measure because it puts into perspective the probability of 20/20 vision. Several studies have indicated that 20/20 best corrected visual acuity (BCVA) is a likely outcome after PK. Claesson et al,5 in their study of 520 grafts at 2 years after PK, showed a strong association between preoperative visual acuity and the likelihood of obtaining postoperative BCVA of 20/40 or better, which is comparable to that found in other studies.6,7 Another important postoperative measure of PK is the corneal power because a crystal-clear graft with excessively steep or flat curvature can yield significant myopia or hyperopia. One study found that on average, the postoperative corneal power ranges from 41.9 to 42.7 diopters (D) and is stable from one month after surgery up to suture removal; after suture removal the graft steepens slightly at 0.61-0.7 D.8 Although these averages are reassuring, it is common to observe that corneal astigmatism after suture removal often fluctuates considerably.9

Similarly, an important measure of endothelial graft function is the endothelial cell count. Ing JJ et al10 showed a decline in endothelial cell count after PK in large scale study, with means of 2467/mm2 at 2 months, 1958/mm2 at 1 year and 960/mm2 at 10 years. This endothelial cell data is comparable to that found in other studies.11-14 These figures serve as the standard for any procedures that may attempt to replace PK. PK sometimes produces an unpredictably high or irregular astigmatism which can lead to poor postoperative refraction. Early postoperative astigmatism measures range from 3 to 7 D.14-17 Williams et al study on postoperative astigmatism at 2 or more years in 60 patients showed that 38% of cases had > 5 D of astigmatism in the graft.15 Overall, in approximately 10-20% of PK cases, high amounts of astigmatism (>5.0 D) can actually prevent functional success of the clear graft.17 Most patients can be visually rehabilitated with spectacles or contact lenses, however, if these modalities fail, several surgical and suture adjustment/removal options exist that may improve the patients vision. A number of factors are thought to contribute to the vexing problem of PK induced astigmatism, one of which is suture tension. The tension necessary to achieve a watertight seal of the full-thickness vertical incision of PK produces a gathering of the donor-recipient junction. This suture-induced tissue stretching yields a topographically “unsmooth” corneal surface that has higher and/or more irregular astigmatism than either the donor or recipient corneas had preoperatively. Irregularities of the trephination margins, donor/recipient thickness disparity and irregular suture technique are other important factors for high astigmatism after penetrating keratoplasty.17,18 Interestingly, the full-thickness vertical incision of PK apparently never heals to preoperative levels of strength. This is supported by the fact that many years after a corneal transplant, a seemingly stable cornea can rupture with trivial trauma leading to possible loss of the eye.19 This is likely because blood vessels are necessary to meet the nutritional needs of the wound healing process, but the cornea is avascular and does not adequately supply these needs. Furthermore, slow recovery of vision,13,20 risk of suture related problems,21 and other interface complications,22 are all untoward outcomes that plague corneal surgeons. The leading causes of corneal transplant failure are allograft rejection and endothelial decompensation.23-27 Since the cornea is usually avascular, the overall cumulative probability of corneal graft rejection at 10 years

Novel Approach for Corneal Endothelial Cell Transplantation is 21%.10 Most of these rejections occur within the first few years after keratoplasty. 28 Once a graft rejection is suspected, it can sometimes be blocked by the use of topical corticosteroids, but up to 49% are irreversible.29

Posterior Lamellar Keratoplasty While PK remains the gold standard for surgical corneal diseases over the past decade, posterior lamellar keratoplasty (PLK) has gained increasing clinical acceptance in the treatment of corneal diseases that involve endothelial dysfunction. This is groundbreaking for corneal surgeons because corneal endothelial dysfunction is the most common indication for PK in developed countries. 30-32 PLK selectively targets the corneal endothelium by replacing some contiguous circular portion of recipient endothelium, Descemet’s membrane and posterior stromal tissue with that of donor tissue. In this manner, the functional donor endothelial cells are able to appropriately transport fluid and solutes away from the stroma leading to an optically transparent cornea. PLK offers several advantages over PK. The concept of PLK was first introduced by Barraquer wherein the posterior corneal tissue was accessed by creating a hinged anterior corneal flap. From this conception, two separate PLK techniques evolved: one which improved upon this hinged-flap approach and another which changed the approach such that the recipient’s anterior chamber is entered from a smaller curvilinear incision. The hinged-flap approach was named microkeratome-assisted PLK (MAPK also called endokeratoplasty and endothelial lamellar keratoplasty) while the small incision approach was designated deep lamellar endothelial keratoplasty (DLEK). The MAPK approach is technically straightforward, wherein a microkeratome is used to create a hinged corneal flap exposing the underlying posterior stroma which is then trephined and transplanted. Though promising results have been obtained, case series have shown that microkeratome-assisted PLK carries the possibility of flap problems in addition to several of the same problems that plague PK and DLEK. The DLEK approach was developed by Melles et al33 who proposed a limbal incision approach to manually dissect a pocket in the midstroma and then trephine the posterior stroma. In 2000, Terry et al31,34 slightly modified Melles’ technique re-designing the instrumentation and performed the first DLEK in the United States. Due to the high difficulty level and other concerns, DLEK has been the subject of several investigations and modifications over the past six years.

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A dilemma common to both MAPK and DLEK is postoperative mismatch between donor and recipient stromal thickness. Even if there is a perfect preoperative match between the thickness of the donor tissue and that of the recipient’s, a discrepancy may be measured with the passage of time. This discrepancy occurs because the thickness of donor tissue in corneal preservation media is greater than the thickness of normal physiological corneal tissue due to edema. Thus, if tissue deturgescence is not done before the donor disk is prepared, the donor stroma may become thinner than anticipated during the postoperative period when the donor’s endothelial cells begin to function. This thinning of the donor disk may cause same degree of corneal flattening which can potentially lead to poor postoperative refraction.35 Consequently, donor tissuedeturgescence is usually calculated preoperatively to lessen or possibly prevent unanticipated postoperative donor thinning. Another problem shared by PLK and MAPK is the issue of haze in the horizontal interface between the donor and recipient stroma.36 This distortion is thought to lead to a limited maximum average Snellen visual acuity that can be attained with these procedures. While PK can attain visual acuities of 20/20, there has been a relative dearth of these cases in MAPK and DLEK; the average highest acuity attained in these procedures has been 20/40 to 20/50.37-42 Terry et al. had only 1 patient who could be fully correctable with spectacles to 20/20 vision in this DLEK series of 98 cases.37 What is interesting is that there is not yet a means for measuring this optical distortion of the transplant interface, and thus several investigations are underway. Clearly, PLK has not yet been fully standardized and will continue to be modified and improved upon. Any procedure that replaces the endothelium ideally should accomplish the following goals: 1. Smooth surface topography without significant change in astigmatism from preoperative to postoperative. 2. A highly predictable and stable corneal power. 3. A healthy donor endothelium that resolves all edema. 4. A tectonically stable globe, safe from injury and infection. 5. An optically pure cornea. An additional sixth goal is technical ease with a reasonable learning curve such that a wide array of corneal surgeons can successfully carry out the procedure.34 The further the instrumentation and technique of any given procedure strays from those classically used by corneal surgeons, the greater the technical difficulty. With technical difficulty comes increased inaccuracies, which can yield intersurgeon variability in success rates and possibly unhappy patients. These 6 goals will be referred to throughout the remainder of the text.

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Microkeratome-assisted PLK (MAPK) In microkeratome-assisted PLK, the opening incision is a hinged anterior stromal flap made with a microkeratome, similar to the flap made during laser-assisted in situ keratomileusis (LASIK) procedures. This flap approximates 130-480 μm in thickness and 8.5-9.5 mm in diameter. The flap is retracted with a flat spatula and it is used to expose the remaining cornea which is then trephined with a 7.08.0 mm diameter trephine to create a lamellar disk (posterior stroma, Descemet’s membrane and endothelium). This trephine diameter is dependent on the flap diameter and hinge width. This recipient lamellar disk carries with it the dysfunctional endothelial cells and is accordingly removed from the eye. Viscoelastic is then placed in the anterior chamber to stabilize it. The donor disk is then prepared (as will be described later) with a similar diameter trephine and positioned into place in the recipient’s stromal bed. The donor-recipient interface is self-adhering, but 8-16 interrupted absorbable or non-absorbable nylon sutures are used to secure the donor disk into place.43 In clinical studies, 50% of patients had a BCVA greater than or equal to 20/60 (range: 20/30-20/200) at a maximum of 1 year of follow up.43,44 One study reported a 1 month average spherical equivalent of -1.25 D.43 At twelve months, Ehlers et al44 found that the endothelial cell density was in the range of 1200-2300 cells/mm2. None of these studies had episodes of graft rejection or wound dehiscence. An important principle of lamellar refractive and LASIK surgeries is that in order to preserve the tectonic integrity of the cornea, a minimum of half or more corneal tissue must remain after the creation of a flap to prevent refractive instability. As this principle is abided by during the MAPK procedure, Behrens et al.32 showed that average intraocular pressures of up to 88 mmHg could be withstood in laboratory eyes when a 200 μm flap is used for the MAPK procedure. This stability renders the eye resistant to any subsequent injuries that may take place during patient’s daily activities. As microkeratomes have been enhanced, optical properties of corneal flaps have greatly improved. Interface scarring is almost absent after microkeratome dissection in LASIK. When compared with manual dissection, microkeratomes create a smoother donor-recipient interface and more uniform depth than manual dissection. Interestingly, although interface scarring is essentially absent, there is a limit to the maximum best average Snellen visual acuity seen in the small clinical series of MAPK. LASIK has proven that corneal dissection performed by

means of microkeratome produces an optical quality which is compatible with 20/20 vision, thus further investigations are needed to determine why the average maximum visual acuity is limited with MAPK procedure. In comparing PK to MAPK, it is widely accepted that both procedures accomplish goals 5 and 3 above. Both procedures also accomplishes goal 6 of technical ease with a very reasonable learning curve. However, as was indicated previously, PK falls short in terms of goals 1, 2, and 4. Although MAPK preserves the original central corneal surface, when flap and intrastromal sutures are put into place, some astigmatism is induced. The MAPK astigmatism is not nearly as high or irregular as that of PK because less sutures are used; thus, goal 1 is relatively (compared to PK, but not to DLEK as we will see later) achieved by the MAPK procedure. Similarly, literature data shows that the MAPK procedure achieves goal 2. Short term studies have indicated that MAPK also achieves goal 4, but large scale studies are needed to determine if goal 4 still holds true over a longer period of time. The MAPK method has less wound complications than PK because the combined anterior flap/posterior trephine wound is inherently stronger than the full thickness/ complete circumferential wound used in PK. Furthermore, the MAPK flap can be lifted for suture removal (with an argon laser to cut intrastromal sutures or a sharp blade after flap elevation), and although speculative, LASIK over the posterior button may correct residual refractive errors.45 In addition, the decreased amount of sutures used during this procedure improves postoperative visual recovery as well as imparts fewer suture-related problems as compared to PK. Clearly, MAPK specifically has potential benefits, mostly related to its conventionality. It allows for easy automated access to the stromal bed as well as classic trephination and transplantation, making the procedure relatively reproducible.46 This procedure also allows easy access for concurrent intraocular surgeries. Furthermore, with the ease and automation of surgery, MAPK is afforded a shortened surgical time. Some possible pitfalls are also incurred specifically with MAPK. This procedure produces a shift in postoperative average corneal power and astigmatism, similar to the transfer of stromal flattening to the corneal surface in LASIK. In MAPK there is a donut effect of donor-recipient interface due to sutures, which is transmitted to the surface flap. Surface sutures may also contribute to this astigmatism and high absolute power. As with LASIK, flap complications may also occur; epithelial ingrowth in the flap-graft interface can decrease the BCVA and has been reported in clinical cases of MAPK.47 As with LASIK, it is suspected

Novel Approach for Corneal Endothelial Cell Transplantation that corneal melting and micro/macrostriae are also possible adverse outcomes of MAPK.

Deep Lamellar Endothelial Keratoplasty (DLEK) In the DLEK method, a temporal or 12 o’clock curvilinear incision (either 9.0 mm or 5.0 mm) is made into the sclera, 1 mm away from and parallel to the limbus. A specialized spatula is then used to dissect from this incision into the stroma to a corneal depth of about 75-80% so as to create a lamellar pocket. Once the desired depth is reached, a special dissector is used to extend this lamellar plane over the whole cornea. A specialized 7.0-8.0 mm interlamellar trephine is then inserted into the created plane to begin cutting out a lamellar disk (posterior stroma, Descemet’s membrane and endothelium) from the posterior cornea. Once the trephine slightly enters the anterior chamber it is withdrawn from the lamellar plane and specialized interlamellar scissors are inserted to complete the trephination to a full 360 degrees. This completes the creation of the recipient’s posterior corneal lamellar disk which is removed from the eye through the temporal incision. The resultant recipient bed diameter is then measured with external calipers (7.08.0 mm). Air is then insufflated into the anterior chamber through a 0.5 mm limbal stab wound at the 2 o’clock position. This air functions to create a fluid free working space for careful insertion and manipulation of the donor tissue and to enhance self-adhere of the donor tissue to that of the recipient. The donor lamellar disk is then prepared (as will be described later) to match the diameter of the recipient bed and viscoelastic is layered on it’s endothelium. The donor disk is then either folded in a taco-like fashion (endothelial side inside) or placed endothelial side down on a specialized transfer spatula (decision depends on the size of the scleral incision), and inserted into the host anterior chamber through the scleral incision. The donor disk is positioned into the recipient’s posterior corneal defect using the transfer spatula. Minor adjustments to further fit the donor into the recipient bed can then be made with a Sinskey hook through the 2 o’clock limbal stab wound. In this method, no sutures are necessary to secure the donor tissue because it is self-adhering. The scleral incision is also selfsealing and sutures may or may not be used to close it. Several corneal surgeons have performed large volumes of DLEK procedures and sufficient data is available. On average, BCVA is 20/48 at maximum 2 years follow up.48-51 Melles et al reported that all patients operated on

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with the initial PLK procedure who did not have concomitant ocular disease have a BCVA of 20/30 or better; several have 20/20 (Netherlands Institute for Innovative Ocular Surgery, unpublished data). Similarly, these same studies also showed that DLEK yields minimal postoperative astigmatism; on average, astigmatism is 1.46 D. One study showed an average postoperative spherical equivalent of -0.369 D at 1 year.50 Postoperative endothelial cell density averages 1790.5 cells/mm2 at 36 months at a maximum of 3 years.48,51 As with MAPK, there appears to be a limit to the maximum best average Snellen visual acuity with DLEK; this is likely associated with postoperative interface opacities caused by stromal scarring from manual dissection.52 The lack of significant postoperative changes in astigmatism and corneal power is due to lack of disruption of the surface of the cornea in this procedure. This eliminates postoperative need for special corrective contact lenses which is seen with PK. Tectonic stability of the eye is in part provided by the continuity of the cornea and limbus. The DLEK technique does not disrupt this continuity and consequently appears to leave the cornea stable, though there are no long-term studies to confirm this. In Terry et al34 laboratory study stability of completed transplant was crudely tested by manually shaking and “pounding” on the globe which did not dislocate the donor disk from the recipient bed. This apparent increased stability renders the eye more resistant to any subsequent injuries that may take place during patient’s daily activities. The lack of sutures to secure the donor into place imparts some risk of graft dislocation with the DLEK procedure. Terry experienced a 6% donor detachment rate in his first 90 DLEK cases (Ophthalmology Times, May 15, 2005). Sano reported 1 donor detachment in his first 3 DLEK cases.49 Price had a 5% detachment rate in his first 101 PLK surgeries.53 In comparing PK to DLEK, it is widely accepted that both procedures accomplish goals 5 and 3 above. PK also accomplishes goal 6 of technical ease with a very reasonable learning curve, but it falls short in terms of goals 1, 2, and 4 (the reasons for this were discussed earlier). In contrast, DLEK leaves the corneal surface untouched and consequently achieves goals 1 and 2. Short-term studies have indicated that this procedure achieves goal 4, but large scale studies are needed to determine if goal 4 still holds true for DLEK over a longer period of time. Clearly, DLEK specifically has potential benefits, mostly due to its lack of corneal incisions and suture use. There are less suture-related complications as compared with PK

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because the sutures are placed on the sclera rather than the cornea. Most outstanding, there is negligible postoperative astigmatism or changes in corneal power because the corneal topography is essentially unchanged. In addition, because DLEK leaves the corneal surface untouched, the recovery time is considerably hastened such that the astigmatic and corneal power advantages are seen immediately after surgery. Moreover, unlike PK and MAPK, no postoperative suture removal visits are necessary which keeps visual fluctuations at a minimal during the healing process. However, some possible pitfalls are also incurred specifically with DLEK, which largely lie in it’s difficulty level. During the DLEK procedure, manual lamellar dissection, interlamellar trephination and scissor excision are difficult and tedious skills which could potentially traumatize the anterior chamber structures or the lens. Similarly, graft folding, and transplantation of donor corneal disk supported only by an air bubble are difficult surgical skills. The accuracy (and thus success) of these techniques is strongly dependent on the surgeon’s skills, making the procedure difficult to reproduce because the learning curve is quite steep. On the other hand, Seitz et al have demonstrated in a laboratory model the feasibility of using the femtosecond laser for DLEK to create both the opening incision as well as the stromal dissection, which may allow an easier clinical procedure.55 Other authors have also corroborated this with further experimental studies.56-58

Donor Tissue Preparation For both the MAPK and DLEK methods, donor tissue is prepared a few different ways using an artificial anterior chamber (AAC). In one method, the donor corneoscleral button is mounted on the AAC endothelial side down and a lamellar pocket is dissected similar to the DLEK recipient procedure. Then the button is turned epithelial side down and trephined at a diameter that is dependent on the measured diameter of the recipient posterior lamellar disk. The donor posterior disk is then separated from its anterior corneal layers and used as indicated in the respective procedures. In another method, the donor corneoscleral button is mounted onto the artificial anterior chamber endothelial side down and trephined to approximately 75% depth with a 9.0 mm diameter suction recipient trephine. A special blade and dissector are then used to carefully excise the 9.0 mm anterior corneal disk and to extend the trephinate depth peripherally throughout the remainder of the corneal diameter. The donor tissue is then removed from the anterior chamber and placed endothelial side up on a donor

punch block where it is trephined at a diameter that is dependent on the measured diameter of the recipient posterior lamellar disk. The donor posterior disk is then carefully separated from its anterior corneal layers and used as indicated in the respective procedures. Yet another method involves the use of a microkeratome wherein a flap is made (as in the MAPK method) and a posterior corneal disk is trephined and used as indicated in the respective procedures. Femtosecond laser preparation of donor tissue from the endothelial side has been done in the laboratory and shows promising results.57 The concerns with donor preparation techniques lie in ensuring endothelial cell survival and technical ease. All procedures have shown comparable endothelial cell survival and no particular method is favored over the other in terms of standardization. Note that viscoelastic is used liberally on the endothelium in all donor preparation techniques.

