Age-Related Changes of the Human Eye (Aging Medicine)

Age-Related Changes of the Human Eye

Aging Medicine Age-Related Changes of the Human Eye, edited by Carlo A. P. Cavallotti and Luciano Cerulli, 2008 Classic Papers in Geriatric Medicine, edited by Robert Pignolo, Mary Anne Forciea, and Monica Crane, 2008 ElderCare Technology: A Guide for Physicians, edited by Robin Felder and Majd Alwan, 2007 Handbook of Pain Relief in Older Adults: An Evidence Based Approach, edited by Michael F. Gloth, 2004

Series Editors Robert J. Pignolo Division of Geriatric Medicine, University of Pennsylvania Health System, Philadelphia, Pennsylvania Mary Ann Forciea Division of Geriatric Medicine, University of Pennsylvania Health System, Philadelphia, Pennsylvania Jerry C. Johnson Division of Geriatric Medicine, University of Pennsylvania Health System, Philadelphia, Pennsylvania

Advisory Board 1. W. Malinovska Brno, Czech Republic 2. J. Bockova London, UK 3. M. Von Pinoci New York, USA 4. R. Kovacs Budapest, Hungary 5. P. Roger Montreal, Canada 6. M. Mancone Rome, Italy Many Chapters have been mailed to the editors after the deadline. These Chapters have not been revised by the Advisory Board, but have been edited in the format in which they have been submitted by the Authors—who are, obviously, responsible for their content and their form.

Age-Related Changes of the Human Eye Edited by

Carlo A. P. Cavallotti, MD, PhD Department of Cardiovascular, Respiratory, and Morphological Sciences University of Rome, La Sapienza Rome, Italy

Luciano Cerulli, MD, PhD Department of Ophthalmology University of Rome, Tor Vergata Rome, Italy

Editors Carlo A. P. Cavallotti, MD, PhD Department of Cardiovascular, Respiratory and Morphological Sciences University of Rome, La Sapienza Rome, Italy [email protected] Series Editors Robert Pignolo Division of Geriatric Medicine University of Pennsylvania Health System Philadelphia, Pennsylvania

Luciano Cerulli, MD, PhD Department of Ophthalmology University of Rome, Tor Vergata Rome, Italy [email protected]

Mary Ann Forceia Division of Geriatric Medicine University of Pennsylvania Health System Philadelphia, Pennsylvania

Jerry C. Johnson Division of Geriatric Medicine University of Pennsylvania Health System Philadelphia, Pennsylvania

ISBN: 978-1-934115-55-8 e-ISBN: 978-1-59745-507-7 DOI: 10.1007/978-1-59745-507-7 Library of Congress Control Number: 2007939892 © 2008 Humana Press, a part of Springer Science + Business Media, LLC All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, 999 Riverview Drive, Suite 208, Totowa, NJ 07512 USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Cover illustration: Figure 1, Chapter 2, by Janos Feher and Zsolt Olah, “Electron microscopy of aged orbicular muscle fibers.” Figure 3a, Chapter 12, by Susanne Binder and Christiane I. Falkner-Radler, “Early face angiogram of a classic CNV.” Printed on acid-free paper 9 8 7 6 5 4 3 2 1 springer.com

Preface

Research on the aging of the human eye crosses all areas of ophthalmology and relies on biological, morphological, physiological, and biochemical tools for its study. Putting together a volume that attempts to cover all the aspects of the aging of the human eye was a daunting task. In fact, some areas may have been invariably overlooked and not every viewpoint may have been included. Despite its shortcomings, we hope Age-Related Changes of the Human Eye will serve as a useful broad-based overview for all the people involved in research and/ or disease on the aging of the human eye. The authors of each chapter were selected for their expertise and prominence in the specific field. Therefore, this book is appropriate for students and graduate students, as well as for postdoctoral and/or professional ophthalmologists. Readers will benefit greatly from the significant revision of material related to the aging of the human eye. The highlights of Age-Related Changes of the Human Eye are its: 1. 2. 3. 4.

Ease of use, Inclusion of numerous personal experiments and data, Versatility, and Bibliography.

The key elements of this volume are the descriptions of age-related changes in almost all the structures of the human eye. The contributors are researchers, physicians, clinicians, technicians, engineers, and members of famous and leading research groups. It should be understood that the eye represents a functional unit, and any modification of one of the structures considered will lead to changes and/or dysfunction of the whole ocular globe. Moreover, we would like to stress that visual function is not only related to the eye, but is a complex activity that is strongly and intimately connected to the brain. Any anomaly, dysfunction, or disease of the ocular globe determines relevant changes in the structure and thus, the function of the brain. Our hope

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is that Age-Related Changes of the Human Eye will give an exhaustive panorama of what happens during the aging process of the eye, thus contributing to the understanding of the physiology and pathology of eye diseases. Carlo A. P. Cavallotti Luciano Cerulli Rome, Italy

Contents

Preface .............................................................................................................

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Chapter 1 Aging as Risk Factor in Eye Disease ...................................... Luciano Cerulli

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Chapter 2 Age-Related Changes of the Eyelid......................................... Janos Feher and Zsolt Olah

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Chapter 3 Aging Effects on the Optics of the Eye. .................................. Pablo Artal

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Chapter 4 Aging of the Cornea ................................................................. Luciano Cerulli and Filippo Missiroli

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Chapter 5 The Aging of the Human Lens ................................................ Jorge L. Aliò, Alfonso Anania, and Paolo Sagnelli

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Chapter 6 The Extracellular Matrix of the Aged Human Trabecular Meshwork: Changes of Glucosaminoglycans ............................................................. Carlo A. P. Cavallotti Chapter 7 Glial and Mobile Cells in the Iris of the Aging Human Eye ................................................................................ Carlo A. P. Cavallotti and Angelica Cerulli Chapter 8 Age-Related Diseases of the Vitreous ..................................... Curtis E. Margo Chapter 9

Age-Related Changes and/or Diseases in the Human Retina ................................................................ Nicola Pescosolido and Panagiotis Karavitis

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Chapter 10 Aging of the Retinal Pigmented Epithelium ........................ Carlo A. P. Cavallotti and Marcus Schveoller

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Chapter 11 The Aging of the Choroid ...................................................... Angelica Cerulli, Federico Regine, and Giuseppe Carella

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Chapter 12 Age-Related Macular Degeneration I: Types and Future Directions ............................................................. Susanne Binder and Christiane I. Falkner-Radler

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Chapter 13 Age-Related Macular Degeneration II: Idiopathic Macular Holes ....................................................... Christiane I. Falkner-Radler and Susanne Binder

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Chapter 14 Age-Related Macular Degeneration III: Epiretinal Membranes ............................................................. Christiane I. Falkner-Radler and Susanne Binder

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Chapter 15 Macular Degeneration: Ultrastructural Age-Related Changes .............................................................. Illes Kovacs, Janos Feher, and Carlo A. P. Cavallotti

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Chapter 16 Non-Exudative Macular Degeneration and Management .................................................................... Thomas R. Friberg and Kenneth T. Wals

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Chapter 17 Treatment of Intraocular Pressure in Elderly Patients .................................................................. Monika Schveoller, Iliana Iliu, Nicola Pescosolido, and Angelica Cerulli Chapter 18 Aging of the Lachrymal Gland ............................................. Hiroto Obata

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Chapter 19 The World According to Blink: Blinking and Aging ................................................................................. Frans Van der Werf and Albertine Ellen Smit

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Chapter 20 Age-Related Changes in the Oculomotor System ..................................................................................... J. Richard Bruenech

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Chapter 21 Rehabilitation of Low Vision in Aged People ...................... Corrado Balacco, Elena Pacella, and Fernanda Pacella

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Chapter 22 Many Suggestions to Protect the Eyes in Aging People ....................................................................... Panagiotis Karavitis, Nicola Pescosolido, and Fernanda Pacella Index ...............................................................................................................

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Contributors

Jorge L. Aliò, MD, PhD Professor and Chairman of Ophthalmology, University “Miguel Hernandez” of Helce, Alicante, Spain Alfonso Anania, MD Diagnostic Centre of Ophthalmic Micro-surgery, Rome, Italy Pablo Artal Laboratorio de Optica (Departamento de Fisica), Universidad de Murcia, Murcia, Spain Corrado Balacco, MD, PhD Department of Ophthalmologic Sciences, University of Rome “La Sapienza,” Rome, Italy Susanne Binder, MD Department of Ophthalmology, The Ludwig Boltzmann Institute for Retinology and Bio-microscopic Laser Surgery, Rudolf Foundation Clinic, Vienna, Austria J. Richard Bruenech, PhD Biomedical Research Unit, Buskerud University College, Kongsberg, Norway Giuseppe Carella, MD, PhD San Raffaele Hospital, Department of Ophthalmology and Vision Sciences, University of Milan, Milan, Italy; University of Rome “Tor Vergata,” Rome, Italy Carlo A. P. Cavallotti, MD, PhD European Ophthalmic Neuroscience Program (Local Research Unit), University of Rome “La Sapienza,” Rome, Italy Angelica Cerulli, MD Department of Ophthalmology, University of Rome “Tor Vergata,” Rome, Italy Luciano Cerulli, MD, PhD Department of Ophthalmology, University of Rome “Tor Vergata,” Rome, Italy

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Christiane I. Falkner-Radler, MD Department of Ophthalmology, The Ludwig Boltzmann Institute for Retinology and Bio-microscopic Laser Surgery, Rudolf Foundation Clinic, Vienna, Austria Janos Feher, MD, PhD Nutripharma Hungaria Ltd., Budapest, Hungary Thomas R. Friberg, MD, PhD UPMC-Eye Centre, University of Pittsburgh, Pittsburgh, PA Panagiotis Karavitis, MD Ophthalmology, University of Athens, Athens, Greece; Department of Ophthalmologic Sciences, University of Rome “La Sapienza,” Rome, Italy Illes Kovacs, MD, PhD Department of Ophthalmology, Semmelweis University, Budapest, Hungary Iliana Iliu, MD Geriatric Centre University of Salonica, Salonica, Greece; Ophthalmology, Department of Aging Sciences, University of Rome “La Sapienza,” Rome, Italy Curtis E. Margo, MD, MPH Ophthalmic Pathology Laboratory, Ophthalmology and Pathology, University of South Florida College of Medicine, Tampa, FL Filippo Missiroli, MD Department of Ophthalmology, University of Rome “Tor Vergata,” Rome, Italy Hiroto Obata, MD, PhD Department of Ophthalmology, Jichi Medical University, Tochigi, Japan Zsolt Olah, BSc School of Sport Medicine, Semmelweis University, Budapest, Hungary Elena Pacella, MD Department of Ophthalmologic Sciences, University of Rome “La Sapienza,” Rome, Italy Fernanda Pacella, MD Department of Ophthalmologic Sciences, University of Rome “La Sapienza,” Rome, Italy Nicola Pescosolido, MD Senior Researcher Section of Ophthalmology, Department of Aging Sciences, University of Rome “La Sapienza,” Rome, Italy Federico Regine, MD Department of Ophthalmology, University of Rome “Tor Vergata,” Rome, Italy Paolo Sagnelli, MD European Ophthalmic Neuroscience Program (Local Research Unit), University of Rome “La Sapienza,” Rome, Italy

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Marcus Schveoller, MD, PhD Department of Ophthalmology, European Ophthalmic Neuroscience Program, University of Pécs, Pécs, Hungary Monika Schveoller, MD Department of Ophthalmology, University of Pécs, Pécs, Hungary Albertine Ellen Smit, MD Department of Neuroscience, University of Rotterdam, Rotterdam, The Netherlands Frans Van der Werf, PhD Department of Neuroscience, University of Rotterdam, Rotterdam, The Netherlands Kenneth T. Wals, MD UPMC-Eye Centre, University of Pittsburgh, Pittsburgh, PA

Chapter 1

Aging as Risk Factor in Eye Disease Luciano Cerulli, MD, PhD

“Une des tristesses de la vie est que toutes les évaluations chiffrées des performances visuelles montrent qu’elles déclinent progressivement avec l’âge.” Kline, 1987

Abstract The major causes of blindness and reduced vision are related to cataracts, glaucoma, age-related macular degeneration, and diabetic retinopathy—all of which recognize aging as the major risk factor. The burden of visual impairment is not distributed uniformly through the world. The least developed regions carry the largest share. Visual impairment is also unequally distributed across age groups, with incidence largely confined to adults 50 years of age and older (83%). A distribution imbalance is also found with regard to the gender throughout the world—females have a significantly higher risk of developing visual impairment than males because their life expectancy is higher and their economic possibilities may be less. Notwithstanding the progress in surgical intervention that has been made in many countries over the last several decades, cataracts remains the leading cause of visual impairment in all regions of the world, except in the most developed countries.

Keywords Cataract, Glaucoma, ARM D, Corneal opacity, Diabetic retinopathy

Over the last few years, aging has become the prevalent risk factor in the overall world population. In the 1980s, this was true only in the European Countries but it has now become a major cause of morbidity and mortality worldwide. Infective agents are, in a certain sense, loosing their primary station as the most relevant cause of illness and death, while degenerative conditions are growing all over the world. This is true for general diseases and also in ophthalmology. It has been estimated that there are 161 million visually impaired individuals in the world, and of this figure, 37 million of them are blind. Fig. 1.1 and 1.2 list the most recent data available from World Health Organization (WHO) reports on the world prevalence of blindness and reduced vision.

From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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Fig. 1.1 Prevalence of Blindness

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Fig. 1.2 Prevalence of Low Vision

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The major causes of blindness and reduced vision are related to cataracts, glaucoma, age-related macular degeneration, and diabetic retinopathy—all of which recognize aging as the major risk factor. The burden of visual impairment is not distributed uniformly through the world. The least developed regions carry the largest share. Visual impairment is also unequally distributed across age groups, with incidence largely confined to adults 50 years of age and older (83%). A distribution imbalance is also found with regard to the gender throughout the world—females have a significantly higher risk of developing visual impairment than males because their life expectancy is higher and their economic possibilities may be less. Notwithstanding the progress in surgical intervention that has been made in many countries over the last several decades, cataracts remains the leading cause of visual impairment in all regions of the world, except in the most developed countries. Other major causes of blindness are (in order of frequency): glaucoma, ARMD, diabetic retinopathy, and trachoma.1 More specifically, Table 1.1 provides the available data on the estimated prevalence of eye diseases as causes of blindness and reduced vision. These diseases do recognize that aging is the major risk factor. WHO has examined and forecast the distribution of the world population at the year 2000, 2025 and 2050, as displayed in Table 1.2. Table 1.1 Estimated prevalence of the eye diseases Cataract 47,9% Glaucoma 12,3% ARMD 8,7% Corneal opacity 5,1% Diabetic retinopathy 4,8%

Table 1.2 Distribution of world population at the years 2000, 2025, 2050

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Table 1.3 % Increase of over 50 in different sub-groups of Countries % increase WLD MDC LDC

2000 1081211 366777 714435

2025 2095593 492345 1603248

2050 3138029 503099 2634932

2000vs2025 194 134 224

% increase 200vs2050 290 137 369

The dramatic increase in the number and percentages of the world’s population is clearly noted as definitively more evident in less-developed countries, while the financial allocation of resources and health services availability and health technology are weaker in those same countries. Without proper interventions, the increase in the number and percentage of people over the age of 50 will lead in the near future to a significant increase in the number and percentage of eye diseases that recognize age as the major risk factor. It has been estimated that without extra interventions, the total number of blind individuals worldwide would increase from 44 million in the year 2000 to 76 million in 2020 Table 1.3. A successful intervention by the VISION 2020 initiative could result in only 24 million people suffering from blindness by 2020 and lead to a reduction of 429 million cases of blindness per year. A conservative estimate of the economic gain is 103 billion dollars.2 A proper approach in the treatment of these clinical pictures will lead to a reduction in numbers and percentage of the blind but also to an increase of reduced vision patients with a dramatic need for rehabilitation activities. How many glaucoma patients are currently affected, how many of them are blind or severely impaired by this disease, what are the geographical and temporal distributions, and what are the main risk factors? These are questions without real scientific answers.3 As we have seen, the WHO estimates that this pathology represents the second cause of blindness and reduced vision worldwide after cataracts. It is estimated that in the year 2000, 66.8 million people are affected by open angle glaucoma. Of these, 6.7 million were affected by a bilateral blindness secondary to this disease, according to Quigley and Vitale.4 In the same year, 2.47 million patients were affected by this pathology just in the United States—1.84 million were Caucasians and 619,000 were people of color. Numerous considerations can be made from the results of this study. Taking into account the person’s age at the beginning of the illness, together with the death rate, the illness lasts longer by 27% in colored people in comparison to patients of the Caucasian race. In industrialized countries, less than 50 percent of the population with glaucoma realize that they are affected. This percentage is much lower in developing countries. As for other disease, epidemiological studies have considered several risk factors, such as age, sex, race, social and economical factors, working activities, climate, the use of tobacco or alcohol, genetic, and ocular factors. Undoubtedly, the Intraocular Pressure (IOP) represents the major risk factor for the development of

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the disease, but together with other factors, we should consider that the prevalence of glaucoma increases with age, with higher baseline IOP values in the elderly.5 This can be explained by the physiological reduction in time of the retinal ganglion cells, associated with a prolonged exposure to nontolerated high values of IOP, together with the action of other risk factors.6 The lack of, or a delay in, a diagnosis may lead to an irreversible blindness. Given the number of patients affected, the social-economical implications related to this pathology are very onerous. In a recent study, for example, it has been calculated that in Germany, 800,000 patients are affected by glaucoma. Every year, Social Security spends more than 1,000 Euro per patient. On the other hand, if we consider the number of patients who are certified blind in connection with this illness, we must consider that the German government spends 150 million Euro in national Social Security and health expenses. These costs are destined to rise due to the fact that the average life span has increased and will continue to increase in the future. In a multicentric study carried out in France, Germany, Italy, and Great Britain, the average cost per person for glaucoma each year was 726 Euro.7 As we have already seen, diabetic retinopathy contributes to 4.8% of blindness and reduced vision worldwide. We must remember that in the United States it is understood that 14 million are affected by diabetes, and of these, 43 percent present a related retinopathy. This complication creates 8,000 new cases of blindness each year. For this pathology—together with other risk factors such as the type of diabetes, race, sex, controlling blood sugar levels and blood pressure, dislipidemia, nephropathy, pregnancy, and the duration of the illness (both for diabetes type 1 and type 2)—aging represents one of the most important risk factors to be considered. It has been estimated that after 20 years, almost all diabetic type 1 patients, and 60 percent of diabetic type 2 patients are affected by this retinal complication. Furthermore, if we consider that cataract surgery—a pathology typical of old age—worsens the course of diabetic retinopathy, we realize that aging represents an important element to be considered for diabetic patients. Taking into account the higher life expectancy for diabetic patients and the aging of the world population, especially in the Southern hemisphere (and thus a major risk to be affected by type 2 diabetes), we can easily understand what sort of dimension this problem can assume in the near future. Aging is not only a risk factor for the previously mentioned diseases (Fig. 1.3). The social changes in our time often leave the elderly in a condition of loneliness and eventually with economical and cultural barriers toward the access to health services. This may lead to a delay in early diagnosis, a delay in the beginning of an appropriate treatment, difficulties in following the medical prescriptions, and the related rehabilitation with a subsequently more significant handicap. This is not only true for eye diseases, but also for uncorrected changes in refraction. WHO has estimated that 153 million people are visually impaired due to uncorrected refractive errors (URE), with 95 percent of them being over 50 years of age. Although this book has been divided into different sections, an effort should be made to understand that the eye represent a functional unit and any modification of any one of its structures will lead necessarily to changes and/or dysfunction of the whole globe.

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Fig. 1.3 Age-related eye diseases as causes of blindness and low vision

In addition, I would like to stress that visual function is not only related to the eye, but is a complex activity that is strongly and intimately connected with the brain. Any anomaly, dysfunction, or disease of the globe can be associated with relevant changes in the structure, and thus the function, of the brain. Our hope is that this book will give an exhaustive panorama of what happens when the eye ages, thus contributing to the understanding of the physiology and physiopathology of eye diseases.

References 1. Resnikoff S and Co. Policy and Practice “Global data on visual impairment in the year 2002” 2. Frick K, et al. (2003) The magnitude and cost of global blindness: An increasing problem that can be alleviated. Am. J. Ophth. April 471-476 3. Quigley HA (1996) Number of people with glaucoma worldwide Br. J. Ophthalmol. May 80(5):389-393 4. Quigley HA, Vitale S (1997) Models of open-angle glaucoma prevalence and incidence in the United States. Invest. Ophthalmol Vis Sci Jan 38:83-91 5. Friedman DS et al. (2006) The prevalence of open angle glaucoma among blacks and whites 73 years old: the Salisbury Eye Evaluation Glaucoma Study. Arch. Ophthalmol. 124:1625-30

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6. Sommer A et al. (1991) A population-based evaluation of glaucoma screening: the Baltimore Eye Survey. Am. J. of Epidemiol. 134:1102-1110 7. Traverso CE et al. (2005) Direct costs of glaucoma and severity of the disease. Br. J. Ophthalmol 89:1245-9

Recommended Bibliographic Resources Recent Books on Vision Disorders in Old Age (www.amazon.com) 1. The Aging Eye by Sandra Gordon, Harvard Medical School, 2001. 2. Communication Technologies for the Elderly: Vision, Hearing & Speech by Rosemary Lubinski, D. Jeffery Higginbotham, 1997. 3. The effects of aging and environment on vision by Donald A. Armstrong, et al., 1991. 4. Treating vision problems in the older adult (Mosby’s optometric problem-solving series) by Gerald G. Melore, 2001. 5. Vision and Aging by Alfred A. Rosenbloom, Meredith W. Morgan, 1993. 6. Age-Related Macular Degeneration by Jennifer I. Lim, 2002. 7. The Impact of Vision Loss in the Elderly (Garland Studies on the Elderly in America) by Julia J. Kleinschmidt, 1995. 8. Vision in Alzheimer’s Disease (Interdisciplinary Topics in Gerontology) by Alice Croningolomb, et al., 2004. 9. The Senescence of Human Vision (Oxford Medical Publications) by R.A. Weale, 2001. 10. Issues in Aging and Vision: A Curriculum for University programs and In-service Training by Alberta L. Orr, 1998. 11. Aging with developmental disabilities changes in vision by Marshall E. Flax, 1996. 12. Trends in vision and hearing among older Americans by U.S. Dept of Health and Human Services, 2000. 13. Optometric gerontology: A resource manual by Sherrell J. Aston, 2003.

Chapter 2

Age-Related Changes of the Eyelid Janos Feher, MD, PhD and Zsolt Olah, BSc

Abstract Changes of the orbicular muscle and its connective tissue play a central role in the aging of the eyelid. Age-related changes of orbicular muscle comprise a decrease of muscular fibers and a disorganization of banding structures (appearance of nemaline bodies, Z-line streaming, cytoplasmic bodies, and Z-line doubling). Mitochondria, particularly in the subsarcolemmal area, showed either a decrease in number and loss of cristae, or enlargement and proliferation of cristae. In combination with both alterations, intramitochondrial crystal formation and altered succinyl-dehydrogenase activity were also a frequent observation. Tubular aggregates originated from the sarcoplasmic reticulum and various sarcoplasmic inclusions were also observed. Intramuscular connective tissue density increased with age, and it was associated with increased glycation of collagen fibers. Neither of these alterations are considered specific for aging, but their particular combination may be responsible for the development of well-known, age-related changes and diseases of the eyelid. In addition, these data may give further information to the pathology of sarcopenia—a devastating age-related muscle disease. Keywords eyelid, aging, orbicular muscle, nemaline body, Z-line streaming, cytoplasmic body, mitochondria, creatine kinase crystal, succinyl-dehydrogenase, sarcoplasmic reticulum, tubular aggregates, electron microscopy.

Introduction Aging of the eyelid is a well-known phenomenon, but it is still a poorly explored interdisciplinary area of medicine. The skin layer belongs mostly to dermatology, the intermediate muscle-connective layer is generally the subject of oculoplastic surgery and neuroophthalmology, while the innermost conjunctiva is reserved for subspecialities of ophthalmology—i.e., for experts of dacryology and the tear filmocular surface. This paper is dedicated to the age-related changes of the orbicular muscle and its connective tissue for two reasons. First, age-related changes of these structures From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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are responsible for the development of malpositions of the lower eyelid known as senile entropion and ectropion.1 In the former case, the margin of the lower eyelid turns inward, eyelashes irritate the corneal surface continuously, and keratoconjunctivitis develops with potential corneal ulceration. In the latter case, the margin of the lower eyelid turns outward, tarsal conjunctiva and lower lachrymal punt are exposed, and chronic lachrymation (epiphora) and conjunctival inflammation develop. There are two main theories to explain the pathomechanism of senile entropion and ectropion—the spastic and the atonic theory. These malpositions of the lower eyelid are therefore often called spastic entropion or ectropion as well as atonic entropion or ectropion. Both theories ascribe essential importance to alterations of the orbicular muscle and neighboring connective tissues.1 Although several morphological and functional observations have been carried out to reveal the pathologic background of abnormal muscle activity, we are far from knowing the exact mechanisms that cause the senile involution of the orbicular muscle, and far from explaining the variable clinical picture. In both conditions, plastic surgery is the choice of treatment. Until now, however, more then 120 original and modified surgical procedures have been introduced, suggesting the poor effectiveness of any of them. In fact, recidivates are quite frequent after each procedure. Early electron microscopy of eyelid aging and its relation to senile entropion and ectropion revealed significant ultrastructural abnormalities in the orbicular muscle fibers,2 but no differences related either to entropion or ectropion.3 Some abnormalities of the mitochondria4 and sarcoplasma5,6 were also described. Here we present a completed ultrastructural morphology of orbicular muscle aging and design putative correlations between these abnormalities. Aging of the orbicular muscle may be generally related to a part of muscle aging known as sarcopenia. The second aim of this chapter is to reveal the earliest ultratstructural alterations related to this poorly explored and uncurable pathology. Sarcopenia is a slowly progressive and complex process that appears in aged muscle that is associated with a decrease in mass, strength, and velocity of contraction. This process is the result of many molecular, cellular, and functional alterations. With the advancement of age, type I muscle fibers decrease in number and increase in size.7 Type I fiber predominance seen in older subjects could be related to a selective decrease of type II fibers as the body ages. It also suggests a possible conversion of type II fibers to type I fibers.8 In the elderly, central nuclei, ring fibers, fiber splitting, scattered highly atrophic fibers, moth-eaten fibers, and vacuoles were also observed. Ring fibers were most easily identified with antidesmin labeling, and highly atrophic fibers exhibited a rough network of labeling. An increased content of actin and spectrin was also observed at the periphery of ring fibers. A qualitative ultrastructural analysis also showed obvious changes, including some myofilament loss, collections of lipofuscin that were also observed in satellite cells, proliferation of the sarcoplasmic reticulum, and increased wrinkling of nuclear membranes and sarcolemma.9 Interestingly, satellite cell populations were not significantly lower in healthy, sedentary older adults compared to young adult men and women.10 Over time, mitochondrial size and mitochondrial percentage per fiber area decrease, and the cristae of mitochondria became irregularly spaced, disrupted,

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and replaced by lamellar, myelin-like structures. Giant mitochondria are often visible. They contain lipid droplets and lipofuscin in the myofibrils, which are often in close relationship with the damaged mitochondria.11 It has been suggested that sarcopenia may be triggered by reactive oxygen species (ROS) that have accumulated throughout a person’s lifetime. In fact, a significant increase in the oxidation of DNA and lipids was found in elderly muscle— more evident in males—along with a reduction in catalase and glutathione transferase activities. Experiments on Ca2+ transport showed an abnormal functional response of aged muscle after exposure to caffeine, which increases the opening of Ca2+ channels, as well as reduced activity of the Ca2+ pump in elderly males. These results proved that oxidative stress plays an important role in muscle aging, and that oxidative damage is much more evident in elderly males, suggesting a gender difference that may be related to hormonal factors. The progression of sarcopenia is directly related to a significant reduction of the regenerative potential of muscle normally due to a type of adult stem cells known as satellite cells. These cells lie outside the sarcolemma and remain quiescent until external stimuli trigger their re-entry into the cell cycle as growth factors. One possibility is that the anti-oxidative capacity of satellite cells could also be altered and this, in turn, can determine the decrease of their regenerative capacity. Data concerning this hypothesis are discussed.12

Changes in Anatomy and Kinematics Topographic anatomy of the eyelids is affected by aging and sex. Normal aging processes may cause laxity of eyelid tissues (skin, muscle, connective tissue) and atrophy of the orbital fat. These changes are responsible for the well-known aesthetic changes, but they may also contribute to the aetiology of several eyelid disorders, such as ectropion, entropion, dermatochalasis, and blepharoptosis. Such aging changes may also affect the position of the eyelids, eyeball, and eyebrow. Aging primarily affects the size of the horizontal eyelid fissure. In adolescence, between the ages of 12 and 25 years, the horizontal eyelid fissure lengthens 3 mm, while the position of other eyelid structures remains virtually unchanged. Between the average ages of 35 and 85 years, the horizontal eyelid fissure gradually shortens again by about 2.5 mm. While the lengthening of the horizontal eyelid fissure between the ages of 12 and 25 years probably reflects growth of the facial structures, the shortening from the age of 35 years onwards is likely due to progressive laxity of the medial and lateral canthal structures. With aging, the distance between the lateral canthal angle and the anterior corneal surface decreases almost 1.5 mm. which means that the shortening of the horizontal eyelid fissure can at least partially be attributed to medial displacement of the lateral canthus. The positions of the lateral canthus and the center of the pupil are identical in men and women, and remain fairly stable throughout life. Aging causes the sagging of the lower eyelid,

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especially in men, and a higher skin fold and eyebrow position in both sexes. Laxity of the lower eyelid causes an increase of the distance between the pupil center and the lower eyelid of about 1 mm in men, and 0.5 mm in women. In a prospective consecutive observational case series, 32 normal adult subjects—comprised of 12 younger (aged 29+/-5 years) and 20 older subjects (aged 74+/-6 years)—underwent lower eyelid tensometry. Younger males had higher eyelid tension than females, and there was no significant reduction in tension with age.13 Aging also causes raised eyebrows and increased skin creases in men and women, but in the study it did not affect the position of the pupil center and the lateral canthus. Men showed a 0.7 mm larger horizontal eyelid fissure than women. However, the eyebrows in women were situated about 2.5 mm higher than in men. Aging does not affect the position of the eyeball proper, or of the lateral canthus.14 Cutis laxa is an uncommon entity characterized by laxity of the skin, which hangs in loose folds, producing the appearance of premature aging. Histological analysis and ultrastructural examination of skin biopsy revealed reduction and fragmentation of elastic fibers. Dermatochalasis is a severe degree of the aging process in the eyelid and orbital soft tissue complex. It can lead to extreme weakness or even dehiscence of the supporting fascia and other surrounding soft tissue—rarely leading to free mobility of the orbital fat pads and hence postural herniation into the eyelids, as seen in this unusual case. Recent studies on the changes in the kinematics of blinking over time demonstrated that disorders of blink systems typically seen in persons 50 years of age or older occur against a backdrop of normal age-dependent changes in eyelid kinematics. Alterations in main sequence slope imply that the operation of central adaptive systems during aging. Reduction in main sequence slope is interpreted as a reduction in aggregate orbicular muscle motoneuron activity. Such a central neurologic adjustment in the motor output of blink systems may serve to compensate for an age-related increase in blink reflex excitability. Compensatory reduction in the main sequence relationship may offset a potentially hyperexcitable blink reflex, thereby reducing the likelihood of disorders such as blepharospasm. These authors described passive and active changes in the kinematics of blinking with age. Passive changes in blink amplitude-peak velocity reflect age-related changes in static eyelid position that can be attributed largely to either weakness of the Muller’s muscle and superior levator muscle, or to the laxity of the connective tissue in the superior transverse ligament, palpebral ligaments, or dehiscence of the levator aponeurosis. Active changes in main sequence relationships demonstrate that blink plasticity interacts with aging processes. The active changes observed in the neural control of blink kinematics do not, by themselves, represent a trend toward the development of blink disorders in an aging population. If the aging of blink systems actively contributed to an age-related trend toward diseases of elevated blink excitability, an increased main sequence slope would have been expected. By contrast, the opposite result suggests that the active, central changes in eyelid kinematics may represent an adaptive response to the established, age-associated increase in blink reflex excitability.15

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Epidermal innervation according to age and anatomical site was evaluated in 82 biopsy samples from surgical procedures. Eyelid epidermis showed the highest ratio of nerve fiber surface to epidermal surface. A trend exhibiting age-associated decreased epidermal innervation of facial skin was found. Epidermal innervation of abdominal skin did not change with age, and an age-associated increased innervation was observed in mammary and palpebral skin.16 In another study, trigeminal blinks in normal human subjects between 20 and 80 years of age were characterized. In normal humans over 60 years of age, lid-closing duration, and the excitability and latency of the trigeminal reflex blink, increase significantly relative to younger subjects. Reflex blink amplitude, however, does not change consistently with age. For subjects less than 70 years of age, a unilateral trigeminal stimulus evokes a 37 percent larger blink in the eyelid ipsilateral to the stimulus than in the contralateral eyelid. Subjects who are 70 years old, however, exhibit blinks of equal amplitude. In all cases, blink duration is identical for both eyelids.17

Myofiber Abnormalities We have performed electron microscopic and histochemical studies on surgical specimens of 86 patients, aged 42-88 years, and affected by various pathologies (tumors, enctropin, ectropin, trauma). Orbicular muscle contains mainly type I fibers. Age-related myofiber abnormalities comprise the decrease of the filamentary structure and disorganization of the normal banding structure, particularly the Z line. The decrease of filaments were the most common aging changes in the orbicular muscle. In some cases, simple quantitative changes were observed without any qualitative alterations (see Fig. 2.1). In most cases, however, decrease of myofilaments were accompanied with disorganization of both fibrillary and banding structures. In some sites, there were only small focal changes, whereas at other places, the damage extended over many sarcomeres and even many fibers. From a morphological point of view, Z-line alterations serve as a reference and four subtypes of myofiber changes related to aging can be distinguished. Nemaline bodies (rods) are by far the most common and most widely studied alteration of the skeletal muscle. This alteration is characterized by rod-form accumulation of Z-line material. They consist of a lattice-like arrangement of squares measuring about 1 nm on each side (see Fig. 2.2). Nemaline bodies are clearly detectable at the light-microscopy level, and ultrastructurally, they originate from the Z-disks of the sarcomeres. These paracrystalline structures stained positively for alpha-actinin.18 In fact, one of the main components of rods is alpha-actinin—an actin-binding protein that localizes to the Z-disk.19,20 More than 70 mutations in the skeletal muscle alpha-actin (ACTA1) gene have now been identified. By and large, mutations are associated with three muscle diseases: a) nemaline myopathy, b) congenital actin myopathy, and c) intranuclear rod

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Fig. 2.1 Electron microscopy of aged orbicular muscle fibers. An apparently normal fiber in the center is surrounded with myofibers of highly variable diameter, but well preserved banding structure can be seen in each fiber (x32 K 68y)

Fig. 2.2 Nemaline bodies (rods). They show a typical paracrystalline structure. Several thin filaments are in connection with the rods. Next to these alterations, the myofibrillary structure is more or less irregular and the normal banding pattern is disrupted (87y years x 22 K)

myopathy. The majority of ACTA1 mutations are dominant, a small number are recessive, and most isolated cases with no previous family history have de novo dominant mutations.21 Nemaline myopathy is a rare autosomal dominant skeletal muscle myopathy characterized by severe muscle weakness and the subsequent

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appearance of nemaline rods within the muscle fibers. The intrafamilial variability suggests that the ACTA1 genotype is not the sole determinant of phenotype.22 Recently, a missense mutation in TPM3, which encodes the slow skeletal alphatropomyosin, was linked to nameline myopathy in a large kindred group with an autosomal-dominant, childhood-onset form of the disease.23 The primary defect, caused by expression of the mutant alpha-tropomyosin, was a decrease in the sensitivity of contraction to activating Ca2+, which could help explain the muscular hypotonia seen in this disease. Interestingly, this mutation did not directly result in nemaline rod formation, which suggests that rod formation is secondary to contractile dysfunction and that load-dependent processes are likely involved in nemaline rod formation in vivo.24 Although a number of genes have been identified in which mutations can cause nameline myopathy, the pathogenetic mechanisms leading to the phenotypes are poorly understood. All these together suggest a common process or mechanism operating in nemaline muscles independent of the variable degrees of pathology. More recently, electron micrographs showed elevated focal repair in nemaline muscles, suggesting that in nameline myopathy, a novel repair feature may operate.25 Sporadic late onset nemaline myopathy was also found to be associated with monoclonal gammapathy.26 Z-line streaming is a focal widening of the Z line, or sometimes widening of the whole Z line in one or more sarcomeres. These alterations were apparently common in both normal and abnormal orbicular muscle (see Fig 2.3). In excessive Z-line streaming, a longitudinal fibrillary structure was displayed, which continued into the I band next to the abnormal Z line (see Fig. 2.4). The sarcomeres around Z-line streaming showed either a normal or an abnormal banding pattern. Although both Z-line streaming and rods contain electrodense Z-line material, and that material enters into both fine filaments, the paracrystalline structure of the

Fig. 2.3 Z-line streaming in an apparently normal muscle. The normal banding pattern, sarcoplasmic reticulum, and mitochondria can be seen (x40 K 73y)

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Fig. 2.4 Excessive Z-line streaming: The longitudinal pattern is highly disturbed, and most Z lines are widened. Between the irregular mitochondria fibers, glycogen particles and tubules of sarcoplasmic reticulum can be see (x40 K 69y)

rods is clearly different from the filamentary structure of Z-line streaming. Z-line streaming may occur in normal aged muscle, but it is more common in muscle disease such as muscle dystrophies, denervation atrophy, collagen vascular disease, hypothyroid myopathy, and central core disease. In rats on a low-protein diet, Z-line streaming and disintegration of sarcomeric striation was associated with depletion of glutathion. The depletion of glutathione by protein malnutrition is responsible for inducing myofibrillary damage through the excess leaking of Ca2+ into the cytosol.27,28 Electron microscopy of biopsy specimens from the gastrocnemius muscles of volunteer human marathon runners showed evidence of inflammation and muscle fiber alterations, including Z-line streaming.29 Cytoplasmic bodies are curious structures that vary widely in size and shape, but have a characteristic appearance. It consists of a round or oval, amorphous electrodense central area surrounded by a halo of less electrodense amorphous material (see Fig. 2.5). Fine filaments from the adjacent muscle fibers pass through this halo and enter into the electrodense central area. Between the fine filaments, a few T tubules, glycogen particles, and a rich network of sarcoplasmic reticulum appear. The origin, the chemical composition, and significance of cytoplasmic bodies are unknown, but in our material they appear to contain Z-line material as well as myofilaments. Cytoplasmic bodies were found in muscle dystrophies, periodic paralysis, and collagen vascular diseases. Double Z lines or duplication of the Z line rarely occurred and it appeared in several neighboring fibers (see Fig. 2.6). Although similar alterations were reported in regenerating muscle and in hypothyreosis, neither the cause nor significance of double Z lines are known.

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Fig. 2.5 Cytoplasmic body formation in the sarcoplasma. The electrodense oval area is surrounded by a halo of fine filaments—tubules of sarcoplasmic reticulum and T tubules (x22 K 82 yrs)

Fig. 2.6 Duplication of the Z line in some neighboring fibers. On both sides of each pair of Z lines, fine fibrils can be seen. No other ultrastructural alterations are observed in association with double Z line (x26 K 63 yr)

Mitochondrial Alterations In normal conditions, mitochondria are located between myofibers and in the subsarcolemmal area. Their number is higher in the type I fibers. Numerous morphometric studies on skeletal muscle describe an age-related decline in the number of mitochondria

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and the loss of cristae. We found similar changes in our material, but it was not a generalized phenomenon. In some specimens, we found a significant increase in the number of mitochondria. In both these cases, however, the surrounding myofibers and mitochondria showed apparently normal structure (see Fig. 2.7). Proliferation of inner mitochondrial membrane/cristae, usually accompanied with the enlargement of mitochondria, was the most characteristic change in aged orbicular muscle. These alterations were found exclusively in the subsarcolemmal area and almost all mitochondria showed more or less abnormalities—i.e., the proliferation of mitochondrial cristae may be accompanied with loss of cristae, even in the same mitochondria, and a decrease of matrix density. (see Fig. 2.8). Mitochondrial inclusions of crystalline structure are very common age- and diseaserelated alterations of mitochondria. In our material, they were mostly observed in the subsarcolemmal area, but rarely in the interfibrillary mitochondria (see Fig. 2.9). Two distinct types of crystals can be distinguished on the basis of shape, size, pattern, unit cell dimension, specific location of the crystals in the mitochondrial intermembrane space, and occurrence in different muscle fiber types. Type I crystals (see Fig. 2.10) are usually present in the intracrystal space, and they occur in type I muscle fibers, whereas the type II crystals (see Fig. 2.11) are preferentially located in the intermembrane space between outer and inner mitochondrial membranes, and occur in type II muscle fibers. In humans, type I crystals are on average 200 nm wide and 2 mm long, while the type II crystals are more cubic in their dimensions—usually 100-300 nm in all three dimensions.30 Immunoelectron microscopy revealed that these inclusions react heavily

Fig. 2.7 Accumulation of mitochondria between myofibers. Both mitochondria and myofibers show well-preserved structure, although there is a light difference between the upper-left part of the picture, where normal number and normal myofiber structure can be seen, and the lower-right part, where an accumulation of mitochondria is associated with compromised myofibers (x22 K 66 yrs)

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Fig. 2.8 Numerous large mitochondria in the subsarcolemmal area. Some of them are extremely large (mega or giant mitochondria) containing enormous cristae arranged either regularly or randomly. Some of the cristae are highly electrodense, some of them have lost membrane structure, and some contain electrodense granules—presumably unsaturated lipids. The diameter of myofibers apparently decreases, but their banding structure is well-preserved and shows a state of contraction. Interfibrillary mitochondria are slightly enlarged but show normal structure (x25 K 70 yrs)

Fig. 2.9 Crystalline mitochondrial inclusion. Almost all mitochondria in the subsarcolemmal area show electrodense inclusions—in some of them the crystalline structure is clearly visible even in this magnification, but some of them contain electrodense amorphous or granular inclusions. In the neighboring myofibers, only Z line can be distinguished (x19 K 79y)

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Fig. 2.10 Morphological features of mitochondria with type I crystals. The basic structural unit of this type appears in thin sections as a rectangular unit—about 32 nm wide and of varying length— always located within the intracrystal space or between the inner and outer membrane (x70 K 71y)

Fig 2.11 Morphological features of a mitochondria with type II crystals. This structure is surrounded by the membranes of cristea—i.e., they are located in the intermembrane space but not in the mitochondrial matrix (x70 K 74y)

with specific antibodies against mitochondrial creatine kinase.31,32 Mitochondrial creatine kinase is located in the intermembrane compartment and is functionally coupled to oxidative phosphorylation. It shuttles high-energy phosphates, formed in the mitochondria, to the cytosol where they are utilized. Recent concepts suggest that mitochondrial creatine kinase has the dual role of a) functioning as a key enzyme in

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energy metabolism, and b) as a structural protein inducing the formation of mitochondrial contact sites between inner and outer membaranes. This leads to membrane cross-linking and an increased stability of the mitochondrial membrane architecture, thereby contributing to the organization of the entire organelle.33 These structural changes in mitochondria were associated with functional changes because it was demonstrated with enzyme-histochemical studies. Succinodehydrogenase activity showed a particular pattern with aging—some of the fibers or some areas in these fibers showed decreased activity (see Fig. 2.12). This irregular distribution of oxidative enzymes and focal decrease of activity has been described as target-fiber, targetoid-fiber, moth eaten-fiber, central core disease, and multi-core disease in various muscular diseases, myopathies, dystrophies, neurogenic atrophy, and hormonal diseases—all in which the above described ultrastructural alterations were observed. These morphological and enzyme-activity alterations together suggest an age-related decline in metabolic activity. From a pathophysiologic point of view, these findings can hardly be interpreted. We suppose that an increase in number of mitochondria, enlargement of mitochondria, and proliferation of cristae are morphologic manifestations of a hypermetabolic state, in which myofiber structure is usually well preserved, while loss of cristae and appearance of osmiophilic or paracrystalline inclusions are signs of mitochondrial dysfunctions, which is more frequently associated with myofiber abnormalities. Early electron microscopic studies of skeletal muscle cells of young and old humans showed the cristae of mitochondria became irregularly spaced, disrupted, and replaced by lamellar, myelin-like structures. Giant mitochondria were often visible. They contained lipofuscin in the myofibrils, too, which was often in close relationship with the damaged mitochondria.34 A substantial fall in mitochondrial oxidative capacity was also observed in ageing muscles.35

Fig. 2.12 Irregular distribution of the succino-dehydrogenase activity in aged muscle in the transversal section (71 yrs, x200)

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Biopsies of skeletal muscle from 2- to 39-year old rhesus monkeys showed that the number of individual fibers containing electron transport chain abnormalities (predominately negative for cytochrome c oxidase activity and/or hyper-reactive for succinate dehydrogenase) increased with age. These alterations associated with the deletions of the mitochondrial genome were observed in 89 percent of these electron transport chain abnormal fibers.36 The correlation between these two findings, however, remains unclear. Mitochondrial volume density was significantly lower in elderly compared with adult muscle, and these alterations were accompanied by a 50 percent reduction in oxidative capacity in the elderly vs. the adult group. In addition,. elderly subjects had nearly 50 percent lower oxidative capacity per volume of muscle than adult subjects. The cellular basis of this drop was a reduction in mitochondrial content, as well as a lower oxidative capacity of the mitochondria with age.37 In sections of orbicular muscle from aged patients, intramitochondrial inclusions of different sizes can be seen in addition to numerous morphologically abnormal and enlarged mitochondria. The formation of inclusions is always preceded by marked changes in cristae membrane disposition, with these membranes often taking a concentric arrangement. The question as to whether lipid peroxidation—presumed to be increased in patients with mitochondrial myopathies—is involved in this reorganization of the membrane system remains to be answered.

Sarcoplasma Age-related alterations of the orbicular muscle also comprise accumulation of tubular structures called tubular aggregates (TA) in the subsarcolemmal region (see Figs. 2.13 and 2.14). These densely packed tubular or tubulo-vesicular structures were apparently derived from the sarcoplasmic reticulum (SR) because it was clearly seen in some of our electron microscopic pictures, thus confirming the previous observations coming from other laboratories. The SR is an internal membrane system of the striated muscle. SR is a type of smooth endoplasmic reticulum specially adapted to surround the myofibrils, and it forms triads with invaginations of the plasma membrane called T-tubules. The sarcoplasmic reticulum contains large stores of calcium that it sequesters and then releases when the cells become depolarized. This has the effect of triggering a muscle contraction.38 Activation of muscle contraction is a rapid event that is initiated by electrical activity in the surface membrane and T-tubules. This is followed by release of calcium from the SR. Relaxation is mediated by the transport of calcium into the lumen of SR by a Ca-ATPase. Calcium then binds to calsequestrin in the lumen of the SR. For the initiation of contraction, calcium is released through the calcium channels or ryanodine receptors, which are under regulation by junctin, triadin, and calsequestrin. Thus, the SR is the major regulator of Ca2+-handling and contractility in muscles.39 It was later found that all types of cells contain cell-specific forms and/or analogues of these three proteins that are responsible for handling calcium. In all cells, these

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Fig. 2.13 Tubular aggregates. Transversal section of muscle fibers (left) and tightly packed tubules in the subsarcolemmal area (right). They show nearly identical diameter. Glycogen particles appear as electrodense granules between myofibers and tubules (x32 K, 65 yrs)

Fig. 2.14 Tubular aggregates. Transversal section of tubules in the subsarcolemmal area with numerous glycogen particles between them. Mitochondria show severe alterations, loss and/or proliferation of cristae, as well as type II crystal inclusion in some of them (x27 K, 67 yrs)

proteins tend to be grouped together. In fact, the SR is an extensive and specialized form of endoplasmic reticulum (ER) in muscle cells. Conversely, all cells contain SR-like specialized domains, but in much smaller amounts. Tubular aggregates are the most common alterations of the SR observed in various pathological conditions, but also in apparently healthy persons. Tubular aggregates

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are ultrastructural abnormalities characterized by the accumulation of densely packed tubules in skeletal muscle fibres, usually beneath the sarcoplasma membrane and rarely between the myofibers. In human skeletal muscle, they are especially rich in patients suffering from tubular aggregate myopathy. We found various forms of tubular aggregates in normal aged orbicular muscle. Tubular aggregates are characterized as more or less densely packed aggregates of vesicular or tubular membranes of variable forms and sizes that may contain amorphous material, filaments, or inner tubules. Various types of tubular aggregates were reported—namely, proliferating terminal cisterns, vesicular membrane collections either with double-walled tubules or with single-walled tubules, aggregates of dilated tubules with inner tubules, aggregates of tubulo-filamentous structures, and filamentous tubules.40 We have also observed tubulo-reticular structures (see Fig. 2.15). Tubular aggregates were immunopositive for the ryanodine receptor (RYR 1) of the SR, the SR Ca2+ pump (SERCA2-ATPase), and the intraluminal SR Ca2+ binding protein calsequestrin, indicating an SR origin of these aggregates. All of these proteins, calsequestrin, RyR, triadin, SERCAs, and sarcalumenin are involved in calcium uptake, storage, and release. These findings support the hypothesis that tubular aggregates form a tubular arrangement of a complete SR containing the junctional, cisternae, and longitudinal components of SR implicated in calcium homeostasis.41,42 They also showed decreased respiratory chain enzyme complex I and complex IV activity. These findings indicate a functional link between mitochondrial dysfunction and the presence of TAs originating from the SR.43 Lipid composition of TAs showed that the predominant lipid of the aggregates is an acetone and alcohol-soluble acidic phospholipid containing a high proportion of plasmalogens and unsaturated fatty acids—a pattern compatible with the lipid composition of SR and mitochondrial membranes in skeletal muscle.

Fig. 2.15 Tubulo-reticular aggregates. Honeycomb appearance of tubular aggregates and a severely altered mitochondria can be seen in the subsarcolemmal area (x50 K, 74 yrs)

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In mouse models of skeletal muscle aging, a persistent decrease in the expression of the calcium binding protein calreticulin, as well as a continuous increase in calsequestrin-like protein expression, both appear unrelated to the tubular aggregate formation.44 In vitro studies using SR-enriched membrane vesicles isolated from the slow-twitch soleus muscle, and the relatively fast-twitch gastrocnemius muscle isolated from adult and aged rats, showed muscle-specific impairment in SR Ca2+ pump function and skeletal muscle contractile properties, which may contribute to the age-associated slowing of relaxation in the soleus muscle.45 An impairment of the mechanisms controlling the release of calcium from internal stores (excitation-contraction coupling) has been proposed to contribute to the age-related decline of muscle performance that accompanies aging. Excitation-contraction coupling in muscle fibers occurs at the junctions between sarcoplasmic reticulum and transverse tubules, in structures called calcium release units. Recent studies showed significant alterations in the calcium release unit morphology and cellular disposition, and a significant decrease in their frequency between control and aged samples. These data indicate that in aging humans, the excitation relaxation coupling apparatus undergoes a partial disarrangement and a spatial reorganization that could interfere with an efficient delivery of Ca2+ response to the contractile proteins.46 TAs should be seen as dynamic structures that commence and cease, progress and retreat, and change their structure, functionality, and composition under multifactorial, yet not well-defined influences. The structural and functional development of tubular aggregates remains also unknown. Tubular aggregates frequently occur with mitochondrial alterations supported by growing evidence of participation of mitochondria in the development of TAs. Factors affecting formation of TAs in skeletal muscle fibers may, however, have different structural and/or functional influences on other cell types. Sarcoplasmic inclusions were also observed in the sarcoplasma of orbicular muscle. They showed filamentary, paracrystalline, or fingerprint structure and usually located beneath the plasma membrane.(see Figs. 2.16, 2.17, 2.18, and 2.19). The origin and pathological significance of these sarcolemmal inclusions are mostly unknown. They may come from abnormal SR and mitochondria, or from both. They may also be signs of degeneration or regeneration, or both. Most probably, they are identical with the desmin-containing sarcolemmal inclusions described in other instances. Desmin is an intermediate filament protein that, in striated muscle, is normally located at Z-line, beneath the sarcolemma, and prominently at neuromuscular junctions. It is abundant during myogenesis and in regenerating fibers, but decreases in amount with maturation. Desmin is coexpressed with vimentin in regenerating and denervated muscle fibers. Aggregates of desmin occur as nonspecific cytoplasmic bodies similar to the aggregates of keratin filaments in Mallory bodies, or the neurofilament aggregates in Lewy bodies. There are now increasing numbers of neuromuscular disorders in which abnormal amounts of desmin are in myopathic muscle fibers.47 Myofibrillar, or desmin-related, myopathies encompass neuromuscular disorders with abnormal deposits of desmin and myofibrillar alterations. In a recent case report on three unrelated patients presenting with proximal and distal myopathy, muscle biopsies shared sarco-

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Fig. 2.16 Fingerprint inclusion. Typical electron microscopic feature of a fingerprint inclusion in the subsarcolemmal area. Note the paucity of other organelles (x22 K, 77 yrs)

Fig. 2.17 Filamentary sarcoplasmic inclusion. This inclusion is located in the subsarcolemmal area, formed by numerous thin filaments without any apparent substructure, and surrounded by numerous mitochondria with well-preserved cristae. However, some electodense granules—presumably lipofuscin—can be seen (x22 K, 71 yrs)

plasmic inclusions—either plaque-like or amorphous, strongly immunoreacted on dystrophin, and variably for desmin, alphaB crystalline, and ubiquitin. In addition, cyclin-dependent kinases were overexpressed in affected fibers. In conclusion, myofibrillar destruction occurs in heterogeneous conditions and may overlap with features of inclusion body myopathy and mitochondrial myopathy

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Fig. 2.18 Paracrystalline sarcoplasmic inclusion. This inclusion is located in the unusually empty subsarcolemmal area, formed by tightly packed, electrodense filaments showing paracrystalline substructure, and surrounded by fine granular material. Some mitochondria with normal cristae can also be seen (x22 Km 67 yrs)

Fig. 2.19 Nuclear inclusions. Two intranuclear inclusions are surrounded by nuclear membrane, suggesting their location in the invaginations of the nucleus (i.e., pseudoinclusions). They contain heterogeneous materials (amorphous, granular, fibrillary, and membraneous). Similar materials can also be seen in the surrounding subsarcolemmal area (x26 K, 65 yrs)

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and with tubulo-filamentous inclusions and sarcolemmal inclusions.48,49 Now, we may also add aging, which may be associated with similar alterations of myofibers, mitochondria, and sarcolemma. We considered alterations of the muscle caused by various genetic and acquired factors as elementary.

Connective Tissue Changes The extracellular matrix (ECM) consists of a variety of substances, of which collagen fibrils and proteoglycans (PG) are ubiquitous. In addition to the PG, the hydrophilic ECM includes a variety of other proteins such as noncollagen glycoproteins and lipids. It is known that the force transmission of the muscle-tendon complex is dependent on the structural integrity between individual muscle fibers and the ECM as well as the fibrillar arrangement of the tendon and its allowance for absorption and loading of contraction-generated energy. Furthermore, it is well-described that the tensile strength of the matrix is based on intra- and intermolecular crosslinks, and the orientation, density, and length of both the collagen fibrils and fibers. The signals triggering the connective tissue cells in response to mechanical loading, however, and the subsequent expression, synthesis, and turnover of specific ECM components—as well as its coupling to the mechanical function of the tissue—are only partly described. Intramuscular connective tissue accounts for one to ten percent of the skeletal muscle and varies quite substantially between muscles. Based on their localization and organization, three types are distinguished: 1) endomysium encloses each individual muscle fiber with a random arrangement of collagen fibrils to allow for movement during contraction, 2) the multisheet-layered perimysium is multisheetlayered and runs transversely to fibers and holds groups of fibers in place, and finally 3) epimysium is formed from two layers of wavy collagen fibrils to form a sheet-like structure at the surface of the tendon. Intramuscular connective tissue has several functions: a) it provides a basic mechanical support for vessels and nerves, b) the connective tissue ensures the passive elastic response of muscle, and c) it contribute to the force transmission from the muscle fibers to tendons and subsequently to bone. The perimysium is especially capable of transmitting tensile force. Up to seven collagen types have been identified in intramuscular connective tissue. The fibrillar collagen type I (from 30% and up to 97% of total collagen) and III (and to some extent type V) dominates the epi-, peri-, and endomysium, but type IV dominates the basement membrane adjacent to the plasma membrane of the sarcolemma.50 In orbicular muscle, an age-related decrease in the diameter of muscle fibers seen in electron microscopy was accompanied with a significant increase in density of interstitial connective tissue. This was particularly evident in transversal sections of orbicular muscle (see Fig. 2.20). Detailed histo-chemical analysis of connective tissue changes by means of polarization microscopy revealed two types of changes: 1) an increase of collagen fibers,

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Fig. 2.20 Transversal section of the interstitial connective tissue of orbicular muscle stained with phenol and seen in polarization microscopy. Thinner septa in a 61-year old patient (A) is compared to those of a 80-year old patient (B) (x200)

and 2) an increase of glycosaminoglycan (GAG) content of fibers. In other words, more glycated fibers accumulated in the interstitial connective tissue (see Fig. 2.21). These observations on the aging of intramuscular connective tissue in orbicular muscle are in full accordance with the previous data that—with aging the nonspecific cross-linking mediated by condensation of a reducing sugar with an amino group—result in accumulation of advanced glycation end products (AGEs) in the connective tissue.51 The accumulation of AGEs with aging thus indicates a stiffer and more load-resistant tendon and intramuscular ECM structure. On the other hand, it reduces the ability to adapt to altered loading because the turnover rate of collagen is markedly reduced. Furthermore, AGEs upregulate connective tissue growth factors in fibroblasts that therefore favor the formation of fibrosis over time in elderly individuals and patients with diabetes.52 Besides reduced physical activity, diet with low albumin concentration may be a risk factor for both muscular loss and connective tissue changes.53 These age-related alterations of the connective tissue may also contribute to the age-related malposition of the eyelid, particularly in senile entropion and ectropion. In these diseases, muscular alterations were always associated with relaxation of ligaments and other connective tissue structures. There is increasing evidence that changes in quantity and quality of intermuscular connective tissue due to aging may influence at least two different types of muscle function. First, partial replacement of contractile muscle fibers with connective tissue essentially modifies muscle contraction-relaxation. This alone may explain

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Fig. 2.21 Transversal section of the interstitial connective tissue of orbicular muscle stained with blue toluidine and seen by polarization microscopy. With aging, an evident increase of collagen density can be seen when comparing specimen A (61 yrs) and B (80 yrs) (x200)

the age-related decline of muscle function and is essentially similar to those seen in various forms of congenital or acquired myopathies. Second, changes in the interstitial connective tissue also influence metabolic exchange between capillaries and muscle cells, as well as modulate neurotransmission. These changes, may also be indirectly responsible for impaired function of aged muscle.

References 1. Stefanyszyn MA, Hidayat AA, Flanagan JC (1985) The histopathology of involutional ectropion. Ophthalmology Jan. 92(1):120-7 2. Feher J (1977) Myofibre abnormalities of orbicular muscle in malposition of the eyelid. Acta Morphol Acad Sci Hung. 25(4):205-18 3. Manners RM, Weller RO (1994) Histochemical staining of orbicularis oculi muscle in ectropion and entropion. Eye. 8 ( Pt 3):332-5 4. Radnot M (1973) Mitochondrial crystals in muscles of a patient with spastic entropion. Am J Ophthalmol. Apr. 75(4):713-9 5. Radnot M, Follmann P (1974) Ultrastructural changes in senile atrophy of the orbicularis oculi muscle. Am J Ophthalmol. Oct. 78(4):689-99 6. Feher J (1978) Tubuloreticular structures in the orbicularis oculi muscle of the human eye. Acta Morph.Acad Sci Hung. 26:3-10 7. Sato T, Akatsuka H, Kito K, Tokoro Y, Tauchi H, Kato K (1986) Age changes of myofibrils of human minor pectoral muscle. Mech Ageing Dev. May 34(3):297-304

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8. Poggi P, Marchetti C, Scelsi R (1987) Automatic morphometric analysis of skeletal muscle fibers in the aging man. Anat Re. Jan. 217(1):30-4 9. Jakobsson F, Borg K, Edstrom L (1990) Fibre-type composition, structure and cytoskeletal protein location of fibres in anterior tibial muscle. Comparison between young adults and physically active aged humans. Acta Neuropathol (Berl). 80(5):459-68 10. Roth SM, Martel GF, Ivey FM, Lemmer JT, Metter EJ, Hurley BF, Rogers MA (2000) Skeletal muscle satellite cell populations in healthy young and older men and women. Anat Rec. Dec 1. 260(4):351-8 11. Beregi E, Regius O (1987) Comparative morphological study of age related mitochondrial changes of the lymphocytes and skeletal muscle cells. Acta Morphol Hung. 35(3-4):219-24 12. Fulle S, Belia S, Di Tano G (2005) Sarcopenia is more than a muscular deficit. Arch Ital Biol. Sep. 143(3-4):229-34 13. Francis IC, Stapleton F, Ehrmann K, Coroneo MT (2006) Lower eyelid tensometry in younger and older normal subjects. Eye. Feb. 20(2):166-72 14. van den Bosch WA, Leenders I, Mulder P (1999) Topographic anatomy of the eyelids, and the effects of sex and age. Br J Ophthalmol. Mar. 83(3):347-52 15. Sun WS, Baker RS, Chuke JC, Rouholiman BR, Hasan SA, Gaza W, Stava MW, Porter JD (1997) Age-related changes in human blinks. Passive and active changes in eyelid kinematics. Invest Ophthalmol Vis Sci. Jan. 38(1):92-9 16. Besne I, Descombes C, Breton L (2002) .Effect of age and anatomical site on density of sensory innervation in human epidermis. Arch Dermatol. Nov. 138(11):1445-50 17. Peshori KR, Schicatano EJ, Gopalaswamy R, Sahay E, Evinger C (2001) Aging of the trigeminal blink system. Exp Brain Res. Feb. 136(3):351-63 18. Weeks DA, Nixon RR, Kaimaktchiev V, Mierau GW (2003) Intranuclear rod myopathy, a rare and morphologically striking variant of nemaline rod myopathy. Ultrastruct Pathol. May-Jun. 27(3):151-4 19. Wallgren-Pettersson C, Jasani B, Newman GR, Morris GE, Jones S, Singhrao S, Clarke A, Virtanen I, Holmberg C, Rapola J (1995) Alpha-actinin in nemaline bodies in congenital nemaline myopathy: immunological confirmation by light and electron microscopy. Neuromuscul Disord. Mar. 5(2):93-104 20. Blanchard A, Ohanian V, Critchley D (1989) The structure and function of a-actinin. J Muscle Res Cell Motil 10:280-289 21. Schroder JM, Durling H, Laing N (2004) Actin myopathy with nemaline bodies, intranuclear rods, and a heterozygous mutation in ACTA1 (Asp154Asn). Acta Neuropathol (Berl). Sep. 108(3):250-6 [Epub 2004 Jun 24] 22. Ilkovski B, Cooper ST, Nowak K, Ryan MM, Yang N, Schnell C, Durling HJ, Roddick LG, Wilkinson I, Kornberg AJ, Collins KJ, Wallace G, Gunning P, Hardeman EC, Laing NG, North KN (2001) Nemaline Myopathy Caused by Mutations in the Muscle a-Skeletal-Actin Gene. Am J Hum Genet. Jun. 68(6):1333-43 [Epub 2001 Apr 27] 23. Ryan MM, Ilkovski B, Strickland CD, Schnell C, Sanoudou D, Midgett C, Houston R, Muirhead D, Dennett X, Shield LK, De Girolami U, Iannaccone ST, Laing NG, North KN, Beggs AH (2003) Clinical course correlates poorly with muscle pathology in nemaline myopathy. Neurology. Feb 25. 60(4):665-73 24. Michele DE, Albayya FP, Metzger JM (1999) A nemaline myopathy mutation in alphatropomyosin causes defective regulation of striated muscle force production. J Clin Invest. Dec. 104(11):1575-81 25. Sanoudou D, Corbett MA, Han M, Ghoddusi M, Nguyen MA, Vlahovich N, Hardeman EC, Beggs AH (2006) Skeletal muscle repair in a mouse model of nemaline myopathy. Hum Mol Genet. Sep 1. 15(17):2603-12 [Epub 2006 Jul 28] 26. Chahin N, Selcen D, Engel AG (2005) Sporadic late onset nemaline myopathy. Neurology. Oct 25. 65(8):1158-64 [Epub 2005 Sep 7] 27. Oumi M, Miyoshi M, Yamamoto T (2000) The ultrastructure of skeletal and smooth muscle in experimental protein malnutrition in rats fed a low protein diet. Arch Histol Cytol. 63(5):451-7

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28. Oumi M, Miyoshi M, Yamamoto T (2001) Ultrastructural changes and glutathione depletion in the skeletal muscle induced by protein malnutrition. Ultrastruct Pathol. Nov-Dec. 25(6):431-6 29. Hikida RS, Staron RS, Hagerman FC, Sherman WM, Costill DL (1983) Muscle fiber necrosis associated with human marathon runners. J Neurol Sci. May 59(2):185-203 30. Farrants GW, Hovmoller S, Stadhouders AM (1988) Two types of mitochondrial crystals in diseased human skeletal muscle fibers. Muscle Nerve. Jan. 11(1):45-55 31. Schnyder T, Winkler H, Gross H, Eppenberger HM, Wallimann T (1991) Crystallization of mitochondrial creatine kinase. Growing of large protein crystals and electron microscopic investigation of microcrystals consisting of octamers. J Biol Chem. Mar 15. 266(8):5318-22 32. Hanzlikova V, and Schiaffino S (1977) Mitochondrial changes in ischemic skeletal muscle. J. Ultrastuct. Res. 60:121-133 33. Speer O, Back N, Buerklen T, Brdiczka D, Koretsky A, Wallimann T, Eriksson O (2005) Octameric mitochondrial creatine kinase induces and stabilizes contact sites between the inner and outer membrane. Biochem J. Jan 15. 385(Pt 2):445-50 34. Beregi E, Regius O, Huttl T, Gobl Z (1988) Age-related changes in the skeletal muscle cells. Z Gerontol. Mar-Apr. 21(2):83-6 35. Trounce I, Byrne E, Marzuki S (1989) Decline in skeletal muscle mitochondrial respiratory chain function: possible factor in ageing. Lancet. Mar 25. 1(8639):637-9 36. Lee CM, Lopez ME, Weindruch R, Aiken JM (1998) Association of age-related mitochondrial abnormalities with skeletal muscle fiber atrophy. Free Radic Biol Med. Nov 15. 25(8):964-72 37. Conley KE, Jubrias SA, Esselman PC (2000) Oxidative capacity and ageing in human muscle. J Physiol. Jul 1. 526 Pt 1:203-10 38. Toyoshima C, Nakasako M, Nomura H, Ogawa H (2000) Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6A resolution. Nature. June 8. 405(6787):647-55 39. Franzini-Armstrong, C (1999) The sarcoplasmic reticulum and the control of muscle contraction. FASEB J. 13 (Suppl.), S266-S270 40. Pavlovicova M, Novotova M, Zahradnik I (2003) Structure and composition of tubular aggregates of skeletal muscle fibres. Gen Physiol Biophys. Dec. 22(4):425-40 41. Chevessier F, Marty I, Paturneau-Jouas M, Hantai D, Verdiere-Sahuque M (2004) Tubular aggregates are from whole sarcoplasmic reticulum origin: alterations in calcium binding protein expression in mouse skeletal muscle during aging. Neuromuscul Disord. Mar. 14(3):208-16 42. Chevessier F, Bauche-Godard S, Leroy JP, Koenig J, Paturneau-Jouas M, Eymard B, Hantai D, Verdiere-Sahuque M (2005) The origin of tubular aggregates in human myopathies. J Pathol. Nov. 207(3):313-23 43. Vielhaber S, Schroder R, Winkler K, Weis S, Sailer M, Feistner H, Heinze HJ, Schroder JM, Kunz WS (2001) Defective mitochondrial oxidative phosphorylation in myopathies with tubular aggregates originating from sarcoplasmic reticulum. J Neuropathol Exp Neurol. Nov. 60(11):1032-40 44. Chevessier F, Marty I, Paturneau-Jouas M, Hantai D, Verdiere-Sahuque M (2004) Tubular aggregates are from whole sarcoplasmic reticulum origin: alterations in calcium binding protein expression in mouse skeletal muscle during aging. Neuromuscul Disord. Mar. 14(3):208-16 45. Narayanan N, Jones DL, Xu A, Yu JC (1996) Effects of aging on sarcoplasmic reticulum function and contraction duration in skeletal muscles of the rat. Am J Physiol. Oct. 271(4 Pt 1): C1032-40 46. Boncompagni S, d’Amelio L, Fulle S, Fano G, Protasi F (2006) Progressive disorganization of the excitation-contraction coupling apparatus in aging human skeletal muscle as revealed by electron microscopy: a possible role in the decline of muscle performance. J Gerontol A Biol Sci Med Sci. Oct. 61(10):995-1008 47. Goebel HH, Bornemann A (1993) Desmin pathology in neuromuscular diseases. Virchows Arch B Cell Pathol Incl Mol Pathol. 64(3):127-35

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48. Wanschit J, Nakano S, Goudeau B, Strobel T, Rinner W, Wimmer G, Resch H, Jaksch M, Akiguchi I, Vicart P, Budka H (2002) Myofibrillar (desmin-related) myopathy: clinico-pathological spectrum in 3 cases and review of the literature. Clin Neuropathol. Sep-Oct. 21(5):220-31 49. Stojkovic T, Maurage CA, Moerman A, Hurtevent JF, Krivosic-Horber R, Pellissier JF, Vermersch P (2001) Congenital myopathy with central cores and fingerprint bodies in association with malignant hyperthermia susceptibility. Neuromuscul Disord. Sep. 11(6-7):538-41 50. Kjaer M (2004) Role of extracellular matrix in adaptation of tendon and skeletal muscle to mechanical loading. Physiol Rev. Apr. 84(2):649-98 51. DeBacker CM, Putterman AM, Zhou L, Holck DE, Dutton JJ (1998) Age-related changes in type-I collagen synthesis in human eyelid skin. Ophthal Plast Reconstr Surg. Jan. 14(1):13-6 52. Twigg SM, Chen MM, Joly AH, Chakrapani SD, Tsubaki J, Kim H-S, Oh R, and Rosenfeld RG (2001) Advanced glycosylation end products up-regulate connective tissue growth factor (insulin-like growth factor binding protein related protein 2) in human fibroblasts: a potential mechanism for expansion of extracellular matrix in diabetes mellitus. Endocrinology 142:1760-1769 53. Visser M, Kritchevsky SB, Newman AB, Goodpaster BH, Tylavsky FA, Nevitt MC, Harris TB (2005) Lower serum albumin concentration and change in muscle mass: the Health, Aging and Body Composition Study. Am J Clin Nut. Sep. 82(3):531-7

Chapter 3

Aging Effects on the Optics of the Eye Pablo Artal, MD, PhD

Abstract Different factors contribute to the increase in optical aberrations with age: possible modifications in the aberrations of the cornea, the lens, or even their relative contributions. The aberrations associated with the anterior surface of the cornea slightly change with age in a normal population, but the aberrations of the crystalline lens change due to the continuous modification of the lens shape with age. As the lens grows, its dimensions, curvatures, and refractive index change, altering the lens aberrations. Glasser and Campbell found a large change in the spherical aberration of excised older lenses measured in vitro. Another important factor to be considered is the nature of aberration coupling within the eye. It was shown that, in young subjects, the lens tends to compensate part of the corneal aberrations to produce an improved retinal image. As the aberrations of the lens change with age, it is quite plausible that this compensation is partially or completely lost. This explains the overall increase in aberration and the reduction of retinal image quality throughout the life span. This chapter will review the current ideas on the change of ocular aberrations with age and the possible impact this will have on the design of some ophthalmic devices, such as intraocular lenses. Keywords Optics of the eye, Increase in optical aberrations, Aging of the eye, Refractive index, Ophthalmic devices.

Introduction Normal aging affects the performance of visual system from many different aspects.1,2 For example, contrast sensitivity function (CSF) declines throughout the life span.3 This deterioration of spatial vision occurs for several reasons, ranging from purely optical degradation to retinal and neural losses. The relative contribution of optical and post-optical factors to this deterioration is not completely stated, although it is now accepted that optical factors play the major important role in normal eyes. In the aging eye, there is larger light absorption by the ocular media with different spectral contribution, a smaller pupil diameter (senile miosis), an increment of intraocular scattering, and a nearly complete reduction of the accommodation capability. In addition, Artal et al.4 first showed From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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that the mean ocular modulation transfer function (MTF)—the ratio of contrast between object and image for a given spatial frequency—in a group of older subjects was lower than the average MTF for a group of younger subjects. This result, although it was obtained in a rather small population, suggested that the ocular aberrations—in addition to intraocular scattering—increase with age. Burton et al.5 compared CSFs obtained both with laser interferometry and conventional gratings also supporting the idea of a decreasing optical image quality with age. Measurements in a larger population showed a nearly linear decline of retinal image quality with age 6. This suggested a significant increase in the optical aberrations of the eye with age, in agreement with other studies in which aberrations were measured directly.7,8,9 In addition, intraocular scatter also increases noticeably in older eyes10 All this research indicates that the degradation in the quality of the retinal image in older eyes may play an important role in limiting spatial vision. Different factors contribute to this increase in optical aberrations with age: possible modifications in the aberrations of the cornea, the lens, or even their relative contributions. The aberrations associated with the anterior surface of the cornea slightly change with age in a normal population,11 but the aberrations of the crystalline lens change due to the continuous modification of the lens shape with age. As the lens grows, its dimensions, curvatures, and refractive index change, altering the lens aberrations. Glasser and Campbell found a large change in the spherical aberration of excised older lenses measured in vitro.12,13,14 Another important factor to be considered is the nature of aberration coupling within the eye. It was shown that, in young subjects, the lens tends to compensate part of the corneal aberrations to produce an improved retinal image.13,14 As the aberrations of the lens change with age, it is quite plausible that this compensation is partially or completely lost. This explains the overall increase in aberration and the reduction of retinal image quality throughout the life span.15 In this chapter, I will review the current ideas on the change of ocular aberrations with age and the possible impact this will have on the design of some ophthalmic devices, such as intraocular lenses. The optical performance of the eye can be measured by different, and in most cases complementary, procedures. By direct recording of the double-pass retinal image, 16,17,18 an overall estimate of the eye optics is obtained, usually expressed through the point-spread function (PSF) or the modulation transfer function (MTF). By using wave-front sensors,19,20,21,22, the optical aberrations of the whole eye are obtained and the retinal image or the MTF are calculated afterwards. Furthermore, by using computer ray-tracing techniques, the aberrations produced by the anterior surface of the cornea alone can be determined from the corneal shape.23 Finally, by comparing the corneal aberrations and the overall retinal image quality, it is possible to establish the relative contributions to aberrations of the different ocular elements. By combining these results with customized modeling of the eye, it is possible to predict the retinal images under many different conditions.24

Retinal Image Quality as a Function of Age Figure 3.1 shows the average double-pass images for three age groups obtained for a 4 mm pupil diameter.

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A direct comparison of the spread of these double-pass images shows a decrease of the optical performance with age. Fig. 3.2 presents the averaged MTFs for every age group, showing a consistent decline of the average MTF for the two older groups compared to the younger group. Figure 3.3 shows the Strehl ratio—an image-quality parameter—as a function of every subject’s age for the same 4 mm pupil diameter, together with a linear regression. It shows an approximately linear decline from 20- to 70-years old, on average (the slope and regression coefficient are -0.0032, 0.83). The previous results were obtained at best focus in every subject. It is quite common, however, that the presence of small refractive errors produces a slightly defocused retinal image. Fig. 3.4 presents the Strehl ratio for the three age groups at best focus and for 0.5 D defocus. This figure shows that the average retinal image quality declines more rapidly with age at best focus, while the reduction with age is less significant for small defocusing. This suggests that the older eye is more

Fig. 3.1 Average double-pass images in subjects of 20–30, 40–50, and 60–70 years of age with 4 mm diameter pupils

Fig. 3.2 Average MTFs for each age group: 20–30 (solid line), 40–50 (short dashed line), and 60–70 (long-dashed line) for 4 mm pupil diameter

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Fig. 3.3 Strehl ratio as a function of every subject’s age for 4 mm pupil diameter. The line is the best linear fitting

Fig. 3.4 Average Strehl ratio for the three age groups, with 4 mm pupil diameter at best focus and at 0.5 D defocus. Error bars indicate the standard deviation

tolerant to small amounts of defocusing. This is quite consistent with the accepted idea of a smaller reduction in image quality with defocusing in systems with reduced overall performance. That is, possible small refractive errors should reduce the MTF relatively more in a younger subject (with good image quality at best focus) than in an older subject (with poorer image quality at best focus). In addition, due to the effect of senile miosis—a smaller pupil size for similar luminance levels in older subjects—the average MTF is approximately constant over time at low luminance levels with natural pupils. These two factors—senile miosis and a better tolerance of defocusing in the older subjects—indicate that the differences in image quality among young and

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older subjects, found under controlled laboratory conditions at fixed pupil diameter and at best focus, will be smaller under normal viewing conditions, especially at low luminance. These two mechanisms may play a protective role against the increases of ocular aberrations with age.

Aberrations of the Eye as a Function of Age The previous results using the double-pass apparatus suggested an increase of aberrations with age. This has been confirmed by measuring wave-aberrations using the Hartmann-Shack wavefront sensor. Fig. 3.5 shows, as an example, wave-aberrations and their associated point-spread functions (PSFs) for one group of young (left panels) and one group of older (right panels) subjects. Fig. 3.6 shows the magnitude of total aberrations (represented as the RMS of the wave-aberration) for the complete eye as a function of the age of the subjects. The pupil size was 5.9 mm and defocus and astigmatism—i.e., sphere and cylinder—were not included in the RMS calculations, because they are normally corrected with spectacles. The magnitude of the aberrations is well correlated with age, although there is variability within the older individuals. In the older subjects, even though the error bars are

Fig. 3.5 Wave-aberrations with associated point spread functions (PSF) for one normal young subject and one normal old subject

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Fig. 3.6 RMS of the aberrations of the eye expressed in microns as a function of age (5.9 mm pupil diameter; defocus and astigmatism not included)

Fig. 3.7 RMS of the aberrations of the cornea expressed in microns as a function of age. Larger symbols with error bars indicate mean values and standard deviations for each age group

generally larger, they are still much smaller than the differences found when comparing the old with the young subjects. On average, we can expect an increase of 0.01 microns of aberration per year.

Aberrations of Cornea as a Function of Age The shape and aberrations of the cornea change with age. It is well known that the radius of curvature slightly decreases with age, and the asphericity also changes. On average, the cornea becomes more spherical with age and, as a consequence, spherical aberrations tend to increase. Fig. 3.7 shows the RMS of the wave aberration of the

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cornea in microns for a 4-mm diameter pupil, again as a function of the age of the subjects. Although there is an increase in the aberrations of the cornea, it is small and cannot completely explain the decrease observed in the optical performance of the complete eye with age. This fact leads us to another very important issue. How does the relationship between the aberrations of the cornea and the internal surfaces of the eye—and, in particular, the lens—change with age? In other words, are the increases in the aberrations of the whole eye with age due to an increase of the aberrations of each ocular component, or rather to progressive decoupling of the aberrations? We have extensively addressed this question in several studies by comparing both corneal and ocular aberrations as a function of age.

Coupling of Corneal and Internal Aberrations as a Function of Age The amount of aberrations for both the cornea and internal optics was found to be larger than for the complete eye in young subjects, indicating a significant role of the internal ocular optics to compensate for the corneal aberrations producing an improved retinal image. During normal aging, the relatively small corneal changes can not account for the degradation in retinal image. The lens changes both its shape and the effective refractive index dramatically with age, however, and its aberrations as a consequence. In this context, it seems possible that, at least in part, the increase in aberrations of the eye with age could be due to the loss of the aberration balance between cornea and lens that seems to be present in the younger eye. Figure 3.8 shows both the RMS of the wave-aberration for the complete eye and for the anterior surface of the cornea. Corneal aberrations in the younger subjects are larger than the total ocular aberrations, indicating that the internal optics compensates for the corneal aberrations. The opposite occurs in older subjects, however, where the cornea has lower aberrations than the complete eye. This indicates that the lenses in the older eyes do not compensate, but in fact add aberrations to those of the cornea. As an example, Fig. 3.9 shows examples of wave-aberrations for the cornea, internal surfaces and the eye—for a typical young eye (upper plots) and for a typical old eye (bottom). In the young eye, the cornea and the internal optics aberrations have a similar magnitude and shape, but are opposite in sign, producing an eye with overall lower aberrations. However, in the older eye, this finely tuned compensation is not present.

Optics of the Aging Eye and Intraocular Lenses This better understanding of the ocular optics in the aging eye was used for designing new and more effective ophthalmic optics. For instance, the ideal substitute for the natural lens in a cataract eye is not an intraocular lens with the best isolated

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Fig. 3.8 RMS of the aberrations of the eye (squares) and the cornea (circles) as a function of age

Fig. 3.9 Examples of wave aberrations for the anterior corneal surface, the internal surface, and the complete eye in a young and older eye, respectively

optical performance, but rather one designed to compensate for the aberrations of the cornea (see schematic example in Fig. 3.10). An improved design for an intra-ocular lens would have an aberration profile that compensates (at least partially) for the corneal aberrations in the older eye, to maximize the quality of the retinal image. A first approximation is the use of aspheric intraocular lenses to correct for the corneal spherical aberration. Other type of lenses—for instance, correcting corneal coma—have also been proposed.25

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Fig. 3.10 Schematic representation of the coupling of the cornea and an intraocular lens. A lens without aberrations will produce an eye with the aberrations of the cornea and relatively poor retinal images. However, a lens with aberrations approximately contrary to those of the cornea will produce an eye nearly free of aberrations

In the future, it may also be possible to have customized lenses correcting most corneal aberrations in situ and maximizing retinal image quality. In addition to aberrations, other optical (mainly intraocular scatter) and post-optical (neural) factors further influence visual performance of older subjects. All those factors have to be considered together when predicting or analyzing visual performance. Acknowledgments Part of the research described in this chapter has been supported by the Ministerio de Educación y Ciencia (MEC) in Spain, and by AMO_Groningen (The Netherlands). The author also wishes to thank all his collaborators in his laboratory at Murcia University and elsewhere who greatly contributed in many of the aspects of the research briefly described here.

References 1. Weale RA (1992) The senescence of human vision. Oxford University Press, Oxford 2. Owsley C, Sloane ME (1990) Vision and aging. In: Boller F, Grafman J (eds). Handbook of Neuropsychology, vol 4. Elsevier Science Publishers B.V. (Biomedical Division), pp 229-249 3. Owsley C, Sekuler R, and Siemsen D (1983) Contrast sensitivity throughout adulthood. Vision Res 23:689-699 4. Artal P, Ferro M, Miranda I, and Navarro R (1993) Effects of aging in retinal image quality. J. Opt. Soc. Am. A 10:1656-1662 5. Burton KB, Owsley C, Sloane ME (1993) Aging and neural spatial contrast sensitivity: photopic vision. Vision Res. 33:939-946

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6. Guirao A, González C, Redondo M, Geraghty E, Norrby S, and Artal P (1999) Average optical performance of the human eye as a function of age in a normal population. Invest. Ophthalmol. Vis. Sci. 40:197-202 7. Jenkins TCA (1963) Aberrations of the eye and their effects on vision: part 1.Brit. J. Physiol. Opt. 20:59-91 8. Calver R, Cox MJ, and Elliot DB (1999) Effect of aging on the monochromatic aberrations of the human eye. J. Opt. Soc. Am. A, 16(9):2069-2078 9. McLellan JS, Marcos S, and Burns SA (2001) Age-related changed in monochromatic wave aberrations of the human eye. Invest. Ophthalmol. Vis. Sci. 42:1390-1395 10. Ijspeert JK, de Waard PWT, van den Berg TJTP, and de Jong PTVM (1990) The intraocular straylight function in 129 healthy volunteers; dependence on angle, age and pigmentation. Vision Res. 36:699-707 11. Guirao A, Redondo M, and Artal P (2000) Optical aberrations of the human cornea as a function of age. J. Opt. Soc. Am. A. 17(10):1697-1702 12. Glasser A, and Campbell MCW (1998) Presbyopia and the optical changes in the human crystalline lens with age. Vision Res. 38:209-229 13. Artal P and Guirao A (1998) Contribution of the cornea and the lens to the aberrations of the human eye. Optics Letters 23:1713-1715 14. Artal P, Guirao A, Berrio E, and Williams, DR (2001) Compensation of corneal aberrations by the internal optics in the human eye. Journal of Vision, 1(1):1-8 15. Artal P, Berrio E, Guirao A, Piers P (2002) Contribution of the cornea and internal surfaces to the change of ocular aberrations with age. J. Opt. Soc. Am. A. 19:137-143 16. Santamaría J, Artal P, Bescós J (1987) Determination of the point-spread function of the human eye using a hybrid optical-digital method. J Opt Soc Am A. 4:1109-1114 17. Artal P, Marcos S, Navarro R, Williams DR (1995) Odd aberrations and double-pass measurements of retinal image quality. J Opt Soc Am A. 12:195-201 18. Díaz-Doutón F, Benito A, Pujol J, Arjona M, Güell JL, Artal P (2006) Comparison of the Retinal Image Quality with a Hartmann-Shack Wavefront Sensor and a Double-Pass Instrument. Invest. Ophthalmol. Vis. Sci. 47:1710-1716 19. Liang J, Grimm B, Goelz S, and Bille JF (1994) Objective measurement of the WA’s aberration of the human eye with the use of a Hartmann-Shack sensor. J. Opt. Soc. Am. A. 11:1949-1957 20. Liang J and Williams DR (1997) Aberrations and retinal image quality of the normal human eye. J. Opt. Soc. Am. A 14:2873-2883 21. Prieto PM, Vargas-Martín F, Goelz S, and Artal P (2000) Analysis of the performance of the Hartmann-Shack sensor in the human eye. J. Opt. Soc. Am. A. 17:1388-1398 22. Iglesias I, Berrio E and Artal P (1998) Estimates of the ocular wave aberration from pairs of double-pass retinal images. J. Opt. Soc. Am. A. 15:2466-2476 23. Guirao A and Artal P (2000) Corneal wave-aberration from videokeratography: accuracy and limitations of the procedure. J. Opt. Soc. Am. A. 17:955-965 24. Tabernero J, Piers P, Benito A, Redondo M and Artal P (2006) Predicting the optical performance of eyes implanted with IOLs to correct spherical aberration. Invest. Ophthalmol. Vis. Sci. 47:4651-4658 25. Tabernero J, Piers P and Artal P (2007) Intraocular lens to correct corneal coma. Opt. Lett. 32 (4):406-408

Chapter 4

Aging of the Cornea Luciano Cerulli, MD, PhD and Filippo Missiroli, MD

Abstract Unlike other ocular structures, as well as most tissues in the body, the cornea does not show important changes with normal aging. A variety of corneal aging changes have, however, been reported. Few of them are clinically evident, while others are demonstrated by chemical, biological, and structural studies. Distinction has to be made between conditions considered within the normal limits of aging and those of true disease processes that commonly affect the cornea in the elderly. The difference with other ocular structures is that changes of cornea due to aging are mostly asymptomatic and do not usually affect vision, hence they do not require treatment. However, some changes occur and, for example, the aged cornea becomes more susceptible to infection because of a decreased ability to resist a variety of physiological stresses. Furthermore, it is sometimes difficult to distinguish age specific deterioration from degenerations modified by environmental and genetic factors. The well-known clinical conditions that occur with age in the cornea will be described first. Then, a review of the effect of age on shape and different aspects of the cornea and its structural (anatomical) changes will be reported. Keywords Aging, cornea, corneal arcus, deep crocodile shagreen, astigmatism, corneal thickness, stroma, keratocyte, endothelium

Clinical Conditions Corneal Arcus Also known as gerontoxon is the most common bilateral manifestation of the aged cornea. It is characterized by a white ring around the peripheral cornea that is separated from the limbus by a clear zone 0.3 to 1 mm in width. It consists of deposits of cholesterol, cholesterol esters, phospholipids, and triglycerides. When it is seen in younger people, it is not an age-related condition (arcus senilis) but is in association with hyperlipoproteinemia types 2 and 3. Initially the superior and inferior peripheral

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Fig. 4.1 Peripheral ring of corneal arcus as seen at slit lamp

cornea is affected and, with time, a complete annulus forms. Slit lamp biomicroscopy reveals the extensive involvement of the subepithelial zone, the corneal stroma, and Descemet’s membrane, as well as fine opacities in the so-called lucid interval of Vogt which is optically clear on naked eye examination (see Fig. 4.1).1

Prevalence In one study, the estimated prevalence was measured at 8 percent for those 40 to 49 years of age, 45 percent for those 50 to 59 years of age, and 75 percent for those 70 to 79 years of age. In another study, the prevalence was measured at 6 to 12 percent in a cohort of insulin-dependent patients with diabetes who were less than 30 years of age, and was measured at 49 to 54 percent for patients with diabetes who were more than 30 years of age. The tendency toward increasing prevalence with age explains the usage of the popular nomenclature arcus senilis, instead of the more technically correct term corneal arcus. In general, ar cus senilis is more common in men than it is in women. It is also more common among patients of African descent, and when it occurs in these patients, it tends to occur earlier in life. Arcus senilis may also be more common in patients who regularly consume alcoholic beverages, with the prevalence in one study increasing as the amount of alcohol consumption increased.3,4,5

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Biochemical Aspects Palmitic, stearic, oleic, and linoleic acids are among the fatty acids that make up many of the deposited lipid molecules. Lipids are normally deposited in the cornea, but with aging, the amount of deposited lipids increases—in some cases it will result in arcus senilis. This supports the assumption that arcus senilis may represent an extension or exaggeration of the natural process of lipid deposition in the cornea. A structural study showed extracellular solid spherical lipid particles (< 200 nm in diameter) enmeshed between collagen fibers. Immunostaining showed significant apoE and apoA-I, but very little apoB in the peripheral cornea. Cholesteryl ester-rich spherical particles accumulate in the extracellular spaces of the peripheral cornea. Most of these lipid particles are 40-200 nm in diameter, and are therefore similar in size to one type of cholesteryl ester-rich lipid particle that accumulates in the extracellular spaces of human atherosclerotic lesions. These extracellular lipid droplets seem to derive from direct deposition of plasma lipoproteins.2

Deep Crocodile Shagreen Also known as mosaic degeneration, deep crocodile shagreen consists ofn bilateral, polygonal, grayish-white opacities that are interrupted by clear spaces that are usually asymptomatic (see Fig. 4.2). Two types of this condition exist:

Fig. 4.2 Both crocodile shagreen (white arrow) and limbal gridle of Vogt (black arrow)

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Anterior shagreen (Vogt’s anterior mosaic crocodile shagreen) are seen in the deep layers of the epithelium or in Bowman’s layer, and become more apparent after instillation of fluorescein stain Posterior shagreen are generally seen in the central deep cornea, which makes it difficult to differentiate with a central cloudy dystrophy of the cornea

Both anterior and posterior forms do not require treatment. Deep crocodile shagreen is sometimes seen in association with peripheral band keratopathy or following trauma.6 Familial type may occur with x-linked megalocornea or in a juvenile form of anterior mosaic crocodile shagreen.