Descemet’s Stripping with Endothelial Keratoplasty (DSEK) A very revolutionary refinement of the DLEK procedure was realized when Melles et al proposed a means of stripping Descemet’s membrane from the recipient’s stromal bed so as to create a smooth recipient surface. This smooth recipient surface would theoretically decrease the interface haze that was incurred with the DLEK technique so that there would no longer be a limited average Snellen visual acuity.59-61 Price slightly modified Melles’ technique and was the first surgeon in the United States to carry out such a procedure.62 In this procedure, there are two methods available, manual dissection and microkeratome-assisted which has been coined Descemet’s stripping automated endothelial keratoplasty (DSAEK). During manual dissection, the donor is mounted epithelial side up on an AAC and dissected 8090% corneal depth over the whole area of cornea with blades.61 In the DSAEK donor preparation method, a microkeratome is used to dissect to a 300-350 μm stromal plane. In both techniques, the donor tissue is then placed endothelial side up on a donor punch block and trephined at 8.0-9.0 mm diameter. The donor disk is then placed in storage media for later use. After marking the recipient cornea with a slightly larger trephine than used for donor tissue, the recipient surgery is then performed. Through a 5.0 mm scleral tunnel, modified Price-Sinskey hook is used to score Descemet’s membrane in a circular pattern beneath the area of the epithelial reference mark. Then, a 45 or 90-degree Descemet’s

Novel Approach for Corneal Endothelial Cell Transplantation stripping instrument is used to strip Descemet’s membrane and endothelium from the recipient’s stroma within the scored area. Once fully stripped, Descemet’s membrane and the endothelium were removed from the anterior chamber with a forceps. The donor disk is then folded and transplanted into the recipient stromal bed as was done in the graft folding method of DLEK. DSEK offers all of the benefits of DLEK in addition to technical ease and less trauma to anterior chamber structures.62 This procedure achieves goals 1 through 6 of the ideal corneal transplant procedure and recovery of useful vision occurs within weeks. Furthermore, in DSEK, there is no problem with donor/host thickness mismatch because Descemet’s membranes with its endothelial cells are not subject to edematous changes and consequently are all approximately the same thickness. Six months after DSEK, mean manifest cylinder was 1.5 ± 0.94 diopters, basically unchanged from the preoperative value of 1.5 ± 1.0 D. Mean manifest spherical equivalent refraction was 0.15 ± 1.5 D, also statistically comparable to the preoperative value. Preoperative mean BSCVA was 20/100. Statistically significant improvement in BSCVA was noted at the 3-month and 6-month examinations; six months after DSEK, 62% of the eyes refracted to = 20/40 and 76% saw = 20/50.63 A further study in DSEK compared microkeratomedissected and manually dissected donor tissue.64 Mean refractive astigmatism was 1.5 D preoperatively and 6 months postoperatively in both groups. Spherical equivalent refraction did not change in the microkeratome group but increased by 0.66 D in the hand dissection group. There were 7 primary graft failures. The major drawbacks of DSEK lie in graft dislocation. It was theorized in the DLEK procedure that good adhesion of donor to recipient tissue is dependent on inherent adhesive quality of bare stromal surfaces when pressed together when assisted by intraocular pressure and stromal bed dissection,65 however, in DSEK there is no stromal bed (perpendicular cuts) thus dislocation may occur. The dislocation rate of DSEK has been quoted as high as 50%,63 making it a significant area of investigation for corneal surgeons. Several successful efforts have been made to improve this high dislocation rate.64 Using techniques to remove fluid from the donor–recipient graft interface ultimately reduced the detachment rate to <1% (1 in the last 140 cases). Another method is to leave the anterior chamber filled with air for 8 minutes in the operating room, after which most of the air was removed; patients were then sent to the recovery room to lie with their face up for 30 to 60 minutes to allow the remaining air to push the donor tissue up against the

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recipient cornea. Furthermore, the corneal surface can be massaged with a Lindstrom LASIK roller to help remove fluid from the graft interface to help prevent dislocation. In the event of a dislocated or malpositioned graft, a repositioning procedure is undertaken. In this procedure, some fluid is drained from the anterior chamber and air is again insufflated into the chamber. The graft is then manipulated back into place through a virgin posterior limbal wound. Postoperatively, the air in the anterior chamber is allowed to further enhance the donor-recipient corneal adherence. In the event of graft failure, the patient’s eye must be reoperated on to implant a new donor graft. There are also other challenges incurred with the DSEK technique. In some eyes it may be more difficult to position the graft under the support of an air bubble. In addition, since Descemet’s membrane/endothelium is very thin and friable, donor preparation may be a difficult task with the looming risk of perforation especially if a deepcutting microkeratome is used such as a 350 µm head. In some cases, Descemet’s membrane fragments during Descemetorhexis. In efforts to remedy this risk Price et al, compared microkeratome donor preparation to manual dissection. 64 His group found that microkeratome dissection significantly reduced the risk of donor tissue perforation, provided faster visual recovery after DSEK, and did not alter the refractive outcome. Furthermore, the incidence of early graft failures was also significantly less with use of microkeratome-dissected tissue (0.5%), compared with use of hand-dissected tissue (5%). Thus, microkeratome-dissection has pretty much replaced the manual dissection technique, though further investigations are still underway to further improve the delicate donor harvesting process.

What Comes Next? Descemet’s Membrane and Endothelial Cell Layer Transplantation From our experience with lamellar corneal surgery (both anterior and posterior) and the results published in the literature, optical performance in both seems to be affected by factors not yet clearly understood. The final BCVA in both approaches appears to be consistently limited despite appropriate graft clarity, good slit-lamp appearance, and optimal postoperative topography. It seems that there are certain optical properties of the cornea that are not quite measurable with conventional methods in these patients, which still restrict their ultimate visual resolution. However, we have hypothesized, based on certain facts

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and observations, that the cornea has particularities in its architecture that should not be disregarded. The BCVA in patients after keratolamellar transplantation is usually 20/40 or 20/30. We hypothesize that this limitation is due to optical distortions originated in the different distribution and orientation of collagen fibers from different corneas when apposed in a transplant. Seitz et al reported a few years ago the necessity of “harmonization” of the donor and recipient corneas in penetrating keratoplasty to minimize the postkeratoplasty corneal astigmatism.66 We believe that this “harmonization” may be even more determinant in cases of lamellar surgery, although perfect orientation of the cornea might still not solve the problems related to specific collagen fiber distribution encountered in corneas from different patients. However, transplantation of endothelial cell layer and Descemet’s membrane alone may warrant these distortions to be avoided. In the current literature we found clinical data to support our hypothesis. In addition, surgical observations during LASIK surgery may validate our impressions. Trials reporting excellent visual acuity after performing the procedure of “deep lamellar keratoplasty” up to Descemet’s membrane level are available in the literature. In this technique, the whole epithelium, Bowman’s layer and stroma are removed from the recipient cornea, leaving the Descemet’s membrane and endothelium intact.67,68 This fact speaks of the minimal optical distortion induced by Descemet’s membrane and endothelial cell layer alone. On the other hand, it is widely accepted that “free” caps after LASIK surgery may create severe optical problems when not repositioned in the exact original orientation. It seems that, other than the expected astigmatic change induced by different thicknesses in the cap, there is an additional optical distortion created by the change in orientation of the collagen fibers, which affects the BCVA. Based on the previous observations, we propose that the ideal treatment for endothelial cell dysfunction would be the replacement of this layer alone. Endothelial cell transplantation is technically difficult in present time, since these cells are very labile and would be challenging to seed them uniformly on the posterior part of the cornea using a surgical approach. However, our approach is based on the transference of these cells using a special carrier that could be thin enough to permit the passage of light without distortions. The natural candidate as a carrier in this case is the Descemet’s membrane, and certain properties of the membrane make it possible to obtain an intact detachment of the structure for endothelial cell transplantation. We have previously published a study describing a technique to dissect the Descemet’s membrane while preserving the viability of most endothelial cells.69

Harvesting of the Descemet’s Membrane with Endothelial Cells To prepare the donor tissue, the corneoscleral button is placed on the metal base of a commercially available artificial anterior chamber (ALTK system, Moria, Antony, France) with the endothelial side up (Figure 35-1). Negative pressure is then applied within the artificial anterior chamber so that the donor corneoscleral button takes on a concave shape (in reference to the endothelium) and the cornea maintains its centration in the chamber due to the suction from central opening (Figure 35-2). It is important

Figure 35-1: The corneoscleral disk is placed upside down on the metal base of the artificial anterior chamber as the first step of the Descemet membrane harvesting.

Figure 35-2: Suction is performed through the opening of the artificial anterior chamber (folds in the stroma are shown with the arrows) to secure the corneoscleral disk in place when the second ring is placed.

to cover the endothelial surface with viscoelastic material to protect the cells during the whole procedure. A hand trephine is used to create a circumferential cut of Descemet’s membrane just inside the Schwalbe’s line and into a small portion of the posterior stroma (Figures 35-3A and B). Positive pressure is then applied to the AAC such that the cornea takes on a convex shape (in reference to the

Novel Approach for Corneal Endothelial Cell Transplantation

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A Figure 35-4: Margins of the Descemet’s membrane (arrows) are peeled off with the bent tip of a 27-gauge needle.

B Figures 35-3A and B: (A) Prior partial thickness trephination, viscoelastic material is placed on the surface of the endothelium to protect the cells (arrow). (B) Trephine is placed on the endothelial surface to mark the area to be dissected for the Descemet transplant.

endothelium). This maneuver allows the margins of the trephination to become clearly visible to facilitate easy separation of the trephined Descemet’s membrane (Figure 35-4). A 27-gauge needle with a bent tip may be used to separate the edges of the Descemet’s membrane at the level of the trephination. Using a cyclodialysis spatula or a Sinskey hook, the Descemet membrane is then carefully separated from the posterior stroma, thus providing a donor disk devoid of the accompanying donor stroma (Figures 35-5A and B). The donor disk is left on the cornea for later transferal to the recipient stromal disk. The average endothelial cell loss in the six samples analyzed was 8.46% just after the harvesting procedure.

Transfer of Descemet’s Membrane to Posterior Stromal Disk The recipient procedure is then begun in the modified MAPK fashion to retrieve a posterior lamellar recipient disk.70 Once the disk is removed from the recipient’s cornea, it is carefully marked to maintain the original orientation

A

B Figures 35-5A and B: A Sinskey hook is used in this case to detach the Descemet’s membrane. See the tension lines of the membrane (arrows) representing the instrument in the correct dissection plane. No resistance should be noted during this step when inserting the instrument, any resistance is a sign of a stroma-stroma dissection (incorrect plane).

in the recipient bed at the time of reinsertion in recipients bed. The recipient stromal disk will carry the endothelial cell layer and Descemet’s membrane from the donor, as an autologous carrier. The posterior lamellar disk is placed endothelial side up on a trephination punch block and

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through Descemetorhexis the diseased endothelium is removed. Once the whole recipient Descemet’s membrane is removed, it is replaced by the one we previously dissected from the donor cornea, carefully sliding the membrane over the recipient stromal disk (Figures 35-6A and B). The membrane is allowed time to adhere so that the disk is now composed of recipient posterior stroma and donor Descemet’s membrane/endothelium (Figures 35-7A and B) (will be called a compound disk hereafter). After adherence is assured, the compound disk is then transplanted into the recipient’s stromal bed with special attention to preserve the original orientation of the disk through the marks previously performed, and allowed to self-adhere. The flap will then be closed and sutured securely. Although a stromal lamellar interface does occur in this procedure, it is foreseen that its distortion effect will be negligible because the interface will be recipient-recipient in nature rather than donor-recipient, and the stromal disk orientation is preserved. It is believed that each person’s

A

B Figures 35-7A and B: (A) Descemet’s membrane completely transferred to the recipient disk before insertion in recipient stroma. (B) Microscopic view on endothelial cells showing few damaged cells (trypan blue stain) after the procedure. A

B Figures 35-6A and B: (A) Transfer of the Descemet’s membrane to the recipient disk by sliding the disk underneath the membrane. (B) Histology of the dissection plane showing an almost complete Descemet’s detachment with no stroma attached (PAS 200X).

stroma has a unique micro-orientation of collagen fibrils in such a way that when a donor-recipient interface is created, there is distortion created by the differing orientation. With this procedure, this is not expected to occur. There are several potential benefits to this approach: 1. In preparing the donor button, the endothelial side facing upwards during the whole harvesting process makes the procedure easy and protective to the donor endothelium. 2. The “Descemetorhexis” of both donor and recipient will likely decrease any optical interface distortion. As it was previously shown that the recipient’s posterior stromal surface is smooth after Descemetorhexis, combining it with donor Descemetorhexis should further eliminate any possible distortions caused by stromal collagen remnants. We expect to obtain 20/20 or better BCVA (if permitted by retinal status) in most cases, which would be a considerable improvement over more recent techniques.

Novel Approach for Corneal Endothelial Cell Transplantation 3. Technical ease is afforded to the recipient procedure by use of a modified microkeratome-assisted corneal flap technique. 4. Fast recovery is warranted by current MAPK technique. The possible challenges of this novel procedure are: 1. Descemet’s membrane folds. 2. Presence of minor postoperative astigmatism as is usual in modified MAPK. 3. Flap complications as indicated previously with the MAPK procedure. 4. Larger opening affecting the corneal stability compared to small incision approaches.

Future With the many possible options for corneal transplantation, its future seems promising. The advent of adhesives and advances in materials science will continue to foreward the various corneal transplantation techniques. In particular, the femtosecond laser provides promise because it creates smooth stromal cuts with consistent interfaces. Studies are underway using this technology. In addition, the possibility of culturing endothelial cells and reinserting them in patients in an autologous fashion may be feasible soon. We are exploring new methods of culturing these cells pointing in this direction for the near future. For all of the newer procedures, long-term follow-up data is needed to determine whether these techniques will become safe and effective alternatives to penetrating keratoplasty.

References 1. Moffatt SL, Cartwright VA, Stumpf TH. Centennial review of corneal transplantation. Clin Experiment Ophthalmol 2005;33:642-57. 2. Fanta H. Eduard Zirm (1863-1944). Klin Monatsbl Augenheilkd 1986;189:64-66. 3. Kang PC, Klintworth GK, Kim T, Carlson AN, Adelman R, Stinnett S, Afshari NA. Trends in the indications for penetrating keratoplasty, 1980-2001. Cornea 2005;24:801-3. 4. Kirkwood BJ. A short history of corneal transplantation: commemorating 100 years. Insight 2006;31:15. 5. Claesson M, Armitage WJ, Fagerholm P, Stenevi U. Visual outcome in corneal grafts: A preliminary analysis of the Swedish Corneal Transplant Register. Br J Ophthalmol 2002;86:174–80. 6. Beckingsale P, Mavrikakis I, Al-Yousuf N, Mavrikakis E, Daya SM. Penetrating keratoplasty: Outcomes from a corneal unit compared to national data. Br J Ophthalmol 2006;90:728-31. 7. Pineros O, Cohen EJ, Rapuano CJ, Laibson PR. Long-term results after penetrating keratoplasty for Fuchs’ endothelial dystrophy. Arch Ophthalmol 1996;114:15-18. 8. Isager P, Hjortdal JO, Ehlers N. Stability of graft refractive power after penetrating keratoplasty. Acta Ophthalmol Scand 2000:78:623-6.

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9. Touzeau O, Borderie VM, Allouch C, Scheer S, Laroche L. Effects of penetrating keratoplasty suture removal on corneal topography and refraction. Cornea 1999;18:638-44. 10. Ing JJ, Ing HH, Nelson LR, Hodge DO, Bourne WM. Ten-year postoperative results of penetrating keratoplasy. Ophthalmology 1998;105:1855-65. 11. Zacks CM, Abbott RL, Fine M. Long-term changes in corneal endothelium after keratoplasty. A follow-up study. Cornea 1990;9:92–97. 12. Zaidman GW, Goldman S. A prospective study on the implantation of anterior chamber intraocular lenses during keratoplasty for pseudophakic and aphakic bullous keratopathy. Ophthalmology 1990;97:757–62. 13. Muraine M, Sanchez C, Watt L, Retout A, Brasseur G. Long-term results of penetrating keratoplasty. A 10-year-plus retrospective study. Graefes Arch Clin Exp Ophthalmol 2003;241:571-6. 14. Maeno A, Naor J, Lee HM, Hunter WS, Rootman DS. Three decades of corneal transplantation: Indications and patient characteristics. Cornea 2000;19:7-11. 15. Williams KA, Ash JK, Pararajasegaram P, Harris S, Coster DJ. Long-term outcome after corneal transplantation. Visual result and patient perception of success. Ophthalmology 1991;98:6517. 16. Riddle HK, Parker DA, Price FW. Management of postkeratoplasty astigmatism. Curr Opin Ophthalmol 1998;9:15-28. 17. Bigar F, Uffer S. [The unsolved problem of transplant astigmatism]. Klin Monatsbl Augenheilkd 1992;200:401-3. 18. Rowsey JJ. Prevention and correction of corneal transplant astigmatism. Trans New Orleans Acad Ophthalmol 1987; 35:35-51. 19. Elder MJ, Stack RR. Globe rupture following penetrating keratoplasty: How often, why, and what can we do to prevent it? Cornea 2004;23;776-80. 20. Tuft SJ, Gregory W. Long-term refraction and keratometry after penetrating keratoplasty for keratoconus. Cornea 1995;14:614-7. 21. Bartels MC, van Rooij J, Geerards AJ, Mulder PG, Remeijer L. Comparison of complication rates and postoperative astigmatism between nylon and mersilene sutures for corneal transplants in patients with Fuchs endothelial dystrophy. Cornea 2006;25: 533-9. 22. Renucci AM, Marangon FB, Culbertson WW. Wound dehiscence after penetrating keratoplasty: Clinical characteristics of 51 cases treated at Bascom Palmer Eye Institute. Cornea 2006;25:524-9. 23. Williams KA, Muehlberg SM, Lewis RF, Coster DJ. Long-term outcome in corneal allotransplantation. The Australian Corneal Graft Registry. Transplant Proc 1997;29:983. 24. Bishop VL, Robinson LP, Wechsler AW, Billson FA. Corneal graft survival: A retrospective Australian study. Aust N Z J Ophthalmol 1986;14:133-8. 25. Dandona L, Naduvilath TJ, Janarthanan M, Ragu K, Rao GN. Survival analysis and visual outcome in a large series of corneal transplants in India. Br J Ophthalmol 1997;81:726-31. 26. Price FW Jr, Whitson WE, Collins KS, Marks RG. Five-year corneal graft survival. A large, single-center patient cohort. Arch Ophthalmol 1993;111:799-805. 27. Vail A, Gore SM, Bradley BA, Easty DL, Rogers CA, Armitage WJ. Conclusions of the corneal transplant follow up study. Collaborating Surgeons. Br J Ophthalmol 1997;81:631-6. 28. Pleyer U, Steuhl KP, Weidle EG, Lisch W, Thiel HJ. Corneal graft rejection: Incidence, manifestation, and interaction of clinical subtypes. Transplant Proc 1992;24:2034-7. 29. Naacke HG, Borderie VM, Boureir T, Touzou O, Moldovan M, Laro M. Outcome of corneal transplantation rejection. Cornea 2001;20:350-3. 30. Melles GR, Eggink FA, Lander F, et al. A surgical technique for posterior lamellar keratoplasty. Cornea 1998;17:618-26.

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31. Terry MA, Ousley, PJ. Deep lamellar endothelial keratoplasty in the first United States patients: Early clinical results. Cornea 2001;20:239-43. 32. Behrens A, Ellis K, Li L, Sweet PM, Chuck RS. Endothelial lamellar keratoplasty using an artificial anterior chamber and a microkeratome. Arch Ophthalmol. 2003;121:503-8. 33. Melles GR, Eggink FA, Lander F, et al. A surgical technique for posterior lamellar keratoplasty. Cornea 1998;17:618-26. 34. Terry MA. Deep lamellar endothelial keratoplasty (DLEK): Pursuing the ideal goals of endothelial replacement. Eye 2003;17:982-8. 35. Azar DT, Jain S, Sambursky R, Strauss L. Microkeratome-assisted posterior keratoplasty. J Cataract Refract Surg 2001;27:353-6. 36. Price MO, Price FW Jr. Cataract progression and treatment following posterior lamellar keratoplasty. J Cataract Refract Surg 2004;30:1310-5. 37. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty visual acuity, astigmatism, and endothelial survival in a large prospective series. Ophthalmology 2005;112:1541-9. 38. Amayem AF, Terry MA, Helal MH, Turki WA, El-Sabagh H, ElGazayerli E, Ousley PJ. Deep Lamellar Endothelial Keratoplasty (DLEK): Surgery in complex cases with severe preoperative visual loss. Cornea 2005;24:587-92. 39. Fogla R, Padmanabhan P. Initial results of small incision deep lamellar endothelial keratoplasty (DLEK). AJO 2006;141:34651. 40. Batlle JF, et al. Endothelial Lamellar Keratoplasty. Presented at the 103rd Annual Meeting of AAO, Orlando, Florida, October 1999. 41. Terry MA, Ousley PJ. Rapid visual rehabilitation with deep lamellar endothelial keratoplasty. Cornea 2004;23;143-53. 42. Terry MA, Ousley PJ. Replacing the endothelium without corneal surface incisions or sutures: The first United States clinical series using the deep lamellar endothelial keratoplasty procedure. Ophthalmology 2003;110:755-64. 43. Busin M, Arffa RC, Sebastiani A. Endokeratoplasty as an alternative to penetrating keratoplasty for the surgical treatment of diseased endothelium: Initial results. Ophthalmology 2000;107:2077-82. 44. Ehlers N, Ehlers H, Hjortdal J, Moller-Pedersen T. Grafting of the posterior cornea. Description of a new technique with 12-month clinical results. Acta Ophthalmol Scand 2000;78:543-6. 45. Azar DT, Jain S, Sambursky R, Strauss L. Microkeratome-assisted posterior keratoplasty. J Cataract Refract Surg 2001;27;353-6. 46. Busin M, Zambianchi L, Arffa RC. Microkeratome-assisted lamellar keratoplasty for the surgical treatment of keratoconus. Ophthalmology 2005;112:987-97. 47. Perez VL, Colby KA, Azar DT. Epithelial ingrowth in the flapgraft interface after microkeratome-assisted posterior penetrating keratoplasty. J Cataract Refract Surg 2003;29:2225-8. 48. Ousley PJ, Terry MA. Stability of vision, topography and endothelial cell density from 1 year to 2 years after deep lamellar endothelial keratoplasty surgery. Ophthalmology 2005;112:5057. 49. Sano Y. Corneal endothelial transplantation: Results of a clinical series using deep lamellar endothelial keratoplasty (DLEK). Cornea 2004;23:S55-S58. 50. Melles GR, Lander F, van Dooren BT, Pels E, Beekhuis WH. Preliminary clinical results of posterior lamellar keratoplasty through a sclerocorneal pocket incision. Ophthalmology 2000;107:1850-6. 51. Van Dooren B, Mulder PG, Nieuwendaal CP, Beekhuis WH, Melles GR. Endothelial cell density after posterior lamellar keratoplasty

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(Melles techniques): 3 years follow-up. Am J Ophthalmol 2004;138:211-7. Soong HK, Katz DG, Farjo AA, Sugar A, Meyer RF. Central lamellar keratoplasty for optical indications. Cornea 1999;18:249-56. Price FW, Price MO. Descemet’s stripping with endothelial keratoplasty in 200 eyes Early challenges and techniques to enhance donor adherence. J Cataract Refract Surg 2006;32:411-8. Busin M. A new lamellar wound configuration for penetrating keratoplasty surgery. Arch Ophthalmol 2003;121:260-5. Seitz B, Langenbucher A, Hofmann-Rummelt C, SchlotzerSchrehardt U, Naumann GO. Nonmechanical posterior lamellar keratoplasty using the femtosecond laser (femto-plak) for corneal endothelial decompensation. Am J Ophthalmol 2003;136:76972. Sarayba MA, Juhasz T, Chuck RS, Ignacio TS, Nguyen TB, Sweet PM, Kurtz RM. Femtosecond laser posterior lamellar keratoplasty: A laboratory model. Cornea 2005;24:328-33. Sikder S, Snyder RW. Femtosecond laser preparation of donor tissue from the endothelial side. Cornea. 2006;25:416-22. Soong HK, Mian S, Abbasi O, Juhasz T. Femtosecond laser-assisted posterior lamellar keratoplasty: Initial studies of surgical technique in eye bank eyes. Ophthalmology 2005;112:44-49. Melles GR, Lander F, Rietveld FJ. Transplantation of Descemet’s membrane carrying viable endothelium through a small scleral incision. Cornea. 2002;21:415-8. Melles GR, Wijdh RH, Nieuwendaal CP. A technique to excise the Descemets’ membrane from a recipient cornea (descemetorhexis). Cornea 2004;23:286-8. Melles GR, Rietveld FJ, Beekhuis WH, Binder PS. A technique to visualize corneal incision and lamellar dissection depth during surgery. Cornea 1999;18:80-86. Price FW, Jr, Price MO. Descemet’s stripping with endothelial keratoplasty in 50 eyes: A refractive neutral cornea transplant. J Refract Surg 2005;21:339-45. Price FW Jr, Price MO. Descemet’s stripping with endothelial keratoplasty in 200 eyes: Early challenges and techniques to enhance donor adherence. J Cataract Refract Surg 2006;32:4118. Price MO, Price FW, Jr. Descemet’s stripping with endothelial keratoplasty comparative outcomes with microkeratomedissected and manually dissected donor tissue. Ophthalmology 2006;113:1936-42. Terry MA, Ousley PJ. Endothelial replacement without surface corneal incisions or sutures: Topography of the deep lamellar endothelial keratoplasty procedure. Cornea 2001;20:14-18. Seitz B, Langenbucher A, Naumann GO. The penetrating keratoplasty. A 100-year success story. Ophthalmology 2005;102:1128-36. Shimazaki J, Shimmura S, Ishioka M, Tsubota K. Randomized clinical trial of deep lamellar keratoplasty vs penetrating keratoplasty. Am J Ophthalmol 2002;134:159-65. Fontana L, Parente G, Tassinari G. Clinical outcomes after deep anterior lamellar keratoplasty using the big-bubble technique in patients with keratoconus. Am J Ophthalmol 2007;143:117-24. Ignacio TS, Nguyen TT, Sarayba MA, Sweet PM, Piovanetti O, Chuck RS, Behrens A. A technique to harvest Descemet’s membrane with viable endothelial cells for selective transplantation. Am J Ophthalmol 2005;139:325-30. Pirouzmanesh A, Herretes S, Reyes JM, Suwan-apichon O, Chuck RS, Wang DA, Elisseeff JH, Stark WJ, Behrens A. Modified microkeratome-assisted posterior lamellar keratoplasty using a tissue adhesive. Arch Ophthalmol 2006;124:210-4.

True Endothelial Cell (TEnCell) Transplantation

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Panagiotis Georgoudis Michael J Tappin

True Endothelial Cell (TEnCell) Transplantation

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Introduction The goal of most transplant surgery is the specific replacement of the diseased structure, with as little disruption to neighboring structures as possible. Corneal surgery is no exception. In the rapidly evolving field of posterior lamellar keratoplasty (PLK) the progression from penetrating keratoplasty (PK) to PLK is an excellent example. Now the thin layers of stroma carrying endothelial cells in many variations of PLK first described by Melles1 and further developed by Terry and Oulsey2 and Price3 -5 go a long way to satisfying this aim. However, the goal of specific endothelial cell transplantation6, 7 is not fulfilled. In this chapter a technique enabling specific endothelial cell transplantation with no corneal stroma is described. Although in its infancy, in comparison to other techniques of PLK, true endothelial cell (TEnCell) transplantation, transplants only donor’s Descemet’s membrane (DM) as a carrier for the endothelial cell layer.8

Indications • Fuchs’ endothelial dystrophy • Pseudophakic bullous keratopathy.

Aim of Surgery • Pain relief • Visual recovery.

Developing the Endothelial Cell Harvesting Technique

iv. Pollock forceps v. Modified Sinskey hook/Custom bent needle for DMrhexis vi. Tappin’s endothelial cannula (ALTOMED®) vii. Insulin syringe.

Practicing Harvesting The surgeon can practice and master the technique of DM harvesting in a single piece, when preparing the DALK tissue, without compromising its quality.

Harvesting Technique The donor button is positioned epithelial side down in a block. The DM peel is initiated at Schwalbe’s line using plain microforceps. In this case, iris root remains attached (Figure 36-1). A circumferential arc of about four clock hours of Schwalbe’s line is dissected. Usually this is found after removing iris root (Figure 36-2). The microforceps are used to grip the free edge of DM and this is extended for three millimetres towards the center of the cornea (Figure 36-3). This loose flap is then placed back on the corneal stroma (Figure 36-4). The hand held trephine is used to cut the donor button (Figure 36-5). The free edge of DM is picked up at the edge of the trephined button using the same two pairs of plain microforceps (Figure 36-6) and the disk of DM is peeled as a single sheet (Figures 36-7 and 36-8).

Histological Analysis, Vital Staining and Viability Histological analysis of the harvested tissue demonstrates that this technique provides a viable endothelial cell layer supported only by DM. There is no attached stroma.

The essential and challenging step of the procedure is a reliable method of successful harvesting of the endothelial cell sheet on the DM. The initial technique was developed during the preparation of the donor material for a deep anterior lamellar keratoplasty (DALK). The DM and endothelial cell layer of the donor tissue is usually removed prior to anterior lamellar corneal grafting. This is often done by scrapping it or removing it in pieces. Rather than destroying the endothelial tissue, there is opportunity to remove DM and endothelial cells in one piece.

Instrumentation i. Trephine block ii. Two pairs of plain microforceps iii. Handheld, long handled 7.5 – 7.75 mm trephine

Figure 36-1: Initiation of DM peel. The peel begins by picking up Schwalbe’s line. In this example some of the iris root remains attached. Schwalbe’s line can be seen as the white line grasped by a pair of nontoothed forceps.

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Figure 36-2: Dissection of an arc of four clock hours.

Figure 36-5: Manual trephination.

Figure 36-3: Extension of free edge towards the center of the cornea.

Figure 36-6: Free edge of Descemet’s membrane picked up with microforceps.

Figure 36-4: Loose flap placed back on the stroma.

Figure 36-7: Peeling of Descemet’s membrane.

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Figure 36-8: Completion of Descemet’s membrane peel.

Figure 36-9: Cross section of endothelial cells on Descemet’s membrane.

Figure 36-11: Vital staining of the harvested tissue. The non-viable cells have taken up staining. The cell walls of the viable cells can faintly be seen.

harvested endothelial cell sheet 9-11 shows viability of the cells after harvesting (Figure 36-11). Vital staining involves staining the harvested tissue with Trypan blue and Alizarin Red-S. Trypan blue stains only the nuclei of non-viable cells and Alizarin Red-S stains the extracellular space, demonstrating the outline of all cells. Figure 36-11 shows a small number of non-viable cells. Under higher power, the cell walls of the viable cells can faintly be seen. The most important measure of final endothelial cell viability after transplantation is the postoperative endothelial cell function as evidenced by the final corneal clarity, visual acuity, central corneal thickness and endothelial cell count.

TEnCell Transplantation

Figure 36-10: Methylene blue staining of intact endothelial cell sheet.

Harvested tissue is stained with haematoxylin and eosin (Figure 36-9) and methylene blue (Figure 36-10). Harvesting DM involves tractional forces that may pose a threat to the viability of the endothelial cells and compromise the final outcome. Vital staining of the

Once the skill of reliably harvesting the endothelial cell layer on DM has been attained, the surgeon can proceed to performing TEnCell transplantation. The harvested sheet requires introduction into the eye, and this is achieved by placing it, endothelial side down on Tappin’s cannula (Figure 36-12) upon which is a layer of 2% hydroxypropyl methylcellulose (HPMC) (Figure 36-13). The patient’s pupil is dilated using tropicamide 1% to facilitate visualization of the host Descemet’s membrane excision (Descemetorhexis).12 An 8.0 mm partial thickness (2/3 of corneal depth) superior corneal incision or limbal incision is made with a 15 degree blade (Figure 36-14). A 27 gauge bent needle or a modified Sinskey hook is introduced into the anterior chamber (AC) in order to perform a modified Descemetorhexis which is necessary to remove the diseased endothelium (Figure 36-15). The procedure is performed like an inverted capsulorhexis, avoiding the use of viscoelastic as this may

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Figure 36-12: Tappin’s cannula (Altomed ®).

Figure 36-14: An 8.0 mm partial thickness corneal incision.

Figure 36-13: Tappin’s cannula with a layer of HPMC and the harvested endothelial sheet.

Figure 36-15: Descemetorhexis.

interfere with adherence of the transplanted endothelial cell sheet. The Descemetorhexis can be performed in a curvilinear pattern or it can be scraped off in small segments. Care should be taken not to destroy the posterior stromal surface. The procedure can be difficult as the detached DM tends to fold back to its initial position in the absence of viscoelastic. It is useful to gently apply a 7.5 mm trephine on the epithelium in order to outline the area of DM to be excised. Alternatively calipers and Jensen violet can be used. Once the Descemetorhexis is performed, a Simco cannula is introduced into the AC to remove the remains of the detached tissue (Figure 36-16). At this point, the initial 8 mm incision is made full-thickness and the AC is formed with balanced salt solution (BSS) (Figure 36-17). The edge of the cornea is lifted with a pair of microtoothed forceps and the cannula with the donor DM is introduced into the AC of the recipient eye (Figure 36-18).