White Limbal Gridle of Vogt Limbal gridle of Vogt is a very common, bilateral, age-related corneal degeneration. It is always asymptomatic and requires no therapy. It affects more than 50 percent of the population over age 40 and is characterized by a subepithelial degeneration and may include calcium deposits. The lesions look like white opacities of the peripheral cornea, forming a half moon-like arc running concentrically with the limbus—usually in the interpalpebral zone along the nasal and temporal limbus (only the horizontal meridian is affected). The opacities may be separated from the limbus by a clear zone of about 1 mm, or without a clear zone in between, and lie at the level of Bowman’s membrane and the immediately subjacent stroma. Histopathology of the lesions show a destruction of Bowman’s membrane and superficial lamellae of the stroma in association with deposition of calcium and areas of hyaline and elastotic degeneration with hypertrophy of the overlying epithelium.6

Hassall-Henle Bodies Also known as Hassall-Henle warts or Henle’s warts, these bodies are small hyaline excrescences on the posterior surface of Descemet’s membrane at the periphery of the cornea. Averaging 0.07 to 0.08 mm in diameter, they are constantly present in adults—occasionally they become confluent and macroscopically visible. They contain bounded material that is believed to be collagen, in which numerous cracks and fissures are filled with extrusions of the corneal epithelium. They represent an over activity of the formation of hyalin by the endothelial cells. They are found in large quantities in degenerations and chronic inflammations. When they become larger and more numerous, they invade the central area (cornea guttata). The condition is probably associated with the aging process.6,7

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Cornea Shape Astigmatism One of the most common ocular changes in the elderly is the variation of manifest refraction. Often this happens because a change in corneal curvature causes alteration in refraction—usually a change from the with-the-rule astigmatism to againstthe-rule astigmatism. The vertical meridian of the cornea is steeper than the horizontal meridian in with-the-rule astigmatism, so the eye has more refractive power (plus cylinder) along the vertical axis. In against-the-rule astigmatism, the horizontal meridian is steeper than the vertical and the eye has more refractive power (plus cylinder) along the horizontal axis. Several studies confirmed that a decrease in the vertex radius occurs with aging, thus demonstrating a steepening of the cornea. A Japanese study of 2,161 subjects found that the prevalence of astigmatism increases and the axis turns to againstthe-rule with age. The result of the linear regression analysis indicates that the age-related change in astigmatism is mainly associated with changes in the cornea.8 The corneal astigmatism was found to change in Hong Kong’s Chinese population, where both the corneal and spectacle astigmatism demonstrated a shift from with-the-rule to against-the-rule with age (keratometer and a computer-assisted videokeratoscope were used).9 Another Japanese study that used a autokeratometer revealed that the cylindrical diopter of with-the-rule astigmatism decreased, and against-the-rule astigmatism increased with aging.10 A study from Turkey suggested that the normal cornea becomes steeper in the horizontal line and superior vertical quadrant, and shifts from with-the-rule to against-the-rule astigmatism. The amount of physiological corneal astigmatism, however, does not change with age.11 A topographic analysis of the changes in corneal shape due to aging was carried out in 734 volunteers in Japan. The maps of subjects in their 70s and > 80 revealed a horizontal, oval-shaped steep area, suggesting against-the-rule astigmatism. The average-of-difference map demonstrated a marked corneal steepening at the horizontal meridians. In the data analysis of the averaged map, the mean refractive powers of the cornea increased with age.12 While corneal topography gives data of the anterior surface of the cornea, Scheimpflug photography is a useful tool with a non-contact technique that allows researchers to determine the shape and astigmatism of the posterior corneal surface. A study where Scheimpflug camera was used to measure the cornea of the right eye in six meridians of 114 subjects ranging in age from 18 to 65 years showed that, with aging, the asphericity of both the anterior and the posterior corneal surface changes significantly—a significant average change in the k value was found for the posterior surface, which indicates a shift to a more aspherical surface. The same study revealed that peripheral thickness along the perpendicular line at 3.75 mm from the apex showed an average difference of 19 micron between the young and the old subjects, while no difference in central corneal thickness was noted.13

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Corneal Thickness The thickness of corneal tissue is an important parameter in refractive surgery, and its measurement is essential in the assessment and management of corneal diseases. In the last few years, it became an important clinical parameter for correct interpretation of Goldmann applanation tonometry results. There are different instruments for determining corneal thickness, but the most widespread method currently in use is still ultrasonic pachymetry, even though it can be measured with other modalities—scanning-slit topography/pachymetry, specular and confocal microscopy, optical low-coherence reflectometry and rotating Scheimpflug camera. So far, ultrasonic pachymetry, scanning-slit topography/ pachymetry and rotating Scheimpflug cameras are considered more reliable tools for determining corneal thickness. In a recent study, Amano and others found that corneal thicknesses were comparable for the rotating Scheimpflug camera (Pentacam), ultrasonic pachymetry, and scanning-slit topography (Orbscan), with the acoustic equivalent correction factor. The measurements taken with the three instruments had significant linear correlations with one another, and all methods had highly satisfactory measurement repeatability.14 Several studies analyzed corneal thickness and its variation with aging, but the age-dependent difference in corneal thickness values remains unclear. Age-related changes in central and peripheral corneal thickness were analyzed by the mean of the Orbscan II topography system. A thinning of the peripheral cornea was noted with increasing age. It was found that the mean corneal thickness was reduced with age (0.38 mm/year), whereas the central corneal thickness was unaltered. These data concord with results of the previously mentioned study of Dubbelman obtained from the Scheimpflug camera.15 Rufer and others found slight variations in the mean central corneal thickness measured by the mean of Orbscan II system. In their work, no significant constant age-related trend was identified. However, there were raised values in the 50- to 59-year olds and 70- to 79-year olds when compared with all the other decades.16 In a Mongolian population where the central corneal thickness was measured using an optical pachymeter, there was a highly significant decrease in central corneal thickness with age—5 microns/decade in men, and 6 microns/decade in women.17 In a large series of eyes undergoing myopic refractive surgery, central corneal pachymetric measurements did not correlate with age.18 A large study where 1,699 Latino participants aged 40 or more years were included, central corneal thickness was measured by ultrasonic pachymeter. The most clinically significant finding was that when compared with normal Latinos aged 40 to 49 years, Latinos aged 70 or more years had substantially thinner corneas on average.19 Among participants of the European Glaucoma Prevention Study (EGPS), the central corneal thickness was higher in younger patients, male patients, and diabetic patients.20 In a study population of different races, where central corneal thickness was measured by ultrasound pachymetry, an inverse relationship between age and CCT

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was found, with a reduction of about 3 micron per decade.21 Sanchis-Gimeno and others have analyzed the changes of central corneal thickness values of Caucasian emmetropic subjects in accordance with their age—Caucasian emmetropic aged subjects have reduced corneal thickness values when compared to young emmetropic subjects.22 In conclusion, it seems there is no substantial change in corneal thickness during aging—nevertheless, there is evidence suggesting a significant age-dependent decrease in corneal thickness later in life for some ethnic groups.

Corneal Aberration Normally, the cornea has positive spherical aberrations that compensate for the negative aberrations attributed to the lens. Around 20 years of age, ocular spherical aberration is almost zero, and it gradually increases with age (positive values). This change in total spherical aberration with aging is because of aberration changes in the lens, which may be induced by the age-related changes of anterior and posterior lens radius.23,24,25 There is wide individual variability in anterior corneal aberrations, and little of this is attributable to age-related changes. Analyzing data from different studies, it is possible to see that while corneal spherical aberrations do not show change with aging, there is a positive correlation between corneal coma aberration with age and evidence that the increase of ocular coma with aging is mainly because of the increase of corneal coma.26 Because coma-like aberrations consist of tilt and/or asymmetry, the corneas become less symmetric with aging. Increases in corneal coma-like aberrations in elderly do not directly indicate the deterioration of visual function in their eyes for different reasons—one of these is that pupils of older subjects tend to be more miotic, thus with smaller influence of the corneal wavefront aberrations on visual performance.

Structure Epithelium The corneal epithelium acts as barrier from environmental agents and contributes to movement of water and molecules through the cornea. With age, this function seems to undergo some deterioration resulting in a breakdown of epithelial barrier function. This can be quantified by fluorophotometric determination of corneal epithelial permeability to fluorescein.27 A breakdown of epithelial barrier function28 and the increased tear contact time29 may explain the increase in epithelial permeability with age that renders the aging cornea more susceptible to infection. Changes in distribution of integrin subunits in the epithelium could also reduce the epithelial

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barrier function. The α6 subunit and the β4 subunit—components of hemidesmosomes—become discontinuous with age.30 A reduced ability of corneal cells to upregulate adhesion molecules, and a reduced phagocytic ability of reactive polymorphonucleocytes in response to infection, also occur with aging.31 This could impair the ability to eliminate a bacterial infection.

Bowman’s Membrane Bowman’s membrane (epithelial basement membrane) is the layer that separates the epithelium from the stroma. It is about 10-16 µm thick and is acellular, except for the nerves that perforate it. Taylor and Kimsey studied the basal membranes of corneas in 12 diabetic patients by transmission electron microscopy (TEM). They found no clear relation between the corneal epithelial basement membrane thickness and age.32 Alvarado et al. published an ultrastructural evaluation and morphometric analysis of the basement membrane on a large number of specimens from subjects ranging in age from 17 weeks of gestation to 93 years of age. They found that structural changes occur with aging in the basement membranes. There is a progressive thickening of the corneal epithelial basement membrane that is caused by two different processes—membrane deposition (forming unilaminar membranes) and membrane reduplication (forming multilaminar membranes). Membrane deposition appears to be the only process involved in membrane thickening in the prenatal and early postnatal period. Later in life, the process of membrane reduplication plays a more prominent role than thickening by deposition. In middle-aged individuals, areas of reduplication are focal (mixed membrane type). With increasing age, a greater proportion of the basement membrane becomes multilaminar.33

Subbasal Nerve Plexus The cornea is one of the most innervated tissues in the body. Its innervation is provided by the ophthalmic and maxillary branches of the trigeminal nerve. The nerve bundles enter the peripheral cornea at the limbus in the anterior third of the stroma, and then penetrate the Bowman’s layer where they form the subbasal nerve plexus. The fibers run parallel to the cornea’s surface between the Bowman’s layer and the basal epithelial layer, and then terminate in the superficial epithelium as free nerve endings. Because of the fast degeneration of nerve fibers after death, the morphology of the corneal nerve based on histological and microscopic studies is limited and unclear. In vivo confocal microscopy is able to visualize and measure subbasal nerve fibers. Confocal microscopy studies have shown that the human subbasal nerve plexus is primarily oriented in a superior-to-inferior direction at the central corneal apex.34

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Grupcheva et al. found a small age-associated decrease in subbasal nerve density in clinically healthy corneas.35 In a recent study, Erie also demonstrated that the density and orientation of the subbasal nerve plexus do not show changes with age.36 Despite this morphological observation, several studies demonstrated that corneal sensation decreases with age.37,38,39 Corneal sensitivity is important in the maintenance of function and structure of the cornea, and it is also crucial in the healing process after injury or surgery. Roszkowska evaluated central and peripheral corneal sensitivity in a population that ranged from 20 to 90 years of age. In this study, they found that corneal sensitivity is maintained throughout life—corneal sensitivity remains stable in the central zone until the age of 60 when it begins to decrease, while the peripheral sensitivity starts to decrease earlier and progresses at a fast rate.40

Stroma Transparency of the cornea is due to the uniform size of the constituent collagen fibrils and to the degree of ordering in their packing in the stroma. Changes in the corneal stromal structure occur with aging for both the collagen fibrils and the cellular component. Studies on these stromal changes were obtained from both in vivo and in vitro observations. Human collagen undergoes progressive changes with age, including a decrease in elasticity. In vitro studies suggest that the physical changes involve progressive crosslinking between collagen molecules. Daxer et al. performed x-ray scattering experiments on corneas of various ages to investigate the three-dimensional structural properties of collagen fibrils in the human corneal stroma. Analyzing fibril diameter, intermolecular Bragg spacing, and axial collagen period, they found that aging is related to a three-dimensional growth of collagen fibrils in the human corneal stroma. The age-related growth of the fibril diameter was mostly a result of an increased number of collagen molecules and, in addition, to some expansion of the intermolecular Bragg spacing, probably resulting from glycationinduced crosslinking.41 The expansion of the collagen intermolecular Bragg spacing within the fibrils suggests that molecules other than collagen are deposited in the fibrils during aging and push the collagen molecules further apart. It confirms recent studies that have demonstrated glycation-induced expansion of the intermolecular spacing and subsequent crosslinking of the molecules with age.42 Advanced glycation end products (AGEs) play a significant role in many age-related disorders. AGEs accumulate in the aging cornea and mediate crosslinking of molecules in the stroma. Such age-related crosslinking occurs largely on the collagen component of the cornea. Two changes with age were identified by Malik: 1) an increase in the cross-sectional area associated with each molecule in corneal collagen, which may be due to an increase in nonenzymatic crosslinking between collagen molecules, and 2) a decrease in the stroma interfibrillar spacing, which could be related to changes in the proteoglycan

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composition of the interfibrillar matrix. The decrease in stromal interfibrillar spacing with age was also found by Kanai and Kaufman using an electron microscopy study.43 These findings related to stromal fibrillar changes can, in part, explain the result obtained by Elsheikh et al. on corneal stiffness. They evaluated the stress-strain behavior of corneal tissue and how the behavior was affected by age. In this study, the cornea demonstrates considerable stiffening with age with the behavior closely fitting an exponential power function typical of collagenous tissue. The increase in stiffness could be related to the additional nonenzymatic crosslinking that affects the stromal collagen fibrils that occurs with age, and to the age-related increase in collagen fibril diameter.44 A new instrument that is starting to be used in clinical practice is the ocular response analyzer that measures the corneal biomechanical response to a rapid indentation obtained by an air jet. This corneal response is called corneal hysteresis (CH), and is a new parameter that can help to better understand the behavior of the cornea on the intraocular pressure measurement obtained by Goldmann applanation. Kotecha et al. investigated the association between CH and both age and central corneal thickness, as well as the agreement between ocular response analyzer and Goldmann applanation tonometer IOP measurements. Analyzing the data, they found a correction factor that describes a biomechanical property of the cornea that is independent from the intraocular pressure and that increases with thicker cornea and decreases with age. This factor is a measure of the corneal material properties, which include both stiffness and viscoelasticity. The observed negative association between corneal viscoelastic properties with advancing age may be further evidence of an increase in crosslinkage of collagen fibrils within the cornea, making it a stiffer and less viscoelastic structure.45 Keraticytes are the principal cellular components of corneal stroma—they are fibroblast-like cells that produce, degrade, and remodel the stroma, and are therefore important in corneal wound healing. Keraticyte density in a normal human anterior corneal stroma has been reported to be around 20.000–24.000 cells/mm3, being highest posterior to the Bowman’s layer and then decreasing towards posterior stroma (see Fig. 4.3).46,47 Using biochemical measurements of the stromal DNA/ mass content within the central 7-mm diameter zone, Møller-Pedersen48 found a direct correlation between keratocyte density and donor age, with a physiologic decline of 0.3 percent per year throughout life. Møller-Pedersen and Ehlers49 describe a 30 percent decrease in cell density in the subendothelial region. The DNA method is invasive and cannot be used to study keratocyte density in vivo. Recent use of Confocal microscopy in vivo allowed quantification of stromal cell density without the need of tissue processing that can alter the tissue. In their work, Patel and coworkers found that full-thickness central keratocyte density was negatively correlated with age and decreased 0.45 percent per year. Keratocyte densities in all anteroposterior regions were negatively correlated with age, except the posterior 67 to 90 percent region of the stroma. The number of keratocytes in

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Fig. 4.3 Confocal images of stroma: A) anterior stroma, and B) posterior stroma with different keratocyte density

the full-thickness stroma was also negatively correlated with age and decreased 0.43 percent per year.47 Using same modality with confocal microscopy, Berlau et al. found that keratocyte density was lower in patients older than 50 years than in those younger than 50 years.50

Descemet’s Membrane Changes in Descemet’s membrane with aging are well-characterized in humans. Before birth it is a very thin basement membrane and different in appearance from the adult Descemet’s membrane. It grows by deposition of a series of similar “membrane units,” which are stacked to form a lamellar structure consisting of at least 30 layers by the end of gestation. At birth, it has an average thickness of 3 µm and exhibits an electron-dense, banding pattern with 110 nm periodicity.51,52 This portion of Descemet’s membrane is referred to as the anterior banded zone. Over the ensuing decades of life, the anterior banded zone remains well-demarcated and stable in thickness and appearance. In postnatal life the membrane continues to grow in thickness by deposition of a non-striated, non-lamellar material posterior to the striated prenatal layer. This posterior portion of the membrane directly subjacent to the endothelium progressively thickens as a non-banded, homogeneous substance referred to as the posterior non-banded zone. Thickening of the posterior

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non-banded zone contributes to the age-dependent growth of Descemet’s membrane from approximately 3 µm at birth to 5-6 µm at 20 years of age and to 13 µm at 80 years of age.51,52

Endothelium The corneal endothelium and Descemet’s membrane play a vital role in maintaining corneal structure and clarity. The endothelium is a monolayer of hexagonal cells located on the posterior surface of the cornea, which functions to pump water out of the stroma to maintain transparency. (see Fig. 4.4) One of the most known effects of age in the human cornea is that endothelial cell density decreases progressively during aging, but its measurement is not a reliable index of the chronological age of the cornea because there is wide range of endothelial cell density in normal populations.52-61 In the absence of a proliferative response to cell loss, endothelial cover of the posterior corneal surface is maintained by a gradual increase in the size of the remaining cells, resulting in increased cellular pleomorphism and a decrease in the percentage of hexagonal cells with age. Endothelial cell analysis provides important clinical information on corneal function and viability. If we look at a picture of endothelial specular microscopy from a healthy young adult of 25-30 years of age, we can tell that the cells are uniform in their shape and size, but not as much as we can see from corneas aged less then 10 years, where the cells are much smaller and rounded. Specular microscopy of corneas older then 70 years of age shows much variety in the size and shape of cells. Fewer cells are exagonal, while the number of pentagonal, six-agonal, and seven-agonal cells increases significantly. Numerous studies were published on the reduction of endothelial cell density and the change in cellular morphology with age. They demonstrate in different races that cellular polymegethism and cellular pleomorphism increases with age.53-61 In 1976, Bourne and Kaufman documented a decrease in the number of central endothelial cells with age by using a clinical specular microscope. They found a rate of reduction of 0.39 percent per year, while Hollingsworth et al. calculated that endothelial cell density decreases at a rate of 0.33 percent per year. The estimated

Fig. 4.4 Specular microscopy image of an normal adult endothelium

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annual reduction found by Murphy et al. averages approximately 0.56 percent per year.57,58,59 Blatt et al. found with the clinical specular microscope that cell density decreases significantly with age when the endothelial cells are regular in size, but that if the endothelial cells are irregular in size and arrangement, the endothelial cell densities do not correlate with age.60 The topographical distribution of the corneal endothelial cell density in different age ranges has been studied by Roszkowska et al. They evaluated 300 eyes of 204 healthy subjects aged from 20 to 83 years. Age-related changes involve both center and periphery. In particular, they observed a higher peripheral decrement in the ancient subjects resulting in a topographical disparity in the elderly. They concluded that the central density evaluation is sufficient to provide the exact information about an entire endothelial surface, but only in the young subjects. It did not work with elderly patients where a topographical disparity might occur and the only central density determination could provide insufficient results.61 The reason why a gradual loss of endothelial cells occurs with age remains unclear. Green hypothesizes that aging processes in the eye occur as a consequence of degradation of enzymes that normally metabolize and detoxify hydrogen peroxide and other free radicals. The loss of enzyme activity allows hydrogen peroxide—which normally occurs within eye fluids—and free radicals to induce irreversible deleterious effects on different eye tissues. These processes may lead to cataract formation in the lens, as well as loss of corneal endothelial cells. This hypothesis is partially supported by the results obtained by Cejkova et al.62 from the analysis of the activities of superoxide dismutase, glutathione peroxidase, and catalase (the enzymatic scavengers of reactive oxygen species) in rabbit corneas. They found that in aged corneas, the activities of all antioxidant enzymes were dramatically decreased, suggesting that the cornea of aged rabbits are more susceptible to oxidative injury in comparison to the corneas of young adult animals.63

References 1. Phillips CI, Tsukahara S, Gore SM (1990) Corneal arcus: some morphology and applied pathophysiology. Jpn. J. Ophthalmol 34:440-442 2. Gaynor PM, Zhang WY, Salehizadeh B, Pettiford B and Kruth HS (1996) Cholesterol accumulation in human cornea: evidence that extracellular cholesteryl ester-rich lipid particles deposit independently of foam cells. J Lipid Res. 37(9):1849-61 3. Cooke NT (1981) Significance of arcus senilis in Caucasians. J R Soc Med 74:201-4 4. Moss SE, Klein R, Klein BE. (2000) Arcus senilis and mortality in a population with diabetes. Am J Ophthalmol 129:676-8 5. McAndrew GM, Ogston D (1965) Arcus senilis and coronary artery disease. Am Heart J 70:838-40 6. Duke-Elder S (1965) System of Ophthalmology, vol VIII, Part 2, Disease of the Outer Eye. London, Kimpton, p 869 7. Hogan MJ, Zimmermann LE (1962) Ophthalmic pathology. An Atlas and textbook. 2nd ed. Philadelphia, Saunders, pp 288-289 8. Asano K, Nomura H, Iwano M, Ando F, Niino N, Shimokata H (2005) Miyake Relationship between astigmatism and aging in middle-aged and elderly Japanese. Jpn J Ophthalmol 49(2):127-33

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9. Lam AK, Chan CC, Lee MH, Wong KM (1999) The aging effect on corneal curvature and the validity of Javal’s rule in Hong Kong. Chinese Curr Eye Res. 18(2):83-90 10. Hayashi K, Masumoto M, Fujino S, Hayashi F (1993) Changes in corneal astigmatism with aging. Nippon Ganka Gakkai Zasshi. 97 (10):1193-6 11. Topuz H, Ozdemir M, Cinal A, Gumusalan (2004) Age-related differences in normal corneal topography. Y Ophthalmic Surg Lasers Imaging 35(4):298-303 12. Hayashi K, Hayashi H, Hayashi F (1995) Topographic analysis of the changes in corneal shape due to aging cornea. 14(5):527-32 13. Dubbelman M, Sicam VADP, Van der Heijde GL (2006) The shape of the anterior and posterior surface of the aging human corne.a Vision Research 46:993-1001 14. Amano S, Honda N, Amano Y, Yamagami S, Miyai T, Samejima T, Ogata M, and Miyata K (2006) Comparison of central corneal thickness measurements by rotating Scheimpflug camera, ultrasonic pachymetry, and scanning-slit corneal topography. Ophthalmology. 113(6):937-41 15. Jonsson M, Markstro K and Behndig (2006) A Slit-scan tomography evaluation of the anterior chamber and corneal configurations at different ages. Acta Ophthalmol. Scand. 84: 116-120 16. Rufer F, Schroder A, Bader C, Erb C (2007) Age-related changes in central and peripheral corneal thickness: determination of normal values with the Orbscan II topography system. Cornea. 26(1):1-5 17. Foster PJ, Baasanhu J, Alsbirk PH, Munkhbayar D, Uranchimeg D, Johnson GJ (1998) Central corneal thickness and intraocular pressure in a Mongolian population. Ophthalmology. 105(6):969-73 18. Price FW Jr, Koller DL, Price MO (1999) Central corneal pachymetry in patients undergoing laser in situ keratomileusis. Ophthalmology. 106(11):2216-20 19. Hahn S, Azen S, Ying-Lai M, Varma R (2003) Los Angeles Latino Eye Study Group. Central corneal thickness in Latinos. Invest Ophthalmol Vis Sci. 44(4):1508-12 20. Pfeiffer N, Torri V, Miglior S, Zeyen T, Adamsons I, Cunha-Vaz J (2007) European Glaucoma Prevention Study Group: Central corneal thickness in the European Glaucoma Prevention Study. Ophthalmology. 114(3):454-9 21. Aghaian E, Choe JE, Lin S, Stamper RL (2004) Central Corneal Thickness of Caucasians, Chinese, Hispanics, Filipinos, African Americans, and Japanese in a Glaucoma Clinic. Ophthalmology 111:2211-2219 22. Sanchis-Gimeno JA, Lleo-Perez A, Alonso L, Rahhal MS (2004) Caucasian emmetropic aged subjects have reduced corneal thickness values. Int Ophthalmol. 25(4):243-6 23. Brown N (1974) The changes in lens curvature with age. Exp Eye Res 19:175-183 24. Dubbelman M, Van der Heijde GL (2001) The shape of the aging human lens: curvature, equivalent refractive index and the lens paradox. Vis Res 41:1867-1877 25. Smith G, Atchison DA, Pierscionek BK (1992) Modeling the power of the aging human eye. J Opt Soc Am A 9: 2111-2117 26. Guirao A, Redondo M, Artal P (2000) Optical aberrations of the human cornea as a function of age. J Opt Soc Am A Opt Image Sci Vis 17:1697-1702 27. de Kruijf EJ, Boot JP, Laterveer L, van Best JA, Ramselaar JA, Oosterhuis JA (1987) A simple method for determination of corneal epithelial permeability in humans. Curr Eye Res. 6(11):1327-34 28. Chang SW, Hu FR (1993) Changes in corneal autofluorescence and corneal epithelial barrier function with aging. Cornea 12:493-499 29. Nzekwe EU, Maurice DM (1994) The effect of age on the penetration of fluorescein into the human eye. J Ocular Pharm. 10:521-523 30. Trinkaus-Randall V, Tong M, Thomas P, Cornell-Bell A (1993) Confocal imaging of the alpha 6 and beta 4 integrin subunits in the human cornea with aging. Invest Ophthalmol Vis Sci 34:3103-3109 31. Hazlett LD, Kreindler FB, Berk RS, Barrett R (1990) Aging alters the phagocytic capability of inflammatory cells induced into cornea. Curr. Eye Res. 9:129-138 32. Taylor HR and Kimsey RA (1981) Corneal epithelial basement membrane changes in diabetes. Invest Ophthalmol Vis Sci. 20:548

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33. Alvarado J, Murphy C, Juster R (1983) Age-related changes in the basement membrane of the human corneal epithelium. Invest Ophthalmol Vis Sci. 24(8):1015-28 34. Oliveira-Soto LM, Efron N (2001) Morphology of cornea nerves using confocal microscopy. Cornea 20:374-384 35. Grupcheva CN, Wong T, Riley AF, et al. (2002) Assessing the sub-basal nerve plexus of the living healthy human cornea by in vivo confocal microscopy. Clin Exp Ophthalmol. 30:187-190 36. Erie JC, McLaren JW, Hodge DO, Bourne WM (2005) The effect of age on the corneal subbasal nerve plexus. Cornea. 24(6):705-9 37. Boberg-Ans J (1955) Experience in clinical examination of corneal sensitivity. Br J Ophthalmol 39:705-726 38. Lawrenson JG, Ruskell GL (1993) Investigation of limbal touch sensitivity using a CochetBonnet aesthesiometer. Br J Ophthalmol. 77:339-343 39. Millodot M (1977) the influence of age on the sensitivity of the cornea. Invest Ophthalmol Vis Sci. 16:240-242 40. Roszkowska AM, Colosi P, Ferreri FM, Galasso S (2004) Age-related modifications of corneal sensitivity. Ophthalmologica. 218(5):350-5 41. Daxer A, Misof K, Grabner B, Ettl A, Fratzl P (1998) Collagen fibrils in the human corneal stroma: structure and aging. Invest Ophthalmol Vis Sci. 39(3):644.8 42. Malik NS, Moss SJ, Ahmed N, et al. (1992) Ageing of the human corneal stroma: structural and biochemical changes. Biochim Biophys Acta 1138:222-228 43. Kanai A, Kaufman HE (1973) Electron microscopic studies of corneal stroma: aging changes of collagen fibers. Ann Ophthalmol. 5(3):285-292 44. Elsheikh A, Wang D, Brown M, Rama P, Campanelli M, Pye D (2007) Assessment of corneal biomechanical properties and their variation with age. Curr Eye Res. 32(1):11-9 45. Kotecha A, Elsheikh A, Roberts CR, Zhu H, Garway-Heath DF (2006) Corneal thickness- and age-related biomechanical properties of the cornea measured with the ocular response analyzer. Invest Ophthalmol Vis Sci. 47(12):5337-47 46. Erie JC, Patel SV, McLaren JW, Maguire LJ, Ramirez M, Bourne WM, (1999) Keratocyte density in vivo after photorefractive keratectomy in humans. Trans. Am. Ophthalmol. Soc. 97:221-236 47. Patel S, McLaren J, Hodge D, Bourne W (2001) Normal human keratocyte density and corneal thickness measurement by using confocal microscopy in vivo. Invest Ophthalmol Vis Sci. 42(2):333-9 48. Moller-Pedersen T (1997) A comparative study of human corneal keratocyte and endothelial cell density during aging. Cornea. 16(3):333-8. 49. Møller-Pedersen T, Ehlers N (1995) A three-dimensional study of the human corneal keratocyte density. Curr Eye Res 14:459-464 50. Berlau J, Becker HH, Stave J, Oriwol C, Guthoff RF (2002) Depth and age-dependent distribution of keratocytes in healthy human corneas: a study using scanning-slit confocal microscopy in vivo. J Cataract Refract Surg. 28(4):611-6 51. Johnson DH, Bourne WM, Campbell RJ, (1982) The ultrastructure of Descemet’s membrane. I. Changes with age in normal corneas. Arch. Ophthalmol. 100:1942-1947 52. Murphy C, Alvarado J, Juster R (1984) Prenatal and postnatal growth of the human Descemet’s membrane. Invest. Ophthalmol. Vis. Sci. 25:1402-1415 53. Laing RA, Sanstrom MM, Berrospi AR, et al. (1976) Changes in the corneal endothelium as a function of age. Exp Eye Res. 22:587-594 54. Yee RW, Matsuda M, Schultz RO, Edelhauser HF (1985) Changes in the normal corneal endothelial cellular pattern as a function of age. Curr Eye Res. 4(6):671-8 55. Hashemian MN, Moghimi S, Fard MA, Fallah MR, Mansouri MR (2006) Corneal endothelial cell density and morphology in normal Iranian eyes. BMC Ophthalmol. 6;6:9 56. Yunliang S, Yuqiang H, Ying-Peng L, Ming-Zhi Z, Lam DS, Rao SK (2007) Corneal endothelial cell density and morphology in healthy Chinese eyes. Cornea. 26(2):130-2

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57. Bourne WM, Kaufman HE (1976) Specular microscopy of human corneal endothelium in vivo. Am J Ophthalmol. 81(3):319-23 58. Hollingsworth J, Perez-Gomez I, Mutalib HA, Efron N (2001) A population study of the normal cornea using an in vivo, slit-scanning confocal microscope. Optom Vis Sci. 78(10):706-11 59. Murphy C, Alvarado J, Juster R, Maglio M (1984) Prenatal and postnatal cellularity of the human corneal endothelium: a quantitative histologic study. Invest Ophthalmol Vis Sci 25:312-22 60. Blatt HL, Rao GN, Aquavella JV (1979) Endothelial cell density in relation to morphology. Invest Ophthalmol Vis Sci. 18(8):856-9 61. Roszkowska AM, Colosi P, D’Angelo P, Ferreri G (2004) Age-related modifications of the corneal endothelium in adults. Int Ophthalmol. 25(3):163-6 62. Green K (1995) Free radicals and ageing of anterior segment tissues of the eye: a hypothesis. Ophthalmic Res 27 (Suppl):143-9 63. Cejkova J, Vejrazka M, Platenik J, Stipek S (2004) Age-related changes in superoxide dismutase, glutathione peroxidase, catalase and xanthine oxidoreductase/xanthine oxidase activities in the rabbit cornea. Exp Gerontol. 39(10):1537-43

Chapter 5

The Aging of the Human Lens Jorge L. Aliò, MD, PhD, Alfonso Anania, MD, PhD, and Paolo Sagnelli, MD

Abstract Age-related lens changes include: a) the progressive increase in lens mass with age, b) changes in the point of insertion of the lens zonules, and c) a shortening of the radius of curvature of the anterior surface of the lens. With age, there is also decreased light transmission by the lens associated with increased light scatter, increased spectral absorption—particularly at the blue end of the spectrum—and increased lens fluorescence. Besides these physiological modifications, we must take into consideration the additional effects caused by exposure to external physical and chemical agents such as ultraviolet rays and drugs, which lead to considerable densitometric changes and consequently to modifications in optical lens quality. At present, new instruments allow the analysis, in clinical practice, of qualitative and quantitative alterations of the lens that occur with aging, confirming objectively the degradation of the optical quality of the crystalline lens. Keywords crystalline, Lens, age related changes, human eye, cataract.

Introduction The crystalline lens of the eye is a principal component in the process of vision. To perform its role, the lens must be transparent and also have the capacity to rapidly alter its shape as it transitions between focusing on near and distant objects. Gross (light and scanning confocal microscopy) and ultrastructural (scanning, transmission, and freeze-etch electron microscopy) analysis of all vertebrate lenses reveals that lenses are composed of exceedingly long fiber-like cells that are of uniform crosssectional shape (hexagonal) and size. These microscopic techniques also show that, in general, as these fibers are formed throughout life, they are overlain, in register, as age-related concentric growth shells. Thus, it has been proposed that the highly ordered arrangement of lens fibers contributes to lens transparency by transforming the individual fibers into a series of coaxial refractive surfaces. Water and protein loss, modifications to membrane lipids, and protein modifications can result in the progressive increase in compaction folds. It follows that substantial senescent alterations in the structure of the embryonic and fetal nuclear fibers From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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would lead to degradation of lens optical quality, especially since these fibers are located entirely within the region defined by the pupillary margin. While significant compaction of nuclear fibers occurs along the antero-posterior axis with aging, an even greater degree of compaction occurs in nuclear cataract formation. Therefore, there is convincing evidence that a senile cataract is an exaggerated final stage of age-related lens changes. Clinical observations of aged lenses show increased light scatter even without overt visual impairment, and it has been demostrated that there is a degradation of the optical quality of the crystalline lens with aging that is associated with morphological changes such as thickness and density. The process of nuclear fiber compaction is probably multifactoral, as the lens is exposed to the cumulative effects of radiation, oxidation, and post-translational protein modifications. Additional changes include: a) the progressive increase in lens mass with age, b) changes in the point of insertion of the lens zonules, and c) a shortening of the radius of curvature of the anterior surface of the lens. With age, there is also decreased light transmission by the lens associated with increased light scatter, increased spectral absorption—particularly at the blue end of the spectrum—and increased lens fluorescence. Besides these physiological modifications, we must take into consideration the additional effects caused by exposure to external physical and chemical agents such as ultraviolet rays and drugs, which lead to considerable densitometric changes and consequently to modifications in optical lens quality. At present, new instruments allow the analysis, in clinical practice, of qualitative and quantitative alterations of the lens that occur with aging, confirming objectively the degradation of the optical quality of the cristalline lens.

Lens Embryology An in-depth study of lens embryology facilitates the understanding of fibers and suture development. 1-4 Lens formation is the result of a series of inductive processes.5,6 The lens placode appears on the optic vesicle that protrudes from the forebrain, around the 25th day of gestation.7 It is a thickening of the surface ectoderm8—a single layer of cuboidal cells—that invaginate into the neural ectoderm of the optic vesicle as the lens pit, becoming free from the surface by the 33rd day9 (see Fig.5.1). The cells at the anterior pole of the lens vesicle remain as epithelial cells—the cell number is controlled by apoptosis.10 The posterior cells elongate as primary lens fibers that obliterate the lumen of the lens vesicle11—the retina largely determines this cytodifferentiation. The tiny developing lens is surrounded by a basement membrane that will become the lens capsule and is filled with nearly structureless primary lens fibers.12 These cells expel their nuclei, mitochondria, Golgi bodies, and endoplasmic reticulum. This structure becomes a spherical, optically clear embryonic nucleus of 0.35 mm in diameter,11 which stays unchanged throughout life7 and is seen inside the Y sutures in the fully developed eye. In an embryo of

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Fig. 5.1 Scheme to show the development of the lens. (A) Lens thickening. (B) Lens pit. (C) Lens pit closing. (D) Lens vesicle. E) Elongation of cells of the posterior wall of the lens vesicle. (F) Obliteration of the cavity of the lens vesicle by cells of the posterior wall. (G) Formation of lens sutures by the meeting of fibers developed in the equatorial region (Mann I: The Development of the Human Eye. Grune Stratton, New York, 1950)

23 mm, equatorial secondary lens fibers derived from the anterior epithelium migrate forward under the anterior epithelium and backward directly beneath the capsule—meeting at the sutures that can be seen easily with slit-lamp microscopy as an upright anterior Y and an upside-down7 posterior Y. The limbs of the Ys are often branched. A large number of recent studies have focused on the involvement of polypeptide growth factors and cytokines in lens differentiation. These factors include fibroblast growth factors (FGFs), insulin and insulin-like growth factors (IGFs), transforming growth factors (TGFs),13 platelet-derived growth factors (PDGFs), epidermal growth factors (EGFs) and several cytokines, including macrophage-migration inhibitory factor (MIF), and tumour necrosis factor-alpha (TNFα).14 After birth, the equatorial fibers grow to form the cortex, meeting at more complex and less well-marked sutures—this growth continues until very shortly after death. The tertiary vitreous condenses within the space between the ciliary body and the lens equator, forming the suspensory ligament of the lens at the fifth month of gestation.7 The developing lens requires nutrition that is obtained through the tunica vasculosa lentis, which is a vascular network supplied posteriorly by the hyaloid artery (a branch of the primary dorsal ophthalmic artery) and anteriorly from an anastomosis with vessels in the pupillary membrane.7 The tunica vasculosa lentis is first seen at about 35 days, and is most prominent at 65 days. It gradually regresses at about 85 days, and by term birth, only whispy remnants of the pupillary membrane are left, with a vestigial hyaloid artery (known as a Mittendorf’s dot) attached to the axial posterior surface of the lens.7,15

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Morphology of the Human Lens Roughly speaking, vertebrate lenses are asymmetrical, oblate spheroids of variable size and spheroidicity (see Fig. 5.2). The lens, encaved in an elastic capsule, consists of: a) an anterior monolayer of epithelial cells, the pre-equatorial members of which exhibit mitotic activity throughout life; b) a superficial layer of elongating, differentiating, and maturing secondary fibers, and c) the main lens body that consists of fully matured primary (embryonal nucleus) and secondary fibers16 (see Fig. 5.3). These fibres are characterized by a high protein content (35-40%), by the absence of nuclei, mitochondria, lysosomes, ribosomes and endoplasmatic reticulum, and are surrounded by increasingly less permeable membranes. All lens fibers are mutually anchored, securing minimal extracellular space, thus minimizing differences in the refractive index from fiber to fiber. Many electron microscopic studies have been performed: initial investigations were undertaken by Wanko and Gavin17 and Cohen18 on mammals and humans, respectively. Many more followed describing fibers from a variety of mammals, including rat,19,20 rabbit,21 pig,22 monkey,23-26 and human.24,27-32 Recent technical advances in fixation methods for scanning electron microscopy (SEM)25 and transmission electron microscopy (TEM)33 have made it possible to analyze differentiated fibers in all regions of the lens. The cross-sectional area of fiber cells varied from region to region, with the smallest areas found in the compressed adult nuclear region and the largest found in the central embryonic

Fig. 5.2 Diagrammatic section of the eye

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capsule epithelium cortex adult nucleus juvenile nucleus

fetal nucleus

embryonic nucleus

Fig. 5.3 Diagram of an aged normal human lens, approximately to scale. The complex suture pattern is not shown. The epithelium and capsule are enlarged for clarity. (Modified from: Morphology of the Normal Human Lens. VL Taylor, KJ Al-Ghoul, CW Lane, VA Davis, JR Kuszak, and MJ Costello Investigative Ophthalmology & Visual Science, June 1996, Vol. 37, No. 7)

nucleus. Cellular organization was most ordered in the cortex, where radial cell columns were found. By contrast, cells were more irregularly packed in the center of the lens, where no apparent arrangement was observed. The cytoplasm of intact cells was smooth and homogeneous in all regions analyzed. The hardened nuclear core corresponds to the fetal and embryonic nuclei, the outer soft layer corresponds to the cortex, and the layer between, with intermediate hardness, corresponds to the adult and juvenile nuclei. The term epinucleus has been used to describe this intermediate layer of tissue corresponding to the adult plus juvenile nuclei.34 In the adult nucleus, the deep cortical fibers are compacted as they are internalized and become part of the adult fiber mass. Changes in shape occurred in the absence of significant membrane loss or turnover, which resulted in an increase of membranous undulations. The small variation in size of the adult nuclear fibers implies that cells seem to be affected equally by this compaction. Both mechanical compression (caused by the continual deposition of new fibers) and dehydration (regulated by the osmotic properties of the crystallins) are involved in this process of compaction.35-37 Cells in the juvenile nucleus were, on average, twice the size of adult cells and approximately half the size of deep cortical cells. This suggests that juvenile cells are also compacted, but not to the degree that the adult cells are. Different developmental events or protein modifications may occur during the formation of juvenile cells, in contrast to adult cells, because they are formed before puberty. The crystallins of the juvenile cells may resist compaction because initially they may be more dehydrated than those of the adult cells.16 Alternatively, some cells may grow in a larger form, or cells may fuse together during elongation.38,39 Both these events would produce cells with larger areas. Radial cell columns could be detected in adult and juvenile regions but were difficult to visualize because of the intricate folding of the membranes. Together,

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the adult and juvenile nuclei comprise an annular ring that undergoes significant compaction with age and, importantly, is flanked by regions that are compacted relatively less with age. Biochemically, a correlation was observed between the protein modifications of lens crystallins and the lens regions.40 At the molecular level, packing of lens crystallins has been shown in the last decade to be random, with no evidence of crystalline regularity. Based on x-ray diffusion measurements in vitro, monomers (or small aggregates of crystallins) are thought to be associated closely. Therefore, at a critical distance, the light scattering of the concentrated protein solution decreases significantly, leading to transparency.41 These long-term changes in radial cell thickness place constraints on cell shape, size, and packing. Possible cell-to-cell fusion also influences packing, especially near the poles and at the sutures. The simplest explanation for the observed changes in this annular ring is that the cytoskeleton is lost in the last stages of cortical differentiation and that the nuclear fibers are squeezed under pressure against a harder nuclear core (fetal and embryonic nuclei), resulting in gradual dehydration of crystallins and cellular compaction. Alternatively, self-association of the crystallins and the resultant decrease in osmotic pressure may induce dehydration of the cytoplasm.37

Lens Capsule Anatomy of the Lens Capsule The position of the lens in the optical system of the eye is assured by the attachment of the zonular fibers to the lens capsule, as well as the support provided by the vitreous and iris. The lens capsule and the zonular fibers constitute the link between the lens fiber substance and the ciliary muscle, and thus play an important role in transmitting the force of ciliary muscle contraction to change the shape of the lens fiber substance that is essential for accommodation. The geometrical pattern of the zonules is complex and varies significantly with age. The zonular fibers attach to the lens in three separate groups: an anterior, an equatorial and a posterior group.42 The attachment of the zonules to the lens is known to involve penetration of zonular fibers into the superficial lens capsule.43,44 The anterior zonules attach to the outer surface of the lens capsule around the lens periphery in a rather broad zone, which increases with age from about 0.25–1.2 mm due to a relative inward displacement of the zonular insertion in the lens capsule.45 A zonular lamella has been described by many observers as a thin membrane surrounding the lens capsule.46,47 Its existence, however, has been a matter of dispute and, according to Hogan,43 the zonular lamella exists only in the equatorial region where the zonular fibers attach to the capsule. In an electron microscope study, Seland44 confirmed the presence of a fibrillar surface layer in newborns, but showed that this fibrillar surface layer retracted from the anterior pole between the ages of 6 and 17. On the posterior capsule, a fibrillar surface layer does not seem to be a

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constant feature, apart from the posterior zonular attachment.43,44 The lens capsule encloses the lens fibers. The inner surface of the anterior lens capsule is in immediate contact with the lens epithelium, while the posterior lens capsule is in contact with the most superficial part of the posterior lens fibers.

Ultrastructure of the Lens Capsule The lens capsule is the thickest basement membrane in the body. When studied under light microscopy the lens capsule appears dense and homogeneous. Under the electron microscope, the lens capsule is found to be made up of parallel lamellae, more tightly packed toward the outer surface.48 The lamellar structure of the lens capsule seems to disappear with age. In the posterior capsule, it disappears in childhood. In the anterior capsule it starts disappearing from the anterior pole in adulthood but persists in the equatorial and preequatorial regions corresponding to the metabolically most active part of the lens epithelium.43,44,49 Ultrastructurally, the support of the lens capsule is type IV collagen, which interacts with other glycoproteins and proteoglycans to form an extracellular matrix.50-53 Type IV collagen is found only in basement membranes, and it is the only collagen that has been shown definitively to be present in basement membranes. Immunoelectron microscope studies of the lens capsule, however, also seem to show the presence of collagen types I and III.54,55 Type IV collagen plays an important role in the formation of a resilient, three-dimensional molecular network.56,57 Compared to the fiber-forming collagens, the type IV collagen molecule is longer, more flexible, and contains frequent interruptions by non-collagenous sequences. The type IV collagen molecule possesses distinct end-region domains and exhibits several binding interactions that enable formation of a stable lattice-like network.

Growth and Thickness of the Lens Capsule The lens capsule continues to grow throughout most of life, growing in thickness anteriorly and increasing in surface area to adjust to the increasing volume of the lens. The anterior lens capsule is produced by the lens epithelium58-60 and therefore reflects the activity of the epithelial cells, which undergo apparent morphological changes with aging. The epithelial cells become flattened—the number of organelles is reduced and become more difficult to distinguish because of an increasing density of the cytoplasmatic matrix.49,61 The regional variation in thickness of the lens capsule changes markedly with age, which suggests a continuous modeling of the lens capsule with age. In contrast to the anterior lens capsule, which is synthesized by the lens epithelium and continues to grow and increase in thickness throughout most of life,44,62,63 the human posterior lens capsule loses its epithelial cells in fetal life.43 It has been suggested that the posterior lens capsule is synthesized and secreted by nucleated cortical lens fibers, or synthesized by anterior epithelial cells and

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secreted into the posterior aspects of the lens during the first part of life, after which the production of the posterior lens capsule is supposed to cease. Capsular thickness is not uniform but varies according to age and the location at which the measurements are taken.44,62-64 Fisher and Pettet62 examined unfixed lens capsules and found that thickness of the neonatal lens capsules varied from 3–5 µm at the posterior pole to approximately 11 µm at the equator. Thickness of the anterior pole and thickness beneath the insertion of the anterior zonules were found to increase with age, whereas thickness of the posterior lens capsule was found to be constant. In pre-presbyopic eyes, the lens capsule was found to be thickest at the equator, whereas in old adults, the lens capsule was found to be thickest beneath the insertion of the anterior zonular fibres. Seland44 examined fixed lens capsules and found that the neonatal lens capsule had a uniform thickness of about 4 µm and that thickness of the anterior as well as the posterior lens capsule increased gradually with age— most markedly in the peripheral region of the anterior lens capsule where thickness reached as much as 30 µm. In all age groups, the thinnest part of the lens capsule was found at the posterior pole. The thickest part of the lens capsule in pre-presbyopic lens capsules was found to be in the mid-periphery of the anterior and posterior lens capsule. Thickness of the anterior peripheral zone was found to increase with age throughout the lifespan, whereas thickness of the posterior peripheral thickened zone was found to increase only in pre-presbyopic lens capsules and to decrease in the older age group.

Mechanical Properties of the Posterior Lens Capsule The anterior and posterior lens capsules differ in several aspects. The lamellar structure of the lens capsule disappears earlier with age in the posterior lens capsule than in the anterior lens capsule,44 and differences have been described in the relative proportion of macromolecular components such as heparansulfate, proteoglycans, and fibronectin.65,66 This could indicate that the mechanical qualities of the anterior and posterior lens capsule are different. Mechanical strength of the posterior lens capsule (ultimate strain, ultimate stress, ultimate elastic modulus) was found to decrease markedly with age in a range similar to that of the anterior lens capsule.48 The age-related loss of mechanical strength, however, seemed to begin earlier in the posterior lens capsule than in the anterior lens capsule. Ultimate load, which reflects the breaking strength of the lens capsule in situ, was significantly lower for the posterior lens capsule than for the anterior lens capsule.48 This is in accordance with the fact that the posterior lens capsule is much thinner than the anterior lens capsule. When looking at data pertaining to the accommodative function range (low strains), the mechanical quality of the posterior lens capsule was found to be similar to that of the anterior lens capsule in all age groups. This indicates that the mechanical properties of the lens capsule in situ vary proportionally with the regional variation in capsular thickness. The age-related loss of mechanical strength, however, seems to begin earlier in the posterior lens capsule than in the anterior lens capsule.

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Aging of the Lens Capsule Studies of the human lens capsule indicate concurrently that aging of the human lens capsule is associated with a progressive loss of mechanical strength, which seems to parallel morphological changes in the lens capsule. Formed elements (inclusions) accumulate in the anterior lens capsule with age.44,67 The laminated structure of the lens capsule disappears with age,44,49 and the optical density of the lens capsule increases with age.68,69 Peczon et al.70 investigated age-related changes in the amino acid composition of the lens capsule and found a relative increase of noncollagenous amino acids and a decrease of collagenous amino acids (hydroxyproline) with age. Because collagen seems to be responsible for the mechanical strength of other soft connective tissues,71 the age-related changes in the amino acid composition of the lens capsule also may have significance in the loss of mechanical strength. The major structural component of the lens capsule is basement membrane type IV collagen, which is organized into a three-dimensional molecular network.72 As discussed previously, the mechanical properties of the lens capsule correlate well with a network structure. The lens capsule is easily deformed at low deformations due to reorientation and alignment of the molecular network structure in the direction of deformation. As the elastic stiffness of the lens capsule at low deformations increases with age in pre-presbyopic eyes, and the extensibility correspondingly, decreases with age, this suggests geometrical changes in the molecular network structure with age. One factor may be the increasing volume of the lens with age, which may cause stretching of the collagen network structure, thus limiting further deformation. Another factor may be an increased crosslinking of the molecular network structure with age, which also may limit deformation.48 The collagen molecules in the lens capsule seem to be extremely long-lived. This provides great opportunity for posttranslational modifications of the molecules, such as nonenzymatical glycosylation,73,74 which can change the mechanical properties of the lens capsule through the formation of stable crosslinks.75-77

The Ocular Lens Epithelium Ultrastructure of the Lens Epithelium A single layer of cells—the lens epithelium—covers the anterior face of the lens that faces the cornea. The lens epithelium ends on the rims of the anterior surface. It contains cells in the central region that do not divide and are essentially quiescent, surrounded by a germinative-dividing zone of cells and followed (at the equatorial fringe) by the dividing cells that differentiate into fiber cells (see Fig. 5.4). A remarkable feature of this epithelium is its capacity to divide and differentiate almost all through the life of an individual. This feature of sustained growth is very

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Fig. 5.4 Simplified drawing of the ocular lens. Note that the epithelium has three regions of cells in nondividing phase (central epithelium), in dividing phase (germinative), and differentiating phase (equatorial). The anterior and posterior sutures are formed by the meeting of the elongating fiber cells that make the bulk of the lens mass. The surrounding capsule (shaded area around the lens) indicates that the basal surface of the epithelium and the fiber cells is on the outside, while the apical surface faces the inside of the lens. apical interface—an area of contact between the apical surfaces of the epithelial cells and the fiber cells is not indicated. It is the area just below the epithelial layer

much similar to its closest embryological sibling—the cells in the skin. The cells in the lens epithelium represent typical epithelial morphology: they are cuboidal, presenting a cobble-stone-like appearance in their native state and in vitro, if cultured without excessive passaging. The diameter of human lens epithelial cells ranges from 9–17 µmm.78 The cell size has been reported to increase with age,79,80 which suggests a change in the cell density. Females have been reported to have higher cell density in the human lens epithelium than males.81,82 Francois and Rabaey83 observed lens epithelium under a phase-contrast microscope. They reported the presence of pale polyhedral and dark, star-shaped cell types. A recent in vivo study84 using the noncontact specular microscopy recognized four morphological features of the live human lens epithelium. These were categorized as linear furrows, columnar organization, puffy clouds, and black holes. The relationship between cell density and age is interesting, although controversial. An earlier report82 that there is an age-related decrease in the cell density in the lens

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epithelium has been recently confirmed.84 This recent study calculated the loss of 675 cell/mm2 in a 75-year life span (that amounts to a loss of 14% of the cells). This estimate is based on the unproven assumption that the rate of loss is linear with age, however it is not very different from that reported in the aging monkey lens central epithelium.85 Others have found no such relationship.86,87 Karim et al.87 reported a decrease in the mitotic index of lens epithelial cells under normal, as well as cataractous, conditions. Harocopos et al.88 concluded that there was no relationship between cell density and the severity of cataracts, or between cell density and age. They did report, however, that the epithelium directly over the opaque area in cataractous lenses had higher cell density when compared to that overlaying the transparent regions. It is possible that a loss of a patch of cells overlying a cataractous fiber cell area may lead to the activation of cell division, and therefore higher cell density.

The Epithelium as the Major Site of Transport, Metabolism and Detoxification The overall metabolic status of the fiber cells in the absence of endoplasmic reticulum, mitochondria, and a nucleus, is comparatively very low.89 There is no vascular system as we know it that would take nutrients to the fiber cells and remove metabolic/physiologic waste to replenish the intra- and intercellular milieu of the lens. Mere diffusion as a process to sustain the slow but substantial physiology of the ocular lens will be insufficient to accomplish this efficiently. A study of relative rates of transport across the anterior and posterior surfaces of the lens has led to the model of the pump-leak system.90-92 Lens epithelium is also a major site of detoxification and defense against oxidative insults93,94 and is able to detoxify physiological concentrations of H2O2 enzymatically involving glutathione reductase, glutathione peroxidase, and the hexose monophosphate shunt.94

Programmed Cell Death and the Lens Epithelium The interest in the study of programmed cell death in the lens epithelium was generated recently by investigators probing the role of epithelium in cataractogenesis.95 These studies are based on the hypothesis that the integrity of the lens epithelium is essential for the normal functioning of the lens, and that a decrease in the cell number of the epithelium may lead to changes in homoeostasis that may in turn lead to cataractogenesis. The role that apoptosis plays in tissue development and morphogenesis is well established.96 Apoptosis or cell death has been morphologically documented in the very early stages of the lens vesicle formation during development of the eye.97 A role for apoptosis in regulating the size of the lens by controlling the number of cells that reach terminal differentiation into fiber cells remains a possibility.

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Different metabolites98 and drugs99 have been reported to initiate apoptosis in lens epithelial cells in culture.100 The research of Zigman,101 Li and Spector,95 Michael et al.102 and Shui et al.103 suggest that apoptosis may occur discretely in the lens epithelium and only in isolated cells that become susceptible. Such a thesis connotes two interesting corollaries: ●

●

Any isolated apoptotic cell may be quickly eliminated by the surrounding cells as a protective response against the spread of cell death. Importantly, therefore, it points to the existence of a potent mechanism that strictly controls and inhibits cell death from spreading to neighboring cells. The presence or absence of apoptosis in the lens epithelium can be interpreted optimistically as a process that eliminates the dys functional cells to keep the rest of the epithelium healthy. A pathological state may precipitate when this ability to remove dysfunctional cells is compromised—for example, by aging or by exposure to harmful metabolites or environmental insults.

Lens Fiber Cells Fiber Cell Organization and Development of Lens Sutures The cells of the lens vesicle that were not induced to form primary fibers remain as a monolayer—the lens epithelium—that covers the anterior surface of the primary fiber mass. From this point on, further lens development and growth occurs throughout life in a manner similar to other stratified epithelia. The lens epithelium constitutes the basal layer—however, whereas typically stratified epithelia have their progenitor cells distributed throughout the basal layer, the lens is unique in that its progenitor cells are sequestered as a distinct subpopulation within the lens epithelium known as the germinative zone, which comprises a narrow, peripheral, latitudinal band of the lens epithelium located just above the equator.104 These cells undergo mitotic division, and selected daughter cells are induced to terminally differentiate and form secondary fibers. As with primary fiber formation, the most apparent structural consequence of secondary fiber formation is the transformation of a cuboidal cell into a long fiber. While forming, however, primary fibers are fixed in position as they elongate essentially unidirectionally. Secondary fiber formation requires the forming fibers to rotate about their polar axis while simultaneously migrating posteriorly and elongating bidirectionally. Fiber rotation is complete when the long axis of a forming fiber is aligned parallel to the antero-posterior axis of the lens, and when the center of a forming fiber reaches the mid-point between the poles, which—by virtue of the fact that all vertebrate lenses are asymmetric, oblate spheroids—is posterior to the equator. As secondary fibers elongate, their anterior ends are insinuated between the lens epithelium and the primary fiber mass, while at the same time their posterior ends are insinuated between the primary fiber mass and the posterior lens capsule.

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Maturation of secondary fibers is complete when they detach from the lens epithelium anteriorly, and the capsule posteriorly, to subsequently overlap with other newly mature fibers to form lens sutures.105

The Contribution of Major Fiber Proteins to Sutural Development and Growth In vertebrate lenses, MP26, gap junction (GJ) connexins, and MP19 are the major fiber intrinsic membrane proteins. The major intrinsic protein (MIP), or aquaporin0 (AQP0), is generally described as constituting more than 60 percent of the total fiber membrane protein.106,107 The connexins (Cxs)108,109 that form the lens GJ communicating channels, display a variety of expression patterns, channel regulation, and posttranslational modifications during differentiation and aging of the lens cells.110,111 Lens epithelial GJs consist mainly of 1 connexin (Cx.1 or Cx43).112 The Connexin50 is essential for normal postnatal lens cell proliferation.113,114 Cortical fiber GJs consist of Cx.3 (or Cx46) and Cx.8 (or Cx50)—often coexisting in the same junctional plaques.115,116 MP19 (also referred to as MP17, MP18 and MP20 in the literature) has also been described as the most abundant intrinsic membrane protein of lens fiber cells.106 However, unlike MIP or the connexins, MP 19 bears no striking resemblance to any other reported protein family and, to date, has no defined structural or functional role. Both MIP and MP 19 co-localize with GJs in distinct regions of the lens.117,118 Thus, it has been proposed that both MIP and MP 19 play some role in GJ formation, maintenance, or organization. While it is well-documented and irrefutable that all vertebrate lenses contain the above described major fiber proteins, a review of the literature suggests that their density and distribution varies is species-specific—varying along fiber length and as a function of fiber depth and therefore of age. GJs primarily conjoin the midsegment of fibers, or those segments of fibers not involved in sutures.119,120 The function of these proteins is probably coordinated during fiber development.

Lens Sutural Anatomy Numerous studies have established that the vast majority of fibers are hexagonal in cross section,121-126 with two broad faces oriented parallel to the lens surface and four narrow faces oriented at acute angles to the lens surface. During differentiation and maturation, the lens fiber membranes undergo typical changes. The lateral and apical surfaces of the hexagonal fibres change from smooth and studded with small ball-and-socket junctions in the superficial cortex, to covered with groovesand-ridges in deeper cortical regions and the nucleus. Freeze fracture studies127 revealed that these surface changes are paralleled by changes in the internal organization of the fiber membranes. Epithelial and superficial fiber membranes are studded with a multitude of intramembrane particles (IMPs) and gap junctions (GJs). The IMPs represent intrinsic membrane proteins

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Fig. 5.5 Formation of anterior Y and offset posterior Y sutures. In this view of the anterior and posterior surface, a number of curved fibers have been highlighted. The anterior ends of these fibers are paired (a-b-c-d-f) to form parts of anterior suture branches. By following these fibers along their length, it can be seen that as a result of opposite end curvature, the posterior ends of these fibers are paired with different fibers to form offset posterior suture branches (Modified from: Development of Lens Sutures. JR Kuszak, RK Zoltoski and CE Tiedemann Int. J. Dev. Biol. 48: 889–902, 2004)

that function as receptors, ion channels, transporters, and pores. GJs allow a direct cell-to-cell exchange of molecules up to 1500 Da. Biochemical studies128 showed that, upon maturation, the cholesterol-tophospholipid ratio of lens membranes dramatically changes from 0.6-0.8 in superficial to over 5.0 in deep cortical and nuclear membranes. All this indicates that lens membranes, apart from those in the most superficial cortex, deviate from most cell membranes in the body. This is in line with electrophysiological studies showing that deep cortical membranes are non-leaky, have a high resistance and low capacitance, and have no or restricted cell-to-cell communication.129,130 In the human lens, fibers are partial or incomplete meridians—that is to say, upon completion of elongation, the vast majority of fibers do not have ends that extend to the poles (see Fig. 5.5). During gestation primary fibers are neither uniform in shape nor size.122,123,126,131,132 As such, the primary fiber cell mass, or embryonic lens nucleus, does not consist of growth shells overlain in the register to form ordered radial cell columns. The initial secondary fibers are similarly nonuniform in shape and size, and also lack an ordered arrangement. Only as lens development proceeds are the additional secondary fibers formed progressively more uniform in shape. The establishment of growth shells comprised of uniform fibers overlain in register as radial cell columns occurs

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within 250–750 µM of the equatorial center of the lens. Coincidentally, this marks the beginning of suture formation.133

Suture Formation after Birth After birth, there are fundamental changes in the fiber differentiation program that result in the formation of progressively more complex iterations of star sutures during infancy, adolescence, and adulthood.

Simple Star Suture Formation Shortly after birth, a new (or secondary) anterior suture branch, and a pair of new (or secondary) posterior suture branches, begin to develop in relation to the extant suture branches within the infero-nasal quadrant. At the same time, the anterior ends of curved fibers that bracket the posterior suture branch are added to the extant primary anterior suture branches. Similarly, the posterior ends of the same curved fibers that bracket the anterior suture branch as a consequence of opposite end curvature, are added to the extant offset primary posterior suture branches. By the end of the infantile period, the anterior suture consists of three enlarged primary branches, and three new secondary suture branches—one completely formed, and two partially formed. The offset simple star posterior suture consists of three pairs of new secondary branches—a pair that are completely formed, two pairs that are only partially formed. All of the suture branches are arranged in a symmetrical, but nonidentical, simple star suture pattern.133 Star and Complex Star Suture Formation The essential parameters of star sutures formed during adolescence, and complex star sutures formed throughout adulthood, are demonstrated in Fig. 5.6.

Fig. 5.6 Key stage in the development of the complex star suture formed throughout adulthood. These sutures are progressively more complex through adolescence and adulthood

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Fig. 5.7 Intermediate-magnification SEM micrographic image of a portion of suture branches. The width and evenness of a suture branch is related to the degree of irregularity between fiber cell ends (Modified from: JR Kuszak, JG Sivak, JA Weerheim, Lens optical quality is a direct function of lens sutural architecture. Invest. Ophthalmol. Vis. Sci.S, vol. 32, 7:2119–2129, 1991)

These sutures are progressively more complex iterations (second and third rows, polar projections of anterior and posterior, respectively) of the simple star sutures formed through infancy. Throughout adolescence, the nine branched star sutures are formed as tertiary anterior suture branches, and tertiary pairs of posterior suture branches sequentially supplement the extant primary and secondary branches.134 The different suture patterns formed during gestation, infancy, adolescence, and adulthood are the anatomical basis of the zones of discontinuity revealed by slit-lamp biomicroscopy.134,135 Throughout life, anterior and posterior suture branch formation continues, and their distal ends extend to confluence at their respective poles. Numerous structural studies confirm that the uniformly shaped fibers are arranged in highly ordered growth shells—however, the ends of the fibers are very nonuniform in shape.133 Thus, their end-to-end arrangement to form suture branches produces naturally occurring regions of disorder aligned directly along the visual axis. In fact, by overlying suture branches in concentric growth shells, line and Y suture lenses produce disordered suture planes aligned directly along the visual axis (see Fig 5.7).

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The Physical Basis for Transparency of the Crystalline Lens To perform its role in the process of vision, the lens must be transparent. The analysis of transmission in terms of the physical interactions between light and the structures of the lens is fundamental to understanding the changes in optical quality with aging. The first author to study the physical concept of transparency in the lens was Trokel in 1962.136 The physical interactions between light and the known structures of the lens (cortical fibers surrounded by cell membranes and protein fraction that comprises most of their cytoplasm) and the manner in which these molecular and microscopic structures affect the traversing light wave determines the transmission characteristics of the lens. These characteristics depend upon the two processes of absorption and scattering. Absorption is the conversion of light from the incident beam to other forms of energy, such as heat or chemical energy. Scattering takes place when light passes over the elastically bound electrons in the atoms and molecules. The scattering interaction may be thought of as producing elastic vibrations that result in the emission of secondary light in all directions—thus, scattering also removes energy from the traversing beam. A distinction is made between light scattering by small and by large particles. Scattering by small particles occurs when the objects are smaller than the wave length of light, such as the soluble proteins of the lens. Large particles are larger than several wave lengths in size,137 and are the structures that can be resolved by the light microscope. Microscopic and submicroscopic structures cause the extinction of light, which determines the transmission characteristics of the lens. This extinction derives from the many processes summarized in Fig. 5.8 that show the absence of absorption and the major role of scattering in the extinction of visible light by the

Fig. 5.8 Summary of the processes that produce extinction of light in the crystalline lens. (Modified from: S Trokel The physical basis for transparency of the crystalline lens. Invest Ophthalmol 1:493, 1962)

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lens. The physics of light scattering must be examined to understand the extinction characteristics of the lens.

Small Particle Scattering Completely regular crystalline matter will change only the velocity of traversing light without removing energy by scattering. It can be concluded that the high degree of light transmission of the intact lens fibers results from the spatial order of lens proteins in their normal state. The spatial order of protein molecules can be described by p(r)—the probability that two protein molecules are a distance r apart. The reduction in scattering due to local order has been derived by Zernike and Prins138 in a general form: ⎡ 4pN ∞ sin ksr ⎤ (1 − r(r ))r 2 dr ⎥ ⎢1 − ∫ V ο ksr ⎣ ⎦ This formula expresses the reduction of scattering of N particles in a volume (V), where k = 2 π/l, and s = 2 sin q /2. The distribution function p(r) is normalized to unity when all r’s are equally probable. This is the dilute solution in which this factor reduces to one, and no external interference of scattered light occurs. Quantitative application of the Zernike-Prins factor to the lens proteins in the intact state is not now feasible because the exact dimensions and the spatial order of the proteins in the intact fiber are unknown. Qualitatively, the high concentration of the soluble proteins in the lens fiber must be accompanied by a degree of local order approaching a paracrystalline state. This results in the interference of scattered light and the transparency of the fibers.

Large Particle Scattering Although the nature of the physical interaction is the same, large particle scattering calls for mathematical treatment different from that of small particle scattering. Incident rays on an isotropic particle give rise to the phenomena of diffraction and reflection. The reflection is accompanied by refraction at the large particle surface. Diffraction and reflection can be considered special cases of scattering. The phase contrast photomicrograph of an unstained section emphasizes those structures that cause large particle scattering (Figs. 5.9). Thus, the lens transparency is made possible by a number of factors, including the regular arrangement of the lens fibers, the nonparticulate fiber cytoplasm, and the uniform distribution and paracrystalline state of proteins within the cells.136-140 Kuszak et al.141 have proposed that the arrangement of lens fibers depends strongly on the ability of newly formed cells to elongate in a pattern that meshes precisely

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Fig. 5.9 Comparison with a phase-contrast photomicrograph of a lens section made perpendicular to the fibers, and a diagram that represents the membrane pairs of the lens fiber walls as a single refractive discontinuity. r1 is a ray reflected back to the light source, and r2 is reflected at a 60-degree angle from the path of the transmitted beam. The cluster of short arrows represents scattering of light by the soluble proteins that comprise the fiber cytoplasm (Modified from: S Trokel S The physical basis for transparency of the crystalline lens. Invest Ophthalmol 1:493, 1962)

with the underlying cells. The lamellar conformation of lens proteins rather than helical structure may also contribute to transparency.142 In addition, it has been proposed that a short-range, liquid crystal-like order of the crystallins is important for transparency of lens cytoplasm.143 In addition to the state of lens crystallins, the tight packing of the lens cells and the regulation of ion and water balance also play significant roles in maintaining the transparency of the normal lens. Consequently, the development of protein aggregates, cell membrane degeneration, the appearance of vacuoles, and the distortion of lens structure can all produce light scatter and the clinical observation of cataracts.139

The Influence of Sutural Architecture on Lens Optical Quality The morphology of lens sutures should be considered when evaluating the optical quality of crystalline lenses.A quantitative analysis of optical quality in line and Y suture lenses confirms that suture planes significantly degrade lens function (sharpness of focus).144-146 Comparable studies in star suture lenses, however, show that the staggering of suture branches in concentric growth shells effectively minimizes the negative influence of suture planes on optical quality.147

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Spherical aberration is a monochromatic anomaly that can be defined as the difference in focal length for light rays that pass through different points of a lens. Generally, light rays that pass through the periphery in vertebrate crystalline lenses can have longer focal lengths than those that pass through the center. This situation is known as negative or over corrected spherical aberration, and it is partially the result of lens structure. The net result of the additional growth shells of lens fiber cells throughout life is an ever-increasing lens mass.144-147 Unlike other stratified epithelia, the strata of the lens (growth shells) are never sloughed off. Rather, they become more internalized as the lens grows. The plasma membrane, the cytoplasm with specialized crystalline proteins, and the extracellular space between the lens fiber cells of the growth shells have different refractive indices. Thus, as the lens grows, a gradient of refractive index is established from the center of the lens to the periphery on the basis of variation in protein content. This gradient of refractive index, possibly in combination with the asphericity of lens shape, neutralizes positive spherical aberration.144 Numerous studies have shown that lens fiber cells are uniform in shape and are overlaid in precise alignment to produce radial cell columns between growth shells.148-152 Thus, it has been proposed that the radial cell columns are a system of coaxial refractive surfaces that are partially responsible for lens transparency.153,154 In contrast, scanning electron microscopy studies147,152,155,156 show that the ends of lens fiber cells are variable in shape and are overlaid in imprecise alignment to produce irregular suture planes between growth shells. If the ordered alignment of uniform fiber cells into precise radial cell columns contributes to negative or corrected-spherical aberration, then the alignment of variably shaped lens fiber cell ends into imprecise suture planes could contribute to nonmonotonic spherical aberration. More importantly, the negative influence of sutures on optical quality increases with age.157-160

Nuclear Fiber Compaction as a Function of Aging and Cataractogenesis The substantial senescent alterations in the structure of the embryonic and fetal nuclear fibers can lead to degradation of lens optical quality, especially because these fibers are located entirely within the region defined by the pupillary margin. In fact, clinical observations of aged lenses show increased light scatter even without overt visual impairment.161 Excessive senescent changes in the morphology of the nuclear lens fibers are likely to be most detrimental to lens optics, because these fibers are located directly along the visual axis. Therefore, age-related fiber compaction resulting in an increase in the membrane complexity along the light path may be a source of increased large particle scatter and ultimately, reduced lens optical quality with age. Light scatter in the lens has been attributed to the interaction of the incident beam with both the cell membranes and the cytoplasmic proteins producing

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respectively, large and small particle scatter.162 It has been suggested that in normal lenses, the majority of light scatter originates from interactions with fiber membranes, which have a higher refractive index as compared to the cytoplasm.163 The cytoplasm is virtually transparent due to the close association of the crystallin proteins that minimizes refractive index fluctuations.164,165 Although numerous biochemical modifications have been noted in the cytoplasmic and membrane components of lenses with age-related nuclear cataracts, the sources of excessive light scatter have yet to be definitively identified. In nuclear cataracts, the signficantly increased fiber compaction may be one of the factors contributing to the excessive scatter in nuclear opacification. The size and shape of the human lens changes dramatically during development and maturation. Assessments of human lens growth have established that the equatorial dimension of lenses increases at a greater rate than the polar (A-P axis) dimension.166-168 While significant compaction of nuclear fibers occurs along the A-P axis with aging, an even greater degree of compaction occurs in nuclear cataract formation.169 In most age-related nuclear cataracts, opacification begins in the lens center, and enlarges gradually. It has been noted clinically that cataracts often have reduced antero-posterior thickness in comparison to age-matched normal lenses.170 However, the rate of compaction is not constant. Morphometric analysis indicates that, in general, more compaction occurrs between young and middle-aged lenses than between middle-aged and aged lenses. Although initially surprising, this finding is temporally consistent with the onset of presbyopia near age 40. It is likely that condensation and compaction of nuclear fibers in early adulthood contribute to the lens hardening and loss of accommodative ability that characterize presbyopia. The process of nuclear fiber compaction is probably multifactoral. The most obvious structural change is the formation of accordion-like folds, which account for much of the compaction along the A-P axis. These folds begin in early adulthood and increase in both frequency and amplitude with age. The early onset of structural changes may be due to controlled modifications in the cytoskeletal171-174 and crystallin175178 proteins that accompany fiber cell maturation, and are probably necessary for long-term maintenance of fibers. In the fourth through eighth decades, cumulative age-related changes—such as water and protein loss,179,180 modifications to membrane lipids,181,182 and protein modifications183—could result in the progressive increase in compaction folds. The further increase in nuclear fiber compaction in age-related nuclear cataracts is consistent with the extensive protein modifications,184 dehydration,185 and lipid peroxidation186-188 known to occur in human cataracts. The major factor influencing compaction is most likely the loss of cytoplasmic water, which necessarily results in the loss of cell volume without reduction in cell surface area. The driving force for the loss of water may be the reported tendency of the crystallins to self-associate into larger aggregates with time, causing the nuclear cytoplasm to have a reduced osmolarity.189,190 Such changes are essential for the high concentrations of proteins in nuclear cytoplasm to exist adjacent to cortical fiber cells with relatively high water content.

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Further changes in the proteins and membrane lipids during cataract formation, specifically by oxidative damage,191 may result in more extensive condensation of cytoplasmic proteins, as well as loss of protein and membrane fragments that lead to increased fluctuations in refractive index at cellular interfaces and increased light scattering.

Biometric, Optical and Physical Changes in the Human Crystalline Lens with Aging Optical and physical properties in the lens are closely related. The crystalline lens focal length and spherical aberration are profoundly influenced by the lens surface curvatures and gradient refractive index. The continued linear growth in mass and volume of the human lens after the age of five years and throughout the remainder of the normal life-span has been well documented.192,193 Glasser found the following results: a) the human lens grows throughout life and becomes heavier and larger in cross sectional area; b) there is a significant linear increase in lens weight with age; c) the lens equatorial diameters tend to increase up to age 70 and then decrease beyond this age; d) there is a significant linear increase in anterior lens surface radius of curvature up to age 65 and then a significant linear decrease after age 65; e) there is a tendency for an increased lens thickness with age; and f) the posterior lens surface curvature has a tendency to flatten with increasing age (see Fig. 5.10). The thickness shows no significant age dependence, although it has a tendency to increase.194 Moreover, the human lens shows an exponential increase in resistance to mechanical deformation with age from birth. Even though the predominant increase in hardness occurs after the age at which accommodation is completely lost, the increasing resistance demonstrates increased hardness of the human lens, which can account for the loss of accommodation. The age-dependent changes in the responses of lenses to mechanical deformation suggest that the human lens may loose elasticity and increase viscosity with age, and that this may account for the loss of accommodation with the development of presbyopia.194

Fig. 5.10 Changes in the lens equatorial and pole-pole dimensions with age. At the time of birth, (A) the lens is an asymmetric ellipse with an equatorial diameter approximately 1.5 times the anteriorposterior dimension. In the young adult (B) the equatorial diameter is close to twice the length of the anterior-posterior dimension, illustrating the unequal growth rate in the two lens axes. (C) and (D) also show that throughout adult life, the equatorial dimension of the lens increases faster than the polar dimension. (Modified from: Al-Ghoul KJ, Nordgren RK, Kuszak AJ et al Structural evidence of human nuclear fiber compaction as a function of ageing and cataractogenesis. Exp Eye Res 72:199, 2001)

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The Change in Equivalent Refractive Index and the Lens Paradox Using Scheimpflug photography in a cross-sectional study of 100 subjects of various ages, Brown195 demonstrated that the aging lens becomes more convex. He found a substantial decrease of the radius of the anterior lens surface from about 15 mm to approximately 8.5 mm between the age 20 and 80 years of age—the posterior lens radius declined from 8.5 to about 7 mm. This would imply an increase in lens power and a tendency toward myopia in the older eye, because other dimensions of the eye do not change significantly with age.195 Between the ages of 30 and 65, however, a hypermetropic shift can be observed.196 This paradoxical feature of the decrease of the radius of curvature of the crystalline lens with age without the eye becoming more myopic has been called the lens paradox.197,198 To explain the lens paradox, there must be a compensating mechanism in the eye that prevents the eye from becoming myopic. Because neither the axial length nor corneal curvature show considerable changes with age, a decrease of the refractive index of the lens has been suggested as such a compensating mechanism. Nevertheless, so far no empirical study has been able to show a decrease of the gradient refractive index with age. Pierscionek199 found no significant age-dependent changes in the refractive gradient index measured in isolated lenses. Glasser and Campbell200 also found no evidence in support of the lens paradox in isolated human lenses or in decapsulated human lenses. Dubbelman and Vander Heiide201 confirm the existence of the lens paradox in the sense that they also found a decrease of the radius of curvature with age, but there are two major differences between their results and the results obtained by Brown.195 The first difference concerns the extent of the paradox. The average decrease of the anterior radius is 57 µm per year according to Dubbelman,201 while Brown195 found a value of about 100 µm per year, which is almost twice as large. The slight decrease of the radius of the posterior lens surface, approximately 17 µm per year, corresponds to the findings of Brown.195 The second difference concerns the absolute value of the anterior and posterior lens radius, which are both smaller than the values found by Brown. According to Dubbelman,201 the difference for the anterior surface is more than 3 mm at the age of 18, which decreases to 0.9 mm at the age of 65. During aging, the posterior radius remains on average 2.3 mm smaller, but this can be explained by the fact that Brown did not correct for the refraction of the lens itself. However, Dubbelman’s findings closely resemble the results of recent phakometric studies202,203 the findings about the lens radii correspond with the radii of the Gullstrand nonaccommodated schematic eye, and also with the earlier measurements listed by Duke-Elder and Wybar.204 To explain the lens paradox, it was suggested that, with age, the increased sharpness of curvature was balanced with increased lens thickness.197 However, Dubbelman’s calculations demonstrated that thickening of the lens only cancels out 15 percent of the decrease of the equivalent refractive index needed to prevent the eye from becoming myopic with age.201 According to the results of Brown,195 Dubbelman201 registered a small decrease of the refractive index with age. The origin of the decrease of the refractive index with age has remained unclear.

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It is suggested that the water content of the lens should increase resulting in a decrease of the refractive power. However, most recent studies on this topic do not support each other. Siebinga et al.205 measured an increase of water content in the nucleus with age, whiles Lahm et al.206 measured the opposite. Clarke et al.207 reported an increase of refractive index in the center of the lens, and another hypothesis is a change in the variation of the index gradient of the lens with age. Using the results of Brown,195 Smith et al.208 proposed a model to describe this variation. They showed that slight changes in lens refractive-index profile would be sufficient to negate the more convex shape of the lens with age. Yet, if the lens radii of the present investigation were used, an even slighter change would suffice. In conclusion, then, recent studies confirm the existence of the lens paradox, although the decrease of the radius of the anterior lens surface, using Scheimpflug photography, is smaller than in earlier studies. There is a highly significant, but small decrease of the equivalent refractive index of the lens, which explains the lens paradox.

Crystalline Lens Position Modification with Age Using Purkinje images, Tscherning209 first reported in 1898 that the human lens deviated 0.25 mm in the upper part and tilted six degrees in the infero-temporal direction. Yu Hu et al.210 showed that the crystalline lens was not aligned perfectly along the visual axis, but its effect on refraction was limited. Aging, associated with an increase in lens thickness211,212 and a more anterior position,213,214 and combined with a complex of anatomical predisposition (a short axial length, a shallow anterior chamber, and a small corneal diameter) and subsequent physiological factors, is conducive to anterior chamber angle closure and is considered to play a major role in the pathogenesis of Primary Angle-Closure glaucoma.215,216 The greater the contact between the anterior surface lens and the posterior surface of the iris, the greater the impediment to the anterior flow of the aqueous humour. In the lens position modification, the zonular apparatus plays an important role.

Zonular Apparatus Synthesis and Structural Organization of Zonular Fibers During Development and Aging Zonular fibers are a specific form of elastic extracellular matrix composed mainly of fibrillins. The major role of the zonule is to anchor the lens in the eyeball between the anterior and posterior chambers, holding the lens in the optical axis. A secondary role of the zonule is the transmission of accommodation forces from

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the ciliary body to the lens. Clinical observations and a preliminary study have shown that zonules are more fragile during aging.217

Zonular Fibers Electron Microscopy Zonular fibers are composed of large bundles of microfibrils, each with a diameter of 12 nm. Microfibrils are more or less in contact with each other, depending on their location in the zonular apparatus. In the human eye, zonular microfibrils appear to become shorter and increasingly disorganized during aging.217 This is supported by the in situ hybridization data, which clearly show an age-dependent decrease of fibrillin-1 mRNA expression as previously observed in the human aorta.218 During aging, a new fibrillar structure of fibrillin microfibrils appears with a 56-nm periodicity.217 This new structure does not have the same periodic pattern as that of classical microfibrils. Banded elastin, however, has a periodicity of less than 50 nm and has never been described in the zonular bundles of microfibrils.219 Other molecules could be involved in this structure, such as type VI collagen, which is a frequent partner of fibrillin-containing microfibrils in other organs (e.g., the nuchal ligaments). Hanssen220 suggested that these modifications could only be the result of cross-linking between fibrillin microfibrils. Crosslinks are often known to appear in long-lived proteins.221 The formation of these putative crosslinked structures, which may be due to the transglutaminase activity demonstrated in zonular fibers,222 may decrease the putative elastic properties of microfibrillar bundles. The low turnover of microfibrillar components may also act to increase this age related modification. In this regard, the appearance of these structures coincides with a physiological age-related modification of accommodation correlated with presbyopia. Gradual loss of elasticity, sclerosis of the lens, and concomitant atrophy of the ciliary muscles have all been proposed as the causes of this dysfunction.

Anterior Shift of Zonular Insertion onto the Anterior Surface of Lens with Age Anatomical Changes of the Zonular Insertion Several studies have investigated the anatomy of zonular fibers in relation to lens structure.223-227 In 1979, Farnsworth and Shyne223 showed that: ● ●

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The distance between the zonular insertion and the lens equator increased with age The circumlental space (the distance from the equator to the ciliary body) decreased with age The distance between the zonular insertion and the ciliary body remained relatively constant Sakabe228 supported the first two findings, but he found that the

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distance between the anterior zonular insertion and the ciliary sulcus increased with age. A possible causal factor suggested is the change of lens contour. With lens growth, there is an increase in both frontal and sagittal diameters,226-231 and a consequent decrease in radius of curvature of the anterior lens surface.232 Sakabe228 found that the diameter of the zonular-free zone decreased with age. Assuming that the attachment position of the zonules remains unchanged throughout life,223 and that thickness of the anterior lens capsule increases (not decreases or is stretched or both) with age,233 apparent decrease in the diameter of the zonular-free zone may be explained by increased convexity of the lens surface. This would result in an increase of anterior zonular insertion, because ciliary sulcus diameter does not change with age. The location of anterior zonular insertion appears to have clinical importance in the practice of cataract surgery, in which continuous curvilinear capsulorhexis is the technique of choice for the majority of surgeons. To create the capsulorhexis within the zonular-free zone, one should stay within the central 6.86-mm area of the anterior capsule, which is the area expected to be completely free from zonular fibers.228 More importantly, this size decreases with age. If the capsulorhexis is not located in the center of the anterior capsule, the edge of the capsular opening can extend more easily to the position of anterior zonular insertion, resulting in a failure to accomplish continuous curvilinear capsulorhexis.

Lens Metabolic Changes with Age and the Effects of External Agents Introduction The lens is exposed to the cumulative effects of radiation, oxidation and postranslational modification. The alteration of proteins and other lens molecules impairs membrane functions and perturbs protein (particularly crystallin) organization, so that light transmission and image formation may be compromised. Damage is minimized by the presence of powerful scavenger and chaperone molecules. Progressive insolubilization of the crystallins of the lens nucleus in the first five decades of life, and the formation of higher molecular weight aggregates, may account for the decreased deformability of the lens nucleus which characterises presbyopia. Additional factors include the progressive increase in lens mass with age, changes in the point of insertion of the lens zonules, and a shortening of the radius of curvature of the anterior surface of the lens. With age, there is also a decrease in light transmission by the lens, associated with increased light scatter, increased spectral absorption (particularly at the blue end of the spectrum), and increased lens fluorescence. A major factor responsible for the increased yellowing of the lens is the accumulation of a novel fluorogen—glutathione-3-hydroxy kynurenine glycoside— which makes a major contribution to the increasing fluorescence of the lens

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nucleus that occurs with age. Because this compound may also crosslink with the lens crystallins, it may contribute to the formation of high-molecular-weight aggregates and the increases in light scattering that occur with age. Focal changes of microscopic size are observed in apparently transparent, aged lenses, and may be regarded as precursors of cortical cataract formation.234,235

Age-related Changes in Calcium, Sodium, Potassium and Lens Membrane Permeability The lens optical density increase with age, and the rate of increase, is much more apparent after the age of 40 years.236 The lens also becomes increasingly colored (yellow) with age, and the intrinsic fluorescence also increases—all of these changes tend to degrade the optical properties of the lens.237 The smallest opacities are the so-called retrodots, which are present in normal, noncataractous lenses, and the frequency of their occurrence increases exponentially after 40 years of age.238 They appear to be formed from multilayered membrane vesicles and have a surprisingly low protein content, but correspondingly high calcium concentration.239,240 When there is a larger, but still localized breakdown in lens fiber structure, the formed opacities disturb the normal visual acuity, especially when they are located on or near the visual axis. Such lenses have near normal sodium and potassium concentrations but have elevated calcium levels.241 Calcium ions appear to have the ability both to disrupt the structure of the lens and also to protect the transparent, unaffected areas by sealing off the damaged fibers. The disrupting properties probably arise through activating the cysteine protease calpain, and several proteins of the structurally important lens cytoskeleton appear to be excellent substrates for degradation by the enzyme.242 The membrane potential of the normal lens appears to decline with age, particularly after the age of 40 years. The decline in voltage is accompanied by a decrease in membrane resistance, indicating that some channel mechanism is being activated in the aging lens.243 This channel is present in lens membranes and appears to permit Na+, K+, and Ca2+ to pass.244,245 It is interesting in this respect that the lens sodium and free calcium content also appear to increase after the age of 40.243 There is a remarkable agreement between the relative increase in permeability to sodium and the increase in lens optical density measured at the wavelength of peak sensitivity of the eye. Both increase more rapidly after the age of 40 and again indicate a common mechanism between alterations in the ionic and structural protein contents of the human lens.

Lens Phospholipid Changes with Age and Cataracts Human lens membrane lipid composition is related to the membrane’s organization,246 structure,247-251 and function.252-256 Age-related changes in human lens lipid composition may serve as a marker for oxidative stress and may reflect systemic oxidative

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insult, providing a window into the health of an individual.257 Species-related phospholipid differences support the idea that humans have adapted so that their lens membranes have a high sphingolipid content that confers resistance to oxidation, allowing these membranes to stay clear for a relatively longer time than is the case in many other species.257 The changes observed in the phospholipid composition of the human lens with age and cataracta were substantial—greater than that reported for any organ or disease. Biochemical studies show that, upon maturation, the cholesterol-tophospholipid ratio of lens membranes dramatically changes from 0.6-0.8 in superficial to over 5.0 in deep cortical and nuclear membranes.258 All this indicates that lens membranes, apart from those in the most superficial cortex, deviate from most cell membranes in the body. This is in line with electrophysiological studies showing that deep cortical membranes are non-leaky, have a high resistance and low capacitance and have no or restricted cell-to-cell communication.259,260 The relative and absolute amount of sphingolipids (including dihydrosphingomyelin and sphingomyelin) increase with age, while glycerolipids (including phosphatidylcholine and two phosphatidylethanolamine-related phospholipids) decrease.266 These changes are exacerbated by the presence of cataracts and are substantial—greater than the changes in lipid levels reported in any organ in association with any disease. The changes in the amount of lipids with age and cataracts support the idea that glycerolipids are selectively oxidized over lipids with fewer double bonds, such as sphingolipids. As a result of the elevation of sphingolipid levels with species, age, and cataracts, lipid hydrocarbon chain order (or stiffness) increases. Increased membrane stiffness may increase lightscattering, reduce calcium pump activity, alter protein-lipid interactions, and perhaps slow fiber cell elongation.261-264 The cause of the changes may be due to lipid oxidation. Lens glycerolipids are approximately three to four times more unsaturated than lens sphingolipid, and consequently they can be selectively oxidized more than unsaturated lipids. Conversely, de Vries265 calculated that the amount of sphingolipid per wet weight of lens—a relatively unsaturated lipid—increases with age up to approximately 45 years. Because phospholipid and cholesterol synthesis do not change within the ages studied,265 the relative and absolute changes between the sphingolipid and glycerolipid with age must be due to degradation. Recent research266 showed that the relative amount of sphingolipids (dihydrosphingomyelin and sphingomyelin) increased from 48 percent at 22 years of age to 57 percent at 69 years of age, in agreement with previous studies.261-264 With cataracts, the relative amount of sphingolipid increased to 78 percent. However, an increase in sphingolipid content in the human lens with age and cataract may indicate deleterious phospholipid oxidation. Human lens lipid composition versus age curves, exhibiting a plateau at 45 years, are remarkably similar to the curves of accommodative amplitude versus age267 and human lens membrane cation passive permeability versus age.268 Correlation does not necessarily indicate causation—however, scenarios can be envisioned in which lens membrane stiffness induced by phospholipid composi-

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tional changes directly or indirectly contribute to presbyopia and/or passive membrane permeability of cations. Recent studies suggest that, as a result of increased sphingolipid content in cataractous lenses compared with age-matched clear lenses, light-scattering increases.269 Lipids scatter 2 to 95 times more light in vitro than do crystallin proteins, indicating that they may contribute to the light-scattering intensity of the lens in vivo.269 Because lipids with ordered hydrocarbon chains have higher polarizabilities, they scatter 2.5 times more light than lipids with disordered hydrocarbon chains.269 An increase in lipid hydrocarbon chain order may also contribute to cataractogenesis indirectly by reducing the activity of the sarco/endoplasmic reticulum isoform of the calcium pump.254 Reduced pump activity could cause an increase in lens calcium levels, wich is elevated in all cataracts,272-275 and maintenance of the calcium homeostasis is essential to lens clarity. The higher sphingolipid content of cataractous lenses may also change protein-lipid interaction276,277 and slow fiber cell elongation261—two factors that could contribute to cataracts.252-261

Age-related Changes in Ganglioside Composition Lens tissues are enriched in the plasma membranes and are known to contain a relatively high concentration of gangliosides among non-neural tissues.278 Because gangliosides are mainly located at the outer leaflet of the plasma membranes, changes in their content and composition may disrupt the functions of the plasma membranes, such as ion transport, cell-to-cell interactions, transmembrane signaling, and so on.279 Ogiso et al.280 reported that human lens accumulates gangliosides in association with aging and senile cataract progression. Structural analysis reveals that gangliosides in human cataractous lenses were composed of ganglio-series gangliosides, such as GM3, GM2, GM1 and GDla, and sialyl-Lewisx containing neolacto-series gangliosides.280 Although Lewisx-containing, neolacto-series glycolipid was found to accumulate in association with aging and cataract progression, the sialyl-Lewisx gangliosides did not show much accumulation in individual lenses from subjects between 16 and 80-years of age.281,282 The content of sialyl-Lewisx gangliosides was about two to four times higher than that of Lewisx glycolipids, suggesting the possibility that the increase in Lex glycolipid is partly due to the desialylation of sialyl-Lex gangliosides.282 On the other hand, the expression of ganglio-series gangliosides increased in an agerelated manner.282 The age dependent, cataract-related increase in ganglioside content in the human cataractous lens is largely derived from the increase in ganglioseries, GM3, GM1 and GD1a.280 Age-related changes in some gangliosides and neutral GSLs, for example, GM3, GM1 and Gb3, appear to be attributable to the accumulation of lens fibers.282 Thus, age-related changes in lens glycolipids may modify the cell-to-cell interaction induced by cell surface sugar chains, leading to the initiation and progression of cataract.282

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Water Content Modifications in Lens with Aging The age-dependance of lens hydration has been studied by a number of techniques in human, as well as in animal, lenses. Lahm et al.283 found a slight age-dependent increase in total water of the intermediate and nuclear regions of human lenses, even if none of these were statistically significant. Nunnari et al.284 and Bours et al.285 also reported no significant changes in the total water content of aging human lenses. On the other hand, the decrease in bound (nonfreezable) water as a percent of the total water with age was statistically significant in each segment.283 This indicates that syneresis286 is involved in aging. In syneresis, bound water is released from the hydration layer of byopolymers and becomes free water.286 The physical process itself has a number of potential consequences. In the eye, syneresis accounts for the liquid pocket formation in the aging vitreous.287 The amount of bound water decreases with age, which supports the existence of syneresis as a factor in aging and in cataract formation286 as inferred from light-scattering measurements,288,289 and shown by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) techniques.284-290 In aging and cataractous lenses, irreversible syneresis contributes to the turbidity of the lens by increasing the amplitude of refractive index fluctuations.291,292 Besides light scattering and thermal studies, the role of syneresis in cataractogenesis has been proven by NMR.293-296 Recent research confirmed that the amount of bound water decreases with age, which supports the existence of syneresis as a factor in aging.297 The implication is that in normal lenses without apparent turbidity, aging causes tighter packing of protein molecules, possibly leading to higher molecular species. The remaining bound water layer, however, becomes tighter, more immobilized, and therefore, potentially still a sufficient barrier to prevent aggregation and cataract formation.297

Oxidative Stress in the Aging Lens Due to its constant exposure to light and oxidants, oxidation is a major insult to the lens.298-300 Oxidative stress corresponds to an imbalance between the rate of oxidant production and the rate of its degradation.301 The complete four-electron reduction of oxygen occurs within the mitochondria, and the end product is water. A partial reduction produces superoxide and various reactive oxidative intermediates (free radicals and reactive oxygen species, or ROS including hydroxyl radicals, singlet oxygen radicals, and hydrogen peroxide).302 Besides these endogenous oxidants, other sources are food, air pollutants, tobacco smoke, exercise, ionizing radiation, IR and, of course, the sun.303 Although the organism adapts by preventing undesirable reactions with its endogenous and partly redundant antioxidant defense (glutathione superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase) and repairing

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damaged molecules and tissues, the few molecules and undesirable reactions that are not prevented or repaired will accumulate over time and be deleterious in the long term.303 All of these conditions will lead to the formation of excessive oxidants and oxidative stress. Oxidative stress is countered by antioxidants that are defined as substances that, at low concentrations relative to the substrate, inhibit the damage to the structural and functional molecules of the body, namely proteins, lipids, carbohydrates, and DNA.303 Antioxidants function by several possible mechanisms: ●

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Scavenging of free radicals involved in chain reactions; tocopherol acting in the lipid phase Regeneration of other antioxidants; ascorbate reduces tocopheryloxy radical to tocopherol by donating an atom Reacting with initiating radicals or oxidants (catalase with hydrogen peroxide) Chelating or sequestering transition metal catalysts which are pro-oxidants; albumin or polyphenols with cupric ion Inhibiting or activating an enzyme; tocopherol and polyphenols inhibit tyrosine kinase and ascorbate activates nitric oxide synthase303

There is a considerable body of evidence to indicate that the ability of the human lens to withstand oxidative attack actually declines with age because the overall level of glutathione decreases and the important enzyme glutathione reductase becomes less stable.304 Because it is well-known that lens nuclear cataracts involve protein oxidation,305 there is now, therefore, the possibility that nuclear and cortical cataracts, with their totally different aetiology and morphological appearance, may both arise from oxidative mechanisms—one taking place primarily at the surface membranes, and the other within the nuclear proteins. This may help explain why the majority of senile cataracts are, in fact, mixed in form, with contributions from both nuclear and cortical changes.306 Recent epidemiological studies of cataracts do suggest that a high intake of antioxidants—either in the diet, or in the form of supplements—does confer a considerable protective effect.307 Age-related nuclear (ARN) cataracts are associated with a loss of glutathione in the center of the lens and extensive modification of the nuclear proteins that include coloration, oxidation, insolubilization, and crosslinkin g.308Accumulation of oxidatively damaged proteins is causally related to the formation of cataracts298,304,309,314 and many other age-related debilities.315-317

Age-related Decline in Ibiquitin Conjugation The extent of accumulation of oxidatively damaged proteins depends on both the rate of production and on the efficiency of removal of the oxidatively damaged proteins.318,319 In most cells, intracellular proteolytic enzymes selectively remove the oxidized or damaged proteins.320-324 Therefore, proteolytic capabilities are considered as secondary defense systems, which can avert or delay the accumulation of damaged proteins.318,322,325,328

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The ubiquitin-dependent proteolytic pathway is a primary proteolytic system which is involved in the selective degradation of oxidatively damaged proteins in various types of cells or cell-free systems,329-333 and a substantial amount of literature indicates that the ubiquitin-dependent proteolytic system functions in lens cells, as well.332-334 Shang et al.335 showed that: ●

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lenses—especially the nuclei of lenses—undergo dramatic changes with aging, including a decreased level of ubiquitin conjugates and decreased ubiquitin conjugation activity there is an increase in endogenous ubiquitin-protein conjugates and enhanced ubiquitin conjugation activity in response to oxidative stress in each developmental zone of lenses the ability to mount a ubiquitin-dependent response to oxidative stress decreases in the old lens—especially in the nucleus of old lenses

This attenuated ability to enhance the ubiquitin conjugation activity with oxidative damage may be associated with the observed accumulation of damaged proteins in old lenses. The progression of cataractogenesis in the normal aging population can be characterized as a continual increase in the intensity of light scattered from the lens. An important molecular mechanism for such light scattering is, in fact, the condensation of protein into aggregates.335 Protein insolubilization in human lenses during aging and cataracts is well documented.336-339 Garner et al.340 showed the association of gamma crystallin with the membrane protein component of human cataract lenses. Recent studies341,342 indicate that the fiber cell plasma membrane has a high capacity to bind a-crystallin in a nonsaturable manner. This association may play an important role in triggering the further interaction of crystallins with plasma membranes in normal aging and cataract formation, which results in massive protein insolubilization.343 The basis for the great association of crystallins with lens membranes during aging and cataractogenesis is unknown, but might involve the interplay of two broad mechanisms. Modification of membrane structures could enhance its protein-binding characteristics, modification of crystallin structure could increase their affinity to bind, and a combination of altered membranes and crystallin structure might be important for association. This increase is an exponential function of age and has a time constant that, on average, is approximately 35 years.344

Hormonal Influence on Lens with Aging The human lens continues to grow throughout life and in all decades from 10 to 70 years—the male lens is heavier than its female counterpart.345 These age-related differences between males and females are interesting because not only do their relative susceptibilities to cataract change with age, but their response to physical trauma also does.

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A role for female hormones in protecting against cataracts has been suggested by recent epidemiological studies. Below the age of 50, the prevalence of cataracts seems to be similar in males and females,346,347 but it increases in postmenopausal women.346-348 Moreover, postmenopausal women on hormone replacement therapy, or younger women taking oral contraceptives, display a decreased prevalence and severity of cataracts.349-352 In addition, the prevalence and severity of certain forms of cataracts are lower in postmenopausal women on hormone replacement therapy involving administration of estrogen, with or without progesterone, than in those who are not undergoing hormone replacement therapy. Hales et al.353 showed that transforming growth factor-β (TGFβ)—a multifunctional growth factor that is present in the aqueous and vitreous humours354—induces rat lenses in culture to develop opacities and other changes that have many features of human subcapsular cataracts. Hales also showed that estrogen protects against cataracts. Interestingly, lenses from male rats are more susceptible than those from female rats and, furthermore, the latter receive added protection from TGFβ if estrogen is also present in the medium.355 The molecular mechanisms underlying the cataractogenic effect of TGFβ are poorly understood, but TGFβ is known to induce transdifferentiation of lens cells so that they produce at least two types of foreign protein—smooth muscle actin and collagen types 1 and 3.356 Neither of these is synthesized in significant amounts by normal lens cells, but can be detected in certain cataracts122 and in cells, giving rise to PCO.358 The TGFβ stimulated production of abnormal intracellular and extracellular proteins disrupts the homogeneous structure of the anterior epithelium, and lightscattering, multilayered cell aggregates are produced.355 Not only do male and female lenses differ in their relative sensitivity to TGFβ, but they also respond differently to mechanical stress. Weale359,360 carried out a quantitative study of the birefringence of male and female lenses, and although the overall pattern is the same, the effect of external stress on the birefringence pattern measured in vitro is different in males and females. Weale360 measured the greatest stress that could be given before an irreversible change in birefringence occurred and, although in both cases the magnitude of the reversible stress declines with age, the rate of decline appears to be steeper with female lenses. Furthermore, Weale identified a number of female lenses in which the merest mechanical stress induced irreversible birefringence changes, and he concluded that this pointed to a subtle structural difference between male and female lenses.360

The Effect of Physical Agents on the Aging Lens Ultraviolet Radiation Sunlight is the principal source of ultraviolet radiation (UVR) for most of the world’s population. Depletion of the stratospheric ozone increases the intensity of UVR. UVR is considered one of the major risk factors for cataracts,361-364 and

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several studies have shown that sunlight increases the risk of cortical cataracts.365-368 Effects of UVR may be analyzed from different perspectives (e.g., at the molecular, cellular, tissue, individual, population, and ecosystem levels).369 UVR damages the lens by disturbing cell proliferation in the lens epithelium,370 by altering kinetic properties of enzymes in the energy metabolism,371 by increasing insoluble and decreasing soluble protein,372,373 by inducing unscheduled DNA synthesis,374 and by disturbing the sodium potassium balance and thereby the water balance in the lens.375,376 One of the major difficulties in epidemiologic studies has been quantification of exposure to UVR from the sun. The consequences of UVR exposure on the epithelium must be considered both in terms of mutagenic as well as cytotoxic effects.377,378 The single layer of epithelium is the first physical and cellular (biological) defense against electromagnetic radiation in the ocular lens.379 Some of the direct effects of UVR exposure on cultured cells have been reviewed in detail.380 UVR exposure results in unscheduled DNA synthesis and repair.381-383 The human lens epithelium accumulates insults due to UVR exposure in its genome over a period of time that are manifested in the aged lens.384 There is, of course, an age dependence of UVR damage to different molecular species, including enzymes such as hexokinase, phosphofructokinase, isocitrate dehydrogenase, and malate dehydrogenase.385 Loss of hexokinase386 would result in the inability of the lens to produce NADPH and downstream antioxidants. It is conceivable that proteins (such as Na+ / K+) ATPase, cytoskeletal elements, membrane proteins), which are dependent on –SH function will be damaged by exposure to increased oxidants. In addition to intensity of sunlight, the ocular dose depends on other factors, such as the amount of time spent outdoors, the environment, the use of ocular protection, and the use of hats.363,364,387-389 In earlier studies, safety limits for UVRB induced cataract have been based on a dichotomous dose-response model, assuming that the outcome of UVR-B exposure is limited to a binary response: cataract/no cataracts.390 In those studies, cataracts were measured qualitatively with a slit lamp, with a grading scale. It has recently been shown with quantitative measurements of cataracts, however, that UVR-B-induced cataracts has a continuous dose-response function.391 For this reason, a new concept—maximum acceptable dose (MAD) for avoidance of UVR-B cataract—was developed for estimation of UVR-B toxicity in the lens.392 Based on the dose-response function, MAD is defined as the dose corresponding to a limit for pathologic forward light scattering. The limit for pathologic forward light-scattering is settled arbitrarily, based on the frequency distribution of light scattering in normal unexposed lenses. The limit is defined so that a certain fraction (α) of normal unexposed lenses scatter light in the forward direction to an intensity above the limit. The magnitude of the fraction is a parameter that has to be settled and is given as an index to MAD1-α. The high rate of cell division in the germinative zone in the young lens may render the young lens more sensitive to UVR-B–triggered DNA fragmentation. Further, the young lens requires more protein synthesis that includes a part of the young lens that is biologically more important than that of the older lens.372,393 Lerman394 exposed young (first decade) and old (seventh decade) normal human lenses to low level (< 0.1 kJ/cm2) broad band UVR-B (300–400 nm), and found that

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the γ-crystallins were significantly affected by UVR-B in young lenses, while the aged lens proteins appeared to be relatively unaffected by this degree of UVR-B exposure. The finding that MAD for avoidance of UVR-B-induced cataracts strongly depends on age implicates that, in the future, age should be considered. Until better data are available, the current data should be considered in toxicity estimates for avoidance of UVR-B cataracts after exposure to the sun, as well as to artificial sources.395

Medications and Cataracts Oral corticosteroids are known to cause cataracts, but the role of many other systemic medications in cataract etiology is uncertain.396 Several case reports suggest that allopurinol may cause cataracts,397-399 but epidemiologic studies are inconsistent.400-402 Case series of institutionalized patients suggest that phenothiazines are associated with cataract development,403,404 but only one population-based study has examined these medications.405 There have been several studies of diuretics and cataracts, with some finding a protective effect,406 and others a harmful one.407-409 In one case series,410 a high proportion of patients on amiodarone had cataracts, but this association does not appear to have been studied by other investigators. There are biological reasons why some drugs used to lower serum cholesterol might cause cataracts, but such an effect has not been shown.411 Finally, the possibility that aspirin lowers the risk of cataracts has received a great deal of attention in recent years, but studies are far from consistent.396 Different types of cataracts have different etiologies, and so it is important to distinguish between types of cataracts when studying cataract risk factors. The Blue Mountains Eye Study is a large population-based study in which cataract diagnosis was based on grading of lens photographs.412 An association between inhaled steroids and cataracts was found in this study population. Four medications were associated with increased cataract prevalence—phenothiazines were associated with nuclear cataract; amiodarone with cortical cataract; and aspirin and mepacrine (an antimalarial medication that was used extensively during World War II) were associated with posterior subcapsular cataract.412 Aspirin is the only one of these four medications that is used extensively in the community. Most medications studied were not associated with cataracts, including allopurinol, cholesterollowering medications, thiazide diuretics, frusemide, beta blockers, calcium-channel blockers, benzodiazepines, and nonsteroidal antiinflammatory drugs.412

Aspirin and Nonsteroidal Anti-inflammatory Drugs There have been at least 15 previous studies of the association between aspirin use and cataracts,396,414-427 including three randomized trials.420-422 None of the randomized trials found any protective effect of aspirin. Of the 12 observational studies,

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six found that cataracts were less frequent among aspirin users.414-418 These positive studies had methodological flaws that could explain their findings, including failure to adjust for prior steroid use and other confounders, inadequate control groups, and use of cataract surgery cases. In the The Blue Mountains Eye Study, long-term aspirin use was associated with increased prevalence of posterior subcapsular cataract.412 At least three other studies have found a slightly higher risk of cataract in aspirin users.405,426,427 Based on the combined evidence from nearly 20 years of research, it is possible to conclude that aspirin does not protect against the development or progression of cataracts. Hankinson et al.428 found some evidence that nonsteroidal anti-inflammatory medications might be associated with increased risk of cataracts, but The Blue Mountains Eye Study found no such association. Interestingly, this study did find that persons with self-reported osteoarthritis were more likely to have had cataract surgery than persons without osteoarthritis.

Diuretics and Antihypertensives Harding and van Heyningen406 reported that thiazide diuretics were used less frequently by patients who underwent cataract surgery than control subjects. More recently, the Beaver Dam Eye Study found that use of thiazides was associated with lower prevalence of nuclear cataracts and increased prevalence of posterior subcapsular cataract.429 Several other studies have found that use of diuretics was associated with increased risk of cataracts.407-409 The Blue Mountains Eye Study did not find convincing evidence of any harmful or beneficial effects of diuretics on the lens.412 Although frusemide was associated with increased prevalence of cortical and posterior subcapsular cataracts in age- and gender-adjusted analyses, these associations appeared to be because of confounding. The Blue Mountains Eye Study found that long-term users of potassium-sparing diuretics might be at increased risk of cataract. The Beaver Dam Eye Study also found a raised incidence for potassium-sparing diuretics, but this was not statistically significant.429 A cataractogenic effect of potassium-sparing diuretics is biologically plausible, because these diuretics disturb sodium transport across the lens fiber membrane.430,431 The calcium-channel blocker nifedipine has been associated with increased risk of cataract extraction406 and angiotensin-converting enzyme inhibitors with decreased risk of nuclear cataracts.429 Neither of these medication types was associated with cataract in the Blue Mountains Eye Study. Confounding by hypertension and other cardiovascular conditions is a potential problem in studies of cataracts and antihypertensive medications,409,429 including diuretics. The Blue Mountains Eye Study addressed this problem by using statistical techniques to check for history of cardiovascular disease, and by repeating analyses in normotensive persons. After confounding had been adjusted for, none of the antihypertensive medications studied were statistically significantly associated with cataracts.

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Cholesterol-lowering Medications The normal lens membrane contains a very high concentration of cholesterol, most of which is actually synthesized in the lens.411 Hence, drugs that reduce cholesterol synthesis could cause cataracts. Laboratory experiments have found that simvastatin has a particularly strong inhibitory effect on lens cholesterol synthesis.411 The Blue Mountains Eye Study, however, found no association between any type of cataract and use of simvastatin (or any other cholesterol-lowering drug).412 Previous epidemiologic studies of simvastatin and lovostatin have similarly found no association with cataracts.411,432

Allopurinol Several case series have noted that persons treated with allopurinol seem to have characteristic lens changes,397-399 perhaps due to photobinding by allopurinol in the lens.398 In the Lens Opacities Case-Control Study, persons using gout medications (most likely to be allopurinol) had increased prevalence of cataracts.400 Two other epidemiologic studies found no relationship between allopurinol and cataract extraction.401,402 In The Blue Mountains Eye Study, use of allopurinol for 10 or more years was associated with posterior subcapsular cataracts in the initial analyses, but there was no association after adjusting for confounders. Previously observed associations between the use of allopurinol and cataracts may have been because of the higher prevalence of risk factors for cataracts among these persons.

Antimalarials The Blue Mountains Eye Study found a strong association between posterior subcapsular cataract and use of mepacrine—a 9-aminoacridine that was used extensively for malaria prophylaxis by Australian soldiers in the Pacific during World War II—and these data support studies conducted in the 1950s, which reported a high prevalence of cataracts in persons taking chloroquine.433

Phenothiazines An association between the phenothiazine chlorpromazine and cataracts was first reported in the 1960s in patients living in psychiatric institutions.434,435 Two epidemiologic studies have found that use of psychotropic medications is associated with cataracts, but these studies did not investigate specific classes of medications.434,435 The only epidemiologic study to date of phenothiazines and cataracts among persons living in the community was conducted by Isaac et al.405

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The increased frequency of cataracts among phenothiazine users in that study could have been caused by selection bias, because only cataract extraction cases were studied (phenothiazines have an anticholinergic effect that can cause blurred vision, which might lead to increased eye examinations and detection of cataract). In The Blue Mountains Eye Study, which avoided this selection bias by basing cataract diagnosis on grading of lens photographs, phenothiazines were associated with increased prevalence of nuclear cataracts.412

Amiodarone This antiarrhythmic drug was associated with an increased prevalence of cortical cataracts in the Blue Mountains Eye Study. This is consistent with reports by Flach et al.410 of high rates of subcapsular cataracts in patients treated with amiodarone.

A Clinical Approach to Lens Modifications with Aging Introduction With the new instruments available today in clinical practice, it is possible to study the correlation between bio-densitometric changes, optical high-order aberrations (HOAs), and modulation transfer function (MTF) of the crystalline lens that take place during the aging process. We have presented a comprehensive study436 in which these changes have been measured in different age groups of patients without cataracts, to evaluate ways in which morphology and optical performance of the human crystalline lens degrade with age. All the measurements are simple, objective, and performed quickly, requiring minimum cooperation from the subject.

Lens Bio-densitometric Changes Through Aging Scheimpflug Photography Features To evaluate lens morphology and densitometric data, a Scheimpflug slit lamp (EAS 1000, Nidek, Japan) was used.437 In this technique, slit-lamp photography measures light that is reflected anteriorly from the lens to the camera. To record a slit image, an alignment system is coupled to a television monitor, and a fixation light is placed to lie along the optical axis of the slit projection lens. A photograph is taken using a flash intensity of 200 W-seconds. Density is measured by optical density units that are EAS 1000-specific. The resulting cross-sectional image of the anterior chamber and lens is displayed on a monitor for evaluation by the operator. If satisfactory, the image can be transferred to the computer for analysis. To quantify nuclear lens density, linear densitometric analysis of the image was performed in

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Fig. 5.11 Photographs using a flash intensity of 200 W-seconds in a Scheimpflug slit lamp. (A) Eight-year-old subject. (B) Eighty-year-old subject. Linear densitometric analysis to quantify nuclear density. 1, 2, 3 = densities at the embryonic nucleus, anterior fetal nucleus, and posterior fetal nucleus, respectively

Fig. 5.12 Photographs using a flash intensity of 200 W–seconds in a Scheimpflug slit lamp. (A) Eight-year-old subject. A. = anterior; R = radius. (B) Eighty-year-old subject. Axial biometric analysis to quantify lens thickness. 1, 2, 3 = densities at the embryonic nucleus, anterior fetal nucleus, and posterior fetal nucleus, respectively

our study. Density was measured at the embryonic, anterior, and posterior fetal nuclei (see Fig. 5.11). To quantify lens thickness, an axial biometric analysis of the image was performed (see Fig. 5.12). Results from Scheimpflug Photography The correlation with age on nucleus density is represented in Figs. 5.13 to 5.15. Densities of embryonic, anterior fetal, and posterior fetal nuclei show a positive correlation with aging after the age of 40. The scatterplots of embryonic and anterior fetal nuclei clearly show a turning point around the age of 40 years, after which densities of the nuclei show an increase with age. The relationship between age and crystalline lens thickness is shown in Fig. 5.16. As exhibited, crystalline lens thickness increases significantly with age in a linear mode.

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Fig. 5.13 (A) Embryonic nucleus (flash intensity, 200 W–seconds) as a function of age. A positive correlation was found after the age of 40 years (r=0.762, P < 0.0001). (B) Embryonic nucleus (flash intensity, 200 W–seconds) in four age groups. The mean difference using Bonferroni multiple comparison is statistically significant for Groups 2 and 3 (P < 0.002) and for Groups 3 and 4 (P < 0.0001). Error bars, minimum and maximum of the 95 percent confidence interval

Fig. 5.14 (A) Anterior fetal nucleus (flash intensity, 200 W-seconds) as a function of age. A positive correlation was found after the age of 40 years (r =0.764, P < 0.0001). (B) Anterior fetal nucleus (flash intensity, 200 W-seconds) in four age groups. The mean difference using Bonferroni multiple comparison is statistically significant for Groups 2 and 3 (P < 0.0001) and for Groups 3 and 4 (P < .0001). Error bars, minimum and maximum of the 95 percent confidence interval

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Fig. 5.15 (A) Posterior fetal nucleus (flash intensity, 200 W-seconds) as a function of age. A positive correlation was found after the age of 40 years (r = 0.756, P < 0.0001). (B) Posterior fetal nucleus (flash intensity, 200 W-seconds) in four age groups. The mean difference using Bonferroni multiple comparison is statistically significant for Groups 2 and 3 (P < 0.0001) and for Groups 3 and 4 (P < 0.0001). Error bars, minimum and maximum of the 95 percent confidence interval

Fig. 5.16 (A) Crystalline lens thickness as a function of age. A positive linear correlation was found (r = 0.679, P < 0.0001). (B) Crystalline lens thickness in four age groups. The mean difference using Bonferroni multiple comparison is statistically significant for Groups 1 and 2 (P < 0.002) and for Groups 2 and 3 (P < 0.004). Error bars, minimum and maximum of the 95 percent confidence interval

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Optical Changes of the Human Crystalline Lens Through Life Modulation Transfer Function The MTF value quantitatively characterizes the performance of the optical system of the eye. For years, the photography industry has used MTF values to measure the optical quality of lenses. The MTF is the ratio of the image wave contrast to the object wave contrast. The higher the MTF value is, the higher the quality of the image is after it passes through a lens. Optical quality was studied using the MTF for monochromatic light. In our study, the MTF was measured436 with the Optical Quality Analysis System (Visiometrics S.L., Terrassa, Spain)—a recent instrument based on the double-pass technique and developed to perform an objective optical quality-ofvision evaluation. The double-pass technique is based on recording images of a point source after reflection in the retina and a double pass through the ocular media.438 With this configuration, therefore, the ocular point-spread function (PSF) can be obtained. The point spread function (PSF) defines the propagation of electromagnetic radiation or other imaging waves from a point source or point object. The degree of spreading (blurring) of the point object is a measure for the quality of an imaging system. From the point-spread function images, the MTF that yields the relationship between the contrast of an object and its associated image as a function of spatial frequency was obtained, computing the modulus of the 2-dimensional Fourier transformations of the point spread function. The 1-dimensional MTF was calculated as the radial projection (averaging over all orientations) of the 2-dimensional MTF (see Figs.5.17 to 5.19). Measurements were done with a 5-mm pupil. Data at 0.5 MTF represent the spatial frequency (cycles per degree) in which the image contrast is degraded 50

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0.00 0.00 5.96 11.91 17.87 23.82 29.78 35.74 c/d

0.00 0.00 8.12 16.24 24.36 32.49 40.61 48.73 c/d

0.00 0.00 8.12 16.24 24.36 32.49 40.61 48.73 c/d

Fig. 5.17 Curves of spatial frequency and modulation transfer function (MTF) obtained using the Optical Quality Analysis System in (A) an eight-year old subject, (B) a 30-year old subject, and (C) an 80-year old subject. c/d = cycles per degree

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Fig. 5.18 A graphic correlation between 2D - 3D PSF and MTF of the crystalline lens with the OQAS

percent relative to the object contrast. Data at 0.1 MTF represent the spatial frequency in which the image contrast is degraded 90 percent relative to the object contrast, and correspond to the maximum resolution of the optical system. The OQAS creates two- and three-dimensional retinal images (or maps) that describe a patient’s total optical system (Fig. 5.20).

MTF Results Measured with the Optical Quality Analysis System The error bar graphs shown in Fig. 5.21 represent 0.1 and 0.5 MTFs in different age groups. The 0.5 MTFs are 4.317 for Group 1, 5.384 for Group 2, 3.501 for Group 3, and 3.046 for Group 4. A significant difference is seen between the age groups of 21 to 40 and 41 to 60 for 0.1 and 0.5 MTFs. The 0.1 MTF decreased with age from 18.557 to 10.100 cycles per degree.

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Fig. 5.19 Graphic representation of two- and three-dimensional retinal images (or maps) that describe a patient’s total optical quality in a cataractous eye

Wavefront Analysis Ocular and corneal wavefront errors were measured (see Fig. 5.22) with a Hartmann–Shack aberrometer (Wavefront Analyzer, Topcon, Tokyo, Japan). Measurements were taken for 4- and 6-mm pupils. The Wavefront Analyzer gives us the total ocular and corneal aberrations for 4- and 6-mm pupils, coma-like Zernike polynomials (Z3i+Z5i) and Z4i+Z6i for a 6-mm pupil, and ocular and corneal Zs for 4- and 6-mm pupils. Zernike mode Z3-3 through Z33 plus a fifth-order Z (Z5-5 through Z55) corresponds to coma-like aberrations. From ocular and corneal aberrations, intraocular aberrations can be obtained. Intraocular aberrations result from the difference between ocular and corneal aberrations, and they are due more to the crystalline lens and less to the posterior corneal surface.

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Fig. 5-20 OQAS 2-D and 3-D representation of PSF in normal and cataractous eyes

Results on Ocular and Corneal Wavefront Errors Measured with a Hartmann–Shack Aberrometer Total ocular and corneal HOAs of a 6-mm pupil are measured as a function of age. Corneal HOA shows a weak statistically significant variation with age. Ocular HOA increases linearly with age. As shown in the scatterplot, ocular HOA is smaller than corneal HOA until 30 to 40 years of age. In the 40s, ocular HOA is similar to corneal HOA, and it increases in older subjects. The same result can be seen for ocular and corneal Z4i+Z6i aberrations. Corneal coma-like aberrations were not statistically significant, and ocular coma-like aberrations show a positive linear correlation with age (see Fig. 5.23). Intraocular spherical aberration (Z40) for a 6-mm pupil (see Fig. 5.24) shows a positive linear correlation with age. Intraocular coma aberration (Z3-1), on the contrary, shows a negative linear correlation with age (see Fig. 5.25).

Conclusions In our study, nucleus density showed a positive correlation with age, after 40 years, for embryonic, anterior fetal, and posterior fetal nuclei. When different age groups

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Fig. 5.21 (A) Spatial frequency for 0.1 modulation transfer function (MTF) in four age groups for a 5-mm pupil. The mean difference with Bonferroni multiple comparison is statistically significant for groups 2 and 3 (P < 0.009). (B) Spatial frequency for 0.5 MTF in 4 age groups for a 5-mm pupil. The mean difference using Bonferroni multiple comparison is statistically significant for groups 2 and 3 (P < 0.004). c/d = cycles per degree. Error bars, minimum and maximum of the 95 percent confidence interval. (Group 1 included subjects from 8 to 20-years old (n = 15); Group 2, subjects from 21 to 40 (n = 20); Group 3, subjects from 41 to 60 (n = 21); and Group 4, subjects from 61 to 80 (n = 16)

are analyzed, we can see that nucleus density does not increase before the age of 40, after which nucleus density increases linearly with age. As a result of the continuous production of new fibers, the aging lens becomes thicker. We found a correlation between age and overall lens thickness, as was also found by Kashima et al.449 In our study, crystalline lens thickness increases from eight years of age to the age of 40, after which the increase in lens thickness is not statistically significant. Due to the anatomical changes that take place with aging, scattering and aberrations of the crystalline lens are expected to increase. The main contributors to the overall aberrations in the eye are the tears, anterior and posterior surfaces of the cornea, and crystalline lens. So, if the aberrations of the crystalline lens increase, total ocular aberrations will increase as well. Several previous studies have reported an increase in overall eye aberrations with aging.439-442 In our study, overall eye HOAs increased linearly with aging, as previously reported in the literature. This increment in overall ocular HOAs is not due to corneal HOA, which shows a very weak correlation with age. Before the age of 30 years, overall HOA and Z4i+Z6i were significantly larger for the cornea than for the entire eye, which suggests that the lens compensates for part of the corneal aberrations. The corneal and lens aberrations show, in fact, a trend to compensate each other.443 In our study, we found that this mechanism is disrupted in the older eye as a consequence of normal aging.

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Fig. 5.22 Total ocular and corneal high-order aberrations (HOAs) as a function of age for a 6-mm pupil. For ocular HOA, a positive linear correlation was found (r = 0.511, P < 0.0001). For corneal HOA, a weakly positive correlation was found (r = 0.248, P < 0.036)

According to our data, the turning point for the coupling of these two optical systems (cornea and the entire eye) seems to appear around 40 years of age. The changes in the optical performance of the crystalline lens with aging should be related to the anatomical changes (nucleus density and thickness) found. With previous studies, authors have investigated the correlation of the development of aberrometric changes with aging.439-442 In such studies, the Zernike polynomials that were used differed from those analyzed in this study. We found a linear correlation between intraocular spherical aberration and age. Because the main contributor to intraocular aberration is the crystalline lens, we can assume that spherical crystalline lens aberration increases with age. On the other hand, intraocular coma aberration (Z3-1) decreases with age. In this study, we investigated corneal, ocular, and intraocular HOAs in the same patient. We studied the overall corneal and ocular HOAs, corneal and ocular Z4i+Z6i, intraocular spherical aberration (Z40), and intraocular Z3-1. We found a positive linear correlation for all the aberrations studied except for intraocular Z3-1, which shows a negative linear correlation. Our results confirm that the increase in corneal aberration is too small

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to account for the increase in ocular aberrations and support the theory that the crystalline lens must be responsible for the increase in the ocular aberrations that take place with aging. To the best of our knowledge, the correlation between crystalline lens aberration changes and the increase in the densitometric values and thickness of the lens has not been previously reported. Changes in crystalline lens morphology are responsible for the degradation of the optical performance of the human eye through aging. Such anatomical changes are also related to the degradation of the MTF with age, as shown by double-pass imaging in the present study, in agreement with other previous reports on the subject.447,448 We observed degradation in the MTF in different age groups. The highest MTF is observed in Group 2 and corresponds to subjects between 21 and 40 years. Between 41 and 60 years, the MTF declines. The turning point for crystalline lens changes seems to be around the age of 40, when presbyopia appears. Nuclear crystalline lens density increases around the age of 40, and this anatomical change

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implies an increase in intraocular aberrations. The increase in scattering and aberrations around the age of 40 decreases the optical quality of the eye, which we measure in our study using the MTF. We can conclude that there is a degradation of the optical function of the crystalline lens measured as changes in the MTF and in the aberration pattern through aging and, also, that those changes are associated with morphological changes in the thickness and density of the lens. The turning point for these changes is shown to be around the age of 40 years. Further morphological changes in the crystalline lens, and the consequent degradation in the eye’s optical quality with aging, should decrease the normal performance of the human eye before the development of evident cataracts. Such visual deterioration would continue further with the development of cataract that is evident at a clinical level.449 The decrease in the eye’s optical performance through aging, shown as a continuous process related to morphological changes at the level of the crystalline

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lens, may have clinical implications in the future. The use of Scheimpflug Photography and MTF striation can help to develop new guidelines for cataracts (see Fig. 5.26). New intraocular lens (IOL) technology is being used to try to improve the optical performance of the eye using customized lens optical design.450 The increase in spherical aberrations associated with aging that were observed by us can be compensated for either by the induction of negative spherical aberration at the corneal level, as in hyperopic excimer laser procedures, or by an adequately designed customized IOL.451 If the optical performance of an eye that is implanted with a customized IOL reaches a level that is superior to that of an aged eye, crystalline lens substitution may have a clinical indication, especially if improvements in other lens functions (such as accommodation) can also be implemented and the complication rates for the surgery are minimal and acceptable. Future research in this area seems to be of utmost importance.

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Fig. 5.26 Scheimpflug Photography analysis provides objective data (B) compared with slit lamp image (A). A wide range of numeric information can help to develop new guidelines for cataracts

References References – Lens Embryology 1. O’Rahilly R and Meyer DB (1959) The early development of the eye in the chick Gallus Domesticus (stages 8 to 25). Acta Anatomica 36:20-58 2. Coulombre JL, Coulombre AJ (1963) Lens development: fiber elongation and lens orientation. Science 142:1489

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51. Mohan PS, Spiro RG (1986) Macromolecular organization of basement membranes. J. Biol. Chem. 261:4328-4336 52. Cammarata PR, Cantu-Crouch D, Oakford L, Morrill A (1986) Macromolecular organization of bovine lens capsule. Tissue Cell 18:83-97 53. Hettlich H-J, Wenzel M, Janssen M, Mittermayer C (1990) Immunhistochemische untersuchungen der menschlichen linsenkapsel. Fortschr. Ophthalmol. 87:147-149 54. Marschall GE, Konstas AGP, Bechrakis NE, Lee WR (1992) An immunelectron microscope study of the aged human lens capsule. Exp. Eye Res. 54 :393-401 55. Majo F, Montard M, Delbose B, Kantelip B (1997) Immunolabelling of collagen types I, III, and IV, laminin and fibronectin in the human lens capsule. J. Fr. Ophthalmol. 20:664-670 56. Timpl R, Wiedemann H, van Delden V, Furthmayr H, Kuhn K (1981) A network model for organization of type IV collagen molecules in basement membranes. Eur. J. Biochem. 120:132-203 57. Kucharz EJ (1992) The Collagens: Biochemistry and Pathophysiology. Springer, New York, p 12-15 58. Young RW, Ocumpaugh DE (1966) Autoradiographic studies on the growth and development of the lens capsule in the rat. Invest. Ophthalmol. 5:583-593 59. Von Sallmann L, Grimes PA, Albert DM (1969) Histogenesis of the lens capsule in tissue culture. Am. J. Ophthalmol. 68:435-438 60. Rafferty NS, Goossens W (1978) Growth and aging of the lens capsule. Growth 42:375-389 61. Rafferty NS (1985) Lens morphology. In: Maisel H. (ed) The Ocular Lens: Structure, Function, and Pathology. Marcel Dekker Inc, New York, p 1-53 62. Fisher RF, Pettet BE (1972) The postnatal growth of the capsule of the human crystalline lens. J. Anat. 112:207-214 63. Seland JH (1974) Ultrastructural changes in the normal human lens capsule from birth to old age. Acta Ophthalmol. 52:688-706 64. Krag S, Olsen T, Andreassen TT (1997) Biomechanical characteristics of the human anterior lens capsule in relation to age. Invest. Ophthalmol. Vis. Sci. 38:357-363 65. Salzmann M (1912) The Anatomy and Histology of the Human Eyeball in the Normal State, its Development and Senescence. Chicago University Press, Chicago, p 165 66. Fukushi S, Spiro RG (1969) The lens capsule. Sugar and amino acid composition. J. Biol. Chem. 244:2041-2048 67. Mohan PS, Spiro RG (1986) Macromolecular organization of basement membranes. J. Biol. Chem. 261:4328-4336 68. Dark AJ, Streeten BW, Jones D (1969) Accumulation of fibrillar protein in the ageing human lens capsule. Arch. Ophthalmol. 82:815-821 69. Sasaki H, Kasuga T, Ono M, Sakamoto Y, Kojima M (1997) Aging changes of lens transparency in subjects with noncataratous eyes. In: Sasaki K, Hockwin O (eds) Cataract Epidemiology, Developmental Ophthalmology, vol. 27. Karger, Basel, p 102-108 70. Nakashima Y, Yoshitomi F, Amano S, Oshika T (2001) Presumed interference colours at the level of the human anterior lens capsule. Eye 15:206-208 71. Peczon BD, Pezcon JD, Cintron C, Hudson BG, (1980) Changes in chemical composition of anterior lens capsules of cataratous human eyes as a function of age. Exp. Eye Res. 30:155-165 72. Oxlund H, Andreassen TT (1980) The roles of hyaluronic acid, collagen and elastin in the mechanical properties of connective tissues. J Anat. 131 :611-620 73. Barnard K, Burgess SA, Carter DA, Woolley DM (1992) Threedimensional structure of type IV collagen in the mammalian lens capsule. J. Struct. Biol. 108 :6-13 74. Cohen MP, Yu-Vu V (1983) Age-related changes in non-enzymatic glycosylation of human basement membranes. Exp. Gerontol. 18:461-469 75. Garlick RL, Bunn HF, Spiro RG (1988) Nonenzymatic glycation of basement membrane from human glomeruli and bovine sources. Diabetes 37:1144-1150 76. Andreassen TT, Oxlund H, Danielsen CC (1988) The influence of non-enzymatic glycosylation and formation of fluoscent reaction products on the mechanical properties of rat tail tendons. Connect. Tissue Res. 17:1-9

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References – Epithelium 78. Brown NAP, and Bron AJ (1987) An estimate of the human cell size in vivo. Exp. Eye Res. 44:899-906 79. Perry MM, Tassin J, and Courtois YA (1979) Comparison of human lens epithelial cells in situand in vitro in relation to ageing: an ultra structural study. Exp. Eye Res. 28:327-341 80. Robinson Jr. WG, Holder N, and Kinoshita JH (1990) Role of lens epithelium in sugar cataract formation. Exp. Eye Res. 50:641-646 81. Konofsky K, Naumann GOH, and Guggenmoos-Holtzmann I (1987) Cell density and sex chromatin in lens epithelium of human cataracts. Ophthalmol. 94:875-880 82. Guggenmoos-Holtzmann I, Engel B, Henke V, and Naumann GOH (1989) Cell density of human lens epithelium in women higher than in men. Invest. Ophthalmol. and Vis. Sci. 30:330-332 83. Francois J and Rabaey M (1951) Examination of the lens by phase-contrast microscopy. Br. J. Ophthalmol. 35:352-355 84. Balaram M, Tung WH, Kuszak JR, Ayaki M, Shinohara T, and Chylack Jr, LT (2000) Noncontact specular microscopy of human lens epithelium. Invest. Ophthalmol. and Vis. Sci. 41:474-481 85. Kuszak JR (1997) A re-examination of primate lens epithelial cell size, density and structure as a function of development, growth and age. Nova Acta Leopoldina 57:45-66 86. Fagerholm PP and Philpsin BT (1981) Human lens epithelium in normal and cataractous lenses. Invest. Ophthalmol. and Vis. Sci. 21:408-414 87. Karim AJA, Jacob TJ, and Thompson GM (1987) Cell density, morphology and mitotic index in normal and cataractous lenses. Exp. Eye Res. 45:865-874 88. Harocopos GJ, Alvares KM, Kolker AE, and Beebe DC (1998) Human age-related cataract and lens epithelial cell death. Invest. Ophthalmol. and Vis. Sci. 39:2696-2706 89. Lieska N, Krotzer K, and Yang HY (1992) A reassessment of protein synthesis by lens nuclear fiber cells. Exp. Eye Res. 54:807-811 90. Kinsey VE and Reddy DVN (1965) Studies on the crystalline lens. XI. The relative role of the epithelium and capsule in transport. Invest. Ophthalmol. and Vis. Sci. 4:104-116 91. Becker B and Cotlier E (1962) Distribution of rubidium-86 accumulated in the rabbit lens. Invest. Ophthalmol. and Vis. Sci. 1:642-645 92. Harris JE and Becker B (1965) Cation transport of the lens. Invest. Ophthalmol. and Vis. Sci. 4:709-722 93. Reddy VN, Giblin FJ, and Matsuda H (1980) Defense systems of the lens against oxidative damage. In: Srivastava S (ed.) Red Blood Cell and Lens Metabolism. Elsevier, North Holland, p 139-154 94. Giblin F (2000) Glutathione: A vital lens antioxidant. J. Ocular Pharmacol. Therapeutics 16:121-135 95. Li W and Spector A (1996) Lens epithelial cell apoptosis is an early event in the development of UVBinduced cataract. Free Radical Biol. Med. 20:301-311 96. Stellar H (1995) Mechanisms and genes of cellular suicide. Science 267:1445-1449 97. Garcia-Porrero JA, Colvee E, and Ojeda JL (1984) The mechanism of cell death and phagocytosis in the early chick lens morphogenesis: a scanning electron microscopy and cytochemical approach. Anat. Rec. 208:123-136 98. Inoue K, Kubota S, Tsuru T, Araie M, and Seyama Y (2000) Cholesterol induces apoptosis of corneal endothelial and lens epithelial cells. Invest. Ophthalmol. and Vis. Sci. 41:991-997

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Chapter 6

The Extracellular Matrix of the Aged Human Trabecular Meshwork: Changes of Glucosaminoglycans Carlo A. P. Cavallotti, MD, PhD

Abstract Glucosaminoglycans play a central role in maintaining the normal functions of the extracellular matrix of the trabecular meshwork of the human eye. Therefore, we evaluated the possible morphological, histo-chemical, and ultrastructural agerelated changes in glucosaminoglycans of this important zone of the human eye. Small samples of the trabecular meshwork were drawn from 24 eyes after exitus from young and old humans. The samples were harvested from the same places of the eye, without any aesthetic damage of the face. Samples were divided in three fragments, each used for morphological, histo-chemical, and ultrastructural staining. Quantitative analysis of images was performed to evaluate morphometrical data that were statistically analyzed. Our findings demonstrate the following age-related changes: ● ● ● ●

deposition of fibrous granular material in the trabecular meshwork increased electron density of the related structures strong decrease of hyaluronic acid content increase of sulphated proteoglycans

Glucosaminoglycans of the extracellular matrix of the human trabecular meshwork, therefore, undergo age-related changes, as demonstrated by our morphological, histo-chemical, and ultrastructural results. Keywords glycosaminoglycans, trabecular meshwork, extracellular matrix, agerelated changes, human eye

Introduction Aging is a general phenomenon to which all humans undergo. While protozoa and microbes have a life that can be considered limitless because they reproduce themselves for division, all the other animal or vegetable organisms have a limited life—immortality is incompatible with the physical life.1 The word aging expresses the slow and fatal evolution of every individual in time. This evolution happens From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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through a series of morphologic and functional modifications of the human organs and tissues.2 Aging is characterized by a progressive decline of the organs and is accompanied with modifications of the connective tissue. Although it is not always possible to distinguish between aging and senility—the pathology versus the physiology in the morphological and/or functional changes of the organs3—the possible age-related changes without pathological modifications are, the following: ●

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Decrease of the mitoses and of the capacity of cellular increase with slowing down of the processes of repair of tissues and reduction of the ability to produce antibodies. Gradual dehydration and progressive cellular atrophy with processes of degeneration and of pigment accumulation. Lessening of the elasticity of the tissues, degenerative modifications of connective elastic tissue and fat infiltration of various parenchyma. Lessening of the oxidation processes and then of the basal metabolism. Deficit of the regulatory mechanisms of homeostasis. Lessening of the enzymatic activities. Slowing down of the neuromuscular reactions and weakening of the muscular force and reduction of the fatigue strength. Degeneration and atrophy of the nervous system. Weakening of the visual acuity, hearing, olfactory sensibility, memory, attention, ability to the intellectual job.

The anatomical features of these histological and/or physiological alterations are always an involution and/or an atrophy that hits, in various degrees, all organs and viscera, including eyes,4 The modifications of the connective tissue, and the increase of the intercellular cementing substance with an increase of the collagen and of the elastic tissue represent the characteristic aspects of aging and/or senility. Bogomoletz and Kavetzky5 think that senility is characterized by the differentiation of the histio-reticular tissue (the last residual of the embryo tissue.6,8 In senility, the cells of the parenchyma develop a progressive atrophy with an increase of the pigment—therefore, this phenomenon is called brown atrophy. The description of normal age-related eye changes is difficult for the frequency of numerous eye diseases in older humans.9,10 The human trabecular meshwork (Tm) contains hyaluronic acid, chondroitin sulphate and dermatan sulphate11 while, in adults, fine fibril-like components are also found. The presence of type IV collagen in the same structures shows the implication in the cell/extra-cell matrix interactions at this site, and its abnormal increase in aging eyes probably reflects a functional defect of Tm in these conditions.12,13 Histo-chemical studies with polarization microscopy showed—in aged eyes—a decrease of hyaluronic acid and an increase of sulphated GAGs.14 With age, collagen tissue becomes more prominent in Tm. At the same time, the hyaluronic acid content decreases and the sulphated GAGs increase.15 Francois16 suggested that an increased amount of GAGs might influence the functional properties of Tm. Quantitative biochemical studies showed a depletion of hyaluronic acid content and an increase of chondroitin sulphates in the trabecular meshwork, ciliary

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processes, and the anterior sclera in age-matched, normal eyes.17,18,19 Sugahara et al 20,21 demonstrated the complex functions of GAGs, and Goes et al.22 characterized GAGs as component of rabbit eye. Rusova et al.23 described a method for sulphated GAGs measurement in tissues. Despite all these data, the actual ocular physiology of GAGs is still not well-known. The aim of the present work is to study the presence of GAGs in the extracellular matrix of Tm in young and older individuals.

Materials and Methods We studied 24 human eyes. Samples of Tm (left eye) were harvested during autopsies. Because post-mortem phenomena may produce early morphologic modifications of the eye structures, our samples were harvested as earliest as possible after death (after 12-18 h). The Ethics Committees of the involved hospitals gave their approval, and the relatives of the dead humans gave their written informed consents. All experiments were performed according to the guidelines of the Declaration of Helsinki and in conformity with the ARVO Statement for the use of human samples in ophthalmic and vision research applied by all Ethics Committees.24 Some characteristics of the dead human eye-donors are reported in Table 6.1. Eight of these patients were classified as young (age range was 201.2 years), while sixteen eye donors were classified as old (age range was 721.6 years). In none of our donors, eyes showed either macroscopic or microscopic abnormalities. Small pieces of the Tm were dissected immediately (< 2 min). All samples were harvested from the same site.25

Light Microscopy Our samples were immediately prefixed in 2 percent osmium tetroxide at pH 7.4 in veronal-acetate buffer for five minutes at 4 ° C. After fixation, the specimens were washed with veronal-acetate buffer (pH 7.4), dehydrated in a graded ethanol series and embedded in paraffin. Thin sections (about 4 m) were made for morphological staining with toluidine blue (0.05% for 1 minute). Lipids were stained by means of special histo-chemical techniques for light microscopic analysis. In order to Table 6.1 Clinical data on individuals from which small fragments of trabecular meshwork were harvested Number of Patients Age Range Sex Eye and General Status 8

20 ± 1.2

Male

16

72 ± 1.6

Male

No ocular, diabetic or vascular diseases No ocular, diabetic or vascular diseases

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determine the composition and distribution of the lipids, three different stains were used: a) Bromine - Sudan black B, which stains all classes of lipids; b) Bromine - acetone Sudan black B, which stains only phosphoric lipids, and c) Oil red O, which stains neutral lipids (especially esters of saturated and unsaturated fatty acids).

Transmission Electron Microscopy (TEM) Samples were pre-fixed in buffered 2 percent gluta-raldehyde for 2 hours, washed in buffer and than post-fixed in buffered 2 percent osmium tetroxyde for 2 hours, dehydrated and embedded in araldite. Ultra thin sections were made with Reichert Ultra microtome. These sections were counterstained by uranyl acetate lead citrate and studied with Zeiss EM 109 electron microscope.26

Staining of Acidic Proteoglycans Acidic proteoglycans are made by using a protein that bounds long and numerous heteropolysaccharidic chains, formed by hexosamine molecules, hexoses, uronic, sialic or sulphuric acid—the latter being usually external to the hydroxylic groups. The best fixative agent for acidic proteoglycans is calcic formalin, and they are PAS negative. The reasons for their PAS negativity are not yet clear. One explanation could be that their powerfully negative electric charge impedes contact with periodic acids. All staining methods for acidic proteoglycans are based on the presence of numerous acidic valences contained in such substances, and so they actually are aspecific methods. In animal tissues, acid groups are essentially represented by carboxylic groups (-COOH) of proteins, by acidic proteoglycans and glycoproteins, by phosphoric groups (==HPO4) of the nucleic acids, and by sulphuric groups (-HSO4) present in the sulphate. To attach themselves with electro polar connections to the basic staining agents (cations), such acidic groups must have a negative electric charge—i.e., must be dissociated in [-COO-] and [H+]. Their dissociation naturally depends on the solution pH the means in which they are placed. In fact, at pH 4, all the acid groups are dissociated; while at pH 2, only the phosphoric and sulphuric acid groups are dissociated; and, finally, at pH 1.8, only sulphuric acid groups are still dissociated and reactive.

ALCIAN-PAS Method This method is realized first by staining with Alcian blue at pH 2.5, and then a normal PAS staining (after Step 3). Acidic proteoglycans appear blue-green, while PAS-positive substances (glycoprotein, glycogen, etc.) are red. The ALCIAN BLUE-CEC Method (Critical Electrolyte Concentration) was performed according to the Pearse method.26

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Quantitative Analysis of Images For a detailed evaluation of the effects of aging on RPE morphology, a quantitative analysis of images (QAI) was performed on slides using a Quantimet Analyzer (Leica) equipped with specific software. Final values were statistically analyzed. The values reported in the current manuscript represent the values of staining for each age group, and are expressed in conventional units (CU) ± S.E.M. CU are arbitrary units furnished and printed directly by the Quantimet Analyzer.27

Statistics Mean values, maximum and minimum limits (experimental values), variations, standard deviation (SD), standard error of the mean (SEM) and correlation coefficients were carried out.28 A correlative analysis of the morphological and histo-chemical data was performed by comparing the significant differences for each group with the corresponding values of the other homogenous groups. The significance of differences between age groups was assessed by the Duncan’s multiple range test.

Results Our results are reported in Figures 6.1 through 6-8 and summarized in Tables 6.1 through 6.4. (The figures had been included in the paper Opthalmic Research 2004; 36:311–217, and have been reproduced with the permission of S. Karger AG, Basel.) The trabecular meshwork has two components: i) the corneoscleral (see Fig. 6.1), formed by trabecular cells (TC), elastic fibers, collagen fibers, and fine fibrilgranular material—the basal membrane is thin in young subjects; and ii) the uveoscleral (see Fig. 6.2), formed by numerous endothelial cells surrounded by other components in the corneoscleral meshwork. Observing a trabecular sheet, we can detect the central elastic fibers surrounded by collagen fibers. Many TC are in close relationships with the basal membrane (see Fig. 6.3). In one eye drawn from an old subject, we could observe the typical age-related changes consisting of a decrease of the fine fibril-granular material, substituted by gross fibril-granular material that causes an increased electron density (see Fig. 6.4). Fig. 6.5 shows other typical age-related changes of the trabecular meshwork of a 71-year old subject—an accumulation of electron-dense material with increased electron density of the whole sample. Other typical age-related changes of the trabecular meshwork are mitochondrial abnormalities in the TC, and swelling and loss of the mitochondrial crystal (see Fig. 6.6). Morphological results regarding the histo-chemical staining of GAGs, observed by means of polarization microscope, are reported in Figs. 6.7 and 6.8 (from a young and old subject, respectively). Schlemm’s canal is embedded

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Fig. 6.1 TEM of a sample of the eye drawn from a young subject (20 years old). Typical structure of the corneoscleral trabecular meshwork formed by trabecular cells, elastic fibers, collagen fibers, fibril-granular material and basal membrane (Magnification 3000x) (Included in the paper Opthalmic Research 2004; 36:311–217, have been reproduced with the permission of S. Karger AG, Basel)

Fig. 6.2 TEM of a sample of the eye drawn from a young subject (21 years old). Typical structure of the uveal meshwork formed by elastic fibers, collagen fibers, basal membrane, fibril-granular material and trabecular cells. (Magnification 5000x) (Included in the paper Opthalmic Research 2004; 36:311–217, have been reproduced with the permission of S. Karger AG, Basel)

into a spongiform tissue rich in GAGs. A positive staining can also be observed in the uveal trabecular meshwork and in the anterior portion of the ciliary muscle. Typical age-related changes are the decrease of the lumen of Schlemn’s canal and the staining increase that corresponds to a GAGs increase.

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Fig. 6.3 TEM of a sample of the eye drawn from a young subject (19 years old). Slanting section of a trabecular sheet: central elastic fibers, surrounded by collagen fibers, basal membrane material, and trabecular cells (Magnification 3000x) (Included in the paper Opthalmic Research 2004; 36:311–217, have been reproduced with the permission of S. Karger AG, Basel)

Fig. 6.4 TEM of a sample of the eye drawn from an older subject (70 years old).Typical age-related changes of corneoscleral trabecular meshwork: increased electron density of the collagen and decrease of fibril-granular material (Magnification 3000x) (Included in the paper Opthalmic Research 2004; 36:311–217, have been reproduced with the permission of S. Karger AG, Basel)

Table 6.1 reports the clinical data relevant in subjects from which the trabecular meshwork was harvested. Eight subjects were young (20 ± 1.2 years) and 16 were old (72 ± 1.6 years). All were male and the left eye was harvested in the same area by the same investigators (n = 6). None of the subjects had any ocular disease. Table 6.2

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Fig. 6.5 TEM of a sample of the eye drawn from an older subject (71 years old). Typical age-related changes of the trabecular meshwork: accumulation of electron-dense material, decrease of fibril-granular material, increased electron density (Magnification 5000x) (Included in the paper Opthalmic Research 2004; 36:311–217, have been reproduced with the permission of S. Karger AG, Basel)

Fig. 6.6 TEM of a sample of the eye drawn from an older subject (72 years old). Typical age-related changes of the trabecular meshwork: mitochondrial abnormalities in the trabecular cells as swelling and loss of cristae (Magnification 5000x) (Included in the paper Opthalmic Research 2004; 36:311–217, have been reproduced with the permission of S. Karger AG, Basel)

shows the amount of fine and dense fibril-granular material in the trabecular meshwork stroma in young and old humans. These results have been compared with the ones obtained by electron density from each sample. As can be seen, we found specific age-related changes, such as a decrease of fine fibril-granular material, an increase of dense fibril-granular material, and an increase in electron density. Table 6.3 shows that hyaluronic acid content decreases in old age, while proteoglycans sulphate content increases with aging.

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Fig. 6.7 Polarization microscopic picture of a sample of the eye of a young subject (21 years old) stained for glucosaminoglycans (GAG). It is clearly seen that Schlemm’s canal is embedded into a spongiform tissue rich of GAGs. Positive staining can also be seen at the uveal trabecular meshwork and the anterior portion of the ciliary muscle (Magnification 250x) (Included in the paper Opthalmic Research 2004; 36:311–217, have been reproduced with the permission of S. Karger AG, Basel)

Fig. 6.8 Polarization microscopic picture of a sample of the eye of an old subject (72 years old) stained for glucosaminoglycans (GAGs). Typical age-related changes: it is clearly seen that Schlemm’s canal is reduced in diameters and is surrounded by a spongiform tissue very rich in GAGs. Intensively positive staining can also be seen at the uveal trabecular meshwork and the anterior portion of the ciliary muscle (Magnification 250x) (Included in the paper Opthalmic Research 2004; 36:311–217, have been reproduced with the permission of S. Karger AG, Basel)

Finally, by performing a differentiated Vialli-PAS staining at various pHs and analyzing the various GAGs types with QAI, we can confirm that hyaluronic acid decreases in older subjects while condroitin, dermatan, keratin, and heparan sulphate

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Young (n = 8)

Old (n = 16)

Fibril-granular fine material 46.6 ± 2.4 12.1 ± 1.4 Fibril-granular dense material 7.9 ± 0.8 61.5 ± 3.1 Electron density 20.7 ± 1.5 49.8 ± 2.1 Note: Results are expressed as CU ± SEM (see Material and Methods section).

Table 6.3 Histo-chemical staining of the trabecular meshwork, quantitative analysis of images in young and old humans Findings Young (n = 8) Old (n = 16) Hyaluronic acid content 34.4 ± 1.8 7.8 ± 1.1 Proteoglycan sulphate content 18.1 ± 2.1 38.2 ± 2.3 Note: Results are expressed as CU ± SEM (see Material and Methods section).

Table 6.4 Measurement of GAGs with quantitative analysis of images in the trabecular meshwork of young and old humans Findings Young (n = 8) Old (n = 16) Hyaluronic acid 32.1 ± 2.3 18.2 ± 1.4 Chondroitin sulphate 16.4 ± 1.6 19.8 ± 1.4 Dermatan sulphate 24.5 ± 1.6 29.7 ± 2.4 Keratan sulphate 19.3 ± 1.8 27.3 ± 2.2 Heparan sulphate 16.3 ± 1.6 21.8 ± 1.9 Note: Results are expressed as CU ± SEM (see Material and Methods section).

increase (Table 6.4). Therefore, hyaluronic acid corresponds to the fine fibrilgranular material, while the other substances correspond to the dense fibril-granular material that increases with age in correlation to the basal membrane.

Discussion Our results show that the major morphological age-related changes of the trabecular meshwork are an increase of extra-cellular material and/or an increase in its electron density. The most abundant extracellular material in young eyes is a fine granular or fibril material. However, with age increase, the fibrillo-granular material decreases, while an electron-dense material becomes prominent. Increased amounts of electron-dense material deposition were found also in the juxta-canalicular zone.

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Stroma of the Trabecular Meshwork The stroma is formed by loosely arranged collagen fibers that are condensed around the vessels. The inter-fibril spaces are rich in glycosaminoglycans. Cells are mostly fibroblasts and melanocytes that often form a plexus and are arranged around the adventitia of a blood vessel. Many moving cells, macrophages, lymphocytes, and clump cells can also be observed in the stroma.

Morphological Changes of the Trabecular Meshwork TC are embedded in various types of extracellular material. The major extracellular material in young eyes is a fine granular or fibril material. However, with age increase, the fibril-granular material occupies less space in the cribriform layer, and an electron-dense material (plaques) becomes prominent.29,31 The corneoscleral meshwork is composed of numerous flattened and perforated sheets. Two types of cells can be distinguished in the trabecular meshwork—TC and trabecular endothelial cells.

Description of Extracellular Matrix We found four components in the extracellular matrix of the human trabecular meshwork: i) elastic fibers located in the trabecular sheets, corresponding to elastic parts of the ciliary muscles; ii) collagen fibers that surround the elastic fibers composed of type I collagen (normal periodicity collagen) with the so-called longspacing collagen in between; iii) basal membrane material (type IV collagen) just beneath TC; iv) fine fibril-granular material located between the trabecular sheets. This material is formed histo-chemically by proteoglycans, and proteoglycans are biochemically composed of hyaluronic acid (29%), chondroitin (14.1%), dermatan (21.5%), keratan (20.3%) and heparan sulphate (15%).32 This material represents the filter responsible for possible outflow resistance to aqueous humor.

Conclusions GAGs of the human trabecular meshwork undergo age-related changes, as demonstrated by our morphological, histo-chemical and morphometric results. Our findings demonstrated the following age-related changes: ● ●

Deposition of fibrous granular material in the trabecular meshwork Increased electron density of the structures

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Strong decrease of hyaluronic acid content Increase of sulphated proteoglycans

References 1. Bourliere F (1948) Biologie de la senescence in Resume des trois journees pour l’etude scientifique du veillissement de la population Paris 2 :5-53 2. Bastai P, Dogliotti GC (1938) Physiopathologie de la vieillesse Masson, Paris 3. Binet L (1951) La senescence La via Medicale 9:15-28 4. Bastai P, Dogliotti GC (1934) Considerazioni sulla circolazione nei capillari sanguigni nelle varie età ed in varie condizioni morbose. Boll Soc. It Biol. Sperim 9:118-136 5. Bogomoletz G, Kawetzky R- J (1951) Med Acad Sc URSS 8:643-664 6. Lansing AI (1947) General physiology of aging. J. Gerontology 7:327-338 7. Bouliere F (1950) Senescence et vitesse de la cicatrisation chez le rat- Rev. Medicale Liege 5 :669-671 8. Carrel A (1913) Artificial activation of the growth in vitro of connective tissue. J. Exp Med 17:14-19 9. Carrel A (1914) Mechanism of the growth of the connective tissue. J. Exp Med 18:287-299 10. Karentz WB (1951) Revitalization of tissue and nutrition in older individuals. Ann. of Internal Medicine 35:1055-1068 11. Sugiura T (1992) Demonstration of glycosaminoglycans (GAGs) in fetal human trabecular meshwork. Acta Soc Ophthalmol Jpn 96:57-66 12. Tawara A, Vaner HH, Hollyfield JG (1989) Distribution and characterization of sulphated proteoglycans in the human trabecular tissue. Invest Ohthamol Vis Sci 30:2215-2231 13. Tripathi BJ, Hansen M, Li J, Tripathi RC (1994a) Identification of type VI collagen in the trabecular meshwork and expression of its mRNA by trabecular cells. Exp Eye Res 58:181-187 14. Valu L, Feher J (1968b) Age depended changes of the trabecular meshwork. Albrecht von Graefes Arch Klin Exp Ophthalmol 175:316-321 15. Valu L, Feher J (1969) Some observation on the connections of the trabecular systems and surrounding tissues. Albrecht von Graefes Arch Klin Exp Ophthalmol 177:21-32 16. Fracois J (1975) The importance of mucopolysaccharides in intraocular pressure regulation. Invest Ophthalmol 14:173-176 17. Segawa K (1975) Ultrastructural changes of trabecular tissues in primary open-angle glaucoma. Jpn J Ophthalmol 19:311-338 18. Knepper PA, Goossens W, Hvizd M, Palmberg PF (1996) Glycosaminoglycans of the human trabecular meshwork in primary open-angle glaucoma. Invest Ophthalmol Vis Sci 37:1360-1367 19. Larsson LI, Rettig ES, Brubaker RF (1995) Aqueous flow in open-angle glaucoma. Arch Ophthalmol 113:283-286 20. Sugahara K, Kitagawa H (2000) Recent advances in the study of the biosynthesis and functions of sulfated glycosaminoglycans. Curr Opin Struct Biol 10:518-527 21. Sugahara K, Yamada S, Kitagawa H (2001) Biosynthetic mechanism of sulphated glycosaminoglycans. Seikagaku 73:458-470 22. Goes RM, Laicine EM, Porcionatto MA, Bonciani Nader H, Haddad A (1999) Glycosaminoglycans in components of the rabbit eye: synthesis and characterization. Curr Eye Res 19:146-153 23. Rusova TV, Matyeeva FL, Talashova IA (2000) Measurement of sulphated glycosaminoglycans in tissue extracts. Klin Lab Diagn 7:17-18 24. 18 Declaration of Helsinki (1964) of the World Medical Association (amended in 1975 and 1983), published in: Philosophy and practice of medical ethics. British Medical Association, 1988 25. Pearse AGE (1972) Histochemistry, theoretical and applied. Churchill-Livingstone Ed., London 26. Millonig G (1961) Advantages of a phosphate buffer for OsO4 solutions in fixation. J Appl Physiol 32:1637-1641

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27. Manual of Methods Quantimet (1997). Microsystems Imaging Solutions, Cambridge (UK) 28. Castino M, Roletto E (1992). Statistica applicata. Ed. Piccin, Padova 29. Millard CB, Tripathi BJ, Triphathi RC (1987) Age-related changes in protein profiles of the normal human trabecular meshwork. Exp Eye Res 45:623-634 30. Miyazaki M, Segawa K, Urakawa Y (1987) Age-related changes in the trabecular meshwork of the normal human eye. Jpn J Ophthalmol 31:558-569 31. Murphy CG, Yun AJ, Newsome DA, Alvarado JA (1987). Localization of extracellular protein of the human trabecular meshwork by indirect immunofluore-scence. Am J Ophthalmol 104:33-43 32. Ascott TS, Westcott M, Passo MS, van Buskirk ME (1985) Trabecular meshwork glycosaminoglycans in human and cynomolgus monkey eye. Invest Ophthalmol Vis 6:1320-1329

Chapter 7

Glial and Mobile Cells in the Iris of the Aging Human Eye Carlo A. P. Cavallotti, MD, PhD and Angelica Cerulli, MD

Abstract This chapter describes the glial and mobile cells that can be found in the iris of the aging human eye. The glial cells of the eye can be divided in two principle classes: Macroglia and Microglia. The Macroglia is of neuroectodermic origin and includes olygodendrocytes, Schwann cells, and astrocytes. Macroglia contains cells that regulate the neuronal metabolism and modulate neuronal functions. Moreover, macroglia regulates also the eye blood vessels functions. In the eye bulb, two cell types can be found as part of the macroglia: Müller cells and astrocytes. Microglial cells are similar to the tissue macrophages. These cells are normally resting, but are sensitive to the pathological changes in the homeostasis of the various components of the eye. When the eye tissues undergo pathological changes, the microglial cells rapidly change into phagocytes capable of mobility. Moreover, the eye contains some types of cells, nonstructurally connected with the other, adjacent cells by mean of junctions, capability of migration, mobility, production of cytochines, and phagocytosis. These cells are named mobile or floating cells. Finally, endothelial cells and pericytes can be found in the eye, which flank the nerve cells and glial cells, or arrange themselves around the blood vessel walls. All these cells show strong age-related changes. Keywords human eye, iris, macroglia, microglia astrocytes, floating cells, phagocytesLymphocytes.

Introduction The glia is the non-nervous component of the central nervous system (CNS) and peripheral nervous system (PNS).1 In the nervous system of the vertebrate, glial cells (according to the numerous sites) are 1 to 10 times more than neurons. Even though their name derives from the Greek word glue, glial cells do not generally join nerve cells. As far as we know, the glia does not directly take part in elaboration of information, but it plays a key role in the following vital functions: From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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As support elements of neurons, give form and structure to nervous tissue Two types of glial cells (oligodendrocytes and Schwann cells) produce myelin Some glial cells have phagocytic function Some glial elements can re-uptake neurotransmitters released from neurons During the development of the nervous system, some types of the glial cells guide the migration of neurons and direct the growth of their axons Some glial cells actively operate in the regulation of the properties of the pre-synaptic terminations Some glial cells (astrocytes) form an impermeable barrier around cerebral capillaries and micro-venules, giving origin to the haemato-encephalic barrier2

The glial cells of the nervous system of vertebrates (including the glial cells of the eye) can be divided into two principle classes: Macroglia and Microglia. The Macroglia, of neuroectodermic origin, includes olygodendrocytes, Schwann cells, and astrocytes, while the cells of Microglia are also called Del Rio Hortega cells for the name of the author who first recognized them. These cells are morphologically and embryologically different from other glial cells. They derive from the mesoderma and are present in the central nervous system towards the end of fetal life, when the vessels of the meningal sheets penetrate and develop into nervous tissue. In this period, it is easy to observe near the vessels lots of small cells. These cells migrate with amoebic movements to the nervous substance spreading in both white and grey substance.3 The cells of the microglia are small elements characterized by two or more fine, but short, extensions that are poorly branched and can be only seen with particular methods of silver impregnation. The nucleus is elongated and small with dense chromatin that is not uniform. The primary function of the microglia is phagocytosis. These cells contain lysosomes and vesicles characteristic of macrophages. Microglial cells can exist at rest or activated like macrophages. Not much is known of the function of microglial cells in the rest mode, but the components are activated during infection or following lesions. Once activated, these operate as phagocytes—they swell up and are called bitter cells. These cells present more sturdy extensions that are more branched than the nonactivated cells. They also have a richer variety of antigens, which might indicate that they represent elements with higher antigen properties of the nervous system. The reactions of the microglia are thus classified: proliferation, hypertrophy, lipid phagocytosis, neuronophagia (phagocytosis of neurons in necrosis) and dendrophagia (phagocytosis of astrocytes prolongation in degeneration).4,5,6,7,8 Macroglia contains cells that regulate the neuronal metabolism and modulate neuronal functions. Moreover, macroglia also regulates the eye blood vessel functions. In the eye bulb, two cell types can be found as part of the macroglia: Müller cells and astrocytes. The Müller cells cross the thickness of the retina from the retinal pigmented epithelium to the inner limiting membrane. The bodies of the Muller cells are located in the inner nuclear layer of the retina. These cells are the regulatory cells for the metabolism of glutamate, ion balance, and neuron function. The Müller cell prolongations form an extended net that sustains and surrounds all of the nervous cells. In addition, these prolongations help to form the inner and outer limiting membranes of the retina. The astrocytes, on the contrary, are limited to the nervous cells layers and envelope the blood vessels and ganglion cells with their cellular protrusions. The

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branched protrusions of these cells occur at right angles with respect to the Müller cells prolongations. These two structures are not linked. The astrocytes are starshaped cells, with round nuclei and numerous thin protrusions. They are horizontally placed and surround the blood vessels with a dense net of fibers. They form an arched, honeycomb structure that surrounds and sustains the axons of the ganglion cells. They are firmly anchored to the walls of the blood vessels.9

Microglia The microglial cells are similar to the tissue macrophages.10 These cells are normally resting, but are sensitive to the pathological changes in the homeostasis of the various components of the eye. When the eye tissues undergo the pathological changes, the microglial cells rapidly change into phagocytes capable of mobility. Moreover, the eye contains some types of cells, nonstructurally connected with the other, adjacent cells by mean of junctions, capability of migration, mobility, production of cytochines and phagocytosis. These cells are named mobile or floating cells. Finally, we also find endothelial cells and pericytes in the eye, which flank the nerve cells and glial cells or arrange themselves around the blood vessel walls. Pericytes are modified smooth muscle cells that regulate the vascular flow through dilation and/or contraction of the diameters of the vessels. The endothelial cells regulate the local homeostatic function and form the blood-retinal barrier.11,12

Aging of the Macroglia (Müller Cells and Astrocytes) The functional weakening of the CNS that occurs with aging and age-related neurodegenerative disorders has been partially attributed to a decline in mitochondrial function. In particular, it has been demonstrated that oxidative damage occurs to mitochondrial DNA in elderly human brains. Recently, it has been shown that mitochondrial DNA is particularly sensitive to damage that accumulates due to the loss of protective histones, the reduction in repair systems, and the vicinity of the internal mitochondrial membrane to active oxygen species. The hypothesis that free radicals are involved in the weakening of the mitochondrial function has been confirmed by recent discoveries—i.e., the fact the administration of free radical scavengers, such as extract of ginkgo biloba (Egb761), improves the function of the brain and liver in elderly animals. Astrocytes are interconnecting cells between the neurons and the surrounding connective tissue (fibroblasts, mesenchymal cells, and endothelial cells). Changes to these cells induce modifications to the intercellular relationships and, eventually, to the nervous function. It has been shown that astrocytes are resistant to oxidative stress due to their high antioxidant content and their ability to regenerate glutathione and ascorbate. Astrocytes therefore act as neuronal protectors, defending the neurons against free radicals.

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Astrocytes in Elderly Individuals These astrocytes have large cell bodies and robust protrusions; particularly those found in the neurofilaments (NFL). In a group of subjects aged over 60 years, the astrocytes showed a higher glial fibrillar acidic protein (GFAP) immunoreactivity with respect to the younger subjects, particularly in the NFL. This observation has been confirmed by electron microscopy, which showed a higher density of glia filaments (formed by GFAP) in the astrocyte cytoplasm of the elderly group. In the NFL, the lack of GFAP(+) signal between the astrocyte bundles indicates that in this layer the astrocytes lose their protrusions. Occasionally, GFAP(+) organelles are found in the glial cells (CGL) and NFL—these correspond to decayed astrocytes. The perivasal astrocytes have few thin protrusions and form a thinner astroglial sheath than in younger individuals. Sporadically, reactive astrocytes are found. The honeycomb structure is not easily distinguished in the CGL, and the gaps in the astroglial plexus have variable forms (circular, square, rectangular). The dimensions are, however, larger than those in younger subjects in both zone A (nearer to the optic disk) and in zone B (closer to the periphery). The number of gaps in the astroglial honeycomb plexus in the CGL is lower in the 60-89 age group. This signifies that the gaps are larger due to the disappearance of astrocytes from the vessel walls and the astroglial protrusions that divide the gaps. The reduction in the number of astrocytes increases with age, as is shown by comparing people between 60 and 79 and those over 80. The comparison between young and elderly retinas has again shown that aging causes numerous changes to the retinal astrocytes. There is an increase in the number of intracytoplasmatic organelles (mitochondria, ribosomes, polyribosomes, wrinkled endoplasmatic reticulum) due to higher cellular activity, an increase in lysosomes and dense bodies (which increase also inside the Müller cells), an increase of the glial intermediate filaments, a thickening of the inner limiting membrane whose constituents are less homogeneous, and an increase in the space between the glial protrusions and the basal membrane of the inner limiting membrane.

Glial Cells in Elderly Individuals All glial cells have numerous lysosomes in their cytoplasm. The prolongations of the glial cells contain dense bodies, formed from incompletely digested myelin that causes cellular swelling. In some elderly retinas, astrocytes of large dimensions are found that have very elevated cellular activity and a higher density of intermediate filaments. This type of astrocytes is called reactive astrocytes. The function of the reactive astrocytes is to protect the neurons (in this case, the ganglion cells) from ischemia-producing neurotrophic factors, increasing the expression of antioxidant substances (i.e., glutathione, vitamin C) and increasing the production and transport of glucose. However, it has been observed that astrocytes are more vulnerable

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to oxidative damage during aging. In fact, as the years roll, the reactive astrocytes cause changes in the geometry and volume of the extracellular space that slows the diffusion of neuroactive substances. The extracellular space is not only the microenvironment of the nerve cells, but is also an important channel of communication between the neurons and astrocytes. The changes in the diffusion parameters, which arrive with aging, can bring on a disappearance of the transmission signals and increase the sensitivity of the nervous tissue to ischemia. The ischemia is due to an increase in the extra cellular acidity with an accumulation of potassium and other toxic substances (similar to glutamate) that damage the neurons.

Reactive Astrocytes Reactive astrocytes have an elevated number of organelles (secondary lysosomes and lipofuscin) and an elevated cellular activity. If exposed to visible light (400700 nm) at a high concentration of oxygen (70 mm Hg)—i.e. conditions ideal for the formation of free radicals— the reactive astrocytes (and therefore the lipofuscin contained within) can cause damage to the cellular proteins and the membrane lipids. The reactive oxygen species can cause damage to cellular and nuclear elements. The presence of high concentrations of toxic substances (glutamate) in the extracellular space (coming from the reactive astrocytes), causes a massive increase of hydrogen and potassium with an increased permeability of the cell membranes, worsened by free radicals, which makes the cell swell. This cellular edema causes the breakage of the intermediate filaments of the astrocytes and, therefore, the loss of GFAP immunoreactivity and finally cell death. This fact explains why a reduction of the number of astrocytes in the CGL is observed, and why a disappearance of the protrusions of the NFL astrocytes has been observed in the elderly. In elderly people, it has also been observed that the basal membrane of the inner limiting membrane is thicker than in the younger group. The increase in thickness impedes the interchange of substances between the retina and the vitreous humor that represents a reserve of glucose, amino acids, potassium and glutathione, and so on for the retina, and a deposit for degradation products.

Hypertrophic Astrocytes Madigan et al.11 have described that in retinas with age related macular degeneration (AMD), there are distended hypertrophic astrocytes on the internal surface of the retina. Many studies have been performed on the neovascular membrane that is found in AMD, produced by the migration of endothelial choroidal cells across the Bruch’s membrane in the subretinal space. None, however, have talked about epiretinal glial membranes. Many studies have shown that the epiretinal membranes may derive from inflammatory processes, retinal ruptures, or retinal vascular occlusions.

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The extended retinal ischemia in AMD causes the astrocytes to migrate into the vitreous humor, where they can find metabolic reserves. In this way, the vitreous humor guarantees the nutrition of the remaining astrocytes in the inner retina while the intercellular junctions between the astrocytes remain intact. It is not known what factors cause the astrocytes and Müller cells to migrate into the vitreous humor in AMD. It is not known if these membranes are dangerous—i.e., if they can, after traction, cause a detachment of the retina.

Aging of the Microglia The retinal microglia originate from the hematopoietic cells and enter the retina from the retinal margin and the optic disk through the blood vessels of the ciliary bodies and iris, and of the retina, respectively. The microglial precursors, which are found on the retina before the arrival of the vessels, are positive to some specific immune staining and express the CD45 marker, but are not positive for specific markers of the macrophages. A second category of microglial precursors, which express typical macrophage markers, migrate into the retina together with vascular precursors. These are localized around the blood vessels in the adult retina and are similar to macrophages or mononuclear phagocytes. The microglial cells are found in the outer plexiform layer, the external nuclear layer, the internal plexiform layer, the gangliar layer, and the nervous fiber layer of the retina in humans. The retinal macrophages are involved in the defense against viral, bacterial, and parasitic infection, in immunoregulation, in tissue repair, in the catabolism of neurotransmitters and hormones, and in the lipid turnover of nervous tissue. The microglias play an important role in the defense against microorganisms, in immune regulation, and in tissue repair. The occurrence of degenerative phenomena, but also normal aging, causes the conversion of the microglia from resting to reactive. Reactive microglias have the responsibility of consuming the debris and facilitating the regenerative processes. The morphology and localization of the microglia is not the same for all age groups. It has been seen that in newborn mice, the microglia cells are round and ameboid, with thick, squat psuedo-polipoid protrusions distributed in the ganglion and nervous fiber layers, while their morphology shows some age-related changes.

Lymphocytes Lymphocytes are cells responsible for acquired immune response. These are rather small cells with a poor cytoplasm. Two functional types of antigen-specific lymphocytes can be distinguished: B and T lymphocytes. Lymphocytes are mainly localized in bone marrow, in peripheral lymphoid organs, in mucous surfaces, and in the thymus. They can also be found in blood and lymph nodes. Both B and T

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lymphocytes recognize and bind only the activated antigen. Binding the antigen is necessary, but not enough to activate the lymphocytes. A second signal, coming from other cells, is also needed. For B cells, the second signal comes from T cells. For T cells, the second signal, known as costimulation, can be produced by three different types of cells: B lymphocytes, macrophages, and dendritic cells. B lymphocytes recognize the antigens that they find outside the cells and, once activated, produce specific antibodies against the antigen responsible for activation. T cells manage to recognize antigens generated inside cells to determine a different immune response, called cell-mediated. Once the development and maturation of T cells in the thymus is complete, they enter the bloodstream, migrate to the peripheral lymphatic organs and return to the bloodstream until they meet the antigen. To take part in the acquired immune response, the nonactivated or natïve T cells must first be induced to proliferate and then differentiate in cells capable of contributing in the removal of pathogens. These cells are called effectors-armed T cells. These can be divided in three groups: T cytotoxic cells, lymphocytes (CD8+) that kill the infected cells, and the inflammatory T cells (CD4+ [T helper 2]) that, by secreting IL-4, IL-5, IL-6, andIL-10, activate antigen-specific B lymphocytes that produce antibodies.12 Following an inflammatory process caused by viral or bacterial infections, or damage through injury or altered functionality or complement activation, the lymphocytes accumulate in the inflammatory sites and release cytokines that regulate the time and amplitude of the inflammatory-immune mediated response. In this manner, they destroy intracellular pathogens by killing infected cells and by activating macrophages, but also by destroying extra cellular pathogens through the activation of B cells.13

Macrophages Macrophages are cells with phagocytic activity, coming from the transformation of circulating monocytes. The macrophage is located in various tissues: the liver (Kupffer cells), connective tissue (histiocytes), and nervous tissue (microglia). In the case of destructive or necrotic tissue lesions, the macrophages appear in about 48 hours. These cells come partially from tissue macrophages, but most come from mononuclear cells in the bloodstream. When a macrophage finds the pathogenic agent, it consumes and destroys it. Macrophages release lysosomal enzymes in the phlogistic sites. During phagocytosis, lysosomal enzymes are released in the extra cellular area and, thanks to the wide spectrum of their enzymatic activity, they can degrade a vast gamma of biological substrates, among which are the various components of connective tissue. Among the lysosomal components of phagocytosis, lactoferrin plays a triple role: this protein, together with bacteriostatic activity, increases the function of NK lymphocytes and promotes the production of cytokines, lysozime, A phospholipase, mieloperoxidase, and neutral proteases (serine-protease and metal protease)—all enzymes that are able to degrade the components of interstitial matrix (type IV collagen, elastin, proteoglicans). The endothelial

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Fig. 7.1 Transmission electron microscopy picture of human iris. Some nerve fibers (nf), containing numerous pre-synaptic vesicles, appear in photo 1. The anterior epithelium (E) or myoepithelium in normal conditions contains numerous mobile cells. The smooth muscle cells (sm) contain numerous microfilaments (mf). The stroma (st) in aged people contains numerous electron dense substances (granules of lipofuscin, proteoglycans, etc.) (Magnification 8500x)

cells of the trabeculum are partially differentiated cells and among other functions they have macrophage activity (see Fig. 7.1, personal observation).

References 1. William D, Willis J (1995) Il sistema nervoso. In: Berne RM, Levy MN (eds.) Fisiologia. Milano, Casa Editrice Ambrosiana. p l01-117 2. Kandel E (2003) Principi di Neuroscienze. Milano, Editrice Ambrosiana, p 20-22 3. Ascenzi A (1997) Sistema nervoso. In: Ascenzi A, Mottura G (eds) Anatomia Patologica, vol. 2. Torino, UTET, p1184-1186 4. Barr M, Kiernan JA (1995) Cellule del sistema nervoso. In: Barr M, Kiernan JA (eds) Anatomia del sistema nervoso umano. McGraw-Hill, Milano, p 26-30 5. Burt AM (1996) Trattato di neuroanatomia. Piccin, Milano, p 50-52 6. Monesi V (1998) Tessuto nervoso e neuroglia. In: Monesi V (ed) Istologia. Piccin, Padova, p 830-834 7. Nobak CR, Strominger NL, Demarest RJ (1999) La neuroglia. In: Nobak CR, Strominger NL (eds) Sistema nervoso. Piccano, Milano, p 25-27 8. Fazio C et al (2003) Neuroanatomia. SEU, Roma, p 550-557 9. Vernadakis, A, (1986) Changes in astrocytes with aging. In: Federoff S, Vernadakys A (eds) Biochemistry, Physiology and Pharmacology of Astrocytes. Academic Press, Orlando, USA, p 377-407 10. Chen L, Yang P, Kijlstra A, (2002) Distribution, markers, and functions of retinal microglia. Ocul. Immunol. Inflamm. 10:27-39 11. Madigan WP, Wertz D, Cockerham GC, Thach AB (1994) Retinal detachement in ostegenesis imperfecta. J. Pediatr. Ophthalmol Strabismus 31:268-269 12. Janeway C, Travers P (1996) Immunobiologia. Piccin, Padova

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13. Salerno A (1996) Le immunodeficienze. In: Pontieri M (ed) Patologia generale. Piccin, Padova p 567-612

Other Linked and Recent References 1. McMenamin PG, Holthouse I (1992) Immunohistochemical characterization of dendritic cells and macrophages in the aqueous outflow pathways of the rat eye. Exp Eye Res. August 55(2):315-24 2. Camelo S, Shanley AC, Voon AS, McMenamin PG (2004) An intravital and confocal microscopic study of the distribution of intracameral antigen in the aqueous outflow pathways and limbus of the rat eye. Exp Eye Res 79(4):455-64 3. McMenamin PG, Crewe J (1995) Endotoxin-induced uveitis. Kinetics and phenotype of the inflammatory cell infiltrate and the response of the resident tissue macrophages and dendritic cells in the iris and ciliary body. Invest Ophthalmol Vis Sci. 36(10):1949-59 4. Yang P, Das PK, Kijlstra A (2000) Localization and characterization of immunocompetent cells in the human retina. Ocul Immunol Inflamm. 8(3):149-57 6. Butler TL, McMenamin PG (1996) Resident and infiltrating immune cells in the uveal tract in the early and late stages of experimental autoimmune uveoretinitis. Invest Ophthalmol Vis Sci. 37(11):2195-210 7. Diaz-Araya CM, Madigan MC, Provis JM, Penfold PL (1995) Immunohistochemical and topographic studies of dendritic cells and macrophages in human fetal cornea. Invest Ophthalmol Vis Sci. 36(3):644-56 8. Becker MD, Planck SR, Crespo S, Garman K, Fleischman RJ, Dullforce P, Seitz GW, Martin TM, Parker DC, Rosenbaum JT (2003) Immunohistology of antigen-presenting cells in vivo: a novel method for serial observation of fluorescently labeled cells. Invest Ophthalmol Vis Sci. 44(5):2004-9 9. Poulter LW, Campbell DA, Munro C, Janossy G (1986) Discrimination of human macrophages and dendritic cells by means of monoclonal antibodies. Scand J Immunol. 24(3):351-7 10. Takase H, Sugita S, Rhee DJ, Imai Y, Taguchi C, Sugamoto Y, Tagawa Y,Nishihira J, Russell P, Mochizuki M (2002) The presence of macrophage migration inhibitory factor in human trabecular meshwork and its upregulatory effects on the T helper 1 cytokine. Invest Ophthalmol Vis Sci. 43(8):2691-6 11. Romeike A, Brugmann M, Drommer W (1998) Immunohistochemical studies in equine recurrent uveitis (ERU). Vet Pathol. 35(6):515-26 12. Weinstein BI, Iyer RB, Binstock JM, Hamby CV, Schwartz IS, Moy FH, Wandel T, Southren AL (1996) Decreased 3 alpha-hydroxysteroid dehydrogenase activity in peripheral blood lymphocytes from patients with primary open angle glaucoma. Exp Eye Res. 62(1):39-45 13. Ueno H, Tamai A, Iyota K, Moriki T (1989) Electron microscopic observation of the cells floating in the anterior chamber in a case of phacolytic glaucoma. Jpn J Ophthalmol. 33(1):103-13 14. Latina M, Flotte T, Crean E, Sherwood ME, Granstein RD (1988) Immunohistochemical staining of the human anterior segment. Evidence that resident cells play a role in immunologic responses. Arch Ophthalmol. 106(1):95-9 15. Lutjen-Drecoll E, Kaufman PL, Barany EH (1977) Light and electron microscopy of the anterior chamber angle structures following surgical disinsertion of the ciliary muscle in the cynomolgus monkey. Invest Ophthalmol Vis Sci. 16(3):218-25

Chapter 8

Age-Related Diseases of the Vitreous Curtis E. Margo, MD, MPH

Abstract The vitreous gel is a transparent, hypocellular tissue that effectively transmits light with negligible scatter or energy absorption. Although it is relatively resilient to age-related wear and tear, the vitreous is susceptible to injury from inflammatory cells and substances that breech the blood-retinal barrier. Over the course of a lifetime, the vitreous undergoes a variety of poorly understood, degenerative changes that lead to liquefaction—also referred to as syneresis. Vitreous syneresis is the most common predisposing factor for posterior vitreous detachment, which places a patient at risk for retinal detachment. While visionthreatening complications from deposits within the vitreous are uncommon (e.g., amyloid), the formation of vitreous membranes inflict considerable ocular morbidity. Vitreous membranes are a manifestation of a heterogeneous collection of disorders that share a final common pathway. Proliferative vitreoretinopathy (PVR) is the term applied to the uncontrolled growth of fibroglial membranes associated with rhegmatogenous retinal detachments. The most common reason for failed retinal reattachment surgery, PVR appears to exhibit an exaggerated reparative response to injury. Keywords amyloidosis, asteroid hyalosis, synchysis scintillans, proliferative vitreoretinopathy (PVR), vitreous membranes, syneresis, retinal detachment, posterior vitreous detachment (PVD).

Introduction The vitreous is the largest single tissue of the eye, measuring approximately 4 mL in volume, but it is also the least energy demanding. Bound anteriorly by the posterior surface of the lens, laterally and posteriorly by the retina, and axially by the canal of Cloquet, the vitreous has modestly strong attachments at the ora serrata, the optic nerve head, and along some larger caliber retinal vessels.1 Physically, the vitreous is a gel. It behaves like a noncompressible liquid, yet it is able to maintain a three-dimensional shape without support. The vitreous is the major connective tissue in terms of volume in the eye, and functions both as a From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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transparent optical media and as a structural support for the outer tunics of the globe. The vitreous is one of the least cellular tissues in the body, being composed of 99 percent water and several key macromolecules.

Vitreous Composition The two major components of mammalian vitreous are collagen and glycosaminoglycans. Collagen is the major structural macromolecule of the insoluble phase of the vitreous gel.2 Most vitreous collagen is type II, characterized by three alpha polypeptide chains twisted into a right-handed helix.3 Although vitreous collagen shares many physiochemical properties with that of type II collagen in cartilage, there are differences in their degree of hydroxylation.4 Bovine collagen of the vitreous contains significantly more hydroxylated residues of lysine and proline compared to bovine cartilage of the nasal septum.4 Lesser amounts of types V, IX and XI collagen have also been identified in mammalian vitreous.4,5,6 While their roles as nonfibrous collagen are not well understood, type IX collagen fibers appear to lie in close proximity to the surface of type II collagen fibers where they likely affect the three-dimensional interactions with glycosaminoglycans.7,8 The thin, heterotypic fibrils of type IX collagen appear to bond noncovalently to chondroitin sulfate, which in turn, may help maintain appropriate spacing between other larger collagen fibrils.9 When peptide sequences from types V and XI collagen of vitreous are compared, their close homology suggests that they are members of a single collagen family rather than distinct types of collagen.8 The network of collagen fibers in the vitreous is designed to loosely envelope soluble macromolecules collectively known as glycosaminoglycans. Hyaluronic acid, a long, unbranched polymer of repeating sugar units, is the major soluble macromolecule of the vitreous.10,11 When the carboxy groups of its gluconic acid are dissociated in solution, the macromolecule becomes a polyanion. The configuration of hyaluronic acid allows it to hold a large volume of water compared to its weight. Athough hyaluronic acid is dependent on several physical-chemical variables for its final molecular weight, its molecular weight can range upward to one million.12 Once hyaluronic acid is removed from the vitreous or destroyed, it is not replaced. Chondroitin sulfate is the second important high molecular weight glycosaminoglycan of the vitreous. Found at lower concentrations than hyaluronic acid in most mammalian vitreous bodies, chondroitin sulfate differs from hyaluronate in its ability to covalently bind to noncollagenous core proteins.13,14 While both hydrated glycosaminoglycans create highly viscous fluids, they interact differently with their surrounding lattice of collagen fibrils. The soluble proteins are the least well-characterized component of the vitreous, but may prove to exert considerable influence over the interaction of the larger macromolecules. In bovine vitreous, there are small amounts of albumin and globulin (between 0.4 to 0.8 µg/ml). The concentration of serum proteins in the vitreous is

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kept low by the blood-retinal barrier.15 While any breakdown in the blood-retinal barrier allows serum proteins to enter the vitreous with greater ease, the gel itself acts to minimize diffusion of these molecules. Although there is limited information on how the collagen components maintain spacing to form a stable meshwork, water molecules are stabilized or trapped in the latticework of collagen by the two major glycosaminoglycans. A similar mechanism for hydrating cartilage exists, although the amount of water molecules trapped by hyaluronic acid in cartilage is much less than in vitreous. Bos et al.16 observed three morphologically distinct types of single fibrils that form links within the fibrillar network of the vitreous. Hyaluronic acid and chondroitin sulphate aggregrate individually and with one another as they attach to collagen fibrils.17 In this way, the glycosaminoglycans create a potentially infinite meshwork in which water is entwined.14 The negatively charged hyaluronic acid interacts with the collagen lattice at specific sites along the fiber referred to as globules.18 Although there is no apparent direct attachment between the two phases of gel (soluble/insoluble), ultrastructural studies reveal an aggregation of soluble vitreous at the globular regions of the collagen fiber.19 When hyaluronic acid is enzymatically digested within the vitreous, it also destroys the thin filamentous links between the larger type II collagen fibrils, suggesting that hyaluronic acid is necessary for their integrity.15 The greatest concentration of collagen and soluble proteins exist in the vitreous cortex. The hypocellular vitreous contains a small number of cells called hyalocytes, found predominantly in the cortex. Their overall morphology resembles a macrophage, but there is evidence that they can become functional fibroblasts.20 The life span of hyalocytes within the vitreous is relatively brief—approximately one week.11 While the function of the hyalocyte is poorly understood, researchers speculate that the cell plays an important role in intraocular homeostasis. The loose, and seemingly fragile, vitreous gel displays an array of amazing physical properties. Its optical clarity keeps light scatter to a minimum, while its viscoelasticity offers considerable support to the lens without causing mechanical distortion of its surface. Along with the blood-brain barrier, the vitreous acts to inhibit the transgression (diffusion) of foreign macromolecules through the interior of the eye.