Figure 36-16: Removal of detached Descemet’s membrane with Simco cannula.

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Figure 36-17: Full-thickness corneal incision.

Figure 36-19: Injection of air through Tappin’s cannula.

Figure 36-18: Introduction of Tappin’s cannula with endothelial sheet into the anterior chamber.

Figure 36-20: Closure of the corneal incision.

Care should be taken so that the thin sheet of tissue is not folded during insertion. Once the cannula is in the AC, a bubble of filtered air is injected through its center, which elevates the donor tissue and apposes it to the recipient stroma (Figure 36-19). HPMC is used because the air-bubble propagates better than with other viscoelastics, ensuring that the air is evenly distributed under the donor tissue which helps lift the endothelium into place against the posterior surface of the cornea. The cannula is withdrawn from the AC. This is a critical step and care should be taken not to drag the implanted tissue during withdrawal. This can be achieved if the surgeon, while withdrawing the cannula, continues to inject air into the anterior chamber. The corneal incision is closed using 10.0 nylon sutures (Figure 36-20). It is important to avoid the edge of the donor endothelium with

the needle as this can dislodge the edge of the grafted tissue. It is important to maintain an air bubble in the anterior chamber during suturing as this keeps the endothelium in place and allows the surgeon to know where to inflate more air at the end of the operation (Figure 36-21). This avoids inadvertently injecting air between the cornea and donor endothelium. It is important that the air-bubble is the correct size; usually ½ to ¾ that of the AC. If the air-bubble is smaller than this, the DM may not attach successfully. If the air bubble is too big, pupil block glaucoma can be induced. Subconjunctival antibiotic and steroid is injected and a bandage contact lens is applied. Postoperative antibiotic drops and steroids are started. [Editorial Note: Prednisolone acetate 1% (Pred Forte 1%, Allergan Inc., Irvine, CA,) six times daily and levofloxacin 1.5% (Iquix, Vistakon Pharmaceuticals,

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Figure 36-22: Final postoperative result. The edges of grafted tissue are visible in the peripheral cornea. Figure 36-21: Further inflation with air.

Jacksonville, FL) QID may be used. In the USA, chloramphenicol drops are not used]. Patient is instructed to posture prone for 30 minutes. The patient should be examined the next day to ensure that DM is attached to the donor stroma.

Advantages i. Tissue specific grafting and therefore potentially better visual outcome due to better interface quality. Less potential problems secondary to mismatch of corneal curvature. DM without a stromal carrier will conform easier to any corneal shape and adhere well to the host’s posterior stromal surface. ii. As in DSEK, TEnCell transplantation preserves the recipient corneal architecture as it does not involve dissection of the host’s posterior stroma. iii. TEnCell transplantation preserves tectonic form and results in a more robust eye compared to a PK. With present instrumentation, the wound construction is approximately 8.0 mm long and a limbal or scleral section can be used. iv. As TEnCell requires fewer sutures compared to a PK, there is less induced astigmatism. Sutures can be safely removed after 3 months. v. The thin, flexible donor button produced by this technique may enhance the rate of successful attachment onto the recipient cornea. vi. Like PLK, DLEK and DSEK, TEnCell transplantation offers rapid visual recovery with excellent visual results. vii. The procedure can be repeated in cases of primary endothelial cell failure. viii. The procedure can be performed under local anesthesia.

Figure 36-23: Anterior segment photograph demonstrating the difference in corneal clarity and thickness between grafted and diseased endothelium.

ix. Inexpensive equipment is required. There is potential for its use in developing countries.

Disadvantages i. Very new technique. ii. Steep learning curve. iii. Requires good quality donor tissue for successful harvesting. iv. At present, the wound is larger (8 mm) compared to other methods of PLK requiring sutures.

Complications • During harvesting o Tearing of harvested tissue at the edges of Schwalbe’s line. o Drying of DM during harvesting. This can be avoided if the tissue is kept moist with balanced salt solution.

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• During implantation o Loss of tissue during insertion in the AC o Tissue wrinkling o Tissue decentration. • During withdrawal of the cannula o Detachment or loss of tissue during withdrawal from the AC. • During suturing o Detachment of tissue and incarceration into the suture tract. o Loss of air-bubble.

Early Postoperative Complications The most common complication is detachment of the harvested tissue. If the detachment is partial, the tissue can be reattached by reinflation of the AC with air. If the tissue is completely detached, the endothelium tends to form a scroll which can be difficult to reapply. In this situation, we have found it necessary to repeat the procedure. While waiting for repeat surgery, the patient develops marked corneal edema. The application of a bandage contact lens is advisable until further surgery is performed. Pupil block glaucoma can be an early postoperative complication as a result of over-inflation of the AC with air.

Late Complications There have been no cases of endothelial cell rejection so far, although this clearly remains a possibility.

Case Example A 68-year-old male patient with Fuchs’ endothelial dystrophy underwent routine small incision cataract extraction with foldable intraocular lens implantation. Visual rehabilitation was complicated by corneal decompensation. Patient underwent a TEnCell transplantation. His preoperative best corrected visual acuity was 6/18. The central corneal thickness was 750 μm. No endothelial cell count could be obtained due to significant corneal edema. The first TEnCell transplant attempt was aborted because the endothelial harvest was unsuccessful. The second attempt two weeks later was uneventful and the transplantation was successful. Postoperative best corrected visual acuity was 6/6, 19 months later. Central corneal thickness was 574 μm. Endothelial cell count was 649 cells/mm2. Figure 36-22 demonstrates the postoperative result and Figure 36-23

demonstrates the difference in corneal clarity between grafted and diseased endothelium.

The Future TEnCell transplantation is a challenging new technique. Specific endothelial cell transplantation has been the aim of many corneal surgeons13-18 and this is the first in vivo description of such a technique. Subsequent transplantation has shown endothelial cell counts of up to 2000 cells/ mm2. Recent work has shown that cultivation and transplantation of human cultured endothelial cells in an animal model is feasible.19 TEnCell tranplantaion may find an application in the transplantation of layers of cultured endothelial cells into human eyes. The experience is still limited due to the small number of patients treated, but as the numbers increase and as new instrumentation becomes available, our belief is that TEnCell transplantation may become more widely adopted, offering potentially excellent visual outcomes for the patient.

References 1. Melles GR, Eggink FA, Lander F, Pels E, Rietveld FJ, Beekhuis WH, Binder PS. A surgical technique for posterior lamellar keratoplasty. Cornea 1998;17(6):618-26. 2 . Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty in the first United States patients: Early clinical results. Cornea 2001;20(3):239-43. 3 . Price FW Jr, Price MO. Descemet’s stripping with endothelial keratoplasty in 50 eyes: A refractive neutral corneal transplant. J Refract Surg 2005;21(4):339-45. 4 . Price FW Jr, Price MO. Descemet’s stripping with endothelial keratoplasty in 200 eyes: Early challenges and techniques to enhance donor adherence. J Cataract Refract Surg 2006;32(3):411-8. 5 . Price MO, Price FW Jr. Descemet’s stripping with endothelial keratoplasty. Comparative outcomes with microkeratomedissected and manually dissected donor tissue. Ophthalmology 2006; [Epub ahead of print]. 6 . Melles GR, Remeijer L, Geerards AJ, Beekhuis WH. The future of lamellar keratoplasty. Curr Opin Ophthalmology 1999; 10(4):253-9. 7 . Yeh PC, Azar DT, Colby K. Selective endothelial transplantation: Novel surgical techniques for the treatment of endothelial dysfunction. Int Ophthalmol Clin 2004;44(1):51-66. 8 . Tappin M. A method for true endothelial cell (Tencell) transplantation using a custom-made cannula for the treatment of endothelial cell failure. Eye 2006; [Epub ahead of print]. 9 . Spence DJ, Peyman GA. A new technique for the vital staining of the corneal endothelium. Invest Ophthalmol 1976;15(12): 1000-2. 10. Taylor MJ, Hunt CJ. Dual staining of corneal endothelium with trypan blue and alizarin red S: Importance of pH for the dyelake reaction. Br J Ophthalmol 1981;65(12):815-9. 11. Singh G, Bohnke M, von-Domarus D, Draeger J, Lindstrom RL, Doughman DJ. Vital staining of corneal endothelium. Cornea 1985-1986;4(2):80-91.

True Endothelial Cell (TEnCell) Transplantation 12. Melles GR J, Wijdh RHJ, Nieuwendaal MD. A technique to excise the Descemet’s membrane from a recipient cornea (Descemetorhexis). Cornea 2004;23(3):286-8. 13. Jumblatt M, Maurice M, McCulley J. Transplantation of tissuecultured corneal endothelium. Invest Ophthalmol Vis Sci 1978;17:1135–41. 14. Gospodarowicz D, Greenburg G, Alvarado J. Transplantation of cultured bovine corneal endothelial cells to rabbit cornea: Clinical implications for human studies. Proc Natl Acad Sci USA 1979;76:464–8. 15. Maurice D, McCulley J, Perlman M. Development in use of cultured endothelium in corneal transplantation. Doc Ophthalmol Proc Ser 1979;20:151–3.

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16. Behrens K, Ellis L, Li PM, Sweet RS Chuck. Endothelial lamellar keratoplasty using an artificial anterior chamber and a microkeratome. Arch Ophthalmol 2003;21:503-8. 17. Mohay J, Lange TM, Soltau JB, Wood TO, McLaughlin BJ. Transplantation of corneal endothelial cells using a carrier device. Cornea 1994;13:173–82. 18. McCulley JP, Maurice DM, Schwartz BD. Corneal endothelial transplantation. Ophthalmology 1980;87:194–201. 19. Mimura T, Yamagami S, Yokoo S, Usui T, Tanaka K, Hattori S, Irie S, Miyata K, Araie M, Amano S. Cultured human corneal endothelial cell transplantation with a collagen sheet in a rabbit model. Invest Ophthalmol Vis Sci 2004;45(9): 2992-7.

Descemet Membrane Endothelial Keratoplasty (DMEK)

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Massimo Busin

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Introduction

Surgical Technique

Corneal transplantation surgery has achieved new heights in terms of advanced surgical techniques used by corneal surgeons where only the diseased portion of the cornea is replaced by a similar healthy donor corneal tissue. This is in contrast to penetrating keratoplasty where the fullthickness of the cornea is replaced regardless of the corneal layer that is involved in the disease process. A new terminology is called selective tissue corneal transplantation (STCT) that is defined as the selective removal of the diseased portion of the patient’s cornea and its replacement with anatomically similar healthy donor tissue.1 The newer surgical technique of corneal transplantation namely, Descemet stripping automated endothelial keratoplasty (DSAEK)2-4 has gained worldwide acceptance and is rapidly becoming the preferred surgical choice for dealing with corneal endothelial decompensation resulting in corneal edema, bullous keratopathy and loss of best corrected visual acuity. This type of surgical procedure is an example of STCT. This procedure, namely DSAEK, is an additive procedure where the postoperative corneal thickness far exceeds the range of normal corneal thickness and the donor-recipient interface is a stroma-to-stroma interface. The newer technique of STCT described by Melles et al5 is called Descemet membrane endothelial keratoplasty (DMEK) where only the Descemet’s membrane and endothelium of the patient’s cornea is replaced by a similar healthy donor corneal tissue. This is a more “natural” form of STCT where the postoperative corneal thickness should be within the range of normal corneal thickness and donorrecipient interface is Descemet’s membrane to stroma, a more “natural” final anatomic result. However, DMEK has its challenges in terms of the surgical techniques involved in this new procedure. This chapter describes a surgical technique of performing DMEK with emphasis in simplifying the surgical technique, that may help in gradually moving DMEK over time, to the forefront of STCT for endothelial decompensation. The technique is based on the concept of the “big bubble”, which was used by Anwar and Teichman2,3 to dissect the corneal stroma from the underlying Descemet’s membrane and endothelium when performing anterior lamellar keratoplasty for keratoconus. Busin’s technique described in this chapter is substantially different in many ways, but like the “big bubble” technique for keratoconus, air is used to achieve a complete dissection between the donor Descemet’s membrane and corneal stroma. This surgical technique described below, includes both harvesting the donor tissue and delivering it into place and attaching it to the patient’s cornea.

The preferred anesthesia is with a peribulbar block. However, other forms of anesthesia including general anesthesia may be considered by the surgeon.

Step-by-Step Surgery The initial surgical step involves the removal of Descemet’s membrane and endothelium from the central part (8.0 to 9.0 mm in diameter) of the posterior surface of the recipient cornea (Figure 37-1). Then, the donor cornea is mounted in an artificial anterior chamber and about 2/3rd of the anterior corneal stroma is removed using a microkeratome, the same way it is done when performing DSAEK surgery (Figure 37-2). This step is not essential, but allows the

Figure 37-1: Intraoperative photograph showing the removal of patient’s Descemet membrane and endothelium from the central part (8 to 9 mm in diameter) of the posterior surface of the cornea.

Figure 37-2: The donor cornea is mounted within an artificial anterior chamber and about 2/3rd of the anterior corneal stroma is removed using a microkeratome the same way it is done when performing DSAEK surgery.

Descemet Membrane Endothelial Keratoplasty (DMEK)

Figure 37-3: A 25 gauge needle connected to a 5.0 cc sterile syringe is inserted, bevel up, into the peripheral donor cornea, 1.0 mm from the limbus, and advanced in a tangential direction immediately beneath the endothelium for about 2.0 mm.

Figures 37-4 and 37-5: Air is then injected until detachment of Descemet membrane is achieved and a large bubble is obtained.

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Figure 37-6: Part of the air is removed from the bubble by using an empty syringe with a 30 gauge needle and achieves a partial collapse of the big-bubble.

surgeon to convert to DSAEK, in case he does not succeed in preparing the endothelial graft. Then the donor cornea is removed from the artificial anterior chamber and placed with the endothelium facing up. Next, a 25 gauge needle connected to a 5.0 cc sterile syringe is inserted, bevel up, into the peripheral donor cornea at about 1.0 mm from the limbus and advanced in a tangential direction immediately beneath the endothelium for about 2.0 mm (Figure 37-3). Air is then injected until detachment of Descemet membrane is achieved and a large bubble is obtained (Figures 37-4 and 37-5). Next, the surgeon removes part of the air from the bubble by using an empty syringe with a 30 gauge needle and achieves a partial collapse of the big-bubble (Figure 37-6). The same needle is used to inject few drops of trypan blue (Vision Blue) into the air-bubble (Figure 37-7). Finally aspirate all the air still present in the space created by the big-bubble (Figure 37-8). As a result of the total bubble collapse, the trypan blue stain outlines the portion of Descemet membrane that is detached from the donor corneal stroma and allows for subsequent, precise punching (trephination) inside the outer limit of dissection. An 8.0 to 9.0 mm trephine is used to punch the donor corneal tissue. Pressure is maintained, while moving the sclerocorneal rim with forceps, thus achieving total detachment of the central donor tissue from the peripheral part of the donor cornea (Figure 37-9). The donor disk obtained using this technique, consists of deep, often thicker, because air-blown corneal stroma and overlying detached Descemet’s membrane and healthy donor corneal endothelium. The stromal disk is held with forceps, which serves as a carrier for the donor endothelium, and the tissue

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Figure 37-7: A few drops of trypan blue (Vision Blue) is injected into the air-bubble created within the donor cornea.

Figure 37-9: Pressure is maintained, while moving the sclerocorneal rim with forceps, thus achieving total detachment of the central donor tissue from the peripheral part of the donor cornea.

Figure 37-10: The thin donor disk is pulled gently until it protrudes out of the funnel of the Busin glide.

Figure 37-8: All the air is aspirated within the space created by the big-bubble.

as a whole is transported onto the plate of the Busin glide. It is then advanced through the distal part of the glide and the thin donor disk is pulled gently until it protrudes out of the funnel of the Busin glide (Figure 37-10). Finally, a coaxial microincision retinal forceps is used to drag the donor graft into the anterior chamber through a nasal clear-cornea incision with a bimanual “pull-through” maneuver similar to that used for DSAEK surgery (Figure 37-11). This entire surgical step is performed under continuous irrigation through an anterior chamber maintainer placed at the 12 o’clock position. As the endothelium is lying flat on the stroma all the time, the risk of inserting it upside down is minimized with this maneuver. An additional advantage of this technique is that the bubble formation counteracts the natural

Figure 37-11: A coaxial micro-incision retinal forceps is used to drag the donor graft into the anterior chamber through a nasal clear-cornea incision with a bimanual “pull-through” maneuver.

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Figure 37-12: The donor tissue floats as a flat membrane inside the anterior chamber and does not require complex manipulation to unroll it.

Figure 37-13: The recipient anterior chamber is air filled after closing all the corneal incisions with interrupted 10-0 nylon suture.

tendency of the endothelial layer to roll onto itself. As a result, the donor tissue floats as a flat membrane inside the anterior chamber and does not require complex manipulation to unroll it (Figure 37-12). The final step is the air fill, which is completed after closing all the corneal incisions with interrupted 10-0 nylon suture ((Figure 3713). The air in the air-filled anterior chamber is allowed to reabsorb spontaneously over time (usually 2-3 days). To prevent a pupillary block, a peripheral iridectomy is performed inferiorly before delivering the donor tissue into the recipient anterior chamber.