Vitreous Architecture The optical transparency that allows the vitreous to effectively transmit the visible portion of the light spectrum to the retina without adsorption or scatter, also befuddles study of its gross architecture. After the introduction of the slit lamp, there have been a variety of interpretations of vitreous organization. Based on dissections of human eyes, Eisner concluded that the vitreous consisted of concentrically packed funnels separated by delicate fibrous membranes.21 Worst used India ink to study the organization of the vitreous and found tracks or

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membranes similar to those described by Eisner.22 In the process of reporting his findings, he introduced the terms cistern and bursa to describe the spaces and compartments created by these membranes.23 In a series of elegant studies using dark-field slit illumination, Seebag and Balzas showed that the vitreous contains fine fibers that run in an anterior-posterior direction and in parallel to Cloquet’s canal.24 The fibers seen by this technique probably represent packed collagen fibers caused by the exclusion of hyaluronic acid.25 The vitreous base refers to a 3-5 mm band of modified vitreous that overlaps the junction of the inner peripheral retina and ciliary body. Because the vitreous base represents the strongest adhesion between the vitreous and adjacent tissue, it plays an important role in the mechanics of several vitreo-retinal diseases. Because the ability to objectively measure the strength of an adhesion between vitreous and adjacent tissue is limited, terms used to describe the mechanical strength of adhesion need to be interpreted with caution. For the most part, the strength of vitreoretinal adhesion has been inferred from various clinical and morphological observations, and never measured directly. Seebag dissected the vitreous body from the retina in 59 human eyes from donors aged from 33 weeks of gestation to 94 years of age.26 The vitreous gel peeled easily from the inner retina in all 44 eyes from persons older than 21 years. In six of the 15 eyes (40%) from donors under the age of 20, portions of the inner retina remained adhered to the vitreous along the temporal arcades, macula, and peripapillary retina. Electron microscopy confirmed fragments of Müller’s cells clinging to the vitreous, indicating that the strength of the bond between the vitreous and retina exceeded the intrinsic strength of the cell walls of Müller’s cells. The method of attachment between vitreous and retina is not entirely clear, although thin collagen fibrils from the vitreous have been observed inserting into the internal limiting membrane.27,28

Aging The molecular basis of vitreous aging is not completely understood. Central to the aging process is gel liquefaction, which, based on post-mortem studies, is a common phenomenon noted in more than 60 percent of the eyes of persons between 80 to 89 years of age.29 Between the ages of 45 and 50, a clinically detectable decline in the ratio of gel-to-liquid vitreous begins that continues through the tenth decade of life.30 In vivo observations of vitreous morphology using ultrasonography reveal that these changes begin much earlier in life. In a study of more than 400 human eyes, Oksala detected abnormal echographic reflections from the gel-liquid interface in persons with normal slit lamp examinations.31 In a large post-mortem study by Balazas and Flood, evidence of liquefaction was detected in patients as young as four years of age. The authors estimated that approximately 12 percent of vitreous is liquefied by the time the eye attains its adult size at 18 years.32 During each

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decade of life, there is a further decrease in the proportion of gel to liquid. By 90 years of age, there is (on the average) more liquid volume to gel volume.26 The transition from gel to liquefaction can be either abrupt or gradual, with a proportional decline in both collagen fibers and soluble vitreous found in the transitional regions.33 In a study of 13 human eyes by light and electron microscopy, Los et al.,28 found that there are neither cellular remnants nor fragments of collagen in the transitional tissue. The aging vitreous shows rather rapid loss of type IX collagen, along with its chondroitin sulfate side chains. Although type IX collagen is a minor component of the insoluble matrix, it may serve an important role in protecting the surface of type II collagen from exposure.34 It has been proposed that type II collagen may become less resistant to damage and more susceptible to dissolution following the loss of contiguous type IX collagen.29 Laboratory models of vitreous syneresis have shed only limited light onto the mechanism of vitreous degeneration because almost all types of injury, no matter how trivial, induce liquefaction. The introduction of almost any type of foreign substance into the vitreous initiates some degree of syneresis. One of the most potent stimuli for liquefaction is acute inflammation, from any source. Miller et al.35 injected profluoropropaine gas into the vitreous of primates, creating a large syneresis cavity. Despite the size of the cavity, the shell of residual vitreous remained intact, suggesting that size alone may not be a major factor in the development of posterior vitreous detachment. Although the cause of age-related syneresis is uncertain, there is laboratory evidence that photodegradation of hyaluronic acid contributes to the process.36 This process may be mediated by light-induced, free-radical formation, which could damage vitreous collagen and hyaluronic acid over time.37

Detachment of the Posterior Vitreous A variety of different conditions, from trauma to inflammation, will cause the separation of the vitreous from its posterior attachments to the surface of the retina and optic nerve. The most common underlying cause in the general population is aging and age-related syneresis of the vitreous. Like syneresis alone, spontaneous posterior vitreous detachment (PVD) is an age-related phenomenon whose fundamental biomechanisms still await clarification. The clinical importance of PVD resides in its association with retinal tears.38 The prevalence of PVD increases both with advancing age and axial length of the eye. In the era of intracapsular cataract surgery, PVD was a common sequel. Partial or complete PVD has been described in 93 percent of autopsy eyes that have undergone prior intracapsular cataract extraction.39 In a study of 61 postmortem eyes, the concentration of hyaluronic acid in the vitreous was greater in globes having no PVD than those with total PVD, although the concentration of hyaluronic acid in gel and liquefied portions of vitreous was similar.40

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The role of precortical vitreous syneresis in the pathogenesis of PVD is unclear. In an autopsy study of 84 eyes, Kishi and Shimizu examined the posterior vitreous for precortical accumulation of liquefied vitreous.41 The posterior wall of this pocket consisted of a thin layer of vitreous lying just anterior to the retina. Its anterior wall was the formed vitreous gel, which in some eyes mingled with lacunae of synergetic fluid. The vitreous pocket was present among the 48 eyes with no PVD or an incomplete PVD, but was found in only 19 of 36 eyes with a complete PVD. The attenuated region of vitreous gel immediately anterior to the retina (posterior wall of precortical pocket) appeared to separate from the gel component of the vitreous in adult eyes without creating a PVD.42 In a series of routine clinic patients, the prevalence of PVD varied by age with 28 percent of persons overall (N = 100) demonstrating positive findings.43 That proportion increased to 53 percent in patients over the age of 50. The ability to detect a PVD, however, is dependent on the method of examination, including the type of lens.44 During the time of intracapsular cataract extraction, the prevalence of PVD was high.45 Base on clinical and autopsy studies, a minority of spontaneous PVDs appear to present with the sudden onset of flashing lights and floaters (see Fig. 8.1). This discrepancy may be due to the fact that the process of vitreous separation from the retina probably occurs slowly in many patients where it goes unrecognized. The process of separation appears to begin in the macula, where the concentration of

Fig. 8.1 Posterior vitreous detachment in the eye of a 30-year old person who died in a motor vehicle accident. The relatively dense posterior cortical vitreous (arrows) can be seen in the region of the ora seratta (arrowhead). Fixation in formalin, which is hypertonic to vitreous, further collapses the gel through dehydration. A small cyst is noted in the pars plana.(hematoxylin-eosin, 30x original magnification)

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insoluble and soluble vitreous components is the least.46 The abrupt separation of the posterior vitreous face from the retina and optic nerve head likely occurs when a rush of liquefied vitreous pours through a rent in the cortical vitreous. Much like a small hole in a burlap sandbag, the watery vitreous rushes through the space dissecting the remaining posterior vitreous surface from the underlying retina and optic nerve. The force generated by the flow of fluid is likely great enough to break any vitreous attachments to the retina or to superficial blood vessels. A full-thickness hole could conceivably form if the force between the vitreous and retina exceeds that holding the retina to underlying pigment epithelium. The prevalence of retinal tear in persons with acute PVD ranges from 8 percent to nearly 50 percent.47,48,49,50,51,52 This variation reflects differences in examination technique and nature of referral practice. In a general ophthalmology practice, the rate of retinal tear found with symptoms of flashes and floaters runs closer to 10 or 15 percent.53,48,54,55 The type of floater also correlates with the likelihood of retinal tear. Multiple small floaters (diffuse or localized) tend to correspond to red blood cells or pigmented cells, which heightens the risk of retinal break.56 In patients who present with a dense vitreous hemorrhage, approximately two-thirds have a retinal tear and one-third have more than a single tear.57 Of 155 patients with symptoms of acute flashes and/or floaters, 11 percent developed similar symptoms in their other eye within two years.47 The mechanism of retinal tear is related to points of firm attachment between the collapsing vitreous and stationary retina. These critical intersections occur along major vessels, at lattice degeneration, enclosed ora bays, and retinal tufts.58 Other sites of firm attachment, like the juxtapapillary retina and vitreous base, are not prone to full-thickness tears. Vitreous traction can result in avulsion of a retinal vessel in the absence of a full thickness retina break.59,60 Failure to relieve the traction can result in recurrent vitreous hemorrhages.52 Because the identification of retinal breaks by ophthalmoscopy is fallible, and because of the possibly of a new break developing late, some authorities recommend a follow-up fundus examination be performed. In one study of 189 eyes with no retinal tear found after initial symptoms of PVD, three new tears were found (n= 169 [1.8%]) at the six-week follow-up visit.61 Because there are no means of altering the physio-chemical or mechanical factors leading to PVD and rhegmatogenous retinal detachment, the most effective method of reducing the risk of vision-threatening retinal detachment is to screen patients with symptoms of acute PVD.62 The indications for treating symptomatic retinal breaks are not uniformly agreed upon, and have been based on expert opinion and consensus opinion.63 The goal of treating retinal breaks is to establish a firm chorioretinal adhesion surrounding the retinal defect. The strongest evidence for preventative treatment exists for symptomatic horseshoe tears and retinal dialysis. Prophylactic therapy for symptomatic horseshoe tears reduces the risk of retinal detachment from approximately 50 percent to 5 percent.64,65,66 Although retinal dialyses are not caused by PVD, they are occasionally found incidentally when examining a patient for symptoms of acute PVD.67 The issue over whether or not to treat asymptomic retinal tears is complex, because the natural history of these

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conditions must be inferred indirectly. While there is little evidence to support the prophylactic treatment of operculated tears or atrophic holes, the treatment of lattice degeneration is more controversial.68 Symptomatic operculated tears have a low risk, if any, of progression to retinal detachment. Those that do may be caused by vitreous adherent to retina in the area of the break.69,70 Although lattice degeneration generally increases the chance of retinal detachment, there is insufficient evidence-based data to support prophylactic treatment of asymptomatic lattice degeneration.71 Given that the annual incidence of rhegmatogenous retinal detachment is between 10 to 15 cases per 100,000, and the prevalence of lattice degeneration in the general population is 6 to 8 percent, an individual with lattice degeneration has a relatively low lifetime risk of retinal detachment.72,73,74,75

Amyloidosis The majority of patients with vitreous amyloid deposits present after the age of 40, although the disease is generally considered a genetic disorder—either inherited as an autosomal dominant trait or through a spontaneous mutation of the transthyretin gene.76 Amyloidosis refers to a heterogeneous group of disorders characterized by the deposition of various abnormal proteins (i.e., amyloid).77 The amyloids are biologically insoluble and poorly digestible aggregates of low-molecular weight proteins with beta-pleated sheet configurations. The three-dimensional orientation of these aggregates imparts certain highly specific physical features to amyloids. The most clinically useful properties are its affinity for Congo red and birefringence under polarized light.78 Amyloid is dichroic to green light when stained with Congo red, due to the preferential transition of light along certain planes of the molecule. The classification of amyloidosis has changed as new insight into its pathogenesis has been made. Vitreous amyloidosis is essentially a manifestation of a hereditary form of the disease known as familial amyloidotic polyneuropathy (FAP).79 Vitreous amyloid is not a feature of other systemic forms of amyloidosis. Although FAP has been divided into four types, only two of the four types (types I and II) develop vitreous deposits. The disease is caused by the accumulation of mutant transthyretin protein. It is transmitted in an autosomal dominant fashion, with similar patterns of phenotypic expression among both homozygous and heterozygous affected individuals. The genetic mutation that gives rise to FAP is within the chromosome region 18q11.2-q12.1.80 Because of the variable clinical expression of FAP, the few reports of isolated or nonfamilial amyloid deposits of the vitreous may represent sporadic mutation of the transthyretin gene.81,82,83 Transthyretin is a transport protein for both thyroxin and retinal-binding protein. It carries almost all of the circulating retinal-binding protein, but complexes with less than 1 percent of thyroxin.84 Also referred to as prealbumin, transthyretin carries nearly 40 percent of circulating retinal-binding protein. The most frequently reported mutation of transthyretin giving rise to amyloidosis involves the substitution of a methionine for valine at position 30.85

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Vitreous amyloid deposits accumulate at a relatively slow rate, with the onset of symptoms beginning over the age of 40 or 50.86 Floaters and decreased vision are invariably present, usually bilaterally, although substantial asymmetry might be noted. The rate of progression to significant visual loss in 18 patients with vitreous amyloid ranged from several months to several years.87 Clinically, the vitreous opacities have a glass wool appearance (see Fig. 8.2). As the density of vitreous deposits increase, visualization of the retina becomes more difficult. There are few other, if any, ocular manifestations of FAP, with the exception of sheathing of retinal vessels that are noted occasionally.88 Vitrectomy can be used to diagnose the disease when suspected clinically, or for treatment of visual symptoms. Confirmation of the diagnosis is based on the typical staining properties of amyloid with Congo red and behavior under polarized light. Ultrastructural examination of the vitreous biopsy is another diagnostic option, looking for characteristic 7-10 nm amyloid fibrils. These fibrils, however, may be difficult to distinguish from vitreous fibrils with diameters ranging from 10 to 15 nm.89 Clinical symptoms following vitrectomy may signal the reaccumulation of amyloid deposits (see Fig. 8.3).90 Nearly a quarter of patients treated surgically require a second vitrectomy for recurrent amyloid.91 Progressively worsening opacity of residual vitreous has led surgeons to remove as much cortical vitreous as safely possible.92 Systemic therapies for amyloidosis have been generally ineffective, but novel treatments are being developed.93

Fig. 8.2 Vitreous amyloid visualized at the slit lamp just posterior to the lens. The mass of amyloid fibrils appears like glass wool (Photograph courtesy of Scott Pautler, MD)

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Fig. 8.3 The eye from a man with familial amyloidosis obtained at autopsy. Arteriole in the inner retina is surrounded by amyloid (arrow). Extracellular deposits of amyloid are also present in the inner retina adjacent to the internal limiting membrane (arrowheads) and in the cortical vitreous.(hematoxylin-eosin, 290x original magnification) (Glass slide courtesy of Ted Dryja, MD)

Asteroid Hyalosis First described as asteroid hyalitis by Benson in 1894,94 this noninflammatory condition is characterized by variable numbers of buff-colored deposits suspended in the vitreous. The average age of diagnosis in 217 patients with this condition was 64 years.95 Approximately three-quarters of cases occur unilaterally. In a consecutive clinical evaluation of over 12,000 patients, the prevalence of asteroid hyalosis was 0.83 percent.96 Among the patients in this series, there was a positive association between the vitreous deposits and diabetes, atherosclerosis, hyperopia, and hypertension. The biological basis for these associations, however, is far from clear. Several earlier studies failed to find an association between asteroid hyalosis and systemic disease.48,97 In routine hematoxylin and eosin-stained sections, asteroid bodies are roundto-oval amphophilic masses that are visible under polarized light (see Fig. 8.4). They stain positive with alcian blue. Histochemical analysis reveals neutral fats and phospholipids.98 Chemical analysis for inorganic substances has disclosed calcium, phosphorus, and trace amounts of sulfur.99,100,101 Electron microscopic studies have shown twisted and intertwined electron-dense multilaminar membranes. Smaller asteroid bodies were more amorphous and contained fewer multilaminar membranes.53,102,103

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Fig. 8.4 Asteroid bodies of the vitreous appear as gray amorphous deposits when stained with hematoxylin-eosin (left). They literally sparkle like diamonds when viewed under polarized light (right) (hematoxylin-eosin, 390x original magnification)

Given the striking clinical appearance of asteroid hyalosis, there are relatively few clinical symptoms. Most patients are unaware of floaters and few have any measurable decline in visual function due to asteroid hyalosis. Vitrectomy has been used to remove the vitreous deposits in persons who are symptomatic, but the clinical indications for elective surgery are not clearly established. Occasionally, asteroid bodies are found in epiretinal membranes where the mild foreign body reaction they incite may aggravate the severity of the epiretinal membrane (see Fig. 8.5).

Synchysis Scintillans First described in the late nineteenth century, synchysis scintillans refers to the glistening crystals present in the liquefied vitreous. Clinically, the highly reflective crystals are dispersed with movement of the eye. Over time, the crystals will settle to the gravity-dependent portion of the vitreous cavity. Unlike asteroid hyalosis in vitreous gel, synergetic fluid cannot support the crystals of synchysis scintillans. Histologically, the crystals cannot be seen directly because the solvents used in processing the tissue dissolve them. They leave, however, slit-like spaces where

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Fig. 8.5 An asteroid body is inciting a foreign-body giant cell reaction. This focal inflammatory cell reaction is present within an idiopathic epiretinal membrane that was removed in surgery (hematoxylin-eosin, 390x original magnification)

cholesterol once existed. Also known as cholesterolosis bulbi, the presence of synchysis scintillans is a secondary manifestation of intraocular hemorrhage or inflammation.104 Though synchysis scintillans is not strictly a disorder of aging, the underlying causes of intraocular hemorrhage and inflammation steadily increase with age, as does the prevalence of cholesterolosis bulbi. The cholesterol liberated from degenerating red blood cells frequently incites an inflammatory reaction, which includes a foreign body reaction. On histological inspection of globes with synchysis scintillans, there is usually evidence of localized hemosiderosis from remote hemorrhage (also see vitreous hemorrhage).

Vitreous Membranes Vitreous membranes describe the final common pathway of a number of primary ocular diseases—including surgical and accidental trauma. Though arising from diverse causes, these membranes have in common the ability to interfere with the optical transparency of the vitreous and disrupt retinal function through a variety of mechanisms. While there is no standard nomenclature of vitreous membranes, classification is usually based on underlying etiology (diabetic, traumatic, etc.), anatomic location (anterior, epriretinal, cyclitic, etc.), or morphology (acellular, cellular, pigmented, etc.).

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The pathophysiological mechanisms involved in vitreous membrane formation overlap those of wound healing. The vitreous normally inhibits cellular proliferation and growth of blood vessels.105,106,107,108,109,110 Loss of inhibitor function and/or the overriding influence of growth promoters are likely involved in membrane formation.111,112 Diabetic vitreoretinopathy—the prototypical vitreous-membrane disorder—up-regulates a number of growth factors. The majority of putative growth factors, both for stromal and vascular cells, likely originate from the serum. Most membranes are composed of several cell types and, unlike reactive membranes in other tissues, are often composed of both glial cells and fibroblasts. Depending on location and primary injury, the proportion of glial cells (including Muller), fibroblasts, macrophages, lens epithelial cells, pigment epithelial cells, and ciliary non-pigmented epithelial cells will vary. Cyclitic membranes are often the sequela to endophthalmitis, uveitis, or anterior segment trauma. They are notoriously difficult membranes to manage clinically because much of their bulk is hidden from view behind the iris leaflets at their site of origin in the posterior chamber (see Fig.8.6). When mature, the membrane extends from the ciliary body centrally to occlude the visual axis like a diaphragm. Typically well-endowed with fibroblasts and thick bundles of collagen, cyclitic membranes have powerful contractile properties. Myofibroblasts within the membrane contribute to its contractile strength that may secondarily detach peripheral retina, displace the crystalline lens or pseudophakic implant, and promote uveal effusion and hypotony.

Fig. 8.6 A relatively young cyclitic membrane is present in the anterior vitreous, forming between the peripheral retina (arrows) and lens capsule (arrowheads). Delicate spindle cells, small caliber vessels and modest amounts of collagen make up the membrane (hematoxylin-eosin, 160x original magnification)

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Vitreous membranes following trauma, retinal detachment, and complicated diabetic retinopathy usually grow on surfaces and do not invade the vitreous gel directly. Membranes that occupy the vitreous cavity likely use the pre-existing posterior vitreous surface as a scaffolding to grow on as the vitreous body collapses (see Fig. 8.7). They tend to grow parallel to the surface of the posterior hyaloid or tangential to the interface created by transvitreal trauma (see Fig. 8.8). Ultrastructural studies have shown that more than 90 percent of these membranes contain myofibroblasts.113 The contractile properties of the myofibroblast can exert a deleterious affect on the neurosensory retina, including tractional retinal detachment (see Fig. 8.9). Nearly half of epiretinal membranes developing after retinal detachment contain retinal pigment epithelium (RPE). The contraction mediated by myofibroblasts may secondarily induce fibrocytes within the membrane to produce more collagen. Old or so-called burned out membranes may consist of nothing more than mature collagen (see Fig. 8.10). The presence of blood within the vitreous further complicates the healing process by inciting an even greater inflammatory reaction (see Fig. 8.11). The breakdown products from red blood cells not only provoke a foreign body reaction on a macroscopic level, they trigger a multitude of inflammatory cascades on a biochemical level. Hemosiderin-laden macrophages and so-called cholesterol granulomas are frequent findings in end-stage membranes.

Fig. 8.7 Advanced proliferative membranes in the eye of a diabetic patient with extensive tractional retinal detachment. The thick fibrovascular bundles of tissue are adhered to the retina and have used the posterior hyaloid as a scaffold on which to grow

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Fig. 8.8 A thin epiretinal membrane grows on the inner surface of the retina. A few spindleshaped nuclei (arrows) can be seen (hematoxylin-eosin, 290x original magnification)

Fig. 8.9 A diabetic tractional retinal detachment of the macula shows the relationship of internal limiting membrane (arrows) and posterior hyaloid (arrowheads) (hematoxylin-eosin, 290x original magnification)

Idiopathic epiretinal membrane (ERM) is considered a distinct clinicopathologic entity, but it has shared features with other forms of periretinal membranes (see Fig. 8.12). Usually regarded as an age-related degenerative process of the retinal basal lamina, ERM is characterized by small dehiscences in the basement membrane

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Fig. 8.10 A thick collagenous epiretinal membrane in the eye of a diabetic patient. This so-called burnt-out diabetic membrane followed panretinal photocoagulation. The membrane rests on the internal limiting membrane (arrows) and contains only larger caliber vessels (hemotoxylin-eosin, 290x original magnification)

Fig. 8.11 A complex epiretinal membrane containing red blood cells, fibrin, inflammatory cells, and spindle-shaped cells rests on the surface of the retina (arrows). There is evidence that the addition of red blood cells and fibrin into a pre-existent epiretinal membrane enhances its growth (hematoxylin-eosin, 290x original magnification)

of the neurosensory retina that allow glial cells access to the inner surface. The transmigration of RPE through the retina has been documented on optical coherence tomographic studies.114 Once on the epiretinal side of the basement membrane, glial cells can proliferate unimpeded. This rather simple view, however, has been

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Fig. 8.12 Idiopathic epiretinal membrane in an enucleated eye without evidence of a full-thickness retinal tear shows surface wrinkling throughout the posterior pole

challenged after finding remnants of cortical vitreous within ERMs.115 This observation raised the possibility that detachment of the cortical vitreous from its retinal attachments may incite or promote the formation of ERMs. Others contend that hyalocytes in the cortical vitreous are the source of epiretinal membranes.116 When Gass proposed that the tangential forces exerted by cortical vitreous are involved in the formation of idiopathic macular holes, investigators took a more serious look at this obscure anatomic territory.117 Epiretinal membranes tend to occur in areas where the basal lamina of the retina is the thinnest (over the fovea and disc) and, presumably, the easiest to breach. The majority of ERMs contain fibrous astrocytes that grow in a centripetal pattern on the inner retina. The membrane is loosely attached to the underlying basal lamina. They may demonstrate an occasional hemidesmosome-type of structure, but, for the most part, ERMs are not anchored to underlying tissue.118 This situation explains the ease with which most epiretinal membranes can be peeled from the retina, and also their occasional spontaneous detachment. The fact that nearly 50 percent of membranes studied by electron microscopy contain RPE suggests that a full-thickness retinal hole may be present in many of these cases.119

Proliferative Vitreoretinopathy It is unclear if proliferative vitreoretinopathy (PVR)—massive periretinal proliferation or massive preretinal retraction—is a distinct nosologic entity or an exaggerated manifestation of wound healing associated with retinal detachment. The term PVR is used to clinically describe the exuberant membranes associated with rhegmatogenous retinal detachment. As the most common cause of failed retinal reattachment

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surgery, PVR is reported to occur in approximately 5-10 percent of rhegmatogenous retinal detachments.120,121 Because of its detrimental effect on visual function, PVR has been studied extensively over the last three decades.122,123 Preoperative risk factors for the development of PVR include size of retinal tear, multiple retinal tears, inflammation, vitreous hemorrhage, aphakia, and presence of preoperative choroidal effusion.124,125,126 Because PVR is rare in exudative retinal detachments, full-thickness retinal tears are considered to play a crucial role in the pathogenesis of membrane formation. The operative risk factors for postoperative PVR have been difficult to reproducibly quantify, but do include excessive amounts of cryotherapy, laser photocoagulation, and diathermy.127 When membrane contraction is sufficient to distort the retina, the lesion is described as a starfold. Epiretinal membranes in PVR tend to be more severe in the inferior quadrants and posterior to the equator. Proliferative membranes can form on either side of the retina. The more advanced stages of the disorder are characterized by the presence of fixed retinal folds in four quadrants. If left untreated, these detachments will progress to a closed funnel configuration. The cellular composition of PVR has been studied extensively, and generally contains the same cells found in other vitreous membranes, although RPE cells are consistently present. Macrophages and lymphocytes are also commonly seen.128 As in other membranes, their contractile properties have been attributed to the presence of myofibroblasts. The extracellular matrix of PVR is made up of types I, II, IV and V collagen, laminin, and tenascin.129 A variety of clinical observations and experimental studies have emphasized the importance of inflammation in the pathogenesis of PVR.130 The identification of fibronectin (both cellular and plasma-derived), plasma-derived growth factors, macrophages, and help/suppressor lymphocytes reflect how similar PVR is to the prototypical inflammatory-repair process.131 In 1983, the Retina Society developed a classification system for PVR to improve communication among clinicians and to facilitate interpretation of clinical studies.132 Because of certain shortcomings in that system, the Silicone Study Group modified the classification algorithm. The classification system developed by the Silicone Group includes separate descriptions and grading of anterior and posterior forms of PVR.133 It also recognizes three different patterns of proliferation— focal, diffuse, and subretinal.

Treatment Although the management of PVR is beyond the scope of this review, a basic overview is provided. While drugs and drug delivery systems that modulate the natural history of PVR are being developed and tested, the general approach to the treatment of retinal detachment with PVR is still surgical. The basic principles of surgical repair include closure of all retinal breaks, relief of anatomically important traction, and appropriate timing of corrective intervention. The overall approach to surgery is stratified according to the severity of clinical findings.

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The closure of retinal breaks with a chorioretinal adhesion is considered of primary importance because it allows for a more rapid return of homeostatic forces—particularly the function of the RPE pump. Although chorioretinal scar formation can be induced through freeze-thaw injury (cryopexy), diathermy, or laser photocoagulation, these processes also induce breakdown of the blood-ocular barrier, which can exacerbate PVR. The association of PVR with excessive cryotherapy has been used as a long-standing argument for its abandonment.134 Whichever method of scar induction is used, it is imperative to use no more thermal energy than necessary to insure closure of the retinal tear. The relief of retinal traction can be accomplished through external buckling (scleral buckling), and/or removal of contracting bands and membranes through various vitrectomy techniques.135 Subretinal fibrosis, while not uncommon in PVR, does not commonly result in clinically significant traction. In those situations in which subretinal bands of fibrous tissue prevent reattachment of the retina, the tissue can be dissected through a retinotomy.136 After all elements of traction are relieved following vitrectomy, fluid-gas exchange can be performed to insure extended internal tamponade of retinal breaks. In cases of severe PVR, there has been considerable debate over the relative merits of the substances used for tamponade. The two major classes of agents used for intraocular tamponade are long-acting gases and silicone oil. The controversy over their use in PVR led to the Silicone Study, which began recruitment in 1985. Enrollment ended in 1990.137 The study provided a trove of information about the behavior of PVR and the nuances of its management. The main findings were that perfluoropropane gas was equally effective in terms of visual outcome and anatomic reattachment to silicone oil, placing greater weight on secondary outcome measures, like complications. Both perfluoropropane gas and silicone oil produced superior results to sulfur hexafluoride gas, even though both gases were not compared head-to-head in a randomized manner.138,139 Long-term follow-up of 36 months showed there was no significant difference between silicone oil and perfluoropropane gas in terms of achieving visual acuity of 5/200, or in terms of secondary keratopathy.140 In general, visual prognosis in patients with PVR is based on severity of preoperative findings, including initial visual acuity, duration of detachment, and extent of detachment.125,141 Because visual outcome is substantially better in cases requiring only a single surgery, careful planning of the primary procedure is vitally important.

Cells within the Vitreous Under normal clinical conditions, the vitreous appears acellular, or pauci cellular, at most. Any increase in the number of cells in the vitreous beyond this baseline needs to be regarded as abnormal, and an appropriate diagnostic evaluation undertaken. The two major categories of disease that present with vitreous cells are

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inflammatory disease and lymphoma—with the great majority of all cases due to an underlying inflammatory process. While the number of inflammatory disorders that can cause secondary vitritis is large (e.g., differential diagnosis of posterior uveitis), there are relatively few conditions other than lymphoma that present as neoplastic vitritis. Metastatic vitritis from a systemic malignancy in the absence of uveal metastasis is rare.142

Primary Ocular Lymphoma Primary ocular lymphoma (POL) describes a subset of B-cell lymphoma known as primary central nervous system lymphoma. Also know as reticular cell sarcoma in the older literature, POL often co-exists with primary central nervous system lymphoma. When POL presents as an isolated B-cell lymphoma of the retina, optic nerve head, or vitreous, the diagnostic evaluation can be challenging.143 This is particularly true when vitreous cells from POL occur without signs of retinal disease. The incidence of primary central nervous system lymphoma is 1 per 100,000, or approximately 1/6 as common as uveal melanoma.144 POL is a disease of older persons with the majority of patients diagnosed between the late 50s and late 60s. Approximately three-quarters of the persons who present with ocular disease will ultimately have CNS involvement.145,146 The majority of patients with POL present with a picture of uveitis or vitritis that is not responsive to conventional therapy.147 Decreased vision, floaters, and photophobia are common complaints from patients with neoplastic vitritis. While the clinical appearance of the vitreous is nonspecific (similar to inflammatory vitritis), retinal lesions, if present, provide an important clue about the underlying diagnosis. The retinal tumors are cream-colored and covered by varying amounts of clumped RPE. When visualization of the fundus is not impeded by vitreous cells, the appearance of the retina is the most direct means of establishing a strong presumptive diagnosis of POL (see Fig. 8.13). Clinical-pathological correllation studies have shown that the malignant B cells accumulate initially between Bruch’s membrane and the RPE, creating a characteristic detachment of the cellular monolayer (see Fig. 8.14). The transition from normal retina to RPE detachment is usually abrupt. Over days to weeks, the progressive destruction of the overlying RPE reveals the lesion’s cream color. When the diagnosis of POL is considered, neurological examination and appropriate radiological studies should be undertaken to exclude primary central nervous system lymphoma because of the close relationship between the two entities. Both computed tomography and magnetic resonance imaging are useful in this regard. Nearly 70 percent of patients with primary central nervous system lymphoma have a solitary lesion on initial scan. Most patients with POL will develop multifocal disease of the brain as the lymphoma progresses.148

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Fig. 8.13 Retinal fundus in a patient with primary ocular lymphoma shows large cream color lesions. The edges of the lesions are discrete. Their surface reveals small residual clumps of pigment epithelium

Fig. 8.14 Retinal pigment epithelial (RPE) detachments due to primary ocular lymphoma. A larger RPE detachment is present to the left (arrows), and a smaller RPE detachment is noted on the right (arrowheads). Tumor cells are present beneath the RPE, resting on Bruch’s membrane (arrows and arrowheads). Tumor cells are two to four times larger than reactive lymphocytes in the choroids. Lymphoma cells are also in the subretinal space.(hematoxylin-eosin, 390x original magnification)

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When POL is suspected in both the eye and the central nervous system, tissue diagnosis can often be made by examination of the cerebral spinal fluid. In cases where the only manifestation of suspected POL is the eye, a diagnostic vitrectomy is necessary to harvest tissue. Most laboratories prefer to have fresh tissue brought immediately for processing.149 Cytologically, the cells of POL have large hyperchromatic nuclei and scant cytoplasm consistent with large cell lymphoma (see Fig. 8.15). Tumor cells may be associated with vitreous debris, necrosis, and reactive inflammatory cells. When the clinical suspicion of POL is high, and vitreous cytology benign, it is possible that malignant cells exist beneath the RPE but have not yet breached the neurosenory retina. A subretinal biopsy may be necessary to harvest an adequate specimen.150 Chorioretinal biopsy is another option, when vitreous cytology is nondiagnostic.151 Because the rate of false-negative biopsy is high,70 several other diagnostic modalities have been applied in this clinical setting. The immunoglobulin gene rearrangements that normally occur during lymphocyte maturation provide the basis for the molecular diagnosis of lymphoma.152 The demonstration of a predominant gene rearrangement allows for the identification of clonal expansions of malignant lymphocytes in an arrested phase of development. Because lymphoma represents the proliferation of unregulated lymphocytes, most neoplastic lymphocytes are conveniently tagged with a surface marker reflecting their aberrant ontology. The restricted expression of B-cell markers (CD19, CD20, CD22) are a phenotypic characteristic of POL and a useful clinical tool to distinguish these cells from other malignancies.153 The laboratory evaluation of lymphoma is

Fig. 8.15 Vitreous biopsy of primary ocular lymphoma shows large atypical cells, many of which have poorly discernable cytoplasm. The cell nuclei measure up to 40 microns.(Papanicolaou stain, 430x original magnification)

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directed towards identifying the expanded clone of neoplastic cells, either through phenotypic typing or genotypic analysis. Flow cytometry has become a mainstay in the diagnosis of systemic lymphoma, usually enhancing the capabilities of histopathology to classify tumors. Flow cytometry of a vitreous biopsy can yield false negative results if tumor cells remain beneath the RPE and only inflammatory cells are present in the vitreous. Flow studies can test for several markers simultaneously, increasing the likelihood of a positive result and enhancing correlation with histological interpretation.154 Flow cytometry will not replace cytological examination of the vitreous, but does effectively compliment tissue diagnosis.155,156 The role of flow cytometry to diagnosis lymphoma in situations where the cytological diagnosis of POL is negative, or inconclusive, is not established. Genotypic analysis for the germline mutation can also be used to establish the diagnosis of POL. For most B-cell lymphomas, the heavy chain immunoglobulin (IgH) gene sequence is selected. When the Southern blot technique was the standard method of identifying DNA gene rearrangements, the amount of material obtained from a vitreous biopsy was often inadequate for analysis. Problems with small sample size have been overcome with the introduction of polymerase chain reaction (PCR), which requires less than one-tenth the amount of DNA for analysis compared to Southern blot. PCR can be run on fresh or paraffin-embedded tissue, which makes the study of archival cases possible. Several different primers can be used for PCR in this setting.157,158 In one study of 57 samples of POL, all demonstrated the IgH germline mutation at the CDR3 site.159 The use of cytokine concentration in the vitreous to diagnosis POL is more controversial. The theory behind their use is based on the fact that interleukin-6 is produced in high levels by inflammatory cells, while interleukin-10 is released by neoplastic B cells. The ratio of interleukin-10 to interleukin-6 in the vitreous has been promoted as a diagnostic test of POL. Several studies recommend a ratio of greater than 1.0 be used as the cutoff for POL.160,161,162 Not everyone agrees on the sensitivity or specificity of the test. Reports of POL with interleukin ratios less than 1.0 have cast doubt on the predictive value of the test.163,164 Because vitreous interleukin assays are not yet standardized, the reliability of laboratory results must always be considered when weighing clinical evidence.

Treatment The management of patients with POL and primary central nervous system lymphoma is undergoing constant modification. In general, treatment differs from that of systemic lymphoma, whose drug regimens tend to have limited efficacy in penetrating the blood-retinal and blood-brain barriers. Optimal treatments for POL have not been determined. Intravenous methotrexate has been the standard drug used in most regiments because of its ability to penetrate the globe.165 More recently, other trials using Ara-C alone or in combination with methotrexate have been reported.166,167,168 While the management of POL and primary central nervous

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system lymphoma is beyond the scope of this chapter, other options include combinations of whole-brain and ocular radiation in concert with systemic and intrathecal chemotherapy.169 Isolated POL presents several management dilemmas in which there is little evidence-based data to fall back on. How frequently patients with POL need to be monitored for central nervous system disease is one example. Initially, treatment consisted of ocular radiation, but it soon became evident that the rate of local complication was so high that radiation was generally abandoned. An alternative means of delivering localized treatment is through intravitreous injection of methotrexate. Results of several small series have been published and appear encouraging. Initially used as an adjunct to systemic chemotherapy and radiation therapy, intravitreous methotexate was able to induce a local remission in seven of seven patients with follow-up between nine and 19 months.170 Another series used an induction phase of twice weekly intravitreous injections followed by weekly injections (consolidation phase).171 The majority of eyes were free of tumors, but required up to 12 injections. Three patients had recurrences, but all responded to a repeat course of injections. Intravitreous methotrexate seems well-suited for persons with POL isolated to the eye because it minimizes systemic toxicity while delivering a high concentration of the drug to the tumor. Long-term monitoring for central nervous system involvement, however, is mandatory. While a variety of local complications can occur, including cataracts, corneal decompensation, and maculopathy, their overall impact is less severe than the complications of ocular radiation.97,98 Delivering methotrexate by intravitreous injection prolongs the drug’s half-life from several hours to approximately five days, which may explain its effectiveness at local tumor control.172 Stem-cell transplantation is an option for patients with primary central nervous system lymphoma and recurrent disease, or who are refractory to standard therapy.173 The roles of blood-brain barrier disruption and whole-brain radiation are being defined.174,175

Vitreous Hemorrhage Vitreous hemorrhage is a relatively common cause of severe vision loss, having an annual incidence of approximately seven cases per 100,000 in the population.176 Depending on the location of the hemorrhage and its volume, symptoms can range from mild peripheral floaters to profound vision loss. The presence of blood in the vitreous often gives rise to other entopic symptoms. Small hemorrhages can be asymptomatic. A wide variety of underlying disorders can lead to vitreous hemorrhage, but the causes can be condensed into three major categories—bleeding from abnormal or pathological vessels, bleeding from normal vessels, or extension of bleeding into the vitreous from an external tissue.177 The frequency of specific causes of vitreous hemorrhage varies depending on study design and the time it was conducted. More recent studies have found proliferative diabetic retinopathy and retinal tears as the most common primary causes. 175,178

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The risk of spontaneous vitreous hemorrhage from pathologic or normal vessels is difficult to quantify, although massive intraocular bleeding appears to occur more frequently in persons on anticoagulants, including aspirin.179,180 The pathophysiological events following vitreous hemorrhage have been studied in experimental animals and humans. The fate of blood in the vitreous is one of progressive catabolism. The breakdown of blood cells in the vitreous gel parallels that in other tissues, with a few notable exceptions. Clot formation in the vitreous gel develops rapidly with diffusion of red blood cells limited by the latticework of collagen fibers.181 Unlike hemorrhage into soft tissues, fibrin persists in the vitreous, in part due to the lack of neutrophil response. The lack of an early neutrophil response contributes to the delay in red blood cell lysis. Macrophages, which typically enter a fibrin clot within several days of bleeding, may not be able to clear degenerating red blood cells and other debris as efficiently in the vitreous as other tissues.182 The natural history of vitreous hemorrhage has been studied in experimental animals using chromium labeled isotopes. These studies show that blood disappears from the vitreous gel in stages with very little clearing during the first three days.183,184 The degradation of red blood cells proceeds on a cellular level while hemoglobin undergoes metabolic oxidation and conversion to a variety of breakdown products, including hematin, biliverdin, hematoidin, and hemosiderin. Red blood cells in the vitreous lose their normal shape, become fragmented and often lyse (see Fig. 8.16). As hemoglobin degrades, the denatured molecule can adhere to the cell membrane forming a Heinz body. Loss of the cytoplasmic constituents of the red blood cell results in so-called ghost cell formation. As red blood cells are broken down and their carcasses carried off by macrophages, cholesterol from

Fig. 8.16 Enucleated eye with relatively fresh vitreous hemorrhages estimated to be about one week old. The cortical vitreous shows myriad fragment red blood cells and no inflammatory cells. A few nucleated cells are present on the surface of the retinal to the left (hematoxylin-eosin, 290x original magnification)

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the plasmalemma remains within the vitreous where it can incite a moderate inflammatory reaction. This form of localized cholesterolosis within the vitreous is referred to as synchysis scintillans (see section on synchysis scintillans). Free hemogloblin may also coalesce within the vitreous gel forming spherical aggregates. This condition is referred to as hemoglobin spherulosis and consists of spheres ranging in size from 10 to 20 microns. When suspended in the vitreous, they are visible via slit lamp biomicroscopy, appearing as golden brown droplets.185 The iron liberated during the breakdown of red blood cells in the vitreous occurs intracellularly within macrophages (where it is stored as hemosiderin or ferritin), or extracellularly (where it is bound to lactoferrin or transferrin).186 Unbound vitreous iron (Fe+2 and Fe+3) likely exerts toxic effects on a variety of local tissues and contributes to vitreous syneresis.187 Iron from intraocular hemorrhage does not have the toxic effects of iron-containing foreign bodies, which cause severe damage to the neurosensory retina. Part of this discrepancy may be due to the higher amount of free bivalent iron released from iron-containing foreign bodies, which is more likely to interfere with the glycolytic activity of the retina.188 Based on clinical observations and experimental studies, blood within the vitreous may play a role in the development of fibrovascular proliferation.189 It is often difficult, however, to separate the role of red blood cells from other confounding factors, like trauma (accidental or surgical), or the underlying disease process causing the hemorrhage. In experimental studies, preretinal membranes follow the accumulation of red blood cells and macrophages on the retina surface.190 Vitreous hemorrhage appears to have a deleterious effect on pre-existent fibrovascular membranes, mediated through cellular-molecular mechanisms, mechanical disruption of the vitreous gel, or both. Treatment Treatment of vitreous hemorrhage falls into two general categories—treatment of the underlying condition and treatment of the media opacity. The indications and guidelines for the treatment of the multitude of disorders that give rise to vitreous hemorrhage will vary with each disorder, and their review is beyond the intent of this chapter. Many of the algorithms for the management of vitreous hemorrhage have a common final pathway despite variations in the treatment of the underlying condition. Once the reason for vitreous bleeding has been dealt with, visual rehabilitation often requires removal of the media opacity. Nonclearing vitreous hemorrhage is one of the most common clinical indications for pars plana vitrectomy. Its role in diabetic retinopathy has been studied in the Diabetic Retinopathy Vitrectomy Study and in smaller studies.191,192,193,194 While the measured benefits of vitrectomy may vary in different studies, certain trends in surgical outcome have become apparent. Improved instrumentation and possibly better training have resulted in steady improvement in most measures of clinical outcome, including declining rates of complications. As a result, the indications of pars plana vitrectomy have been changing with lower thresholds for surgery.

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Although surgery remains the standard of care for nonclearing vitreous hemorrhages, the concept of pharmacological vitrectomy has been a long-time dream of researchers. Still far from a reality, chemically induced clearing of vitreous hemorrhages has rarely advanced beyond the stage of a pilot study.176,195 Although the primary target of drug therapy has been the red blood cell, an alternative approach to accelerating the removal of blood is to liquefy the vitreous. One potentially promising therapy includes intravitreous injection of highly purified hyaluronidase, which has facilitated visualization of the fundus by liquefying the vitreous gel.196,197

Summary A variety of unrelated disorders affect the vitreous throughout life. Common degenerative conditions due to aging (e.g., vitreous syneresis) place everyone at some risk for PVD, retinal tear, and retinal detachment. While the environmental factors that predispose to vitreous syneresis are not completely understood, posterior segment inflammation of any type readily promotes vitreous liquefaction. The vitreous is often the site of overflow, or secondary, inflammation from primary uveitic disorders. Additionally, primary ocular lymphoma must always be considered in the differential diagnosis of chronic vitreitis. The aging vitreous can loose it transparency from the accumulation of amyloid, phospholipids (asteroid hyalosis), or cholesterol (synchesis scintillans). Among the three disorders, only amyloid causes significant ocular morbidity or carries any implication for general health. Vitreous membranes are the final common pathway for a number of different types of ocular injury. While their pathogenesis likely differs according to underlying cause, they share many features with the nonspecific inflammatory-reparative processes that promote wound healing.

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134. Campochiaro PA, Kaden IH, Vidaurri-Lead J, Glaser BM (1985) Cryotherapy enhances intravitreal dispersion of viable retinal pigment epithelial cells. Arch Ophthalmol 103:434-436 135. Glaser B (1989) Surgery for proliferative viteoretinopathy. In: Glaser B, Michels RG (eds) Retina, vol 3, Ch 138. CV Mosby Company, St Louis, p 385-400 136. Machemer R (1980) Surgical approaches to subretinal strands. Am J Ophthalmol 90:81-85 137. Gentile RC, Abrams GW (1998) The Silicone Study. In: Kertes PJ, Conway MD (eds) Clinical Trials in Ophthalmology, Ch 10. Lippinocott Williams & Wilkins, Philadelphia, 163-184 138. The Silicone Study Group. (1992) Vitrectomy with silicone oil or sulfur hexafluoride gas in eyes with severe proliferative vitreoretinopathy. Results of a randomized clinical trial. Silicone Study Report 1. Arch Ophthalmol 110:770-779 139. The Silicone Study. (1992) Vitrectomy with silicone oil or perfluoropropane gas in eyes with severe proliferative vitreoretinopathy. Results of a randomized clinical trial. Silicone Study Report 2. Arch Ophthalmol 110:780-792 140. Abrams GW, Azen SP, McCuen BW, et al. (1997) Vitrectomy with silicone oil or long-acting gas in eyes with severe proliferative vitreoretinopathy: results of additional and long-term follow up. Silicone Study Report 11. Arch Ophthalmol 115:335-344 141. Tseng W, Cortez RT, Ramirez G, et al. (2004) Prevalence and risk factors for proliferative vitreoretinopathy in eyes with rhegmatogenous retinal detachment but no previous vitreoretinal surgery. Am J Ophthalmol 137:1105-1115 142. Young SE, Cruciger M, Lukeman J (1979) Metastatic carcinoma to the retina: A care report. Ophthalmology 86:1350-1354 143. Freeman LN, Schachat AP, Knox DL, et al. (1987) Clinical features, laboratory investigations, and survival in ocular reticulum cell sarcoma. Ophthalmology 94:1631-1639 144. Schabet M (1999) Epidemiology of primary CNS lymphoma. J Neurooncol 43:199-201 145. Akpek EK, Ahmed I, Hochbert FH, et al. (1999) Intraocular-central nervous system lymphoma: clinical features, diagnosis, and outcomes. Ophthalmology 106:1805-1810 146. Whipcup SM, de Smet MD, Rubin BI, et al. (1993) Intraocular lymphoma. Clinical and histopathologic diagnosis. Ophthalmology 100;1399-1406 147. Chan D-D, Wallace DJ (2004) Intraocular lymphoma: update on diagnosis and management. Cancer Control 11:285-1295 148. Hormigo A, DeAngelis LM (2003) Primary ocular lymphoma: clinical features, diagnosis and treatment. Clin Lymphoma. 4:22-9 149. Davis JL, Solomon D. Nussenblatt RB, et al. (1992) Immunocytochemical staining of vitreous cells. Indications, techniques, and results. Ophthalmology 99:250-256 150. Pavan PR, Oteiza E, Margo CE (1995) Ocular lymphoma diagnosed by internal subretinal pigment epithelial biopsy. Arch Ophthalmol 113:1233-1234 151. Kirmant MH, Thomas EL, Rao NA, et al. (1987) Intraocular reticulum cell sarcoma: diagnosis by choroidal biopsy. Br J Ophthalmol 71:748-752 152. Bagg A, Kallakury BL (1999) Molecular pathology of leukemia and lymphoma. Am J Clin Pathol 112(Suppl 1):S76-S92 153. Davis JL, Viciana AL, Ruiz P (1997) Diagnosis of intraocular lymphoma by flow cytometry. Am J Ophthalmol 124:362-372 154. Coupland SE, Bechrakis NE, Anastassiou G, et al. (2003) Evaluation of vitrectomy specimens and chorioretinal biopsies in the diagnosis of primary intraocular lymphoma in patients with masquerade syndrome. Grafes Arch Clin Exp Ophthalmol 241:362-372 155. Char DH, Lhung BM, Deschenes J, et al. (1988) Intraocular lymphoma: immunological and cytological analysis. Br J Ophthalmol 72:905-911 156. Rothova A, Ooijman F, Kerkhoff F, et al. (2002) Uveitis masquerade syndrome. Ophthalmology 108:386-399 157. Shen DF, Zhuang Z, LeHoang P, et al. (1998) Utility of microdissection and polymerase chain reaction for the detection of immunoglobulin gene rearrangement and translocation in primary intraocular lymphoma. Ophthalmology 105:1664-1669

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158. Gorochov G, Parizot C, Bodaghi B, et al. (2003) Characterization of vitreous B-cell infiltrates in patients with primary ocular lymphoma, using CDR3 size polymorphism analysis of antibody transcripts. Invest Ophthalmol Vis Sci 44:5235-5241 159. Chan CC (2003) Molecular pathology of primary intraocular lymphomas. Trans Am Ophthalmol Soc 101:275-292 160. Chan CC, Whitcup SM, Solomon D, et al. (1995) Interleukin-10 in the vitreous of patients with primary intraocular lymphoma. Am J Ophthalmol 120:671-673 161. Whitcup SM, Stark-Vancs V, Wittes RE, et al. (1997) Association of interleukin 10 in the vitreous and cerebrospinal fluid and primary central nervous system lymphoma. Arch Ophthalmol 115:1157-1160 162. Cassoux N, Merle-Beral H, LeHoang P, et al. (2001) Interleukin-10 and intraocular-central nervous system lymphoma. Ophthalmology 108:426-427 163. Akpek EK, Maca SM, Christen WG, et al. (1999) Elevated vitreous interleukin-10 level is not diagnostic of intraocular-central nervous system lymphoma. Ophthalmology 106:2291-2295 164. Buggage RR, Velez G, Myers-Powell B, et al. (1999) Primary intraocular lymphoma with a low interleukin 10 to interleukin 6 ratio and heterogeneous IgH gene rearrangement. Arch Ophthalmol 117:1239-1242 165. Sandor V, Stark-Vancs V, Pearson D, et al. (1998) Phase II trial of chemotherapy alone for primary CNS intraocular lymphoma. J Clinic Oncol 16:3000-6. Comments: Henson JW, Yang J, Batchelor T (1999) Intraocular methotrexate level after high-dose intravenous infusion. J Clin Oncol 17:1329 166. Baumann MA, Ritch PS, Hande KR, et al. (1986) Treatment of intraocular lymphoma with high-dose Ara-C. Cancer 57:1273-1275 167. Strauchen JA, Dalton J, Friedman AH (1989) Chemotherapy in the management of intraocular lymphoma. Cancer 63:1918-1921 168. Valluri S, Moorthy RS, Khan A, et al. (1995) Combination treatment of intraocular lymphoma. Retina 15:125-129 169. Ferreri AJ, Blay JY, Reni M, et al. (2002) Relevance of intraocular involvement in the management of primary central nervous system lymphoma. Ann Oncol 13:531-538 170. Ishburne BC, Wilson DJ, Rosenbaum JT, et al. (1997) Intravitreal methotrexate as an adjunctive treatment of intraocular lymphoma. Arch Ophthalmol 115:1152-1156 171. Smith JR, Rosenbaum JT, Wilson DJ, et al. (2002) Role of intravitreal methotrexate in the management of primary central nervous system lymphomas with ocular involvement. Ophthalmology 109:1709-1716 172. De Smet MD, Vancs VS, Kohler D, et al. (1999) Intravitreal chemotherapy for the treatment of recurrent intraocular lymphoma. Br J Ophthalmol 83:448-451 173. Soussain C, Suzan F, Hoang-Xuan K, et al. (2001) Results of intensive chemotherapy followed by hematopoietic stem-cell rescue in 22 patients with refractory or recurrent primary CNS lymphoma or intraocular lymphoma. J Clin Oncol 19:742-749 174. DeAngelis LM (2001) Brain tumors N Engl J Med 344:114-123 175. Nasir S, DeAngelis LM (2000) Update on the management of primary CNS lymphoma. Oncology (Huningt) 14:228-234 176. Lindgren G, Sjodell L, Lindblom, B (1995) A prospective study of dense spontaneous vitreous hemorrhage. Am J Ophthalmol 119:458-465 177. Spraul CW, Grossniklaus HE (1997) Vitreous hemorrhage. Surv Ophthalmol 42:3-39 178. Dana WR, Werner MS, Viana MA, Shapiro MJ (1993) Spontaneous and traumatic vitreous hemorrhage. Ophthalmology 100:1377-1383 179. Feman SS, Barlett RE, Roth AM, Foos RY (1972) Intraocular hemorrhage and blindness associated with systemic anticoagulation. JAMA 220:1354-1355 180. El Baba FE, Jarrett WH, Harbin TS, et al. (1986) Massive hemorrhage complicating agerelated macular degeneration. Ophthalmology 93:1581-1592 181. Swann DA, Chesney C, Constable IJ, et al. (1974) The role of vitreous collagen in platelet aggregation in vitro and in vivo. J Lab Clin Med 84:264-274

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182. Wolter JR (1960) The macrophages of the human vitreous body. Am J Ophthalmol 49:1185-1193 183. Greer DR, Benson WE, Spalter HF (1968) A study of simulated vitreous hemorrhages using labeled blood. Arch Ophthalmol 79:755-758 184. Boyer HK, Suran A, Hogan MJ, et al. (1958) Studies on simulated vitreous hemorrhages. I. Rate of disappearance of radiochromium tagged red cells. Arch Ophthalmol 59:232-234 185. Grossniklaus HE, Frank KE, Farhi DC, et al. (1988) Hemoglobin spherulosis in the vitreous cavity. Arch Ophthalmol 106:961-962 186. Van Bockzmeer FM, Martin CE, Constable IJ (1983) Iron-binding proteins in vitreous humour. Biochim Biophys Acta 758:17-23 187. Declercq SS, Meredith CA, Rosenthal AR (1977) Experimental siderosis in the rabbit. Arch Ophthalmol 95:1051-1058 188. Burger PC, Klintworth GK (1974) Experimental retinal degeneration in the rabbit produced by intraocular iron. Lab Invest 30:9-19 189. Ehrenberg M, Thresher RJ, Machemer R (1984) Vitreous hemorrhage nontoxic to retina as a stimulator of glial and fibrous proliferation. Am J Ophthalmol 97:611-626 190. Lean JS, Gregor Z (1980) The acute vitreous hemorrhage. Br J Ophthalmol 64:469-471 191. Thompson JT, de Bustros S, Michels RG, Rice TA (1987) Results and prognostic factors in vitrectomy for diabetic vitreous hemorrhage. Arch Ophthalmol 105:191-195 192. Diabetic Retinopathy Vitrectomy Study Research Group (1988) Early vitrectomy for severe proliferative diabetic retinopathy in eyes with useful vision. Results of a randomized trialreport number 3. Ophthalmology 95:1307-1320 193. Diabetic Retinopathy Vitrectomy Study Research Group. (1985) Early vitrctomy for severe vitreous hemorrhage in diabetic retinopathy. Two-year results of a randomized trial-report number 2. Arch Ophthalmol 103:1644-1652 194. Diabetic Retinopathy Vitrectomy Study Research Group. (1990) Early vitrctomy for severe vitreous hemorrhage in diabetic retinopathy. Four-year results of a randomized trial-report number 5. Arch Ophthalmol 108:958-964 195. Tanaka M, Qui H (2000) Pharmacological vitrectomy. Semin Ophthalmol 15:51-61 196. Kuppermann BD, Thomas EL, de Smet MD, et al. (2005) Pooled efficacy results from two multinational randomized controlled clinical trials of a single intravitreous injection of highly purified ovine hyaluronidase (Vitrase) for the management of vitreous hemorrhage. Am J Ophthalmol 140:573-584 197. Kupperman BD, Thomas EL, de Smet MD, et al. (2005) Safety results of two phase III trials of an intravitreous injection of highly purified ovine hyaluronidase (Vitrase) for the management of vitreous hemorrhage. Am J Ophthalmol 140:585-597

Chapter 9

Age-Related Changes and/or Diseases in the Human Retina Nicola Pescosolido, MD and Panagiotis Karavitis, MD

Abstract As people grow older, changes in the retina occur as part of the natural course of aging. Additional changes in the retina come from pathological causes, for which an ophthalmology specialist may be asked to evaluate and treat. Often patients will present to an eye doctor with complaints of worsening vision or visual distortions that cannot be accounted for by media opacity (cataract) or refractive error. A solid working knowledge of the normal age-related changes of the retina, as well as a firm understanding of clinically relevant age-related pathology, will help any eye physician manage the aging patient population. The aim of this study is not only to fully understand the senile involution of retinal structures that allows us to observe the wonders of the world, but also to keep these structures intact for as long as possible. An increasing number of elderly give a good cause for investigation of age-related changes and/or diseases in the human retina. This chapter describes the major age-related changes and diseases of the human retina in aged people. The first part of the chapter deals with common lesions in the senile retina, while the second part describes the major diseases of the senile retina. Keywords Retina aging, retina age-related changes, retina diseases, senile retina, retina involution, human retina

Senile Lesions of the Retina It is very common to encounter senile lesions of the eyes, and therefore a reduction in the acuity of vision and also peripheral chorioretinal lesions that cause severe retinal lesions, such as senile retinal detachment. The interest in studying geriatric changes to vision lies in identifying the first lesions as early as possible. Not that it is possible (at least up to now) to impede the inexorable evolution of the degenerative phenomena, but an early diagnosis can at least allow us to greatly slow this process. With the passing of the years, the normal architecture of the retina undergoes modifications starting from the ora serrata towards the posterior pole for a variable distance. At the posterior pole of the Bruch’s membrane (drusen), lipid or calcium

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carbonate soap deposits and a strong increase of the number of capillaries (obliterated aneurisms) are seen. Dry and wet macular degeneration are clinical pictures often discussed in pathology. Finally, the cystoids of the macula begin with an oedema with brilliant irregular foveal reflexes that surround the macula. In the macula there may be an isolated vesicle, dark red in color, with sharply defined edges. In the periphery, the most frequent lesions are cystic degeneration and Blessig-Iwanoff holes, especially located in the ora serrata.1 This damage derives from small, highly reactive molecules known as free radicals2 that are produced during normal metabolic processes and are associated with important cellular functions.3 Of these damaging agents, we shall consider reactive oxygen intermediates capable of inducing oxidative stress. In addition, peroxy-nitrites, endogenic alkylating agents and aldehyde products, resulting from the oxidation of lipids, also have the capacity to damage macromolecules (protein and lipid complexes, DNA, etc.).4 Macromolecular damage results in cellular disfunction manifested by nuclear instability, inappropriate cell differentiation, and consequent cell death (necrosis or apoptosis).5 These age-related changes of the retina primarily cause a loss of visual acuity and impairment of color discrimination as well as reduction of the visual field. Impairment of visual functions in aged subjects has long been considered the consequence of opacity of the dioptric media, while little attention has been paid to the retinal age-related changes. In light of our present findings, we can hypothesize that the fall in visual acuity occurring in old age is influenced, at least in part, by the age-related changes observed in human retinal tissues.6

Age-related Changes of the Human Retina Our group studied the Scanning Electron Microscopic (SEM) features of the human retina. For a detailed evaluation of the effects of aging on retinal morphology, a quantitative analysis of images was performed for obtaining morphometric data. Our findings underlined that the human retina can be considered an optimal model for studies of neuronal maturation and/or neuronal aging, with particular sensitivity to age-related changes and senile decay.7

Major Senile Diseases of the Retina From a review of the patients hospitalized in our Clinical Sections of Ophthalmology in the last five years, the major ocular pathologies of elderly people are as follows: ● ●

senile cataracts senile detachment of the retina

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vascular alterations senile ocular pigment degeneration cystic degeneration retinoschisis latex degeneration cobblestone degeneration

Here are briefly described the major characteristics of these diseases.

Senile Cataracts The lens is exposed to the cumulative effects of radiation, oxidation, and translational modification. The alteration of proteins and other lens molecules impairs membrane functions and perturbs protein (particularly crystallin) organization, so that light transmission and image formation may be compromised. Damage is minimized by the presence of powerful scavenger and chaperone molecules. Progressive insolublization of the crystallins in the lens nucleus in the first five decades of life, and the formation of higher molecular weight aggregates, may account for the decreased deformability of the lens nucleus that characterizes presbyopia. Additional factors include the progressive increase in lens mass with age, changes in the point of insertion of the lens zonules, and a shortening of the radius of curvature of the anterior surface of the lens. There is also a decrease in light transmission by the lens with age, associated with increased light scatter, increased spectral absorption (particularly at the blue end of the spectrum), and increased lens fluorescence. A major factor responsible for the increased yellowing of the lens is the accumulation of a novel fluorogen—glutathione-3-hydroxy kynurenine glycoside—which makes a major contribution to the increasing fluorescence of the lens nucleus that occurs with age. Because this compound may also crosslink with the lens crystallins, it may contribute to the formation of high-molecular weight aggregates and the increases in light scattering that occur with age. Focal changes of microscopic size are observed in apparently transparent, aged lenses, and may be regarded as precursors of cortical cataract formation.

Senile Detachment of the Retina It is not easy to define retinal detachment as senile. There are chorioretinal and vitreal alterations that are typically geriatric, and may cause detachment of the retina. Cystic Degeneration of the Periphery of the Retina On the one hand, cystic degeneration causes a thinning and weakening of the retina and on the other hand favors the formation of pathological adherence with the

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vitreo, and therefore creates a predisposition to retinal rupture at the margin or at the operculum. Furthermore, the walls of the cysts may break and therefore retinal holes may form.

Paved, Cobblestone or Pavement Degeneration as Proposed by Straatsma8 or Gonin’s Foci of Atrophic Choroidosis From a histological point of view, these lesions show the disappearance of the choriocapillaries and the pigment epithelium, or atrophy of the inner layers of the retina—i.e. those that depend on the trophism of the choriocapillaries for nutrition. The retina is strongly held to the choroid in these degenerative foci. They almost never form holes or ruptures of the retina. This type of degeneration does not favor the detachment of the retina per se, but shows that there is wear and choroidal and retinal degeneration at the periphery that may favor the onset of retinal detachment. In fact, O’Malley and Allen9 observed that numerous patients who show cobblestone degeneration also have cystic degeneration of the retina, fence degeneration, retinoschisis, retinal holes and ruptures, and cysts of the pars plana near these lesions.

Senile Degeneration of the Vitreous After 50 years of age, a fibrillar and lacunar degeneration of the vitreous begins in emmetropic eyes, slowly progressing and possibly resulting in the posterior detachment of the vitreous with its collapse. This is a typical senile disease that is found in 65 percent of individuals over 65 years old and more or less 100 percent of individuals over 77 years. The posterior detachment of the vitreous may cause, where there is an adherence between the vitreous and the retina, rupture of the retina by traction. The retinal rupture may be secondary to a choroidal exudation of congestive or allergic inflammatory origin that passes the pigment epithelium, applying pressure to the retina towards the inside of the eye bulb, causing its rupture at a weak point. Upon contact with the choroidal exudation liquid, the vitreous then coagulates. Senile retinal detachment seems to be essentially caused by cystic degeneration of the retina, and posterior detachment of the vitreous with collapse. This is due to the fact that in cases of senile detachment, posterior detachment of the vitreous with collapse is nearly always found. If we consider, however, the rarity of retinal detachment with respect to posterior detachment of the vitreous and cystic degeneration of the retina in senility, we must admit that many other factors must be relevant in retinal detachment, such as genetic predisposition, vascularretinal-choroidal factors, and abiotrophic factors. The changes that lead to retinal detachment in senile eyes are very similar to those in myopic eyes. A study performed on 829 patients affected by retinal detachment and surveyed over three years found that about 34 percent were myopic and 66 percent were not. Of these, around 63 percent were individuals more than 40

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years old.10 This shows, therefore, that senility plays an important role in the initiation of retinal detachment. Without doubt, retinal detachment results from degenerative lesions of the retina and the choroid—lesions that normally do not have characteristics appreciably different from degenerative myopic lesions. In the case of senility, the lesions are related to obstructions of the retinal and sometimes choroidal capillaries. The study of the cases in which the retinal detachment occurred in nonmyopic individuals in senile age groups confirms that normally in these degenerative lesions there is evidence of vascular obstruction in the form of thin, obliterated blood vessels that prevail in the superior temporal quadrant. In the eyes of nonmyopic senile-aged subjects, it is possible to see (more frequently than in young subjects) small pigmented equatorial spots that are often hexagonal in appearance. These are often the starting point of horseshoe ruptures, on the borders of which pigment deposits are found. Finally, there can be interruptions in the continuity of the macula deriving from senile alterations. In senile degeneration of the macula, pits may form in the macular lamella, which—although not very frequent—become perforations with consequent retinal detachment. Senility, as well as favoring the arrival of rhegmatogenous retinal alterations, is also responsible for the degenerative vitreol changes that can produce traction, rupture, and therefore detachment of the retina.11

Vascular Alterations The influence of vascular alterations in the pathogenesis of senile retinal detachment must be taken into consideration. Because aging causes a reduction in cardiac and lung performance, it may also cause a decrease of the retinal integrity. A parallel can be seen between the athero-sclerotic lesions of the retinal blood vessels, and those of the other organs. We should remember that aphakia—not often seen these days, as a result of good cataract operations—has a tight correlation with retinal detachment. It originates from small ruptures or holes in the ora serrata. In senile aphakic eyes, retinal degeneration is very often seen at the periphery and particularly in the ora serrata with small holes generally located in the meridional folds of the retina.

Senile Peripheral Pigment Degeneration Senile peripheral pigment degeneration is associated with wartiness of the Bruch’s membrane and sclerosis of the choroid. It is often bilateral. One type of pigment alteration that is not, however, comes from moniliform pigmented scratches of the chorioretina. These are pigment granules that seem to be localized on sclerotic choroidal blood vessels in the nasal inferior quadrant.12

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Cystic Degeneration The retina shows two types of cystic degeneration—typical and reticular. The first originates on the outer plexiform layer, while the second originates in the nervous fiber layer. These changes are very common after 70 years of age. The typical peripheral cystoid degeneration (TPCD), also called Blessig-Iwanoff cysts, is characterized by cysts of the outer plexiform layer containing hyaluronic acid that can also coagulate, producing a globular form with winding channels that branch irregularly. Complications are rare. The retinal holes do not produce detachment of the retina because the vitreous is normally complete over the lesion. The extension of the lesion beyond the equator is also rare. The breakage of the walls of the cysts or gaps causes the formation of peripheral lamellar holes. The retina is not detached and there is not the operculum of real retinal holes. Rare, but possible, is the formation of real retinal holes, with operculum to which vitreous body filaments adhere following a filamentous degeneration of the vitreous body and separating from the ora serrata. This can lead to another periferal alteration—degenerative retinoschisis. The second type of modification is retinal periphery cystoid degeneration, which is almost always continuous and located behind areas of typical peripheral cystiod degeneration, and is usually found in the inferotemporal quadrant. This has a reticular aspect that corresponds to the retinal vessels of the inner layers. A finely punctured inner surface corresponds to the attachment points of the tissue cushions to the inner layer. The cystic spaces are located in the nervous fiber layer. This process occurs in 18 percent of adults, and occurs in bilateral form in 41 percent. It can evolve into degenerative reticular retinoschisis.9

Retinoschisis Two degenerative forms of retinoschisis have been described. They are both most frequently seen in the infero-temporal quadrant, and derive from a pre-existing form of peripheral cystoid degeneration. TPCD can evolve into typical degenerative retinoschisis, while both the typical and reticular forms of peripheral cystoid degeneration can transform into reticular degenerative retinoschisis.Typical degenerative retinoschisis causes a smooth raising of the retina in 1 percent of the adult population (bilateral in 33% of cases). Typical peripheral cystoid degeneration surrounds the lesions. The retina is divided along the outer plexiform layer, and consequently the inner layer comprises the ILM, the nervous fiber layer, the retinal vessels, the ganglion cells, the inner plexiform layer, and the inner nuclear layer. Normally, only the ILM, the nervous fiber layer, and part of the inner nuclear layer are visible. The outer layer is thicker, with cavities, and made up of the external nuclear layer and the photoreceptors.13 Reticular degenerative retinoschisis develops from the concurrent presence of typical and reticular cystoid degeneration of the peripheral retina. It is characterized by oval or round areas of detached retina,

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in which a lump forms in the very thin inner layer, and is observed in 1.6 percent of the adult population (bilateral form in 15% of cases). Normally, typical peripheral cystoid degeneration is located anterior to the reticular degenerative retinoschisis, while the reticular form is adjacent. The detachment occurs in the nervous fiber layer—the inner portion contains only the ILM, some retinal vessels, and a variable portion of the nervous fiber layer. The outer portion contains the remaining relatively complete retinal layers. Sometimes, typical and reticular retinoschisis are seen at the same time. It is not always easy to differentiate between the two forms on a clinical basis, unless there are lumpy aspects. The presence of holes in the outer margin or posterior extension is characteristically more common of the reticular form than the typical form.

Latex Degeneration The frequency of latex degeneration was found to be 8 percent in an ample clinical study, and 10.7 percent in autopsy studies. The lesions are bilateral and symmetrical in 48.1 percent of the cases, and the frequency increases after the second decade of life. The majority of the lesions, located in the pre-equatorial region, are orientated according to the circumference and more frequently in the vertical meridian.14 Latex lesions appear like a thinning of the retina. They can also look like a plait caused by sclerotic vessels, and may have variable pigmentation resulting from hypertrophy of the retinal pigmented epithelium (RPE). Histological latex degeneration is characterized by an overlying sack of vitreous fluid, absence of the ILM, vitreous condensation at the margins, hyperplasia of the glial cells and at the edges of the RPE (in some cases), thinning and the formation of retinal holes at the center of the lesion, sclerosis of the major blood vessels, sclerosis and a-cellularity of the capillaries’ hypertrophy, and hyperplasia of the RPE.

Cobblestone Degeneration This common degenerative chorio-retinal process is found in up to 27 percent of subjects after the 30th year. It is located between the ora serrata and the real side of the equator, and represents a boundary zone between the posterior choroidal circulation and the anterior ciliar circulation. Opthalmoscopically, it looks like a small and discrete yellow-white area, with very visible choroidal blood vessels— sometimes with hypertrophic and dark RPE at the margins. The lesions can join together to form a band of depigmentation behind the ora serrata. Histo-pathologic studies show signs of ischemic atrophy of the outer retina, with attenuation or disappearance of the choriocapillaries and loss of the RPE and the outer retinal layer up to and including the outer part of the inner nuclear layer. These changes are limited to the portion of the retina that is supplied by the choriocapillaries, and

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can be reproduced in rabbits after binding the choriodal blood supply.8 Cobblestone degeneration is a characteristic that suggests peripheral vasculopathy and can be highly extended in carotid stenosis.9

Prevention Considering what has been described above, prevention of the degenerative factors induced by aging points to the application of an anti-apoptotic action to the nervous cells and glia of the human retina. One possible drug to perform this is acetylcarnitine (ALCAR). In recent years, attention has been focused on the advantageous influence of carnitine as an anti-apoptotic agent—above all, as a molecule able to block the mitochondrial pathway in programmed cell death. Moreover, ALCAR seems to have a major protective role in senile retinal decay. The anti-apoptotic action caused by carnitine includes: induction of growth factors, increase in mitochondrial metabolism, protective action on the mitochondrial membrane integrity, inhibition of caspase activity, and, finally, an antioxidant activity.

References 1. Owsley C, Jackson GR, Cideciyan AV, Huang Y, Fine SL, Ho AC, Maguire MG, Lolley V, Jacobson SG (2000) Psychophysical evidence for rod vulnerability in age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 41:267- 273 2. Armstrong D (1984) Free radical involvement in the formation of lipo-pigments. In: Armstrong D (ed) Free Radicals in Molecular Biology, Aging and Disease. Raven Press, New York, p 137-182 3. Brizee KR, and Ordy JM (1981) Cellular features, regional accumulation, and prospects of modification of age pigments in mammals. In: Sohal RS (ed) Ageing Pigments Elsevier/North – Holland Biomedical Press, Amsterdam, p 176-181 4. Dykens JA (1999) Free radicals and mitochondria dysfunction in excyto-toxicity and neurodegenerative disease. In: Koliatos VE and Rantan RR (eds) Cell Death and Diseases of the Nervous System, Humana Press, Totowa, P 45-68 5. Handelman GJ and Dratz EA (1986) The role of antioxidants in the retina and retinal pigment epithelium and the nature of pro-oxidant induced damage. Adv. Free Radicals. Biol. Med. 2:1:89 6. Cavallotti C, Artico M, Pescosolido N, Feher J (2004) Age-related Changes in human retina. Can. J Ophthalmol. 39:61-68 7. Foulds WS (1980) Factors influencing visual recovery in retinal detachment surgery. Trans. Ophthalmol. Soc.U.K.100:72-77 8. Straatsma BR, Foos RY, Feman SS (1980) Degenerative diseases of the peripheral retina. In: Duane TD (ed) Clinical Ophthalmology. Harper & Row, Philadelphia, p 1-27 9. O’Malley PF, and Allen RA (1967) Peripheral cystoid degeneration of the retina. Incidence and distribution in 1,000 autopsy eyes. Arch. Ophthalmol. 77:769-776 10. Curcio CA, Millican CL, Allen KA, Kalina R.E (1993) Aging of the human photoreceptor mosaic: Evidence for selective vulnerability of rods in central retina. Invest. Ophthalmol. Vis. Sci. 34:3278-3296

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11. Marshall J (1987) The ageing retina: physiology and pathology. Eye. 1: 282-295 12. De Laey JJ (1988) Ophtalmologie geriatrique. In: Oosterhosch (ed) Traité de Geriatrie,Sociéte Scientifique de Medicine Generale, Bruxelles, p 263-488 13. Foos RY (1970) Senile Retinoschisis: relationship to cystoid degeneration. Trans. Am. Acad. Ophthalmol. 68:329-403 14. Byer NE (1989) Long-term natural history of lattace degeneration of the retina. Ophthalmology. 96:1396-1402

Chapter 10

Aging of the Retinal Pigmented Epithelium Carlo A. P. Cavallotti, MD, PhD and Marcus Schveoller MD, PhD

Abstract The age-related changes of the human retinal pigmented epithelium cells are listed here. These cells play an important role in nutrition of all retinal cells. Changes in cellular density, granules of lipofuscine, granules of melanin, and complex granule s were found. In our laboratories, eight samples of the human retina (including retinal pigmented epithelium) of young individuals and 16 retinas of older subjects were used for our experiments. These samples were studied with: ● ● ●

Light microscopy for the detection of microanatomical details Histo-chemical techniques for the dye of the lipids Transmission electron microscopy for the detection of the ultra-structural findings

Our results, comparing retinal pigmented epithelium of subjects 21-years old with those of subjects 75-years old, demonstrate a(n): ● ● ● ● ●

Strong depigmentation in old subjects Strong increase of intracytoplasmic residual bodies Strong increase of total lipids Decrease of phospholipids and neutral esters fatty acids Increase of electron density of the sub-cellular structures due to the increase of pigment granules

All results demonstrate that the retinal pigmented epithelium of human eye undergoes specific age related changes. Keywords aging, human eye, RPE, de-pigmentation, lipids, electron-density, lipofuscine.

Retinal Pigmented Epithelium (RPE) The retinal pigmented epithelium (RPE) is formed by a single layer of cells from the root of the iris to the ora serrata. It is continuous with the anterior layer of the iris epithelium. The cellular cytoplasm contains numerous round or oval pigment granules. From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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The mitochondria are smaller and less numerous in these cells than in those in the non-pigmented epithelium. The basal side, facing the ciliary body, has a basement membrane—uniform in thickness—throughout their course.1 The basal cell membrane shows only a few in-folding. The cell membrane on the apical side is like that in the nonpigmented epithelium. There are numerous cell junctions. The lateral surfaces show numerous interdigitations.2

Micro-anatomical Details The cells of the human RPE are post-mitotic cells, with an hexagonal shape, that form a single layer of cubic epithelial cells that separate the external portion of the photoreceptors from the choroid. The RPE provide metabolic and functional support for the external portion of the photoreceptors and all the remaining layers of the retina. Each human eye contains between 4 and 6 million RPE cells. In the central part of the retina, the shape and dimensions of the RPE cells are uniform. They are about 14 µm in diameter and 12 µm in height. At the equator, the cells are taller and larger, and at the extreme periphery lose their uniformity of size and shape. Some cells may contain more than one nucleus, and at the ora serrata, the RPE cells may measure up to 60 µm in diameter These cells are formed from a basal portion, apical portion, and six lateral faces.3 The basal portion shows the cellular membrane with numerous invaginations, which can sink up to 1 µm into the cytoplasm to increase the absorbent surface. The basal membrane of these cells is adjacent to the basal lamina that forms the proximal layer of the Bruch’s membrane. The basal invaginations increase the surface area of the cellular membrane, because this is involved in transport functions. The apical portion of the RPE cells that sits in front of the acromeres of the photoreceptors is folded to form micro-villa of 5-7 µm in length that surround the third terminal part of the acromeres. Because the acromeres of the rods and cones are of different dimensions, the villa that surround the external portion of the rods are smaller (3 µm) than those that surround the cones. From a functional point of view, there are two different types of microvilla—one softer, which is dedicated to transepithelial transport, and the other connected to the distal lamina of the photoreceptors. The lateral portions of the RPE are linked (zonula occludens and adherens) to create the external hematoretinal barrier and are interconnected through intercellular junctions at the same time. The cells have a round basal nucleus, and the cytoplasm is rich in lysosomes, smooth endoplasmic reticulum, mitochondria at the basal level, round pigmented granules, and oval ones containing melanin. The majority of the melanin granules are found in the apical portion or in the villa. The granules measure up to 1 µm in diameter and from 2 to 3 µm in length. The pigment granules adsorb light, preventing diffusion, and also act as free radical scavengers. The RPE cells in the macular and equatorial regions contain a major quantity of pigment.4

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In old subjects, however, the RPE appears depigmented owing to the decrease of the melanin granules. For this reason the samples coming from young subjects can be previously depigmented. Fig. 10.4 shows that the RPE of a young healthy man after depigmentation (for the decrease of the melanin granules) was stained with Sudan Black B and bromine acetone for the detection of the phospholipids. The phospholipids are increased if compared with old subjects (A1). On the contrary, oil red O stains neutral lipids in a young (B) and/or in an old (B1) man. There is a strong increase of neutral lipids with age. Fig. 10.5 shows that Sudan black B dyes the total lipids in a young (A) and/or in an old man (B). The total lipids are increased with age. Table 10.1 shows the values of QAI of lipids in RPE for young and /or old subjects. Three classes of lipids are dyed—total lipids, phospholipids, and neutral lipids. After the specific coloration, a quantitative analysis of images was performed and results were expressed in conventional units (see Methods section). The probability or significance index was calculated, comparing the results obtained in young subjects versus older ones. All the tabled results show a high statistic significance (p < 0.001).

Fig. 10.4 Light microscopy of the RPE in a 19-year old eye donor (A and B) and/or a 75-year old donor (A1 and B1). The two figures A and A1 are stained with bromine-acetone-sudan black B (phospholipids), and those on the bottom (B and B1) are stained with oil red (neutral lipids). It can be seen that the intensity of staining with both systems increases with age. Therefore, both phospholipids and neutral lipids show a progressive age-related increase. (Magnification 400x; Calibration bar 10 µm)

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Increase in Granules of Lipofuscin With advancing years, granules of lipofuscin appear inside the cells of the RPE. These granules represent the lysosomal accumulation of residual bodies, including the nondegradable final products of metabolism and the consumed acromeres of the photoreceptor, as already described.6 Each cell of the RPE is in a continuous process of intracellular renewal. Sometimes this process of molecular degradation is not complete and results in the increase of metabolic debris and interference with other metabolic activity in these cells. The residual materials are useless molecular aggregations—normally called lipofuscin granules—that contain damaged RPE cells and the membranes of rod and cone phagocytes (i.e., incompletely degraded cellular debris).8 The incomplete molecular degradation seems to be due to altered substrates, which are not therefore recognized by the enzymatic systems. These molecular alterations result from the harmful effects that free radicals have on the RPE cells and their photoreceptors—rich in polyunsaturated fatty acids that become peroxides.The damaged molecules are consumed by the RPE cells and accumulate in their cytoplasm, thus compromising their metabolism and inducing cell death. The acromeres of the retinal photoreceptors can be abnormal due to oxidative damage, or the abnormality can be hereditary. The reason why the acromeres of the photoreceptors are sensitive to oxidative stress is their richness in polyunsaturated fats. Exposure to light, and particularly short wavelength radiation, increases the production of free radicals and accumulation of lipofuscin in the macular RPE. In addition, environmental factors such as cigarette smoke reduce the level of antioxidants and promote the formation of free radicals, contributing to the accumulation of cellular debris.9 Lipofuscin exists inside the RPE cells in the form of granules that appear yellow-green under UV light excitation. Topographically, the majority of the granules of lipofuscin store in the posterior pole of the eye bulb while their quantity decreases in the fovea. This distribution of lipofuscin with respect to the location remains constant during the entire life, and correlates with the density of photoreceptors. The lipofuscin granules contain lipids and proteins. The cytoplasmic volume of the RPE occupied by lipofuscin increases with age, from 8 percent at age 40 to 29 percent at age 80. Furthermore, the cytoplasmatic volume occupied by lipofuscin in the macula is greater than that in the periphery— 19 percent in the macula as compared to 13 percent in the periphery. The excessive accumulation of lipofuscin in the RPE cells slows the metabolic activity of the cells and predisposes them to age-related macular degeneration (AMD).10 Lipofuscin is the generic name given to a heterogenic group of lipid-protein aggregates that are found in aging cells in all human tissues. Different from the other tissues, the lipofuscin does not derive from the degradation of intracytoplasmatic organelles in the retina, but from the incomplete degradation of the products derived from the phagocytosis of the outer portions of the photoreceptors following the peroxidation of the unsaturated fatty acids. Once accumulated, the lipofuscin causes the death of the RPE cells because it acts as a true generator of free radicals11—lipofuscin can release lysosomotropic amines. Finally, other

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authors consider lipofuscin to be an inert substance that acts directly through the congestion of the cytoplasm (in some tissues lipofuscin can occupy up to 30% of the cell volume).12

Increase in Melanin Granules Melanin has a double role in the retina—to reduce the chromatic aberration, increasing the visual acuteness, and to protect against oxidative stress by acting as a cellular antioxidant. Its concentration increases from the equator to the posterior pole, reaching a peak in the macula. The increased concentration of melanosomes in the macula is due to the fact that the RPE cells here are larger and concentrated into a smaller area with respect to the smaller extra-macular RPE cells dispersed in a much larger area.13 The differential distribution of melanosomes is maintained during the first 40 years of life, but a significant reduction in the melanin granules is seen in all regions of the retina afterwards.14 To make a comparison considering three age groups (10-20; 21-60; 61-100), the reduction in the quantity of melanosomes between the first and the third class is 35 percent. Talking in terms of cell volume, therefore, around 8 percent is occupied by melanin in the first two decades of life, which reduces to 6 percent in the second age group and finally further diminishes to 3.5 percent in the third age group.15 Melanin is a complex heterogenic biopolymer, containing free radicals which can be identified using electron spin resonance spectroscopy. Using this technique a 40 percent reduction in melanin content is observed with aging.16 Three possible mechanisms may explain the loss of melanin from RPE cells—expulsion of the granules, lysosomal degradation, and chemical damage. The expulsion of the granules may be a possibility, notwithstanding the fact that the granules are not found in the Bruch’s membrane nor in the interphotoreceptor space. Lysosomal degradation is highly elevated due to its function of degrading the acromeres of the photoreceptors.17 With aging comes an increase in the number of melano-lysosomes, accompanied by a change in the appearance of the melanin granules. Notwithstanding that the morphology of the melanosomes changes following an interaction with the lysosomes, it is likely at the melanin is not degraded and that the changes derive from the degradation of the proteins of the matrix on which the melanin is deposited. The third mechanism is that of chemical degradation. The irradiation of human eyes with intense blue light induces a nonuniform photobleaching of the melanosomes. The lack of uniformity of the bleaching seems to be due to the fact that lipofuscin is also found in the complex granules of aged RPE cells and that it is more photoreactive than melanin, and may act as a photosensitizer. Blue light, therefore, would induce oxidative photodegradation of melanin by the formation of superoxide anion and hydrogen peroxide. If, on one hand, the photodegradation of melanin (oxidation or irreversible bleaching) does not have any biological significance in tissues with high turnover (such as hair and skin), then, on the other hand, this event gains a high importance when it occurs in those tissues with low turnover, such as in RPE cells that are post-mitotic cells.17

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Increase of Complex Granules With the increase in age, melanin, lipofuscin, and lysosomes can join together to form complex structures (melanolipofuscin and melanolysosomes) inside the RPE. These granules have a regional distribution similar to that of lipofuscin—i.e., the highest concentration is in the macula and it decreases in the periphery and in the fovea. In regard to the percentage of cellular volume occupied by complex granules, this varies from 3.3 percent in the first decade of life and reaches to 8-10 percent in the sixth decade. The association of lysosomes with pigment granules (for example, melanosomes, lipofuscin granules, and complex pigments) explains their age-dependant variation observed in - lysosomal enzyme levels and the activity of RPE cells. The increase in pigment granules with age causes an increase in lysosomes and in the activity of various enzymes, such as acid phosphatase and Cathepsin D, to maintain the normal degradation cycle that follows the ingestion of the acromeres of the photoreceptors by the RPE cells.17

Personal Results In our experiments, only samples of the human retina coming from autopsies, including RPE, were used. Because post-mortem phenomena may bring about early modifications in the data obtained from the eye tissues, the samples were harvested in the same ocular side and at the same time after the death. Our studies were approved by the local Ethical Committee and patients or their relatives gave their informed written consent. The investigations were performed according to the guidelines of the Declaration of Helsinki.18 After removal, the eye-bulb was dissected with a razor blade and samples of intact retinal tissue (located precisely in the same site, equatorially in the nasal region) were harvested. The presence of melanin granules can interfere with morphological ad/or histo-chemical observations on RPE. For these reasons, it is necessary to wish for the strong age-related decrease of melanin granules as it happens in older individuals. On the contrary, in young individuals it is necessary to fade the melanin granules by means of light (exposure for 60 min at a ultra-violet lamp ) or by immersion in an oxidant solution of 3 percent H2O2 for 60 min.

Light Microscopy Samples of the human retina were immediately prefixed in 2 percent osmium tetroxide at pH 7.4 in veronal-acetate buffer for 5 minutes at 4 °C. After fixation, the specimens were washed with veronal-acetate buffer (pH 7.4), dehydrated in a graded ethanol series and embedded in paraffin. Thin sections (about 4 µm) were made for morphological staining with toluidine blue (0.05% for 1 minute).19

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Staining of Lipids Lipids were stained by means of special histo-chemical techniques for light microscopic analysis. To determine the composition and distribution of the lipids, three different stains were used: the bromine–Sudan black B stain, which stains all classes of lipids; Bromine–acetone–Sudan black B, which stains only phospholipids; and Oil red O, which stains neutral lipids (especially esters of saturated and unsaturated fatty acids).20

Transmission Electron Microscopy (TEM) Samples from the autopsy were fixed in buffered 2 percent glutaraldehyde for two hours, washed in buffer and then post-fixed in buffered 2 percent osmium-tetroxyde for two hours, dehydrated, and embedded in araldite. Ultra-thin sections were made using a Reichert Ultra-microtome. These sections were counterstained by uranylacetate and lead citrate21 and observed with a Zeiss EM 109 electron microscope.

Quantitative Analysis of Images For a detailed evaluation of the effects of aging on retinal morphology, a QAI was performed on slides and microphotographs using a Quantimet Analyzer (Leica®) equipped with specific software. This software made it possible to determine (see Table 10.1): a) the thickness of the retina; b) the thickness and the number of the cells of RPE; c) the number of granules in the RPE cells; and d) the electron density of the intracellular sub-structures. Final values must be submitted to statistical analysis of data. The values reported in this paper represent the values of staining for each age group, and are expressed in conventional units (CU) ± S.E.M. CU are arbitrary units furnished and printed directly by the Quantimet system.22

Statistical Analysis of Data The statistical methods used throughout this study must be interpreted as an accurate description of the data rather than as a statistical inference of such data. The preliminary studies of each value were performed with the aid of basic sample statistics. Mean values, maximum and minimum limits, variations, standard deviation (SD), standard error of the means (SEM), and correlation coefficients were calculated. Correlation coefficients denote a significant level less than 0.001 (P < 0.001), while it is not significant when P > 0.05 (n.s.).23

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Results All our morphological results are reported in Figs. 10.1 through 10.5, while the histochemical results are summarized in Table 10.1. Fig. 10.1 shows the normal structure of the human retina in a young subject. The trineuronal intraretinal chain is male by photoreceptors, bipolar and ganglion cells. The RPE is detached, by experimental manipulations, from the other retinal layers. Fig. 10.2 shows, as appears in a digital angiography, the retinal fundus in a young subject. In this image, we can see the nasal superior and inferior branches of the ophthalmic artery. These vessels show a normal caliber, without signs of aging or diseases. Fig. 10.3 shows the same image as in Fig. 10.2, but comes from an old man. The retinal vessels show an increased caliber, a snake-like running and numerous dystrophic zones in comparison with RPE. All these findings can be considered as age-related changes. The major age-related changes concern the metabolism of the lipids. In fact, as said above, the melanin in young subjects can alter the results because of the high pigmentation due to the melanin granules and it is present in all the cells of RPE.

Fig. 10.1 Light microscopic image of a normal human retina in young and healthy subjects . The RPE is detached from the other retina layers. RPE = retinal pigmented epithelium, P = photoreceptors, B = bipolar cells, and G = ganglion cells (Magnification 1600x; Calibration bar 100 µm)

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Fig. 10.2 Retinal fundus in a young (22 years of age) and healthy subject as appears in a digital angiography of the ophthalmic artery. In comparison with the head of the optic nerve, the ophthalmic artery is branched in four divisions—one for each quadrant of the retina. In this image, the nasal superior and inferior branches are evident. (field 40° corresponding to a magnification of about 5x)

Fig. 10.3 Retinal fundus in an older (70 years of age) subject. The retinal vessels show an increased caliber. We can see many dystrophic zones in comparison with the RPE (field 40° corresponding to a magnification of about 5x)

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In old subjects, however, the RPE appears depigmented owing to the decrease of the melanin granules. For this reason the samples coming from young subjects can be previously depigmented. Fig. 10.4 shows that the RPE of a young healthy man after depigmentation (for the decrease of the melanin granules) was stained with Sudan Black B and bromine acetone for the detection of the phospholipids. The phospholipids are increased if compared with old subjects (A1). On the contrary, oil red O stains neutral lipids in a young (B) and/or in an old (B1) man. There is a strong increase of neutral lipids with age. Fig. 10.5 shows that Sudan black B dyes the total lipids in a young (A) and/or in an old man (B). The total lipids are increased with age. Table 10.1 shows the values of QAI of lipids in RPE for young and /or old subjects. Three classes of lipids are dyed—total lipids, phospholipids, and neutral lipids. After the specific coloration, a quantitative analysis of images was performed and results were expressed in conventional units (see Methods section). The probability or significance index was calculated, comparing the results obtained in young subjects versus older ones. All the tabled results show a high statistic significance (p < 0.001).

Fig. 10.4 Light microscopy of the RPE in a 19-year old eye donor (A and B) and/or a 75-year old donor (A1 and B1). The two figures A and A1 are stained with bromine-acetone-sudan black B (phospholipids), and those on the bottom (B and B1) are stained with oil red (neutral lipids). It can be seen that the intensity of staining with both systems increases with age. Therefore, both phospholipids and neutral lipids show a progressive age-related increase. (Magnification 400x; Calibration bar 10 µm)

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Fig. 10.5 Light microscopy of the RPE in an 19-year old eye donor (A ) and/or a 75-year old donor (A1). Both A and A1 are stained with bromine-sudan black B. This method stains all classes of lipids. It can be seen that the intensity of staining increases with age. Therefore, the total lipids show a progressive age-related increase. (Magnification 400x; Calibration bar 10 µm)

Table 10.1 QAI of lipids in the RPE of Young and old objects Class of lipids and staining Young

Old

Total Lipids Sudah Blach B Phospholipids Sudan Black B plus Bromine acetone

44.5 ± 3.1* 25.7 ± 2.3*

31.3 ± 2.2 62.6 ± 4.4

Neutral lipids Oil Red O 30.4 ± 2.9 All the value are expressed as Conventional Units (C.U.) ± SEM (see methods) *P<0,001 young versus old.

49.6 ± 2.6*

Discussion Our results demonstrate that the old subjects undergo the following age-related changes in RPE cells: a) a strong decrease of the granules of melanin with cellular de- pigmentation; b) a strong increase of intra-cytoplasm cell bodies; c) a strong increase of the total lipids while phospolipids and fatty acids are decreased; and d) an increase of the electron density of all cellular substructures. RPE cells take part of the so-called choroids retinal complex (named also chorio-retina) formed by choroids, Bruch’s membrane, basal membrane, and RPE. This complex supplies blood and metabolic support to the other cellular layer of the retina (including photoreceptors bipolar and multi-polar cells). The RPE play a fundamental role in the transport and storage of the retinoids, essential for maintaining the visual cycle. Another function of the RPE is to eliminate components of the acromeres of the photoreceptors through phagocytic activity mediated by cathepsin D and by the integrins that act as membrane receptors mediating the phagocytosis. The phagosomes are linked to lysosomes to form phago-lysosomes. These cellular substructures are digested by the intracellular digestive enzymes located at the basal surface of the cell. The final metabolic products are expelled into the choroidal circulation by exocytosis through the Bruch’s membrane. If they are not completely digested, they can accumulate inside the cells

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as granules of lipofuscin. These granules increase with age,24 especially in the macular area. The constant exposure of the RPE to light, at elevated concentrations of oxygen, together with the high metabolic activity of these cells, creates an environment favorable (especially in the macular area) to the formation of toxic reactive oxygen radicals. The reactive radicals can induce an oxidative stress. In defense against oxidative stress, the RPE cells contain antioxidants such as superoxide dismutase, catalase, reduced glutathione, melanin, and carotenoid. The quantity of these substances decreases with age and, therefore, the antioxidant defenses also decrease with age. The RPE cells, together with the other retinal cells, allow us to observe the wonders of the world. Unfortunately, all these cells undergo to a senile involution with a strong decrease of their functions. It is well-known that the level of senescence is determined by the interaction between two types of factors: a) promoting aging, and b) counteracting it (theory of senescence). The cellular regeneration capacity is under genetic control and is able to counteracting the senescence. On the other hand, the theory of the biological clock asserts that the senescence of each species is genetically determined, and that every change is the result of environmental influences and/or some mutations. To fully understand the senile involution of RPE, further research is needed.

References 1. Cavallotti C, Pescosolido N (2006) Age-related changes in the human retina. In: Conn PM (ed) Handbook of Models for Human Aging. Academic Press Ed. Elsevier, San Diego, USA p 793-812 2. Panda-Jonas S, Jonas J, Jakobczyk-Kmija M (1996) Retinal pigment epithelial cell count distribution and correlations in normal human eyes. Am. J. Ophthalmol.121:181-189 3. Chader GJ (2002) Animal model in research on retinal degenerations. Past progress and future hope. Vision Res. 42:393-399 4. Katz ML, and Robison WG (1984) Age-related changes in the retinal pigment epithelium of pigmented rats. Exp. Eye Res. 38:137-151 5. Young RW (1982) The Bowman Lecture: Metabolism of the pigment epithelium. In: Shimizu K, Oosterhuis JA (eds) Proceedings of the XXIII International Congress Kyoto,14-20 May, 1978. Excerpta Med., Amsterdam, p 159-166 6. Burns RP, and Feeney-Burns L (1980) Clinico-morphologic correlations of drusen of Bruch’s membrane. Trans. Am. Acad. Ophthalmol. Soc. 78:206-255 7. Dorey CK, Wu G, Ebestein D, Garsd A, Weiter JJ (1989) Cell loss in the aging retina: relationship to lipofuscin accumulation and macular degeneration. Invest. Ophthalmol. Vis. Sci. 30:1691-1699 8. Armstrong D, (1984) Free radical involvement in the formation of lipo-pigments. In: Armstrong, D (ed) Free Radicals in Molecular Biology, Aging and Disease. Raven Press, New York, p 137-182 9. Lerman S, (1988) Ocular photo-toxicity. N. Engl. J. Med. 319:1475-1477 10. Bird AC (1997) What is the future of research in age-related macular degeneration. Arch. Ophthalmol. 115:1311-1313 11. Boulton M, Dontsov A, Jarvis- Evans J, Ostrovsky M, Svistunenko D (1993) Lipo- fuscin is a photo-inducible free radicals generator. J. Photochem. Photobiol. 19:201-204 12. Eldred GE, and Lasky MR (1993) Retinal age pigments generated by self-assembling lysomotrophic detergents. Nature 361:724-726

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13. Handelman GJ, and Dratz EA (1986) The role of antioxidants in the retina and retinal pigment epithelium and the nature of pro-oxidant induced damage. Adv. Free Radicals. Biol. Med. 2, 1:89 14. Weiter JJ, Delori FC, Wing GI, Fitch KA (1986) Retinal pigment epithelial lipofuscin and melanin and choroidal melanin in humans eyes. Am.J. Ophthalmol. 27:145-152 15. Feeney-Burns L, Hilderbrand ES, Eldridge S (1984) Aging of human PRE: morphometric analysis of macular, equatorial and peripheral cells. Invest. Ophthalmol. Vis. Sci. 25: 195-200 16. Sarna T, Burke JM, Korytowski W, Rozanowska M, Shumatz CM, Zareba A, Zareba M (2003) Loss of melanin from human RPE with aging: possible role of melanin photooxidation. Exp. Eye Res. 76:89-98 17. Boulton M and Wassel J (1998). Ageing of the human retinal epithelium. In: Coscas G and Piccolino FC (eds) Retinal Pigment Epithelium and Macular Disease Doc. Ophthalmologica. 62:20-28 18. Declaration of Helsinki (1964) of the World Medical Association (amended in 1975 and 1983). In: Philosophy and practice of medical ethics. British Medical Association, 1988 19. Spandrio L (1988) Manuale di laboratorio: Metodi di colorazione Piccin Ed., Padua, p 34 20. Pearse AGE (1972) Histochemistry, theoretical and applied. Churchill-Livingstone Ed., London 21. Millonig G (1961) Advantages of a phosphate buffer for OsO4 solutions in fixation. J Appl Physiol 32:1637–1641 22. Leica Manuale dei Metodi Quantimet 500 (1997) Microsystems Imaging Solutions, Cambridge, UK 23. Castino M, Roletto E (1992) Statistica applicata. Ed. Piccin, Padova 24. Iwasaki M, and Inomata H (1988) Lipofuscin granules in human photoreceptor cells. Invest. Ophthalmol. Vis. Sci. 29:671-679

Recent Books on RPE Disorders in Old Age (www.amazon.com) 1. The Aging Eye by Sandra Gordon. Harvard Medical School, 2001. 2. Communication Technologies for the Elderly: Vision, Hearing & Speech by Rosemary Lubinski and D. Jeffery Higginbotham, 1997. 3. The Effects of Aging and Environment on Vision by Donald A. Armstrong, et al. 1991. 4. Treating Vision Problems in the Older Adult (Mosby Optometric Problem-Solving Series) by Gerald G. Melore, 2001. 5. Vision and Aging by Alfred A. Rosenbloom and Meredith W. Morgan, 1993. 6. Age-Related Macular Degeneration by Jennifer I. Lim, 2002. 7. The Impact of Vision Loss in the Elderly (Garland studies on the Elderly in America) by Julia J. Kleinschmidt, 1995. 8. Vision in Alzheimer’s Disease (Interdisciplinary Topics in Gerontology) by Alice CroninColomb, et al., 2004. 9. The Senescence of Human Vision (Oxford Medical Publications) by R.A. Weale, 2001. 10. Issues in Aging and Vision: A Curriculum for University Programs and In-service Training by Alberta L. Orr, 1998. 11. Aging with Developmental Disabilities Changes in Vision by Marshall E. Flax, 1996. 12. Trends in Vision and Hearing among Older Americans, by U.S. Dept of Health and Human Services, 2000. 13. Optometric Gerontology: A Resource Manual by Sherrell J. Aston, 2003.

Chapter 11

The Aging of the Choroid Angelica Cerulli, MD, Federico Regine, MD, and Giuseppe Carella, MD, PhD

Abstract The first section of this chapter describes the anatomy and the physiology of the choroid and the vascular pattern of the choroidal vessels. The choroid is of fundamental importance for nourishment of the retina so that all the alterations of the choroid lead to a disfunction of the retinal pigment epithelium (RPE), Bruch’s membrane, and choriocapillary complex. The various methods used to study the choroid and its pathologies in post-mortem studies and in vivo are described—injection of chromopolimer, Indocyanine Green Angiografy, and Doppler flow studies. Age-related changes of choroid are analyzed. Alterations have been described in the various layers of the choroid, which are part of the physiologic aging process. In certain cases, they can cause disease, but sometimes the border between physiologic and pathologic age-related changes is very hard to identify. The choroid represents the preferential target of certain age-related diseases. In particular, we describe the physiopathology of age-related pathologies such as hypertension, diabetes, AMD, and atherosclerosis—so common in the elderly. In particular, diabetes and AMD represent the main causes of blindness in industrialized countries. Keywords choroid aging, age-related macular disease, choroidopaty hypertensive choroidopathy

Anatomy of the Choroid The choroid is the middle tunic of the eye. It lies between the fibrous outer tunic, whose function is support, and the inner neural tunic (the retina) that provides visual function. The choroid appears as a reddish-brown membrane extending from the ora serrata to the optic nerve. Its color is due to the presence of pigment cells (melanocytes) and blood vessels. The thickness ranges from 90-100 microns near the ora serrata to 300 microns at the posterior pole. With advancing age, these measurements may change as a result of vessel sclerosis and decreases in the collagen content of the membrane.1

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Histology The choroid is composed of four layers (proceeding outermost to innermost): ● ● ● ●

the suprachoroidal layer, or lamina fusca the choroidal stroma the choriocapillaris the lamina vitrea or Bruch’s membrane (BM)

The Suprachoroidal Layer or Lamina Fusca This layer is composed of elastic fibers and branched, syncytial elements with flattened, oval-shaped nuclei, all immersed within in a network of thin collagen lamellae. The nerves and the long posterior ciliary arteries run through the lacunae situated between the lamellae. These structures, unlike the short posterior ciliary arteries and the vorticose veins, do not penetrate the lamina fusca. This layer also contains giant melanocytes whose cytoplasm is full of granules. Posteriorly, the lamellae insert perpendicularly into the sclera, producing rigid cohesion; anteriorly, adherence to the sclera is looser because the lamellae lie parallel to the sclera. This layer has a thickness of 10-15 microns, and it extends anteriorly to the scleral spur.

Choroidal Stroma This layer is composed of collagen fibers (isolated or bundled); thin elastic fibers (0.3-0.4 microns); fibrocytes; large, star-shaped melanocytes filled with cytoplasmic pigment granules; macrophages; and blood vessels. The vessels are arranged in three layers: 1) the outermost, or Haller’s layer, which contains large-caliber vessels; 2) the middle layer, or Sattler’s layer, which contains medium-caliber vessels; and 3) the inner layer, or the choriocapillaris (also known as Ruysch’s membrane).

The Choriocapillaris This layer is composed of a dense network of broad-lumened capillaries that are devoid of pericytes. The capillary endothelium is thin with relatively few nuclei. The cytoplasm of the cells contains pores closed by a thin membrane measuring 3 nanometers in the thickness in the part of the wall facing Bruch’s membrane, while the more external capillaries are characterized by endothelial cells whose cytoplasm is filled with vesicles. The thickness of the choriocapillary layer ranges from 10 to 30 microns. The capillaries of the choriocapillary layer are arranged to form lobules, each of which has its own terminal blood supply and is functionally

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independent from the others. Each afferent arteriole reaches one unit and divides to form orderly, polygonal lobules at the posterior pole, and fan-shaped lobules in the peripheral regions.

The Lamina Vitrea or Bruch’s Membrane This thin (2-4 microns), elastic mucoprotein membrane lies between the metabolically active tissue of the retinal pigment epithelium (RPE) and the choriocapillaris, which is the source of RPE’s blood supply. In young subjects, the three layers are tightly adhered. The portion facing the choroid is mesodermal in origin, while that part facing the RPE is derived from the ectoderm. Bruch’s membrane can be schematically divided into five layers: ●

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The external choroidal layer consisting of the endothelial cells of the choriocapillaries in the basement membrane, which is frequently interrupted by endothelial buds The outer collagenous zone (OCZ) A layer of fenestrated elastic fibers The inner collagenous zone (ICZ) The basement membrane of the RPE

The complex formed by the choriocapillaris, Bruch’s membrane, and the RPE represents an important connection system for exchanges with the retina. The thickness of Bruch’s membrane is greatest at the posterior pole.

Vascularization The choroid receives its blood supply from the anterior ciliary arteries and from the long and short posterior ciliary arteries.

The Anterior Ciliary Arteries These vessels arise from the muscular branches of the ophthalmic artery; and their numbers range from six to eight. They run alongside the tendons of the ocular muscles—generally two arteries per tendon. They give rise to conjunctival and episceral branches before perforating the sclera to reach the choroid at the level of the ciliary muscle, which they supply with a few branches. They continue on, anastomosing with the long posterior ciliary arteries to form the greater circle of the iris. This circle is composed of the anterior and posterior ciliary arteries and is located near the ciliary margin of the iris. It gives rise to branches that run posteriorly to supply the ciliary muscle and ciliary processes and others that run through the iris toward the pupillary margin, where they form anastomoses with one another and give rise to the small arterial circle of the iris.

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The Posterior Ciliary Arteries The posterior ciliary arteries (PCA) are the main source of blood supplying the head of the optic nerve. They also supply the pre-equatorial portion of the choroid (the RPE), 130 µ of the outer retina (or, when the cilioretinal artery is present, the full thickness of the retina in this zone), and the lateral and medial aspects of both the ciliary bodies and iris. The PCA circulation is therefore the most important component of the ocular vasculature and that of the optic nerve. Disturbances in the PCA circulation can lead to a variety of disorders involving the ocular vasculature and the head of the optic nerve, which can produce different degrees of visual impairment. The ophthalmic artery gives rise to one to five posterior ciliary arteries that are distinguished as short and long PCAs.

Short Posterior Ciliary Arteries (SPCA) Subdivision of the PCAs gives rise to 10-20 short ciliary arteries—the paraoptic branches, which penetrate the sclera near the optic nerve, and the distal branches, which penetrate the sclera a short distance from the optic nerve and run radially toward the equator. The distal SPCAs that penetrate the sclera temporally to the optic nerve supply blood to the macular region.

Long Posterior Ciliary Arteries (LPCA) There are generally two LPCAs—one medial and one lateral. They penetrate the bulb on the horizontal plane, not far from the distal branches of the SPCAs—one on the lateral side, and the other on the medial aspect—and run radially on the horizontal meridian forward to the iris.2,3,4,5,6,7

Vascular Patterns of the Posterior Ciliary Arteries and their Branches The earliest structural descriptions of the choroid plexus were based on postmortem studies in which material injected into the plexus solidified, providing a three-dimensional reconstruction of the tributary complex of the vessel being examined. Professor Carella has conducted numerous studies on the anatomy of the choroid using chromopolymer injections. The scheme below summarizes the method used to study the choroid in cadavers: 1. Cross-section craniotomy (360° frontal-parietal-occipital) 2. Exposure and removal of the brain

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3. Isolation of the chiasm and carotid arteries at their point of entry into the cavernous sinus 4. Removal of the ceiling and contents of the orbital cavity, contents of the intracanalicular and intracranial portions of the ophthalmic artery, and the carotid siphon 5. Chromopolymer injection 6. Formalin fixation 7. Isolation of the ocular membrane under stereo-microscopy 8. Clearing and diaphanization of the tissue In Fig. 11.1, we reported the physiologic aspect of the choriocapillaris lobules in a normal macula. Watershed zones between lobules are clearly visible. Experimental occlusion of the PCA in monkeys, and the spontaneous occlusion seen in patients with giant-cell arteritis, cause segmental infarcts at the level of the choroid plexus. These studies indicate that each PCA has a segmental distribution in the choroid and in the optic nerve, and the PCAs do not anastomose with adjacent PCAs or the anterior ciliary arteries.4,5,8,9,10,11,12,13,14,15,16,17,18,19,20 Similarly, when the anterior ciliary artery is occluded during surgery on the rectus muscles of the eye in monkeys or humans, the PCAs do not supply the anterior uvea that is normally supplied by the occluded artery.21,22 ICG angiography was first used in ophthalmology in 1969. Thereafter, it was gradually introduced into clinical practice. It allows excellent visualization of the vessels of the external choroid, but the choriocapillaris is more difficult to examine due to the high level of background fluorescence. In vivo angiographic studies have shown that the entire choriocapillary bed is composed of small, independent

Fig. 11.1 Choriocapillaries’ architecture in the normal macula as obtained from chromopolymer casting studies of human choroid. Irregular polygonal lobules separated by watershed zones are clearly visible

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lobules.21,23,24 Each lobule is supplied by a terminal arteriole located at its center. Drainage is provided by a network of venules located at the periphery of the lobule. There are no anastomoses between adjacent lobules. The lobules are arranged in a mosaic-like pattern. The shape and size of the various lobules varies depending on their location within the choroid (polygonal at the posterior pole, fan-shaped in the peripheral regions). The choriocapillaris is much thicker at the posterior pole and becomes progressively thinner toward the periphery. In conclusion, all these studies clearly demonstrate that there are no anastomoses between adjacent segments, and the PCAs and their branches thus behave as terminal arteries in vivo. The discrepancies between data from in vivo and postmortem studies could be due to the fact that the vascular bed of the choroid is richly innervated by autonomic fibers in vivo. The influence of these nerves on blood flow and circulation is naturally absent in postmortem studies. The true physiological behavior of this vascular bed can be evaluated only by in vivo fluorescent angiography. In contrast, when the material is injected under pressure in postmortem studies, it fills the entire vascular bed without any neuronal control. These studies can therefore provide information only on the morphology of the choroid plexus and not on the physiology of choroidal blood flow. In vivo studies are thus able to explain the localized nature of inflammatory, ischemic, and metastatic lesions of the choroid.

Areas Supplied by the Anterior Ciliary Arteries The anterior ciliary arteries run along the four rectus muscles in a posteroanterior direction and then, at the level of the bulbar tendon insertion, they provide blood to the bulbar conjunctiva before penetrating the sclera. During their intrascleral course, they give rise to small branches that interweave to form the intrascleral arterial plexus. After crossing the sclera, they anastomose with branches of the long PCAs to form the greater arterious circle of the iris. The vessels that branch off from this arterial circle extend to the iris, the ciliary body, and the ciliary muscle. In addition, some recurrent branches of the circle run to the anterior portion of the choroid.

Areas Supplied by the Short Posterior Ciliary Arteries The paraoptic branches of the PCAs supply blood to the optic nerve, the peripapillary region of the choroid, and the circle of Zinn-Haller. Each of the distal short ciliary arteries supplies a sector of the choroid that generally extends from the posterior pole to the equator. The sectors vary considerably in shape, size, and location. Their margins are irregular, and they fit together like the pieces of a jigsaw puzzle. Further subdivisions of the short PCAs correspond to smaller segments, irregular in shape and size, so that the blood supply from the various choroidal arteries has a geographic distribution—the smaller the artery, the smaller the area it supplies.

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Areas Supplied by the Long Posterior Ciliary Arteries Each artery runs radially within the horizontal meridian—one on the medial side, one on the lateral side. On the temporal side, the long PCA supplies a sector that is temporal to the macula and whose apex is oriented posteriorly. Each artery also supplies small sectors of the ciliary body and the iris, on both the medial and lateral sides.

The Watershed Zone in the Vascular Bed of the PCAs The watershed zone marks the divide between the distribution territories of two terminal arteries. This zone is the most vulnerable to ischemic damage when there is a drop in the perfusion pressure of the vascular bed of one or more of the terminal arteries. There are watershed zones between the areas supplied by the various ciliary arteries—those supplied by the short PCAs, and those supplied by the anterior and posterior ciliary arteries.17,21,23 In the presence of two long PCAs (medial and lateral), the watershed zone between these two vessels consists of a vertical band whose location is highly variable—it can be situated temporally to the peripapillary choroid; it may cross the temporal peripapillary choroid or a segment of the optic nerve; it may surround the entire optic disk; or it may cross the nasal peripapillary choroid. Various combinations of the previous pictures are also possible. In the presence of three or more long PCAs, the watershed zones between various PCAs play an important role in ischemic optic neuropathies because the optic nerve lies in a watershed zone, which renders it highly vulnerable to ischemic damage. It is important to recall that the temporal branches of the distal short PCAs enter the bulb and supply blood to the macular region, where their watershed zones are located. The studies of Hayreh have shown that there are no anastomoses between the long and short PCAs. Therefore, the long and short PCAs are separated by a watershed zone.

Watershed Zones Between the Territories Supplied by the PCAs and the Anterior Ciliary Arteries Experimental and clinical studies involving the occlusion of the posterior ciliary artery 11-20,25 or the anterior ciliary artery have clearly demonstrated that there are no in vivo anastomoses between the anterior and posterior ciliary arteries. Using ICG, Takanashi et al.26 also showed that there are no functional anastomoses between the terminal branches of the PCAs and those of the anterior ciliary arteries. Therefore, there is a watershed zone between the PCAs and the anterior ciliary artery, and this zone is located in the equatorial region of the choroid.23,29 From a clinical point of view, it is well-known that the watershed zones play a fundamental role in the development of ischemic lesions. Therefore, it is clear that portions of the optic nerve and choroid lying in a watershed zone are more subject to ischemic damage. These data are supported by various studies.29,27,28,29,30,31,32,33,34,35,36

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Venous Drainage of the Choroid The veins that drain the choroid empty into the 4-6 vorticose veins at the equator. Hayreh’s studies of primates subjected to vorticose-vein occlusion showed that each quadrant of the choroid is independently drained by a single vorticose vein.37 With angiography, the resulting watershed zones form a cross-shaped zone of hypofluorescence that runs horizontally across the optic nerve and the fovea and vertically through the papillomacular region.40,38 A study conducted by Mori et al., in which the venous phases of ICG angiography were analyzed in healthy subjects, revealed asymmetric venous drainage of the macula in 50 percent of the subjects studied. In two-thirds of these patients, the direction of drainage was predominantly superotemporal, while inferotemporal or superonasal drainage was observed in the remaining cases. It may be that the preferential direction of venous outflow from the choroid is influenced by closure of the embryonic cleft, which occurs in an asymmetric fashion.39

Physiology of the Choroid The choroid is of fundamental importance for nourishment of the retina—it supplies approximately 70 percent of the oxygen and glucose needed by the retina.40 The choroid is characterized by extremely high flow rates (around 1800 microliters /min / 100 gr) that are ten times greater than that of the retina. The flow is regulated by the autonomic nervous system via vascular adrenergic fibers that extend only as far as the lamina cribrosa. Blood flow through the choroidal circulation is mainly dependent on three factors: intraocular pressure, mean arterial pressure, and peripheral vascular resistance. The choroid has no systems for autoregulation, and sudden changes in intraocular pressure are not compensated by pressure changes in the choroidal vasculature. After it circulates within the lobule, the blood drains through the venous system, which forms a ring consisting of postcapillary venules—this arrangement results in rapid, efficient outflow. This complex vascular structure serves multiple functions: 1) transport of nutrients to the overlying layers, Bruch’s membrane, the RPE, and the neuroepithelium of the retina; and 2) dissipation of the heat produced by the retina during photochemical and metabolic reactions.41

Growth and Aging Alterations have been described in the various layers of the choroid, which are part of the physiologic aging process. In certain cases, they can cause disease. The choroid is also the preferential target of certain age-related diseases.

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Age-related Changes in the Choroid The most important data come from cadaver studies and in vivo imaging studies (ICG angiography, Doppler, and laser Doppler flow studies).

Histological Studies of Age-related Changes in the Choroid The choroids of young subjects are characterized by a higher number of cell nuclei, which is thought to reflect the presence of inflammatory infiltrates that are especially common in newborns (term and preterm alike). With advancing age, small, homogeneous, focal deposits of anomalous material appear in Bruch’s membrane. These deposits, known as Drusen, are surrounded by degenerated pigment epithelium and are sometimes calcified. In the choroid of an elderly subject, granular deposits can also be seen on Bruch’s membrane. They have proved to be the result of lipid-degeneration phenomena. The intima of the vessel wall and Bruch’s membrane are targets of many age-related changes. These changes affect the endothelial surface of the vascular wall, and both the endothelial and epithelial surfaces of Bruch’s membrane. In both cases, the main effect of aging is an increase in thickness. The thickness of Bruch’s membrane increases by approximately 135 percent over ten decades.42,43 The thickening occurs mainly in the outer collagen zone.44 Age-related anatomical changes in Bruch’s membrane include the progressive accumulation of debris, lipid deposits, and alterations involving the extracellular matrix.

Accumulation of Debris There are three types of deposits beneath the pigment epithelium: Drusen, basal linear deposits, and basal laminar deposits.45,46,47 Drusen can be seen on ophthalmoscopy. They are extracellular deposits situated between the basement membrane of the pigment epithelium and the inner collagen zone of Bruch’s membrane. Their composition is very similar to that of atherosclerotic deposits. The second type of deposit has been defined by Green and Enger as the basal linear deposit. They form a thin membranous layer beneath the RPE.50 Sharks et al. maintain that they are made of a material released from the basement membrane of the RPE and are incapable of passing into the inner collagen zone.50,48,49 The third type is the basal laminar deposit, which is found within the basement membrane of the RPE and is primarily composed of collagen.50 With age, this debris accumulates and eventually involves all collagen layers of the basement membrane.47 All of these deposits can represent waste products produced by an altered RPE51,52,53,54 or the sequelae of endothelial dysfunction at the level of the choriocapillaris.55

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Lipid Deposits Aging is accompanied by an exponential increase in lipid deposits within Bruch’s membrane. In the eyes of a patient under 60, these deposits contain small, round, solid particles that are scattered throughout the various layers of collagen. In elderly subjects, lipid droplets occupy over one third of the inner collagen zone, and they form a thin lipid layer external to the basement membrane of the RPE.56 These deposits lead to an exponential decrease with age in the hydraulic conductivity of Bruch’s membrane.57,58 The lipid deposits in Bruch’s membrane are composed of esterified and nonesterified cholesterol, and for this reason they have been compared to the atherosclerotic deposits found in blood vessels.59,59,60

Changes in the Extracellular Matrix Collagen synthesis increases with age in both the choroid and in the arteries, and the excess collagen is also less soluble than normal.61,62 The fibers of the elastic membrane increase in number, and pin-point crystals are deposited between the fibers.45 Bruch’s membrane undergoes calcification and fragmentation, which reduces its elasticity.

Age-related Changes in ICG-angiographic Findings in Normal Subjects ICG angiography allows dynamic studies of the choroidal circulation. Comparison of the angiograms of subjects from different age groups has revealed certain differences between young and elderly subjects. In young persons, arteriolar filling begins in the subfoveal region and extends radially toward the periphery of the ocular fundus. In the macular area, the choroidal system appears fine, tortuous, and multibranching. The vertical watershed zone that passes through the optic nerve is clearly visualized (see Fig. 11.2). With age, small areas of hypofluorescence appear at the posterior pole, and arteriolar fluorescence becomes less intense. The choroidal arterioles become thinner and more tortuous, and there is a decrease in the number of collateral vessels. The vertical watershed zone becomes harder to detect (see Fig. 11.3). These changes could be related to reduced blood flow through the macular choroid and to delayed arterial filling.46 These findings have been confirmed by laser Doppler flow studies, which also demonstrated age-related decreases in the choroidal blood volume.63 Ocular blood flow can be studied with various methods, including fluorescein angiography, scanning laser ophthalmoscopy, scanning laser Doppler flowmetry, Heidelberg retinal flowmetry, magnetic resonance imaging, transcranial Doppler, and color Doppler ultrasound. The latter method can provide precise information on certain hemodynamic parameters for each case studied. Color Doppler provides three types of information—simultaneously and in real time (see Fig. 11.4):

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Fig. 11.2 Early phase ICG in a young subject: watershed zone between the long posterior ciliary arteries can be easily seen passing just temporally of the optic disk

Fig. 11.3 ICG image in an old normal subject: watershed zone is hardly defined and the choroidal arterioles are thinner and more torturous. The veins appear dilated

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morphology of the tissues based on conventional B-mode sonography color-coded visualization of the vessels vessel hemodynamics based on pulsed Doppler analysis

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Color Doppler is noninvasive and does not require the use of contrast agents—it is also easy to perform, can be repeated as needed, and is well-tolerated by patients. It provides specific topographic information and data-characterizing blood flow. Color Doppler furnishes morphological information as well as quantitative data on ocular blood flow, including peak systolic velocity, telediastolic velocity, mean velocity, and the resistance index. Atrophy and depigmentation in the peripheral

a.

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Fig. 11.4 Color Doppler imaging of the choroidal vasculature. In (a), the ophthalmic artery presents a low resistance index in a young subject. At the opposite, in an elderly patient (b), the opthalmic artery shows a high resistance index. In (c), we report the color Doppler image of the normal choroid

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Fig. 11.4 (continued)

choroid are common findings in senescence. Similar atrophic changes are frequently seen in the peripapillary region, as well. In both settings, the lesions are associated with (and perhaps caused by) fibrosis, hyalinization, and occlusion of the choroidal vessels—especially those of the choriocapillaris. ICG angiography can detect aneurysms of the choroidal arteries, which appear as focal sac-like dilatations. Various mechanisms have been implicated in their pathogenesis. Aneurysms of the retinal artery are associated with hypertension, arteriosclerosis, and aging.64,65,66 Similar aneurysms can develop in cerebral vessels as a result of hypertension.67 In a study conducted by Schneider et al., ICG angiography was performed on patients with geographic atrophic lesions caused by AMD. The studies revealed68,48 aneurysms of the choroidal vessels that are typically observed in areas of geographic atrophy with reduced choroidal perfusion.69,70,68,71,48

Age-related Diseases that Preferentially Target the Choroid This category includes age-related macular degeneration (AMD)—vascular changes associated with systemic diseases such as hypertension, diabetes, and atherosclerosis that are typically found in elderly subjects.

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Age-related Macular Degeneration (AMD) AMD is one of the main causes of blindness in industrialized countries. There are exudative and nonexudative forms. The latter is clinically defined by the presence of Drusen and pigmentation changes in the RPE, including geographic atrophy. The exudative form has the same characteristics, plus signs of choroidal neovascularization (CNV) (choriocapillaris vessels that perforate Bruch’s membrane and grow into the subretinal spaces and under the RPE), hemorrhages, hard exudates, and serous detachment of the neuroepithelium or of the RPE caused by CNV. AMD is a multifactorial, degenerative disease that affects the RPE, Bruch’s membrane, the photoreceptors, and the choroid. The pathophysiological mechanisms underlying its development are clearly related to aging. The anatomical structures mainly affected by the disease are the complex consisting of the choriocapillaris, the RPE, and Bruch’s membrane.71 Bruch’s membrane is a semipermeable complex that allows intense metabolic exchange between the choroidal and retinal sides. It contributes to the creation of a barrier between the blood and the external retina since the negative electrical charge of the membrane proteoglycans acts as a filter for macromolecules coming from the choroidal circulation. Some of the most important histopathological and pathogenetic changes of AMD occur in this membrane. The outer retina receives oxygen and metabolites that diffuse across Bruch’s membrane and the RPE from the choroidal vessels. For the inner retinal layers, the supply comes from the capillary layers of the retinal circulation. The photoreceptor cells located in the foveal zone are characterized by oxidative metabolism that is four times higher than that of central nervous system cells. The high flow through the choriocapillaris ensures an adequate supply of metabolic products and effective clearance of the catabolites produced. With time, lipid and protein substances and catabolites can accumulate in Bruch’s membrane. Increases in the thickness of the membrane diminish its permeability and elasticity, which reduces supplies of oxygen and metabolites. The substances that accumulate originate in the vessels of the choriocapillaris and in the cells of the RPE, which is the main site of phagocytosis and elimination of the outer segment and of photoreceptor catabolites. From a functional point of view, these morphologic changes reduce the passage of nutrients and the elimination of catabolic products and ultimately lead to the formation of Drusen. The next step involves progressive decreases in the production of pigment epithelium-derived growth factor (PEDF). Compared with healthy subjects, AMD patients have significantly lower levels of this growth factor in cells of the RPE, Bruch’s membrane, and the choroidal stroma. As a result of these changes, the resistance and integrity of the membrane are threatened. Breats can form that are an important stimulus for subretinal neovascularization. The changes observed during the course of AMD may therefore represent an extreme variant of the normal aging process. Disturbances of the ocular circulation in patients with AMD have been documented by various studies.72 Comparative studies of ICG angiograms of AMD patients and healthy, age-matched controls

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have revealed that arterial filling is slowed in the former group. Arteriolar fluorescence is characterized by asymmetric distribution with focal filling deficits at the foveal level. The arterial watershed zones are poorly defined. In general, they appear larger and have darker margins than those of the controls. The choroidal arterioles are thinner and more tortuous, and the number of collateral vessels is reduced. The veins appear dilated. The number of macular arterioles is reduced, and fluorescence is diminished in the arterial filling phases. These changes, however, are also noted in healthy elderly subjects. At least part of the diminished arterial perfusion observed in the choroids of AMD patients might be attributed to choroidal aging. Data from histo-pathological studies suggest that choroidal circulation is reduced in these patients, which supports the view that AMD is associated with hemodynamic anomalies.73,74,46,75 Laser Doppler flowmetry is a noninvasive method for measuring choroidal blood flow, blood volume, and flow velocity with a diode laser. In a study conducted by Grunwald, these three parameters were investigated with laser Doppler flowmetry in normotensive and hypertensive AMD patients. Compared with the patients who had no history of hypertension, those in the hypertensive subgroup had significantly diminished choroidal blood flow. Several studies have demonstrated reductions in choroidal blood flow in patients with AMD and Drusen.75 The decreases observed in the parameters of choroidal blood flow are inversely proportional to the severity of the AMD, and they are also associated with an increased risk for choroidal neovascularization.76 The findings of Ross et al.36 show that choroidal neovascularization develops more frequently at the borders of the watershed zones, which is consistent with the view that ischemic areas can play a role in its development. These watershed zones, which are the last areas labeled during the early phases of ICG angiography, are the areas most likely to develop ischemia and hypoxia if choroidal flow decreases. The association between diminished choroidal blood flow, AMD, and arterial hypertension may reflect an abnormal autoregulatory response caused by those histopathological changes that are now known to be typical of AMD, such as the reduced density and calibers of the capillaries in the macular region of the choroid.46

Disorders Affecting the PCAs Circulation These changes can occur during the course of several systemic diseases (hypertension, arteriosclerosis, and diabetes). They have been reproduced in primates by occlusion of the PCAs or experimental induction of malignant hypertension.12,14, 77,78,79

When there is an occlusion in a branch of the PCAs, which supply the peripheral regions of the choroid, fluorescein angiography reveals a triangular defect in the zone of the obstruction. The location and extension of the damage depends on the site of the occlusion itself. Triangular syndromes can also develop after laser photocoagulation or diathermy. The long posterior ciliary arteries originate temporally and laterally to the macula; diathermy or photocoagulation in this region produce a

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typical peripheral triangular syndrome. Triangular syndromes caused by obstruction of the short ciliary arteries are smaller and more irregular. When they are confluent, they can lead to the atrophy of an entire quadrant.

Acute Ischemic Lesions of the Choroid The appearance of these lesions depends on their stage of evolution, the size of the artery or arteriole involved, and the severity of the ischemia.12,14,82-80 Evolutionary Stage of the Lesions Recently developed lesions are white—the RPE and retina are no longer perfused.12,14,82 About a week after onset, their appearance begins to change. After one to two weeks, they are grayish-white and granular, and they gradually take on the appearance of a nonspecific pigmented chorioretinal lesion. Initially, histopathology reveals coagulative necrosis of the RPE and outer retina associated with disruption of the blood-retinal barrier secondary to RPE damage. Even in eyes with severe ischemic damage, the bloodretinal barrier resumes its activity after about three months.82 In the acute stage, fluoroangiography reveals initial hypofluorescence due to a filling defect and late hyperfluorescence related to stain uptake. Advanced chorioretinal lesions are associated with hyperfluorescence (window effect) with no staining. Size of the Artery or Arteriole Involved in the Occlusion Occlusion of the long PCAs produce large choroidal infarcts.12,14,82 The lesions are often triangular and located in the periphery of the ocular fundus with the base facing the equator and the apex pointing toward the posterior pole.12,14-16,81 Focal occlusions of the terminal arterioles of the choroid result in small, roundish, localized lesions that represent infarcts of the lobules of the choriocapillaris. Elschnig’s spots, which are seen in advanced hypertensive choroidopathy, are a typical example of these focal ischemic lesions.84 Severity of the Ischemia Severe forms of ischemia cause true infarcts of the choroid, RPE, and outer retina. Moderate or mild ischemia causes ischemic dysfunction of the RPE, with or without overt RPE infarction. The barrier function of the RPE is lost, and liquid passing from the choroid to the retina causes serous detachment of the retina, similar to that seen in hypertensive choroidopathy and in eclampsia84 (see Fig. 11.5).

Ischemic Lesions of the Macular Choroid The temporal branches of the distal short PCAs supply blood to the macular choroid. These arteries subdivide to form numerous terminal branches. The result

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Fig. 11.5 Chromopolymer casting studies of the choroidal vasculature in a diabetic patient. Ischemic damage zones are identified as areas of absence of the choriocapillaries

is a high number of watershed zones, which render the macular choroid one of the zones at highest risk for ischemic injury.82 It is important to recall that, when there is a drop in the perfusion pressure in the vascular bed of one or more of these terminal arteries, the watershed zone (which is poorly perfused) is the area most vulnerable to ischemic damage. These findings are confirmed by fluorescein angiographic studies in monkeys with malignant hypertension, which reveal delayed filling in the macular region of the choroid where lesions associated with hypertensive choroidopathy are most commonly found.84 When the perfusion pressure is experimentally reduced in the same studies, fluorescein angiography reveals a marked delay in filling that involves the watershed zones of the macular region of the choroid.27,87 The watershed zones represent the most common site of ischemic damage. Therefore, it is reasonable to expect that lesions will frequently be found in the macular region. For similar reasons, patients with atherosclerosis frequently present with reticular pigmented degeneration in the equatorial region, which represents the watershed zone between territories supplied by the anterior and posterior ciliary circulations.27,87,83

Hypertensive Choroidopathy The characteristics of the vascular bed of the choroid explain the pathophysiology of the damage that occurs during the course of malignant hypertension. The choroidal bed is not equipped with a blood-eye barrier because the walls of the choriocapillaries contain large fenestrations. The vascular bed of the choroid has no autoregulatory

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system. It tends to respond passively, like a sponge, to changes in IOP (like those that occur during open-bulb surgery) or arterial blood pressure. The following scheme has been proposed to explain the pathogenesis of the choroid damage caused by hypertension—in the absence of a barrier in the vessels of the choriocapillaris, hypertension causes seepage of plasma containing angiotensin II and other vasoconstrictors into the choroidal space. These substances cause vasoconstriction, which leads to ischemia—first in the choroid and then in the RPE. Damage to the RPE disrupts the barrier between the blood and the outer retina and leads to serous detachment of the neuroretina. This hypothesis is confirmed by the fact that the following changes are observed in hypertensive choroidopathy. Anomalies Involving the Choroidal Vascular Bed In the initial stages, fluorescein angiography reveals a delayed choroidal filling that is particularly marked in the macular region.84 Histo-pathologic studies have revealed true occlusion of the choroidal arteries.83 Lesions of the RPE Focal ischemic lesions are found. For simplicity’s sake, these lesions can be classified as acute or degenerative. Those of the acute phase are pale white, pin-point lesions located predominantly in the macular region of the RPE, and less frequently in other areas of the RPE. They are associated with focal serous detachments of the retina and produce late staining on fluorescein angiography.84 Within two to three weeks, these lesions are inevitably transformed into degenerative focal lesions. As time passes, they tend to become confluent, producing progressively larger areas of RPE atrophy or diffuse pigmented alterations. The vessels of the choroid become sclerotic.84 Fluorescein angiography reveals hyperfluorescence (window defects), but there is no staining because the blood-retina barrier is intact. Serous Detachment of the Neuroretina Detachments may be focal, flat, bullous, or total. They can be located in the macular and/or peripapillary region and/or in the periphery. The subretinal fluid is initially clear, but it becomes increasingly turbid and occasionally proteinaceous. Subretinal fibrosis may develop. In the macular region, the detachment sometimes has a pseudocystic appearance.84

Conclusion The choroid is a complex system that receives its blood supply from the anterior and posterior ciliary arteries and serves as transport for nutrients to high metabolic tissues and dissipation of the heat produced by the retina during photochemical and

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metabolic reactions. We reported choroid alterations that are part of the physiologic aging process, and the physiopatology of age-related pathologies such as hypertension, diabetes, atherosclerosis, and age-related macular degeneration. In all cases, the choroid seems to be a major target of the aging process, and may be due to its high metabolic activity. Acknowledgment We are happy to acknowledge Prof Fernando Galassi (Clinica Oculistica, Università di Firenze) for his contribution to the color Doppler imaging section of this chapter.

References 1. Olver JM (1990) Functional anatomy of the choroidal circulation. Eye 4:262-272 2. Hayreh SS (1962) The ophthalmic artery III. Branches. Br J Ophthalmol. 46:212–247 3. Hayreh SS (1995) The 1994 Von Sallman Lecture. The optic nerve head circulation in health and disease. Exp Eye Res. 61:257–272 4. Hayreh SS (2001) The blood supply of the optic nerve head and the evaluation of it: myth and reality. Prog Retin Eye Res. 20:563–593 5. Ducournau D (1979) Systematisation vasculaire de la choroide. Lyon, France: Association Corporative des Etudiants en Medecine de Lyon 17–22 6. Olver JM (1990) Functional anatomy of the choroidal circulation: methyl methacrylate casting of human choroid. Eye. 4:262-272 7. Hayreh SS (1974) The long posterior ciliary arteries: an experimental study. Graefes Arch Clin Exp Ophthalmol. 192:197-213 8. Hayreh SS, Baines JAB (1972) Occlusion of the posterior ciliary artery I. Effects on choroidal circulation. Br J Ophthalmol. 56:719-735 9. Hayreh SS, Baines JAB (1972) Occlusion of the posterior ciliary artery II. Chorio-retinal lesions. Br J Ophthalmol. 56:736-753 10. Hayreh SS, Baines JAB (1972) Occlusion of the posterior ciliary artery III. Effects on the optic nerve head. Br J Ophthalmol. 56:754-764 11. Hayreh SS, Chopdar A (1982) Occlusion of the posterior ciliary artery V. Protective influence of simultaneous vortex vein occlusion. Arch Ophthalmol. 100:1481-1491 12. Hayreh SS (1974) Anterior ischaemic optic neuropathy I. Terminology and pathogenesis. Br J Ophthalmol. 58:955-963 13. Hayreh SS (1974) Anterior ischaemic optic neuropathy II. Fundus on ophthalmoscopy and fluorescein angiography. Br J Ophthalmol. 58:964-980 14. Hayreh SS (1985) Inter-individual variation in blood supply of the optic nerve head: its importance in various ischemic disorders of the optic nerve head, and glaucoma, low-tension glaucoma and allied disorders. Doc Ophthalmol. 59:217-246 15. Hayreh SS (1990) Anterior ischaemic optic neuropathy: differentiation of arteritic from nonarteritic type and its management. Eye. 4:25-41 16. Hayreh SS (1996) Acute ischemic disorders of the optic nerve: pathogenesis, clinical manifestations and management. Ophthalmol Clin North Am. 9:407-442 17. Hayreh SS, Podhajsky PA, Zimmerman B (1998) Ocular manifestations of giant cell arteritis. Am J Ophthalmol. 125:509-520 18. Hayreh SS (1975) Segmental nature of the choroidal vasculature. Br J Ophthalmol. 59:631-648 19. Hayreh SS (1983) Physiological anatomy of the choroidal vascular bed. Int Ophthalmol. 6:85-93 20. Hayreh SS (1990) In vivo choroidal circulation and its watershed zones. Eye. 4:273-289

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21. Hayreh SS, Scott WE (1978) Fluorescein iris angiography II. Disturbances in iris circulation following strabismus operation on the various recti. Arch Ophthalmol. 96:1390-1400 22. Virdi PS, Hayreh SS (1987) Anterior segment ischemia after recession of various recti: an experimental study. Ophthalmology. 94:1258-1271 23. Hayreh SS (1974) The choriocapillaris. Graefes Arch Clin Exp Ophthalmol. 1974;192:165–179 24. Hayreh SS (1981) Controversies on submacular choroidal circulation. Ophthalmologica. 1981;183:11–19 25. Hayreh SS (1981) Anterior ischemic optic neuropathy. Arch Neurol. 1981;38:675–678. 26. Takahashi K, Muraoka K, Kishi S, Shimizu K (1996) Watershed zone in the human peripheral choroid. Ophthalmology. 103:336-342 27. Ernest JT (1976) Stern WH. Archer DB. Submacular choroidal circulation. Am J Ophthalmol. 81:574-582 28. Torczynski E, Tso MO (1976) The architecture of the choriocapillaris at the posterior pole. Am J Ophthalmol. 81:428-440 29. Stern WH, Ernest JT (1974) Microsphere occlusion of the choriocapillaris in rhesus monkeys. Am J Ophthalmol. 78:438-448 30. Hayashi K, de Laey JJ (1985) Indocyanine green angiography of submacular choroidal vessels in the human eye. Ophthalmologica. 190:20-29 31. Giuffre G (1989) Main posterior watershed zone of the choroid: variations of its position in normal subjects. Doc Ophthalmol. 72:175-180 32. Giovannini A, Mariotti C, Ripa E, Scassellati Sforzolini B, Tittarelli R (1994) Choroidal filling in age-related macular degeneration: indocyanine green angiographic findings. Ophthalmologica. 208:185-191 33. Ross RD, Barofsky JM, Cohen G, Baber WB, Palao SW, Gitter KA (1998) Presumed macular choroidal watershed vascular filling, choroidal neovascularization, and systemic vascular disease in patients with age-related macular degeneration. Am J Ophthalmol. 125:71-80 34. Sato Y, Tomita G, Onda E, Goto Y, Oguri A, Kitazawa Y (2000) Association between watershed zone and visual field defect in normal tension glaucoma. Jpn J Ophthalmol. 44:39-45 35. Ito YN, Mori K, Young-Duvall J, Yoneya S (2001) Aging changes of the choroidal dye filling pattern in indocyanine green angiography of normal subjects. Retina 21:237-242 36. Oto S, Yilmaz G, Cakmakci S, Aydin P (2002) Indocyanine green and fluorescein angiography in nonarteritic anterior ischemic optic neuropathy. Retina 22:187-191 37. Hayreh SS, Bauines JA (1973) Occlusion of the vorteix veins: an experimental study. Br J Ophthalmol 57:217-38 38. Hayreh SS (1974) Submacular choroidal vascular pattern: experimental fluorescein fundus angiographic studies. Graefes Arch Clin Exp Ophthalmol 192:181-96 39. Keisuke M (2004) Asimmetry of choroidal venous vascular patterns in the human eye Ophthalmology 111:507-512 40. Yanoff M, Duker JS (2005) Ophthalmology 3:1-4 41. Parver LM, Auker C, Carpenter DO (1980) Choroidal blood flow as a heat dissipative mechanism in the macula. Am J Ophthalmol 89:641 42. Hogan MJ, Alvarado J (1967) Studies on the human macula. IV. Aging changes in Bruch’s membrane. Arch Ophthalmol 77:410–20 43. Ramrattan RS, van der Schaft TL, Mooy CM, de Bruijn WC, Mulder PG, de Jong PT (1994) Morphometric analysis of Bruch’s membrane, the choriocapillaris, and the choroid in aging. Invest Ophthalmol Vis Sci 35:2857-64 44. Killingsworth MC (1987) Age-related components of Bruch’s membrane in the human eye. Graefes Arch Clin Exp Ophthalmol 225:406-12 45. Sarks SH (1976) Ageing and degeneration in the macular region: a clinico-pathological study. Br J Ophthalmol 60:324-41 46. Feeney-Burns L, Ellersieck MR (1985) Age-related changes in the ultrastructure of Bruch’s membrane. Am J Ophthalmol 100:686-97 47. Green WR, Enger C (1993) Age-related macular degeneration histopathologic studies. The 1992 Lorenz E. Zimmerman Lecture. Ophthalmology 100:1519-35

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48. Sarks SH, Arnold JJ, Killingsworth MC, Sarks JP (1999) Early drusen formation in the normal and aging eye and their relation to age related maculopathy: a clinicopathological study. Br J Ophthalmol 83:358-68 49. Loeffler KU, Lee WR (1986) Basal linear deposit in the human macula. Graefes Arch Clin Exp Ophthalmol 224:493-501 50. Curcio CA, Millican CL (1999) Basal linear deposit and large drusen are specific for early agerelated maculopathy. Arch Ophthalmol 117:329-39 51. Burns RP, Feeney-Burns L (1980) Clinico-morphologic correlations of drusen of Bruch’s membrane. Trans Am Ophthalmol Soc 78:206-25 52. Ishibashi T, Sorgente N, Patterson R, Ryan SJ (1986) Aging changes in Bruch’s membrane of monkeys: an electron microscopic study. Ophthalmologica 192:179-90 53. Young RW (1987) Pathophysiology of age-related macular degeneration. Surv Ophthalmol 31:291-306 54. El Baba F, Green WR, Fleischmann J, Finkelstein D, de la Cruz ZC (1986) Clinicopathologic correlation of lipidization and detachment of the retinal pigment epithelium. Am J Ophthalmol 101:576-83 55. Friedman E, Smith TR, Kuwabara T (1963) Senile choroidal vascular patterns and drusen. Arch Ophthalmol 69:220-30 56. Ruberti JW, Curcio CA, Millican CL, Menco BP, Huang JD, Johnson M (2003) Quick-freeze/ deep-etch visualization of age related lipid accumulation in Bruch’s membrane. Invest Ophthalmol Vis Sci 44:1753-9 57. Starita C, Hussain AA, Pagliarini S, Marshall J (1996) Hydrodynamics of ageing Bruch’s membrane: implications for macular disease. Exp Eye Res 62:565-72 58. Fisher RF (1987) The influence of age on some ocular basement membranes. Eye 1:184-9 59. Lakatta EG, Levy D (2003) Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: Part I: aging arteries: a ‘set up’ for vascular disease. Circulation 107:139-46 60. Haimovici R, Gantz DL, Rumelt S, Freddo TF, Small DM (2001) The lipid composition of drusen, Bruch’s membrane, and sclera by hot stage polarizing light microscopy. Invest Ophthalmol Vis Sci 42:1592-9 61. Barnes MJ, Farndale RW (1999) Collagens and atherosclerosis. Exp Gerontol 34:513-25 62. Karwatowski WS, Jeffries TE, Duance VC, Albon J, Bailey AJ, Easty DL (1995) Preparation of Bruch’s membrane and analysis of the age-related changes in the structural collagens. Br J Ophthalmol 79:944-52 63. Grunwald JE, Hariprasad SM, DuPont J (1998) Effect of aging on foveolar choroidal circulation. Arch Ophthalmol 116:150-154 64. Raab MF, Gagliano DA, Teske MP (1988) Retinal arterial macroaneurysms. Surv Ophthalmol 33:73-96 65. Ring HG, Fujino T (1967) Observations on the anatomy and pathology of the choroidal vasculature. Arch Ophthalmol 78:431-444 66. Robertson DM (1973) Macroaneurysms of the retinal arteries. Trans Am Acad Ophthalmol Otolaryngol 77:55-67 67. Russell RWR (1963) Observations on intracerebral microaneurysms. J Pathol 93:393-398 68. Sarks SH (1973) Senile choroidal sclerosis.Br J Ophthalmol 57:98-109 69. Friedman E, Smith TR, Kuwabara T, Beyer C (1964) Choroidal vascular patterns in hypertension. Arch Ophthalmol 71:842-850 70. McLeod DS, Lutty GA (1994) Highresolution histologic analysis of the human choroidal vasculature. Invest Ophthalmol Vis Sci 35:3799-3811 71. Pauleikhoff D (1990) Aging of Bruch’ s membrane: histological, morphologic and clinical correlation. Proc int soc eye res VI 337:101 72. Ciulla TA, Harris A, Kagemann et al. (2002) Choroidal perfusion perturbations in nonneovascular age related macular degeneration. Br J Ophthalmol 86:209-13 73. Sarks SH (1978) Changes in the region of the choriocapillaris in aging and degeneration. XXIII Concilium Ophthalmologicum, Kyoto, 1978. Amsterdam: Excerpta Medica 228-38

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74. Sarks JP, Sarks SH, Killingsworth MC (1988) Evolution of geographic atrophy of the retinal pigment epithelium. Eye 2:552-77 75. Korenzweig AB (1977) Changes in the choriocapillaries associated with senile macular degeneration. Ann Ophthalmol 9:753-64 76. Grunwald JE, Metelitsina TI, Dupont JC, et al. (2005) Reduced foveolar choroidal blood flow in eyes with increasing AMD severity. Invest Ophthalmol Vis Sci 46:1033-8 77. Loeffler KU, Hayreh SS, Tso MOM (1994) The effects of simultaneous occlusion of the posterior ciliary artery and vortex veins. Arch Ophthalmol. 112:674-682 78. Kishi S, Tso MOM, Hayreh SS (1985) Fundus lesions in malignant hypertension I. A pathologic study of experimental hypertensive choroidopathy. Arch Ophthalmol. 103:1189-1197 79. Hayreh SS, Servais GE, Virdi PS (1986) Fundus lesions in malignant hypertension VI. Hypertensive choroidopathy. Ophthalmology 93:1383-1400 80. Hayreh SS (1980) Acute choroidal ischaemia. Trans Ophthalmol Soc UK. 100:400-407 81. Hayreh SS (1975) Anterior Ischemic Optic Neuropathy. Springer-Verlag, Heidelberg, Germany 82. Hayreh SS (1983) Macular lesions secondary to choroidal vascular disorders. Int Ophthalmol 6:161-170 83. Foos RY, Trese MT (1982) Chorioretinal juncture. Arch Ophthalmol 100:1492-1503

Chapter 12

Age-Related Macular Degeneration I: Types and Future Directions Susanne Binder, MD and Christiane I. Falkner-Radler, MD

Abstract Age-related macular degeneration is the leading cause of severe visual impairment in industrialized countries in patients over 50 years of age. In its natural course, AMD leads to progressive loss of central vision, leaving the patients with only orienting vision and the peripheral visual field at its final stage. This chapter deals with the description of future trends in therapy. In fact, combination therapies will become more tailored to the stage and severity of the disease. To provide long-term effects, long-acting delivery systems for drug combinations need to be developed. In addition, combinations with surgical therapies, laser, or photodynamic treatment (PDT) might be reasonable to decrease dosage and treatment intervals. For non-responders or advanced cases of AMD, cell-derived therapies will be necessary—like retinal transplantation or gene therapies— for better restoration of a more normal foveal condition to restore the vision in an aging patient. Keywords AMD, PTD, Therapy, human eye future directions, retinal transplantation.

Introduction Age-related macular degeneration (AMD) is currently the leading cause of severe visual impairment in industrialized countries in patients over 50 years of age.1 Although prevalence varies, the two largest studies show that AMD occurs between 2.8 and 20.9 percent in patients over 55, and 15.5 - 41.7 percent over the age of 75.2,3 In its natural course, AMD leads to progressive loss of central vision, leaving the patients with only orienting vision and the peripheral visual field in its final stage. Within two years, a loss of six lines (30 letters) will occur in more than 60 percent of patients.4 AMD represents a major medical and social problem, with 2025 million people affected worldwide. This number is expected to triple in the next 30 years because of the increase in aging populations.5 In addition, higher expectations of better quality of life (including the ability to read and drive) are being demanded by elderly patients, which makes AMD an even more significant

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problem. Furthermore, numbers of cases in developing countries do increase with better screening of patients, although AMD might manifest itself in various forms according to environmental and nutritional circumstances on different continents.6

Etiology and Pathogenesis The etiology of AMD is not fully understood. Genetic factors, oxidative stress, ischemia, and aging of the retinal pigment epithelium are proposed etiologic factors.7 Most likely, genetic and environmental factors determine whether the onset of the disease is earlier or later in life.8 AMD is believed to be caused by progressive deterioration of the retinal pigment epithelium (RPE), Bruchs’membrane (BM), and the choriocapillaris choroidal complex that consequently leads to subsequent damage of the photoreceptor (PR) cells.9 The RPE plays a central role in maintaining retinal function by assuming a strategic position as the metabolic gatekeeper between PRs and the choriocapillaris that suffer cumulative damage over lifetime and—in susceptible individuals—induce AMD.10

RPE RPE is a cuboidal hexagonal monolayer comprising the outermost layer of the retina. Its apical portion faces the outer segments of the PRs and its basolateral surface interacts with the choriocapillaris.11 The RPE is a post-mitotic cell that does not proliferate under normal conditions, and its tight junctions represent the outer blood retinal barrier. Besides its function as a metabolic coordinator for PR cells, including the digestion of PR outer segments, it participates in vitamin A metabolism (visual circle), melanin synthesis, extracellular matrix synthesis, and molecule transport, and secretes and responds to numerous growth factors and other cytokines. Among them, RPE expresses several fibroblast growth factors (bFGF, acidic FGF, and FGF5), as well as ciliary neurotrophic factor (CNTF)12. In addition, vascular endothelial growth factor (VEGF-A)—a very potent angiogenic growth factor—is secreted to act as a paracrine trophic factor for the epithelium of the choriocapillaris, and to maintain its fenestrations.13,14 In hypoxia, hyperglycemia, advanced glycation end products (AGE), and other pathologic stimuli, VEGF expression is up-regulated, thus playing a central role in ocular neovascularisation.15,16 Insulin-like growth factor (IGF-1) and its binding protein (IGF-BP) are synthesized also by the RPE and were found to be up-regulated in various ischemic retinal conditions.17 Most important, however, is the secretion of pigment epithelial derived factor (PEDF), which acts as the key coordinator of retinal neuronal and vascular function, and is a potent inhibitor of angiogenesis.18,19 An equilibrium shift in VEGF and PEDF secretion ratios might be a possible cause of development of choriodal neovascularization (CNV) in AMD.20 With age, the number and density of RPE decreases, and accumulation of lipofuscin—a yellowishbrownish autofluorescent lipofuscin granula with lipid membranes that contain toxic biomolecules that interact with normal function—occurs.

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Bruch’s Membrane Bruch’s membrane (BM) is a 1-4 mm thick structure wwere the basal RPE rests. Its five different layers are: 1) the basal membrane of the RPE, 2) the inner collagenous layer, 3) the elastine layer, 4) the outer collagenous layer, and 5) the basal membrane of the choriocapillaris endothelial cells. BM increases in thickness with age for some time and becomes distorted.

Basal Lamina Deposits Basal lamina deposits (BlamD, BlinD) consist of amorphous material located between the basement membrane of the RPE and its cytoplasm. They are considered the earliest changes of AMD found in histopathologic examinations,21 and clinically they are almost invisible.

Drusen Drusen represent the first clinically visible changes in the ocular fundus. They appear as yellowish-white dots that can occur in the macula, the paramacular area, and in the retinal periphery as signs of senescence or early AMD. In relation to size and margin, they are divided into hard Drusen—consisting of hyaline material— and soft Drusen—consisting of a granular, amorphous vesicular structure—with indistinct margins and a size larger than 63 µm (see Fig. 12.1). In some Drusen, neutral lipids prevail while others are predominately composed of phospholipids.22 If Drusen are numerous (more than five), they become an independent

Fig. 12.1 Diffuse soft and hard Drusen in the macular (a) and and perimacular area (b)

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risk factor for AMD.23 If soft Drusen coalesce, they form a serous pigment epithelial detachment (PED) or RPE atrophy. Patients with Drusen and good vision in both eyes will experience new atrophic or exudative lesions annually in 8 percent over three years.24 The reason for the foveal location of AMD might be explained by topographical differences in the structure and composition of BM in the macula,25 higher oxidative stress of the central area related to light overload, and stress and other factors. Recent access to the human genomic sequence has made more powerful analytical methods possible, including haplotype mapping and single nucleotide polymorphism (SNP) analysis.26,27,28,29 It was reported that a variation of the factor H gene (HF1/CFH) dramatically increases the likelihood of developing AMD. In an earlier report of the same group, the formation of Drusen with deposits of inflammatory proteins was implicated with the complement cascade—a pathway associated with the innate immune system,30,31 indicating that a low grade chronic inflammation is an important factor in the pathogenesis of AMD. Because AMD is a complex disease that occurs in different forms and stages, it would seem likely that multiple genes are involved with varying penetration for the different forms of AMD.

Risk Factors for AMD Age Age is associated with an increased incidence, prevalence, and progression of AMD.32

Family History Family history has become the second largest risk factor during the last few years. First-degree relatives of involved patients have, for example, a 3-fold increased risk of developing exudative AMD.33 An overall inheritability of early AMD of 45 percent was demonstrated in a twin study, showing an 81 percent inheritability with the presence of 20 or more hard Drusen, 57 percent for large soft Drusen, and 46 percent for pigment changes.34

Gender According to a report from the Blue Mountain Eye Study, the female sex shows twice the incidence of CNV as men,35 although there is no gender significance for AMD in general.

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Race Caucasians seem to have a higher prevalence of AMD.36 However, recent studies suggest that colored people develop early signs of AMD quite frequently, but seem to have a low incidence of advanced AMD at all ages.37,38

Smoking Cigarette smoking became the third highest risk factor, and both current and prior smokers are at increased risk for AMD.39 This might be related to effects on the antioxidant metabolism as well as the blood flow.40 Interestingly, female smokers seem to be at higher risk for progression to advanced AMD, while male smokers may be of higher risk for non-exudative AMD.35

Hypertension Hypertension turns out to be even more important than arteriosclerosis, although similar compositions of Drusen and arteriosclerotic deposits are present.41 It was found that Angiotensin,2 a main risk factor for systemic hypertension, induces VEGF and angiopoetin expression, which both play an important factor in angiogenesis.42

Nutrition Diet and body mass index (BMI) have been carefully studied because they could be influenced through nutrition and lifestyle. For example, the intake of high linolenic acid is associated with a 49 percent increased risk of AMD, and high docosahexaenoic acid is associated with a 30 percent lower risk of AMD.43 The analysis of the BMI found that obese individuals are at higher risk for both dry and neovascular AMD, while very lean individuals are at higher risk for dry AMD.44

Comorbidity Comorbidity of AMD with other age-related diseases was examined in few studies. In the Beaver Dam Population,45 a significant correlation between hearing loss and late AMD was documented. In the Rotterdam Study,46 a striking correlation was

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found in patients with late AMD who developed Alzheimer’s disease. Cardiovascular diseases, which have smoking and hypertension as common risk factors with AMD, are also discussed today.

AMD Types AMD is divided into two main subtypes—the primary neovascular (exudative, wet) NV-AMD, and the non-neovascular (non-exudative, atrophic, dry) NNV-AMD.

Neovascular AMD NV-AMD occurs only in about 20 percent of all AMD forms, but is responsible for rapid and severe visual loss in the majority of patients. The hallmark of NV-AMD is choriodal neovascularization (CNV), which can either grow directly from the choriocapillaries into the sub-RPE or subretinal space, or be preceded by a serious PED after soft Drusen coalesce and become vascular. If the CNV connects with retinal neovascularization, it can form a retinal choriodal anastomosis termed as retinal angiomatous proliferation (RAPs).47,48 The main stimulus for neovascularization is imbalance of the angiogenic vascular endothelial growth factor (VEGF) versus the angioinhibitours pigment epithelial derived factor (PEDF) secretion from trans-differentiated RPE cells. The route of new vessels seem to go through areas of lesser resistance—for example, defects in Bruch’s membrane and RPE irregularities.49 Clinically, these CNVs are seen as elevated grey-green or pinkishyellow lesions, sometimes with pigment, exudates, and blood (see Fig. 12.2). In the later stage, chronic leakage and bleeding leads to the formation of a central fibrovascular scar with further loss of RPE and photoreceptors. In this terminal form of AMD, only irreversible, orientating vision remains. Besides visual acuity testing and biomicroscopic examination, the two most important additional examination methods are the angiography and optical coherence tomography of the retina.

Flouresceine Angiography With fluoresceine angiography, 5 mg of natrium-fluorescein are injected into the cubital vein of the patient, and series photographs are taken after the pupil is dilated. CNVs can be detected and categorized either as classic or occult, or a combination of the two, depending on the leakage patterns they present at various time points on the angiogram. This differentiation was imperative for laser treatments where well defined margins for treatment decision were necessary. Today the differentiation is still important to evaluate disease activity and to decide on drug selection in the area of intravitreal applications of antiinflammatory and anti-VEGF medication (see Fig. 12.3).

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Fig. 12.2 Neovascular AMD shows yellow-greyish cental area (arrow ) and small hemorrharges on the edges

Fig. 12.3 Fluoresceine angiography of classic and occult CNV. Early (a) and late face (b) angiogram of a classic CNV, showing immediate hyperfluorescence in the early phase. Early (c) and late face (d) angiogram of occult CNV, showing hyperfluorescence of dye only in the late phase

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Because fluorescein is a hydrophilic molecule, only Drusen rich in phospholipids will stain with this dye, while Drusen composed mainly of neural lipids will not stain.50

Optical Coherence Tomography Optical coherence tomography (OCT) has brought additional insights in the diagnosis and follow-up of AMD. It provides precise information about the retinal layers in the macular region—intra-, subretinal, and subpigment and the epithelial fluid dynamics—showing PR-atrophy as well as RPE- behavior and scarring. In close proximity to the retina, the posterior hyaloid behavior, and the underlying choriocapillary-choroid condition can be examined, too. Compared to fluoresceine angiography, it is a noninvasive examination technique that can be repeated without additional stress on the patient (see Fig. 12.4).

Fig. 12.4 Optical Coherence Tomography (OCT). (a) OCT shows Drusen as irregularities in the area of the retinal pigment epithithelium—Bruch’s membrane (arrows)—and the foeveal contour is normal (red arrow). (b) Choroidal neovascularisation (arrows) with massive subretinal edema (red arrow) and loss of foveal contour

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Non-neovascular AMD While the first changes for NN-AMD are mostly invisible basal lamina deposits, they can already affect vision. As already mentioned, Drusen are the first clinically visible changes in the ocular fundus. Immuno-histochemistry shows that these deposits, which later develop into hard or soft Drusen, contain apolipoproteins B and E, faxtor X, different immunoglobins, amyloid P component, complement C5 and C5b-9 terminal complexes, fibrinogen, vitronectin, and other participants in humoral and cellular immunity.30 These findings strongly suggest a role for immunological and inflammatory processes in the pathogenesis of AMD. Drusen can occur simultaneously with pigment epithelial mottling, or disappear later in the disease with a confluent area of pigment epithelial atrophy developing. If this atrophy exceeds 175 mm in diameter, it is called geographic atrophy. This atrophy not only increases on its borders, but also increases vertically, so that finally RPE loss leads to apoptosis of the overlying metabolically dependant PRs primarily in the outer—but finally also in the inner—nuclear layers. The choriocapillarischoroid complex simultaneously becomes atrophic because of the withdrawal of trophic secretion of the RPE, leaving a white, deep, sharply demarcated area. Loss of vision over time is slower than in N-AMD but NN-AMD involves about 80 percent of all cases of AMD and is responsible for 20 percent of legal blindness. Calculations show the cumulative incidence of severe visual loss in the presence of bilateral atrophy at baseline as 9 percent after two years and 17 percent after four years.51 It occurs bilateral in over 50 percent of patients52 and may also result in about 20 percent of cases after flattening of a PED53 (see Fig. 12.5).

Fig. 12.5 Atrophic-nonneovascular AMD, map-like central whitish- yellow defect of the pigment epithelium with sharp margins (arrows ), and Drusen in close proximity

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The most informative additional examination in NN-AMD is fundus autofluorescence, obtained with a confocal scanning laser ophthalmoscope (Heidelberg Retina Angiograph) that gives information about the distribution of the intensity of the autofluorescence signal derived from lipofuscin contents in the RPE cell. In the presence of NN- AMD, it clearly shows new areas of atrophy (decreased autofluorescence) and estimates of areas that might undergo further atrophy (increased autofluorescence), showing increased lipofuscin content before cell death occurs.

Current Treatment Options NV-AMD Laser Photocoagulation Laser photocoagulation was the only available treatment for many years for NVAMD.54 Because of its immediate damage to all retinal layers, scarring, and considerable collateral damage at the borders, it is now only indicated for extrafoveal classic CNV at a minimal distance of 200 µm from the fovea.55

Photodynamic Treatment Photodynamic treatment (PDT) aims to inactivate the CNV, leading to reduction of size and cessation of exudation of fluid under the neurosensory retina. In a two-step procedure, a photosensitizer (Visudyne) is first administered intravenously and accumulated in the CNV. In the second step, the sensitizer is activated by light irradiation of a specific wavelength appropriate for dye absorption.56 The treatment is repeated in three-month intervals as long as activity of the CNV is present. PDT has its best results for subfoveal classic CNVs, or smaller occult lesions with no classic component.57 However, its effect on the patients’ vision was limited—only 14 percent of patients treated have an improved visual acuity of one or more lines after two years. While in former laser studies differences only higher than two lines gained or lost were considered real changes in VA, a gain of one line in the new PDT studies is already considered a success, and a loss of three or more lines is considered a failure. Stabilization of vision since that time has been defined as between constant VA and three lines of loss. Stabilization of vision with PDT has been shown in large, randomized clinical trials.58 Transpupillary Thermotherapy Transpupillary thermotherapy (TTT) was evaluated in one large study, and several small studies. In relation to PDT, TTT is easy to perform and relatively

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Fig. 12.6 Transpupillary Thermotherapy (TTT): occult CNV before (a) and after (b) TTT treatment, note increased lesion size after treatment although inactivity

inexpensive. Primarily developed by Osterhuis and Korver for the treatment of choroidal melanoma,59 it was also considered for treatment of occult CNV in AMD.60 Unfortunately, a multi-center trial failed to show significance, and longterm results show stabilization but rarely improvement in vision61 (see Fig. 12.6). To summarize, treatment options with different forms of laser treatments have so far focused on prevention of progression and stabilization of vision in susceptible eyes. Fortunately, with a greater understanding of the molecular mechanisms of the disease and CNV development, therapeutic strategies have been developed with the advantage of halting or improving the disease without the collateral damage of thermal laser treatment or PDT.

Anti-angiogenic Therapies Anti-angiogenic therapies for trans-scleral, intravitreal application have been introduced in AMD treatment and are currently used for all subgroups of CNV. The first drug approved was Pegaptanib (Macugen*)—a 28-base ribonucleotid aptamer designed to bind and block specifically the activity of the extracellular VEGF 165 amino acid isoform—thus the main responsible VEGF for ocular neovascularization.62 Ranibizumab (Lucentis*) was designed for ophthalmic use for better retinal penetration with a molecular size of 48 kD. It is a humanized monoclonal VEGF antibody fragment (rhu-fab V2) that binds all isoforms of VEGF. In several different multicenter clinical trials, it has been demonstrated

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that 20-25 percent of patients treated monthly did gain vision, and 90 percent remained stable. Its effectiveness was demonstrated as a sole therapy, and in combination with PDT. When compared with PDT, Ranibizumab was superior (Marina-, Anchor-, Focus-, Pier- and Excite-Study).63 The basic substance, however, is Bevacicumab (Avastin*), a recombinant, humanized, full-lengh anti-VEGF monoclonal antibody with a molecular size of 150 kD. It binds all forms of VEGF-A, and has been approved for cholorectal cancer in addition to cytostatic therapy. For ophthalmic use, the molecule was considered too large to sufficiently penetrate the retina. Therefore, it was primarily used intravenously for a small series of AMD patients, showing positive results.64 To avoid side effects like thromboembolic events and increased blood pressure, Rosenfeld and his group decided to apply Bevacicumab intraviteally and soon presented efficacy and tolerability of the drug.65 Because of the much lower costs, Bevacicumab is injected today worldwide in patients with AMD in a dosage between 1.25 to 2-5 mg. Early studies have shown no side effects, and visual improvements, retinal penetration, and a lack of toxicity were also confirmed in an experimental study.66 Besides the three anti-VEGF substances used today, it should be mentioned that many other stimulators of angiogenesis do exist and will need further exploration.

Cortisone and Cortisene Triamcinolone in dosages of 4, 8 and 25 mg was used intravitreally, mainly as an adjunct to PDT to minimize inflammation, exudation, and VEGF production. It was reported to reduce the needed number of PDT retreatments, and allows PDT treatment in eyes with a primarily unfavorable prognosis.67,68 Because of its side effects—namely, high eye pressure up to 40 percent and more rapid cataract development—it is considered outdated today. However, the anti-inflammatory effect of Cortison in AMD should not be underestimated. To avoid an increase in pressure and cataracts and the risk connected with intravitreal application like endophthalmitis, bleeding, and cataracts, Anecortave (Retaane*)—a cortisene—was developed for iuxtascleral application. While one study had demonstrated that its effect is similar to PDT for classic CNV,69 its effect is rather slow and most likely not sufficient for active disease. However, it is being tested in a current study for patients with CNV and second eye Drusen as a preventative drug rather than a curative one.

Surgical Removal Surgical removal of CNV was successfully performed in young patients and eyes with CNV related to histoplasmosis, but failed to show a beneficial effect on vision in elderly patients with AMD.70,71 The surgery includes pars plana vitrectomy, a small retinotomy close to the neovascular membrane, careful mobilization of the membrane, and gentle removal with subretinal forceps. The retina is reattached by

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fluid-gas exchange, and the retinotomy is sealed with laser application. In a meta-analysis evaluating 26 studies and a total of 647 cases of subretinal membrane excision in AMD patients, it was shown that visual improvement was achieved in 33 percent and deterioration observed in 27 percent of the cases.72 Furthermore, progression of atrophy was demonstrated after surgery because of the simultaneous removal of the RPE on and around the CNV during surgery, leading to subsequent PR and choriocapillaris dysfunction.73,74 In two prospective studies comparing subretinal surgery with laser treatment and the natural course, there was only an advantage for surgery found in AMD patients with large pathologies including hemorrhages.75,76

Retinal Rotation Retinal rotation techniques have given us proof of principle that extrafoveal RPE can maintain foveal function. A 360° full rotation was performed for the first time by Machemer and Steinhorst in 1993.77 Although very good successes have been demonstrated by few groups,78,79 the surgery has not been widely adopted for several major reasons: the length and complexity of the surgery, an initial association with a high rate of retinal detachment and PVR, lack of evidence from clinical comparative trials, and finally uncertain management of postoperative diplopia.80

Transplantation Transplantation of the autologous RPE seems to be a logical approach to restore normal retinal function81 after homologous transplants have shown an immune reaction82 It is performed in two different ways—the transplantation of a freshly harvested RPE suspension immediately after membrane removal83,84 and transplantation of a full thickness RPE-choroidal patch excised from the midperiphery of the retina and translocated subfoveally.85,86 While the suspension technique is a relatively easy technique with complications similar to membrane removal alone, and a one-step procedure, best results were observed in AMD patients with small lesions. The flap technique makes silicone tamponade and removal necessary, and PVR rates of up to 40 percent were reported.87 However, the transplantation of a homogenous layer of polarized cells on their basal lamina is intriguing and seems to be more suitable for eyes with very large lesions that are the only candidates for surgery today. Still, this surgery is considered experimental, and although it was demonstrated that better reading vision results can be obtained with RPE suspensions than with membrane removal alone,85 visual improvements are limited so far—although visual improvement in some cases can be remarkable. With further improvement of technique, and the combination of recent knowledge in molecular biology and genetic modifications of cells, cell-derived therapies might soon become a reasonable treatment option for eyes with AMD where other therapies have failed.

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NN-AMD Vitamins Although there is evidence that high doses of antioxidative vitamins (500 mg Vit. C, 15 mg ß-carotene, 400IU Vit. E ) and zinc (80 mg zinc combined with 2 mg copper for deficiency prophylaxis) did lead to a significant lower incidence of NVAMD. It did not show a significant reduction for NN-AMD.88

Lutein and Zeuxanthin Lutein and Zeaxanthin have antioxidative and blue-light filtering effects and are considered as the two most potent vitamins for light protection. However, to date there is not a prospective randomized study that can prove its effects.

Rheopheresis Rheopheresis for extracorporal blood filtration is used in patients with early AMD— namely Drusen and small pigment epithelial atrophies. It is applied in eight sessions, four weeks apart, and one session consisting of two treatments, two or three days apart. Its effect is currently evaluated in multi-center prospective trials.89

Laser Application Laser application—either grid or focal—and also in a sub-threshold manner in patients with Drusen has shown no beneficial effect on patients’ vision, and results in higher numbers of CNVs in the treated group.90

Future Directions Future directions for AMD treatment will concentrate on early detection and prevention. As more drugs are invented, available combination therapies will become more tailored to the stage and severity of the disease. To provide long-term effects, long-acting delivery systems for drug combinations need to be developed. In addition, combinations with surgical therapies, laser, or PDT might be reasonable to decrease dosage and treatment intervals. For non-responders or advanced cases of AMD, cell-derived therapies will be necessary—like retinal transplantation or gene therapies for better restoration of a more normal foveal condition in an aging patient to restore vision.

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References 1. Ambati J (2003) Age-related macular degeneration: etiology, pathogenesis and therapeutic strategies. Surv Ophthalmol 48:257-93 2. Klein R (1992) Prevalence of age- related maculopathy. The Beaver Dam Eye Study. Ophthalmology 99:933-43 3. Mitchell P (2002a) Five year incidence of age-related maculopathy lesions: the Blue Mountain Eye Study. Ophthalmology 109:1092-97 4. Fine SL (2000) Age-related macular degeneration. N Engl J Med 342:483-492 5. Tasman W, Rovner B (2004) Age- related macular degeneration: treating the whole patient. Arch Ophthalmol 122:648-649 6. Brown MM, Brown GC, Stein JD et al. (2005) Age-related macular degeneration: economic burden and value based medicine analysis. Can J Ophthalmol 40:277-87 7. Spaide RF, Amstrong D, Brown R (2003). Continuing medical education review: Choroidal neovascularisation in age-related macular degeneration-what is the cause? Retina 23:595-614 8. Holz, FSchütt F, Pauleikoff et al. (2003) Pathophysiology. In: Holz et al. (eds) Age-related Macular Degeneration, p 31-46 9. Vingerling JR (1995) The prevalence of age-related maculopathy in the Rotterdam Study. Ophthalmology 102:205-10 10. Steinberg RH (1979) The relationship of the retoinalö pigment epithelium to photoreceptor outer segments in human retina. In: Zinn KM, Marmor MF(eds) The Retinal Pigment Epithelium. Harvard University Press, Cambridge, MA p 32-44 11. Hogan MJ (1971) Histology of the Human Eye. WB Saunders, Philadelphia 12. Campociaro PA (1993) Cytokine production by retinal pigmented epithelial cells. Int Rev Cytol 146:75-82 13. Blaauwgeers HG (1999) Polarized vascular endothelial growth afctor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaries. Evidence for a tropic paracrine relation. Am J Pathol 155:421-28 14. Roberts WG (1995) Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor. J. Cell Sci 41:2438-44 15. Adamis AP (2005) The role of vascular endothelial growth factor in ocular health and disease. Retina 25:111-18 16. Campociaro PA (2004) Ocular neovascularisation and excessive vascular permeability. Expert Opin Biol Ther 4:1395-1402 17. Meyer-Schwickerath R (1993) Vitreous levels of the insulin-like growth factors I and II, and the insulin growth factor binding proteins 2 and 3, increase in neovascular disease. Studies in non diabetic subjects. J Clin Invest 92:2620-25 18. Dawson DW (1999) Pigment epithelium derived factor: a potent inhibitor of angiogenesis. Science 285:245-8 19. King GL (2000) Pigment epithelium derived factor: a key coordinator of retinal neuronal and vascular functions. N Engl J Med 342:349-51 20. Ohno-Matsui K (2001) Novel mechanism for age-related macular degeneration : an equilibrium shift between the angiogenesis factors VEGf and PEDF. J Cell Physiol 189:323-33 21. Green WR (1993) Age-related macular degeneration histopathologic studies. The 1992 Lorenz E. Zimmerman Lecture. Ophthalmology 100:1519-35 22. Pauleikoff D, Barondes MJ, Minessian D et al. (1990) Drusen as risk factors in age related macular disease.Am J Ophthalmol 109:38-43 23. Macular Photocoagulation Study Group (1997) Risk factors for choroidal neovascularisation in the second eye of patients with iuxtafoveal or subfoveal choriodal neovascularisation secondary to age-related macular degeneration. Arch Ophthalmol 115:741-47 24. Holz FG (1994b) Bilateral macular drusen in age-related macular degeneration. Prognosis and risk factors.Ophthalmology 101:1522-28

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25 Chong NHV.(2005) Decreased thickness and integrity of the macular elastic layer of Bruch’s membrane correspond to the distribution of lesions associated with age-related macular degeneration. Am J Pathol 166:241-51 26. Daiger SP (2005) Genetics: was the human genome project worth the effort? Science 308:362-364 27. Edwards AO (2005) Complement factor H polymorphism and age-related macular degeneration. Science 308:421-424 28. Haines JL (2005) Complement factor H variant increases the risk of age-related macular degeneration. Science 308:419-421 29. Klein RJ (2005) Complement factor H polymorphism in age-related macular degeneration. Science 308:385-89 30 Hagemann GS (2001) An integrated hypothesisthat considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch’s membrane interface in aging and age-related macular degeneration. Prog Ret Eye Res 20:705-732 31. Anderson DH (2002) A role for local inflammation in the formation of drusen in the aging eye. Am J Ophthalmol 134:411-31 32. Klein R (2002a) Ten-year incidence and progression of age-related maculopathy. The Beaver Dam Eye Study. Ophthalmology 109:1767-79 33. Klaver CC (1998) Genetic risk of age-related maculopathy. Population based familial aggregation study. Arch Ophthalmol 116:1646-51 34. Hammond CJ (2002) Genetic influence on early age-related maculopathy: a twin study. Ophthalmology; 109: 730-36 35. Mitchell P (2002a) Five-year incidence of age-related maculopathy lesions: the Blue Mountains Eye Study. Ophthalmology 109:1092-97 36. Klein R.J (2003) Early age-related maculopathy in the cardiovascular heath study. Ophthalmology 110:25-33 37. Schachat AP (1995) Features of age-related macular degeneration in a black population. The Barbadous Eye Study Group. Arch Ophthalmol 113:728-35 38. Klein R (1999) Age related maculopathy in a multiracial United States population: the national Health and Nutrition Examination Survey III Ophthalmology 106:1056-65 39. Smith W (1996) Smoking and age-related maculopathy. The Blue Mountains Eye Study. Arch Ophthalmol 114:1518-23 40. The Eye Disease Case-Control StudyGroup (1992) Risk factors for neovascular age-related macular degeneration. Arch Ophthalmol 110:1701-08 41. Mullins RF (2000) Drusen associated with aging and age-related macular degeneration contain proteinscommon to extracellular deposits associated with arteriosclerosis, elastosis, amyloidosis, and dense deposit disease. Faseb J 14:835-46 42. Fijiyama S (2001) Angiotensin AT (1) and AT (2) receptors differentially regulate angiopoetin2 and vascular endothelial growth factor expression and angiogenesis by modulating heparin binding-epidermal growth factor (EGF)-mediated EGF receptor transactivation. Circ Res. 88:22-29 43. Cho E (2001) Prospective Study of dietary fat and the risk of age-related macular degeneration. Am J Clin Nitr. 73:209-18 44. Schaumberg DA (2001) Body mass index and the incidence of visually significant age-related maculoptathy in men. Arch Ophthalmol 119:11259-65 45. Klein R (1998c) Is age-related maculopathy related to hearing loss? Arch Ophthalmol 116:360-65 46. Klaver CC (1999) Is age- related maculoptahy associated with Alzheimer’s disease ? The Rotterdam Study. Am J Epidemiol 150:963-68 47. Hartnett ME (1996) Deep retinal vascular anomaloius complexes in advanced age-related macular degeneration. Ophthalmology 103:2042-53 48. Slatker JS (2000) Retinal choroidal anastomoses and occult choroidal neovascularisation in age-related macular degeneration. Ophthalmology 107:742-53 discussion 753-4

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49. Gass JD (1994) Biomicroscopic and histopathologic considerations regarding the feasibility of surgical excision of subfoveal neovascular membranes. Am J Ophthalmol 118:285-98 50. Pauleikoff D (1992) Correlation between biochemical composition and fluorescein binding of deposits in Bruch’s membrane. Ophthalmology 99:1548-53 51. Sunnes JS (1999) Geographic Atrophy. In: Berger JW, Fine SL, Maguire MG (eds) Agerelated Macular Degeneration. Mosby, St. Luis p 155-172 52. Sarks JP 1988. Evolution of geographic atrophy of the retinal pigment epithelium. Eye 2 (Part 5): 552–77 53. Braunstin RA (1979) Serous detachments of the retinal pigment epithelium in patients with senilemacular disease. Am J Ophthalmol 88:652-60 54. Macular Photocoagulation Study Group for senile macular degeneration (1982) Results of a randomised clinical trial. Arch Ophthalmol 100:912-918 55. Macular Photocoagulation Study Group (1991a) Argon laser photocoagulation for neovascular maculopathy. Five year results from randomised clinical trials. Arch Ophthalmology 109:1109-114 56. Henderson BW, Dougerthy TJ (1992) How does photodynamic therapy work? Photochem Photobiol 55:145-57 57. Bressler NM (2001) Photodynamic therapy of subfoveal choroidal neovascularisation in agerelated macular degeneration with verteporfin: two year results of 2 randomized clinical trials –tap report 2. Arch Ophthalmol 119:198-207 58. Treatment of Age-related macular degeneration with photodynamic therapy (TAP) Study Group (1999) One year results of two randomised clinical trials-Tap report 1. Arch Ophthalmol 117:1329-45 59. Osterhuis JA (1995) Transpupillary therapy in choroidal melanomas Arch Ophthalmol 113:315-321 60. Reichel E (1999) Transpupillary thermotherapy of occult subfoveal choroidal neovascularisation in patients with age-related macular degeneration. Ophthalmology 106:1908-14 61. Stolba U (2006) Long-term results after transpupillary thermotherapy in eyes with occult choroidal neovascularisation associated with age related macular degeneration: a prospective trial. Brit J Ophthalmol 90:158-61 62. Gragoudas Se (2004).Pegaptanib for neovascular age-related macular degeneration. N Engl J Med 351:2805-16 63. Heier JS (2006) Ranibizumab for treatment of neovascular age-relatedmacular degeneration a phase I/II multicenter, controlled, multidose study. Ophthalmology 113:642e1-642e4 64. Michels S (2005) Systemic bevacizumab (Avastin) therapy for neovascular age-related macular degeneration.Twelve-week results of an uncontrolled open-label clinical study. Ophthalmology 112:1035-47 65. Rosenfeld PJ (2005) Optical coherence tomography findings after an intravitreal injection of Bevacizumab (Avastin) for neovascular age-related macular degeneration. Ophthalmic Surg Lasers Imag. 36:331-35 66. Fisher SK (2006) Electrophysiologic and retinal penetration studies following intravitreal injection of bevacizumab (Avastin). Retina 26(3):262-9 67. Augustin AJ (2006) Verteporfin therapy combined with intravitreal triamcinolone in all types of choroidal neovascularisation due to age-related macular degeneration. Ophthalmology 113:14-22 68. Krebs I (2006) A new treatment regime in combined intravitreal injection of triamcinilone acetonide and photodynamictherapy.GraefesArcxh clin Exp Ophthalmol 244:863-63 69. Slatker JS (2006) Anecortave acetate (15 milligrams)versus photodynamictherapy for treatment of subfoveal neovascularisation in age-related macular degeneration. Ophthalmology 113:3-13 70. Thomas MA (1991) Surgical removal of subfoveal neovascularisation in the presumed ocular histoplasmosis syndrome. Am J. Ophthalmol. 11:1-7 71. Thomas MA (1994a) Visual results after surgical removal of subfoveal choroidal neovascular membranes. Ophthalmology 101:1384-96

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72. Falkner CI (2007) The end of submacular surgery for age-related macular degeneration A meta-analysis. Graefes Arch Clin Exp Ophthalmol (online) 73. Castellarin AA (1998b) Progressive presume choriocapillaris atrophy after surgery for age related macula degeneration. Retina 18:143-49 74. Nasir MA (1997) Decreased choriocapillaris perfusion following surgical excision of choroiodal neovascular membranes in age-related macular degeneration. Brit J Ophthalmol 81:481-89 75. Lambert HM (1992) Surgical excision of subfoveal neovascular membranes in age-related macular degeneration. Am J Ophthalmol 113:257-62 76. Bressler NM (2000) Submacular surgical trials randomized pilot trial of laser photocoaguilation versus surgery for recurrent choroidal neovascularisation secondary to age-related macular degeneration: Ophthalmol outcomes submacular surgery trials pilot study report no. I. Am J Ophthalmol 130:387-407 77. Machemer R (1993) Retinal separation, retinotomy and macular relocation: II. A surgical approach for age-related macular degeneration. Graefes Arch Clin Exp Ophthalmol 231:635-41 78. Eckardt C (1999) Macular rotation with and without counter rotation of the globe in patients with age-related macular degeneration. Graefes Arch Clin Exp Ophthalmol 237:313-25 79. Toth C (2001) Macular translocation with 360-degree peripheral retinectomy impact of technique and surgical experience on visual outcome. Retina 21:293-303 80. Freedman SF (2000) Combined superior oblique muscle recession and inferior oblique muscle advancement and transposition for cyclotorsion associated with macular translocation surgery. JAAPOS 4:75-83 81. Gouras P (1984) Transplantation of cultured human retinal cells to monkey retina. Ann Acad Bras Cienc 56:431-443 82. Grisanti S (1997) Immunity and immune privilege elecited by cultured retinal pigment epithelial cell transplants. Invest Ophthalmol Vis Sci 38:1619-26 83. Binder S (2002) Transplantation of autologous retinal pigment epithelium in eyes with foveal neovascularisation resulting from age-related macular degeneration. A pilot study. Am J Ophthalmol 133:215-25 84. Binder S (2004) Outcome of transplantationof autologous retinal pigment epithelium in agerelated macular degeneration: a prospective trial. Invest Ophthalmol Vis Sci 5:4151-4160 85. van Meurs JC (2004b) Autologous retinal pigment epithelium and choroid translocation in patients with exudative age-related macular degeneration: short term follow-up. Am J Ophthalmol 136:688-95 86. Joussen AM (2006) Autologous translocation of the choroid and retinal pigment epithelium in age-related macular degeneration. Am J Ophthalmol 142:17-30 87. Age-related Eye Disease Study Group (2001) A randomized, placebo controlled, clinical trial of high dose supplementation with vitamins C and E and beta-carotene for age-related cataract and vision loss. AREDS report No 9. Arch Ophthalmol 119:1439-52 88. Klingel R (2002) Rheopheresis for age-related macular degeneration: a novel indication for therapeutic apheresis in ophthalmology. Ther Apher 6:271-81 89. Pulido JS (2002) Multicenter prospective, randomised, double masked placebo controlled study of reopheresis to treat non-exudative age-related macular degeneration: interim analysis Trans Am Ophthalmol Soc. 100:85-106 (discussion 106-7) 90. Olk RJ (1999) Therapeutic benefit of infrared (810 nm) diode laser grid photocoagulatoinin prophylactic treatment of non-exudative age-related macular degeneration: two year results of a randomised pilot study. Ophthalmology 106:2082-2090

Chapter 13

Age-Related Macular Degeneration II: Idiopathic Macular Holes Christiane I. Falkner-Radler, MD and Susanne Binder, MD

Abstract This chapter describes the macular holes as various histopathological modifications in the macular area of the retinal fundus of the eye. Advances in OCT technology provide promising objective methods for analyzing morphological and functional aspects of idiopathic macular holes. The UHR-OCT and the threedimensional UHR-OCT may help to elucidate all structural changes associated with macular holes, as well as improve early diagnosis, monitoring of disease progression and response to treatment. The longer imaging times of these new diagnostic tools have to be refined to make them applicable for the routine clinical setting.

Keywords AMD, macular Holes, Diagnostic tools, Fluorescein angiography, laser, Treatment options.

Macular Holes The term macular hole has been used to describe various clinical and histopathological disorders in the macular area of the fundus. A full-thickness macular hole (FTMH) is a round break that involves all retinal layers, from the internal limiting membrane (ILM) through the outer segments of the photoreceptor layer. Macular pseudoholes (MPH) and lamellar macular holes (LMH) are two differential diagnoses for FTMH. These entities mimic retinal excavation without actual tissue loss. Their only common feature is the round and reddish appearance of the macula. The MPH is directly associated with the centripetal contraction of an epiretinal membrane (ERM), while the LMH usually results from partial opening of macular cysts. Metamorphopsia may be present in both forms.1 The exact cause of idiopathic FTMH still remains unknown. Older theories for macular hole development—namely association with trauma or with the presence of some kind of cystoid macular edema (CME)—have not been proven as the main cause.2 According to the theory of Gass, macular hole formation is strongly associated with vitreomacular traction. Earliest stages of macular hole formation usually begin with perifoveal vitreous detachment (PVD) resulting in a foveal dehiscence. From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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Further anterior-posterior and dynamic vitreous traction progresses from foveal detachment to macular hole formation. Gass historic theory could be partially confirmed with optical coherence tomography (OCT) findings.3 However, macular hole formation and spontaneous macular hole closure were documented in eyes with and without complete PVD.4 The etiology of macular holes seems to be multifactorial, including vitreomacular traction, foveolar dehiscence, and other factors. Trauma and high myopia are the causative factors most frequently seen.5 Most patients presenting FTMHs are older than 55 years, and female patients are predominately affected (male: female = 1: 2-3). The overall prevalence is approximately 3.3 cases in 1000 in this age group.6 The risk for development of an idiopathic FTMH in the fellow eye is approximately 6.5 percent within five years. In Gass classification of the development of macular holes, Stage 1 is foveal detachment characterized by loss of foveal depression and the appearance of a yellow spot (stage 1A, 50-100 µm) or a yellow ring (stage 1B) due to the distribution of xanthophylls. Stage 2 results in early hole formation with a full-thickness macular opening. Vitreous separation in the macular area then leads to Stage 3. Complete and obvious posterior vitreous separation, along with a full-thickness macular hole, constitutes Stage 4.7

Diagnostic Tools In most cases, careful biomicroscopy slit-lamp examination with a contact lens allows to us diagnose a FTMH. Clinically, an early stage idiopathic macular hole is often undetectable. With progression, a round excavation with punched-out borders interrupting the slit-lamp beam can be seen. A grayish halo corresponding to serous detachment surrounds an often red area with a yellow spot or ring corresponding to accumulation of xanthophylls. An operculum may be seen as sign of vitreous separation. During the examination, the patient notices a discontinuity in the slit-lamp beam as it passes over the hole (positive Watzke-Allen test). Some false-positive results, however, are possible. Alternatively, a 50 mm spot laser-aiming beam, shown directly in the center of the macular, will not be seen in patients with macular holes (positive laser-aiming beam test). Fluorescein angiography (FAG) is rarely necessary in patients with FTMH. The angiographic findings present a hyperfluorescence in the macular area. Other techniques, such as the scanning laser ophthalmoscope (SLO) and fundus autofluorescence have also been used to evaluate the extent of the lesion. The SLO has shown that the visual loss in eyes with macular holes is related to the absence or reduction of function in the area of the neurosensory defect.8 Autofluorescence imaging demonstrates a marked hyperfluorescent spot in the foveolar region, which disappears after successful surgical treatment.9 During the last few years, the OCT has become the gold standard for the diagnosis of FTMHs. On OCT images, a Stage 1 macular hole appears as a distinct foveal thickening often with a large intraretinal cyst present under the fovea (Stage 1A between 100200 µm; Stage 1B between 200-300 µm). Linear reflections corresponding to the

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posterior vitreous face are typically noted inserting at the central fovea. Stages 2 through 4 represent an enlarging full thickness defect through the neurosensory retina (Stage 2 less than 400 µm; Stage 3 and 4 more than 400 µm) and an increasing anterior-posterior vitreous traction resulting in separation of the vitreous body from the surface of the retina (Stage 3 no signs of PVD, often pseudo-operculum; Stage 4 complete PVD). The advantage of the OCT over biomicroscop g of retinal layers, new technologies with increased resolution have been introduced. The ultrahigh resolution OCT (UHR-OCT) provides improved imaging of epiretinal, intraretinal, and subretinal layers. The UHR-OCT is useful in distinguishing between FTMH and MPH or LMH, and enables the visualization of changes in the photoreceptor morphology. MPHs have a nearly normal, or slightly increased, central foveal thickness and a steepened foveal pit with thickened retinal edges. In contrast, LMHs have a thinner central foveal thickness, and an irregular foveal center with split edges.1 The visualization of the junction between the photoreceptor’s inner and outer segments was found to be an important indicator of photoreceptor integrity. Consequently, their integrity or impairment seems to be an indicator of macular hole progression and surgical outcome.10 Three-dimensional UHR-OCT imaging enables new imaging protocols—comparable with optical biopsy—that improve visualization and mapping of retinal microstructure11 (see Figs. 13.1a, 13.1b, and 13.1c). Most patients with FTMHs notice a gradual decrease in visual acuity (VA), blurred vision, and/or metamorphopsia in the affected eye. Rarely, they complain of a central scotoma. Vitreomacular traction ma y result in photopsia and the sudden onset of floaters. The VA of patients varies according to size, location, and the stage of the FTMH. Patients with Stage 1 FTMHs usually retain a good VA, ranging between 1.0 and 0.33. Once a macular hole represents a full-thickness defect through the neurosensory retina, the usual range of VA is from 0.25 to 0.05, averaging at 0.1.5 Approximately 34 percent of eyes with idiopathic macular holes show an increase in the lesion size, and 45 percent show a decrease in VA of two or more lines during a follow-up of several years.6 Generally, the vision loss stabilizes at the 0.1 to 0.05 level, and retinal pigment epithelial atrophy surrounding the macular hole occurs.12 Randomized clinical trials have reported that Stage 1 FTMHs with a baseline VA of 0.4 or worse showed a significantly higher risk of progression (66%) compared to Stage 1 macular holes with a baseline VA of 0.5 or better (33%).13 Most Stage 2 FTMHs, especially centric Stage 2 macular holes with pericentral hyperflourescence and eccentric holes, were found to progress to Stage 3 or 4.14 A spontaneous closure rate of the FTMHs has been described to occur in 11.5 percent of cases with a VA remaining stable.15

Treatment Options Compared with observation alone, surgical intervention in Stage 2 macular holes result in a significantly lower incidence of hole enlargement and appear to be associated with a better functional outcome.16 Since the introduction of vitreous surgery followed by intraocular tamponade for the treatment of idiopathic FTMHs in 1991,

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advances in vitreoretinal surgery resulted in a steady improvement of the anatomical and functional results.17 The current overall closure rate, as reported in a randomized clinical trial, is over 80 percent, with approximately 45 percent of eyes having a VA of 20/40 or greater. Additionally, surgical eyes present with a better near VA than observation eyes.15 Because the surgical outcome is influenced by the duration of symptoms, the preoperative macular function, and the preoperative hole diameter, early diagnosis and intervention in FTMHs should be emphasized.18 However, the benefit of vitrectomy for Stage 1 macular holes is still controversial.19,20 Patients with MPHs and LMHs usually have a favorable natural history with retaining a good VA, and surgery is only indicated for symptomatic ERM formation and/or vitreomacular traction and for decreasing VA to 0.25 level or less.21 ERMs are common in eyes with FTMHs. The presence of ERM does not correlate with postoperative VA, but excessive ERM growth contributes to hole reopening after surgery. Therefore, ERM peeling is recommended to be performed during surgery for FTMHs.22 A meta-analysis showed that removal of the ILM appears to significantly increase the anatomical, as well as the functional success rate in macular hole surgery.23 Other studies suggest that ILM peeling does not seem to be beneficial for macular holes less than 400 µm in diameters.24 For better visualization and surgical manipulation, vital dyes for staining the ERM and/or the ILM have been introduced. Trypan blue (TB) and triamcinolone acetonide (TA) have proven to be effective and safe, and offer a favorable anatomic and functional outcome.25,26 Intravitreal application of indocyanine green (ICG) may cause retinal damage, resulting in less improvement of VA and unexpected visual field defects. The underlying mechanisms are still unclear and are the subject of ongoing investigations.25,27 To improve the hole closure rate, various biological adjuncts like transforming growth factor-β (TGFβ), autologous serum, thrombin, and autologous platelet concentrate have been introduced but not yet proven to have any added benefit compared to controls.15,28,29,30 After vitrectomy and membrane peeling, a intraocular tamponade is recommended for successful macular hole surgery. Perfluoropropane (C3F8) has proven to be a more effective tamponade than silicone oil with respect to initial closure rate and final VA in eyes with FTMHs.31 Additionally, long-acting gas tamponade (C3F8) gives a higher success rate compared to short-acting gas tamponade (SF6).