Postoperative Follow-up/Drug Regimen

Surgical Pearls and Tips 1. DMEK graft tends to curl with the endothelium inward. It is important to recognize the correct tissue orientation prior to attaching the donor tissue to the recipient cornea. 2. Trypan blue staining helps in the proper orientation of the donor tissue and in the centration of the donor disc to the patient’s cornea.

Postoperative medications are the same as for penetrating keratoplasty or DXEK/DSAEK as per surgeon’s choice, namely, topical steroid and antibiotic drops qid.

References 1. John T (Editorial). Selective tissue corneal transplantation: A great step forward in global visual restoration. Expert Rev Ophthalmol 2006;1:5-7. 2. John T. Surgical Techniques in Anterior and Posterior Lamellar Corneal Surgery. New Delhi, India: Jaypee Brothers Medical Publishers; 2006. 3. John T. Step by Step in Anterior and Posterior Lamellar Keratoplasty. New Delhi, India: Jaypee Brothers Medical Publishers; 2006. 4. Ide T, Yoo SH, Kymionis GD, Goldman JM, Pereez VL, O’Brien TP. Descemet-stripping automated endothelial keratoplasty: Effect of anterior lamellar corneal tissue-on/-off storage condition on Descemet-stripping automated endothelial keratoplasty donor tissue. Cornea 2008;27:754-7. 5. Melles GR, Ong TS, Ververs B, van der Wees J. Descemet membrane endothelial keratoplasty (DMEK). Cornea 2006; 25:987-90.

Corneal Endothelial Reconstruction with a Bioengineered Cell Sheet

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Jui-Yang Lai Ging-Ho Hsiue

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Abstract Cellular organization of foreign grafts constructed from cultivated cells is critical to successful graft-host integration and tissue repair. This chapter described a novel human corneal endothelial cell (HCEC) therapeutic method, where cultivated adult HCEC sheet with uniform orientation was prepared and transplanted to a rabbit cornea. Having a correct morphology and intact barriers, the HCEC sheet was made by the temperature-modulated detachment of monolayered HCECs from thermo-responsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted surfaces and was delivered with proper polarity to the corneal posterior surface by a bioadhesive gelatin disk. Results of the in vivo studies, including the follow-up clinical observations and histological examinations showed the laminated HCEC sheet was successfully integrated into rabbit cornea denuded with endothelial layer after the biodegradation of gelatin carrier. These data indicate the feasibility of the proposed procedure in cell therapy for corneal endothelial cell loss.

Introduction Human corneal endothelial cells (HCECs) maintain corneal clarity by means of a barrier function and pump-leak mechanism.1 Although the number of HCECs decreases with aging, ocular trauma/surgeries, contact lens wearing or inflammations, HCECs do not proliferate in vivo to compensate for the cell loss.2 In more than half of all global cases involving penetrating keratoplasty (PK, a full-

thickness corneal transplantation), the only corneal component that requires replacement is the endothelial cell layer. Given insufficient supplies of donor corneas and complications of PK, there would be a substantial advantage in being able to replace the endothelium alone by delivering cultured HCECs to the recipient. Corneal endothelial cell transplantation was attempted to repopulate rabbit cornea with unhealthy endothelium by directly injecting a cell suspension into the anterior chamber.3 However, that trial was limited because of only scattered clumps of endothelial cells randomly attached to the targeted cornea and to other normal ocular tissues such as the iris and lens. In recent years, numerous investigators have reported a method to transplant corneal endothelial cells by seeding and cultivating them on different carriers made of either natural tissue materials4,5 or artificial polymeric materials.6-9 Although a monolayered architecture of cultured cells was maintained, the intraocular grafting of these engineered tissue replacements may possibly cause problems such as unstable attachment of the cell carrier membrane to the host corneal stroma and fibroblastic overgrowth between the membrane and stroma.3 By avoiding the permanent residence of foreign carrier materials in the host, our group has recently presented a novel cell sheet-based therapy for corneal endothelial reconstruction (Figure 38-1). Bioengineered

Figure 38-1: A novel strategy for corneal endothelial reconstruction with a bioengineered cell sheet by utilizing functional biomaterials. Schematic illustration shows that the cultivated human corneal endothelial cell (HCEC) sheet was harvested via temperature modulation of a thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted surface (A-C), and delivered to corneal posterior surface without endothelium using a bioadhesive gelatin hydrogel disk (D). After swelling (E) and biodegradation (F) of the cell carrier, a gelatin disk, the transplanted HCEC sheet with uniformly proper polarity was attached and integrated onto the denuded cornea to allow regeneration of endothelial monolayer (G).

Corneal Endothelial Reconstruction with a Bioengineered Cell Sheet HCEC sheets were fabricated from thermoresponsive culture supports and were delivered by using multifunctional gelatin hydrogel disks.10-12 The advantage of this strategy is that it allows the HCEC sheet grafts with their deposited extracellular matrix (ECM) can be directly transplanted onto the damaged corneas without biomaterial barriers. Cell sheet engineering is a novel technology for harvesting cultivated cell sheets via temperature modulation of thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted culture surfaces.13 By using this method, it is feasible to create transplantable tissue/organ sources without using a three-dimensional biomaterial scaffold, which may elicit host inflammatory responses after in vivo implantation of tissue-engineered replacements into damaged sites.14 Recently, this powerful technology has been proven to be effective for cardiac tissue repair15,16 and corneal epithelial reconstruction.17-19 It has been reported that cells could adhere and proliferate on the hydrophobic PNIPAAm-grafted surfaces at 37°C, and spontaneously detached from the switched hydrophilic surfaces when the culture temperature was reduced to a level below the lower critical solution temperature of PNIPAAm (i.e. 32°C in water).20 To harvest the cell cultures as whole sheets instead of as isolated suspensions, we have fabricated the PNIPAAm-grafted

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culture supports by means of plasma chemistry, a unique technique that previously allowed us to develop artificial corneas.21-23 In this study, untransformed adult HCECs were cultivated on the nanostructured PNIPAAm-grafted surfaces for 3 weeks at 37°C, and confluent monolayers were obtained at 20°C (Figure 38-2). The characteristics of bioengineered HCEC sheets were determined in vitro by evaluating their viability and by scanning electron microscopy, immunohistochemistry, and histological studies. Evaluations of native corneal endothelium from human eye bank donors were conducted simultaneously for comparison. After removal of the PNIPAAm-grafted surfaces at a low culture temperature, the HCEC sheets are usually soft and fragile due to the loss of anchorage dependence. It may be necessary to apply supporting materials to strengthen these cell monolayers for transportation and surgical handling. Gelatin is obtained by thermal denaturation or physical and chemical degradation of collagen. It has been reported that a gelatin membrane substrate cross-linked with glutaraldehyde could be used to support the growth of cultivated rabbit corneal endothelial cells for transplantation.6 Our previous study has also demonstrated the feasibility of fabricating native gelatins into sandwich-like encapsulating membranes for retinal sheet transplantation.24 In this investigation, given the bioadhesive and

Figure 38-2: Fabrication of bioengineered human corneal endothelium from thermoresponsive culture supports. Schematic illustration shows that the cultured HCEC monolayers could be harvested from thermoresponsive supports by a mechanism of temperature-dependent switch in surface hydrophobicity/hydrophilicity for controlling cell adhesion and detachment.

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prompt degradable properties, gelatin disks were used for the delivery of HCEC sheet to the corneal posterior surface, the denuded Descemet’s membrane (i.e. the basement membrane of corneal endothelium). We performed an in vivo study in a rabbit model to examine the efficacy of the use of bioengineered HCEC sheets for improving corneal endothelial reconstruction.

Experimental Materials N-isopropylacrylamide (NIPAAm) was purchased from Acros (Fairlawn, NJ, USA). OPTI-modified Eagle’s medium (OPTI-MEM), Hank’s balanced salt solution (HBSS, pH 7.4), gentamicin, and trypsin-EDTA were obtained from Gibco BRL (Grand Island, NY, USA). Dispase II was purchased from Roche Diagnostics (Indianapolis, IN, USA). Fetal bovine serum (FBS) and antibiotic/antimycotic (A/A) solution were obtained from Biological Industries (Kibbutz Beit Haemek, Israel). Human recombinant epidermal growth factor (EGF) was purchased from Upstate Biotechnology (Lake Placid, NY, USA). Nerve growth factor (NGF) was obtained from Biomedical Technologies (Stoughton, MA, USA). Pituitary fibroblast growth factor (FGF), ascorbic acid, chondroitin sulfate, calcium chloride, human lipid fraction, RPMI 1640 vitamin solution, and PKH26 red fluorescent dye were purchased from SigmaAldrich (St. Louis, MO, USA). All the other chemicals were of reagent grade and used as received without further purification.

Preparation of Thermoresponsive Culture Surfaces Polyethylene (PE) dish substrates (35 mm in diameter) were ultrasonically cleaned in ethqanol for 1 hour before use. The detailed procedure for the plasma modification process has been described in a previous work.25 Briefly, a model PD-2 plasma deposition reactor with a bell jar-type glow discharge cell (Samco, Kyoto, Japan) was used. Under argon atmosphere, plasma was generated to activate the surfaces of PE substrates. The samples were immersed in a 10 wt% NIPAAm monomer aqueous solution. Photografting polymerization of NIPAAm onto the peroxidized sample surfaces was performed by ultraviolet light irradiation. The modified surfaces were washed for 3 days with cold deionized water to remove the NIPAAm homopolymers and dried under nitrogen atmosphere. An atomic force microscopy (AFM) (Veeco Digital, Santa Barbara, CA, USA) was also utilized to scan surface

topography. All measurements were made in tapping mode with a silicon cantilever and a scan rate of 0.6 Hz. AFM images were recorded with a scan size of 1 µm and a data scale of 30 nm. Three measurements were done on different surface sites to calculate the mean surface roughness for each sample.

Preparation of Gelatin Hydrogel Disks Gelatin (Nitta Gelatin, Osaka, Japan), manufactured through an alkaline process of bovine bone collagen, was used to prepare the multi-functional hydrogel carriers for HCEC sheet transfer. The isoelectric point (IEP), weightaverage molecular weight (MW), and polydispersity index of the gelatin sample, reported by the manufacturer are respectively 5.0, 100 kDa, and 2.3. Gelatin hydrogel disks (7 mm in diameter, 700-800 µm thick) were prepared by solution casting methods as described elsewhere.11 Briefly, an aqueous solution of 10 wt% gelatin was cast into a polystyrene planar mold, and air-dried for 3 days at 25°C to obtain hydrogel sheets. Using a corneal trephine device, the hydrogel sheets were cut out to create small gelatin disks (0.4 cm2, 700-800 µm thick). Afterwards, the gelatin disks were sterilized by gamma irradiation using a cobalt60 source located at the National Tsing Hua University (Hsinchu, Taiwan, ROC). According to our earlier report,24 irradiation was performed in the presence of air at a dose of 16.6 kGy, applied at a dose rate of 0.692 kGy/h; irradiation temperature, 25 ± 1°C.

Cell Preparation This study adhered to the tenets of the Declaration of Helsinki involving human subjects and was approved by Institutional Review Board. Twenty-five corneas from human donors (age, 55-80 years) stored in Optisol-GS at 4°C were obtained from National Disease Research Interchange (Philadelphia, PA, USA). Primary culture of adult HCECs was performed in our laboratory as described elsewhere.12 Briefly, the corneal endothelium-Descemet’s membrane complex was digested using a 1.2 U/mL of dispase II in HBSS for 1 hour at 37°C. Afterwards, the solution was collected and centrifuged, and the resulting HCEC pellet was resuspended and cultured in growth medium containing OPTI-MEM, 15% FBS, 40 ng/mL of FGF, 5 ng/mL of EGF, 20 ng/mL of NGF, 20 µg/mL ascorbic acid, 0.005% human lipids, 0.2 mg/mL of calcium chloride, 0.08% chondroitin sulfate, 1% RPMI 1640 vitamin solution, 50 µg/mL of gentamicin, and 1% A/A solution. By this method, around 3 × 103 to 105 cells could be acquired from one donor cornea.

Corneal Endothelial Reconstruction with a Bioengineered Cell Sheet Cultures were then incubated in a humidified atmosphere of 5% CO2 at 37°C. Medium was changed every other day. After 1 week in culture, confluent cell monolayers were subcultured by treating with trypsin-EDTA for 2 minutes, and seeded at a 1:3 split ratio. Only secondpassage HCECs were used during all experiments.

Harvest of HCEC Sheets from Thermoresponsive Culture Supports For the purpose of in vivo tracking, HCECs were labeled with PKH26 red fluorescent dye following manufacturer’s instructions.26 Thermoresponsive supports grafted with PNIPAAm at an optimal density of 1.6 µg/cm2 were used in this study. After surface sterilization with ultraviolet light for 2 hours in the laminar flowhood,27 HCECs were plated on PNIPAAm-grafted culture dishes at a density of 4 × 104 cells/cm2 and cultivated under the same conditions as mentioned for cell preparation. Cell morphology was observed by inverted phase-contrast microscopy (Nikon, Melville, NY, USA). To estimate the cell density, a micrometer scale was used to determine area for calculation of endothelial cell numbers in confluent cultures after 3 weeks of incubation. Six regions on each culture surface were randomly selected and cell nuclei within each area were counted manually at 40× magnification. For the harvest of cell sheets, the thermoresponsive supports containing confluent cultures were rinsed twice with warmed phosphate-buffered saline (PBS) and replenished with serum-free OPTI-MEM. The HCEC monolayers were detached from PNIPAAm-grafted surfaces by changing the culture temperature from 37°C to 20°C.

Viability Bioassay Cell viability of harvested HCEC monolayers was determined by a membrane integrity assay, using the LIVE/ DEAD Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR, USA), which contains calcein acetoxymethyl and ethidium homodimer-1. Briefly, after washing three times with PBS, the HCEC sheets were stained with a working reagent, which is composed of 4 µL of ethidium homodimer-1, 2 mL of PBS, and 1 µL of calcein acetoxymethyl. The samples were incubated for 30 minutes at 37°C and viewed under an inverted fluorescence microscope (Eclipse TS100 equipped with an epifluorescence attachment; Nikon).

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glutaraldehyde in 0.1 M cacodylic acid buffer (pH 7.4) overnight at 4°C. After rinsing with 0.1 M cacodylic acid buffer three times for 10 minutes each time, the specimens were post-fixed in 1% osmium tetroxide for 30 minutes and dehydrated in ethanol solutions 50%, 70%, 90%, and 100%, twice and during 10 minutes for each concentration. The samples were further dried with CO2 in a critical point dryer (Hitachi, Tokyo, Japan), and gold coated by ion sputtering (Structure Probe, PA, USA) before examination under SEM (Jeol, Tokyo, Japan) at an accelerating voltage of 10 kV.

Immunohistochemistry Control samples and HCEC sheets were fixed with 4% paraformaldehyde for 10 minutes at 4°C. After washing with PBS, the fixed specimens were then permeabilized in 0.3% Triton X-100 for 15 minutes, and blocked with 4% bovine serum albumin in PBS for 30 minutes. The samples were incubated with primary antibodies overnight at 4°C in a moist chamber. The antibodies, diluted in PBS containing 4% bovine serum albumin, were directed against zonula occludens-1 (ZO-1) (1:100; Zymed Laboratories, South San Francisco, CA, USA) or Na+,K+-adenosine triphosphatase (ATPase) (1:150; Upstate Biotechnology). The negative controls were incubated without a primary antibody. The specimens were washed in PBS, and incubated with fluorescein (FITC)-conjugated or rhodamine (TRITC)-conjugated donkey anti-mouse IgG secondary antibodies (1:200; Chemicon International, Temecula, CA, USA) for 2 hours at room temperature in the dark. Unbound excess labels were removed by rinsing in PBS. The samples were viewed under fluorescence microscopy (Axioplan 2; Carl Zeiss, Oberkochen, Germany or BX51; Olympus, Tokyo, Japan).

Histology Control samples and HCEC sheets were mounted onto precooled chucks in OCT embedding medium (Tissue-Tek, Sakura Finetek, Torrance, CA, USA) and frozen at -70°C. Frozen specimens were cut into 5-µm sections at -20°C using a cryostat. After the fixation with 4% paraformaldehyde for 1 minute, the sections were stained with 4’,6-diamidino2-phenylindole (DAPI; Vector, Peterborough, United Kingdom) for visualization of cell nuclei and examined using a fluorescence microscope (Axioplan 2; Carl Zeiss).

Scanning Electron Microscopy

HCEC Sheet Transplantation in a Rabbit Model

Whole corneas from human eye bank donors (control groups) and detached HCEC sheets were fixed with 2%

Animals were treated according to the ARVO (Association for Research in Vision and Ophthalmology) Statement for

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the Use of Animals in Ophthalmic and Vision Research. Eighteen New Zealand white rabbits (National Laboratory Animal Breeding and Research Center, Taipei, Taiwan, ROC) weighing 3.0 to 3.5 kg were used in this study. Surgery was performed in the single eye of animals, with the normal fellow eye. As previously described, rabbit corneal endothelium was treated with 0.1 mg/mL of mitomycin-C (Sigma-Aldrich, St. Louis, MO) for 2 weeks to establish an animal model mimicking human corneas.10 For HCEC sheet transplantation, the rabbits were anesthetized intramuscularly with 10 mg/kg body weight of xylazine hydrochloride (Chanelle, Loughrea, Co. Galway, Ireland) and 60 mg/kg body weight of ketamine hydrochloride (Merial, Lyon, France), and topically with two drops of 0.5% proparacaine hydrochloride (Alcon-Couvreur, Puurs, Belgium). After disinfection and sterile draping of the operation site, the pupil was dilated with one drop of 1% atropine sulfate (Oasis, Taipei, Taiwan, ROC), and a lid speculum was placed. In the right eye of each rabbit, the cornea was penetrated near the limbus with a slit knife under the surgical microscope (Carl Zeiss, Oberkochen, Germany). The central 7 mm of corneal endothelium was removed using a silicone-tipped cannula. Once detached and floating in the medium, the HCEC sheets were immediately attached with the gelatin disks (cell apical side up). The gelatin-HCEC sheet constructs were subsequently implanted in the anterior chamber of the eye (cell apical side down) through a 7.5 mm sclerocorneal incision (HCEC sheet group, n = 6). The incision site was closed with 10-0 nylon sutures. Traumatized rabbit corneas received gelatin disk transplantation only (gelatin group, n = 6) or no transplantation (wound group, n = 6) were the controls. After surgery, 1% chlortetracycline hydrochloride ophthalmic ointment (Union Chemical & Pharmaceutical, Taipei, Taiwan, ROC) was immediately applied to the ocular surface in all three groups. For topical administration of corticosteroids, each surgical eye received two drops of 0.3% gentamicin sulfate ophthalmic antibiotic solution (Oasis, Taipei, Taiwan, ROC) and one drop of 1% prednisolone acetate ophthalmic steroid suspension (Allergan, Westport, Co. Mayo, Ireland) four times a day during the follow-up.

An average of ten readings was taken. The results of CCT were expressed as mean ± standard deviation (SD). Comparative studies of means were analyzed using Student’s t-test (two-tailed) with a statistical significance at p < 0.05. At 2 weeks postoperatively, the surgical corneas of the three groups were excised. The integrity of tight junctions of grafted HCEC monolayers were studied by flat-mount preparations. Immunostaining of ZO-1 was performed as mentioned above. For histological examinations, the corneal samples were mounted onto pre-cooled chucks in OCT embedding medium and frozen at –70°C. Frozen specimens were cut into 5 µm sections at –20°C. After the fixation with 4% paraformaldehyde, the sections were stained with hematoxylin and eosin (H&E) and examined using a microscope equipped with fluorescence (Carl Zeiss).

Results Harvest of HCEC Sheets from Thermoresponsive Culture Supports Representative AFM images of the PNIPAAm-grafted PE surfaces are shown in Figure 38-3. The surfaces of PNIPAAm-grafted substrates became rougher after covering with grafted PNIPAAm homogeneously. The mean surface roughness of PNIPAAm-grafted samples was determined to be 14.6 ± 3.2 nm, indicating the PNIPAAm coating on the culture surface is nanostructured.

Postoperative Evaluations The corneal conditions were examined daily for 1 month postoperatively. Corneal clarity was assessed using slitlamp biomicroscopy (Topcon Optical, Tokyo, Japan). Central corneal thickness (CCT) was determined using an ultrasonic pachymeter (DGH Technology, Exton, PA, USA).

Figure 38-3: Representative topographic image of PNIPAAm-grafted surface was observed by tapping-mode atomic force microscopy (scan size = 1 µm, data scale = 30 nm). The mean surface roughness of nanostructured supports (n = 3) with a homogeneous covering of PNIPAAm was determined to be 14.6 ± 3.2 nm.

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After 4 hours of plating, isolated HCECs attached and spread well on the PNIPAAm-grafted surfaces (Figure 38-4A). Cells grew readily to reach confluence after 1-week cultivation at 37°C. By a further incubation for 2 weeks in medium, a thick layer of extracellular matrix (ECM) was deposited at the basal cell surface to allow the formation of a fully confluent monolayer. Under a phase-contrast microscope, confluent HCECs on the PNIPAAm-grafted surfaces showed a generally polygonal morphology and a high cell density, around 2500 cells/mm2 that was almost the same as found in vivo (Figure 38-4B). This indicated that the ex vivo proliferation rate of HCECs on the thermoresponsive supports was maintained. By lowering the culture temperature to 20°C, the detachment of monolayered HCECs from the switched hydrophilic PNIPAAm-grafted surfaces forms a sheet-like movement (Figure 38-4C). During the sheet-like movement, each endothelial cell at the leading edge assembles by contracting fan-shaped lamellipodia. In addition, cell release from the PNIPAAm-grafted surfaces was observed (Figure 385). At the beginning of the low-temperature treatment, the HCEC monolayers were rolled up at the margin of the culture surfaces and centripetally detached owing to the gradual hydration of the PNIPAAm-grafted chains (Figure 38-5A). Such a process of cell separation from thermoresponsive supports is a mode of sheet-like movement (Figure 38-5B). After 45 min of incubation, a laminated HCEC sheet with a size of around 0.75 cm2 was harvested from completely hydrated PNIPAAm-grafted surfaces, and was wrinkled because of a contracting force of this cell lamella (Figure 38-5C). These results suggest that the cell sheet detachment from thermoresponsive supports correlates closely with temperature-modulated surface wettability.

Viability Bioassay

C Figures 36-4A to C: Phase-contrast micrographs of HCEC cultures on the thermoresponsive supports. (A) At 4 hours after seeding, the HCECs were attached on the PNIPAAm-grafted surfaces. (B) After incubation at 37°C for 3 weeks, a fully confluent endothelial monolayer was consisted of small, polygon-shaped cells. (C) At 20°C, the detachment of monolayered HCECs exhibited a sheet-like movement. Scale bars, 100 µm.

Cell viability of harvested HCEC monolayers from PNIPAAm-grafted surfaces was determined by the LIVE/ DEAD Viability/Cytotoxicity assay (Figure 38-6). It depends on the intracellular esterase activity to identify the living cells, which cleaves the calcein acetoxymethyl to produce a green fluorescence. In dead cells, ethidium homodimer-1 can easily pass through the damaged cell membranes to bind to the nucleic acids, yielding a red fluorescence. Nearly all of the cells were vital throughout the central region of detached HCEC sheets (Figure 38-6A). Only very few dead cells were interspersed between the live cells of endothelial monolayers. However, the results for the peripheral region of HCEC sheets showed a large number of green-stained cells and red-stained nuclei in the margin of cell monolayers (Figures 38-6B and C). This was probably due to the loss of

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A A

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C Figures 38-5A to C: Gross observations of cultured HCEC monolayer detachment from thermoresponsive supports after incubation at 20°C for 10 minutes (A), 25 minutes (B), and 45 minutes (C). Scale bars, 5 mm.

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C Figures 38-6A to C: Cell viability of harvested HCEC monolayers was determined by staining with a LIVE/DEAD Viability/Cytotoxicity Kit in which the live cells fluoresce green and the dead cells fluoresce red. (A) Merged green and red fluorescence image (central region of cell sheets). (B and C) Color-separated images (peripheral region of cell sheets). Scale bars, 100 µm.

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anchorage-dependence for marginal cells caused by overgrowth of a fully confluent cell sheet during 3 weeks of culture. These data clearly demonstrated that the majority of monolayered HCECs remained viable after detachment at 20°C, suggesting the low-temperature incubation does not compromise cell viability.

Scanning Electron Microscopy

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SEM studies examined the morphological characteristics of native endothelium in human eye bank corneas and of monolayered cells in cultured HCEC sheets (Figure 38-7). In control groups, HCECs on the Descemet’s membrane from the eye bank corneas (donors = 50 years of age) packed together and formed a single continuous monolayer (Figure 38-7A). The individual cells were intact, had distinct borders, and possessed a hexagonal morphology with minor irregularities. By contrast, after immediate separation from thermoresponsive supports, the HCEC monolayers remained well-organized. The monolayered cells within HCEC sheets exhibited a polygonal phenotype and had multiple cellular interconnections (Figure 38-7B). The absence of clear boundaries between these single cells was probably due to the cell contraction caused by detachment at a low culture temperature. In addition, a thick ECM layer was observed on the basal cell surface of HCEC sheets (Figure 38-7C).

Immunohistochemistry B

C Figures 38-7A to C: Scanning electron microscopy (SEM) micrographs of human eye bank corneas (control samples) and HCEC sheets. (A) Native endothelium in whole corneas showed a normal hexagonal cell shape with minor irregularities. (B) Within HCEC sheets, cells exhibited a polygonal morphology with multiple cellular interconnections (arrow). (C) A layer of extracellular matrix (ECM) (arrow) was deposited at the basal cell surface of cell sheets. Scale bars, 50 µm.

Immunohistochemical staining of ZO-1, a tight junctionassociated protein, was used to determine whether the cells within HCEC sheets formed tight junctions. In human donor corneas, the cells of native endothelium retained the tight junctions that are responsible for establishing the passive permeability properties of the endothelial barrier (Figure 38-8A). Similar to that of control samples, ZO-1 was located at the cell boundaries of HCEC sheets suggesting the formation of focal tight junctional complexes (Figure 38-8B).28 At higher magnification, the discontinuity of ZO-1 localization in HCEC sheets, which is a normal feature in corneal endothelium, was observed with gaps occurring at the Y-junctions between three adjacent cells (Figure 38-8C). In addition, there was no evidence of apoptotic cell death in the HCEC monolayers after detachment via thermal stimulus (i.e. the low- temperature incubation), as reflected by the maintenance of the integrity of cell nuclei (Figure 38-8D). On the other hand, Na+,K+ATPase, an integral membrane protein complex responsible for regulating ionic pump functions, was located at the basolateral membrane of the HCECs within both control samples and detached cell sheets (Figures 38-8E and F).29

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Figures 38-8A to F: Fluorescence micrographs of immunolocalization of zonula occludens-1 (ZO-1) and Na+,K+-adenosine triphosphatase (ATPase) in HCECs within detached cell sheets compared to control samples. A typical pattern of lateral membrane interdigitation of ZO-1 (arrows) was presented at the endothelial cell boundary in both control samples (A) and HCEC sheets (B). (C) At higher magnification, a discontinuous tight junction was detected by immunostaining for ZO-1 protein (arrow), which indicated barrier formation. (D) Nuclear morphology of cells within HCEC monolayers labeled with 4’,6-diamidino-2-phenylindole (DAPI). Distribution of Na+,K+-ATPase (arrows) was also detected in control samples (E) and HCEC sheets (F), respectively. TRITC: red fluorescence (A), FITC: green fluorescence (B, C, E, and F), and DAPI: blue fluorescence (D). Scale bars, 50 µm (A, B, and D); 10 µm (C); 20 µm (E and F).

Corneal Endothelial Reconstruction with a Bioengineered Cell Sheet Histology The cross-sections of control samples and HCEC sheets were stained for DAPI to examine the histological structure of endothelium. The endothelial cells from the human donor corneas were organized on the Descemet’s membrane as a monolayer (Figure 38-9A). The detached cell sheets also showed a monolayered architecture of cells that mimicked native endothelium (Figure 38-9B).

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to correspond to the cell polarity as in vivo (Figures 38-1D and E). After cell separation from thermoresponsive culture substrates at 20°C, a bioadhesive gelatin disk was placed on the apical surface of the harvested HCEC sheets, and the gelatin-HCEC sheet constructs were spontaneously formed by a 5-min incubation at room temperature (Figure 38-10).

A Figure 38-10: Gelatin carriers for intraocular delivery of thermally detached HCEC sheets. After cell release from PNIPAAm-grafted culture surfaces at 20°C, the gelatin-HCEC sheet constructs were made by using the 7-mm diameter gelatin disks (arrow) as a transparent and bioadhesive supporter for HCEC sheets with a size of around 0.75 cm2. Scale bar, 5 mm.

Postoperative Evaluations

B Figures 38-9A and B: Histological examination of control samples (A) and HCEC sheets (B) with DAPI labeling show a monolayer of endothelial cells (arrows) was located on corneal stroma (asterisk) and cell carrier membrane (double asterisk), respectively. Scale bars, 20 µm.

Gelatin Carriers for HCEC Sheet Transplantation Given that HCECs in vivo possess polarity and pump water from corneal stroma into the anterior chamber, a correct orientation of the transplanted HCECs must be maintained with the apical side facing the aqueous humor in anterior chamber. Accordingly, the detached HCEC sheets were delivered using the 7 mm gelatin disks (700-800 µm thick, MW = 100,000, IEP = 5) with the HCECs apical side down

For in vivo transplantation studies, the central 7 mm of corneal endothelium was removed with a silicone-tipped cannula (Figure 38-11A). In HCEC sheet groups, after surgery, slit-lamp biomicroscopy revealed that the anterior chamber was filled up with the gelatin-HCEC sheet constructs (Figure 38-11B). Moreover, an intact, roundshaped layer of HCECs was positioned onto the denuded corneal posterior surface. The following day, severe corneal swelling was noted, and persisted until completion of the experiment in wound (Figure 38-11C) and gelatin groups. At postoperative 2 weeks, the gelatin disks largely dissolved and HCEC sheets were attached onto the denuded surface of Descemet’s membrane in the HCEC sheet groups. The swollen cornea returned to clarity and a nearly normal corneal thickness after implantation of HCEC sheets 4 weeks postoperatively (Figure 38-11D). The corneal thickness of traumatized corneas with transplanted HCEC sheet improved more significantly than that of the control groups during the first postoperative 2 weeks (Figure 38-12). All corneas in the control groups did not return to

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Figures 38-11A to D: Representative slit-lamp biomicroscopic images revealed that the native rabbit corneal endothelium (arrow) was mechanically stripped from Descemet’s membrane (wound groups) (A), an intact monolayer of HCECs was positioned onto the cornea denuded of endothelial cells after the gelatin-HCEC sheet construct (arrow) was inserted into the anterior chamber (HCEC sheet groups) (B). At postoperative 4 weeks, the corneas in the wound groups were seriously edematous and cloudy (C). However, in the HCEC sheet groups, corneal opacity and edema were significantly improved (D). Scale bars, 5 mm.

Figure 38-12: The mean central corneal thickness (CCT) in the HCEC sheet groups gradually decreased from 911 ± 20 µm to 552 ± 18 µm, i.e. slightly higher than those before transplantation, 517 ± 30 µm. In the traumatized corneas implanted with a gelatin disk only (gelatin groups) and wound groups, the mean CCT remained at a high level, i.e. greater than 1000 µm. Above data indicated that corneal edema was significantly improved due to the transplanted bioengineered HCEC sheet. Wound, gelatin, and HCEC sheet, n = 6 for each group. *P < 0.05, **P < 0.001 versus wound or gelatin groups (t test).

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C Figures 38-13A to C: (A) After transplantation for 2 weeks, the excised cornea in the HCEC sheet groups was examined by fluorescent microscopy. Immunohistochemistry for ZO-1 protein revealed that tight junctions (arrow) within the HCEC sheet were maintained. (B and C) At postoperative 2 weeks, histological examinations indicated the implanted HCEC monolayer (arrow) was attached onto Descemet’s membrane by hematoxylin-eosin (H-E) staining (B). Integrated HCECs (PKH26 red fluorescent dye-positive) (arrow) on the Descemet’s membrane was shown by fluorescent microscopy (C). Scale bars, 20 µm.

normal during the follow-up. Figures 38-13A to C illustrate the images of flat-mounts and cross-sections of the grafted corneal samples in the HCEC sheet groups. Under fluorescence microscopy, the HCEC sheet grafts were shown on the recipient corneas and maintained intact tight junctions at 2 weeks postoperatively (Figure 38-13A). Histological examination under light and fluorescent microscopy revealed that, after surgery for 2 weeks, the implanted HCECs labeled with PKH26 red fluorescent dye remained attached (Figures 38-13B and C).