Fig. 13.1 Comparison of three optical coherence tomography (OCT) technologies for the evaluation of full-thickness macular holes (FTMHs)—FTMH Stage 4. The patient is a 77-year old man who presented a best corrected baseline visual acuity (VA) of 0.1 in the left eye. (a) The standard resolution Stratus OCT 3000 image (Zeiss) demonstrates a full-thickness macular hole with intraretinal cystic changes and irregularities in the photoreceptor layer. The posterior hyaloid is detached and an epiretinal membrane (ERM) is visible. (b) The ultrahigh resolution OCT (UHROCT), according to Drexler W et al., allows an improved visualization and structural analysis of the intraretinal changes. Additionally, the UHR-OCT shows irregularities in the inner and outer segments of the photoreceptor layer. (c) The three-dimensional UHR-OCT (3D UHR-OCT) image, according to Glittenberg C. et al, is comparable with a virtual biopsy. The photoreceptor inner and outer segments are clearly delineated in configuration and size, with a characteristic peak in the subfoveal area. The architecture of choroidal vascularization is distinctly imaged.

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Postoperative face-down positioning is considered to be essential for short-acting gas, while the significance of posturing remains uncertain for long-acting gases.32 Complications of vitreoretinal surgery for FTMHs include a reopening of holes (2%), cystoid macular edema (1%), choroidal neovascular membrane (1%) and endophthalmitis in (1%). More common surgical complications are retinal pigment epithelium alterations in 33 percent of the cases and retinal detachment in 11 percent, which result in a significantly reduced postoperative VA.33 Therefore, the vitreoretinal procedure should be followed by a careful examination of the peripheral retina— particularly the sclerotomy incisions—to detect iatrogenic retinal tears and avoid entry site retinal detachments. Because of the high incidence of significant cataract formation after surgery, most surgeons perform a combined cataract surgery and vitrectomy. Combined surgery for FTMHs is a safe and effective procedure, allows for better intraoperative visualization, facilitates the use of a large gas bubble (which may contribute to hole closure without postoperative prone posturing), and prevents patients from having to return for cataract surgery.34 The treatment of FTMHs continues to evolve because modifications to the standard surgical procedure have been proposed and evaluated. These innovations include sutureless 25-gauge and 23-gauge vitrectomy systems, the introduction of novel intraocular dyes, and the use of macular-protecting, yellow-tinted intraocular lenses for combined surgery.35,36,37,38 There is still no gold standard in macular hole surgery and some surgical procedures are controversial, including the benefit of ILM peeling, the use of ICG, and the details of endotamponades and post-operative positioning. Advances in OCT technology provide promising objective methods for analyzing morphological and functional aspects of idiopathic macular holes. The UHR-OCT and the three-dimensional UHR-OCT may help to elucidate all structural changes associated with macular holes, as well as improve early diagnosis, monitoring of disease progression, and response to treatment. The longer imaging times of these new diagnostic tools have to be refined, however, to make them applicable for the routine clinical setting.

References 1. Haouchine B, Massin P, Tadayoni R, et al. (2004) Diagnosis of macular pseudoholes and lamellar macular holes by optical coherence tomography. Am J Ophthalmol 138:732-739 2. Smiddy WE, Flynn HW, Jr (2004) Pathogenesis of macular holes and therapeutic implications. Am J Ophthalmol 137:525-537 3. Schumann RG, Schaumberger MM, Rohleder M, et al. (2006) Ultrastructure of vitreomacular interface in full-thickness idiopathic macular hole. A consecutive analysis of 100 cases. Am J Ophthalmol 141:1112-1119 4. Lo WR, Hubbard GB (2006) Macular hole formation, spontaneous closure, and recurrence in a previously vitrectomized eye. Am J Ophthalmol 141:962-964 5. Ezra E (2001) Idiopathic full thickness macular hole: natural history and pathogenesis. Br J Ophthalmol 102-108 6. Chew EY, Sperduto RD, Hiller R, et al. (1999) Clinical course of macular holes: The Eye Disease Case-Control Study. Arch Ophthalmol 117:242-246

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7. Gass JD (1995) Reappraisal of biomicroscopic classification of stages of development of macular hole. Am J Ophthalmol 119:752-759 8. Guez JE, Le Gargasson JF, Massin P, et al. (1998) Functional assessment of macular hole surgery by scanning laser ophthalmoscopy. Ophthalmology 105:694-699 9. Framme C, Roider J (2001) Fundus autofluorescence in macular hole surgery. Ophthalmic Surg Lasers 32:383-390 10. Ko TH, Fujimoto JG, Duker JS, et al. (2004) Comparison of Ultrahigh- and standard-resolution optical coherence tomography for imaging macular hole pathology and repair. Ophthalmology 111:2033-2043 11. C. Glittenberg, B. Povazay, B. Hermann (2007) Three dimensional reconstruction of tomographic images of the retina. Spektrum Augenheilkd 21:13-16 12. Casuso LA, Scott IU, Flynn HW, et al. (2001) Long-term follow-up of unoperated macular holes. Ophthalmology 108:1150-1155 13. Kokame GT, de Bustros S (1995) Visual acuity as a prognostic indicator in stage 1 macular holes. The Vitrectomy for Prevention of Macular Hole Study Group. Am J Ophthalmol 112-114 14. Kim JW, Freemann WR, el-Haig W, et al. (1995) Baseline characteristics, natural history and risk factors to progression in eyes with stage 2 macular holes. Results from a prospective randomized clinical trial. Vitrectomy for Macular Hole Study Group. Ophthalmology 102:1818-1828 15. Ezra E, Gregor ZJ (2004) Surgery for idiopathic full-thickness macular hole: Two-year results of a randomized clinical trial comparing natural history, vitrectomy and vitrectomy plus autologous serum: Moorfields Macular Hole Study Group Report No.1. Arch Ophthalmol 122:224-236 16. Kim JW, Freeman WR, Azen SP, et al. (1996) Prospective randomized trial of vitrectomy or observation for stage 2 macular holes. Vitrectomy for Macular Hole Study Group. Am J Ophthalmol 605-614 17. Kelly NE, Wendel RT (1991) Vitreous surgery for idiopathic macular holes. Results of a pilot study. Arch Ophthalmol 109:654-659 18. Kang SW, Ahn K, Ham DI (2003) Types of macular hole closure and their clinical implications. Br J Ophthalmol 87:1015-1019 19. De Butros S (1994) Vitrectomy for prevention of macular holes. Results of a randomized multicenter clinical trial. Vitrectomy for Prevention of Macular Hole Study Group. Ophthalmology 101:1055-1059 20. Subramanian ML, Truong SN, Rogers AH, et al. (2006) Vitrectomy for stage 1 holes identified by optical coherence tomography. Ophthalmic Surg Lasers Imaging 37:42-46 21. Smiddy WE, Gass JD (1995) Masquerades of macular holes. Ophthalmic Surg 26:16-24 22. Cheng L, Azen SP, El-Brady MH (2002) Effects of preoperative and postoperative epiretinal membranes on macular hole closure and visual restoration. Ophthalmology 109:1514-1520 23. Mester V, Kuhn F (2000) Internal limiting membrane removal in the management of full-thickness macular holes. Am J Ophthalmol 769-777 24. Tadayoni R, Gaudric A, Haouchine B, et al. (2006) Relationship between macular hole size and the potential benefit of internal membrane peeling. Br J Ophthalmol 90:1239-1241 25. Beutel J, Dahmen G, Ziegler A, et al. (2007) Internal limiting membrane peeling with indocyanine green or trypan blue in macular hole surgery: a randomized clinical trail. Arch Ophthalmol 125:326-323 26. Karacorlu M, Ozdemir H, Karacorlu A (2005) Does intravitreal acetonide-assisted peeling of the internal limiting membrane effect the outcome of macular hole surgery? Graefe’s Arch Clin Exp Ophthalmol 243:754-757 27. Gass CA, Haritoglou C, Schaumberger M, et al. (2003) Functional outcome of macular hole surgery with and without indocyanine green-assisted peeling of internal limiting membrane. Graefe’s Arch Clin Exp Ophthalmol 241:716-720 28. Duker JS, Wendel R, Patel AC, et al. (1994) Late re-opening of macular holes after initially successful treatment with vitreous surgery. Ophthalmology 101:1373-1378 29. Vine AD, Johnson MW (1996) Thrombin in the management of full-thickness macular holes. Retina 16:474-478

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30. Korobelnik JF, Hannouche D, Belayachi N, et al. (1996) Autologous platelet concentrate as an adjunct in macular hole healing: a pilot study. Ophthalmology 103:590-594 31. Tafoya ME, Lambert HM, Vu L, et al. (2003) Visual outcomes of silicone oil versus gas tamponade for macular hole surgery. Semin Ophthalmol 18:127-131 32. Szurman P, Di Tizio FM, Lafaut B, et al. (2000) Significance of postoperative face-down positioning after surgery for idiopathic macular holes: consecutive case-control study. Klin Monatsblatt Augenheilkd 217:351-355 33. Banker AS, Freeman WR, Kim JW (1997) Vision-threatening complications of surgery for full-thickness macular holes. Vitrectomy for Macular Hole Study Group. Ophthalmology 104:1442-1452 34. Simcock PR, Scalia S (2001) Phacovitrectomy without prone posture for full-thickness macular holes. Br J Ophthalmol 85:1316-1319 35. Schuettauf F, Haritoglou C, May CA, et al. (2006) Administration of novel dyes for intraocular surgery: an in vivo toxicity animal study. Invest Ophthalmol Vis Sci. 47:3573-3578 36. Kellner L, Wimopissinger B, Stolba U, et al. (2007) 25-gauge versus 20-gauge system for pars plana vitrectomy: a prospective randomized clinical trial. Br J Ophthalmol Jan 3 (Epub ahead of print) 37. Rizzo S, Belting C, Cresti F, et al. (2007) Sutureless 25-gauge vitrectomy for idiopathic macular hole repair. Graefe’s Arch Clin Exp Ophthalmol Mar15 (Epub ahead of print) 38. Falkner CI, Binder S (2006) UV-filter iol versus blue light-filter iol in combined cataract surgery with vitrectomy: a prospective randomized clinical trial. Invest Ophthalmol Vis Sci ARVO Abstract 1484

Chapter 14

Age-Related Macular Degeneration III: Epiretinal Membranes Christiane I. Falkner-Radler, MD and Susanne Binder, MD

Abstract This chapter deals with the description of the epiretinal membranes, which are common in patients over 50 years of age. Moreover, clinical studies have found that these membranes increase with age. Modifications of diagnosis and treatment of epiretinal membranes have resulted in a steady improvement of the patients’ outcome. Most cases show very satisfying functional results with a mean improvement in VA of 2 or more lines in approximately 72 percent. A less favorable outcome is found in cases with underlying or accompanying diagnoses, like trauma, retinal vascular disorders, or retinal detachment. The clinical implication provided by the UHROCT and three-dimensional UHR OCT has yet to be established. New techniques, like sutureless vitrectomy or novel dyes may further improve the patients’ outcome. Keywords AMD, Epiretinal membranes, vitrectomy, diagnostic tools, histological evaluation.

Introduction Epiretinal membranes (ERM) are common in patients over 50 years of age. Clinical studies have found that ERM formation is increasing with age, that the prevalence of ERMs is approximately 5.3 percent, and that bilateral involvement occurs in approximately 14 percent of the cases.1 Most cases of ERMs are idiopathic. They present no underlying ocular disease or disorder and have a direct association with posterior vitreous detachment.2 ERMs can also be associated with various ocular pathologies, including surgical causes, like cataract surgery, retinal detachment surgery, and laser photocoagulation, as well as nonsurgical causes like blunt ocular trauma, uveitis, retinal vascular disorders, and macular holes.3 The prevalence of ERMs in idiopathic macular holes has been reported to be in the range of 30 to 73 percent. Two hypotheses exist regarding the association of ERM in macular holes. Some investigators believe that the ERM contributes to the macular hole formation and reopening, while others suggest that the ERM develops as a result of macular hole formation. The role of the ERM in the

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pathophysiology and surgical outcome of idiopathic macular holes is still not well-defined.4 ERMs arise from proliferation of glial cells on the inner retinal surface. The formation of the ERM is related to migration of astrocytes through breaks in the internal limiting membrane. The membranes are also composed of retinal pigment epithelium cells, fibrocytes, and myofibroblasts.5 Besides, investigations of the ultrastructure of the vitreoretinal interface in the vitreomacular traction syndrome suggest two different forms of epiretinal fibrocellular proliferation, namely ERMs interposed in native vitreous collagen as well as cell proliferation directly on the internal limiting membrane (ILM). A predominance of myofibroblasts may explain a high prevalence of cystoid macular edema and progressive vitreomacular traction characteristics for this disorder.6 According to Gass,7 translucent membranes without retinal distortion are classified as grade 0 (cellophane maculopathy), membranes with irregular wrinkling of the inner retina are classified as grade 1 (crinkled cellophane maculopathy), and membranes with distortion of the retinal vasculature are classified as grade 2 (macular pucker). The grading system adopted from Klein et al.8 divides ERMs into the relatively mild form without visible retinal folds (cellophane macular reflex), and the more severe form with retinal folds (preretinal macular fibrosis). However, no standard nomenclature is used at present, and ERMs are commonly classified according to their density, the severity of retinal distortion, and associated biomicroscopic changes. ERMs have been reported to be slowly progressive and to rarely cause a severe visual impairment. Effects on vision vary depending on the severity and extent of the membrane. Patients with a mild ERM usually report no symptoms or may notice mild metamorphopsia like blurred or distorted vision, while a central photopsia often indicates traction on the macular area. If the ERM progresses to severe macular pucker, the central vision is affected.9 Occasionally, in less than 1 percent of cases, an ERM spontaneously separates from the retina, which may result in visual improvement.

Diagnostic Tools The clinical examination of ERMs includes near and distance visual acuity testing, and evaluation of metamorphopsia with the standard Amsler grids and funduscopy. In the last few years, the optical coherence tomography (OCT) has become a standard diagnostic tool for preoperative and postoperative evaluation of ERMs. The OCT allows visualization of the full extent and localization of structural changes, as well as vitreoretinal adhesion or traction of ERMs. The main diagnostic characteristics for ERMs using the OCT are: 1) the ERM is separated or adherent, 2) a loss of the foveal depression, 3) a diffuse swelling of the retina, and 4) the presence of intraretinal cystic spaces. The OCT is highly specific and highly sensitive for measurements of the foveal thickness and for the structural assessment of the macula. OCT findings have shown that ERMs are more frequently fully adhered, rather than separated with focal points of adherence.

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Furthermore, the OCT plays an important role in the differential diagnosis of a macular pseudohole in ERMs and of a full-thickness macular hole. OCT data have shown that the VA correlates with measurements of foveal thickness. The mean foveal thickness in a normal eye is 150 µm. Eyes with ERMs have a mean increased foveal thickness of 419 µm. After surgery, the mean foveal thickness decreases to 300 µm, but the macular profile rarely returns to normal. Marked foveal thickening, nonexisting foveal depression, and extensive cyst formation are supposed to correlate with a rather poor visual outcome10. Fluorescein angiography (FAG) is not commonly performed to make the ERM diagnosis. FAG particularly allows evaluating the topography of leakage. For assessment of macular function and morphology Goldmann perimetry, multifocal electroretinography (mfERG) or autofluorescence can be used. For enhanced visualization of epi-, intra- and subretinal structures, ultrahigh-resolution tomography (UHR-OCT) and three-dimensional UHR-OCT technology have been developed.11,12. The UHR-OCT improves visualization of photoreceptor and pigment epithelium morphology, as well as subtle intraretinal and epiretinal changes associated with vitreoretinal disease. The three-dimensional UHR-OCT system offers an interactive possibility to plastically visualize, objectively quantify, and perform a virtual biopsy of retinal structures. With its improvement in image quality and comprehensive analysis of the ERM and all structural levels, this new technology has the potential to become a useful tool for elucidating disease pathogenesis and improving disease diagnosis, surgical planning, and outcome. Comparison of three optical coherence tomography (OCT) technologies for evaluation of epiretinal membranes (ERMs): Macular pucker. The patient is a 73-year old man who presented a best corrected baseline visual acuity (VA) of 0.4 in the right eye.

Fig. 14.1 a The standard resolution Stratus OCT 3000 image (Zeiss) demonstrates all four main diagnostic characteristics for ERMs, which are labeled 1) the ERM is separated or adherent, 2) a loss of the foveal depression, 3) a diffuse swelling of the retina, and 4) the presence of intraretinal cystic spaces. The posterior hyaloid is detached and a clear visualization of overall wrinkling of the internal limiting membrane (ILM) and the thickened gliotic membrane is obtained

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Fig. 14.1 b The ultrahigh resolution OCT (UHR-OCT) image, according to Drexler W et al., shows that the ERM is attached to the foveal center and that no signs of imminent hole formation are present. Distortion of the retinal architecture is visible, with intact photoreceptor layer

Fig. 14.1 c The three-dimensional UHR-OCT (3D UHR-OCT) system, according to Glittenberg C. et al., allows a comprehensive analysis of focal and diffuse diseases and provides 3-D images of topographic dynamics from the retinal surface down to the level of photoreceptor segments

Treatment options and prognosis The principal indication for ERM surgery is a significant disturbance in vision resulting from a decreased visual acuity (VA) with or without metamorphopsia. Usually, patients have a baseline VA of less than 0.6. Since the 1970s, a standard three-port pars plana vitrectomy has been used for the removal of ERMs in patients with symptomatic visual disturbances.13 Vitreoretinal surgery has been found to be

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effective in removing ERMs from the macula, improving VA and decreasing metamorphopsia. Favorable visual outcome can be achieved postoperatively in the majority of cases. A significant improvement in VA after surgery has been reported in more than 80 percent of the cases, with more than 90 percent having a subjective improvement in reduction of distortion.14 The surgical complications described include intraoperative detection of retinal breaks (5%), a small amount of intraretinal bleeding after removal of the ERM and postoperative progressive nuclear sclerosis (12%-68%), retinal detachment (5%), macular edema, retinal pigment epitheliopathy, and recurrence of ERM (10%).15 Histological evaluations of tissue removed during vitrectomy have shown that the ILM, as a potential scaffold for cellular proliferation, seems to be associated with recurrence of ERMs. Therefore, the application of dyes such as indocyanine green (ICG), trypan blue (TB) and triamcinolone acetonide (TA) have been introduced to assist the removal of ERMs, the ILM, or both. The use of these dyes improves the visualization of the ILM and ERMs, and consecutively allows a more controllable, easier, and less traumatic membrane peeling. Investigations of functional results after ERM surgery suggest a higher percentage of improved or stable VA and a lower recurrence rate in eyes in which an additional ILM peeling has been performed. Staining of the ERM and/or the ILM has proven to reduce the recurrence rate, but no significant difference was found for VA, reduction of macular edema, postoperative Amsler grid test, or subjective improvement.16 While the use of TB and TA during vitreoretinal surgery seems to be safe, and no adverse effects have been described, the use of ICG to assist the ILM peeling is controversial, and the potential retinal toxicity of ICG—particularly the presence of postoperative visual field defects—is currently under discussion.17,18,19,20 For refining the intraoperative conditions for the surgeon and improving the patients’ outcome, new dyes for intraocular surgery have been developed and investigated in animal studies. Brophenol blue (BPB) or lightgreen SF yellowish (LGSF) produced no significantly detectable toxic effects on the retina in vivo, but the safety and benefits of these novel dyes must be established in preclinical studies.21 Most patients with symptomatic ERM formation are older than 50 years and present different forms of coexisting lens opacities. As vitrectomy generally increases cataract formation, the vitreoretinal procedure is often combined with phacoemulsification and intraocular lens (IOL) implantation. Several studies have reported that a combined vitreoretinal procedure is a safe and effective way to manage cases with vitreoretinal diseases and cataracts, with the functional outcome comparable to those of sequential surgery.22 New types of blue-light filter IOLs have been designed for macular protection, and the implantation of these yellow-colored IOLs has become increasingly common in cataract surgery. Initially, there were concerns about adverse effects on the surgeon’s ability to perform specific vitreoretinal procedures when using these yellowtinted lenses in combined surgery. Nevertheless, a randomized, controlled trial indicates that there is no significant influence on the intraoperative conditions for the surgeon or on the patients’ outcome, and suggests that the routine use of the yellow-tinted IOL in vitrectomy combined with cataract surgery can be recommended.23

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For improvement of the surgical technique, investigations covering the minimal invasive methods of vitreoretinal surgery have been conducted. Sutureless 25gauge and 23-gauge vitrectomy systems have been introduced and compared to standard 20-gauge systems for pars plana vitrectomy. According to recent reports, the 25-gauge system has proven to be safe and efficient for ERM surgery. The duration of surgery was comparable to the 20-gauge system, because the shorter time for wound opening and closure is equalized by longer vitrectomy duration. Advantages of the 25-gauge vitrectomy include the minimized surgery-induced trauma, and the reduction of postoperative inflammation and of patients’ discomfort. The 25-gauge vitrectomy seems to allow earlier postoperative visual rehabilitation than conventional 20-gauge vitrectomy for patients presenting ERMs, and it may be preferable to 20-gauge vitrectomy in these cases.24,25 The 23-gauge vitrectomy system is still under investigation. Modifications of diagnosis and treatment of ERMs have resulted in a steady improvement of the patients’ outcome. Most cases show very satisfying functional results with a mean improvement of VA of two or more lines in approximately 72 percent of the cases. A less favorable outcome is found in cases with underlying or accompanying diagnoses, like trauma, retinal vascular disorders, or retinal detachment. The clinical implication provided by the UHR-OCT and three-dimensional UHR-OCT has yet to be established. New techniques, like sutureless vitrectomy or novel dyes, may further improve the patients’ outcome.

References 1. Fraser-Bell S, Guzowski M, Rochtchina E, et al. (2003) Five-year cumulative incidence and progression of epiretinal membranes. The Blue Mountains Eye Study. Ophthalmology 110:34-40 2. Hirokawa H, Jalkh AE, Takahashi M, et al. (1986) Role of the vitreous in idiopathic preretinal macular fibrosis. Am J Ophthalmol 101:166-169 3. Appiah AP, Hirose T (1989) Secondary causes of premacular fibrosis. Ophthalmology 96:389-392 4. Cheng L, Freeman WR, Ozerdem U, et al. (2000) Prevalence, correlates, and natural history of epiretinal membranes surrounding idiopathic macular holes. Ophthalmology 107:853-859 5. Gandorfer A, Rohleder M, Kampik A (2002) Epiretinal pathology of vitreomacular traction syndrome. Br J of Ophthalmol 86:902-909 6. Smiddy WE, Maguire AM, Green WR, et al. (1989) Idiopathic epiretinal membranes. Ultrastructural characteristics and clinicopathologic correlation. Ophthalmology 96:811-820 7. Gass JDM. (1987) Stereoscopic Atlas of Macular Diseases. Mosby, St Louis, 671-726 8. Klein R, Klein BEK, Wang Q, et al. (1994) The epidemiology of epiretinal membranes. Trans Am Ophthalmol Soc 92:403-430 9. Wise GN (1975) Clinical features of idiopathic preretinal macular fibrosis. Am J Ophthalmol 79:349-357 10. Massin P, Allouch C, Haouchine B, et al. (2000) Optical coherence tomography of idiopathic macular epiretinal membranes before and after surgery. Am J Ophthalmol 130:732-739 11. Drexler W, Sattmann H, Hermann B (2003) Enhanced visualization of macular pathology using ultrahigh resolution optical coherence tomography. Arch Ophthalmol 121:695-706 12. Glittenberg C, Hermann B, Povazay B, et al. (2006) Creating a steroegraphic three dimensional ultra high resolution optical coherence tomography display system using high-end raytracing software algorithms. Invest Ophthalmol Vis Sci. ARVO Abstract 4054

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13. Marchemer R (1974) A new concept for vitreous surgery.7. Two instrument techniques in pars plana vitrectomy. Arch Ophthalmol 92:407-412 14. Wong JG, Sachdev N, Beaumont PE (2005) Visual outcomes following vitrectomy and peeling of epiretinal membrane. Clin Experiment Ophthalmol 33:373-378 15. Pournaras CJ, Donati G, Brazitikos PD, et al. (2000) Macular epiretinal membranes. Semin Ophthalmol 15:100-107 16. Hillenkamp J, Saikia P, Gora F, et al. (2005) Macular function and morphology after peeling of idiopathic epiretinal membrane with and without the assistance of indoyanine green. Br J Ophthalmol 89:437-443 17. Haritoglou C, Kampik A (2006) Staining techniques in macular surgery. Ophthalmologe 103:927-934 18. Hillenkamp J, Saika P, Herrmann WA, et al. (2007) Surgical removal of epiretinal membranes with or without the assistance of indocyanine green: a randomised controlled clinical trial. Graefes Arch Clin Exp Ophthamol 245:973–979 19. Haritoglou C, Gandorfer A, Schaumberger M, et al. (2004) Trypan blue in macular pucker surgery: an evaluation of histology and functional outcome. Retina 24:582-590 20. Tognetto D, Zenoni S, Sanguinetti G, et al. (2005) Staining of the internal limiting membrane with intravitreal triamcinolone acetonide. Retina 25:462-467 21. Schuettauf F, Haritoglou C, May CA, et al. (2006) Administration of novel dyes for intraocular surgery: an in vivo toxicity animal study. Invest Ophthalmol Vis Sci; 47:3573-3578 22. Ling R, Simcock P, McCoombes J, Shaw S (2003) Presbyopic phacovitrectomy. Br J Ophthalmol 87(11):1333-1333 23. Falkner CI, Binder S (2006) UV-filter IOL versus blue light-filter IOL in combined cataract surgery with vitrectomy: a prospective randomized clinical trial. Invest Ophthalmol Vis Sci. ARVO Abstract 1484 24. Kellner L, Wimopissinger B, Stolba U, et al. (2007) 25-gauge versus 20-gauge system for pars plana vitrectomy: a prospective randomized clinical trial. Br J Ophthalmol 91:945–948 25. Kadonosono K, Yamakava T, Uchio E, et al. (2006) Comparison of visual function after epiretinal removal by 20-gauge and 25-gauge vitrectomy. Am J Ophthalmol 142:513-515

Chapter 15

Macular Degeneration: Ultrastructural Age-Related Changes Illes Kovacs, MD, PhD, Janos Feher, MD, PhD, and Carlo A. P. Cavallotti, MD, PhD

Abstract The aim of this chapter is to reveal the contribution of mitochondria and peroxisomes to the turnover of the photoreceptor outer segment and to describe the subsequent alteration of the retinal pigment epithelium and Bruch’s membrane in normal aging and in age-related macular degeneration (AMD). Fifty-two surgically removed human eyes were involved in these histo-pathologic studies (25 female and 27 male, aged 56 to 87 years—mean age 68 years). Twenty-six of them were affected by early AMD, and 26 eyes were used as age-matched normal controls. For better visualization of lipids, osmium tetroxid postfixation was added to the standard electron microscopic technique. Polarization microscopy was also applied for the study of extracellular matrix components. Age-related changes of anisotropy were statistically analyzed using linear regression, and Fisher’s transformation in both control and early AMD groups. Electron microscopy of retinal pigment epithelium both aged and early AMD showed a) accumulation of lipofuscin in the cytoplasm, b) focal or rarely diffuse alterations of mitochondrial cristae and matrix, and (c) accumulation of peroxisomes of variable size and electron density distributed throughout the cytoplasm. Electron and polarization microscopy of extracellular matrix showed a) an accumulation of amorphous, vacuolated and granular material in the collageneous layers of Bruch’s membrane, b) the appearance of soft (rarely hard) Drusens, and c) a thickening of the basement membrane of retinal pigment epithelium (RPE) and choriocapillaries due to addition of axiparallel-oriented glycated fibrils and transversally oriented lipids. Although these processes were observed in both normal aging and early AMD, statistical analysis of anisotropy suggested that deposition of lipids and glycated fibrils was significantly different in AMD compared to normal aging. Keywords mitochondria, peroxisomes, age-related macular degeneration, retinal pigment epithelium, basement membrane

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Introduction Age-related macular degeneration (AMD) is a progressive neurodegenerative disease of the central retina. and represents the most common cause of legal blindness in industrialized countries.1 Current pathophysiologic concepts suggest that this multifactorial disease affects primarily the RPE and the thin connective tissue layer (Bruch’s membrane) interposed between the RPE and choriocapillaries. Electron microscopy revealed accumulation of lipofuscin granules in the cytoplasm of RPE, and deposition of randomly distributed amorphous or granular material in the Bruch’s membrane.2 Histo-chemically, these letters contain mainly extracellular matrix-associated lipids and glycoproteins.3 Frequently, similar materials build up clinically detectable focal deposits—so-called soft Drusen—located just beneath the RPE.5 The subsequent impairment of metabolic exchange between choriocapillaries and RPE leads to secondary degeneration of photo-receptor cells resulting in further deposition of abnormal metabolites in the Bruch’s membrane, and finally, permanent loss of the visual functions at the affected macular area.5,6 Biochemical studies have shown increasing lipid deposits in Bruch’s membrane exponentially correlated with age. The macular area was preferentially affected.7 Further studies on normal donor eyes showed that the Bruch’s membrane contains significant amount of lipids derived from long-chain polyunsaturated fatty acids normally found in the photoreceptor outer segment, providing support for the cellular, but not plasma origin of these lipids.8 These findings suggested that incomplete catabolism of the photoreceptors results in intra- and extracellular accumulation of undigested materials—first of all lipid peroxides. Recent studies showed that Bruch’s membrane is also enriched in cholesterol, particularly in esterified cholesterol, which increases significantly with age. Photoreceptors are poor in unesterified cholesterol, and RPE can not esterify cholesterol. Thus, cholesterol deposition in human Bruch’s membranes may rather have plasma than photoreceptor origin.9 All these findings justify an assumption that both catabolism and uptake of lipids by RPE are compromised in AMD. Although the critical role of mitochondria and peroxisomes in the abnormal lipid metabolism of RPE was suggested several years ago, this concept was never confirmed in humans.5,10,11 Bruch’s membrane is intriguingly similar to the arterial intima because both of them contain extracellular matrix molecules that potentially can interact and bind lipids. Furthermore, Bruch’s membrane alterations in AMD and atherosclerotic vascular lesions possess several common features.9 These observations are in accordance with epidemiologic studies that showed an association between atherosclerosis and the presence of early AMD and their risk factors.12,13 Ultrastructural and histo-chemical studies showed that alterations of the Bruch’s membrane may also include focal or diffuse thickening of the basement membrane of the RPE and choriocapillaries.2,14 Similar thickening of capillary basement membrane is a well known feature of age- and diabetes-related microangiopathy. Although electron microscopy shows homogeneous appearance of basement membrane, histochemically it contains type IV collagen, heparan sulfate proteoglycans, and

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laminin. Polarization microscopy revealed that collagen filaments are present in axiparallel linear order in the basement membranes, and the carbohydrate components of proteoglycan macromolecules are also in linear order, which is correlated with that of the collageneous framework.15 A lipid layer formed by transversally oriented hydrocarbon chains of lipids has also been described in the basement membranes.16,17 Although extracellular matrix (ECM) alterations in aging and in several diseases (neurodegenerative diseases, metabolic diseases, and atherosclerosis) have been widely studied, neither the origin of these structural lipids nor their pathologic significance were explored. Currently, the role of peroxisomes in the intracellular accumulation of lipids is under exploration in several laboratories, but its contribution to extracellular lipid deposition is unknown. The central role of mitrochondria in aging and in the pathogenesis of the above mentioned diseases is well-established.18 In spite of that, these mitochondrial diseases are usually associated with marked changes in content and composition of ECM. The correlation between mitochondrial pathology and ECM alterations have not yet been studied. For the present studies, all of our specimens were obtained from surgically removed human eyes. The time between enucleation of eyeball and fixation was extremely short, thus post-mortem changes of membrane lipids were insignificant. We also used these specimens for polarization microscopy for the study of ECM alterations. This is a very sensitive technique for revealing macromolecular organization of proteoglycans and lipids. It may also give quantitative information on age- or disease-related changes of each basement membrane component.19 This unique human material and special microscopic techniques permitted an excellent insight into the pathology of a common eye disease, which has neither animal model nor treatment, and which may be considered as a paradigm for neurodegenerative diseases. Furthermore, these studies also added new information to the pathology of ECM—apparently suitable for learning more on aging, atherosclerosis, diabetic microangiopathy, and infiltrative neoplastic diseases.

Methods Selection of Materials Fifty-two human eyes were involved in these histo-pathologic studies (25 female and 27 male, aged 56 to 87 years—mean age 68 years). Twenty-six of them were affected by early AMD, and 26 eyes were used as age matched normal controls. Clinical criteria for AMD was pigment irregularities and/or soft Drusen by ophthaimoscopy, while histological criteria for AMD wasa presence of soft Drusen and/or basement membrane thickening. These eyes were surgically removed because of malignant tumor or severe ocular trauma—neither of them affected the posterior pole of the eyeball.

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Transmission Electron Microscopy Small pieces of the retina and choroid were dissected at the posterior pole, immediately (< 2 minutes) after the removal of the eyeball, and fixed at 40C in 2 percent buffered glutaraldehyde for two hours, and post-fixed in 2 percent osmium tetroxide for another two hours. The post-fixation with osmium tetroxide has been found to be effective for demonstrating lipid peroxides as tetramethylbensidine.20 The specimens were dehydrated, embedded in araldite, sectioned with Reichert ultramicrotome, contrasted with lead-citrate and uranyl-acetate, and studied with Zeiss 109 Electron Microscope.

Light Microscopy Other small pieces of the retina and choroids were fixed in 10 percent buffered formaldehyde for 48 hours, then dissected, One half of the eyeball was dehydrated, embedded in paraffin, and the 6-micron thick sections were stained with hematoxyline-eosine and PAS-Hale staining.

Polarization Microscopy This technique, by the use of histochemical reactions to selectively modify the anisotropy (topo-optical reactions), is a unique microscopic approach for the study of membrane lipids, proteoglycans, and collagen fibrils at macromolecular levels.

For the Study of Lipids Other small pieces of the retina and choroids after fixation were embedded in gelatin. Ten-micron thick frozen sections were made with cryostat, and the unstained sections were mounted in gum arabic. This simple technique suppresses the birefringence of all structures except lipids, and thus permits the study of lipid structures. For lipid extraction, metanol-chlorophorm 1:3 mixture was used for 24 hours.

For the Study of Proteoglycan Structures Aldehyde-Bisulfite-Toluidine Blue (ABT) staining reactions were used.15 This technique allows the polarization microscopic studies of vicinal OH groups, based on the aldehyde-bisulfate addition reaction followed by toluidine blue staining at pH1.0. During the procedures, vicinal OH groups are transformed into dialdehydes

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by periodic acid, and then transformed into negatively charged groups by the bisulfite addition reaction. In this way, they are rendered capable of binding toluidine blue at low pH, which results in basophilia and anisotropy indicating linear order of the OH groups in the reacting macromolecules. In detail, the deparaffined sections were first treated with 0.5 percent periodic acid for 30 minutes, then for 30 more minutes with a saturated solution of sodium bisulfate. After short rinsing with water, the slices were stained for five minutes with toluidine blue at pH 1.0 (0.1% toluidine blue in 0.1 normal HCl). The controls were stained with toluidine blue at pH 4.5 (0.1% toluidine blue in McIlvain buffer). After staining, the dye solution was blotted off with filter paper and then 1 percent potassium ferricyanide solution was dropped onto the slides. This resulted in stabilization of the dye binding in the oriented state in which the dye has been bound by the structures originally and in maintaining its optical effects—the metachromatic basophilia and anisotropy. Without being rinsed in water, the slices were mounted in gum arabic containing 1 percent potassium ferricyanide. This gum arabic layer was allowed to dry for two to three days.

For the Study of Collagen Fibrils The deparaffined sections were mounted in 50 percent phenol-containing canada balsam. Addition of phenol specifically inverses and increases the anisotropy of collagen fibers, but not the other protein fibrils. These slices were studied with a Leitz-Ortoplan-Pol microscope. The light retardation (anisotropy) was measured with a Brace-Kohler rotary compensator in 580 nm light.19 Five serial sections for every specimen were used and the light retardation was measured in ten different areas of each section—thus, every date point of patients represented a mean value of 50 measurements.

Statistical Analysis For the study, the relationship between age and anisotropy of the ECM components was analyzed using a simple linear regression model, and Fisher’s transformation was used for the calculation of correlation coefficients.

Results Alterations of the RPE Numerous lipofuscin granules can be seen in the RPE—some of them are fused with melanosomes forming melanolipofuscin granules. Mitochondria in the RPE show focal loss of cristae and increased translucency of matrix. Some of the mitochondria show normal structure. Several microsomes and peroxisomes of various

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density, shape, and size are distributed randomly in the cytoplasm of RPE, even among the lipofuscin granules.

Bruch’s Membrane Changes Most of the mitochondria show focal loss of cristae, and a decrease in matrix density. Numerous peroxisomes and single membrane-bound, empty vacuoles can also be seen. In the upper part of the RPE, accumulation of lipofuscin and melanolipofuscin can be observed. The basement membrane of RPE is slightly thickened. Electrodens fibrillo-granular material and several small profiles of vacuoles can be seen in the Bruch’s membrane.

Age-related Changes in the Macular Region The Drusen is located just beneath the RPE basement membrane, it contains electrodense granules, various forms of membrane-like structures, and amorphous material. A few electrodense granules can also be seen in the collagenous layers of the Brush’s membrane. The RPE cell contains some small mitochondria with normal appearance, but some of them show a seriously altered structure.

Basement Membrane of RPE The basement membrane shows several focal, or worth-like thickenings with electrodense areas. The inner collagenous layer of Bruch’s membrane contains numerous profiles of vacuoles and patches of fibrillo-granular material. The elastic layer is interrupted, while the outer collagenous layer shows normal appearance in this place. Cytoplasm of the RPE contains numerous abnormal mitochondria—characterized by loss of cristae and translucent matrix. Several microsomes and peroxisomes can also be seen.

Thicking of Capillary Basement Thickening of the capillary basement was due to the addition of mostly homogeneous material, a few fibrils, and electrodense granular material

Peroxisomal Contribution to Basement Membrane Thickening of RPE Typical thickening of the Bruch’s membrane due to the addition of abnormal basement membrane material to the basement membrane of RPE. The inner collagenous layer of Bruch’s membrane contains numerous small profiles of vacuoles, and patches of

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fibrillo-granular material. Almost all mitochondria of the RPE show focal electrontranslucent areas of the matrix and loss of cristae. Elastic layer, interposed between inner and outer collagenous layers, shows various thickness and electron density, as well as several interruptions. In a transversal section, the abnormal basement membrane forms numerous worth-like deposits in which cytoplasmic processes of the basal cell membrane can be seen. In an oblique section of the Brach’s membrane, numerous peroxisomes can be seen in the basal laminal deposits and in the RPE. Cytoplasmic processes of RPE form labyrinths between basal laminal deposits. Filamentary structure of abnormal basement membrane can be observed in longitudinal section and/or cross-section of abnormal basement membrane with exocytosis of vesicles.

Histochemical Composition of Bruch’s Membrane PAS positive staining of Bruch’s membrane. In some places the pericapillary connective tissue also shows strong Anisotropy of the collagen fibrils in the Bruch’s membrane as shown by phenol reaction: basement membrane of the choriocapillaries shows more intensive anisotropy compared to RPE. Anisotropy of the lipids in the Bruch’s membrane and in the pericapillary wall are bright in the dark background. The basement membrane of RPE and choriocapillaries show intensive anisotropy after ABT reaction due to presence of vi.cinal OH groups in linear order.

Statistical Analysis of Anisotropy Statistical analysis showed that aging and AMD affect basement membrane composition differently. The averaged values (in nm) of the anisotropy of collagen, GFs, and lipids are summarized in the following table, along with the statistical significance of comparisons between controls and patients with AMD (see Table 15.1). The relationships between age and the anisotropy of the ECM components were analyzed using the simple linear regression model. The estimated equations are reported in the following table in which the coefficients of determination R2 and the correlation coefficients r between age and the anisotropy of the observed substances are also indicated (see Table 15.2). Using the Fisher’s transformation, the correlation coefficients calculated in the two groups were compared. As concerns the anisotropy of

Table 15.1 Collagen, glicans and lipids in the basament membranes of normal and AMD eyes structures Mean ± Mean ± 95% C.I. of sd AMD sd Control t-value P-Value Mean Diff Collagen 28.0 ± 4.4 25.1 ± 3.2 2.88 GFs 33.8 ± 3.9 28.1 ± 2.6 5.73 Lipid 16.2 + 5.6 9.88 ± 2.5 6.31 AMD Group (n = 26); CONTROL Group (n = 26).

2.68 (P < 0.01) 6.18 (P < 0.0001) 6.31 (P < 0.0001

0.72 - 5.05 3.87 - 7.59 3.91 - 8.71

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Table 15.2 Statistical analysis of anisotropy in normal and AMD eyes structures r Control Group(26) R2 AMD Group (26) R2 Collagen y = −9.42 ± 0.50 0.67 0.82 GFs y = −1.13 ± 0.44 0.66 0.81 Lipid y = −39.8 ± 0.75 0.95 0.98 AMD Group (n = 26); CONTROL Group (n = 26).

y=−4.85 ± 0.40 y=−7.51 ± 0.27 age y = −11.7 t 0.29 age

0.81 0.57 0.72

R 0.90 0.76 0.85

collagen and GFs, no statistical significance was found. On the other hand, the coefficients of correlation between lipids and age were found significantly different (P < 0.001) in the two groups—they were higher in the AMD group.

Discussion Photoreceptor cells have a very high turnover rate, probably the highest among neuronal cells. It comprises a continuous shedding of the apical plasma membranes (discs) of the photoreceptors’ outer segment (POS) and their renewal in the cell body. Each RPE cell opposes around 24-44 POS, and 10-15 percent of each outer segment is renewed daily—thus, each RPE cell consumes and degrades about 20,004,000 discs per day.21 This phagocytosis is mediated by CD36 receptors of RPE that have developed adequate cellular systems to metabolize this enormous quantity of membranes.22 Lipid components of disc membranes may have two distinct metabolic fates—retinol and docosahexaenoic acids are recycled to photoreceptor cells through the interphotoreceptor matrix.23 The rest of the lipids are further catabolized by mitochondrial beta oxidation. Recent studies show that RPE cells may also uptake low-density lipoprotein (LDL) from the choriocapillaries using a similar receptor mediated pathway.24 These plasma lipids enter the POS turnover and are either used for the renewal of disc membranes or catabolized by mitochondrial beta oxidation.25 An accumulation of residual bodies or lipofuscin granules in the cytoplasm of RPE cells are common findings, and it is thought to be a sign of incomplete catabolism of POS disc membranes. Lipofuscin is a heterogeneous material composed of a mixture of lipids low-density lipoprotein (LDL) particularly lipid peroxides, proteins, and different fluorescent compounds derived mainly from vitamin A. Lipofuscin granules appear at the early26 age of 18 months, and until the age of 80, their amount increases approximately twenty fold.27 However, recent studies have shown that lipofuscin accumulation is not an end stage of failed lipid metabolism—instead, it is a manifestation of the current balance between production and elimination of lipid peroxides.28 There is a considerable body of evidence that well-established detoxifying mechanisms exist in RPE to eliminate lipid peroxides. Besides the common cytosolic antioxidant substances (glutathion, ascorbat, and alpha tocopherol), certain subcellular structures (mitochondria, peroxisomes, and melanosomes) and mechanism (exocytosis) serve to maintain RPE homeostasis.

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First, accumulation of lipofuscin in RPE cells greatly reduces their phagocytic capacity in vitro.29. If this negative feedback works in vivo, it may be an important protective mechanism to prevent lipid overload of RPE. Accumulation of empty vacuoles in the Bruch’s membrane, which are cholesterols and cholesterol esters, may be a sign of arrested lipid uptake from choriocapillaries.9 Second, melanosomes of the RPE are connected to the phagolysosomal degradation pathway and are involved in many important functions, such as protection from photo-stress and detoxification of peroxides. The lipofuscin content of RPE seems to be inversely related to the melanin content, which suggests that melanin may have a protective role in preventing lipofuscin formation. Third, through radiation and by directly scavenging free radicals,30 early histological studies show that RPE appears to discharge partially degraded membrane material from their basal region toward Bruch’s membrane and choriocapillaries. This mechanism seems to be part of the lipid transportation into the choriocapillaris, as shown by experimental studies.5,31 Finally, mitochondrial beta oxidation is considered to be the major pathway metabolizing fatty acids.32 High mitochondrial enzyme-activity was found in the RPE.33 Currently, no in vivo date on the saturation threshold of mitochondrial beta oxidation in RPE. However, the unmetabolized lipid peroxides certainly can compromise mitochondrial functions. Particularly inner membranes, where electron transport enzymes and mitochondrial DNA (mtDNA) are located, are sensible for oxidative damage.34 Lipofuscin granules of the RPE cells, which are toxic to these cells, probably act through mitochondrial damage, thus supporting a role for lipofuscin in aging and AMD.35 Our observations, the first time, showed morphological alterations of mitochondria in early AMD. Focal loss of cristae, associated with focal decrease of electron density of matrix, were common findings. Sometimes, more extensive alterations of cristae and matrix were seen, but no early apoptotic alterations (swelling, blebs of external membrane) were found. We considered these mitochondrial abnormalities, besides infra- and extracellular accumulation of partially metabolized lipids, to be morphological manifestations of saturated lipid catabolism in RPE—due to lipid overload either from photoreceptor shedding or from the plasma. Peroxisomes are small, round or oval single membrane-bound organelles, and are found in almost all eukaryota cells. Peroxisomes derive from endoplasmic reticulum, and their functions are related to lipid metabolism. They contribute to the beta oxidation of fatty acids. This pathway is essential for the catabolism of a variety of substances that are not oxidized by mitochondria. Peroxisomes are also involved in the metabolism of hydrogen peroxide. Finally, peroxisomes catalyze the initial steps in the biosynthesis of some lipids, such as plasmalogens. A peculiar characteristic of peroxisomes is their inducibility by a variety of compounds, which can activate a new member of the nuclear receptor superfamily called peroxisome proliferator-activated receptors (PPARs). The ligand-activated PPARs heterodimerize with the 9-cis retinoic acid receptor (RXR) and the complex bind-to-response elements located in the regulatory region of target genes. Intracellular elevation of naturally occurring fatty acids and eicosanoids activate PPARs, resulting in peroxisome proliferation and activation of lipid metabolism. PPAR alpha up-regulates genes of

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lipid catabolism, while PPAR gamma up-regulates genes of lipogenesis and enhances intracellular lipid storage.36 Lipid peroxides, particularly oxidized LDL, can also activate37 PPAR gamma. Our observations confirmed the abundance of peroxisomes in the RPE in both normal aging and in AMD. The functional interpretation of these finding suggested that peroxisomes may be involved in the lipid turnover of POS. We suppose that presence of peroxisomes in RPE may be a morphologic manifestation of the activation of an alternative pathway for lipid degradation. This hypothesis is supported by observations that the induction of peroxisome proliferation is associated with a strong stimulation of the enzymes involved in peroxisomal beta oxidation.36 Recently, similar results were described in the RPE indicating peroxisomal contribution to POS turnover.38 Basal laminar deposits appear as diffuse or worth-like thickening of the basal membrane of RPE and choriocapillaries. In contrast to the normal basement membrane, these thickenings usually show fibrillary structure with focal electrodensity.2,39 Immunohisto-chemical studies show that BLD contains type IV collagen, heparan sulfate proteoglycans, and laminin and it is now considered to be an accumulation of an abnormal basement membrane.14 Our electron and polarization microscopic studies confirmed both fibrillary structures and the highly glycated nature of BLD, and we suggested that these structures be denominated as glycated fibrils(GFs) to distinguish from the normal basement membrane constituents, which usually do not show fibrillary appearance. In addition, polarization microscopy clearly showed a lipid layer just beneath the RPE and in the wall of choriocapillaries, suggesting that lipids are also added to the excessive basement membrane. Furthermore, these lipids, at least in part, are in organized form as structure lipids. Their hydrocarbon chains are oriented perpendicular to the length of basement membrane. Similar deposition of lipids were found in the adult human Descemet’s membrane, which is the basement membrane of the corneal endothelium.17 This phenomena was particularly evident at the periphery of aged corneas.40 All these findings justify an assumption that aging of the basement membrane is associated with deposition of structure lipids in it. Our observations showed an unexpected topographic correlation between peroxisomes and basement membrane alterations in AMD. At the basal region of RPE cells, peroxisomes seem to fuse with basal plasma membranes and extrude their content into basement membrane. These peroxisome-derived materials showed a high degree of electron density similar to those of the lipid peroxides in the peroxisomes. It was recently demonstrated that Bruch’s membranes from white donors aged 40-78 years contain significant amounts of lipids and lipid peroxides.7,8 These authors suggested that lipid peroxides may accumulate within Bruch’s membrane by a combination of both discharge from RPE cells and from oxidized LDL coming from the plasma. Our observations showed a third possibility that lipid peroxides may also come from peroxisomes of the RPE cells. These observations suggested that proliferation of peroxisomes may also be associated with deposition of lipids or lipid peroxides in the ECM. This may be a new feature of peroxisome functions. The colocalization of GFs and structure lipids remains an intriguing question. Two mechanisms may be involved in this procedure. There is accumulating evidence that intracellular lipid peroxides may also influence production of proteoglycans

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and their extracellular deposition. High-cholesterol diets for rats resulted in thickening of the endothelial basement membrane of the choriocapillaries.41,42 Exposure of human arterial smooth muscle cells to linoleic acid increased 2-10 times the expression of mRNA for the core proteins of proteoglycans (versican, decorin, and syndecan) compared to control cells. Darglitazone—a PPARgamma ligand—neutralizes the linoleic acid induction of the decorin gene. This suggests that some of the linoleic acid effects are mediated by PPAR gamma.43 Intracellular accumulation of lipid peroxides may also enhance production of advanced glycation end products (AGE).44,45 Three stages can be distinguished during the glycation process: 1) nonenzymatic glycation of proteins (formation of Amadori product); 2) glycoxidation reactions; and 3) fibril formation of AGEs. Recent data46 suggests that lipid peroxides play an essential role in glycoxidation in aging, atherosclerosis, and diabetes . Biochemical and immunohisto-chemical studies show a linear age-dependent increase in AGEs in human Bruch’s membranes, basal membranes, and Drusen; in choroidal extracellular tissue in both diabetic and nondiabetic eyes, and in surgically removed subretinal membranes of patients with AMD.47,48,49 Vitronectin—a multifunctional glycoprotein and a major constituent of Bruch’ membrane thickenings—is synthesized by RPE.50 It is of interest that AGEs colocalizing with vitronectin were detected in diabetic retina,51 suggesting that GFs (seen in basement membranes) may also contain both glycoprotein and AGEs. However, colocalization of lipid peroxides and GFs, in the abnormal basement membrane of RPE and choriocapillaries, may represent a convergence between structure lipids and GFs synthesis—both of them are regulated by nuclear receptor activation and performed by endoplasmic reticulum-derived organelles. Whether the extracellular deposition of structure lipids and GFs represents a glycolipid and/or glycoxilipid formation, and whether these structures are formed inta- or extracellularly, remains to be determined. Polarization microscopy of Bruch’s membrane revealed that all basement membrane components (collagen, GFs, and lipid) increased with age in both control and AMD retinas. The anisotropy of each components was higher in AMD compared to the control group (see Table 15.1). The simple linear model is adequate to explain the dependence of anisotropy on age in both groups. Aging influences collagen and GFs in the same manner—in both AMD and the control group—while anisotropy of lipids in AMD increases at a much higher rate (see Table 15.2, correlation coefficient 0.98 vs 0172). It is of interest that the difference between aging and AMD was also significant for GFs, but over 75 years saturation or even decline of GF-induced anisotropy was found in AMD but not in the control group. Based on these observations, we may conclude that one of the significant differences between aging and AMD is manifested by more marked deposition of lipids and GFs in the ECM Current concepts on aging and age-related diseases asssigned a primary role to mutation of mtDNA due to cumulative effect of reactive oxigen species.52 An impaired mitochondrial function results in further generation of reactive species and subsequent oxidative damage to the mitochondria itself and to other cellular or extracellular constituents. Besides reduced ATP generation, which affects almost all cell functions, activation of caspase cascade for cell death may take place.18

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Taking our electron microscopic and polarization microscopic findings together, we suppose that mitochondria may be involved in the activation of an alternative pathway for ECM pathology. By-products of mitochondrial lipid metabolism (lipid peroxides, oxiLDL, and eicosanoids), in addition to the ortodox peroxisomal functions, may also activate peroxisomes to deposit abnormal basement membrane material. Thus, the peroxisomal contribution to basement membrane deposition may be one of the pathophysiologic links between mitochondrial and ECM pathology. Both are common in aging, in age-related diseases, and in several other diseases. Further studies are certainly needed to verify this hypothesis.

Conclusions Morphological evidences suggested that a) mitochondria and peroxisomes of RPE contribute to the abnormal lipid metabolism of photoreceptor outer segments, and b) peroxisomes may be involved in the deposition of abnormal basement membrane material. Thus, extracellular matrix alterations, besides being due to entrapped abnormal metabolites (lipid peroxides, free radicals), may also come from activation of peroxisomes. Contribution of peroxisomes to the extracellular matrix formation, this new function of peroxisomes, may be a pathophysiologic link between mitochondrial and extracellular matrix abnormalities seen in aging, age-related neurodegenerative diseases, atherosclerosis, and diabetic microangiopathy. Acknowledgements The authors thank Ida Bozso for her excellent technical assistance, and Livia Feher and Alessandro Mariani for their contribution to this chapter.

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35. Winkler BS, Boulton ME, Gottsch JD, Stemberg P (1999) Oxidative damage and age-related macular degeneration. Mol Vis 5:32 36. Kersten S, Desvergne B, Wahli W (2000) Roles of PPARs in heath and disease. Nature 405:4214 37. Nagy L, Tontonoz P, Alvarez JGA, Chen H, Evans RM (1998) Oxidized LDL regulates macrophage gene expression through ligand activation of PPAR-gamma. Cell 93:22940 38. Ershov AV, Bazan NG (2000) Photoreceptor phagocytosis selectively activates PPARgamma expression in retinal pigment epithelial cells. J Neurosci Res 60:328-37 39. Young RW (1987) Pathophysiology of age-related macular degeneration. Sury Ophthalmol 31:291-306 40. François J, Feher J (1973) Arcus senilis. Doc. Ophthalmol 34:165-82 41. Tokura T, Ito S, Nishikawa M, Yamane A, Miki H. (1999) Changes in Bruch’s membrane in experimental hypercholesteremia in rats. Nippon Ganka Gakkai Zasshi 203:85-91 42. Miceli MV, Newsome DA, Tate DJ, Sarohie TG (2000) Pathologic changes in the retinal pigment epithelium and Bruch’s membrane of fat-fed atherogenic mice. Current Eye Res. 20:8-16 43. Olsson U, Bondjers G, Camejo G (1999) Fatty acids modulate the composition of extracellular matrix in cultured human arterial smooth muscle cells by altering the expression of genes for proteoglycan core proteins. Diabetes 48:616-22 44. Nerlich AG, Schleicher ED (1999) N(epsilon)-(carboxymethyl)lysine in atherosclerotic vascular lesion as a marker for local oxidative stress. Atherosclerosis 144:41-7 45. Oak J, Nakagawa K, Miyazawa T (2000) Synthetically prepared Amadori-glycated phosphatidylethanol-amine can trigger lipid peroxidation via free radical resction. FEBS Lett 481:26-30 46. Fu MX, Requena JR, Jenkins AJ, Lyons TJ, Baynes JW, Thorpe SR (1996) The advanced glycation end product, Nepsilon-(carboxymethyl)lysisne, is a product of both lipid peroxidation and glycoxidation reaction. J Biol Chem 27:9982-6 47. Ishibashi T, Murata T, Hangai M, Nagai R, Horiuchi S, Lopez PF, Hinton DR, Ryan SJ (1998) Advanced glycation end products in age-related macular degeneration. Arch Ophthalmol 116:1629-32 48. Handa JT, Verzijl N, Matsunaga H, Aotaki-Keen A, Lutty GA, Koeppele JM, Miyata T, Hjelmeland LM (1999) Increase in advanced glycation end product pentosidine in Bruch’s membrane with age. Invest Ophthalmol Vis Sci 40:755-9 49. Hammes HP, Hoerauf H, Alt A, Schleicher E, Clausen JT, Bretzel RG, Laqua H (1999) N(ep silon)(carboxymethyl)lysin and AGE receptor RAGE colonize in age-related macular degeneration. Invest Ophthalmol Vis S6 40:1855-9 50. Hagemann GS, Mullins RF, Russell SR, Johnson LV, Anderson DH (1999) Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells. FESEB J. 13:477-84 51. Hammes HP, Weiss A, Hess S, Araki N, Horiuchi S, Brownlee M, Preissner KT (1998) Modification of vitronectin by advanced glycation alters functional properties in vitro and in the diabetic retina. Lab Invest. 75:325-38 52. Ozawa T (1997) Genetic and functional changes in mitochondria associated with aging. Physiol Rev. 77: 425-64

Chapter 16

Non-Exudative Macular Degeneration and Management Thomas R. Friberg, MS, PhD and Kenneth T. Wals, MD

Abstract Non-exudative, age-related macular degeneration, which accounts for 85 percent of AMD cases, includes a spectrum of macular pathology. In its most mild form, the macula possesses few Drusen or retinal pigmentary epithelial (RPE) change with no visual disability. At the other end of this spectrum, the macula is affected by large areas of RPE atrophy, with devastating loss of central vision. This chapter examines normal and pathologic changes in the macular leading to non-exudative macular degeneration, and reviews the clinical trials that have been performed on patients with this disease. Keywords macular degeneration, Drusen, AREDS, PTAMD, CAPT, CNVPT, rheophoresis, MIRA, laser, AMD

Introduction Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the United States.1 AMD affects approximately 8 million Americans2 and tens of millions worldwide. As the world population continues to age, the number of people with this disease will rise dramatically. Non-exudative age-related macular degeneration, which accounts for 85 percent of AMD cases, includes a spectrum of macular pathology. In its most mild form, the macula possesses few Drusen or retinal pigmentary epithelial (RPE) change with no visual disability. At the other end of this spectrum, the macula is affected by large areas of retinal pigment epithelial atrophy, with devastating loss of central vision.

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Normal Aging Changes The macula undergoes an array of changes with aging that affect the photoreceptors, the RPE, and Bruch’s membrane. Photoreceptors become reduced in both density and distribution. The RPE cells accumulate lipofuscin granules, also called residual bodies. Within Bruch’s membrane, basal laminar deposits—long-spacing collagen located between the basal lamina of the RPE cell and the inner aspect of the basement membrane of the RPE—accumulate.

Non-neovascular Abnormalities in AMD The lipofuscin-laden RPE cells that degenerate with age may be phagocytosized by neighboring RPE cells. If sufficient in number, they become visible in the fundus as a diffuse mottling of small pigment clumps. In addition to the basal laminar deposits described above, basal linear deposits—phospholipid vesicles and electron-dense granules within the inner aspect of Bruch’s membrane—develop. These two deposits comprise Drusen, which are visible as round, dull yellow lesions lying deep in the retina. Drusen are believed to be by-products of photoreceptor metabolism, related to a decreased ability of the eye to recycle outer segment debris. This may be a consequence of alterations or a reduction of enzymes that are intrinsic to this recycling. Drusen are generally categorized into hard and soft types, with a number of subtypes. Hard Drusen, which appear discreet and well demarcated, consist of globular deposits of hyalinized material within Bruch’s membrane. Soft Drusen are usually larger and have indistinct margins, and consist of both basal laminar and basal linear deposits. Ophthalmoscopically, soft Drusen possess an obvious thickness and tend to become confluent, and therefore vary more in both size and shape. Further confluence of Drusen leads to an appearance that resembles a pigment epithelial detachment (PED), and is termed Drusenoid PED. Cuticular Drusen are round small or large Drusen that are usually innumerable and homogeneous, and are more apparent on angiography with a characteristic starry night appearance. Drusen size can be approximated by noting that the width of a major vein as it appears at the edge of the optic disc is 125 µm. Small Drusen are defined as less than ½ a vein width (63 µm), and are considered to be hard.3 This vague definition, however, is a morphologic one and has little relevance. Drusen greater than 125 µm are large, and these are considered to be soft unless they are in the process of regressing. Drusen between 63 µm and 125 µm (intermediate) can be either hard or soft. Geographic atrophy is a sharply delineated round or oval area of hypopigmentation or depigmentation, or apparent absence of the RPE, in which choroidal vessels are more visible than in surrounding areas. Most cases of geographic atrophy develop in eyes with prominent Drusen or Drusenoid PED’s as the Drusen material regresses. Less commonly, atrophy occurs unrelated to the location of individual

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Drusen. In this case, the atrophy typically begins around the perimeter of the fovea in a band of microreticular hyperpigmentation.

Stages of Non-Exudative AMD Different grading schemes have been used in various studies for both non-exudative and exudative AMD. All of these have limitations, though, and can be misleading. By describing varying degrees of AMD in stages, these studies imply that earlier stages of AMD inexorably progress to more advanced stages. This is not true. In fact, many patients who possess only Drusen continue with a stable ophthalmic exam for the rest of their lives. However, the presence of Drusen does represent a risk for the development of visual loss and neovascular AMD, particularly if the Drusen are large and numerous.4 In general, early non-exudative AMD is defined as the presence of several medium-sized Drusen with either RPE hyper- or hypopigmentation. Patients usually have no visual complaints (see Fig. 16.1). Intermediate non-exudative AMD consists of numerous medium and/or large Drusen. Visual acuity is typically normal, and these patients may be asymptomatic, although they commonly complain of nyctalopia (see Fig. 16.2). Advanced non-exudative AMD is defined as geographic atrophy. As long as the area of geographic atrophy does not involve the fovea, visual acuity generally remains normal, although patients may complain of nyctalopia or difficulty reading a line of print. Once geographic atrophy involves fixation, visual acuity typically drops to 20/200 or worse (Fig. 16.3).

Fig. 16.1 Category 1 – Few or No Drusen

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Fig. 16.2 Category 2 – Intermediate Size Drusen

Fig. 16.3 Category 3 – Large Drusen

Management of Non-Exudative AMD: Clinical Trials Several multi-centered randomized controlled trials have been performed on subjects who possess non-exudative AMD. Because the presence of Drusen increases the risk of developing choroidal neovascularization (CNV) or visual loss, it was postulated that if Drusen could be induced to resorb, the CNV event rate of those eyes would be altered. Hence, Drusen reduction might have

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a prophylactic benefit. In addition, if Drusen were prominent in the fovea, such a reduction might also have therapeutic implications and allow visual improvement. This rational was the impetus for several of the studies discussed below.

Age-Related Eye Disease Study (AREDS)6 This pivotal trial evaluated the effect of high-dose vitamins C and E, beta-carotene, and zinc supplements on the progression of AMD and visual acuity. Subjects were randomly assigned to receive daily tablets containing antioxidants (500 mg vitamin C, 400IU vitamin E, 15 mg beta carotene), 80 mg zinc oxide with 2 mg copper, antioxidants plus zinc, or placebos. Copper was added to prevent zinc-induced anemia. More than 4,000 subjects were involved and were followed for five years or more. Subjects with high-risk non-exudative AMD—defined as extensive intermediate-size Drusen, at least 1 large Drusen, noncentral geographic atrophy, or advanced AMD in at least one eye—benefited from supplementation with antioxidants plus zinc. Individuals in the group assigned to the combination supplement had a 25 percent reduction of risk for progression to advanced AMD, and a 19 percent risk reduction in the rate of moderate vision loss (≥ 3 lines of visual acuity) after five years. The AREDS trial substantiated the benefit of vitamin and mineral therapy in patients with non-exudative AMD.

Complications of Age-Related Macular Degeneration Prevention Trial (CAPT)8 CAPT evaluated the effectiveness and safety of low-intensity grid treament with 532 nm argon laser in the prevention of visual acuity loss among participants with bilateral large Drusen. Participants were followed for five years. After five years, there was no difference in visual acuity between treated and observed eyes (20.5% losing at least three lines in both groups). There also was no difference between groups for the development of choroidal neovascularization (13.3% in both groups).

Prophylactic Treatment of Age-Related Macular Degeneration (PTAMD)7 This study sought to determine whether a grid of mild subthreshold, 810 nm diode laser would have a prophylactic benefit on preventing non-exudative AMD from progressing to the neovascular form, and if laser treatment would improve the

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long-term visual acuity compared with the natural history of the disease. The treatment regimen differed from the CNVPT and CAPT (see below) studies in two important ways. The first was that the laser applications were intentionally very mild, so that the spots could not be seen directly upon placement (subthreshold). (see Fig. 16.4.) The second was that no retreatments were allowed. The PTAMD study was divided into two arms—patients with multiple large Drusen in one eye and neovascular AMD in the other, and patients with multiple large Drusen in both eyes. Participants were followed for three years. In the unilaterally eligible group, laser treatment placed the patients at higher risk of developing neovascular AMD than with observation alone (15.8% vs. 1.4% after 1 year), without significant difference in visual acuity loss. In the bilaterally eligible group, diode laser treatment had no effect on the development of neovascular AMD (11% vs. 9%), and had a negligible effect on visual acuity (mean improvement of 1.5 letters compared to the observation group). A subset of patients whose vision was impaired at study entry did, however, have a modest benefit in visual acuity, greater than the mean of 1.5 letters.

Choroidal Neovascularization Prevention Trial (CNVPT)5 This study aimed to describe the short-term effects of low-intensity 532 nm argon laser treatment to eyes with Drusen that were at risk of developing choroidal neovascularization. The study consisted of two arms—the Bilateral Drusen Study and the Fellow Eye Study. The Bilateral Drusen Study included patients with non-exudative AMD with more than 10 medium or large Drusen in each eye. The Fellow Eye Study included patients with one eye as described above and one eye with exudative AMD. The treatment consisted of grid laser within the macula that spared the foveal avascular zone. Retreatment was applied whenever the area of Drusen had not been reduced by 50 percent or more. Subjects were followed for 18 months.

Fig. 16.4 PTAMD – Annular grid pattern of subthreshold laser

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In both arms of the study, the area of Drusen decreased with laser treatment. Although the incidence of CNV was negligibly increased in the treatment group versus the observation group in the Bilateral Drusen Study (2.6% vs. 1.3%), the rate of CNV was higher in the Fellow Eye Study (16.9% vs. 3.3%). Visual acuity remained similar between both treatment and observation groups in the Bilateral Drusen Study, but was worse in the treatment group of the Fellow Eye Study. Results from the Bilateral Drusen Study laid the foundation for the Complications of Age-Related Macular Degeneration Prevention Trial (CAPT), which will be described later.

Multi-center Investigation of Rheophoresis I (MIRA I)9 Rheophoresis, a form of double-filtered plasmaphoresis, is an extracorporeal blood filtration procedure, and an established method for reducing certain molecules in plasma. The rationale for its use for AMD is based on the potential to modify the diffusion through Bruch’s membrane, improve microcirculation, and change the complement cascade. MIRA I failed to demonstrate a significant difference in visual acuity between a treatment and placebo group at 12 months. A gain of 0.8 lines was only demonstrated when post-hoc analysis removed any participants who had cataract surgery, cataract progression, laser capsulotomy, fewer than four rheophoresis treatments, or central geographic atrophy. A proposal for an additional Phase III clinical trial has been submitted to the FDA, and this new trial is anticipated to be launched in 2007.

Age-related Eye Disease Study II (AREDS II) AREDS II will evaluate supplements of macular xanthophylls (lutein and zeaxanthin) and omega-3 long-chain polyunsaturated fatty acids (docosahexaenoic acid and eicosapentaenoic acid) on the development of neovascular AMD and geographic atrophy, and is currently enrolling participants. Although lutein is widely advocated as an eye supplement, it was unavailable in sufficient commercial quantities to be studied in AREDS I.

Conclusion Non-exudative AMD is a multi-factorial disease that will undoubtedly be seen in increasing numbers as the world population continues to age. Non-exudative AMD includes a wide spectrum of macular changes, with a similarly wide spectrum of

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effects on vision. Laser treatment and rheophoresis have proven ineffective to-date for preventing the progression of non-exudative AMD. Currently, the only proven treatment is with high-dose antioxidants and zinc, which has shown modest benefits. The results of AREDS II will hopefully shed new light on treatment modalities for this common and potentially devastating disease.

References 1. National Advisory Eye Council (1993) Vision research: a national plan 1994-1998. NIH publication no. 93-3186. US Department of Health and Human Services, Bethesda, MD 2. Age-Related Eye Disease Study Research Group. (2003) Potential public health impact of Age-Related Eye Disease Study results: AREDS report no. 11. Arch Opthhalmol 121:1621-24 3. Klein R, Klein BEK, Linton KLP (1992) Prevalence of age-related maculopathy: the Beaver Dam Eye Study. Ophthalmol 99:933-43 4. Bressler SB, Maguire MD, Bressler NB, Fine SL (1990) Relationship of Drusen and abnormalities of the retinal pigment epithelium to the prognosis of neovascular macular degeneration. Arch Ophthalmol 108:1442-7 5. Age-Related Eye Disease Study Research Group. (2001) A randomized, placebo-controlled clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss: AREDS report no. 8. Arch Ophthalmol 119(10):1417-36 6. Complications of Age-Related Macular Degeneration Prevention Trial Research Group. (2006) Laser treatment in patients with bilateral large Drusen. Ophthalmol 113:1974-86 7. Friberg TR, Musch, DC, Lim JI, et al. (2006) PTAMD Study Group. Prophylactic treatment of age-related macular degeneration report number 1: 810-nanometer laser to eyes with Drusen. Unilaterally eligible patients. Ophthalmol 113:612-22 8. Choroidal Neovascularization Prevention Trial Research Group. (1998) Laser treatment in eyes with large Drusen. Ophthalmol 105:11-23 9. Pulido JS, Sanders K, Klingel R (2005) Rheophoresis for age-related macular degeneration: clinical results and putative mechanism of action. Can J Ophthalmol 40(3):332-40

Chapter 17

Treatment of Intraocular Pressure in Elderly Patients Monika Schveoller, MD, Iliana Iliu, MD, Nicola Pescosolido, MD, and Angelica Cerulli, MD

Abstract The aim of this paper is to study the effect of many systemic antihypertension drugs on intraocular pressure and on the visual field. Six hundred patients were enrolled in this experiment with the approval of the Ethical Committee of our hospital. All patients were divided into four groups: the first group of 200 patients was treated with local or systemic administration of a calcium channel blocker; the second group of 200 patients was treated with oral or systemic administration of β-blockers; the third group of 100 patients was treated with systemic administration of ACE inhibitors; and the fourth group of 100 patients was treated with a diuretic drug (acetazolamide). All patients were subjected monthly to measurements of their systemic blood pressure, intraocular pressure, and visual field. Our results confirm that the oral administration of a calcium channel blocker (nitrendipina) in subjects with moderate essential hypertension and without ocular hypertonia causes systemic effects with a moderate decrease of ocular pressure, while the ocular instillation of the same drug causes a remarkable general hypotensive effect. The scotoma in glaucomatous subjects with normal pressure gets better after the administration of local calcium channel blockers, showing that the peripheral vascular reaction enhances the optical nerve blood flow. The oral administration of β-blockers is also correlated with a reduction of the intraocular pressure, especially if the β-blocker also reduces the systemic blood pressure. Nadolol, a long half-life, nonselective β-blocker, in a single oral dose of 20 or 40 mg, may result in a remarkable decrease of the intraocular pressure during the whole day. It has been demonstrated that the systemic administration of ACE inhibitors is also effective in reducing intraocular pressure by some mechanisms which, although not known yet, seem to act on the posterior ciliary arteries, shunting the blood to the ciliary body. Finally, acetazolamide, a diuretic usually used to reduce systemic blood pressure, is also able to reduce intraocular pressure. On the other hand, if perfusion pressure is reduced as a consequence of antihypertensive treatment, damage to the visual field could be accelerated. Keywords Low-tension glaucoma, open-angle glaucoma, antihypertension drug, calcium channel blockers, β-blockers, ACE inhibitors, diuretics.

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Introduction The relationship between the antihypertension drugs and ocular pressure has been well studied. In fact, the international literature (see PubMed [antihypertension drugs and ocular pressure]) contains more then 1537 articles on this field in the last 20 years. Although the etiology of increasing intraocular pressure (IOP) remains unknown, several local and systemic risk factors for the developing of this disease have been considered.1 Among ocular factors (and besides the IOP and the cup/disk ratio), severe myopia2 and presence of Krukemberg’s spindle3 are regarded as important etiologic factors for the development of an open angle primary glaucoma. Systemic pressure, athero-sclerosis, vasospasm, acute hypotension, diabetes mellitus, and blood dyscrasies are judged important systemic risk factors for the development of glaucoma (Goldeberg et al 1981). Arterial pressure has received specific attention because it seems to play both a protective and a harmful role for the survival of retinal ganglion cells. The hypothesis is that, in people younger than 60 years, when the small vessels are damaged by hypertension, high blood pressure enhances blood flow through a dynamic modification of the vessels’ diameter, so acting as a protective factor for ganglion cells and their axons. Later on, when small vessel damage is established, as in old age, flow resistance is increased with possible damage to the optical nerve’s head.4 It is important to notice that there is no linear relationship between systemic blood pressure and perfusion pressure (PP) because of the ocular vessels’ autoregulation.5 Using the echo-color-Doppler scan it is possible to demonstrate that retinal flow does not change despite a 41 percent increase of arterial pressure or an increase of ocular pressure that reduces the perfusion pressure by 50 percent.6 At the microstructural level, choroid vessels are richly innervated, and there are still doubts about the existence of an autoregulation system. The vessels of the optical nerve’s head have the same autoregulation capability as the retinal vessels, as demonstrated in several experiments on primates and cats.6 Other studies have shown a relationship between nontreated hypertension and hyperbaric glaucoma and found the same association7-9 when systolic blood pressure (SBP) was greater than 165 mmHg and diastolic blood pressure (DBP) was greater than 95 mmHg. For every 10 mmHg increase above those levels there is an increase of 0.23 and 0.24 respectively of the IOP. A 100 mmHg increase of blood pressure would cause only a 2 mmHg increase in the IOP. This relationship is stronger in people aged 55–69 years compared to people over 70 years. In the first group the relation seems to be stronger in women, whereas in the latter it seems to be stronger in men. The people treated with antihypertension drugs do not show an increase in the IOP.10 It seems so far that hypertension per se causes an increase in the IOP, but this relation is not found in treated patients. Studies on primary and secondary open angle glaucoma don’t find differences in the prevalence of diabetes and hypertension in cases and controls of the same age.11

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Tielsh and coworkers1 demonstrated that IOP is more related to low blood pressure than to hypertension. This finding was later confirmed by other studies.12-15 Moreover, it seems that the relation involves DBP; and the strongest relation between systemic blood pressure and chronic simple glaucoma (POAG) is found when DBP is lower than 50 mmHg. A further decrease in the perfusion pressure causes a further increase in the prevalence of POAG. Blood pressure is not the only risk factor for glaucoma, since only 28 percent of glaucoma patients have DBP lower than 50 mmHg. We can therefore say that data on the correlation between IOP and blood pressure are not conclusive. Some authors found this correlation only in normal pressure glaucoma.4,16,17 Normal pressure glaucoma is characterized by a progressive ipoplasia and atrophy of the optical nerve’s head with progressive visual field loss, similar to POAG but without ocular hypertonia.4,7 In this regard, a recent hypothesis is that normal pressure glaucoma depends on a reduced perfusion of the optical nerve’s head because of vascular diseases or other factors that can influence blood viscosity.18 Epidemiological studies on elderly patients with normal pressure glaucoma evidenced a higher incidence of cerebral ischemia and cardiovascular diseases; these data could explain the ischemic degeneration of the optical nerve.19 Further studies have confirmed the ischemic theory of normal pressure glaucoma, and have taken into account age, presence of diabetes, myocardial infarction, and hypertensive crisis.18,20,21,22 The difference between systemic pressure and intraocular pressure (perfusion pressure) seems much more important than blood pressure per se. Phelps and Corbett22 hypothesized a relation between hemicrania and normal pressure glaucoma, since hemicrania affects 86 percent of the elderly with normal pressure glaucoma, compared to 64 percent of elderly controls, and 95 percent of patients with ocular hypertonia. To some authors hemicrania and glaucoma could share the same pathogenesis, hemicrania being an ischemic disorder. In patients with normal pressure glaucoma, the reduction of diastolic values estimated through ophthalmodynamometry, with or without a decrease in the systolic pressure, suggests a role for perfusion pressure reduction of the optical nerve’s head in glaucomatous damage.23 Antihypertensive treatment could accelerate the loss of the visual field.24,25 In spite of all these researches, little attempt has been made to compare the different therapies through very frequent medical examinations. For these reasons we have performed the present experiments.

Material and Methods Six hundred patients were enrolled in this experiment. The Ethical Committee of our hospital gave their approval and the patients gave their written informed consent. All experiments were performed according to the guidelines of the Declaration

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of Helsinki and in conformity with the ARVO Statement on the Use of Human Subjects in Ophthalmic and Vision Research applied by all ethical committees. All patients were divided into four groups: the first group of 200 patients was treated with local or systemic administration of a calcium channel blocker; the second group of 200 patients was treated with oral or systemic administration of β-blockers; the third group of 100 patients was treated with systemic administration of ACE inhibitors; and the fourth group of 100 patients was treated with a diuretic drug (acetazolamide). All patients were subjected monthly to measurements of their systemic blood pressure, intraocular pressure, and visual field.

Results Our results are summarized in Table 17.1. The oral administration of a calcium channel blocker (nitrendipina) in subjects with moderate essential hypertension and without ocular hypertonia causes systemic effects with a moderate decrease of ocular pressure, while local instillation causes a remarkable hypotensive effect. The scotoma in glaucomatous subjects with normal pressure gets better after the administration of calcium channel blockers, showing that the peripheral vascular reaction enhances the optical nerve blood flow. The oral administration of β-blockers is also correlated with a reduction of the IOP, especially if the β-blocker reduces systemic blood pressure. Nadolol, a long half-life, nonselective β-blocker, in a single oral dose of 20 or 40 mg, may result in a remarkable decrease of the IOP during the whole day. It has been demonstrated that the systemic administration of ACE inhibitors is also effective in reducing the IOP by some mechanisms which, although not known yet, seem to act on the posterior ciliary arteries, shunting the blood to the ciliary body. Finally, acetazolamide, a diuretic usually used to reduce systemic blood pressure, can be used to reduce the IOP. On the other hand, if perfusion pressure is reduced as a consequence of hypertension treatment, the damage to the visual field could be accelerated.

Table 17.1 Values of arterial blood pressure, intraocular pressure, and visual field before and after a treatment of six months with an antihypertension drug Drug Administration Blood pressure Ocular pressure Visual Field No treatment NO 180/110 23 18 dB Ca++ channel blocker systemic 160/90 19 20 dB Ca++ channel blocker local 140/90 17 20 db Beta-Blocker systemic 170/90 20 22 dB Beta-Blocker local 160/80 18 24 dB ACE inhibitor systemic 160/90 20 21 dB Acetazolamide systemic 170/100 21 24 dB

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Discussion Among all possible risk factors for glaucoma, particular attention is given to systemic hypertension. Several studies have shown a relation between nontreated systemic hypertension and ocular hypertension, even if there is not a direct relationship between blood pressure and ocular perfusion pressure because of the autoregulation of the ocular vessels. Moreover, researchers have demonstrated a higher correlation between low diastolic blood pressure and intraocular pressure (IOP), with prevalence of a low pressure in chronic glaucoma. The influence of antihypertensive drugs on the IOP should not be underestimated, since these drugs can influence the progression of glaucoma damage. The vascular autoregulation is the vessels’ capability of keeping blood flow constant despite modification in PP. Retinal and choroid blood flow depends on perfusion pressure and vascular resistance, i.e., the vessels’ diameter. The PP can be considered as the difference between the mean arterial pressure (MAP) and the intraocular pressure (IOP). MAP is the sum of the diastolic pressure (DBP) and a third of the difference between systolic (SBP) and diastolic blood pressure: MAP = DBP + (SBP - DBP)/3. Vessel diameter depends on smooth cells’ contractility and the action of pericytes, which are in turn regulated by several factors, including neurotransmitters and systemic or local vasoactive substances such as endothelins and nitric oxide. At a microstuctural level, retinal arterioles have no precapillary sphincters and receive no innervation from the autonomic nervous system, although α and β adrenergic and angiotensine receptors have been found on pericytes. Retinal autoregulation is based on changes in resistance that are obtained through changes in contractility of retinal arterioles. It depends on metabolic (pCO2, pO2, pH …) and myogenic mechanisms. It seems clear, from what we said about the role of blood pressure as a risk factor for glaucoma, that the possible influence of antihypertensive drugs on the IOP and in the progression of the glaucomatous damage should not be underestimated. Hypertensive patients with glaucoma can consume drugs belonging to different classes. Calcium channel blockers, β-blockers, ACE inhibitors, and diuretics are of particular importance in cases of concomitant cardiovascular diseases. Since the early 1980s, experiments on animals and humans to estimate the efficacy of calcium channel blockers in glaucoma patients have been performed. In 1983 Monica et al26 demonstrated that, in patients with moderate hypertension27 and without ocular hypertonia, oral administration of nitrendipine 20 mg caused systemic effects (a reduction in peripheral resistance, cardiac output, and ejection fraction) as well as a significant, though moderate, decrease in intraocular pressure (despite the absence of a basal hypertonia).28 On the other hand, in 1988 Abelson29 didn’t confirm the different activity of oral and topical use of calcium channel blockers other studies had found.30 In fact, while in humans a dose of nitrendipine consumed per os has a hypotensive effect at the

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ocular level, a local instillation caused marked ocular hypertension. Abelson et al29 showed that an instillation of verapamil 1.25 mg/ml caused a significant reduction of the intraocular pressure lasting about 10 hours. The efficacy of topical medication was not dependent on cardiovascular changes, suggesting that the observed effects were not consequences of the systemic vasodilatation, as happens with the oral route. The hypotensive effect seemed to be linked to local dilatation of veins and arteries.31 Verapamil inhibits intracellular calcium uptake by inactivation of its ATP-dependent channels, located on the inner side of the cells’ membrane. This reduction of calcium entrance in muscular cells inhibits contraction, so causing vasodilation.32 A local reduction of blood pressure causes a reduction in aqueous production that induces hypotonia. Verapamil can also interfere with the calciumdependent gap junctions between pigmented and unpigmented cells of the ciliary epithelium, so altering permeability and inhibiting aqueous outflow.32 It seems clear so far that calcium has many effects on the aqueous dynamic. Among them, a hydrostatic component mediated by an effect on blood pressure and ciliary body perfusion, and an osmotic component caused by an effect on active secretion of sodium, calcium, and other ions from the ciliary epithelium. It is important to notice that different subclasses of calcium channel blockers can have different effects: for example, diltiazem doesn’t reduce intraocular pressure as do verapamil and nitrendipine, these sharing the same ocular effect despite different cardiovascular effects.33 The only difference between the last two drugs (that have a hypotensive effect when instilled) and diltiazem is that diltiazem doesn’t have a negative inotropic effect. How this is related to the lack of ocular hypotensive effects is not known. Beside these effects on ocular tone, calcium channel blockers can enhance the optical nerve’s blood flow, an effect that can be positive in patients with normal pressure glaucoma. In recent studies calcium channel blocker effects were evaluated in patients with normal pressure glaucoma and chronic simple glaucoma who were receiving calcium channel blockers for extraocular reasons. These studies are characterized by a long follow-up. Netland34 found a significant difference in the progression of visual field defects and in the optical nerve damage between subjects with normal pressure glaucoma, who had a better prognosis, and those with chronic simple glaucoma. The authors and Kitazawa suggested using calcium channel blockers, in patients with normal pressure glaucoma, to slow the progression of the campimetric damage. Patients with normal pressure glaucoma are more exposed to vascular damage of the optical nerve than those with chronic simple glaucoma. The alteration of the optical disk found in these patients is caused by an ischemic degeneration. However, it is not clear if ischemia is mechanically induced by the high intraocular pressure or by a primary vascular damage of the optical nerve. The presence of ischemic damages and the association to migraine and Raynaud’s phenomenon, as already noticed, suggest an associated or primary vascular anomaly. This is the reason why the use of calcium channel blockers could prevent vasospasm and enhance blood flow in the head of the optical nerve.22

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In a number of patients with defects of the visual field of unknown etiology, the history of very cold hands is rather characteristic. In these cases the study of finger circulation, through the topical cold test and the capillaroscopy, is advisable.19 These tests, inducing an intense vasospasm, will confirm the hypothesis that visual defects are related to a vasospastic syndrome. Glasser and Flammer35 noticed that visual field defects worsened as hands were dipped into cold water and scotoma improved after the administration of calcium channel blockers, so demonstrating that a peripheral vasculature reaction to the drug accompanied an increase in the optical nerve’s blood flow. To evaluate the effects of β-blockers on blood and ocular pressure, these drugs were used in patients with both systemic and ocular hypertension. When blood pressure was within normal values (SBP<160 mmHg and DBP< 90 mmHg) the IOP was significantly reduced,36 but when systemic hypertension did not respond to β-blockers, even the IOP was scarcely modified. Nadalol is a nonselective β-blocker that has no intrinsic adrenergic activity and no effect as a membrane stabilizer, four times stronger than propranolol, with the longest half-life (20–24 hours) among β-blockers. It is easily absorbed by the whole gastrointestinal tract, has its maximun effect in 3–4 hours and can be administered once daily.37,38 IOP reduction is dose-related: 20–40 mg/day per os can have a therapeutic role in glaucoma. The nadolol ocular hypotensive effect in normotensives is considerable for as long as 24 hours, at all dosages used. The reduction of the IOP at 24 hours with both dosages is lower than that observed after 3 hours following drug intake. This suggests that a single 20 mg or 40 mg dose of nadolol can completely block the adrenergic receptors for a 24-hour period. According to Williamson,39 20 mg of nadolol twice daily, instead of 40 mg once daily, could result in a substantial decrease of the IOP. Studies on β-blockers in chronic simple glaucoma have compared the effects of a single dose per os with those of a topical application of timolol twice daily.40 The absolute reduction of the IOP obtained with the oral route was the same as with the topical route of timolol. Higher doses, such as those used for systemic hypertension (80 mg) were not necessary. Nadolol efficacy was well maintained for rather a long period. The oral route for the treatment of glaucoma would be more suitable than the topical one in patients who already receive β-blockers for cardiovascular diseases and in those who, for any reason, cannot instill ocular drops. Using autoradiographic techniques, β2 receptors have been identified in the optical nerve’s head. Since stimulation of β2 receptors causes vasodilatation and their inhibition blocks vasodilatation, there is concern about the possibility that a prolonged use of β-blockers could have an ischemic effect on the ocular disk with a reduced vasodilatation response to tissue needs (altered autoregulation). The third antihypertensive class we will focus on are the ACE inhibitors. The presence of precursors and enzymes that are necessary for angiotensin II production in the eye suggests that this organ could have its own renin-angiotensin system.41,42,43 This hypothesis has physiologic and pathophysiologic implications. Kaufman and Barany44 demonstrated that angiotensin I increases the aqueous outflow. This in turn suggested that angiotensin II could be involved in IOP regulation,

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in that its inhibition could cause an IOP reduction. Costagliola et al45 demonstrated that captopril reduced the IOP in all patients examined: 10 controls; 10 hypertensives with normal IOP; 10 normotensives with open-angle glaucoma; and 10 hypertensives with open-angle glaucoma. In this study, hypertensive patients were selected to evaluate whether captopril modification of the IOP was mediated by a reduction of pressure in the episcleral veins and the consequent increase in trabecular outflow. The absence of a correlation between blood pressure and IOP makes it unlikely. Other mechanisms should explain this ocular hypotensive effect. Moreover, the optical nerve blood flow through the posterior ciliary arteries is sensible to angiotensin II.46 The angiotensin II vasoconstriction effect on ocular vasculature could divert blood to the ciliary body; this would implement its metabolic activities and, in the end, its aqueous production. The captopril-induced block of the octapeptide production would then reduce aqueous production with a consequent IOP reduction. Nonetheless, ACE inhibitors have other properties. For example, they enhance bradikinin cleavage that is involved in endogenous prostaglandin synthesis.47,48 Prostaglandins are powerful ocular hypotensive agents, and captopril effects on the IOP could be mediated by the increased production of endogenous prostaglandins that enhance the uveoscleral defluxion. Among diuretics, which are not commonly used to reduce blood pressure, acetazolamide, an inhibitor of the carbonic anhydrase, reduces the IOP interfering with aqueous production, presumably inhibiting the enzyme of the ciliary epithelium.49 From all we have said, it seems that systemic antihypertensive drugs have a modulatory effect on the IOP and then on the visual field. The IOP also depends, at least in part, on high blood pressure control. If the perfusion pressure is reduced by antihypertensive treatment, visual field loss can be accelerated.