Discussion HCECs in vivo demonstrate an age-related decrease in cell density and cannot be compensated due to their limited regenerative capacity.2 When the cell density is less than a critical level of 1000 cells/mm2, the endothelium no longer functions, causing corneal edema and loss of visual acuity. In these cases, HCEC transplantation aims to restore vision with the hope of reconstituting a structural and functional endothelial monolayer. Central to the tissue reconstruction

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is the cellular arrangement and organization of grafts (i.e. the well-organized cell sheets or isolated cell suspensions). Therefore, the ability to obtain and deliver an intact monolayer of HCECs would be beneficial for developing an effective therapeutic strategy to rescue damaged endothelium. In this chapter, we presented a novel method for harvesting cultured human corneal endothelium for tissue repair. In contrast with traditional enzymatic digestion (i.e. trypsinization), a cell culture system using thermoresponsive supports has been designed to allow the bioengineered endothelial equivalents to retain cellular activity, organization, function, and ECM integrity. Cultivation of adult HCECs from older donors has been proven to be difficult.30 To overcome this, a growth factorenriched medium was used to succeed in mass culturing untransformed adult HCECs. In this study, when cultivated on the nanostructured PNIPAAm-grafted surfaces, the HCECs derived from older donor tissue keep their phenotypic characteristics and proliferative capacity as grown on the commercial tissue culture plates. For strengthening of the harvested HCEC monolayers, the confluent cultures could be stimulated to become thicker by a further 2-week incubation to enhance cell production of ECM. This unique phenomenon of ECM formation in cultured HCECs possibly indicated the same property of increasing thickness of Descemet’s membrane with aging in the human cornea.31 In addition, the cell density of confluent HCEC monolayers is comparable to those as in vivo. It suggests that these bioengineered tissue equivalents have sufficient cell numbers to support their barrier and ionic pump functions. After 45 min of incubation at 20°C, the area of harvested HCEC sheets decreased approximately from 9.6 to 0.75 cm2. These results support the report by Shimizu et al demonstrating that spread cells are compacted after the temperature-modulated detachment of cultured cardiomyocyte sheets.15 One possible explanation for the shrinkage of HCEC sheets is that the reorganization of cytoskeleton is induced by the low-temperature treatment. When autologous transplantation is considered, the HCEC sheet grafts should be designed to fit the size of the initial biopsy in the recipient. Despite the contraction observed in these bioengineered tissue grafts, the size of harvested HCEC sheets can be arbitrarily adjusted by controlling the surface size of thermoresponsive culture supports. It has been reported that the hypothermic preservation (4°C) of cells would result in decreased activity of the Na+ pump 32 or induce apoptosis. 33 Since the cell sheet detachment is performed at 20°C, which is lower than its normal culture temperature (37°C), the effects of lowtemperature treatment on the viability of harvested tissue

equivalents should be investigated. Our data indicate that the detached HCEC monolayers are tolerant of 45 min incubation at 20°C. In this study, we performed viability testing in cell sheets after immediate separation from thermoresponsive supports. Although the HCEC monolayers were detached and preserved in serum-free OPTI-MEM, the storage time (time between detachment and begin of implantation) is not a concern for our study. Using cell-adhesive and transparent gelatin hydrogel carriers, the detached HCEC sheets with good viability can be immediately transplanted through a 7.5 mm sclerocorneal incision to recipient corneas denuded of endothelium.10 In addition to retain their deposited ECM, the harvested HCEC sheets with cellular interconnections are consisted of closely packed, small, polygon-shaped cells when studied morphologically. These findings are consistent with previous publications regarding morphological observations of cultured corneal endothelial cells on carrier substrates5,7-9 or corneas denuded of endothelium.1,34-40 Immunohistochemistry studies demonstrated the proper location of ZO-1 and Na+, K+-ATPase proteins implying the HCEC sheets are capable of maintaining intact barrier functions as well as ionic pump functions. By histological examination, the monolayered architecture of detached cell sheets is also confirmed. It is important to note that these characteristics of cultured HCEC monolayers are similar to those observed on the native endothelium of eye bank donor corneas. In an effort to develop alternative therapy techniques, we have evaluated the feasibility of using HCEC sheets for corneal endothelial reconstruction.10 As a graft source, cell sheets have an intact cellular arrangement and a cellular organization, which are important factors for successful graft-host integration and tissue repair. Our previous report on transplantation of intact retinal sheets has demonstrated that these grafts positioned with correct polarity could grow into ordered and viable laminated retinas.24 However, the dissociated retinal cell suspensions or microaggregates simply develop the rudimentarily differentiated rosettes after being grafted into the subretinal space.41 Despite having a tissue-like architecture, the thermally detached cell sheets were easily wrinkled and folded during removal of the thermoresponsive culture substrates.42 In the field of cell sheet transfer, Okano et al have introduced poly(vinylidene difluoride) (PVDF) membranes as a supporter, which renders for the three-dimensional manipulation of cardiomyocyte sheets into layered constructs.15 Moreover, using a doughnut-shaped PVDF supporter, recent attempts have been made to reconstruct ocular surface by transplantation of cultured epithelial cell sheets originating from autologous corneas17 or oral

Corneal Endothelial Reconstruction with a Bioengineered Cell Sheet mucosae. 18 The results of these works have been encouraging since the visual acuity of patients who received bioengineered cell sheets was significantly improved. However, intraocular grafting is different from the trials in corneal epithelial cell therapy because the unique physiological environments (i.e. anterior chamber and subretinal space) are perfused with large amounts of tissue fluid, which may cause unstable attachment of implanted cell sheets to lesion sites. In these cases, to deliver and retain the cells at the site of injury is important in the design of related cell therapy techniques. Therefore, it is necessary to provide a temporary support structure for enhancing the graft-host integration during tissue reconstruction. Compared with other reports on corneal endothelial cell transplantation studies using different carriers,4-9 we have developed a novel method to deliver the cultivated HCEC monolayers by utilizing a biodegradable and cell-adhesive gelatin disk without permanent residence of carrier materials in vivo. We have shown that the gamma-sterilized hydrogel disks made from raw gelatins (IEP = 5.0, MW=100 kDa) with appropriate dissolution degree and acceptable cytocompatibility are capable of providing stable mechanical support, making these carriers promising candidates for intraocular delivery of cultivated HCEC sheets.11 Results from a short-term study have suggested that the transplanted HCEC sheets could be integrated into rabbit corneas denuded of endothelium.10 Additionally, the corneas have returned to a nearly normal thickness indicating the function of bioengineered HCEC sheets. For intraocular delivery of the cultured cell sheets, the use of gelatin carriers is very attractive because of the highly transparent and deformable nature of this hydrogel material. It is known that surgery with smaller incisions may provide faster rehabilitation and reduce postoperative morbidity. Currently, PK is the most common way to treat corneas that are opacified due to endothelial dysfunction. When compared with PK, the cell therapy techniques proposed in the present study enable intraocular grafting of HCEC sheets to be performed to occur with minimal surgical incisions. Since the foreign supporting materials are substantially completely absorbed in vivo, we believe that this novel approach will have a high success rate in treating corneal endothelial cell loss. Although these data are encouraging, long-term efficacy and safety data need further investigation. In summary, this study described a novel cell therapeutic method for HCEC loss, by mass cultivating HCECs from adult human corneal donors, harvesting HCECs as a cell sheet after detaching from a thermoresponsive PNIPAAmgrafted surface and delivering HCECs with a negatively

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charged, high molecular weighted gelatin disk. Based on the current methodology, the adult HCEC monolayers having normal morphology and viability can be obtained without the need for cell carriers during cultivation. After transplantation, the functional HCEC sheets were integrated into the denuded corneas, with the returned corneal clarity. Results of this study demonstrated the feasibility of transplanting HCEC sheet for corneal endothelial cell loss and as a possible alternative to PK.

References 1. Jumblatt MM, Maurice DM, McCulley JP. Transplantation of tissue-cultured corneal endothelium. Invest Ophthalmol Vis Sci 1978;17:1135-41. 2. Joyce NC. Proliferative capacity of the corneal endothelium. Prog Retin Eye Res 2003;22:359-89. 3. McCulley JP, Maurice DM, Schwartz BD. Corneal endothelial transplantation. Ophthalmology 1980;87:194-201. 4. Lange TM, Wood TO, McLaughlin BJ. Corneal endothelial cell transplantation using Descemet’s membrane as a carrier. J Cataract Refract Surg 1993;19:232-5. 5. Ishino Y, Sano Y, Nakamura T, Connon CJ, Rigby H, Fullwood NJ, Kinoshita S. Amniotic membrane as a carrier for cultivated human corneal endothelial cell transplantation. Invest Ophthalmol Vis Sci 2004;45:800-6. 6. Jumblatt MM, Maurice DM, Schwartz BD. A gelatin membrane substrate for the transplantation of tissue cultured cells. Transplantation 1980;29:498-9. 7. Insler MS, Lopez JG. Microcarrier cell culture of neonatal human corneal endothelium. Curr Eye Res 1990;9:23-30. 8. Mohay J, Lange TM, Soltau JB, Wood TO, McLaughlin BJ. Transplantation of corneal endothelial cells using a cell carrier device. Cornea 1994;13:173-82. 9. Mimura T, Yamagami S, Yokoo S, Usui T, Tanaka K, Hattori S, Irie S, Miyata K, Araie M, Amano S. Cultured human corneal endothelial cell transplantation with a collagen sheet in a rabbit model. Invest Ophthalmol Vis Sci 2004;45:2992-7. 10. Hsiue GH, Lai JY, Chen KH, Hsu WM. A novel strategy for corneal endothelial reconstruction with a bioengineered cell sheet. Transplantation 2006;81:473-6. 11. Lai JY, Lu PL, Chen KH, Tabata Y, Hsiue GH. Effect of charge and molecular weight on the functionality of gelatin carriers for corneal endothelial cell therapy. Biomacromolecules 2006;7: 1836-44. 12. Lai JY, Chen KH, Hsu WM, Hsiue GH, Lee YH. Bioengineered human corneal endothelium for transplantation. Arch Ophthalmol 2006;124:1441-8. 13. Yamato M, Okano T. Cell sheet engineering. Mater Today 2004;7:42-47. 14. Yang J, Yamato M, Kohno C, Nishimoto A, Sekine H, Fukai F, Okano T. Cell sheet engineering: Recreating tissues without biodegradable scaffolds. Biomaterials 2005;26:6415-22. 15. Shimizu T, Yamato M, Isoi Y, Akutsu T, Setomaru T, Abe K, Kikuchi A, Umezu M, Okano T. Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circ Res 2002;90:e40-e48. 16. Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K, Hao H, et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med 2006;12:459-65.

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17. Nishida K, Yamato M, Hayashida Y, Watanabe K, Maeda N, Watanabe H, et al. Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface. Transplantation 2004;77:379-85. 18. Nishida K, Yamato M, Hayashida Y, Watanabe K, Yamamoto K, Adachi E, et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med 2004;351:1187-96. 19. Hayashida Y, Nishida K, Yamato M, Watanabe K, Maeda N, Watanabe H, et al. Ocular surface reconstruction using autologous rabbit oral mucosal epithelial sheets fabricated ex vivo on a temperature-responsive culture surface. Invest Ophthalmol Vis Sci 2005;46:1632-9. 20. Yamada N, Okano T, Sakai H, Karikusa F, Sawasaki Y, Sakurai Y. Thermo-responsive polymeric surfaces; control of attachment and detachment of cultured cells. Makromol Chem Rapid Commun 1990;11:571-6. 21. Hsiue GH, Lee SD, Wang CC, Shiue MHI, Chang PCT. Plasmainduced graft copolymerization of HEMA onto silicone rubber and TPX film improving rabbit corneal epithelial cell attachment and growth. Biomaterials 1994;15:163-71. 22. Lee SD, Hsiue GH, Kao CY, Chang PCT. Artificial cornea: Surface modification of silicone rubber membrane by graft polymerization of pHEMA via glow discharge. Biomaterials 1996;17:587-95. 23. Chang PCT, Lee SD, Hsiue GH. Heterobifunctional membranes by plasma induced graft polymerization as an artificial organ for penetration keratoprosthesis. J Biomed Mater Res 1998;39: 380-9. 24. Hsiue GH, Lai JY, Lin PK. Absorbable sandwich-like membrane for retinal-sheet transplantation. J Biomed Mater Res 2002;61: 19-25. 25. Hsiue GH, Wang CC. Functionalization of polyethylene surface using plasma-induced graft copolymerization of acrylic acid. J Polym Sci Pol Chem 1993;31:3327-37. 26. Horan PK, Slezak SE. Stable cell membrane labelling. Nature 1989;340:167-8. 27. Fischbach C, Tessmar J, Lucke A, Schnell E, Schmeer G, Blunk T, Göpferich A. Does UV irradiation affect polymer properties relevant to tissue engineering? Surf Sci 2001;491:333-45. 28. Petroll WM, Hsu JKW, Bean J, Cavanagh HD, Jester JV. The spatial organization of apical junctional complex-associated proteins in feline and human corneal endothelium. Curr Eye Res 1999;18:10-19. 29. McCartney MD, Wood TO, McLaughlin BJ. Immunohistochemical localization of ATPase in human dysfunctional corneal endothelium. Curr Eye Res 1987;6:1479-86.

30. Senoo T, Joyce NC. Cell cycle kinetics in corneal endothelium from old and young donors. Invest Ophthalmol Vis Sci 2000;41: 660-7. 31. Stiemke MM, Edelhauser HF, Geroski DH. The developing corneal endothelium: Correlation of morphology, hydration and Na/K ATPase pump site density. Curr Eye Res 1991;10:14556. 32. Mathew AJ, Baust JM, Van Buskirk RG, Baust JG. Cell preservation in reparative and regenerative medicine: Evolution of individualized solution composition. Tissue Eng 2004;10: 1662-71. 33. Rauen U, Petrat F, Li T, De Groot H. Hypothermia injury/coldinduced apoptosis: Evidence of an increase in chelatable iron causing oxidative injury in spite of low O2–/H2O2 formation. Faseb J 2000;14:1953-64. 34. Chen KH, Azar D, Joyce NC. Transplantation of adult human corneal endothelium ex vivo: A morphologic study. Cornea 2001;20:731-7. 35. Insler MS, Lopez JG. Extended incubation times improve corneal endothelial cell transplantation success. Invest Ophthalmol Vis Sci 1991;32:1828-36. 36. Insler MS, Lopez JG. Heterologous transplantation versus enhancement of human corneal endothelium. Cornea 1991;10:136-48. 37. Gospodarowicz D, Greenburg G, Alvarado J. Transplantation of cultured bovine corneal endothelial cells to rabbit cornea: Clinical implications for human studies. Proc Natl Acad Sci USA 1979;76:464-8. 38. Gospodarowicz D, Greenburg G, Alvarado J. Transplantation of cultured bovine corneal endothelial cells to species with nonregenerative endothelium. The cat as an experimental model. Arch Ophthalmol 1979;97:2163-9. 39. Aboalchamat B, Engelmann K, Böhnke M, Eggli P, Bednarz J. Morphological and functional analysis of immortalized human corneal endothelial cells after transplantation. Exp Eye Res 1999;69:547-53. 40. Böhnke M, Eggli P, Engelmann K. Transplantation of cultured adult human or porcine corneal endothelial cells onto human recipients in vitro. Part II: Evaluation in the scanning electron microscope. Cornea 1999;18:207-13. 41. Juliusson B, Bergström A, van Veen T, Ehinger B. Cellular organization in retinal transplants using cell suspensions or fragments of embryonic retinal tissue. Cell Transplant 1993;2: 411-8. 42. Hirose M, Kwon OH, Yamato M, Kikuchi A, Okano T. Creation of designed shape cell sheets that are noninvasively harvested and moved onto another surface. Biomacromolecules 2000;1: 377-81.

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Future of Posterior Lamellar Keratoplasty

Thomas John Kenneth R Kenyon

Future of Posterior Lamellar Keratoplasty

39

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Corneal Endothelial Transplant

Introduction Descemet stripping automated endothelial keratoplasty (DSAEK)1-11 is a new form of corneal transplant surgery where the donor corneal disk comprising of deep corneal stroma, Descemet’s membrane (DM) and healthy endothelium is added onto the inner corneal surface of the patient’s cornea. An air-bubble facilitates the initial intraoperative adhesion of the donor disk to the patient’s cornea, followed by the donor corneal endothelium helping in the continued adherence of the donor disk to the patient’s cornea. Further, over time, this adhesion becomes even stronger as there is continued tissue remodeling and integration of the stromal donor-recipient tissues at the level of the interface.

From the Present to the Future In DSAEK surgery, stroma, endothelium and DM are transplanted. However, only the healthy endothelium is necessary to clear a cloudy host cornea. Hence, the elimination of the donor corneal stroma will be beneficial and also will decrease the overall increased corneal thickness of the transplanted host cornea. The donor stromal tissue acts much like a carrier layer for the healthy donor endothelium in this form of transplantation surgery. This progression of transplantation technique without the donor corneal stroma has already been performed12,13 [See also Chapter 36, True Endothelial Cell (TEnCell) Transplantation, and Chapter 37, Descemet Membrane Endothelial Keratoplasty (DMEK)]. In 2006, Melles et al used organ-cultured DM with the endothelium in one patient and in 10 cadaveric eyes and called the procedure Descemet membrane endothelial keratoplasty (DMEK).12 In 2007, Tappin reported using donor endothelial cells with only a Descemet’s carrier in 3 patients with endothelial cell failure, and called it true endothelial cell (TEnCell) transplant.13 However, this technique of transplanting only the healthy donor endothelium with the attached Descemet’s membrane (DM) is called by the author (TJ) “Descemet’s membrane endothelial transplant (DECT)” [See also Chapter 36, True Endothelial Cell (TEnCell) Transplantation, and Chapter 37, Descemet Membrane Endothelial Keratoplasty (DMEK) and Chapter 13, Definition, Terminology and Classification of Lamellar Corneal Surgery] has not been perfected for mass duplication by corneal surgeons at the present time (at the time of writing this chapter). The terms DMEK and TEnCell transplant are synonymous with DECT. However, DMEK is the more commonly known name for this procedure. The future directions in DSAEK surgery may be in way of further improvements and refinements in

this surgical technique for easier duplication. This may also include the designing and manufacturing of new surgical instruments to facilitate this goal. In this type of procedure, some of the hurdles include handling a thin, flexible layer of tissue and attaching such a layer to the recipient cornea without any folds or wrinkles in the transplanted thin circular disk. Another surgical direction in the future would be the possible transplantation of healthy donor endothelial cells without any carrier tissue, onto the inner surface of the patient’s cornea (See also Chapter 38, Corneal Endothelial Reconstruction with a Bioengineered Cell Sheet). This type of surgery is called by the author (TJ) endothelial cell transplantation (ECT) (See also Chapter 13, Definition, Terminology and Classification of Lamellar Corneal Surgery). In this case the healthy donor endothelial cells have to adhere to the host cornea, continue to function and clear the host cornea. Some of the difficulties in ECT would include the preparation of healthy donor endothelial cells, introduction and transfer of these cells into the recipient anterior chamber (AC), and having these endothelial cells adhere to the inner corneal surface of the recipient cornea without being detached and washed away by the aqueous humor. Also, if endothelial cells are detached and dislocated to another region in the AC such as the iris, lens surface or the anterior chamber angle this may have some potential deleterious effects. Additionally, in ECT, the transplanted cells have to be healthy and continue to function in the recipient environment to clear the host cloudy cornea. This type of surgery will eliminate some of the current surgical steps used in DSAEK surgery, including the donor disk preparation using the artificial anterior chamber, the tacofolding of the donor corneal disk, and the use of an airbubble to attach the disk to the host cornea. However, at the present time (time of writing this chapter), ECT is not a reality. The ultimate step in the future directions of DSAEK surgery would be to eliminate the use of all donor corneal tissues and only work with the patient’s own corneal endothelial cell layer. This may mean, some form of technique(s) to activate the decompensated corneal endothelium to function again to clear the patient’s cloudy cornea. Such a type of procedure may be called endothelial cell activation (ECA) (See also Chapter 13, Definition, Terminology and Classification of Lamellar Corneal Surgery). ECA may be limited to those cases of early endothelial cell dysfunction, prior to complete endothelial decompensation and irreversible cellular death. This type of surgery is not possible at the time of writing this chapter. The current advancements in Selective Tisuue Corneal Transplantation (STCT), a term that the author (TJ)

Future of Posterior Lamellar Keratoplasty introduced,1-3 is highly beneficial in the field of corneal transplantation, since it has eliminated a full-thickness corneal wound and the use of any corneal sutures. The future of DSAEK surgery looks even more promising, and such surgical techniques as DECT, ECT, and ECA may become an everyday reality with improved qualitative and quantitative visual outcome for the patient.

References 1. John T. Descemetorhexis with endokeratoplasty. In: Surgical Techniques in Anterior and Posterior Lamellar Corneal Surgery. John T (Ed.). Jaypee Brothers Medical Publishers (P) Ltd., New Delhi, India, 2006;411-20. 2. John T. (Ed.) Selective tissue corneal transplantation: A great step forward in global visual restoration. Expert Rev Ophthalmol 2006;1:5-7. 3. John T. Descemetorhexis with endokeratoplasty (DXEK). In: Step by Step Anterior and Posterior Lamellar Keratoplasty. John T (Ed.). Jaypee Brothers Medical Publishers (P) Ltd., New Delhi, India, 2006;177-96. 4. Melles GR, Wijdh RH, Nieuwendaal CP. A technique to excise the Descemet membrane from a recipient cornea (Descemetorhexis). Cornea 2004;23:286-8.

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5. Busin M, Arffa RC, Sebastiani A. Endokeratoplasty as an alternative to penetrating keratoplasty for the surgical treatment of diseased endothelium: Initial results. Ophthalmology 2000; 107:2077-82. 6. Terry MA. The evolution of lamellar grafting techniques over twenty-five years. Cornea 2000;19:611-6. 7. Melles GR, Lander F, Rietveld FJ. Transplantation of Descemet’s membrane carrying viable endothelium through a small scleral incision. Cornea 2002;21:415-8. 8. Price MO, Price FW Jr. Descemet’s stripping with endothelial keratoplasty: Comparative outcomes with microkeratomedissected and manually dissected donor tissue. Ophthalmology 2006;113:1936-42. 9. Price FW Jr, Price MO. Descemet’s stripping with endothelial keratoplasty in 200 eyes: Early challenges and techniques to enhance donor adherence. J Cataract Refract Surg 2006;32: 411-8. 10. Mearza AA, Quershi MA, Rostron CK. Experience and 12month results of descemet-stripping endothelial keratoplasty (DSEK) with a small-incision technique. Cornea 2007;26:27983. 11. Price MO, Price FW. Descemet’s stripping endothelial keratoplasty. Curr Opin Ophthalmol 2007;18:290-4. 12. Melles GR. Posterior lamellar keratoplasty: DLEK to DSEK to DMEK. Cornea 2006;25:879-81. 13. Tappin M. A method for true endothelial cell (Tencell) transplantation using a custom-made cannula for the treatment of endothelial cell failure. Eye 2007;21:775-9.

Index.................................................. A Accommodation 40 AC-OCT and DSEK complications 55 AC-OCT for DSEK and DSAEK 54 Active Cl- transport 15 Active Na+ transport 15 Air bubble 306 Anesthesia 173,226 Anterior chamber exploration with the Visante™ OCT 40 accommodation 40 anterior chamber measurements 40 cornea 42 crystalline lens 41 Anterior lamellar keratoplasty (ALK) 136 Anterior segment clinical ultrasonic imaging 63 Anterior segment OCT 45 Aperture inlay 51 presbyopia correction 51 Archival system 90 Artifact removal 91 Artificial anterior chambers 124 Barron disposable ACC 127 Bausch and Lomb ACC 126 disposable ACC 127 Moria ACC 124 reusable ACC 124 types 124 Astigmatism after DLEK 181 Automated donor tissue preparation (DSAEK) 229

B Barrier function 14 Barron disposable ACC 127 Basal cells 76 Bowman’s layer 77 confocal microscopy appearance 76 Descemet’s membrane 78 endothelium 78 epithelium 76 stroma 77 superficial epithelial cells 77 wing cells 76 Bausch and Lomb ACC 126 Bi-fold method 292 Bioengineered cell sheet 407 Biomechanical metrics 9 implication 9 modeling 9 surgical planning 9

Biomechanics 3 intraocular pressure 7 Blood 312 anterior chamber 312 Bowman’s layer 77

C Capsulorhexis 92 Cell preparation 408 Classification of LKP 137 age 138 depth of the recipient corneal resection 138 diameter (donor and recipient) 138 donor corneal thickness 138 general 137 location 137 number of procedures 138 stem-cell transplantation 137 substitution or addition of donor tissue 138 surgical approach 138 type 137 use of microkeratome 138 Combined DLEK and phacoemulsification 185 donor tissue preparation 192 phacoemulsification technique 185 recipient surgery small incision DLEK 188 surgical procedure 185 transplantation of the donor tissue 195 Combined surgery 234 with DSAEK 234 Combining with phaco 292 Computer-aided surgery 91 Confocal microscopy 72 development 72 Congenital hereditary endothelial dystrophy (CHED) 27 Control of corneal hydration 18 Cornea 4,42 collagen structure 4 Corneal adhesive 373 Corneal anatomy 76 Corneal avascularity 18 Corneal biomechanical properties 5 in vivo measurement 6 metrics 5 new techniques 6 Corneal characteristics 80 Corneal edema 19 endothelial changes 19

epithelial edema 19 stromal edema 19 Corneal endothelial reconstruction 406 bioengineered cell sheet 407 cell preparation 408 experimental 408 gelatin carriers for HCEC sheet transplantation 415 harvest of HCEC sheets 409 HCEC sheet transplantation 409 histology 409,415 immunohistochemistry 409,413 materials 408 postoperative evaluations 410,415 preparation of gelatin hydrogel disks 408 preparation of thermoresponsive culture surfaces 408 results 410 scanning electron microscopy 413 thermoresponsive culture supports 410 viability bioassay 411 Corneal endothelium 14,16 active Cl- transport 15 active Na+ transport 15 aging 24 Descemet’s membrane 26 development 24 disease 23 disease states 27 congenital hereditary endothelial dystrophy (CHED) 27 diabetes 32 endothelial dystrophies 27 Fuchs’ endothelial dystrophy 29 iridocorneal endothelial (ICE) syndrome 28 mesenchymal dysgenesis 31 posterior polymorphous dystrophy (PPMD) 27 toxic anterior segment syndrome (TASS) 32 electrophysiology of corneal epithelium and ion transport 15 embryology 24 endothelial barrier 17 future directions 32 health 23 morphological characteristics 24 physiology 14,16 pump and barrier function 26 pump functions 17 refractive function 14 ultrastructural characteristics 25 wound repair 24

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Corneal hydration 18 control 18 Corneal hysteresis 3 Corneal innervation 18,79 Corneal metabolism and nutrition 17 Corneal physiology 14 active Cl- transport 15 active Na+ transport 15 control of corneal hydration 18 corneal avascularity 18 corneal endothelium 14,16 corneal epithelium 15 corneal innervation 18 corneal metabolism and nutrition 17 corneal stroma 15 corneal thickness and hysteresis 14 electrophysiology 15 endothelial barrier 17 ion transport 15 pump functions 17 refractive function 14 Corneal slits 304 Corneal stroma 15 physiology 15 Corneal thickness and hysteresis 14 Corneal transparency 243 Crystalline lens 41

D Deep lamellar endothelial keratoplasty (DLEK) 381 Descemet’s membrane 26,78,383 Descemet’s stripping automated endothelial keratoplasty (DSAEK) 227, 311 postoperative complications 316 disk detachment 316 epithelial ingrowth 317 failed graft 317 graft rejection 317 infection 325 interface blood 317 macro-folds 317 recipient peripheral scraping technique 227 Descemet’s stripping with endothelial keratoplasty (DSEK) 382 Diabetes 32 Diameter (donor and recipient) 138 Digital 3D microscope system 86 archival system 90 benefits 89 computer-aided surgery 91 depth of focus 89 diagnostics 91 digital microsurgical workstation 86 display system 88 ergonomics 89 form factor 90

image acquisition 88 image enhancement tools 90 image framing 90 intelligent microscopy 90 low light levels 89 metrology 90 registration 90 working distance 90 Digital microsurgical workstation 86 Disease states 27 congenital hereditary endothelial dystrophy (CHED) 27 diabetes 32 endothelial dystrophies 27 Fuchs’ endothelial dystrophy 29 iridocorneal endothelial (ICE) syndrome 28 mesenchymal dysgenesis 31 posterior polymorphous dystrophy (PPMD) 27 toxic anterior segment syndrome (TASS) 32 Disk detachment 316 Display system 88 Disposable ACC 127 DLEK surgery 172 indications 172 rationale 172 DLEK surgery in a large prospective study 180 astigmatism after DLEK 181 clinical results 180 donor endothelial survival after DLEK 181 large prospective study 180 visual results after DLEK 180 DLEK surgical procedure 159 donor tissue preparation 163 recipient surgery 159 transplantation of the donor tissue 165 DLEK/PLK visual recovery 363 DMEK step-by-step surgery 400 Donor cornea 134 Donor corneal disk insertion 286 Donor corneal preparation 176,285 Donor corneal stroma 352 Donor corneal surgery 352 Donor corneal thickness 138 Donor corneal tissue 220 Donor disk 254 Donor disk adherence to recipient cornea 304 corneal slits 304 surgical technique 304 Donor disk transplantation 353 Donor endothelial survival after DLEK 181 Donor medical review 219 Donor tissue preparation 163, 192, 231, 382 DSEK procedure 231

using pre-cut tissue 177,231 without a microkeratome 178,231 Donor-recipient interface 312 fluid 312 Dropped disk 315 vitreous cavity 315 DSAEK instruments 118 DSAEK visual recovery 363

E Early postoperative complications 396 EK techniques and manifest cylinder outcomes 246 evolution 246 Electrophysiology 15 corneal epithelium 15 ion transport 15 Eligibility determination 219 Embryology and development 24 Endothelial barrier 17 Endothelial cell density 243 Endothelial cell harvesting technique 390 advantages 395 case example 396 complications 395 developing 390 disadvantages 395 early postoperative complications 396 histological analysis 390 instrumentation 390 late complications 396 practicing harvesting 390 TEnCell transplantation 392 viability 390 vital staining 390 Endothelial cell layer transplantation 383 endothelial cells 384 harvesting of the Descemet’s membrane 384 Endothelial changes 19 Endothelial dystrophies 27 Endothelium 78 Epithelial edema 19 Epithelial ingrowth 317 Epithelium 76 Ergonomics 89 Eye banking 219 call 219 consent 219 contact 219 donor medical review 219 eligibility determination 219 evaluation 219 physicial inspection 219 process from donor to recipient 219 recovery 219 release of tissue 219 storgae 219 transport 219

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Index

F Failed graft 317 Femtosecond laser 294 Flipped-disk 314 Form factor 90 Fuchs’ endothelial dystrophy 29

G Gelatin carriers 415 HCEC sheet transplantation 415 Gelatin hydrogel disks 408 Graft rejection 317

H Harvesting technique 390 HCEC sheet transplantation 409 rabbit model 409 Histological analysis 390 Histology 409,415 Host cornea 258 Hysteresis and corneal biomechanics 8 clinical applications 8

I ICG in DLEK 351 completion of donor corneal surgery 352 donor disk transplantation 353 ICG preparation 351 ICG staining of the donor corneal stroma 352 stromal staining 354 surgical technique 354 trypan blue staining of the donor corneal stroma in DSAEK procedure 353 Image acquisition 88 Image enhancement tools 90 Image framing 90 Immunohistochemistry 409,413 Infection 325 Instrumentation 390 Intelligent microscopy 90 Interface blood 317 Intraoperative complications 312 blood in the anterior chamber 312 disk detachment during unfolding 315 dropped disk into vitreous cavity 315 flipped-disk 314 fluid in the donor-recipient interface 312 iris prolapse 312 macro-folds 313

Iridocorneal endothelial (ICE) syndrome 28 Iris prolapse 312

J John dexatome DSAEK spatula 108 John DSAEK 110 Descemet’s stripper 110 fixation hook 114 glider 115 inserting forceps 112 marker (8 mm/9 mm) 117 stromal scrubber 112 John PLK classification 139 optical LKP 139 John retrocorneal super micro forceps 110 John retrocorneal super micro scissors 112 John-Malbran ALK classification 138 optical LKP 138

L Lamellar corneal dissection 49 OCT imaging 49 Lamellar keratoplasty 136 Laser scanning confocal microscope 75 Learning curve and precautions 292 LKP 139 indications 139 Low light levels 89

M Macro-folds 313,317 Mentor system 93 dealing with complications 93 Mesenchymal dysgenesis 31 Metrology 90,93 Microkeratome 138 use of 138 Microkeratome-assisted PLK (MAPK) 369, 380 Microscopy history 72 Modified microkeratome-assisted posterior lamellar keratectomy (MMAPLK) 372 surgical technique 372 Moria ACC 124

N New concepts in DSAEK surgery 108 John dexatome DSAEK spatula 108 John DSAEK Descemet’s stripper 110 John DSAEK fixation hook 114 John DSAEK glider 115

John DSAEK inserting forceps 112 John DSAEK marker (8 mm/9 mm) 117 John DSAEK stromal scrubber 112 John retrocorneal super micro forceps 110 John retrocorneal super micro scissors 112 other DSAEK instruments 118 Normal cornea 3

O Optical coherence tomography 48 accommodation 40 anterior chamber exploration 40 anterior chamber measurements 40 anterior segment 39 aperture inlay 51 cornea 42 corneal implant surgery 47 crystalline lens 41 imaging 49 lamellar corneal dissection 49 presbyopia correction 51 principles 48 refractive inlays 50 Visante™ OCT 40

P Penetrating keratoplasty 136,368,378 Peripheral circular area of recipient corneal stroma 305 Physicial inspection 219 PKP visual recovery 362 Posterior lamellar keratoplasty 136, 369, 379 corneal adhesive 373 last advances 371 microkeratome-assisted posterior lamellar keratoplasty 369 modified microkeratome-assisted posterior lamellar keratectomy (MMAPLK) 372 modified microkeratome-assisted posterior lamellar keratoplasty surgical technique 372 scleral-pocket incision approach 370 Posterior polymorphous dystrophy (PPMD) 27 Posterior stromal disk 385 transfer of Descemet’s membrane 385 Pre-cut corneal tissue for DSEK 333 cutting the pre-cut tissue 337 examination of the tissue 333 surgical technique 333 transplanting the tissue 338 Pump and barrier function 17, 26

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R Rabbit model 409 Real time corneal topography 93 Recipient corneal resection 138 Recipient surgery 159,173 phacoemulsification technique 185 small incision DLEK 188 Refractive function 14 Refractive inlays 50 Removal of unwanted corneal reflections and artifacts 91 Reusable ACC 124

S Scanning electron microscopy 409,413 viability bioassay 409 Scanning-slit confocal microscope 75 Scleral-pocket incision approach 370 Single-sided scanning confocal microscope 74 Small incision DLEK procedure 173 Spherical equivalent outcomes 247 Stem-cell transplantation 137 Stroma 77 Stromal edema 19 Stromal staining 354 Substitution or addition of donor tissue 138 Superficial epithelial cells 77 Surgical applications 91 artifact removal 91

mentor system of dealing with complications 93 metrology 93 real time corneal topography 93 removal of unwanted corneal reflections 91 using a template for capsulorhexis 92 variable magnification within the surgical field 92 Surgical instruments for DSAEK 108 Surgical microscope 89 benefits of 3D 89 Surgical planning 9 implication 9

T Tandem scanning confocal microscope 73 advantages 73 Tandem scanning design 74 advantages 74 prior designs 74 TEnCell transplantation 392 Thermoresponsive culture supports 409 harvest of HCEC sheets 410 Thermoresponsive culture surfaces 408 Third party preparation 332 corneal tissue for DSAEK 332 Tissue examination 333 Toxic anterior segment syndrome (TASS) 32 Transplantation of the donor tissue 165,178,195,232

Transplanting the tissue 338 Trephine diameter 254 Trypan blue 353,357 donor corneal stroma 353 DSAEK procedure 353 staining 353

U Ultrasonic imaging and biometry of the cornea 65 Uniform Anatomical Gift Act 219

V Viability 390 Viability bioassay 409,411 Visante™ OCT 40 Visual and refractive results 241 Visual outcomes 248 Vital staining 390

W Wing cells 76 Wound architecture in LKP 139 Wound repair 24

Z Z-axis scanning 73