References 1. Tielsch JM, Katz J, Sommers A, et al. (1995) Hypertension, perfusion pressure, and primary open-angle glaucoma. Arch. Ophthalmol.,113:216-221 2. Patel R and Abreau R (1970) Topica corticosteroid testing of myopic patients. Orient. Arch. Ophthalmol. 8:208-211 3. Becker B and Podos SM (1966) Krukemberg’s spindles and primary open angle glaucoma. Arch. Ophthalmol., 76:635-639 4. Goldeberg L, Hollowos FC, Lass MA, et al. (1981) Systemic factors in patients with low-tension glaucoma. Br. J. Ophthalmol. 65:56-62 5. Riva CE, Sinclair SH and Grunwald JE (1981) Autoregulation of retinal circulation in response to decrease of perfusion pressure. Invest. Ophthalmol. Vis. Sci. 21:34-38 6. Drance SM (1995) Update to glaucoma, ocular blood flow and drug treatment. Kugler (ed) 7. Leighton DA and Phillips CL (1972) Systemic blood pressure in open-angle glaucoma, low tension glaucoma, and the normal eye. Br. J. Ophthalmol. 56:447-453 8. Rouhiainen HJ and Terasvirta ME (1990) Hemodynamic variables in progressive and non progressive low tension glaucoma. Acta Opthalmol. 68:34-36 9. Dielemans I, Vingerling JR, Algra D, et al. (1995) Primary open-angle glaucoma, intraocular pressure, and systemic blood pressure in the general elderly population. The Rotterdam Study. Ophthalmology 102:54-60

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10. Bulpitt CJ, Hodes C and Everitt MG (1975) Intraocular pressure and systemic blood pressure in the elderly. Br. J. Ophthalmol. 59:717-729 11. Jonas J and Grundler A (1998) Prevalence of diabetes mellitus and arterial hypertension in primary and secondary open-angle glaucomas. Graefe’s Arch. Clin. Exp. Ophthalmol. 236:202-206 12. Kalm HA and Milton RC (1980) Alternative definitions of open-angle glaucoma effect on prevalence and associations in the Framingham Eye Study. Arch. Ophthalmol. 98:2172-2179 13. Klein BE and Klein R (1981) Intraocular pressure and cardiovascular risk factors. Arch. Ophthalmol. 99:837-839 14. Leske MC and Podgot MJ (1983) Intraocular pressure, cardiovascolar risk variables and visual field defects. Am. J. Ophthalmol. 118:280-287 15. Klein BEK, Klein R, Sponsel W, et al. (1992) Prevalence of glaucoma. The Beaver Dam Eye Study. Ophthalmology 99:1499-1504 16. Morgan RW and Drance SM (1975) Chronic open-angle glaucoma and ocular hypertension an epidemiology study. Br. J. Ophthalmol. 59:211-215 17. Katz J and Sommer A (1988) Risk factors for primary open angle glaucoma. Am. J. Prev. Med. 4:110-114 18. Carter CJ, Brooks DE, Doyle DL and Drance SM (1990) Investigation into a vascular etiology for low-tension glaucoma. Ophthalmology 97:49-55 19. Drance SM (1977) Is ischemia the villain in glaucomatous cupping and atrophy? In: Brockhurst RJ, Bonchoff SA, Hutchinson BJ and Lessel S (eds) Controversy in Ophthalmology. Kluger Philadelphia, 292-300 20. Spaeth GL (1975) Fluorescein angiography its contributions towards understanding the mecchanisms of visual loss in glaucoma. Trans. Am. Ophthalmol. Soc. 73:491-553 21. Chumbley LC and Brubaker RF (1976) Low-tension glaucoma. Am. J. Ophthalmol. 81:764-767 22. Phelps CD and Corbett JJ (1985) Migraine and low-tension glaucoma a case control study. Invest. Ophthalmol. Vis. Sci. 26:1105-1108 23. Sisler HA (1972) Comparative ophtalmodinamometry using scleral pressure, suction and corneal pressure units. Am. J. Ophthalmol. 74:964-966 24. Demmailly P, Aubuer G and Abadie P (1987) Timolol and functional perimetric prognosis of primary open angle glaucoma. J. Fr. Ophtalmol. 71:766-771 25. Kaiser HJ, Flammer J, Stumplig D and Hendrickson P (1994) Long-term visual field follow-up of glaucoma patients treated with beta blockers. Surv. Ophthalmol. 38 (Suppl. May):156-160 26. Monica LM, Hesse RJ and Messerli FM (1983) The effect of a calcium channel blocking agent on intraocular pressure. Am. J. Ophthalmol. 96:814 27. Ventura HO, Messerli FH, Oighman W, et al. (1983) Immediate hemodynamic effects of new calcium channel bloking agent (nitrendipine) in essential hypertension. Am. J. Cardiol. 51:783-791 28. Kelly SP and Valley TJ (1988) Effects of calcium antagonist nifedipine on intraocular pressure in normal subjects. Br. J. Ophthalmol. 72:216-223 29. Abelson MB, Gilbert CM and Smith LM (1998) Sub-stained reduction of intraocular pressure in humans with the calcium channel blocker verapamil. Am. J. Ophthalmol. 105:155-159 30. Beatty JF, Krupin T, Nichols PF and Becker B (1984) Elevation of intraocular pressure by calcium channel blocker. Arch. Ophthalmol. 102:172 31. Bill A (1985) Some aspects of the ocular circulation. Invest. Ophthalmol. Vis. Sci. 26:410-424 32. Johansson B (1978) Process involved in vascular smooth muscle contraction and relaxation. Circ. Res. 34 (Suppl. 1):1-14 33. Vanhoutte PM (1987) Expert committee of the world health organization on classification of calcium antagonist. The viewpoint of the rapport. Am. J. Cardiol. 59:3-9 34. Netland PA, Chatuervedi N and Dreyer EB (1993) Calcium channel blockers in the menagement of low-tension and open-angle glaucoma. Am. J. Ophthalmol. 115:608-613 35. Glasser P and Flammer J (1987) Influence of vasospasm on visual function. Doc. Ophthalmol. 66:3-18

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36. Suzuki R, Hanada M, Fujii H and Kuimoto S (1992) Effects of orally administered b-adrenergic blockers and calcium channel blockers on the intraocular pressure of patients with treated hypertension. Ann. Ophthalmol. 24:220-223 37. Rennie DG and Smerdon DL (1985) The effect of a once daily oral dose of nadolol on intraocular pressure in normal volunteers. Am. J. Ophthalmol. 100:445-447 38. Duff GR (1987) The effect of twice daily nadolol on intraocular pressure. Am. J. Ophthalmol. 104:343-345 39. Williamson J, Atta HR, Kennedy PA and Moir JG (1985) Effect of orally administered nadolol on the intraocular pressure in normal voluntaries. Br. J. Ophthalmol. 69:38-40 40. Williamson J, Young JDH, Atta H, et al. (1985) Comparative efficacy of orally and topically administered b-blockers for chronic simple glaucoma. Br. J. Ophthalmol. 69:41-45 41. Ikemoto F and Yamamoto K (1978) Renin angiotensin system in the acqueous humor of rabbits, dogs and monkeys. Exp. Eye Res. 27:723-725 42. Weinreb RN, Dandman R, Ryder ML and Friberg TR (1985) Angiotensin converting enzyme activity in Human acqueous humor. Arch. Ophthalmol. 103:34-36 43. Stamek SJ, Wallow HH, Tewksbury DA, et al. (1992) An ocular renin-angiotensin system. Invest. Ophtahlmol. Vis. Sci. 33:1627-1632 44. Kaufman PL and Barany EH (1981) Adrenergic drug effects on acqueous outflow facility following muscle retrodisplacement in the cynomolgus monkey. Invest. Ophthalmol. Vis. Sci. 20:644-51 45. Costagliola C, Di Benedetto R, De Caprio L, et al. (1995) Effect of oral captopril (SO14225) on intraocular pressure in man. Eur. J. Ophthalmol. 5:19-25 46. Sossi N and Anderson DR (1982) Blockage of axonal transport in optic nerve induced by elevation of intraocular pressure. Arch. Ophthalmol. 101:94-98 47. Regoli D and Batabe M (1980) Pharmacology of bradikinin and related kinins. Pharmacol. Rev. 22:1-45 48. Erdos G and Skidgel RA (1987) The angiotensin I converting enzyme. Lab. Invest. 56:345-348 49. McCannel CA, Heinrich SR and Brubaker RF (1992) Acetazolamide but not timolo lowers aqueous humor flow in sleeping humans. Graefe’s Arch. Clin. Exp. Ophthalmol. 230:518-520

Chapter 18

Aging of the Lachrymal Gland Hiroto Obata, MD, PhD

Abstract The lachrymal gland is an appendage of the ocular surface that secretes tear fluid consisting of water, proteins, and electrolytes, which helps to maintain the cells of the ocular surface. The lachrymal gland and ocular surface form a mucosal immune system, and both are affected by environmental factors. The quality and quantity of tear fluid decreases with age, and dry eye is one of most common problems in elderly patients visiting ophthalmologists. The lachrymal gland is innervated by the autonomic nervous system and the secretory function is very complicated. Few previous studies have examined the aging mechanisms of the lachrymal gland. Histopathological studies of the human lachrymal gland have demonstrated that acinar atrophy, periacinar fibrosis, and periductal fibrosis increase with age. Animal studies have shown that morphological changes, reduced lachrymal secretion of protein, decreased density of innervation, and increased number of inflammatory cells in the lachrymal glands occur with aging. Generally, inflammation and neural dysfunction might be involved in the pathogenesis of agerelated lachrymal gland dysfunction, but the mechanisms linking lachrymal gland dysfunction with aging remain unclear. Keywords lachrymal gland; aging; dry eye; tear fluid; secretion; atrophy; fibrosis; inflammation; neural dysfunction

The lachrymal gland is the primary source for the aqueous portion of the tear film. This organ secretes tear fluid comprising water, proteins, glycoproteins, and electrolytes, all helping to maintain a healthy ocular surface. Tear fluid is supplied from not only the lachrymal gland, but also the Meibomian gland and goblet cells of the conjunctiva. Although every component of tear fluid from these tissues is necessary for a healthy ocular surface, the major source of tear fluid is the lachrymal gland. The committee of the International Dry Eye Workshop recently reported a new definition of dry eye, as follows: “Dry eye is a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. It is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface.”1

From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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The prevalence of dry eye increases with age,2-5 consistent with previous reports that the quality and quantity of tear fluid normally decreases with age.6-13 Moreover, dry eye is one of the most common ocular problems in the world.2-5 Clarification of the mechanisms involved in the associations between aging and the lachrymal gland is thus extremely important.14

Anatomy of the Lachrymal Gland Gross Anatomy of the Lachrymal Gland The human lachrymal gland consists of the main lachrymal gland and the accessory lachrymal gland. The main lachrymal gland resides in the superior temporal orbit and comprises palpebral and orbital lobes, which are continuous with each other at the lateral edge of the aponeurosis of the levator palpebrae superiosis.15-18 The orbital lobe lies in the lachrymal fossa on the anterior lateral part of the orbit. The palpebral lobe lies below the aponeurosis of the levator palpebrae superiosis and is in contact with the superior and lateral fornices of the conjunctiva. Excretory ducts arising from the palpebral and orbital lobes open into the superior conjunctival fornix. The accessory lachrymal gland comprises histologically identifiable small glands located in the lamina propria of the conjunctiva.15,16 Human accessory lachrymal glands are divided into two types: glands of Krause, and glands of Wolfring. Glands of Krause are located in the lamina propria of the fornix, while glands of Wolfring reside in the edge of the tarsus. Ducts of both glands open onto the conjunctival surface. Other vertebrates also have accessory lachrymal glands in the conjunctiva. For instance, the nictitating membrane is well known as a site containing accessory lachrymal glands. Most animal studies have used lachrymal glands from rodents and rabbits. The anatomy of lachrymal glands from those animals differs substantially from that in humans. In rodents, the lachrymal gland consists of the intraorbital, exorbital, and Harderian glands. The exorbital gland is found under the skin on the lateral side of the face near the ear. The rabbit also has a lachrymal and Harderian gland, both located within the orbit. The Harderian gland produces mainly lipids.

Histology of the Lachrymal Gland The lachrymal gland is composed of many lobules separated from one another by loose connective tissue.15-18 Each lobule displays a tubuloacinar structure with numerous acini and intralobular ducts. The acini appear as rosettes of polarized pyramid-shaped acinar cells with a central lumen. Acinar cells include numerous periodic acid-Schiff (PAS)-positive secretory granules, indicating an

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abundance of glycoproteins. Various lachrymal proteins are synthesized and secreted by these acinar cells. Myoepithelial cells are flattened and distributed surrounding the acini and intercalated ducts, and contain many myofilaments in the cytoplasm that are thought to squeeze secretory products down the lumen. The intra- and interlobular ducts comprise 2–3 layers of epithelial cells and lack myoepithelial cells. Ductal epithelial cells have small amounts of granules in the cytoplasm that differ from granules in acinar cells. In rodents, enzyme Na+-K+ATPase involved in fluid and ion transport is present in the basolateral membranes of intra- and interlobular ductal cells, not in acinar cells.19 The connective tissue contains interlobular ducts, vessels, nerve fibers, fibroblasts, plasma cells, lymphocytes, macrophages, and mast cells. Plasma cells secrete immunoglobulin (Ig)A, which is important in protecting the ocular surface from infection. The conjunctiva and lachrymal gland are commonly thought to represent a mucosa-associated lymphoid tissue (MALT), representing the immune system located at mucosal surfaces.20,21 The accessory lachrymal gland is histologically and histochemically identical to the main lachrymal gland.22 However, the gland of Krause is very small and usually comprises only one lobule. The extent to which the accessory lachrymal gland contributes to total tear volume remains unclear, but that main lachrymal gland is generally considered the major fluid-secreting organ.

Innervation of the Lachrymal Gland The lachrymal gland is innervated by both parasympathetic and sympathetic divisions of the autonomic nervous system.23-25 Parasympathetic nerves are predominantly developed in the gland and release acetylcholine, whereas sympathetic nerves are less abundant and release norepinephrine. Parasympathetic cholinergic nerves also contain neuropeptides such as vasoactive intestinal polypeptide (VIP), substance P (SP) and neuropeptide Y. Sympathetic adrenergic nerves also contain neuropeptide Y. Autonomic nerve endings are innervated not only in acinar cells, but also in myoepithelial cells, ductal cells, and blood vessels.26,27 Sensory nerves, as a division of the trigeminal nerve, also innervate the gland and release SP and calcitonin gene-related peptide (CGRP), but have the least dense innervation. Parasympathetic, sympathetic, and sensory innervations play complex stimulatory and inhibitory roles in the secretory function of the lachrymal gland. Although neural control of lachrymal secretions includes emotional responses, as in crying, the most well documented control involves stimulation to the ocular surface, cornea, and conjunctiva, activating afferent sensory nerves on the ocular surface and leading to activation of the efferent sympathetic and parasympathetic nerves in the gland to stimulate secretion. The generally accepted concept is that components of the ocular surface (cornea, conjunctiva, and Meibomian glands), the main and accessory lachrymal glands, and interconnecting innervation act as a functional unit.28,29

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Secretory Function of the Lachrymal Gland Because the complex innervation involves multiple transmitters, secretory function of the lachrymal gland is very complicated and the situation currently contains too many unknowns. However, acetylcholine, norepinephrine, and VIP represent major stimuli for lachrymal gland secretion and activate different signaling pathways.30-34 From the perspective of secretion type, the major thrusts of research are divided broadly into 2 categories: protein secretion, and water secretion.35-37

Protein Secretion Protein secretion basically involves the secretion of granules from acinar cells by exocytosis. Secretion of lachrymal proteins is stimulated by neurotransmitters and neuropeptides released from the neurons innervating the gland. Acinar cells have receptors for acetylcholine (muscarinic M3), norepinephrine, and VIP.30-34 In many cells, membrane receptors are coupled to G proteins that in turn regulate the activity of several second-messenger systems. Muscarinic acetylcholine receptors interact with G proteins, which activate phospholipase C, resulting in increased production of 1,4,5-inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 induces the release of intracellular stores of Ca2+ from the endoplasmic reticulum and the influx of extracellular Ca2+. This intracellular increase of Ca2+ stimulates secretion by activating Ca2+/calmodulin-dependent protein kinases. In contrast to IP3, DAG activates specific isoforms of protein kinase C (PKC), which stimulate further secretion. Norepinephrine binds to both α1- and β-adrenergic receptors. In the lachrymal gland, α1-adrenergic agonists are a potent stimulus for secretion and activate a signaling pathway involving Ca2+ and PKC. In contrast, β-adrenergic agonists offer less stimulus of secretion and activate a 3′,5′-cyclic adenosine monophosphate (cAMP)-dependent pathway. VIP activates a cAMP-dependent pathway. An increase in the level of cAMP activates protein kinase A, which stimulates lachrymal secretion. VIP also increases intracellular Ca2+ levels. An increase in intracellular Ca2+ levels by all three agonists (acetylcholine, α1-adrenergic, and VIP) is necessary to simulate secretion. Other stimuli for lachrymal gland secretion include the EGF family of growth factors.38 For more information about signal transduction and activation of the lachrymal gland, see review articles.30-32 A recent article reviewed the molecular mechanism of exocytosis, including protein and membrane trafficking and transport factors (microtubules, actin filaments, and motor proteins) in lachrymal acinar cells.39

Water Secretion Water is moved mainly from the interstitial spaces of the gland into the lumen of the gland. This water movement is attributed to the osmotic gradient, which depends on the movement of ions from the acinar cells and interstitium into the lumen.35-37

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The basolateral membranes contain numerous sodium pumps, with Na+-K+-ATPase to actively transport K+ into the cell and Na+ from the cytosol into the interstitium and maintain low cytosolic Na+ activities. The apical membranes have Cl− and K+ channels. Activation of cells by increased intracellular Ca2+ drives an outward movement of Cl− into the lumen. The resulting lumen-negative transepithelial voltage drives the paracellular flux of Na+ from the interstitium to the lumen. In turn, the osmotic gradient provides the motive force for the movement of water through the intercellular space. Some studies over the last decade have revealed that the apical membranes contain aquaporin 5 water channels, which facilitate the movement of water intracellularly to the lumen.40,41 In addition, basolateral membranes also have Cl−, K+, and Ca2+ channels. One study using connexin 32-deficient mice showed that gap junctions are essential for optimal fluid secretion by the lachrymal gland.42 See the review articles for more information about water and electrolyte secretion.35-37

Age-Related Changes to Tear Fluid As reported previously, the quality and quantity of tear fluid changes with age.6-13 Reflex secretion of tears as measured by the Schirmer test without anesthesia is known to decrease with age. As for a more detailed picture of tear flow, one study has shown tear volume and flow decline with age, whereas osmolarity and evaporation increase.11 In terms of the quality of tear fluid, both lysozyme and lactoferrin, as major proteins of tear fluid, have been shown to similarly decline with age.9 Interestingly, IgA levels gradually decline with age, but do not significantly correlate with age.9 These results probably correspond to the fact that the cell source and secretory mechanism of IgA differ from those of lysozyme and lactoferrin. Another study has shown that epidermal growth factor (EGF) levels in tear fluid do not correlate with age.12 These studies suggest that not all proteins necessarily decrease with age. Peroxidase activity in tear fluid decreases with age, but differs between men and women. Peroxidase activity in women decreases during and after menopause, but remains constant in men up to middle age, subsequently declining with advancing age.13 This suggests that gender-related differences are accentuated during aging. Corneal innervation is important for reflex tearing. Corneal sensitivity and the density of sensory nerves in the cornea decrease with age, resulting in decreased reflex loop activation of autonomic nerves that innervate the lachrymal gland.43,44

Age-Related Changes in the Lachrymal Gland The mechanisms causing lachrymal gland dysfunction are still poorly understood. Many possible factors have been proposed, including sex hormonal imbalances,45-47 pituitary hormones,48-51 neural dysfunction,52,53 and inflammation.54-64 Inflammation

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includes various factors such as environmental factors, proinflammatory cytokines such as interleukin 1β (IL-1β) and tissue necrosis factor α (TNF-α),56,57,59 autoimmue reaction, 60-62 and infection. 63,64 Compared to the number of physiological, biochemical, and immunological studies of secretory function and dysfunction in lachrymal glands, 65 research into the effects of aging on lachrymal glands has been limited.

Human Studies Some histopathological studies have examined the human lachrymal gland. In general, age-related pathological changes of lachrymal glands include diffuse fibrosis, diffuse atrophy, and periductal fibrosis.66-68 A histopathological study of 80 human lachrymal glands showed significant correlations between age and diffuse fibrosis, diffuse atrophy, and periductal fibrosis in the orbital lobes of females, and periductal fibrosis in the palpebral lobes of males.68 Moreover, diffuse fibrosis and diffuse atrophy in the orbital lobes were more frequently observed in females than in males. This result is of great interest, as it suggests a relationship with the greater frequency of dry eye in elderly women. An example of age-related histopathological change is shown in Fig. 18.1. The acinar area in the lachrymal glands of aged females is clearly much smaller than that of young males. One of the mechanisms behind these age-related changes is speculated to be a loss of nerve branches, causing fibrosis and acinar atrophy in aged glands, as these pathological changes are characteristically observed per unit area of lobule (Fig. 18.2). Another possibility is that these changes might be induced by circulatory disturbance or ischemia. Aging of the vascular system is an important issue, as seen with the most frequent and serious vascular diseases in elderly people, myocardial infarction and brain infarction. Periductal fibrosis may be an important factor related to the decrease in outflow of tear fluids.68 Atrophic ductal epithelium is often associated with periductal fibrosis. These ductal pathologic changes may interfere with electrolyte and water secretion, as ductal epithelial cells are considered responsible for this function.19,35 Periductal lymphocytic infiltration, which is not an age-related finding, is observed as a pathological change in the human lachrymal gland (Fig. 18.3).68 Focal lymphocytic infiltration of the lachrymal gland suggests subclinical dacryoadenitis, but the question of how lymphocytic infiltration might alter secretory function remains unresolved. In Sjögren’s syndrome, the most severe dry eye syndrome, periductal lymphocytic infiltration is thought to be the earliest histopathological finding. As for the relationship between inflammation and fibrosis, fibrosis may become the most prominent feature of chronic inflammation. Liver fibrosis and cirrhosis often follow chronic hepatitis. In the lachrymal gland, however, whether periductal fibrosis may follow periductal lymphocytic infiltration is unclear. Interlobular ductal dilatation is not an age-related finding, but is of importance when considering the pathogenesis of dry eye.68 This suggests stenosis or obstruction

Fig. 18.1 Histological findings of age-related changes A) Normal histological finding from a 17-year old male. B) Histological finding of an 87-year old female shows acinar atrophy and periacinar and periductal fibrosis at the same magnification. Reprinted from Obata et al., copyright 1995, with permission from the American Academy of Ophthalmology

Fig. 18.2 Lobular atrophy with fibrosis Acinar atrophy with periacinar fibrosis is present throughout one lobule. Normal acini are present on the right side of the photograph

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Fig. 18.3 Periductal lymphocytic infiltration Periductal lymphocytic infiltration, which is not age-related, suggests subclinical dacryoadenitis, but the question of how lymphocytic infiltration might alter the secretory function, and whether periductal fibrosis might follow periductal lymphocytic infiltration remains unresolved

of the orifices of excretory ducts in the fornix of the conjunctiva. Conjunctivitis might thus play a crucial role in causing obstructive dry eye. Regarding gross anatomy, magnetic resonance imaging has revealed that the thickness and area of the lachrymal gland decrease with age in women, but not in men.69 Many unknowns remain with regard to the molecular mechanisms of aging in the human lachrymal gland. One reason probably involves the difficulty in obtaining human lachrymal gland tissue for study. The human lachrymal gland is rarely removed at surgery or autopsy, and biopsy is considered unadvisable for such a small organ to prevent iatrogenic dry eye. Implementing a longitudinal study of age-related human lachrymal gland dysfunction is thus quite difficult.

Animal Studies Animal models of lachrymal gland dysfunction would offer a useful tool for investigating pathological mechanisms.70 Although animal models mimicking human age-related dry eye disease have not yet been reported, comparative studies of young and old animals have been described. Morphological studies in rats have shown that the type of acini change initially from serous to seromucous acini, followed by gradual transformation of seromucous acini to mucous acini with age.71-73 Another study in mice has shown that periductal fibrosis and acinar atrophy, as in the case of humans, are observed in glands from 24- and 32-month-old mice.74 Ultrastructural examination has revealed marked reductions in the Golgi apparatus and dilatation of the rough endoplasmic reticulum in the acinar cells of glands from 24-month-old rats.71,73

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Several studies have revealed that age-related dropout of nerves occurs in the lachrymal gland.74,75 The number and intensity of immunoreactive nerves for substances such as acetylcholinesterase, VIP, neuropeptide Y, CGRP, SP, tyrosine hydroxylase, and dopamine β-hydroxylase are reduced in the glands of aged mice and rats.74,75 One study has shown that decreases in innervation start in 24month-old mice, coinciding with a decline in acetylcholine release from the gland.74 Protein secretion in response to several neural agonists also decreases with increasing age.71-74,76,77 Acetylcholine, carbachol, adrenaline, phenylephrine, isoproterenol, SP, VIP, histamine, and 5-hydroxytryptamine were used as stimulants in these studies. However, secretion of peroxidase became highly variable in the aged gland and differed from protein secretion in response to sympathomimetic stimulation,76,77 suggesting a complex etiology of age-related lachrymal gland dysfunction. Stimulated peroxidase secretion decreased in 8-month-old mice and continued for 12–24 months before starting the decrease of innervation in 24-month-old mice.74 This means that impairment of protein secretion in the early stages of aging cannot be explained simply by decreases in innervation. Inflammatory cells such as lymphocytes and mast cells infiltrate the aged lachrymal gland of mice and rats.71,74,75 As seen with Sjögren’s syndrome, lymphocytic infiltration might play a role in inhibiting lachrymal gland secretion during aging. A study in the NZB/W mouse model of Sjögren’s syndrome found no correlation between the extent of lymphocytic infiltration and the degree of tear flow reduction,78 suggesting lymphocytic infiltration alone is insufficient to explain secretory dysfunction in this model mouse. Conversely, another study in NZB/W mice showed age-related decreases in the density of innervation to the acini.79 Of note is the fact that the decrease in innervation density is observed before any lymphocytic infiltration. Taken together, both inflammation and innervation might play crucial roles in causing lachrymal gland dysfunction. Carbonic anhydrase is part of a family of metalloenzymes that catalyze the rapid conversion of CO2 to H+ and HCO3−. An age-related increase in carbonic anhydrase activity was present in the gland from aged male rats, but not aged females,80 suggesting gender-related differences in the lachrymal gland.81,82 The physiological significance of this increase is not yet known. One study showed that aging significantly reduces tyrosine phosphorylation of insulin receptors in the lachrymal gland of rats, suggesting that this reduced phosphorylation may affect later stages of insulin signal transduction.83 Recently, advanced glycation end products (AGEs) have been found to increase with age and contribute to the chronic complications of aging in several tissues. AGE binding to its receptor leads to activation of the transcription factor nuclear factor-κB (NF-κB). AGE, its receptor, and NF-κB are highly expressed in aged lachrymal glands of rats compared to young glands.84 These metabolic events may therefore be related to lachrymal gland dysfunction with aging.

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Conclusion The mechanisms behind age-related lachrymal gland dysfunction remain unclear due to the multifactorial and complex nature of this system. To take a broad view, both inflammation and neural dysfunction may play crucial roles in age-related lachrymal gland dysfunction. The chance of encountering foreign antigens on the ocular surface increases with age, causing subclinical inflammation of the conjunctiva. Thereafter, the lachrymal gland could also be affected and inflamed because both conjunctiva and lachrymal gland are MALTs, and antigens are shared by both tissues. If this immune defense mechanism is dysregulated, the lachrymal gland might undergo some sort of adverse effect, such as excessive inflammation. Conversely, the density of innervation to the cornea and lachrymal gland reduces with age. Alteration of both sensory nerves innervating the cornea and autonomic nerves innervating the lachrymal gland could cause decreases in tear secretion. Moreover, to account for gender differences, since dry eye is observed more frequently in women than in men, hormonal influences also need to be resolved in the pathogenesis of lachrymal gland dysfunction. For instance, androgen level is considered to decrease with increasing age, affecting immunohomeostasis on the ocular surface. Exploring a neuroendocrine immune network in the lachrymal gland is thus absolutely essential, despite being an old issue. Senescence can be activated by both telomere-dependent and telomereindependent pathways. Genetic alterations, genome-wide DNA damage, and oxidative stress have recently been identified as inducers of senescence. Development of aging research will elucidate the most pertinent molecular pathways linking lachrymal gland dysfunction and aging.

References 1. Lemp MA, Baudouin C, Baum J, et al. (2007) The definition and classification of dry eye disease: Report of the Definition and Classification Subcommittee of the International Dry Eye WorkShop. Ocul Surf 5:75-92 2. McCarty CA, Bansal AK, Livingston PM, Stanislavsky YL, Taylor HR (1998) The epidemiology of dry eye in Melbourne, Australia. Ophthalmology 105:1114-1119 3. Moss SE, Klein R, Klein BE (2000) Prevalence of and risk factors for dry eye syndrome. Arch Ophthalmol 118:1264-1268 4. Schaumberg DA, Sullivan DA, Buring JE, Dana MR (2003) Prevalence of dry eye syndrome among US women. Am J Ophthalmol 136:318-326 5. Lekhanont K, Rojanaporn D, Chuck RS, Vongthongsri A (2006) Prevalence of dry eye in Bangkok, Thailand. Cornea 25:1162-1167 6. Henderson JW, Prough WA (1950) Influence of age and sex on flow of tears. Arch Ophthalmol 43: 224-231 7. de Roetth A Sr (1953) Lacrimation in normal eyes. Arch Ophthalmol 49: 185-189 8. Norn MS (1965) Tear secretion in normal eyes estimated by a new method: the lacrimal streak dilution test. Acta Ophthalmol 43:567-573 9. McGill JI, Liakos GM, Goulding N, Seal DV (1984) Normal tear protein profiles and age-related changes. Br J Ophthalmol 68:316-320

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10. Seal DV (1985) The effect of ageing and disease on tear constituents. Trans Ophthalmol Soc UK 104:355-362 11. Mathers WD, Lane JA, Zimmerman MB (1996) Tear film changes associated with normal aging. Cornea 15:229-234 12. Nava A, Barton K, Monroy DC, Pflugfelder SC (1997) The effects of age, gender and fluid dynamics on the concentration of tear film epidermal growth factor. Cornea 16:430-438 13. Marcozzi G, Liberati V, Madia F, Centofanti M, de Feo G (2003) Age- and gender- related differences in human lacrimal fluid peroxidase activity. Ophthalmologica 217:294-297 14. Van Haeringen NJ (1997) Aging and the lacrimal system. Br J Ophthalmol 81:824-826 15. Iwamoto T, Jakobiec FA (1982) Lacrimal glands. In: Jakobiec FA, ed. Ocular Anatomy, Embryology, and Teratology. Harper & Row, Philadelphia, p 761-781 16. Bron AJ, Tripathi RC, Tripathi BJ (1997) The ocular appendages: eyelids, conjunctiva and lacrimal apparatus. In: Bron AJ, Tripathi RC, Tripathi BJ (eds) Wolff’s Anatomy of the Eye and Orbit, 8th ed. Chapman & Hall Medical, London, p 30-84 17. Snell RS, Lemp MA (1998) Lacrimal apparatus. In: Snell RS, Lemp MA (eds) Clinical Anatomy of the Eye, 2nd ed. Blackwell Science, Malden, MA, p 114-124 18. Obata H (2006) Anatomy and histopathology of human lacrimal gland. Cornea 25:S82-S89 19. Winston DC, Hennigar RA, Spicer SS, Garrett JR, Schulte BA (1988) Immunohistochemical localization of Na+, K+-ATPase in rodent and human salivary and lacrimal glands. J Histochem Cytochem 26:1139-1145 20. Wieczorek R, Jakobiec FA, Sacks EH, Knowles DM (1988) The immunoarchitecture of the normal human lacrimal gland. Relevancy for understanding pathologic conditions. Ophthalmology 95:100-109 21. Knop E, Knop N (2005) The role of eye-associated lymphoid tissue in corneal immune protection. J Anat 206:271-285 22. Obata H, Horiuchi H, Dobashi Y, Oka T, Sawa M, Machinami R (1993) Immunohistochemical localization of epidermal growth factor in human main and accessory lacrimal glands. Jpn J Ophthalmol 37:113-121 23. Sibony PA, Walcott B, McKeon C, Jakobiec FA (1988) Vasoactive intestinal polypeptide and the innervation of the human lacrimal gland. Arch Ophthalmol 106:1085-1088 24. Matsumoto Y, Tanabe T, Ueda S, Kawata M (1992) Immunohistochemical and enzyme histochemical studies of peptidergic, aminergic and cholinergic innervation of the lacrimal gland of the monkey (Macaca fuscata). J Auton Nerv Syst 37:207-214 25. Walcott B, Cameron RH, Brink PR (1994) The anatomy and innervation of lacrimal glands. Adv Exp Med Biol 350:11-18 26. Ruskell GL (1975) Nerve terminals and epithelial cell variety in the human lacrimal gland. Cell Tissue Res 158:121-136 27. Lemullois M, Rossignol B, Mauduit P (1982) Immunolocalization of myoepithelial cells in isolated acini of rat exorbital lacrimal gland: cellular distribution of muscarinic receptors. Biol Cell 86:175-181 28. Stern ME, Beuerman RW, Fox RI, Gao J, Mircheff AK, Pflugfelder SC (1998) The pathology of dry eye: the interaction between the ocular surface and lacrimal glands. Cornea 17:584-589 29. Stern ME, Gao J, Siemasko KF, Beuerman RW, Mircheff AK, Pflugfelder SC (2004) The role of the lacrimal functional unit in the pathophysiology of dry eye. Exp Eye Res 78:409-416 30. Dartt DA (1994) Signal transduction and activation of the lacrimal gland. In: Albert DM, Jakobiec FA (eds) Principles and Practice of Ophthalmology. W.B. Saunders, Philadelphia, p 458-465 31. Hodges RR, Dartt DA (2003) Regulatory pathways in lacrimal gland epithelium. Inc Rev Cytol 231:129-196 32. Dartt DA (2004) Dysfunctional neural regulation of lacrimal gland secretion and its role in the pathogenesis of dry eye syndromes. Ocul Surf 2:76-91 33. Nakamura M, Tada Y, Akaishi T, Nakata K (1997) M3 muscarinic receptor mediates regulation of protein secretion in rabbit lacrimal gland. Curr Eye Res 16:614-619

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34. Satoh Y, Sano K, Habara Y, Kanno T (1997) Effects of carbachol and catecholamines on ultrastructure and intracellular calcium-ion dynamics of acinar and myoepithelial cells of lacrimal glands. Cell Tissue Res 289:473-485 35. Alexander JH, van Lennep EW, Young JA (1972) Water and electrolyte secretion by the exorbital lacrimal gland of the rat studied by micropuncture and catheterization techniques. Pflugers Archiv 337:299-309 36. Mircheff AK (1994) Water and electrolyte secretion and fluid modification. In: Albert DM, Jakobiec FA, (eds) Principles and Practice of Ophthalmology. W.B. Saunders, Philadelphia, p 466-472 37. Walcott B (1998) The lacrimal gland and its veil of tears. News Physiol Sci 13:97-103 38. Dartt DA (2004) Interaction of EGF family growth factors and neurotransmitters in regulating lacrimal gland secretion. Exp Eye Res 78:337-345 39. Wu K, Jerdeva GV, da Costa SR, Sou E, Schechter JE, Hamm-Alvarez SF (2006) Molecular mechanisms of lacrimal acinar secretory vesicle exocytosis. Exp Eye Res 83:84-96 40. Raina S, Preston GM, Guggino WB, Agre P (1995) Molecular cloning and characterization of an aquaporin cDNA from salivary, lacrimal, and respiratory tissues. J Biol Chem 27:1908-1923 41. Ishida N, Hirai SI, Mita S (1997) Immunolocalization of aquaporin homologs in mouse lacrimal glands. Biochem Biophys Res Commun 238:891-895 42. Walcott B, Moore LC, Birzgalis A, et al. (2002) Role of gap junctions in fluid secretion of lacrimal glands. Am J Physiol Cell Physiol. 282:C501-C507 43. Millodot M, Owens H (1984) The influence of age on the fragility of the cornea. Acta Ophthalmol (Copenh) 62:819-824 44. Niederer RL, Perumal D, Sherwin T, McGhee CN (2007) Age-related differences in the normal human cornea: a laser scanning in vivo confocal microscopy study. Br J Ophthalmol 27 (Epub ahead of print) 45. Sullivan DA, Wickham LA, Rocha EM, et al. (1998) Influence of gender, sex steroid hormones, and the hypothalamic-pituitary axis on the structure and function of the lacrimal gland. Adv Exp Med Biol 438:11-42 46. Azzarolo AM, Wood RL, Mircheff AK, et al. (1999) Androgen influence on lacrimal gland apoptosis, necrosis, and lymphocytic infiltration. Invest Ophthalmol Vis Sci. 40:592-602 47. Sullivan DA (2004) Tearful relationships? Sex, hormones, the lacrimal gland, and aqueousdeficient dry eye. Ocur Sur 2:92-123 48. Minami A, Kamei T (1959) Sur la glande lacrymale exterieure chez le Rat et ses modifications après hypophysectomie. CR Soc Biol 153:269-273 49. Jahn R, Padel U, Porsch PH, Söling HD (1982) Adrenocorticotropic hormone and alpha-melanocyte-stimulating hormone induce secretion and protein phosphorylation in the rat lacrimal gland by activation of a cAMP-dependent pathway. Eur J Biochem. 126:623-629 50. Azzarolo AM, Bjerrum K, Maves CA, et al. (1995) Hypophysectomy-induced regression of female rat lacrimal glands: partial restoration and maintenance by dihydrotestosterone and prolactin. Invest Ophhtalmol Vis Sci 36:216-226 51. Eckstein AK, Finkenrath A, Heiligenhaus A, et al. (2004) Dry eye syndrome in thyroid-associated ophthalmopathy: lacrimal expression of TSH receptor suggests involvement of TSHR-specific autoantibodies. Acta Ophthalmol Scand 82:291-297 52. Nguyen DH, Beuerman RW, Toshida H (2002) The effects of sensory and parasympathetic denervation on the kinases and initiation factors controlling protein synthesis in the lacrimal gland. Adv Exp Med Biol 506:65-70 53. Song XJ, Li DQ, Farley W, et al. (2003) Neurturin-deficient mice develop dry eye and keratoconjunctivitis sicca. Invest Ophthalmol Vis Sci. 44:4223-4229 54. Williamson J, Gibson AAM, Wilson T, et al. (1973) Histology of the lacrimal gland in keratoconjunctivitis sicca. Br J Ophthalmol 57:852-858 55. Paranyuk Y, Claros N, Birzgalis A, Moore LC, Brink PR, Walcott B (2001) Lacrimal gland fluid secretion and lymphocytic infiltration in the NZB/W mouse model of Sjögren’s syndrome. Curr Eye Res 23:199-205 56. Stern ME, Pflugfelder SC. (2004) Inflammation in dry eye. Ocul Surf 2:124-130

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57. Kublin CL, Hodges RR, Zoukhri D (2002) Proinflammatory cytokine inhibition of lacrimal gland secretion. Adv Exp Med Biol 506:783-787 58. Smith RE (2005) The tear film complex. Pathogenesis and emerging therapies for dry eyes. Cornea 24:1-7 59. Zoukhri D (2006) Effect of inflammation on lacrimal gland function. Exp Eye Res 82:885-898 60. Jabs DA, Prendergast RA, Whittum-Hudson JA (2002) Pathogenesis of autoimmune lacrimal gland disease in MRL/MPJ mice. Adv Exp Med Biol 506:771-776 61. Zhu Z, Stevenson D, Schechter JE, Mircheff AK, Atkinson R, Trousdale MD (2003) Lacrimal histopathology and ocular surface disease in a rabbit model of autoimmune dacryoadenitis. Cornea 22:25-32 62. Trousdale MD, Zhu Z, Stevenson D, Schechter JE, Ritter T, Mircheff AK (2005) Expression of TNF inhibitor gene in the lacrimal gland promotes recovery of tear production and tear stability and reduced immunopathology in rabbits with induced autoimmune dacryoadenitis. J Autoimmune Dis 2:6 63. Rhem MN, Wilhelmus KR, Jones DB (2000) Epstein-Barr virus dacryoadenitis. Am J Ophthalmol 129:372-375 64. Obata H, Yamagami S, Saito S, Sakai O, Tsuru T (2003) A case of acute dacryoadenitis associated with herpes zoster ophthalmicus. Jpn J Ophthalmol 47:107-109 65. Gibson IK, Argüeso P, Beuerman R, et al. (2007) Research in dry eye: Report of the Research Subcommittee of the International Dry Eye WorkShop (2007). Ocul Surf 5:179-193 66. Damato BE, Allan D, Murray SB, Lee WR (1984) Senile atrophy of the human lacrimal gland: the contribution of chronic inflammatory disease. Br J Ophthalmol 68:674-680 67. Roen JL, Stasior OG, Jakobiec FA (1985) Aging changes in the human lacrimal gland: role of the ducts. CLAO J 11:237-242 68. Obata H, Yamamoto S, Horiuchi H, Machinami R (1995) Histopathologic study of human lacrimal gland: Statistical analysis with special reference to aging. Ophthalmology 102:678-686 69. Ueno H, Ariji E, Izumi M, Uetani M, Hayashi K, Nakamura T (1996) MR imaging of the lacrimal gland. Age-related and gender-dependent changes in size and structure. Acta Radiol 37:714-719 70. Barabino S, Dana MR (2004) Animal model of dry eye: A critical assessment of opportunities and limitations. Invest Ophthalmol Vis Sci 45:1641-1646 71. Draper CE, Adeghate E, Lawrence PA, Pallot DJ, Garner A, Singh J (1998) Age-related changes in morphology and secretory responses of male rat lacrimal gland. J Auton Nerv Syst 69:173-183 72. Draper CE, Adeghate EA, Singh J, Pallot DJ (1999) Evidence to suggest morphological and physiological alterations of lacrimal gland acini with ageing. Exp Eye Res 68:265-276 73. Draper CE, Singh J, Adeghate E (2003) Effects of age on morphology, protein synthesis and secretagogue-evoked secretory responses in the rat lacrimal gland. Mol Cell Biochem 248:7-16 74. Rios JD, Horikawa Y, Chen LL, et al. (2005) Age-dependent alterations in mouse exorbital lacrimal gland structure, innervation and secretory response. Exp Eye Res 80:477-491 75. Williams RM, Singh J, Sharkey KA (1994) Innervation and mast cells of the rat exorbital lacrimal gland: the effects of age. J Auton Nerv Syst 47:95-108 76. Bromberg BB, Welch MH (1985) Lacrimal protein secretion: comparison of young and old rats. Exp Eye Res 40:313–320 77. Bromberg BB, Cripps MM, Welch MH (1986) Sympathomimetic protein secretion by young and aged lacrimal gland. Curr Eye Res 5:217-223 78. Paranyuk Y, Claros N, Birzgalis A, Moore LC, Brink PR, Walcott B (2001) Lacrimal gland fluid secretion and lymphocytic infiltration in the NZB/W mouse model of Sjögren’s syndrome. Curr Eye Res 23:199-205 79. Walcott B, Claros N, Patel A, Brink PR (1998) Age-related decrease in innervation density of the lacrimal gland in mouse models of Sjögren’s syndrome. Adv Exp Med Biol 438:917-923 80. Bromberg BB, Welch MH, Beuerman RW, et al. (1993) Histochemical distribution of carbonic anhydrase in rat and rabbit lacrimal gland. Invest Ophthalmol Vis Sci 34:339-348 81. Cornell-Bell AH, Sullivan DA, Allansmith MR (1985) Gender-related differences in the morphology of the lacrimal gland. Invest Ophthalmol Vis Sci 26:1170-1175

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82. Hann LE, Allansmith MR, Sullivan DA (1988) Impact of aging and gender on the Ig-containing cell profile of the lacrimal gland. Acta Ophthalmologica 66:87-92 83. Rocha EM, Carvalho CR, Saad MJ, Velloso LA (2003) The influence of ageing on the insulin signaling system in rat lacrimal and salivary glands. Acta Ophthalmol Scand 81:639-645 84. Alves M, Cunha DA, Calegari VC, et al. (2005) Nuclear factor-kB and advanced glycation end-products expression in lacrimal glands of aging rats. J Endocrinol 187:159-166

Chapter 19

The World According to Blink: Blinking and Aging Frans Van der Werf, PhD and Albertine Ellen Smit, MD

Abstract In this chapter, the neuroanatomical and neurophysiological background of the blink circuit and the consequences of aging will be discussed. Eyelid and eye kinematics are described for healthy subjects and patients with facial movement disorders. Attention will be paid to the blink rate, which can be used as an external parameter of the condition of the blink circuit and of brain structures influencing the circuit in health and disease. Reflex blinks give important information and are excellent experimental models for the assessment of internal networks and nuclei. The reflex blink circuit is the most commonly used neuronal blink circuit model for the study of how relatively simple lid movements are controlled and generated by the central nervous system. Keywords blink, eyelids, blink circuit, facial movement disorders, Bell’s palsy A blink is a brief simultaneous closure and opening of the eyelids and a rotation of both eyes.1 Eyelid closure, together with eye movement, provides optimal tear film distribution over the cornea and is imperative to maintain a transparent cornea and to protect the eye against corneal drying and damage. During aging, the morphology and performance of eyelid structures and the organization of eye muscle fiber types will alter. The lipid profiles in human meibomian gland secretions show significant alterations in older men and women.2 The myofibrous composition of the orbicularis oculi muscle, the eyelid closing muscle, can change during aging,3 and the sarcomeres in the myofibrils of the levator palpebrae superioris muscle, the eyelid opening muscle, can increase due to stretching. The levator aponeurosis may become thinner and its autonomic innervation will be less efficient.4,5 Other morphological structures like fat tissue, collagen, and collagen elastic fibers decrease in and around the eyelids. Eyelid movements are also involved in the expression of emotions such as smiling, grimacing, and winking. Movements of the upper eyelid are closely linked to vertical eye movements, the lid saccade. For instance, the upper eyelids actively follow the eyes during the upward phase and passively during the downward phase of a saccade. Knowledge of the nature and shape of blinking, demonstrated with eyelid kinematics, is very important for a good understanding of eyelid function.

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In this chapter the neuroanatomical and neurophysiological background of the blink circuit and the consequences of aging will be discussed. Eyelid and eye kinematics are described for healthy subjects and patients with facial movement disorders. Attention will be paid to the blink rate, which can be used as an external parameter of the condition of the blink circuit and the brain structures influencing the circuit in health and disease. Reflex blinks give important information and are excellent experimental models for the assessment of internal networks and nuclei. The reflex blink circuit is the most commonly used neuronal blink circuit model for the study of how relatively simple lid movements are controlled and generated by the central nervous system. Several focal dystonia will be discussed in relation to blinking, and finally reflex blinking will be used as a tool to determine the consequences of cerebellar diseases on the precise timing in the onset of eyelid movement after a stimulus. Because it is impossible to discuss in this chapter all the different aspects of brain, brainstem, and cranial nerve function related to blinking and aging, a selection of subjects was made focusing on the most common movement disorders.

Neuroanatomical and Neurophysiological Background of the Blink Circuit In humans, eyelid responses mainly result from the neuronal activity of two different motor systems: the facial and the oculomotor systems.6-8 The facial motor system innervates facial muscles, including the orbicularis oculi muscle. The orbicularis oculi muscle is a sphincter muscle and can be divided into an orbital portion, a preseptal portion, and a pretarsal portion.8 The orbicularis oculi muscle fibers are relatively short and heterogeneous in length.3 The muscle fibers are arranged parallel to the rims of the eyelids. The oculomotor system innervates five of the six extraocular muscles; the inferior oblique muscle, the recti superior, inferior, and medial muscles, and the levator palpebrae superioris muscle. The levator palpebrae superioris muscle contains a unique levator slow-twitch fiber type,9 and the levator aponeurosis connects this muscle with the upper eyelid. Efferently, the motoneurons that innervate the orbicularis oculi muscle are located in the ipsilateral intermediate subnucleus of the facial motor nucleus.8 The motoneurons of the levator palpebrae superioris are located in the central caudal nucleus (CCN) of the oculomotor nucleus.7 A population of CCN motoneurons subserves both levator muscles and is probably involved in synchronizing eyelid movement. Afferently, the orbicularis oculi muscle, like all facial muscles, lacks sensory innervation.7 Sensory innervation via the muscle spindle was found in the levator palpebrae superioris muscle of a human fetus.10 This result confirms the retrograde tracing study in the levator palpebrae superioris muscles of the monkey, in which primary sensory neurons were detected in the gasserian ganglion.7

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The integrity of efferent and afferent pathways of blinking can be examined through blink reflex studies. Reflex blinks can be divided into trigeminal blinks and non-trigeminal blinks. Trigeminal blinks arise from trigeminal stimuli, whilst non-trigeminal blinks are evoked by auditory or visual stimuli or as a component of other motor behaviors. Essentially, the blink reflex circuit is short: through stimulation of the trigeminal blink system, motoneurons of the facial and/or oculomotor nucleus are recruited. Subsequently, the intermediate subnucleus of the facial motor nucleus activates the orbicularis oculi muscle, which is followed by eyelid closure (Fig. 19.1). Attempts were made to disentangle the complete map of the neuronal blink circuit,11 including the eye blink generator regulating the eyelid and eye movement during the blink. However, many open questions still remain about the connections between pathways and the role of intermediate nuclei in the blink circuit. The presence of an “eye blink generator” for all types of blinking has again been proposed recently.12 In the studies of Smit and coworkers (2005, 2006)13,14 the location of an eye blink generator was indicated in the reticular formation. These authors revealed that an area in the pontomedullary reticular formation, the dorsal part of the medullary reticular nucleus, subserves both the facial and oculomotor systems (Fig. 19.2). However, separated areas in the reticular formation and cervical spinal cord were also found that initiate only an eyelid or an eye movement during blinking (Fig. 19.3). Another interesting part of the neuronal blink circuit in the brainstem that needs to be elucidated is the location of the premotor area of neurons innervating the orbicularis oculi muscle and the premotor area of neurons innervating the levator palpebrae superior muscle. Premotor neurons of the facial motor nucleus were mainly seen in the lateral part of the pontine reticular formation; in the lateral part

Fig. 19.1 Through stimulation of the trigeminal blink system, motoneurons of the facial and or/ oculomotor nucleus are recruited. The intermediate subnucleus of the facial motor nucleus activates the orbicularis oculi muscle, which is followed by eyelid closure

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Fig. 19.2 An area in the pontomedullary reticular formation, the dorsal part of the medullary reticular nucleus, subserve both the facial and oculomotor systems

of the medullary reticular nucleus, premotor neurons of the superior colliculus were observed.14 In another study inhibitory glutamic acid decarboxylase (GAD) premotor neurons of the facial motor nucleus were located in the paralemniscal zone of the midbrain tegmentum.15 This study does not describe an exclusive blink premotor area, as the retrograde tracer injections comprised almost the whole facial nucleus. Numerous studies have exposed the mechanisms of coordination of the levator palpebrae superioris muscles during lid saccades. Compelling neuroanatomical and neurophysiological data exist on levator palpebrae superioris muscle innervation during lid saccades.16-18 Little is known about the pathways of premotor neurons of the central caudal nucleus that innervate the levator palpebrae superioris muscle during blinking.

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Fig. 19.3 Separated areas in the reticular formation and cervical spinal cord were found to initiate only an eyelid or an eye movement during blinking

Neuronal tracing studies in humans, monkeys, and cats reveal that the medial longitudinal fasciculus17 and the interstitial nucleus of Cajal serve as premotor areas for lid saccades.18 In cats, retrograde tracer injections in the interstitial nucleus of Cajal did not result in labeling of a specific neuron population in the pontomedullary reticular formation, a candidate location for the “eye blink generator.” This finding indicates that the location of the blink generator for eye movements is entirely different from that of the “eye blink generator.” Further investigations are needed to localize the pathway(s) between the “blink” levator premotor neurons and the “eye blink generator.” The eye movement component during blinking is initiated by neurons of the lateral superior colliculus portion.19 The afferent pathways of the eye movement component run via the gasserian ganglion to the sensory trigeminal complex towards the deep layers of the lateral superior colliculus portion.20-21 The neuronal pathways between the superior colliculus and specific areas of the lateral reticular formation were explored by neuronal tracing studies in rats.13-14 Connections between higher brain areas and the brainstem, including the intermediate stations, are speculative. An extensive neuronal tracing study of the monkey

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motor cortex and facial motor nuclei revealed that the representation regions of the upper and lower face, located in the gyrus precentralis of the motor cortex, project directly to the facial motor nucleus. The upper face region projects mainly bilaterally and the lower face region projects contralaterally to the facial motor subnuclei.22-23 Further analysis revealed that other higher brain structures are involved in blinking. Basal ganglia and probably the noradrenergic system24-25 can modulate the blink reflex. In Parkinson’s patients the dopaminergic system is disturbed, which results in a higher blink rate with deviated blink profiles26 and lid saccadic metrics.27 Other suprasegmental structures, such as the primary motor cortex, supplementary motor area, dorsolateral prefrontal cortex, posterior parietal cortex, visual cortex, central thalamus, and cerebellum are connected with the facial motor nucleus.28 The cerebellum, besides its function in motor learning and memory, is also important for the control of fine motor movements, the balance control.29 The cerebellar cortex, like the motor cortex, contains a homunculus of the body. It is known that the hemispheral lobule VI represents the area of eyelid-related movements.30-31 Direct projections from lobule VI towards brainstem regions involved in blinking have not yet been found. The common opinion is that lobule VI is connected with one of the deep cerebellar nuclei, the interpositus nucleus. The interpositus nucleus is connected with the red nucleus,32 which projects to the reticular part of the blink circuit.11 Recently a neurophysiological study in a group of cerebellar ataxia patients showed aberrant timing of blink reflex (unpublished results).

Blink Rate Humans blink for the first time in the fetal stage at 33 weeks menstrual age. The fetal “spontaneous” blink rate is 6 blinks per hour;33 at birth the blink rate increases up to 4 blinks per minute. In adults the spontaneous blink rate is about 14 blinks per minute in the rest position, and by the age of 89 the blink rate increases to 31 blinks per minute.34 Thus throughout life the blink rate increases about 300-fold! Blink rate can dramatically be reduced to 0 to 3 blinks per minute in patients with the Steel-Richardson-Olszewski syndrome or slightly decreased in Parkinson patients26,35 and remarkably increased to over 50 blinks per minute in patients with cranial dystonia.36 Gender can influence the blink rate; men blink faster and suppress blinks better than women.37 In addition, the blink rate can be changed by numerous other neuropathological conditions. External factors like the time of day, environment, humidity, emotional state, mental load, or activity can also influence blinks and blink rate. A study of the influence of humidity on blinking revealed that an elderly group with a mean age of 71 tended to blink less frequently than a young group with a mean age of 22, although the differences were not significant.38 Because the groups were small and rather variable in age, analysis of larger groups is necessary to gain significant differences.

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Eyelid Kinematics Blinks can occur either voluntarily, spontaneously, or as a reflex in response to external stimulation. They have more or less the same profile (Fig. 19.4), although they differ in total duration, maximal downward amplitude, and velocity. Blinks evoked as a consequence of eye blink conditioning, the so-called conditioned blinks, are different in their profile from spontaneous, voluntary, and reflex

Fig. 19.4 The waves have more or less the same profile, although they differ in total duration, maximal downward amplitude, and velocity

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blinks.6 These eyelid responses are used as a measure of associative motor learning and memory.39 Conditioned blink responses are longer in duration, have longer lid closure duration, the maximal downward amplitude is lower, and the neural mechanisms underlying the eyelid movement are different. Therefore it is questioned whether these “blinks” can be defined as blinks and not as conditioned eyelid movements: CEMs. Eyelid movement during blinking can be translated into three vectors: the vertical and horizontal displacement, and retraction. The vertical displacement is the most important one, but horizontal displacement is also a crucial factor in eyelid movement. The contraction of the eyelid during blinking slightly changes the 3-D position of eyelid tissues and retracts the eye between 1.0 and 2.0 mm.40 The origin and kinematics of horizontal displacement during eyelid movement has recently encouraged a discussion about the performance of eyelid surgery in ptosis patients.41 An important issue in this discussion between ophthalmologists and plastic reconstructive surgeons was agreement on the definition of the primary position of the eye when the subject looks straight forward.42 This center position is crucial for detailed reconstruction of “normal” lid movement in these ptosis patients. The vertical and horizontal eyelid displacement can be observed with an eye tracker or video camera in order to obtain the eyelid movement during a blink. The kinematics of eyelids are best investigated with electromagnetic recordings in combination with orbicularis oculi electromyography (OO-EMG) recordings.43 Surface EMG recordings made with a wide-frequency band allow measuring of the low frequencies involved in eyelid movement. This method may also be useful to characterize blink disturbances.44 There is a risk of signal recording from nearby muscles because of volume conduction.45 Other tools used for examining eyelid kinematics are the high-speed video camera and electroencephalography.46 Of all blinks, reflex blinks are the least variable in duration, maximal downward amplitude, and maximal downward velocity. Blinks elicited by acoustic click have the shortest duration and latency, followed by blinks elicited by electrical stimulation of the supraorbital nerve or an air puff on the cornea. Spontaneous blinks have the longest duration and greatest variability.43 Simultaneous eye movements occur in all three types of blinks.12 In general, about 4 ms after the onset of eyelid closure, the eye movement starts. The direction of the eye movement runs from the initial gaze position, down towards the nose, smoothly followed by a lateral upward movement towards the initial gaze position.1 Another active movement where eyelids and eyes act simultaneously is the lid saccade. During a saccade the eyelid follows the eye independent of the goal position of the saccade. Since saccades require eye and eyelid movement coordination they should be regulated by a common neuronal structure or “saccadic generator.” The kinematics of eyelid closure are good tools to investigate the influence of aging on blinking. Sun and coworkers34 demonstrated disorders of blink systems in a group of subjects aged 50 years or older. These authors observed an age-related reduction in the relationship between the peak velocity and the amplitude during a blink, the so-called main sequence slope. This was interpreted as a reduction in

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efficiency of orbicularis oculi motoneuron recruitment. In a study of healthy subjects over 60 years of age it was shown that the duration of eyelid closure was prolonged, and the excitability and latency of the trigeminal blink reflex increased significantly compared to those in younger subjects.47 The increased blink duration exhibited after blinks and blink oscillations is also observed in patients with dry eye syndrome (Fig. 19.5). The blink adaptations are seen as a possible mechanism for development of blepharospasm.48 In Parkinson’s patients the excitability of blinking is disturbed, probably due to dopaminergic depletion in the basal ganglia neurons. In these patients blink rate and amplitude were increased, though the latency of the blink response (onset) did not differ from that in healthy subjects26 independent of their clinical status.27 When eyelid closure is not optimal due to internal or external factors, the distribution of the tear film is influenced. A study on the effect of soft contact lenses

Fig. 19.5 The increased blink duration exhibited after blinks and blink oscillations are also observed in patients with dry eye syndrome

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revealed that closure of the palpebral aperture, named the blink completeness, is higher in healthy subjects than in soft lens wearers.49 Blepharoptosis can be induced by rigid contact lenses and can reduce eyelid motility. A study of the effect of aging on rigid contact lens wearers revealed that lid saccades in both groups have smaller amplitudes and lower peak velocities.50 In order to study the effect of aging in healthy subjects and in subjects with schizophrenia and other neuropsychiatric disorders, prepulse inhibition of the acoustic startle response was used. The combination of a 30-500 ms weak prestimulus preceding a startle stimulus enables measurements of startle plasticity and habituation. A decrease of the startle amplitude and an increase of the startle latency were measured in a large group of non-psychiatric subjects.51 In Parkinson’s patients and in patients with dry eye, prepulse inhibition experiments caused an increased excitability of the reflex blinks.52 The authors concluded that prepulse inhibition reflects the intrinsic characteristics of the blink reflex circuit. Lid saccades of upper and lower eyelids can also be affected by aging. In a study of two groups, one 20-30 years of age and the other 60-91 years of age, no significant difference in saccade amplitude was found. However, in the elderly group, a clear decline in peak velocity of the upper eyelid and an increase of the amplitude of the lower eyelid were measured.53

Facial Movement Disorders Hemifacial paralysis (Bell’s palsy) Facial nerve palsy may have a variety of causes such as trauma, nerve compression, toxins, and infection. In over 50 percent of facial nerve palsy patients the cause is unknown. The syndrome is then termed idiopathic facial nerve palsy, or Bell’s palsy. The most common infectious agents that may cause facial palsy are the herpes simplex and varicella zoster viruses. However, a recent study about the detection of herpes simplex (HSV-1) and varicella-zoster (VZV) viruses in patients with Bell’s palsy revealed that HSV-1 or VZV DNA was detected in only two of the 20 patients.54 This proportion is much smaller than that found in the study of Murakami and coworkers,55 indicating that it is still unclear whether the assumption of viral involvement in the etiology of Bell’s palsy is valid. Some authors reported an enhanced blink rate in Bell’s palsy patients.56,57 Others postulated that reduced eyelid motility may produce increased trigeminal blink reflex excitability after stimulation of the supraorbital nerve ipsilateral to the palsied eyelid.58 The chance of affliction increases with age but is gender independent. In patients with a substantial distal degeneration of the facial nerve, R1 is absent during electrical supraorbital nerve stimulation, and the M wave is markedly reduced in amplitude for several months, and in some cases for a few years.

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A longitudinal study on blink recovery in severely affected patients with Bell’s palsy, who were treated with prednisolone, revealed that the recovery process of OO-EMG and eyelid kinematics occurs in roughly four phases.1 In the first phase, start times of OO-EMG and lid movement were synchronized between the onset of the affliction and 18 weeks. Remarkably, during this synchronisation the non-palsied side compensates for the palsied side in its OO-EMG activity. The start times are delayed and values of maximal downward amplitude and velocity are lower than has been measured in healthy subjects. During the second phase, which lasted from 18 weeks until 36 weeks after onset of the affliction, the palsied eyelids of these patients showed the first signs of OOEMG activity and active eyelid movement at the same time. The third phase (weeks 36-52) is characterized by overshoot of OO-EMG activity of the palsied eyelid. The overshoot was no longer measurable approximately one year after the onset of the affliction. Together with the increase of OO-EMG activity on the palsied side, a decrease in OO-EMG activity was observed on the non-palsied side. The sum of OO-EMG of both eyelids remained almost constant throughout the study, indicating that compensation mechanisms occur during the affliction (Fig. 19.6). In the fourth phase a subtle increase of maximal amplitude and velocity was found. Except for the start times of the eyelid movements, recovery of eyelid movements at the palsied side during reflex blinking remained incomplete at 84 weeks. In summary, in severely affected Bell’s palsy patients, the OO-muscle activity at the palsied side was normal after one year, whilst the concomitant eyelid movement remained deviated (Fig. 19.7). Bell’s palsy patients also have abnormal eye movements during blinking, directly after the affliction. After onset of the affliction, both eyes rotate during blinking in a deviated lateral upward direction.

Fig. 19.6 The sum of OO-EMG of both eyelids remained almost constant throughout the study, indicating that compensation mechanisms occur during the affliction

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Fig. 19.7 In severely affected Bell’s palsy patients, the OO-muscle activity on the palsied side is normal one year, while the concomitant eyelid movement remains deviated

In the nineteenth century Sir Charles Bell observed in facial nerve palsy patients an upward eye movement during blinking in the palsied eye.59 He noted that only the eye at the palsied side rotates in a delayed upward direction, but not that both eyes rotate! Later this misinterpretation was quoted world-wide. This observation must not be confused with Bell’s phenomenon observed in 1823.60 Bell’s phenomenon exists during forceful voluntary eyelid closure and can be described as a slow upward and outward deviation of the eyes.46 This phenomenon is also seen in healthy subjects during spontaneous blinking.61 In a longitudinal study of severely affected Bell’s palsy patients the direction of the eye movements during voluntary (and often during spontaneous) blinking remained impaired throughout recovery, which was a year and a half after the onset of the affliction.1 Interestingly, the direction of eye movement during reflex blinking was normal after one year, indicating that structural changes may take place in the somatosensory and motor cortex. The preliminary results of a longitudinal recovery study of blink and mouth movements in severely affected Bell’s palsy patients using the functional MRI technique revealed that the representation fields in the motor cortex and probably the somatosensory cortex for blinking and mouth movements change in size. Monitoring the distinct motor cortex regions which are directly or indirectly involved in blinking and mouth movements on the palsied side revealed no significant changes in the first three months. Subsequently, a strong enhancement of the blink area and a less prominent enhancement of the mouth region were observed between four months and one year (unpublished results; see Fig. 19.8). The results of this ongoing study have now been monitored for a year and a half. The plasticity noted in the motor cortex representation areas in the study implies that Bell’s palsy is not purely a peripheral affliction; it should be realized that central reorganizations in the motor cortex can influence an optimal recovery. This is supported by a study about motor cortex plasticity, which revealed that rehabilitation is very important for an optimal facial function after brain injury.62 The major complications of peripheral facial nerve palsy are synkinesia, facial weakness, and the occurrence of corneal ulceration due to an incomplete closure of the palsied eyelid, the lagophthalmos.

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Fig. 19.8 A strong enhancement of the blink area and a less prominent enhancement of the mouth region were observed between four months and one year

Directly after the paresis of the orbicularis oculi muscle, eyelid closure is imperative, and gentle closure of the palsied eyelid by gravity is inhibited by the tonic activity of the levator palpebrae superioris muscle. In addition lid saccades, mainly executed by the levator palpebrae superioris muscle, are also affected in Bell’s palsy patients. This phenomenon, named thixotropy, can be eased by regular stretching of the levator palpebrae superior muscle.45 In order to improve facial symmetry of late or partially recovered Bell’s palsy patients, non-surgical intervention with facial physiotherapy, botulinum toxin injections, or surgical treatment can be necessary63. Botulinum toxin is often used to diminish synkinesia of facial structures. However, the result is temporary and often unsatisfactory; but alternatives are few. Electrical stimulation of facial muscles during recovery may be one of these alternatives although improvement of eyelid movement is often very poor.64 An electrophysiological study in Bell’s palsy patients showed that patients with residual facial weakness showed enhanced blink reflex recovery after electrical SO nerve

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stimulation on the palsied side.57 Cossu and coworkers65 suggested starting “treatment” about 3 months after onset of complete facial nerve palsy, the period when the first connections are made between growing axons and denervated muscles. At this stage very little muscle activity was detected in these patients. Starting therapy at that stage risks an overstimulation (activation) of facial muscles at the non-palsied side, which could result in pronounced facial asymmetry. In order to prevent the overstimulation, prudence should be used regarding the start of the training. VanderWerf and coworkers1 recommend starting therapy directly after the first signs of innervation of both the OO and orbicularis oris muscles.

Apraxia Another effect of aging was seen in a large group of subjects over 60 years of age who suffered from apraxia of eyelid opening or closure. Patients with apraxia of eyelid opening have difficulty initiating the act of eyelid opening on command.66,67 Many of these patients also exhibit an inability to keep the eyelids open for long period of time. Aramideh and coworkers68 found that involuntary inhibition of the palpebrae superioris muscle activity causes an inability to keep the eyelids open or to reopen them after involuntary closure. This form of apraxia accompanies the focal dystonia blepharospasm and is more frequently seen in patients with extrapyramidal disorders. However, dystonia is unlikely to account for all cases of apraxia of eyelid opening.69 In one population study of apraxia of eyelid opening, the affliction coincided with adult-onset blepharospasm in 75 percent and with atypical Parkinsonism in 25 percent of the cases.70 Apraxia of eyelid closure is characterized by the inability of the patient to close the the eyelids on command. However, spontaneous blinking is preserved and several patients deny that the eyelids remain open on attempts to close them. The affliction is often associated with parietal lobe lesions.

Hemifacial spasm Hemifacial spasm is characterized by involuntary, paroxysmal bursts of tonic and clonic contractions of muscles on one side of the face. It is named primary or idiopathic when it does not follow Bell’s palsy, and secondary or postparalytic when it does.71 The most common cause of primary facial spasm is compression of vascular malformation: for instance, a cerebellar artery, impinging on the facial nerve at the exit of the pontine level. Several types of posterior fossa tumours have also been reported in association with hemifacial spasm.72 The “postparalytic” hemifacial spasm, though less frequent, should be differentiated from the “primary” hemifacial spasm, as well as from synkinesia due to aberrant regeneration after Bell’s nerve palsy (post-Bell’s palsy synkinesia).

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Blink reflex recovery curves are used to examine motoneuron excitability of the facial nerve and brainstem interneurons in order to diagnose hemifacial spasm. By application of two shocks (conditioning and test stimuli) to the supraorbital nerve at varying intervals, the size of the test response can be expressed as a percentage of the first conditioning response at each level. An increased R2 recovery curve is often seen in hemifacial spasm patients stimulated on the affected side73 (Fig. 19.9). A

conditioning

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interval (sec) 0.21

0.3

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3

10 time 10 ms/div. R1

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75%

25%

0.1 0.3 1.0 interval S2-S1 (log.sec.)

3.0 10.0 3.0

Fig. 19.9 An increased R2 recovery curve is often seen in hemifacial spasm patients stimulated at the affected side (with courtesy of Dr. M. Aramideh)

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Good treatments for hemifacial spasm are microvascular decompression and botulin toxin injections. The injections can provide relatively long-lasting relief or substantial reduction in symptoms and have the advantage of being simple, safe, and indefinitely repeatable.74

Blepharospasm Blepharospasm is a focal dystonia of the eyelids,75 characterized by tremulous, clonic, and/or phasic discharges in the orbicularis oculi muscle. The disease is chronic and progressive, but the exact pathophysiology of this dystonia is unknown. The initial onset of dystonia at the eyelids manifests in humans at the age of about 50 years.76 The clinical aspects are different and range from frequent and strong blinking to clonic spasm of the eyelids. Simultaneous levator palpebrae superioris and orbicularis oculi muscle EMG recordings reveal impairment in reciprocity and timing of the two eye muscles77 (Figs. 19.10 and 19.11). Botulin toxin injections around the eye are undoubtedly the best choice of treatment for an overall weakening of the orbicularis oculi muscles.74 Injections abolish the spasm and improve spontaneous blinking for 6 to 12 weeks; after that, treatment will be restarted. Evidence is available indicating that disturbances of the trigeminal part of the blink circuit are the precursor of involuntary eyelid movements. Recording of blink

Fig. 19.10 Simultaneous levator palpebrae superioris and orbicularis oculi muscle EMG-recordings revealed impairment in reciprocity and timing of the two eye muscles (with courtesy of Dr. M. Aramideh)

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A LP

OO

B LP

OO

“close eyes”

LP

OO

01mV “open eyes”

200 ms

Fig. 19.11 Another feature is the constant in shape and kinematics of blink profiles after repeated exposure to a given stimulus (with courtesy of Dr. M. Aramideh).

reflex recovery curves in blepharospasm patients is used to determine the abnormal excitability enhancement of interneurons in the brainstem and the motor cortex. Reduction of the inhibition of the R2 response can also be detected.78 A minority of patients also have involuntary levator palpebrae inhibition. In all of these patients the R2 is enhanced in the recovery curves, indicating an abnormal processing of sensory inputs, and leading to excessive activity in premotor circuits.

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Blinking as a Tool in Timing of Reflexes Compelling evidence is presented in the literature that the cerebellum is also involved with leg and blink reflexes.30,32,79 The main functional role of the cerebellum is its involvement with associative learning and memory39 and balance control.80 Associative learning and memory can be studied with eyeblink conditioning using the delay, the trace, or the startle conditioning paradigms. Delay conditioning is used to study the functioning and involvement of the cerebellum. Trace conditioning is used to study the involvement of the hippocampus in learning, and startle conditioning is used to investigate the involvement of the amygdala. For the delay conditioning paradigm, a 500 ms tone is used as the conditioning stimulus, and a 20 ms air puff, as the unconditioned stimulus, is presented at the end of the tone. Both stimuli end simultaneously.39 For the trace conditioning paradigm, a 20 ms air puff is used as the conditioned stimulus, followed 250 ms later by a 100 ms air puff as the unconditioned stimulus,6 Aging influences awareness during the trace conditioning, especially when the trace time is increased.81 This was confirmed by a study on the effects of age and awareness using eyeblink conditioning, which revealed that increased age is associated with a decline in the overall eyeblink conditioned response frequency.82 One of the main features of a reflex blink is the exact timing of the onset of the movement in response to a stimulus. Another feature is the constancy in shape and kinematics of blink profiles after repeated exposure to a given stimulus (Fig. 19.11). Both parameters are modulated by the cerebellum, but knowledge of the blink reflex modulation is limited, and different interpretations of the role of the cerebellum do not always agree with each other. The major studies support the concept that the cerebellum modulates both conditioned and reflex blinks. Two cerebellar structures are most often mentioned, the cortex83 and the nucleus.84 A study of various cerebellar ataxia patients reveals impaired eyeblink conditioning in several subtypes of cerebellar ataxia. Besides their inability to learn during delay eyeblink conditioning and their decreased blink rate, spinocerebellar ataxia (SCA) 3 and 7 and multiple system atrophy patients are unable to time their blink reflex (unpublished results; see Fig. 19.12). In another study of the maximal amplitude of unconditioned eyeblink responses, “the blinks” varied using the same paradigms. This might indicate also that a brainstem structure like the olivary body is involved in the feedback control of reflex blinking, and not the deep cerebellar nuclei as suggested by Welsh.83

Conclusion The world of blinking and blinking-related movements is generated, regulated, and controlled by many brain structures. External factors, such as the environment, or internal factors, such as aging, can have great consequences for the normal function of the blink.

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Fig. 19.12 Spinocerebellar ataxia (SCA) 3 and 7, and multiple system atrophy patients are unable to time their blink reflex

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45. Aramideh M, Ongerboer de Visser BW (2002a) Brainstem reflexes: electrodiagnostic techniques, physiology, normative data, and clinical implications. Muscle nerve 26:14-30 46. Iwasaki M, Kellinghaus C, Alexoppoulos AV, Burgess RC, Kumar AN, Han YH, Lüders HO, Leigh RJ (2005) Effects of eyelid closure, blinks, and eye movements on the electroencephalogram. Clin Neurophysiol 116:878-885 47. Peshori KR, Schicatano EJ, Gopalaswamy, Sahay E, Evinger C (2001) Aging of the trigeminal blink system. Exp Brain Res 136:351-363 48. Evinger C, Bao JB, Powers AS, Kassem IS, Schicatano EJ, Henriquez VM, Peshori KR (2002) Dry eye, blinking and blepharospasm. Mov Disor 17:S75-S78 49. Collins MJ, Iskander DR, Saunders A, Hook S, Anthony E, Gillon R (2006) Blinking patterns and corneal staining. Eye & Contact Lens 32:287-293 50. Wouters RJ, Van den Bosch WA, Mulder PG, Lemij HG (2001) Upper eyelid motility in blepharoptosis and in the aging eyelid. Invest Ophthalmol Vis Sci 42:620-625 51. Ellwanger J, Geyer MA, Braff DL (2003) The relationship of age to prepulse inhibition and habituation of the acoustic startle response. Biological Psychology 62:175-195 52. Schicatano EJ, Peshori KV, Gopalaswamy R, Sahay E, Evinger C (2000) Reflex excitability regulates prepulse inhibition. J Neurosc 20:4240-4247 53. Leite LV, Cruz AA, Messias A, Malbonisson J (2006) Effect of age on upper and lower eyelid saccades. Braz J Med Biol Res 39:1651-1657 54. Stjernquist-Desatnik A, Skoog E, Aurelius E (2006) detection of herpes simplex and varicella-zoster viruses in patients with Bell’s palsy by the polymerase chain reaction technique. Ann Otol Rhinol Laryngol 115:306-311 55. Murakami S, Mizobuchi M, Nakashiro Y, Doi T, Hato N, Yanagihara N (1996) Bell palsy and herpes simplex virus: identification of viral DNA in endoneurial fluid and muscle. Ann Intern Med 124:27-30 56. Pastor P, Munoz E, Valldeoriola F, Valls-Sole J (1998) Enhanced blink rate and involuntary contralateral eye closure in patients with Bell’s palsy. Muscle nerve 21:1596 57. Syed NA, Delgado A, Sandbrink F, Schulman AE, Hallett M, Floeter MK (1999) Blink reflex recovery in facial weakness. An electrophysiological study of adaptive changes. Neurology 52:834-838 58. Schicatano EJ, Mantzouranis J, Peshori KR, Partin J, Evinger C (2002) Lid restraint evokes two types of motor adaptation. J Neurosc 22:569-576 59. Bell C (1830) The Nervous System of the Human Body [Appendix, Case 49]. Longman, Rees, Orme, Brown and Green, London, p 85-87 60. Bell C (1823) On the motions of the eye, in illustration of the uses of the muscles and nerves of the orbit. Philos Trans R Soc London 111:166-186 61. Bender MB (1960) Comments on the physiology and pathology of eye movements in the vertical plane. J Nerv Ment Dis 130:456-466 62. Ramanathan D, Conner JM, Tuszynski MH (2006) A form of motor cortical plasticity that correlates with the recovery of function after brain injury. PNAS 103:11370-11375 63. Gilden DH (2004) Clinical practice. Bell’s Palsy. N Engl J Med. 351(13):1323-1331 64. Gittins J, Martin K, Sheldrick J, Reddy A, Thean L (1999) Electrical stimulation as a therapeutic option to improve eyelid function in chronic facial nerve disorders. Invest Ophthalmol Vis Sci. 40:547-554 65. Cossu G, Valls-Sole J, Valldeoriola F, Munoz E, Benitez P, Aquilar F (1999) Reflex excitability of facial motoneurones at onset of muscle reinnervation after facial nerve palsy. Muscle & Nerve 22:614-620 66. Bour LJ, Aramideh M, Ongerboer de Visser BW (2000) Neurophysiological aspects of eye and eyeylid movements during blinking in humans. J Neurophysiol 83:166-176 67. Esteban A, Traba A, Prieto J (2004) Eyelid movements in health and disease. The supranuclear impairment of the palpebral motility. Neurophysiologie Clinique 34:3-15

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68. Aramideh M, Valls-Sole J, Cruccu G, Ongerboer de Visser BW (2002) Disorders of the cranial nerves, Ch 43. In: Brown WF, Bolton CF, Aminoff MJ (eds) Neuromuscular Function and Disease. WB Saunders, Philadelphia, p 757-780 69. Zadikoff C, Lang AE (2005) Apraxia in movement disorders. Brain 128:1480-1497 70. Lamberti P, De Mari M, Zenzola A, Aniello MS, Defazio G (2002) Frequency of apraxia of eyelid opening in thew general population and in patients with extrapyramidal disorders. Neurol Sci 23(Suppl):S81-S82 71. Zulch KJ (1970) Idiopathic facial paresis. In: Vinken PJ, Bruyn GW (eds) Handbook of Clinical Neurology, Vol 8. Elsevier, Amsterdam p 241-302 72. Digre K Corbett JJ (1988) Hemifacial spasm: differential diagnosis, mechanism, and treatment. Adv Neurol. 49:151-76 73. Eekhof JLA, Aramideh M, Bour Lj, Hilgevord AAJ, Speelman JD, Ongerboer de Visser BW (1996) Blink reflex recovery curves in blepharospasm, torticollis spasmodica, and hemifacial spasm. Muscle Nerve 19:0-15 74. Elston JS, Granje FC, Lees AJ (1989) The relationship between eye-winking, tics, frequent eye blinking and blepharospasm. J Neurol Neurosurg Psychiatry 52:477-480 75. Marsden CD (1976) Blepharospasm-oromandibular dystonia syndrome (Brueghel’s syndrome): A variant of adult-onset torsion dystonia? J Neurol Neurosurg Psychiatry 39:1204-1209 76. Weiss EM, Hershey T, Karimi M, Racette B, Tabbal SD, Mink JW, Paniello RC, Perlmutter JS (2006) Relative risk of spread of symptoms among the focal onset primary dystonias. Movement Disord 21:1175-1181 77. Aramideh M, Cruccu G, Valls-Sole J, Ongerboer de Visser BW (2002b) Cranial nerves and brainstem reflexes: electrodiagnostic techniques, physiology and normative data. In: Brown WF, Bolton CF, Aminoff MJ (eds) Neuromuscular function and disease, Ch 23, WB Saunders, Philadelphia, 433-453 78. Sommer M, Ferbert A (2001) The stimulus intensity modifies the blink reflex recovery cycle in healthy subjects and in blepharospasm. Clinical Neurophysiol 112:2293-2299 79. Kolb TF, Lachauer S, Schoch B, Gerwig M, Timmann D, Kolb FP (2006) Comparison of the electrically evoked leg withdrawal reflex in cerebellar patients and healthy controls. Exp Brain Res Oct 19 (Epub ahead of print) 80. Knuttinen MG, Power JM, Preston AR, Disterhoft JF (2001) Awareness in classical differential eyeblink conditioning in young and aging humans. Behav Neurosc 115:7447-757 81. Bellebaum C, Daum I (2004) Effects of age and awareness on eyeblink conditional discrimination learning. Behav Neurosc 118:1157-1165 82. Yeo CH, Hardiman MJ (1992) Cerebellar cortex and eyeblink conditioning: a reexamination. Exp Brain Res 88:623-638 83. Welsh JP (1992) Changes in the motor pattern of learned and unlearned responses following cerebellar lesions: a kinematic analysis of nictitating membrane reflex. Neuroscience 47:1-19 84. Gerwig M, Dimitrova A, Maschke M, Kolb FP, Forsting M, Timmann D (2004) Amplitude changes of unconditioned eyeblink responses in patients with cerebellar lesions. Exp Brain Res 155:341-351

Chapter 20

Age-Related Changes in the Oculomotor System J. Richard Bruenech, PhD

Abstract This chapter aims to review the most important parameters in oculomotor control and provide information regarding the functional implications of the age-related changes taking place in the oculomotor system. Age-related changes in muscle fibers such as loss of myofilaments and reduction in mitochondrial content will change the length tension curve of the muscle, making the relationship between the degree of contraction and development of muscle force (i.e., the degree of eye rotation), less predictable. Changes in the pattern of innervation is also likely to interfere with muscle dynamics and thus create an additional variable parameter in the length tension curve. The so-called fibrillen-structure fibers were found to be most affected. These muscle fibers may have more functions than previously assumed. The reduction in ocular motility observed in elderly patients may be caused by age-related changes, either directly through a reduced oculorotatory capacity or indirectly through a reduced ability to manipulate the angle of insertion of the distal tendon during eye rotation. Keywords oculomotor system, Motor unit, Nerve fibers, Sensory receptors, age related changes.

Introduction Age-related changes in the oculomotor system contribute to a number of common visual disorders observed in the mature population. The onset and extent of these changes vary considerably between individuals. While some people enjoy the privilege of good binocular vision throughout life, others exhibit restrictions in ocular motility long before they have reached middle-age. The chronological age of the patient is hence not a precise indicator of the process of senescence, although the incidence and diversity of age-related changes inevitably increases with age. The biological mechanisms behind these changes are not fully understood, but some of the factors that determine impairment of somatic motor systems seem to apply to the oculomotor system as well. Lack of cellular reproduction is regarded as one of the most important of these factors. Muscle fibers and neurons do not normally From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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proliferate after terminal differentiation occurs before birth. As a consequence, there will be no replacement of the cellular loss that occurs later in life. Other neurogenic and myogenic factors include reduced conduction capacity, alteration in neuromuscular transmission, and loss of myofilaments.1,2,3 Furthermore, the discharge frequency in the motor neurons serving all striated muscle fibers is influenced by a number of pre-motor areas in the cortex and brainstem. A progressive decline in neural interaction between these areas will influence the efferent signal to the extraocular muscles, as well as to their somatic counterparts. The co-contraction of extraocular muscles and somatic muscles—which is so essential to maintaining good visual perception during head and body movements—will suffer accordingly. This may, in turn, result in modification of behavior, such as changes in posture, balance, and hand-eye coordination that are commonly observed in elderly patients.4,5 Despite the intimate neural relationship between the somatic motor system and the oculomotor system, the functional principals of the respective systems are fundamentally different in many respects. The constant weight of the eye, along with the absences of a variable external load creates a fixed relationship between the efferent innervation to the extraocular muscles, and the resulting rotation of the eyes.6 This renders the demand for sensory feedback virtually redundant, and suggests that the role of proprioception in oculomotor control is different than in other somatic motor systems. The absence of a stretch reflex in extraocular muscles,7 and the complement of unique sensory receptors, seem to support this notion.8,9 In addition to unique physiological and morphological features, there is also a distinct organization of the distal insertion in these muscles. Human extraocular muscles have complex structures of collagen at their distal insertions that are believed to influence the line of pull of the muscle during eye rotations.10 This organization is inconsistent with the distal insertions of conventional somatic muscles, where tendon attaches directly to bone. These, and other factors addressed below, indicate that our current knowledge of the conventional somatic motor system can not serve as a satisfactory model for understanding the functional implication of age-related changes in the human oculomotor system. Several studies have documented age-related changes in saccadic velocity, optokinetic nystagmus, and smooth pursuit eye movements.11 Changes in these complex patterns of eye rotation strongly suggest that it is not only the extraocular muscles that are subjected to age-related changes, but also the central control mechanisms responsible for coordination and tuning of the various oculomotor functions. In other words, both the subnuclear and supranuclear level of the oculomotor system seem to be subjected to age-related changes. Age-related changes can occur simultaneously with pathological changes, and the differentiation between the two conditions can, in some cases, represent a diagnostic challenge. Detailed knowledge of the process of senescence can hence serve as a valuable clinical tool in enhancing the diagnosis and management of a broad spectrum of visual disorders. This chapter aims to review the most important parameters in oculomotor control, and provide information regarding the functional implications of the agerelated changes taking place in the tissues of the oculomotor system.

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Structural and Functional Organization of the Human Oculomotor System In addition to the muscles, the oculomotor system includes the three ocular motor nerves (III, IV, and VI), and all supranuclear structures acting upon their neurons. Through observations of pathological conditions in humans, and from animal experiments, we have found that the supranuclear stimulation arises from several neural components located in the brainstem, and the cerebellar and cortical systems.12 Structures such as the vestibular apparatus, superior colliculus, and frontal and parietal areas of the cortex have neural pathways connecting them with the ocular motor nuclei. They project either directly, through internuclear pathways such as the medial longitudinal fasciculus (MLF), or via immediate premotor structures such as the paramedian pontine reticular formation (PPRF). The sum of stimulation and inhibition from the supranuclear components will dictate the discharge frequency in the motor nerves (Fig. 20.1). Once the motor neuron is stimulated to discharge at a set frequency, the signal cannot be altered before contraction of the receiving muscle fibers has taken place. Any deviation between the predetermined movement and the one actually being performed can only be adjusted by restimulating the muscle in question or its antagonist. This neural arrangement was first observed in skeletal muscle many years ago and is now commonly referred to as the final common pathway. The final

Fig. 20.1 The figure summarizes the connections between the supranuclear structures participating in horizontal eye movement control. The supranuclear connections from the frontal eye fields (FEF) and the parietal eye field (PEF) project to the superior colliculus (SC) and the paramedian pontine reticular formation (PPRF). Drawing by IB Kjellevold Haugen

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common pathway from the nuclei of the three oculorotatory cranial nerves down to the extraocular muscles constitutes the subnuclear level of the oculomotor system.

Age-related Changes in the Supranuclear Level of the Human Oculomotor System Because age-related changes at the supranuclear level have other clinical manifestations compared to those at the subnuclear level, it is of clinical significance to differentiate between the two. The term supranuclear embraces all structures and neural activity that have a direct or indirect impact on the discharge frequency in the motor neurons of the III, IV, and VI cranial nerve nuclei. The ability to coordinate the activity in whole muscle groups rather than single muscles makes the supranuclear structures able to execute complex patterns of eye rotations, such as saccadic, optokinetic, vestibular, convergence, and smooth-pursuit eye movements. Age-related changes in the various supranuclear structures can compromise these gaze functions, and factors such as the weight loss that occurs in the brain as we grow older has been argued to be a contributing factor in the development of oculomotor anomalies. The reduction in weight, which represents approximately 8–10 percent between young and old, is attributed to loss of neurons and changes in intracellular content, extracellular volume, and/or reduction in cell processes.4 All of these neurogenic factors could influence the discharge frequency in the oculorotatory nerves and subsequently lead to the development of concomitant deviations. However, because only a minority of mature patients develop concomitant anomalies, there are clearly some adaptive mechanisms that can tune the system and compensate for the neural loss. This would require accurate sensory feedback from the extraocular muscles or other structures participating in ocular dynamics. One of the supranuclear structures that receive this type of input is the cerebellum. The role of the cerebellum as a coordinator of motor activity is reflected in the large number of ascending fibers in comparison to the rather modest number of fibers descending from it (40:1).

The Cerebellum The cerebellum has been implicated in a variety of oculomotor functions, and plays an essential role in the long-term adaptive process that compensates for oculomotor dysmetria. This function, which is essential for maintaining oculomotor performance throughout the ageing process,13 relies primarily on the neural input from the vestibular system, the proprioceptive system and specific cerebral cortical areas. Information from theses sources terminate in different regions of the cerebellar cortex and make it possible to divide the cerebellum into compartments or modules

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that all have different roles in the adaptive process. The flocculonodular lobe is referred to as the vestibulocerebellum, because fibers from the vestibular system terminate here and help to control balance and initiate compensatory eye movements. The vermis is called spinocerebellum because it receives proprioceptive information from the spinal cord and medulla. It is involved in the control of posture, locomotion, and fine motor coordination. The cerebellar hemispheres are called cerebrocerebellum, because they receive impulses from the cortex via the pons—therefore, also referred to as the as pontocerebellum. The cerebellar hemispheres are involved in planning, practicing, and learning complex movements.14 Regardless of their origin, all the afferent fibers terminate on the highly folded cortex of the cerebellum (Fig. 20.2). The cortex itself consists of three layers: an outer molecular layer, a central layer of Purkinje cells, and an inner granular layer (Fig. 20.3 and 20.4). Most of the axons that carry sensory information pass directly through to the deeper layers of the cerebellum on their way to the Purkinje cells. The Purkinje cells are the main efferent cells in the cerebellum, and axons from these neurons usually terminate on the same structures from which they receive afferent axons. In general this means that the vestibulocerebellum affects the vestibular nuclei, spinocerebellum affects motor neurons in the spinal cord, and cerebrocerebellum affects neurons in the cortex. The pathway from the cerebellum to the respective regions goes through the cerebellar nuclei, with the exception of the vermis. Efferent axons from the vermis descend directly down to the vestibular

Fig. 20.2 The micrograph shows the cerebellum of a Rhesus monkey. The cerebellum consists of two hemispheres divided by the vermis. Each hemisphere is divided into lobules, each of which has a superficial layer of gray matter (cortex) and a core of white matter. The section is stained with toluidine blue

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Fig. 20.3 The micrograph shows the various cortical layers of the cerebellum—the outer molecular layer, the central layer of Purkinje cells, and an inner granular layer. Rhesus monkey stained with toluidine blue

Fig. 20.4 The micrograph shows the Purkinje cells (large cells) in the cortex of the cerebellum of a Rhesus monkey. The section is stained with toluidine blue

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nucleus, where they terminate in the nuclear region that has connections to the oculorotatory nuclei via the MLF. Age-related changes in the neural input to the cerebellum, as described above, would interfere with a variety of visual reflexes, as well as voluntary based eye movements. The role of the vestibular system, proprioception, and cortical input is therefore worthy of some further consideration and will be dealt with.

Age-related Changes in the Vestibulocerebellar Pathway The cerebellum influences posture, balance, and equilibrium through the vestibulocerebellar pathways to axial and proximal limb muscles. Vestibulocerebellar dysfunction, caused by age-related changes or pathological conditions, are hence likely to result in unsteady gait and ataxic movements. The vestibular nuclei also innervate the three oculorotatory cranial nerves, and the role of the cerebellum has therefore been studied in modifiability of the vestibule-ocular reflex (VOR). When the head is turned to the left, there is a reflex tendency for the eyes to turn to the right. A three-neuron arc connects the horizontal semicircular canals to the extraocular motor neurons so that rotation of the head tends to produce an equal and opposite movement of the eyes—thus stabilizing the gaze. If the vestibule-ocular reflex fails to compensate completely for head movements, the image on the retina slips when the head is turned. It has been suggested that a mismatch of this sort between the vestibular input and the eye movement can be detected and forwarded to the flocculus located in the flocculondular lobe of the cerebellum. Purkinje cells in the flocculus can then serve to adjust the vestibule-ocular reflex.15 The sensory cells of the three semicircular canals convey their information through the short preganglionic nerve fibers of the vestibular nerve. The postganglionic fibers have two destinations—the cerebellum and the vestibular nucleus. The neural pathway that terminates directly in the flocculus of the cerebellum constitutes the primary vestibulocerebellar pathway. Those fibers that have synaptic interruptions in the vestibular nucleus constitute the secondary vestibulocerebellar pathway. The cerebellum has efferent fibers returning to the vestibular nucleus and hence can influence the efferent neurons interacting with motor neurons serving extraocular muscles as well as somatic muscles. Although the neural pathways concerned with oculomotor control are the most prominent,16 some Purkinje fibers in the vermis also send fibers to the lateral vestibular nucleus, which in turn is connected to the medulla spinalis. The latter projection—the tractus vestibule spinalis—is concerned with balance and movement of the extremities. Simultaneous contraction of somatic muscles and extraocular muscles are hence facilitated by these neural pathways, and compensatory rotations of the eye can be executed following head or body movements (Fig. 20.5). The vestibular system is capable of affecting both horizontal and vertical eye movements through ascending fibers in the medial longitudinal fasciculus. This long-fiber tract extends from the lower aspects of the brainstem up to the III nerve

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Fig. 20.5 The figure illustrates a vestibulocerebellar pathway. The primary afferent vestibulocerebellar pathway terminates directly in the cerebellum, while the secondary vestibulocerebellar pathway has a synaptic interruption in the vestibular nucleus before it terminates in the cerebellum (blue lines). The efferent fibers returning to the vestibular nucleus can influence those efferent neurons interacting with motor neurons serving extraocular muscles as well as somatic muscles (black lines). Drawing by IB Kjellevold Haugen

nuclear complex in the mesencephalon. Some fibers extend even further and terminate in two distinct nuclei that are referred to as the interstitial nucleus of Cajal and the rostral interstitial nucleus of the medial longitudinal fasciculus (rMLF). These nuclei are both involved in vertical gaze. The majority of the ascending fibers that travel in the ipsilateral aspect of the MLF derive from the superior aspect of the vestibular nucleus, while those fibers that cross over to the contralateral side arise from the medial aspect of the vestibular nucleus. Both components of the MLF supply (to a large extent) the oculomotor nuclei bilaterally via collaterals that cross the midline. The fibers that connect the abducens nucleus with the contralateral medial rectus subnucleus travel in the medial aspect of the MLF (Fig. 20.6). The role of the MLF becomes evident in pathological conditions, such as internuclear ophthalmoplegia, where the patient’s ability to adduct is lost. The fact that most of these patients are still able to converge, which is an ocular movement that also requires adduction, suggests that the neural pathway for convergence does not run through the MLF.17,18 However, clinical observations of reduced ability to perform horizontal conjugate eye movements, as well as convergence insufficiency among elderly patients, suggests that both of these neural pathways may be subjected to neurogenic age-related changes. Studies of eye movements among subjects over the age of 75 have revealed a significant reduction in speed and accuracy of the vestibule-ocular reflex (VOR) in comparison to that of younger age groups. Reduction in speed of the VOR would require a longer period of suppression of the sensory image. The ability to suppress the sensory image during the actual VOR movement is essential to maintaining orientation and focus, especially in situations where head movements and object movements occur simultaneously. The fact that suppression of the VOR movement has been found

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Fig. 20.6 The figure shows how axons from PPRF travel to the ipsilateral abducens nucleus, where they synapse with abducens motor neurons whose axons travel to the ipsilateral lateral rectus muscle (LRM). These axons also synapse with abducens interneurons whose axons cross the midline and travel in the medial longitudinal fasciculus (MLF) to the portion of the oculomotor nucleus concerned with medial rectus (MRM) function in the contra lateral eye. Drawing by IB Kjellevold Haugen

to decline with age may be a contributing factor to why elderly subjects find it difficult to orientate in visual environments with many moving objects. This may well also account for the confusion and dizziness some of them report.19 A significant age-related lag in the initiation of optokinetic nystagmus has also been reported. It has been postulated that there is a correlation between these subjective findings and the decline in sensory receptors in the semicircular canal of the vestibular system. Quantitative analysis suggests that more than 40 percent of the receptors have degenerated by the age of 75.20,21 Loss of these receptors will have implications for activity in all supranuclear components receiving afferents from the semicircular canals, as well as for the discharge frequency in the motor neurons in the oculorotatory nuclei. The cerebellum’s modifiability of the vestibule-ocular reflex may also be compromised with age for the same reasons.

Age-related Changes in the Spinocerebellar Pathway The afferent spinocerebellar pathways ascend from neurons in the spinal cord—either directly or indirectly—to the cerebellum. The direct pathways convey information from sensory receptors in somatic muscles such as muscle spindles and tendon

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Fig. 20.7 The figure illustrates the spinocerebellar pathway from proprioceptors in the somatic muscles up to the cerebellum (blue lines). Efferent pathways descend to the vestibular nucleus and the reticular formation facilitating control of somatic activity (black lines). Drawing by IB Kjellevold Haugen

receptors, while the indirect pathways provide additional information essential for head and eye movements (Fig. 20.7). The spino-olivar pathway, which is synaptically interrupted by the inferior olive nucleus in the medulla oblongata, is one of the essential indirect pathways. The olive nucleus receives a substantial input from the retina and visual cortex through the superior colliculus and the pretectal nucleus. Some of this information is projected to the lobus flocculonodularis and contributes to the tuning of the vestibule-ocular reflex. The cerebellum also receives ocular proprioception through the trigeminal nuclei because receptors in the extraocular muscles convey their sensory information via the ophthalmic division of the trigeminal nerve. Fibers from the trigeminal nuclei will, in turn, terminate in the spinocerebellum. This neural arrangement indicates that the proprioceptive information plays a vital role in tuning interactions between somatic and oculomotor control. Age-related changes altering the morphology of the receptors or associated afferent pathways would cause shifts in the supranuclear input and give rise to oculomotor anomalies. The Muscle Spindle The muscle spindle is regarded as one of the main sources of proprioception and plays a vital role in the motor control of most somatic muscles. Animal experiments have revealed that the sensitivity of the muscle spindles declines with age. The reason for the decline is not known, but it may be associated with age-related deficits in cholinergic signal transduction.22 Deficits in the transport, synthesis, and/or

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release of acetylcholine would interfere with the co-activation of intrafusal and extrafusal fibers, and could explain the delay in the reflex contractions that are initiated following changes in external load on the extremities in the elderly. However, the functions attributed to proprioception in somatic muscles are not necessarily valid for EOM.23 Decline in spindle sensitivity is therefore not a likely explanation for age-related changes in oculomotor functions. The eye’s center of gravity corresponds well with its center of rotation. The effect of gravity will therefore not act upon the muscles during rotation of the eye.24 Furthermore, the EOM load remains fairly constant during normal eye movements. These factors may explain the lack of a stretch reflex in the oculomotor system.25,26 In recent years, histological analysis of the muscle spindle has revealed a number of peculiar features that have raised questions regarding the proprioceptive capacity of this type of receptor. The majority of spindles in human EOM lack the generous periaxial space that provides protection from the mechanical force created by the extrafusal fibers during a muscle contraction. Furthermore, the intrafusal fibers are seldom in register, and often lack a modified region. In a significant number of spindles, large muscle fibers with extrafusal features can be found inside or embedded in the capsule wall. Few of the intrafusal fibers have accumulation of nuclei in the equatorial region that can justify the term nuclear bag fiber. A number of the intrafusal fibers are fragmented and fail to run the full length of the spindle. In some spindles, the periaxial space is left virtually free of intrafusal fibers (Fig. 20.8 and 20.9). These and other peculiar features, which cannot be considered beneficial to the function of a mechanoreceptor, have led to the conclusion that the ocular spindles are incapable of proprioception.23 The notion that these peculiarities

Fig. 20.8 Micrograph of a muscle spindle in human extraocular muscle showing interrupted intrafusal fibers and an empty periaxial space

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Fig. 20.9 Drawing of a muscle spindle with interrupted intrafusal fibers and empty periaxial space. The dotted line illustrates the level from where the micrograph is taken (see Fig. 20-8). Drawing by IB Kjellevold Haugen

could be caused by degeneration of the spindle after their role had been fulfilled in the earlier stages of life could be sustained, but the presence of the same features in infant subjects jeopardizes this view.27 The alternative view is that they have become phylogenetically redundant. However, the presence of redundant muscle spindles does not preclude the possibility that there might be other receptors present that are capable of fulfilling a proprioceptive role.

Tendon Receptors Although putative myotendinous receptors have been noted in the extraocular muscles of various species, the classical Golgi tendon organ (GTO) form has not been reported in man.27 Despite the absence of GTOs and functional muscle spindles, there seems to be a proprioceptive signal arising from the human EOMs. Intracranial ophthalmic neurectomy in monkeys has revealed ophthalmic nerve fibers entering EOMs.28 Most recent papers seem to support this finding and favor the ophthalmic nerve as the main route for proprioception also in man.29 Clinical observations of oculomotor deficits in patients with herpes zoster ophthalmicus add credence to this view.30 The interest in potential sources of proprioception was renewed following reports on alterations in position sense in patients who had undergone surgery in this region of the muscle.31 Recent studies have confirmed the presence of tendon receptors in human EOM, and their resemblance to the nerve endings at the musculotendinous junction in cats has led to the contention that they are of the same

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Fig. 20.10 Micrograph showing a transverse section through a myotendinous cylinder at the distal end of a Felderstruktur fiber

Fig. 20.11 The figure illustrates the structural organization of the myotendinous cylinder. The dotted line illustrates the level from where the micrograph is taken (see Fig. 2010). Drawing by IB Kjellevold Haugen

origin.32 Morphological varieties of this receptor have resulted in a variety of names, such as myotendinous cylinders33,34 and musculo-tendinous complexes.35 In the literature, these terms are often used as synonyms.36 These receptors are innervated by unmyelinated and myelinated nerve fibers with diameters ranging from 1 to 6 µm. These small, afferent nerve fibers are regularly occurring features throughout the length of the myotendinous junction in mature subjects, but infrequent in infants.27, 37,38 The distally located nerve terminals are exclusively associated with the multiply innervated Felderstruktur fibers (Fig. 20.10 and 20.11). Previous studies have demonstrated an age-related change in the number of these fibers, which suggests a corresponding decline in the number of receptors.39 The literature promotes the view that proprioception from extraocular

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muscles contributes to the reflexes that serve to stabilize the visual image on the retina. The notion that reduction in proprioception can cause decline of both the vestibule-ocular reflex and the optokinetic reflex cannot be dismissed. However, several sensory systems contribute to stabilizing the visual image on the retina during head movements, and proprioception from the muscles in the head and neck must also be taken into consideration. A recent study has raised the question of whether the cervico-ocular reflex, which serves to stabilize the retinal image through rotations of the neck, can compensate for the deficits in the vestibuleocular reflex (VOR). The results from this study are intriguing and suggest that there is a synergistic function between the VOR and cervico-ocular reflex, and that the latter reflex can be upgraded to compensate for the decline in VOR that occurs with age.40

Age-related Changes in the Cerebrocerebellum and the Pontocerebellar Pathway The pontine nuclei receive substantial input from the cerebral cortex, which is further projected to the cerebellum through the middle cerebral peduncles. The vast majority of the afferent fibers that terminate in the two hemispheres of the cerebellum originate from the pons, and the cerebrocerebellum is therefore also referred to as the pontocerebellum. The function of the pontocerebellum is primarily planning and control of somatic muscle activity and timing of their contractions, including that of the extraocular muscles. Single-cell recordings from selected cortical regions have revealed neuronal activity prior to conjugate eye movements.12 The most essential of these regions are the frontal eye field and the parietal eye field. These two cortical areas are responsible for saccadic and pursuit eye movements, respectively, and have neural pathways that descend to premotor structures in the pons and associated areas in the brainstem.

The Frontal Eye Field (FEF) The FEF is located in the frontal lobe of each hemisphere and coincides with the cortical area 8 of Brodmann. The neural pathways from the FEFs descend through the capsula interna, and decussate before terminating in premotor regions such as the superior colliculus, pretectal nucleus, and paramedian pontine reticular formation (PPRF). From this, it follows that neural stimulation of the right FEF initiates conjugate eye movements to the left, while the left FEF initiates movements to the right—in both cases, the movements will be saccadic in nature. These jerky discontinuous eye movements have dynamic characteristics that vary to a certain degree according to the nature of the saccade. The basic features, however, can be summarized as rapid accelerating eye movements of short duration with a peak velocity of about 400–600 deg/s and an amplitude of less than 15 degrees.

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Fig. 20.12 Micrograph of the reticular formation of a monkey. The reticular formation is an ill-defined collection of neurons and fibers that extends through several regions of the brainstem, including the medulla, pons, and mesencephalon

Several studies have documented age-related changes in duration, velocity, acceleration, and accuracy of horizontal saccades.40,41,42 This indicates that the subject’s ability to redirect the central retina to a new object of interest declines with age. In general, saccades disturb visual processing because the visual acuity is reduced while the visual image is swept across the retina. If the speed of the saccades is reduced, this disturbance is no longer kept to a minimum. A reduction in accuracy will disturb visual processing even further, because a corrective movement will extend the time allocated to refixate the target. Similar deficits have been found in the control of vertical saccades.43 In a recent study, it was found that age deteriorates the ability to trigger regular volitional vertical saccades, but not the ability to produce reflex initiated saccades.44 These findings were defended to reflect the fact that there is a widespread atrophy of both gray and white matter in the cerebral cortex, affecting both the frontal lobe and the posterior cortex. The neural pathway for volitional vertical saccades originates in the frontal eye field and projects to the rostral interstitial nucleus of the medial longitudinal fasciculus (rMLF). Reflexive saccades, on the other hand, can be generated by the occipitaltectal system and are hence not affected. In a more recent study by the same authors, it was found that aging only has a minimal affect on the overall accuracy of vertical saccades due to control mechanisms in the brainstem and cerebellum.45 The clinical implications of age-related changes in the frontal eye field are not limited to deficits in saccadic eye movements, but may also affect the vergence system. The neural pathway for the vergence system is not fully explored, but neurons in the primary visual cortex are believed to provide input to the frontal eye field. The FEF and possibly other visual areas provide input to cells with vergencelinked activity in the cerebellum. Cerebellar signals go to supraoculomotor areas,

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which in turn go to motor neurons in the abducens nucleus or the medial rectus subnucleus—for divergence or convergence, respectively. Atrophy of cortical neurons could also affect the interaction between the motor neurons in the III nerve nuclear complex and jeopardize the simultaneous contraction of smooth muscles in the ciliary body/iris and the striated muscle fibers in the medial rectus.

The Parietal Eye Field (PEF) Clinical observations of pursuit eye movements in patients of different age groups have shown that older patients have difficulties in following slowly moving targets.42 The control of the smooth pursuit movements arise from the lateral parietal and mid-temporal cortices—also referred to as the parieto-occipito-temporal junction in previous literature. The cluster of cortical neurons that constitute the left parietal eye field controls smooth pursuits to the left, and a similar ipsilateral innervation applies for the right eye field, which controls pursuits to the right. The immediate initial motor signal arises from neurons in the (PPRF), which in turn has neural pathways to the cerebellum and superior colliculus. Disorders of the pursuit system can hence be caused by changes in neurons or the associated neural pathways of the PEF, the PPRF, the cerebellum, or segments of the brainstem. The function of this system can be monitored by making the patient trace a moving target with their eyes. The patient’s inability to perform pursuit movements is usually compensated by a number of small saccades to maintain fixation. These types of saccadic eye movements are frequently observed in elderly patients with age-related changes in their pursuit motor system. However, disorders of the smooth pursuit system in elderly patients may not be exclusively associated with structural changes. They can also be caused by other factors associated with old age, such as medication and fatigue.

The Paramedian Pontine Reticular Formation (PPRF) The reticular formation is responsible for coordination of complex patterns of body movements and facilitates simultaneous contraction of muscles involved in head and neck rotations, as well as eye movements. The ventral reticulospinal pathway is of special importance when it comes to movements of the extremities, while the pathway from the superior colliculus to reticulospinal neurons is of importance for movements initiated to move the head and upper body towards new objects of interest in the visual field. The median region of the reticular formation—the PPRF—is allocated to the control of horizontal conjugate eye movements, and is frequently referred to as the horizontal gaze center. It extends from the level of the trochlear nerve nuclei and up to abducens nuclei, and consists of neurons of variable sizes.12 This seemingly random distribution of neurons and nerve fibers gives the PPRF the reticular appearance from which the name is derived (Fig. 20.14). Other structures—also known to be associated with eye movement control—have extensive efferent projections to the PPRF. These

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Fig. 20.13 The micrograph shows neurons and corresponding nerve fibers in the paramedian pontine part of the reticular formation of monkey

distant premotor structures include the vestibular nuclei, cerebellum, frontal eye field, the pretectal nuclei, and the superior colliculus. The PPRF is therefore the immediate premotor structure responsible for executing conjugate horizontal saccades, and contains neurons that fire immediately before and during ipsilateral saccades. The same neurons are found to be inactive during smooth pursuit eye movements and when the eyes are fixating. Excitatory burst neurons are believed to activate the required number of motor units in the medial rectus subnuclei and contra lateral abducens nucleus. Corresponding inhibitory neurons inhibit the antagonistic muscles during the duration of the saccade. The paramedian reticular formation, which occupies only a part of the whole reticular formation, consists of neurons with large intercellular distances in comparison to other premotor and cortical regions (Fig. 20.13). This indicates that that when one neuron dies, there will be a limited number of neighboring neurons to replace it. Clinical observations of patients with lesions in the PPRF seem to confirm this notion. These patients present large deficits in horizontal eye movements, even in cases where the estimated cellular damage is limited. However, it seems that progressive age-related changes in the PPRF do not cause the same deficits as sudden lesions do—suggesting a certain ability to adapt to the process of senescence.

Superior Colliculus (SC) On the dorsal aspect of the mesencephalon, there are four elevations referred to as the superior and inferior colliculi (corpora quadrigemina). The superior colliculus receives fibers from the optic nerve, visual cortex, and other cortical regions, including the somatosensory cortex. This sensory input enables SC to initiate reflex

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movements in response to various stimuli. Several neurons in the mesencephalon have descending fibers to the spinal cord, and many of these originate from the superior colliculus. This pathway—the tectospinal pathway—provides contraction of somatic muscles in the head and neck. The SC also influences the motor neurons in the oculorotatory cranial nuclei through reticular formation. Together, these pathways provide reflex-based direction of the eyes towards the object of interest. The SC is also likely to participate in auditory reflexes through interneurons from the inferior colliculus, which is responsible for conveying auditory information, to higher levels of the central nervous system. Adaptation of the latter reflex is essential because the auditory input varies over a large scale. The adaptive process is influenced by the facial and trigeminal nuclei, which innervate muscles in the middle ear such as the stapedius muscle and the tensor tympani. These muscles reduce the effect of the auditory signal and protect the system against loud sounds. Facial palsies can hence create oversensitivity to sound because this protective mechanism is then lost. A similar affect can be caused by age-related changes in the neuromuscular arrangement. The muscle fibers in the muscles referred to above have histological features that correspond to the slow contracting multiply innervated muscle fibers found in the extraocular muscles. Previous studies have shown that these fibers are subjected to a variety of age-related changes (described in “Age-related Changes in the Muscle Fiber Population”). Decline in the auditory functions with age may therefore include an inability to adapt to sound, in addition to reduced sensitivity to various frequencies. Declining auditory functions in oculomotor control can also be caused through atrophy of interneurons connecting the superior and inferior colliculus. The SC also participates in the accommodation reflex to a larger extent than previously assumed. Axons from the visual cortex are believed to terminate on the SC in addition to the pretectal nucleus, with further projections to the PPRF. From there, axons travel to the parasympathetic motor neurons in the superior aspect of the III nerve nuclear complex. Stimulation of these neurons will, in turn, initiate contraction of the ciliary muscle through postganglionic parasympathetic nerve fibers.

Age-Related Changes in the Subnuclear System of the Oculomotor System The final common pathway from the motor neurons in the III, IV, and VI cranial nerves, down to their termination on the extraocular muscle fibers, constitutes the subnuclear level of the oculomotor system. This pathway has a generous complement of efferent nerves in comparison to the rather modest number of muscle fibers they supply (i.e., a small motor unit). It also contains a large number of afferent nerve fibers conveying information from a unique complement of sensory receptors not compatible with their somatic counterparts. The extraocular muscles are therefore a diverse muscle mass with a structural and functional organization that differs from skeletal muscle in numerous

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respects. From this, it follows that our current understanding of the aging process in somatic muscles does not necessarily serve as a good model for understanding the age-related changes taking place in the oculomotor system.

Cranial Nerves The nucleus of the third cranial nerve consists of a series of cell columns in pairs on either side of the midline in the mesencephalon. With exception of the medial rectus muscle, which receives input from the contralateral subnucleus, all muscles innervated by the third nerve receive uncrossed input from their corresponding ipsilateral subnuclei. The levator palpebrae muscle, which is also innervated by this nerve, receives a bilateral input. In contrast to the IV and VI cranial nerves, the III nerve also carries parasympathetic innervation to the iris and ciliary body. The fourth cranial nerve—the trochlear nerve—innervates the superior oblique muscle. This cranial nerve decussates shortly after it emerges from the dorsal aspect of the brainstem. The left nucleus innervates the right superior oblique muscle, and vice versa. The sixth cranial nerve innervates the temporal rectus muscle, which, in turn, abducts the eye. The III, IV, and VI cranial nerves carry myelinated and unmyelinated axons in the region of 1–20 µm (Figs. 20.14 and 20.15). Recent studies of human extraocular muscles have revealed that these muscles are innervated by a larger complement of unmyelinated efferent nerve fibers than previously assumed.46 The fibers were traced in serial sections and found to terminate on the Felderstruktur fibers. When all the unmyelinated nerve fibers were taken into account, the efferent innervation

Fig. 20.14 Electron micrograph showing small unmyelinated and myelinated nerve fibers Kjellevold Haugen I-B, Bruenech JR. (2005) Histological analysis of the efferent innervation of human extraocular muscles In: De Faber, J-T. (ed.) 29th European Strabismological Association Meeting Transactions, Izmir, Turkey, June 1–4, 2004, ISBN: 0415372119, Publisher Taylor & Francis

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Fig. 20.15 Histogram illustrating the mean value of all nerve fiber diameter spectrums. The trimodal distribution was apparent in all specimens Kjellevold Haugen I-B, Bruenech JR. (2005) Histological analysis of the efferent innervation of human extraocular muscles. In: De Faber, J-T. (ed.) 29th European Strabismological Association Meeting Transactions, Izmir, Turkey, June 1–4, 2004, ISBN: 0415372119, Publisher Taylor & Francis

of these multiply innervated muscle fibers were found to be profoundly more generous than previously assumed, with a motor unit ranging from 1:1 to 1:3. The ratio for the Fibrillenstruktur fibers was 1:7 to 1:10, depending on the morphology of the associated muscle fiber. The low motor units, which have seemingly been missed in previous studies using lower resolution techniques, have functional implications and suggest that EOMs have the ability to recruit one muscle fiber at a time. Adjusting muscle contraction by adding the force of one single muscle fiber is the most precise motor control theoretically possible. There is an apparent change in the size of the motor unit with age. This is caused by a decrease in muscle fibers with age (addressed next). Because the nerve fibers do not seem to suffer the same extent of degeneration, there are many redundant nerve fibers in adult human extraocular muscles attempting to seek new targets.46 Alterations in the size of the motor unit will have functional implications, not only in terms of a reduction in muscle force, but also in terms of interfering with the correlation between the efferent signal traveling in the final common pathway and the corresponding rotation of the eye. Unless the information from the sensory receptors within the muscle can be tuned to fit the new situation, there will be a neural disagreement between proprioception, efferent innervation, and the general visual information.

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Extraocular Muscles Extraocular muscle fibers do not normally proliferate after terminal differentiation occurs before birth, which, in turn, results in a decline in muscle fibers with age. The lack of a replacement of these fibers is regarded by some workers to be one of the primary causes of decline in ocular motility with age. EOMs proliferate from mononucleated cells into mature multinucleated muscle fibers, and by the 18th week of gestational age, most muscle fibers have differentiated into specific fiber types.47,48 In contrast to skeletal muscles, they also contain nontwitch muscle fibers that are usually the first to develop. They are innervated by small myelinated or nonmyelinated axons arising from motor neurons in the periphery of the three oculorotatory nuclei. Based on previous observations of muscle fibers receiving more than one axon, it has been postulated that polyneural innervation is an early, temporary phenomenon that later changes into a monomer innervation.49 The nature of the multiply innervated muscle fiber in extraocular muscles is not fully explored, and suggested presence of polyneural innervation in adults is still controversial. Previous studies of infant extraocular muscles have shown that although most of the neuromuscular arrangement is genetically predetermined, there is a significant postnatal progressive modification of these fibers. In specimens obtained from infant subjects, numerous muscle fibers with central areas free of contractile material were found in various regions of the muscles.50 The presence of successive centrally placed nuclei with features matching the myotube cell of immaturity will affect the contractile properties of the muscle.16 It is reasonable to assume that developmental delay in the extraocular muscles, with accompanied accumulation of immature muscle fibers, affects the contractile properties of the muscles and development of binocular vision (Fig. 20.16).

Fig. 20.16 Micrograph of human extraocular muscle fibers with immature features

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Failure to establish binocular vision will affect the normal growth and development of the muscle fiber and associated nerves.51 As a result, these factors may contribute to early onset of the structural age-related changes observed in young adults. As the extraocular muscles reache maturity, most of the distinct structural diversities of these muscle fibers can be observed at light microscopic level. The densely stained muscle fibers have sparse amounts of sarcoplasmic reticulum and are innervated by small efferents with diameters in the region of 1–4 µm.52 The pale staining fibers have larger diameters, fine stippled appearances and well-delineated myofibrils. Large myelinated axons are frequently found to terminate on such muscle fibers, displaying motor end plates with terminal boutons clearly indenting the sarcolemma. The two distinct fiber types (Figs. 20.17 and 20.18) have previously been described by others as Felderstruktur and Fibrillenstruktur fibers.53,54,55

Fig. 20.17 Micrograph showing Felderstruktur fibers and Fibrillenstruktur fibers

Fig. 20.18 Electron micrograph showing Felderstruktur and Fibrillenstruktur fibers

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The existence of two morphologically distinct fiber types in extraocular muscles has led to the notion that they may serve different functions in oculomotor control. The nature of the slow fiber system, comprised of the Felderstruktur fibers, has been subject to the most speculation. The existence of such a system must clearly have some functional implications, and the supply of strong tonic activity in eye fixation has been suggested as one plausible function.56 Accepting this view implies that subjects with a low concentration of Felderstruktur fibers in their extraocular muscles would be more subjected to fixation instability and fatigue during prolonged close work. Previous estimates have found that the Felderstruktur fiber constitutes approximately 20 percent of the fiber population in human extraocular muscles.57,58 However, recent studies have found that even though the majority of subjects fall within the range of 18 and 23 percent, significant variations between healthy individuals do occur.52 The potential implications of a low complement of Felderstruktur fibers has been investigated in a recent study where there seems to be a correlation between the number of Felderstruktur fibers and congenital oculomotor abnormalities.50

Age-related Changes in the Muscle Fiber Population Age-related changes in extraocular muscle fibers are well-documented in the ophthalmic literature and there is a consistency in the observations of the senescence process in these muscles.57,59,60 The most commonly observed features were described in a recent paper, and new information regarding the pattern of innervation was added.39 The study in question confirmed many of the morphological features previously described by others. Fragmentation and loss of myofilaments (Fig. 20-19a), along with the presence of lipofuscin (Fig. 20.19b), were regularly occurring features in muscles from subjects over 70 years of age. Muscle fibers with concentric striated annulets of myofibrils, resembling the previously described Ringbinden fibers (Fig. 20.19c), was a regular feature in all mature muscles and increase in number with age.39

Fig. 20.19 Micrographs showing fragmentation of myofilaments (A), accumulations of lipofuscin (B), and a muscle fiber displaying concentric striated annulets of myofibrils (C) Kjellevold Haugen I-B, Bruenech JR. (2006) Age-related neuromuscular changes in human extraocular muscles. In: R. Gomez de Liano (ed.) 30th European Strabismological Association Meeting Transactions, Killarney, Co Kerry, Ireland, June 8–11, 2005, Publisher Taylor & Francis. (reprinted with permission)

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Fig. 20.20 An electron micrograph of a Fibrillenstruktur fiber with modest amounts of mitochondria (A) and a light micrograph of Fibrillenstruktur fibers (B) and corresponding nerves embedded in connective tissue Kjellevold Haugen I-B, Bruenech JR. (2006) Age-related neuromuscular changes in human extraocular muscles. In: R. Gomez de Liano (ed.) 30th European Strabismological Association Meeting Transactions, Killarney, Co Kerry, Ireland, June 8–11, 2005, Publisher Taylor & Francis. (reprinted with permission)

Through electron microscopy, the size and number of mitochondria was found to decline with age (Fig. 20.20a), which also is in agreement with previous reports.61 The nerve fibers were frequently found to share their connective tissue sheet with associated muscle fibers, making their point of termination predictable (Fig. 20.20b). The perineural sheathing of muscle fibers was not observed in tissues from young subjects—an observation consistent with previous reports.62 A counting of fibers revealed a reduction in muscle fiber population with age. The oldest subjects were found to have a decline in muscle fiber content of more than 50 percent in comparison to the youngest subjects. In contrast, the numbers of nerve fibers were sustained. On most Fibrillenstruktur fibers, the nerve fiber terminated in a single, conventional motor endplate (MEP), displaying prominent boutons, postsynaptic folds, and soleplate nuclei. In selected specimens from the oldest subjects, muscle fibers were found to have more than one MEP.39 In a number of these samples, the MEPs were found to be interconnected by single myelinated axons with a neural arrangement best described as multiple innervation (Fig. 20.21), yet with clear structural differences from the innervation of Felderstruktur fibers. In other samples, however, there were no neural connections between the two MEPs, nor was any common origin revealed between the motor nerves when traced backwards to their point of entry into the muscle. These muscle fibers seemed to be served by more than one motor neuron with a seemingly polyneural innervation (Fig. 20.22). Muscle fibers holding more than one MEP suggest that the neuromuscular arrangement in ageing EOM is labile. The polyneural innervation and the observed reduction of muscle fiber content indicate that redundant nerve fibers may seek new targets—subsequently forming more than one MEP. This progressive neural reorganization, along with other age-related changes such as the loss of myofilaments and a reduction in mitochondrial content, will

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Fig. 20.21 Micrographs of two motor endplates associated with the same Fibrillenstruktur fiber. The MEPs were found to be interconnected by a myelinated axon, as described in the middle illustration. The conventional innervation of Felderstruktur fibers is illustrated at the top of the middle figure

Fig. 20.22 Micrographs of two motor endplates associated with the same Fibrillenstruktur fiber. In contrast to micrographs in Fig. 20.21, the two MEPs were not interconnected (middle illustration) and had seemingly no common neural origin. The conventional innervation of Felderstruktur fibers is illustrated at the top of the middle figure

arguably change the length-tension relationship of the muscle, making the correlation between the degree of contraction and development of muscle force less predictable. Furthermore, age-related changes in the neuromuscular arrangement of human extraocular muscles might have other implications than those related to muscle contraction and the length tension curve. Recent observations promote the view that a significant portion of muscle fibers depart from the main bulk of the muscle and terminate on connective tissue related to Tenon’s capsule.63,64 Any potential function these fibers may have on the distal insertion, or the so-called sleeve/pulley system, would therefore suffer accordingly. The reduction in ocular motility observed in elderly patients may be caused by some of the age-related changes described above, either directly through a reduced oculorotatory capacity or indirectly through a reduced ability to manipulate the insertion-angle of the distal tendon during eye-rotation. The latter concept is worthy of some further consideration and is described next.

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Age-related Changes in the Distal Insertions and Associated Structures Collagen consists of long-chain macromolecules produced by fibroblasts. During production of new macromolecules, the new fibers become enmeshed with old fibers and form crosslinks. This process forms the basis for the crosslink theory of aging, which promotes the view that the crosslink process increases the density of the collagen molecules that, in turn, decreases the capacity to transport nutrients into the cells.3 Removal of waste products from the cells is believed to decrease for the same reason. This theory has potential implications for collagen-rich tissues, such as the extraocular muscles that have large amounts of epimysium, perimysium, and endomysium compared with their somatic counterparts. The crosslink theory of aging has been subjected to criticism over the years, and the metabolic consequences of crosslinkage of the macromolecules are still debated. However, regardless of the validity of this theory, it is legitimate to argue that any age-related changes affecting the structural organization of collagen would have great implications for the proposed role of the distal insertion of human extraocular muscles. Histological examination of rectus muscles and magnetic resonance imaging of healthy volunteers has revealed evidence that not all muscle fibers insert onto the globe. A substantial number of fibers insert in the orbital side of the fibroelastic Tenon’s capsule, forming a connective tissue pulley or muscle sleeve.63,64,65 According to the pulley hypothesis, the distal ends of the muscles slide through the sleeves/pulleys, which act, in principal, as the muscle origin. By altering the position of the sleeves through separate adjustment of the orbital fibers, the axis of rotation can be changed.66,67 This theory is attractive in the sense that it explains how pulleys can be manipulated so that the eye can comply with Listings law (any orientation of the eye is attainable by rotating around axes lying in Listing’s plane), and many of the mechanical and neural aspects of the pulley hypothesis have been demonstrated through sophisticated and elaborate mathematical models.68 Over the years, a steadily increasing number of observations have confirmed that the orbital fibers of rectus muscles separate from the global fibers and insert in the muscle sheath.63 The double insertion suggests that orbital fibers are unlikely to contribute significantly to ocular rotation (Fig. 20.23). The functional implications of this observation are still being debated, and the degree of differential contraction between the orbital and global fibers, as well as the muscle fibers’ ability to slide through the sleeve/pulley, remains unresolved. Histological examination of both monkey and human material has only demonstrated a modest separation between the orbital and global layers, and the connective tissue between them seems to be continuous with the collagen of the surrounding pulley/muscle sleeve.63 Although such observations offer little indication of that sliding could occur, they do not preclude the notion that the pulley may fulfill its proposed role. New models (coordinated pulleys, weak differential pulleys, and strong differential pulleys) have been promoted to investigate how different degrees of freedom between the various layers would affect ocular rotation.66 Detailed information regarding the principals of these various models is beyond the scope of this chapter, but if we accept the notion that the pulleys/muscle sleeves

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Fig. 20.23 Distal insertion of human extraocular muscles described by Ruskell et al in: Ruskell GL, Haugen IB, Bruenech JR, Van der Werf F (2005) Double insertion of extraocular rectus muscles in man and the pulley theory. Journal of Anatomy 206, 295–306

play a vital role in human eye movement, then these structures must also be taken into consideration when the process of senescence is addressed. This was done in a recent study where normal functional anatomy of rectus muscles and pulleys in older humans was compared with previously reported findings in younger subjects.69 The results from this study demonstrated significant changes in the functional anatomy of the horizontal rectus muscles with age, suggesting that the pulleys in older people have a different location on the globe than in younger people. The inferior displacement of the pulleys was argued to convert the force of the rectus muscles to depression. These findings could explain the impairment of elevation observed in older people and their predisposition to incomitant oculomotor anomalies. The structural organization of the extraocular connective tissue and its potential role in ocular motility is still a matter of debate and awaits further investigation.70

Restitution of the Oculomotor System The oculomotor system must be able to execute very fine graded and synchronized contractions to maintain binocularity. Even minute structural changes at any level of this system should, in principal, have large functional implications and disrupt ocular motility. Yet, the vast majority of the population enjoys good binocular vision throughout their old age—seemingly unaffected by the progressively increasing number of age-related changes as described in this chapter. This has led to the contention that cells in the oculomotor system must have regenerative properties that counteract the process of senescence.45

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Authorities within the field have recently provided evidence suggesting that extraocular muscles have the unique ability to retain a population of activated satellite cells that provide for continuous myofiber remodeling throughout life.71 The myogenic precursor cells are believed to retain their proliferative properties even in aging muscles, and may be more robust and long-lived than their counterparts in limb muscles. These findings suggest that extraocular muscles have unique abilities to survive aging, injury, functional degeneration and disease. Furthermore, the authors of the same study have raised exciting questions regarding the potential use of this tissue in repair or reconstruction of other tissues, such as nervous tissue and bone.71 This opens new possibilities for treatment of oculomotor anomalies, as well as a broad spectrum of other clinical conditions. Although embryonic origin and histological composition determine much of the regeneration properties of a given tissue, there are other factors that can influence the speed and extent of cellular repair. Experimental studies in cell cultures, and in vivo, show that activity-dependent production of neurotrophic factors plays a role in promoting neuronal survival and growth.4 This is supported by epidemiological studies that suggest that humans with active minds have a reduced risk of developing neural decline as they age4. The use it or loose it concept has prevailed for a long time, and has formed the basis for many treatment regimes, including the management and treatment of binocular anomalies. The fact that muscle function can be restored through systematic activation—such as orthoptic exercises or other forms of neuromuscular stimulation—is well-documented. The literature also offers documentation of the reverse effect in cases of neuromuscular inactivity. Vacuolization, loss of tissue substance, and other evidence of atrophy has been found in extraocular muscles obtained from strabismus patients and other conditions associated with reduced muscle tone. However, activated satellite cells have been found in the same type of muscles, suggesting that there is also a regenerative process taking place.72 The speed and extent of this process seem to vary between individuals suffering from the same oculomotor anomalies. The notion that the unique regenerative properties of extraocular muscles can be enhanced through specific activity or treatment regimes cannot be dismissed. This could account for some of the individual differences in regenerative activity observed in the aged population, and may explain why the chronological age of the patient is not a precise indicator of the process of senescence. Furthermore, it may also explain some of the functional improvements obtained through various unconventional treatment regimes used in clinical practice.

References 1. Jenkyn LR, Reeves AG, Warren T, Whiting RK, Clayton RJ, Moore WW, Rizzo A, Tuzun IM, Bonnett JC, Culpepper (1985) Neurologic signs in senescence. Arch Neurol 42:1154 2. Kokemon E, Bossemeyer RW Jr, Barney J, Williams WJ. (1977) Neurological manifestations of aging. J Gerontol 32:411

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3. Loeser RF, Delbono O (2003) Aging of the muscles and joints. In: Hazzard WR, Blass JP, Halter JB, Ouslander JG, Tinetti ME (eds) Principles of Geriatric Medicine & Gerontology, 5th ed. McGraw-Hill, PUBLICATION CITY, p 905–919 4. Pakkar A, Cummings JL (2003) Mental status and neurologic examination in the elderly. In: Hazzard WR, Blass JP, Halter JB, Ouslander JG, Tinetti ME (eds) Principles of Geriatric Medicine & Gerontology, 5th ed. McGraw-Hill PUBLICATION CITY, p 111–119 5. Tinetti ME (1986) Performance-oriented assessment of mobility problems in elderly patients. J Am Geriatr Soc 34:119 6. Collins CC (1971) Orbital mechanics. In: Bech-y-Rita P, Collins CC, Hyde JE (eds) The control of eye movements. Academic Press, PUBLICATION CITY, p 285–325 7. Ruskell GL (1999) Extraocular muscle proprioceptors and proprioception. Prog Retin Eye Res 18:126–128 8. Breinin GM (1957) Electromyographic. Evidence of ocular muscle proprioception in man. Arch Ophthalmol 176–180 9. Keller EL, Robinson DA (1971) Absence of a stretch reflex in extraocular muscle of monkey. J Neurophysiol 34:908–919 10. Demer JL, Miller JM, Poukems V, Vinters HV, Glasgow BJ (1995) Evidence for fibromuscular pulleys of the recti muscles. Invest Ophth Vis Sci 36:1125–1136 11. Clarc RA, Isenberg SJ (2001) The range of ocular movements decreases with ageing. J AAPOS 5:26–30 12. Büttner U, Büttner-Ennever JA (1988) Present concepts of oculomotor organization. In: Büttner-Ennever (ed) Neuroanatomy of the oculomotor system. Elsevier 1–23 13. Robinson D (1975b) How the oculomotor system repairs itself. Invest Ophthalmol 14:413–415 14. Heines DE, Mihailoff GA, Bloedel JR (2006) The Cerebellum. In: Heines DE (ed) Fundamental Neuroscience for Basic and Clinical Applications, 3rd ed. Elsevier p 432–449 15. Ito M (1982) Cerebellar control of the vestibulo-ocular reflex-around the flocculus hypothesis. Annu Rev Neurosci 5:275–296 16. Lisberger SG (1998) Physiologic basis for motor learning in the vestibule-ocular reflex. Otolaryngol Head Neck Surg. 119: 43–48 17. Anderson TJ, Jenkins IH, Brooks DJ, Hawken MB, Frackowiak RSJ, Kennard C (1994) Cortical control of saccades and fixation in man: A PET study. Brain 117: 1073–1084 18. Horn AKE, Buttner U, Buttner-Ennever JA (YEAR) Brainstem and cerebellar structures for eye movement. PUBLISHER, CITY 19. Kerber KA, Enrietto JA, Jacobsen KM, Baloh RW (1998) Disequilibrium in older people-A prospective study. Neurology 51: 574–580 20. Balaban CD (1999) Vestibular autonomic regulation (including motion sickness and mechanisms of vomiting) 12:29–33 21. Brandt T, Dieterich M (1999) The vestibular cortex:Its location, function and disorders. Ann N Y Acad Sci 871:293–312 22. Matthews PBC (1991) The human stretch reflex and the motor cortex. Trends Neurosci 14:87–91 23. Ruskell GL (1989) The fine structure of human extraocular muscle spindles and their potential proprioceptive capacity. J Anat 167:199–214 24. Collins CC (1971) Orbital mechanics. In: Bach-y-Rita P, Collins CC, and Hyde JE (eds) The Control of Eye Movements Academic Press, New York, p 285–325 25. Breinin GM (1957) Electromyographic evidence of ocular muscle proprioception in man. Arch Ophthal 57:176–180 26. Keller EL, Robinson DA (1971) Absence of a stretch reflex in extraocular muscle of the monkey. J Neurophys 34:908–919 27. Bruenech JR, Ruskell GL (2001) Muscle spindles in extraocular muscles of human infants. Cel Tiss Org 169:388–394 28. Ruskell GL (1983) Fibre analysis of the nerve to the inferior oblique muscle in monkeys. J Anat 137: 445–455

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29. Miller NR (1985) Central trigeminal pathways of the extraocular muscle spindles. In: Miller NR (ed) Walsh & Hoyt’s Clinical Neuro-Ophthalmology Williams & Wilkins, Baltimore, p 1030–1032 30. Campos EC, Chiesi C, Bolzani R (1986) Abnormal spatial localisation in patients with herpes zoster ophthalmicus. Arch Ophthal 104:1176–1177 31. Steinbach MJ, Smith DR (1981) Spatial localisation after strabismus surgery. Evidence for inflow. Sci 213:1407–1409 32. Richmond FJR, Johnson WSW, Baker RS, Steinbach MJ (1984) Palisade endings in human extraocular muscles. Invest Ophthal and Vis Sci 25: 471–476 33. Ruskell GL (1978) The fine structure of innervated myotendinous cylinders in extraocular muscles of rhesus monkeys. J Neurocyt 7: 693–708 34. Bruenech JR, Ruskell GL (2000) Myotendinous nerve endings in human infant and adult extraocular muscles. Anat Rec 260:132–140 35. Sodi A, Corsi M, Faussone Pellegrini MS, Salvi G (1988) Fine structure of the receptors at the myotendinous junction of human extraocular muscles. Histol Histopathol 3:103–113 36. Eberhorn AC, Horn AKE, Fischer P, Buttner-Ennever JA (2005) Proprioception and palisade endings in extraocular eye muscles Ann NY Acad.Sci 1039:1–8 37. Bruenech JR, I-B Kjellevold Haugen (2005) The structural organization of the distal insertion of human extraocular muscles (EOM). Ophthalmic Res 37:2164–98 38. Kjellevold Haugen IB, Bruenech JR (2005) Sensory receptors in extraocular muscles (EOM) and their potential role in oculomotor control. Ophthalmic Res 37:2163–98 39. Kjellevold Haugen I-B, Bruenech JR (2006) Age-related neuromuscular changes in human extraocular muscles. In: Gomez de Liano R (ed) 30th European Strabismological Association Meeting Transactions. Taylor & Francis, Killarney, Ireland, p 141–144 40. Kelders WPA, Kleinrensink GJ, van der Geest JN, Feenstra L, deZeeuwMiller CI, Frens MA (1985) Compensatory increase of the cervico-ocular reflex with age in healthy humans. In: Miller NR (ed) Walsh and Hoyt’s Clinical Neuro-Ophthalmology. Williams & Wilkins, CITY 41. Mulch G, Petermann W (1979) Influence of age on results of vestibular function tests. Review of literature and presentation of caloric test results. Ann Oto Rhinol Laryngol 88:1–17 42. Paige GD (1994) Senescence of human visual-vestibular interactions: smooth pursuit, optokinetic and vestibular control of eye movements with ageing. Exp Brain Res 98:355–372 43. Huaman AG, Sharpe JA (1993) Vertical saccades in senescence. Invest Ophthalmol Vis Sci 34:2588–2595 44. Yang Q, Kapoula (2006) The control of vertical saccades in aged subjects Exp Brain Res 171:67–77 45. Yang Q, Kapoula. (2006) Aging does not affect the accuracy of vertical saccades nor the quality of their binocular coordination. Neurobiol Aging [Need publication data here]. 46. Kjellevold Haugen I-B, Bruenech JR (2005) Histological analysis of the efferent innervation of human extraocular muscles. In: De Faber, J-T (ed) 29th European Strabismological Association Meeting Transactions, Izmir, Turkey, June 1–4, 2004, Taylor & Francis, CITY 47. Eggers HM (1982) Functional anatomy of the extraocular muscles. In: Jakobiec FA (ed) Ocular Anatomy, Embryology and Teratology. Harper & Row, Oxford, p 783–824 48. Sevel D (1981) A reappraisal of the origin of the human extraocular muscles. Ophthal 88:1330–1338 49. Gamble H.J, Fenton J, Allsopp G (1978) Electron microscope observations on human fetal striated muscle. J Anatomy 126:567–589 50. Bruenech JR, Kjellevold Haugen IB (2006) Morphological variations in human extraocular muscles and their functional implications. Acta Ophthal Scan vol 84, Suppl 239 51. Miller JE Ageing changes in extraocular muscle. In: Lennerstrand G, Bach-Y-Rita P (eds) Basic Mechanisms of Ocular Motility and their Clinical implications. Pergamon, Oxford, p 47–61 52. Bruenech JR (2000) Neuroanatomical organization of human extraocular muscles review of the effector organ of the oculomotor system. PUBLISHER, CITY, p 1–58

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53. Locket NA (1968) The dual nature of human extraocular muscles. Br Ort Jour 25:2–11 54. Nunomura S, Hizawa K, Ii K, Sano T (1984) A histochemical study on fibre types in human extraocular muscles. Biomedical Research 5:295–302 55. Peachey L (1971) The structure of the extraocular muscle fibres in mammals. In: Bach-y-Rita P, Collins CC, Hyde JE (eds) The Control of Eye Movements. Academic Press, New York, 47–66 56. Scott AB, (1973) Collins CC, Division of labour in human extraocular muscles. Arch Ophthal 90:319–322 57. Mühlendyck H, and Ali SS (1978) Histological and ultrastructural studies on the ringbands in human extraocular muscles. Graefes Arch Clin Exp Ophthalmol 208:177–191 58. Ringel SR, Wilson B, Barden MT, Kaiser KK (1987) Histochemistry of human extraocular muscle. Arch Ophthal 96:1067–1072 59. McKelvie P, Friling R, Davey K, Kowal L (1999) Changes as the result of aging in extraocular muscles: a post-mortem study. Aust N Z J Ophthalmol 27:420–425 60. Scelsi R, Scelsi L, Poggi P (2002) Microcirculatory Changes and Disuse Are Cause of Damage to Muscle Fibres During Aging. Basic App Myol 12(5):193–199 61. Berard-Badier M, Pellissier JF, Toga M, Mouillac N, Berard PV (1978) Ultrastructural studies of extraocular muscles in ocular motility disorders. II. Morphological analysis of 38 biopsies. Albrecht Von Graefes Arch Klin Exp Ophthalmol 208 (1–3):193–205 62. Ruskell GL (1984) Sheathing of muscle fibres at neuromuscular junctions and at extrajunctional loci in human extra-ocular muscles. J Anat 138(1):33–44 63. Ruskell GL, Kjellevold Haugen IB, Bruenech JR, van der Werf F (2005) Double insertions of extraocular muscles in humans and the pulley theory. J Anat 206:295–306 64. Demer JL (2000) Evidence for active control of rectus extraocular muscle pulleys. Invest. Ophthalmol. Vis Sci 41:1280–1290 65. Oh SY, Poukens V, Demer JL (2001) Quantitative analysis of rectus extraocular muscle layers in monkey and humans. Invest Ophthalmol Vis Sci 42:10–16 66. Miller JM (2007) Understanding and Misunderstanding Extraocular Muscle Pulleys. J Vis vol 7, num 11, art 10, p 1–15 67. Demer JL (2006) Current concepts of mechanical and neural factors in ocular motility. Cur Opin Neurol 19:4–13. 25 68. Quaia C, Optican LM (1998) Commutative saccadic generator is sufficient to control a 3-D ocular plant with pulleys. J Neurophysiol 79:3197–3215 69. Clark RA, Demer JL (2002) Effect of aging on human rectus extraocular muscle paths demonstrated by magnetic resonance imaging Am J Ophthalmol 132(6):872–8 70. McClung JR, Allman BL, Dimitrova DM, Goldberg SJ (2006) Extraocular connective tissues: A role in human eye movements? Invest Ophthalmol Vis Sci 47:202–205 71. McLoon LK, Thorstenson KM, Solomon A, Lewis MP (2007) Myogenic precursor cells in craniofacial muscles. Oral Dis March 13(2):134–40 72. McNeer KW, Spencer RF (1981) The histopathology of human strabismic extraocular muscle. In: Lennerstrand G, Zee DS, and Keller EL (eds) Functional Basis of Ocular Motility Disorders. Pergamon, Oxford, p 27–38

Chapter 21

Rehabilitation of Low Vision in Aged People Corrado Balacco, MD, PhD, Elena Pacella, MD, and Fernanda Pacella, MD

Abstract Early screening, diagnosis, and treatment of age-related eye diseases are emerging challenges for public health professionals. New techniques of evaluation and management are currently being used. The scanning laser ophthalmoscope is used for microperimetry and determination of preferred retinal locus in order to treat absolute central scotomas. Biofeedback in low vision patients using the Improved Biofeedback Integrated System has given promising results in the rehabilitation of patients. Optical aids include optical magnifiers, magnifying eyeglasses, telescopes, monoculars, Galilean, Keplerian, and electronic systems. Public health professionals must increase public awareness about age-related eye diseases and ensure medical management and supportive care for patients. At present the leading causes of low vision and blindness in the elderly population are cataract, glaucoma, age-related macular degeneration, diabetic retinopathy, and degenerative myopia. The specific causes of visual impairment vary greatly by race/ethnicity and geographical location. Keywords visual impairment, elderly, surgical techniques, facoemulsification, visual field, cataract.

Visual Impairment in the Elderly Notwithstanding the progress in surgical intervention over the last few decades, cataracts are the main cause of visual impairment in all the world except the most developed countries. The rapid advances in surgical techniques and modern instrumentation have modified cataract removal from intracapsular to extracapsular extraction with intraocular lens implantation and facoemulsification, dramatically reducing postsurgical recovery and improving the quality of vision. Another major cause of low vision in the elderly is open angle glaucoma. Diagnosis is based on intraocular pressure measurement, detecting changes in the optic nerve head and demonstrating visual field alterations by visual field testing. Early glaucoma detection and treatment significantly delay the progression of disease, reducing the irreversible damage to optic nerve fibers. Topical medication to reduce intraocular From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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pressure is the mainstay of management. At present topical acetazolamide and prostaglandin molecules are also commonly used. Surgical treatment is reserved for cases with poor response to topical drugs and progression of disease with elevated intraocular pressure and optic fiber damage. Age-related macular degeneration (AMD) has several forms. Wet AMD is caused by choroidal neovascularization and if unrecognized and untreated can result in rapid deterioration of vision. Initially laser photocoagualtion was the only clinically proven management in neovascular AMD. At present veteporfin therapy with photodynamic treatment is used in patients with predominantly classic subfoveal choroidal neovascularization. Dry AMD is the most common form and tends to progress more slowly compared to the wet form. The most advanced form of AMD, geographic atrophy, also causes central vision loss. Early detection and prompt treatment will help optimize treatment outcome. New forms of treatment of wet AMD like injecting antivascular endothelial growth factor, pegaptanib (Macugen) e Avastatin, have effectively reduced the progression of disease in some randomized trials. Vision loss from diabetic retinopathy can result from several diabetes related retinal changes but early recognition and treatment, including laser therapy, can prevent blindness. The retinopathy depends on the duration of hyperglycemia rather than age. Therefore, prevention emphasizes the need for blood glucose level control to reduce the incidence, progression, and severity of diabetic retinopathy. Degenerative myopia is another cause of visual impairment not only in the elderly but also in the younger population. Total refractive error correction and healthy nutrition are fundamental in reducing the progression of degenerative retinal alterations. A cataract is a progressive, irreversible opacity of the lens which causes reduction of visual acuity. The lens is a transparent biconvex organ which is situated perpendicular to the optical axis behind the iris and in front of the vitreous body. It can be divided into a central portion, the nucleus; a peripheral portion, the cortical (anterior and posterior); and a capsule. The principle function of the lens is accomodation; that is, to focus images on the retina similar to a camera. Its peculiar optical structure and transparency characteristics allow the passage of light rays to reach the retina. The lens is the only organ that continues variable development throughout life, changing in thickness, elasticity, and above all transparency.1 Aging and any pathogenetic noxae that alter the biochemical and structural integrity of the lens cause opacities,2 resulting in a significant reduction of visual acuity. This pathology, earlier described by the ancient Greeks, Romans, and Arabs as “a rapid fall of humour from the brain,” is defined as a “cataract,” which means “a falling downwards.” It is the primary cause of blindness. The surgical removal of cataracts is the most frequently performed operation in the medical field. The following types of cataract can be described. Senile cataract: This is the most frequent form and is due to aging. A slow but progressive loss of lens transparency is typical of the elderly population. Epidemiological studies have shown that in western countries cataracts first appear among the population over age 50, and the incidence progressively increases with age. Indeed, about 50 percent of the population over 60 years of age and 100 percent of people over 80 have lens opacities.

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Congenital cataract: This form can occur in one or both eyes at birth or appear in the following months. There can be various degrees of opacity, leading to reduced visual acuity and a secondary amblyopia (permanent reduction of vision). Therefore early diagnosis and management are of extreme importance. Congenital opacities may remain stable or worsen over the years. An exception are total cataracts associated with leukokoria (white pupillary reflex) with nistagmus in bilateral forms and strabismus in monolateral cases. Among the causes of congenital cataract are genetic factors, x-rays, the use of medication in pregnancy, and maternal metabolic alterations such as diabetes, hypothyroidism, alimentary defects, or premature birth. However, the most frequent causes are infectious diseases contracted by the mother during pregnancy, such as rubella, systemic herpes infection, toxoplasmosis, etc. Complicated cataract: This term is used when the cataract follows ocular pathology—most frequently, anterior and posterior uveitis, acute glaucoma, high myopia, intraocular tumors, and retinal detachment. Cataract associated with systemic disease: Cataracts arise four times more frequently in patients with diabetes than in the general population. The cataract is similar to the senile form but arises in younger patients with a more rapid evolution. In cases with poor glycemic control it is frequently bilateral.3,4 Cataract associated with drugs: This is the type secondary to long term use of cortisone or topical treatment (drops) used in glaucoma. Traumatic cataract: This occurs following closed or perforating trauma and is generally monolateral. The principle symptom of a cataract is a slow and progressive quantitative and qualitative reduction of visual acuity. This can be described as blurred or unfocused vision, alteration of colour perception, difficulty in night vision, vision of coloured rings around artificial and solar light, double vision, etc. In some elderly patients it can be described as a transitory improvement of near vision due to a myopic shift that delays presbyopia. When the opacity is central, visual acuity improves at night when illumination is dim and there is mydriasis. Slit lamp examination of the anterior segment with pharmacological pupillary dilatation allows detailed examination of the lens. Until the begining of the 1980s topical medication was prescribed to delay the progression of cataract. However, there is no valid pharmacological agent at present. The sole approach to this pathology is the surgical removal of the lens. The increased frequency of cataract extraction results not only from the aging of the population but also from an improvement of surgical success rates. This in turn results from the improvement of surgical methods, with more sophisticated technology and faster postoperative visual recuperation. Among the surgical methods employed are intracapsular cataract extraction (ICCE), which is rarely used nowadays; extracapsular cataract extraction (ECCE); and the latest and most frequent method, facoemulsification, which consists of the use of ultrasound for cataract fragmentation. Once the cataract is extracted, an intraocular lens is usually implanted to replace it. At present facoemulsification can be carried out with topical anesthesia with nearly immediate functional recovery.5-8 This does not imply that the surgical process is simple and without risks; it is extremely sophisticated microsurgery which requires much experience and postoperative follow-up. A severe complication can be internal infection of the eye (endophthalmitis) due to pathogenic germs during

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or after surgery. Fortunately, this has a very low frequency, only about four of 1000 operations. Another complication can be rupture of the posterior capsule, where the intraocular lens is usually placed. This can cause the slipping of cataract fragments into the vitreous fluid. Minor complaints are redness, tearing, and foreign body sensation, which are resolved with local medical applications such as antibiotics and anti-inflammatory agents. A frequent secondary event which can occur both in ECCE and in facoemulsification is secondary cataract formation. This consists of opacification of the posterior capsule. The incidence is 10-50 percent in 3.5 years following surgery.9,10 This opacification causes a progressive reduction of visual acuity. The management at present is a NeoDymion YAG-laser capsulotomy.11-13 A small central opening is created in the posterior capsule, allowing a return to the visual acuity values reached after the original surgery.14 Nd-YAG laser capsulotomy is considererd a safe and effective procedure which can, however, present some complications such as ocular hypertension, cystoid macular edema, and retinal detachment.15

Management of Glaucoma Glaucoma is characterized by damage to the retinal ganglion cells, which is clinically manifested by alterations of the optic disk cup and typical visual field defects. The traditional definition includes high intraocular pressure and damage to the optic nerve shown by defects of the visual field. Glaucoma has a silent progression. The disease progresses and symptoms appear late, when the patient complains of bumping into objects and tripping. This is due to the loss of lateral vision, as central vision is maintained until the terminal stages of the disease. Indeed, visual acuity is good, but the visual field is progressively reduced.16,17 In advanced disease, without adequate treatment, the loss of the visual field becomes so severe as to cause permanent blindness.18,19 Two principle hypotheses for the pathogenesis have been advanced. The first emphasizes the importance of reduced vascular perfusion to the optic nerve and the nerve fibers layer. The second considers mechanical damage to the lamina cribrosa.20-22 At present the physiopathological mechanisms are not completely clear; however in both theories the alterations of the optic nerve seem to be caused by: 1) the intrinsic vulnerability of the optic disk, and 2) the increase of intraocular pressure up to a critical level for the optic disk. The etiopathogenetic cause is an abnormal deflux of aqueous humor from the anterior chamber, with an increase of intraocular pressure (IOP). This is the most important factor,23,24 and the higher the IOP the higher the risk of developing glaucomatous optical neuropathy.25,26 Visual field studies have shown that the optic nerve head is where the most damage occurs.27,28 Patients with optic nerve damage can be divided into two groups: those with IOP higher than 21 mmHg, and those with IOP below 18 mmHg, glaucoma sine hypertension.29 Axonal flux studies have shown that with high IOP, the nerve fiber and axons are vulnerable during the passage from the optical disk.30 Two hypotheses have been advanced to explain the

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damage: 1) indirect ischaemic damage, in whch elevated IOP causes the degeneration of nerve fibers by compromising the microcirculation of the optic disk; and 2) direct mechanical damage, in which high IOP causes direct damage to the retinal nerve fibers. The factors which determine IOP are the amount of aqueous humour production and the resistance of the drainage system. Aqueous humour is secreted from the ciliary processes in the posterior chamber and diffuses through the iris and the pupil to the sclerocorneal trabecular meshwork situated in the angle of the anterior chamber.31,32 About 80 percent of the drainage is through the trabecular meshwork, Schlemm’s canal, and the Fontana spaces to the episcleral vessels; whereas about 20 percent is uveal scleral drainage, where the aqueous humour passes through the ciliary body to the suprachoroidal space and through the venous circulation of the ciliary body, the choroid, and sclera.33 This continuous cycle is programmed to maintain a slight positive pressure in the eye. Alterations of this delicate equilibrium can cause elevated IOP, leading to damage.34-38 The statistical approach indicates 21 mmHg as the value above which glaucoma is suspected.39-41 The diagnosis of disease cannot be made solely on IOP; other factors such as familiarity, myopia, diabetes, race, age, and trauma must be considered. Primary open angle glaucoma: This is the most frequent type of glaucoma and leads to blindness in 13 percent of cases. It occurs with the same frequency in males and females but more frequently in the elderly, afflicting 2 percent of the population over 70 years of age with initial alterations after 40 years of age. Both eyes are generally involved, with initial manifestations in one eye. Subjective symptoms are absent and early management can only be based on prevention, since visual acuity is perfectly conserved even with visual field alterations. It is universally accepted that the risk of irreversible damage of visual function increases in relationship to the increase in IOP. Primary angle closure glaucoma: This form is relatively rare, occurring only in about 0.1 percent of subjects above 40 years of age, though four times more frequently in women. It is also called acute glaucoma, as the anterior chamber angle is rapidly blocked, which causes a sudden increase of IOP up to 60 mmHg or more. The main symptom is extremely intense pain (similar to migraine) accompanied by nausea, vomiting, headache, and rapid deterioration of vision. Secondary glaucoma: Secondary glaucoma follows other ocular diseases such as inflammation, trauma, or drug side effects. This represents 25-30 percent of all glaucomas and can occur in either open or closed angle forms. Secondary glaucoma can also occur in the course of dismetabolic diseases such as diabetes, ocular hypertension, and advanced cataract.42 Congenital glaucoma: Congenital glaucoma is a very rare form that is present from birth due to a congenital malformation of the iridocorneal angle. The increase in intraocular pressure determines the typical aspect of “bufthalmous” by the third year of age; the eyeball is large due to scleral and corneal enlargement, since in children high intraocular pressure causes distortion of the ocular layers. This condition necessitates early surgical management aimed at eliminating the iridocorneal malformation. Examination ophthalmology: The increase of IOP is not always synonymous with glaucoma. Certainly IOP increases the risk, but the amount of pressure that the

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optic nerve tolerates has individual variability. This explains why an IOP between 10 and 21 mmHg (considered normal) can, in some cases, cause damage. The examination of the patient should include slit lamp observation, tonometry, optic disk morphology study, and iris-corneal angle evaluation. Ophthalmoscopy evaluates the relationship between optic excavation and the optic disk diameter or cup/ disk ratio. Signs of pathology are values of about 0.5, and a difference of more than 0.2 between the two eye ratios. Perimetry evaluates visual field alterations; there are typical alterations in the glaucomatous patient. Gonioscopy allows direct observation of the anterior chamber angle and can show shallow chambers and angle closure. Prevention is fundamental in this pathology, as glaucoma is a silent disease with progressive functional damage leading to blindness. The therapeutic management of glaucoma includes systemic or local use of drugs. However, when these do not permit adequate control of disease, parasurgical and surgical treatment is indicated. Medical treatment of glaucoma is aimed at intervening in aqueous humour dynamics on the following systems:43,44 ●

● ●

secretion of aqueous humour; beta-blockers; inhibitors of anhydrase carbonate; alpha2-antagonists; trabecular drainage; miotic agents; epinephrine; venous episcleral perfusion/ciliary body perfusion; alpha-2-agonist with alpha1-agonistic properties.45-48.

Medical treatment must be used constantly, and in this manner the majority of patients maintain IOP under control. Frequently an association of drugs administered once or twice daily is necessary.49,50 The use of laser techniques began in 1956 when Meyer-Schwickerath created an iridotomy without surgical incision using xenon arc photocoagulation.51 Later the pulsed ruby laser,52,53 followed by the argon laser,54 was used with less damage inflicted on the lens and cornea. Today argon laser iridotomy and neodymium:yttrium-aluminium-garnet (Nd:YAG) laser iridotomy have replaced surgical iridectomy as the primary treatment for angle-closure glaucoma.55 Laser iridotomy has advantages over surgical methods and does not have the risks of intraocular surgery. The primary indication is angle-closure glaucoma due to primary or secondary (iris capture of an intraocular lens or pupillary seclusion in iridocyclitis) pupillary block. Indications for laser peripheral iristomy are also acute, intermittent, and chronic pupillary block or in the treatment of one eye in a patient with angle-closure glaucoma in the other. In patients with narrow angles and after an attack of malignant glaucoma in one eye, prophylactic laser iristomy in the other eye may avert a surgical irisectomy or a trabeculectomy. Another important laser technique is laser trabeculoplasty, which is used to control elevated intraocular pressure in those patients with open-angle glaucoma for whom medical therapy has failed to bring about control. It is also useful in some secondary open-angle glaucoma such as pseudoexfoliative and pigmentary glaucoma.56 It can also be used after filtration surgery where intraocular pressure is still high. Many studies have been carried out regarding the indications for surgical management of the glaucomatous patient. Surgical treatment is usually indicated when

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medical and laser treatment is not sufficient to prevent the progression of disease. Surgery in primary glaucoma is essentially aimed at preventing and “correcting” the state of ocular hypertension in order to limit the progression of irreversible damage to the optic nerve, visual field, and therefore visual function. In the literature there are two concepts on which surgery is based: to modify the anatomic structures of the natural drainage systems at various levels, and to restore the natural paths of drainage. The majority of techniques consist of creating a bypass between the posterior and anterior chamber and the subconjunctival space; that is, creating an artificial drainage system. These can be divided into 1) a bypass between the posterior and anterior chamber (sector iridectomy or peripheral iridectomy); 2) a bypass between the anterior chamber and subconjunctival space at times protected by a scleral flap (filtrating tecniques such as scleral-iridectomy of Lagrange, scleral trephination according to Elliot, iridoenclesis, thermosclerostomy according to Schede) protected filtration such as trabeculectomy, or non filtration techniques such as viscocanalastomy and deep sclerectomy. In patients who techniques to enhance outflow are not possible or have not been successful, operations to decrease aqueous production by destroying portions of the ciliary body can be carried out. These cylio-destructive procedures are used on eyes with neovascular, inflammatory, or aphasic glaucoma. They are also useful in providing comfort in patients with severe vision loss due to glaucoma. Cyclodiathermy was first introduced in 1933.57 Bietti described the use of dry ice for cyclocryotherapy in 1950.58 Today cyclocryotherapy with nitrous oxide gas cooling is used. Lasers are also used to cause ciliary body damage and a decrease in aqueous production. Three principal routes of laser energy administration to the ciliary body have been employed: transpupillary cyclophotocoagulation, endophotocoagulation, and transscleral cyclophotocoagulation. This latter method is the most widely adopted. Noncontact Nd:YAG laser energy is administered via a slit lamp delivery system, with or without a contact lens. Age-related macular degeneration (AMD) has always been considered a pathology of the elderly. About 30 percent of adults aged 75 or older present some signs of maculopathy; 6-8 percent of these individuals have advanced stages of disease with severe visual loss.59-61 At present it seems to involve younger patients (45-50 years old). Research suggests that AMD is a very complex disease, caused by a combination of multiple genetic and environmental factors. There seems to be strong evidence for the heredity of AMD based on familiar aggregation studies, twin studies, and segregation analysis. Choroidal neovascularization can represent the initial phase of an eventual choroidal scar.62 This initial stage is called the exudative phase. The later stage is the scar. The increased permeability of neovessels shown with indocyanine green staining and with fluorangiography is defined as leakage. During the neovascularization stage, endothelial growth factor (EGF) is present. The scars are frequently circular; they are called “disciform.” In choroidal neovascular membranes (CNVM), as in all hypo-oxygenation disorders, there is tissue damage or loss followed by tissue repair processes. The cause of tissue destruction in AMD is probably due to apoptosis or programmed cell death. Trauma, laser treatment, and angioid streaks share a common factor, a defect of the retinal pigment epithelium (RPE) and/or of the choriocap-

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illaris. The localized relative hypoxia increases the hydrophobic proprieties of Bruch’s membrane, which could play an important role in the physiopathology. In the United States the prevalence of neovascular AMD in the population over 40 years of age is 1.47 percent. CNVM and the majority of correlated diseases are not associated with higher mortality. Smoking seems to increase the progression of AMD by 350 percent. It is well established that smoking also dramatically increases the incidence of cancer and cardiovascular death. The main symptom of CNVM is loss of central vision, but peripheral vision seems to be preserved unless complications follow surgical management. The prevalence of AMD increases with age; it is common after age 60, and even more so after 70. The frequency of AMD is 90 percent in Caucasian males over 80 years of age and is 16.39 percent in Caucasian females over 80. In males ocular trauma leading to CNVM is more frequent than in females, whereas AMD is more frequent in females. In the past the aging of the retinal epithelial pigment (REP) cells was considered the pathogenetic mechanism responsible for the disease. However, at present, increasing earlier manifestations of disease have led to a consideration of other factors. An important pathogenetic factor is an altered immune system involved in the elimination of epithelial pigment cells which degenerate and encounter cellular death. The REP cells are terminal differentiated cells similar to cells of the cornea, and do not divide. Therefore, from 20 to 90 years of age there is a progressive loss of retinal cells. Near the fovea there is less cellular loss. There is, however, aging of the pigmented epithelium adjacent to the paramacular drusen. In vitro studies show an increase of the B-glactosidosis enzyme precursor to AMD and also of the factor involved in cellular reproduction. In vivo it is difficult to demonstrate the presence of B-galactosidosis. What happens to the cells that encounter apoptosis? There are certainly changes at the cellular level; an increased sensitivity to calcium, binding of the cellular membrane, DNA fragmentation, appearance of the glycoprotein rich in istidine which determines the clearance of apoptotic cells. The systems which eliminate apoptotic cells are humoral and cellular. The former are based on the activation of the complement pathway and that of the C-reactive protein. The latter are based on dendritic cell morphology which, in studies on mice, are localized under the REP to regulate and control inflammation. Dendritic cells function as scavengers for silent elimination of dead epithelial pigment cells. This function at choroidal level is most probably carried out by macrophages which do not activate the complement. A similar mechanism of cellular regulation for the elimination of dead cells has been observed in bone marrow in humans through the production of complement factors.59-62 What occurs when there is an increased vulnerability to AMD? Most probably the complement factors go out of control, leading to cellular necrosis rather than apoptosis, thus generating angiogenesis. Presumably the normal scavenger function does not determine disease. Altered scavenger regulation due to genetic vulnerability can lead to dry AMD. When there is little complement activation, an accumulation of necrotic cells, and an increase of apoptotic cells, geographic atrophy results. Wet AMD arises when complement factors are activated and are out of control and there is activation of vascular endothelial growth factor (VEGF). AMD is being studied

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at the genetic level in order to allow primary prevention in the future; but there is high expressivity of the disease, and further research is warranted. At present secondary prevention is possible by trying to intervene in environmental risk factors and by administering appropriate treatment where the disease is already present. Careful patient follow-up is essential. This is carried out by examining the ocular fundus of patients and by using instrument techniques such as ocular computerized tomography (OCT), electroretinogram (ERG), visual field examination, fluorangiography (FAG), and indocyanine green angiography (ICG). AMD can have a profound impact on quality of life and independence. Due to the loss of central vision, afflicted individuals may be unable to read, write, or drive. 62 percent of patients with AMD lose three lines of visual acuity in one year. The management of AMD is limited. The unique proven treatment for the dry form of AMD is antioxidant/mineral supplementation, which slows AMD progression by 25 percent over five years. The approved treatment options for exudative AMD based on clinical trials are laser photocoagulation, photodynamic therapy, and anti-angiogenic agent injection. The major limitation of laser photocoagulation is the damage to the neurosensory retina that is associated with a sudden decrease in visual acuity. Photodynamic therapy is a nonthermal process based on the targeted photoactivation of an intravenously administrated photosensitive drug. The activated dye results in the creation of oxygen intermediates and free radicals affecting the exposed endothelial cells.63 The era of pharmacological treatment of CNV in AMD has just began. Triamciolone acetonate is widely used. The optamer Na-pegaptanib agonist VEGF is used as intravitreal injections. The effects of ranibizumab and bevacizumab are being studied. A cortisone acetate is also being studied which inhibits endothelial cell migration and also reduces the release of VEGF. In contrast to triamcinolone, it has no effect on the lens or intraocular pressure. Diabetic retinopathy is the most important cause of low vision and blindness in patients of working age in the United States and Europe. The prevalence of diabetes in Europe is estimated at 2.5 percent of the entire population, and of these patients about 40 percent present signs of retinopathy. This pathology afflicts younger patients with respect to senile macular degeneration, thereby presenting higher costs to the society, especially in terms of productivity. Diabetic retinopathy has been studied on anatomic-pathological bases, and the major alterations involve the coriocapillaris. The alterations consist in thickening of the basal membrane, selective loss of intramural pericytes (those surrounding the endothelial cells), development of vascular insufficiency, and passage of liquid. These alterations give rise to local edema and finally development of regional tissue hypoxia. Diabetic retinopathy can be classified in four major groups: 1) nonproliferant, 2) initial, 3) preproliferant, and 4) proliferant. Areas of hypoxia can be evidenced by fluorangiography of the retina. In advanced diabetic retinopathy the retina shows vast areas of hypoxia and capillary nonperfusion. The neovascolarization which involves the optic nerve head and other areas is secondary to hypoxia in the areas of reduced or absent capillary perfusion. The retinal alterations progress slowly over many years. The most precise parameter to

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foresee the incidence and prevalence of retinopathy in the diabetic population is the duration of diabetes. The possibility of developing diabetes increases with age; nearly all patients with diabetes older than 20 present some signs of retinopathy. However, retinal alterations must be studied at all stages, even if the longer the duration of diabetes, the higher the risk of developing retinopathy. The diagnosis of diabetic retinopathy is based on a detailed examination of the fundus oculi with ophthalmoscopy. The fundus examination should be carried out after pupillary dilatation with tropicamide 1 percent or phenilephrine 2.5 percent. The retinal alterations in diabetic retinopathy begin at the capillary level with thickening of the basal membrane and loss of endothelial cells and pericytes. There is an increase in blood density and reduced deformability of blood cells; these alterations lead to occlusion of the capillaries and transudation of liquids. The final stage is the simultaneous presence of edematous and haemorrhagic areas (defined as humid), alternated with ischaemic areas due to reduced perfusion (defined as dry). The damage to vision caused by diabetic retinopathy is represented by direct damage, iatrogenic damage, and damage due to secondary pathologies. Direct damage includes the following: ●

● ● ●

● ●

maculopathy—the exudative form represented by focal macular edema, and the ischaemic or dry form; cystoid macular edema (in the advanced stages); optic neuropathy, with loss of portions of central vision; retinal haemorrhage—the preretinal forms can cover large areas of the central visual field; central or branch venous occlusion; and tractional retinal detachment.

The damage caused by diabetes is associated with iatrogenic damage due to therapeutic stategies aimed at preventing long term effects of diabetic retinopathy. These include the following: ● ● ● ● ● ● ● ● ●

argon laser treatment on central neovascular membranes; central grid with krypton laser; paracentral focal laser treatment; peripheral panphotocoagulation; retinal haemorrhages and cystoid macular edema secondary to panphotocoagulation; photodynamic treatment; vitreoretinal surgery; silicone oil; and surgical complications of cataract and glaucoma.

Among the damage induced by secondary pathologies are the following: ● ● ●

cataracts; neovascular glaucoma; and cerebral damage which arises due to diabetes.

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In the past decade there has been much progress in the management of diabetic retinopathy. The treatment of choice is laser photocoagulation. It is thus possible to control proliferative diabetic retinopathy, which can cause exudative maculopathy. Laser photocoagulation can be carried out in the office with a slit lamp. It is a relatively painless procedure requiring two to four sessions lasting 20-30 minutes each. A total of 1200-1500 spots with diameter of 200-500 microns are placed, avoiding the optic nerve head, the macula, and principal vessels. The neovascularization of the optic nerve head is not directly treated; however, the aim is the involution of neovessels in a variable period of three weeks to three months. Since the principle cause of blindness in diabetic retinopathy is vitreal haemorrhage, caused mainly by neovascularization, the reduction or disappearance of new vessels in the optic nerve head and in other areas reduces the incidence of blindness. In patients with proliferative diabetic retinopathy treated with laser photocoagulation the statistical risk of severe loss of vision is reduced by 50 percent. There are unfortunately patients who present a progression of disease, vitreal haemorrhage, and blindness even after adequate laser photocoagulation. The majority of patients have some reduction of central vision (reading) corresponding to two lines of Snellen tables. At times this loss of vision is permanent, but generally it is temporary. In a significant number of patients there are visual field defects, at times with lateral defects. In the majority of patients there is nocturnal vision reduction, limiting driving at night. In the last decade many microsurgery techniques have been put forward to treat vitreal haemorrhages. In diabetic retinopathy vitreal gel is important in the development of neovessels. If this gel contracts (vitreal separation) or liquefies (syneresis), the delicate neovascular net can break with consequent vitreal haemorrhage. Vitrectomy should be taken into consideration under the following conditions: 1) vitreal haemorrhage which does not resolve in 6 months; 2) tractional retinal detachment (caused by vitreous contraction) which involves the macula; 3) vitreal haemorrhage followed by retinal detachment; or 4) bilateral vitreal haemorrhage. Vitrectomy does present significant risks, but it is the sole hope in selected cases. Visual reduction is defined as slight (5-8/10), intermediate (1-4/10), and severe (less than 1/10). An intermediate state of vision reduction is most frequently encountered. Less visual reduction is seen in nonproliferative retinopathy, whereas in the proliferative phase of disease there is severe loss of vision. Nonproliferative retinopathy causes maculopathy with retinal edema in the foveal area and consequent reduction of central vision. This is usually well diagnosed with fluorangiography. This edema and the typical yellow exudates which are frequently associated with it are caused by exudation from vessels with altered walls and/or microaneurisms. The exudates frequently form a partial or complete ring in, near, or around the macular area (circinate maculopathy). Generally it is thought that adequate

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control of blood glucose and normal levels of glycosylated haemoglobin delay the onsent of retinopathy and/or reduce the progression of vascular alterations.64-66 It is noted that a state of ketoacidosis or hyperosmolarity can cause alterations in refraction. In diabetic patients corrective lenses should not be prescribed until the clinical condition is stable. Other critical systemic diseases for the diabetic patient are: hypertension, atherosclerotic lesions of the carotid arteries, and cardiac or renal alterations. In conclusion, diabetic retinopathy is an ocular pathology, but collaboration with the internist is appropriate in order to offer complete and effective treatment to the patient.

Degenerative Myopia The simple definition of myopia is: a refractive error in which light rays from an infinite point are focused in a static refractive state on a plane in front of the retina. This occurs with a longer antero-posterior length of the eye and/or a higher ocular diopter power.67 Degenerative myopia is considered a true disease, with degenerative pathology of the sclera, choroid, and retina. This form is always due to an abnormal development of the antero-posterior axis of the eye with values which can exceed 30 mm. It has a progressive evolution. Of all patients afflicted with myopia, the percentage of the degenerative form is 6-8 percent and females are more frequently involved. The disease begins around three to four years of age and progresses throughout life. The progression has individual variability. The high myope has large, slightly protruding eyes, blinks frequently, and sometimes presents divergent strabismus. Visual acuity is not always perfect, but near vision is usually maintained. The progressive nature of the disease, however, can reduce visual function over time. The chorioretinal complications usually lead to a negative prognosis.68-70 Even though degenerative myopia is not strictly a disease of the older population, the progressive characteristic makes it one of the principal causes of low vision and blindness in the elderly. The etiopathogenesis of myopia has been much discussed.71 A myriad of hypotheses have been advanced; we would like to present the theory of Balacco Gabrieli, in which degenerative myopia is considered not only a refractive state but a disease which involves many genetic and neuroendocrine mechanisms.72-74 The most important anatomic-pathological condition of the eye is the excessive increase in the antero-posterior length. In the most severe forms there is a scleral elongation of the posterior pole causing the myopic stafiloma. The visual disfunction of the uncorrected or undercorrected myopic eye would influence the encephalic-hypophyseal axis, causing an endocrine alteration responsible for a weakening of the scleral collagen.75,76 The luminous stimulus which reaches the eye is transmitted to the hypothalmus and the hypophysis, and the latter, in turn, through the secretion of melatonin, interferes with gonadal function.77-79 The hypothalamus, on the other hand, regulates the function of other endocrine glands through the hypophysis.80

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The eye is linked to the central nervous system through two paths:81,82 1) the optical, and 2) the retinal-hypothalmic. In the hypothesis of Balacco-Gabrieli the myopic eye, with genetic predisposition, sends an abnormal visual message to the encephalo-hypophysis axis with a consequent alteration of the secretion of some hormones, especially the steroidal hormones.83-85 This hormonal alteration influences the sclera, causing progressive weakening and elongation of the eye. There is also a parallel increase of the urinary excretion of acid mucopolysaccharides, which demonstrates collagen metabolism alteration86 The influence of the luminous stimulus on the retina is of fundamental importance; the myopic eye sends insufficient (or better, abnormal) stimuli to the encephalic-hypophyseal axis.87-92 The encephalic-hypophyseal axis reacts to the modified message with a increase of steroidal hormones and a modified secretion of neurotransmitters and neuromodulators (VIP) of the retinal neurons, causing a further increase in the growth of the eye.93-98 The myope has reduced visual acuity without optical correction by means of spectacles or contact lenses. Over the past two decades photorefractive methods have been refined in the correction of myopia.99-102 The causes of low vision and blindness in degenerative myopia have individual variability. Some complications, such as glaucoma and cataract, can be solved with medical and surgical management.103 Other complications, such as chorioretinal degeneration, can be severe and progressive, with negative prognosis. These alterations can be divided into macular and peripheral retinal alterations. Myopic maculopathy can be classified as atrophic, hemorrhagic, or exudative-haemorrhagic with a neovascular membrane. The final state is almost always dry chorioretinal degeneration. Fluorangiography, indocyanine green angiography, and optical coherence tomography are new instrumental measures which allow early diagnosis and better management methods. Treatment has involved photocoagulative argon laser therapy, but new measures such as photodynamic treatment are now being used. The peripheral degenerations have clinical importance, as some can evolve towards retinal detachment. Laser preventive measures and routine retinal examination is of fundamental importance.104-106

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Bietti. Current Concepts on MYOPIA - Basics, Clinical Research and Surgery; Rome, Sept. 21-24 95. Final program and abstr. book, 59, 1995. 79. C. Balacco Gabrieli, E. Pacella, A. Moramarco, F. Bozzoni, F. Cruciani. Hormonal balance in high myopia: clinical investigation. Arvo: The Association for Research of Vision and Ophthalmology. Annual Meeting Fort Lauderdale, Florida 21-26 Aprile 1996. 80. Hollwich F, Dieckhues B. The effect of natural and artificial light via the eye on hormonal and metabolic balance of animal and man. Ophthlmologica 1980;180:188-197. 81. Hollwich F. Studies on the effect of the “energetic portion” of the optic nerve on functional processes and especially on the water balance. Ber. Dtsch. Ophthal. Ges. 1958;54:326. 82. Hollwich F. Effect of light via the eyes on the color changes of the frog. Klin Mbl Augen; 1958;133:748. 83. Balacco Gabrieli C, Santoro G, Santoro M, Lattanti V, Di Gioia C, Giorgino R. Plasmatic and urinary steroids in the high myopia. III note: cortisol, 17-KS (prolific females). Boll Soc Ital Biol Sper 1978;54:981. 84. Balacco Gabrieli C, Santoro G, Santoro M, Bellizzi M, Giorgino R. Plasmatic and urinary steroids in the high myopia. IV note: progesterone, 17-beta estradiol, 17OH, 17-KS. Boll Soc Ital Biol Sper 1978;54:984. 85. Aveysov ES, Winezkaija NF. Einige stoffwechsel werte fursanfte mukopolysaccaride bei der myopie. Klin Mbl Augen 1976;168:750. 86. Balacco Gabrieli C. the aetiopathogenesis of degenerative miopia. Ann. Ophthlmol 1983;15:312. 87. Wiesel TN, Raviola E. Myopia and eye enlargment after neonatal lid fusion in monkeys. Nature 1997;266:66-68. 88. Raviola E, Wiesel TN. Increase in axial lenght of the monkey eye after corneal opacification. Invest. Ophtalmol Vis Sci 1979;18:1232-1236. 89. Balacco Gabrieli C, Chetri A, Palmisano C, Pacella E, Castellano C. luce e miopia. Atti del Corso Teorico Pratico “Luce e Occhio” Roma Dic. 1989. 90. Balacco Gabrieli C, Chetri A, Pacella E. Light and miopia. V Internetional Conf. On Miopia. Syngapore, marzo 1990. 91. Trachtman J, Wallman J. Estreme miopia produced by modest changes in early visual experience. Science 1978;201:1249-51. 92. Balacco Gabrieli C.: Aetiopathogenesis of degenerative miopia. A hypothesis. Ophthalmologica 1982;185 n.4:199. 93. Balacco Gabrieli C, Santoro G, Santoro M, Scardapane P, Tundo R, Giorgino R. Plasmatic and urinary steroids in the high myopia. II note: cortisol, 17-KS (males). Boll Soc Ital Biol Sper 1978;54:978. 94. Balacco Gabrieli C, Santoro G, Santoro M, Lattanzi V, Di Gioia C, Giorgino R. Plasmatic and urinary steroids in the high myopia. III note: cortisol, 17-KS (prolific females). Boll Soc Ital Biol Sper 1978;54:984. 95. Greene PR. Miopia, the sclera; mechanical stress and the obliques. 3rd Int conf Myopia. Copenhagen. 1980. 96. Young FA. Intraocular pressure dynamics associated with accomodation. 3rd Int Conf on Myopia. Copenhagen 1980. 97. Young FA. Comunication at the Copenhagen myopia conference in relation to own paper, the distribution of myopia, in man and monkey. Doc Ophthalm Proc Ser 1980;28:5-11. 98. Tepperman J. Fisiologia metabolica ed endocrina. Pensiero scientifico edit. Roma 1969. 99. Balacco Gabrieli C, Pacella E, Abdolrahimzadeh S, Giustolisi R Excimer laser photorefractive keratectomy for high miopia. Advance Ophthalmic Laser Surgery an International Training Course, Interlaken, Switzerland 26-28 giugno 1997. 100. Pacella E, Abdolrahimzadeh S, Abdolrahimzadeh B, Mollo R, Balacco Gabrieli C Excimer laser photorefractive keratectomy for hygh myopia and myopic astigmatism. Invest Ophthalmol Vis Sci ( ARVO). 1998; 1608 B489: S347. 101. Balacco Gabrieli C, Pacella E, Abdolrahimzadeh S. Excimer laser photorefractive keratectomy for hig myopia and myopic astigmatism. Ophthalmic Surgery Lasers 1999;30(6):442-448.

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102. Pacella E, Pacella F, Abdolrahimzadeh S, Vulcano C.L, Brandozzi M, Mollo R, Balacco Gabrieli C. Photorefractive keratectomy in the manegement of high myiopic defect, tissuesaving vs planoscan. Invest Ophthalmol Vis Sci ( ARVO). 2006;-3621/B105. 103. Balacco Gabrieli C, Lorusso V, Moramarco A, Pacella E. Myopic cataract and implantation. Acta Of The V° Congr.Intern., On Cataract And Refractive Microsurgery pp. 143-144. Proceedings Of The V International Congress Florence, Italy; Giugno 1989. A.E Maumenee, W.J. Stark, I. Esente. Cappelli Edit. Bologna, 1991. 104. Contestabile MT, Recupero SM, Palladino D, De Stefanis M, Abdolrahimzadeh S, Suppressa F, and C Balacco Gabrieli al“A new method of biofeedback in the management of low vision Eye, Jul 2002; 16 (4):472-80. 105. Giorgi D, Contestabile M, Pacella E, Balacco Gabrieli C.An instrument for biofeedback applied to vision. Applied Psychophysiology and Biofeedback. 2005;30(7):389-395. 106. Balacco Gabrieli C, Giorgi, Dario, Pacella E, Turchetti P, Pacella F, Mascaro T, Vingolo E, Vulcano C.L Biofeedback and visual rehabilitation in patients with macular degeneration. Invest.Ophthalmol. Vis. Sci. (ARVO). 2006;- 798/B719.

Chapter 22

Many Suggestions to Protect the Eyes in Aging People Panagiotis Karavitis, MD, Nicola Pescosolido, MD, and Fernanda Pacella, MD

Abstract Some eye problems do not threaten our eyesight. Others are more serious diseases and can lead to blindness. Some common eye complaints can be treated easily. Sometimes they can be signs of more serious problems. Other eye problems can lead to vision loss and blindness. Often they have few or no symptoms. Having regular eye exams is the best way to protect our eyes. In this chapter we report the major suggestions to protect our eyes in aging. The Greek Society for the Prevention of Vision Loss and Blindness suggests some precautions to prevent age-related changes and/or diseases of the human eyes. These precautions are similar to those established by the National Eye Institute (NEI) in the USA, or the Italian Institute for Vision (IIV) in Italy. Are you holding the newspaper farther away from your eyes than you used to? Join the crowd—age can bring changes that affect your eyesight. Some changes are more serious than others, but no matter what the problem, there are things you can do to protect your vision. The keys are regular eye exams and finding problems early. Keywords eyesight, blindness, protection, prevention, professional care, Check, eye diseases, low vision.

Five Steps for Protecting Your Eyesight 1) Have your eyes checked every one or two years by an eye care professional. This can be an ophthalmologist or an optometrist. He or she should put drops in your eyes to enlarge (dilate) your pupils. This is the only way to find some eye diseases, such as diabetic retinopathy, that have no early signs or symptoms. If you wear glasses, they should be checked too. 2) Find out if you are at high risk for eye disease. Are you over age 65? Do you or people in your family have diabetes or eye disease? If so, you need to have a dilated eye exam.1 3) Have regular physical exams to check for diseases like diabetes and high blood pressure. These diseases can cause eye problems if not treated.2

From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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4) See an eye care professional right away if you suddenly cannot see or everything looks dim, or if you see flashes of light. Also see an eye care professional if you have eye pain, fluid coming from the eye, double vision, redness, or swelling of your eye or eyelid. 5) Wear sunglasses that block ultraviolet (UV) radiation and a hat with a wide brim when outside. This will protect your eyes from too much sunlight, which can raise your risk of getting cataracts.

Common Eye Problems Some eye problems do not threaten your eyesight. Others are more serious diseases and can lead to blindness. The following common eye complaints can be treated easily. Sometimes they can be signs of more serious problems.3,4,5,6,7 Presbyopia is a slow loss of the ability to see close objects or small print. It is a normal process that happens as you get older. Holding the newspaper at arm’s length is a sign of presbyopia. You might also get headaches or tired eyes when you read or do other close work. Reading glasses usually fix the problem. Floaters are tiny specks or “cobwebs” that seem to float across your eyes. You might notice them in well-lit rooms or outdoors on a bright day. Floaters can be a normal part of aging. Sometimes they are a sign of a more serious eye problem such as retinal detachment. If you see many new floaters and/or flashes of light, see your eye care professional right away. This is considered a medical emergency. Tearing (or having too many tears) can come from being sensitive to light, wind, or temperature changes. Protecting your eyes, by wearing sunglasses for example, may solve the problem. Sometimes, tearing may mean a more serious eye problem, such as an infection or a blocked tear duct. Your eye care professional can treat both of these conditions. Eyelid problems can come from different diseases or conditions. Common eyelid problems include red and swollen eyelids, itching, tearing, being sensitive to light, and crusting of eyelashes during sleep. This condition is called blepharitis and may be treated with warm compresses. Other less common eyelid problems, such as swelling or growths, can be treated with medicine or surgery.8,9,10,11

Eye Diseases and Disorders The following eye problems can lead to vision loss and blindness. Often they have few or no symptoms. Having regular eye exams is the best way to protect yourself. If your eye care professional finds a problem early, there are things you can do to keep your eyesight. Cataracts are cloudy areas in the eye’s lens causing loss of eyesight. Cataracts often form slowly without any symptoms. Some stay small and don’t change

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eyesight very much. Others may become large or dense and harm vision. Cataract surgery can help. Your eye care professional can watch for changes in your cataracts over time to see if you need surgery. Cataract surgery is very safe. It is one of the most common surgeries done in Greece. Corneal diseases and conditions can cause redness, watery eyes, pain, lower vision, or a halo effect. The cornea is the clear, dome-shaped “window” at the front of the eye. Disease, infection, injury, and other things can hurt the cornea. Some corneal conditions are more common in older people. Treatments for corneal problems can be simple. You may just need to change your eyeglass prescription and use eye drops. In severe cases, corneal transplantation is the treatment. It generally works well and is safe. Dry eye happens when tear glands don’t work well. You may feel itching, burning, or have some vision loss. Dry eye is more common as people get older, especially among women. Your eye care professional may tell you to use a home humidifier, or special eye drops (artificial tears), or ointments to treat dry eye. In serious cases special contact lenses or surgery may help. Glaucoma comes from too much fluid pressure inside the eye. Over time, the pressure can hurt the optic nerve. This leads to vision loss and blindness. Most people with glaucoma have no early symptoms or pain from the extra pressure. You can protect yourself by having regular eye exams through dilated pupils. Treatment may be prescription eye drops, medicines that you take by mouth, laser treatment, or surgery. Retinal disorders are a leading cause of blindness in Italy. The retina is a thin tissue that lines the back of the eye and sends light signals to the brain. Retinal disorders that affect aging eyes include:

Age-related Macular Degeneration (AMD) AMD affects the part of the retina (the macula) that gives you sharp central vision. Over time, AMD can ruin the sharp vision needed to see objects clearly and to do common tasks like driving and reading. In some cases, AMD can be treated with lasers. Photodynamic therapy uses a drug and strong light to slow the progress of AMD. Another treatment uses injections. Ask your eye care professional if you have signs of AMD. Also ask if you should be taking special dietary supplements that may lower your chances of its getting worse.

Diabetic Retinopathy This is a problem that may appear if you have diabetes. It happens when small blood vessels stop feeding the retina as they should. It develops slowly and there

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are no early warning signs. Laser surgery and a treatment called vitrectomy can help. Studies show that keeping blood sugar under control can prevent diabetic retinopathy or slow its progress. If you have diabetes be sure to have an eye exam through dilated pupils at least once a year.

Retinal Detachment This is when the retina separates from the back of the eye. When this happens, you may see more floaters or light flashes in your eye, either all at once or over time. Or it may seem as though there is a curtain in front of your eyes. If you have any of these symptoms, see your eye care professional at once. This is a medical emergency. With surgery or laser treatment, doctors often can bring back all or part of your eyesight.

Low Vision Low vision affects some people as they age. Low vision means you cannot fix your eyesight with glasses, contact lenses, medicine, or surgery.12,13,14,15 It can get in the way of your normal daily routine. You may have low vision if you: ●

● ● ●

have trouble seeing well enough to do everyday tasks like reading, cooking, or sewing; can’t recognize the faces of friends or family; have trouble reading street signs; or find that lights don’t seem as bright as usual.

If you have any of these problems, ask your eye care professional to test you for low vision. There are special tools and aids to help people with low vision read, write, and manage daily living tasks. Lighting can be changed to suit your needs. You also can try large-print reading materials, magnifying aids, closed-circuit televisions, audio tapes, electronic reading machines, and computers that use large print and speech. Other simple changes also may help: ● ● ● ●

●

●

Write with bold, black felt-tip markers. Use paper with bold lines to help you write in a straight line. Put coloured tape on the edges of your steps to help you keep from falling. Install dark-coloured light switches and electrical outlets that you can see easily against light-coloured walls. Use motion lights that turn on by themselves when you enter a room. These may help you avoid accidents caused by poor lighting. Use telephones, clocks, and watches with large numbers; put large-print labels on the microwave and stove.

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References 1. Balacco GC, Pacella E, and Abdolrahimzadeh S (1998) Neovascular glaucoma. Capitolo XI, Pathophysiology of the eye, 4. Glaucoma, J Feher ed, Akademial Kiado, Budapest 2. Pescosolido N, Pacella E, Sagnelli P, Cavallotti C (2005) Effect of systemic anti-hypertensive drugs on intraocular pressure. Nuova Stampa Medica Ital 25(4):5–14 3. Pacella E, Leopardi S, Abdolrahimzadeh S, Stalfieri M, Mollo R, Balacco Gabrieli C (1997) Terapia topica antiglaucomatosa: prospettive terapeutiche. Boll Ocul, Anno 76 5:713–723 4. Hurst JW (1986) Medicina clinica, ed Masson 5. Goodman & Gilman’s The Pharmacological Basis of Therapeutics (1987) McGraw-Hill, 9th Ed 6. Pecori Giraldi J, Pacella E, Librando A, Gabrielli A, Pacella F, Galasso R, Panichi A (2001) Tre anni di esperienza con il latanoprost nel glaucoma ad angolo aperto. Boll Ocul 80:593–600 7. Pecori Girardi J, Pacella E, Librando A, Gabrieli A, Cofone C (2002) La combinazione latanaprost-timololo e la duplice somministrazione di timololo 0.050% associato o no con latanaprost: efficacia e tollerabilita’ nel gpaa. Boll Ocul Anno 81 5/6:11–15 8. Alm A (1995) Latanoprost administered once daily caused a maintained reduction of intraocular pressure in glaucoma patients treated concomitantly with timolol. Br J Ophthalmol 79:12 9. Hartenbaum D (1996) The efficacy of dorzolamide, a topical carbonic anhydrase inhibitor, in combination with timolol in the treatment of patients with open-angle glaucoma and ocular hypertension. Clin Ther 18:460 10. Meyer-Schwickerath G (1956) Erfahrungen mit der Licht-koagulation der Netzhaut und der Iris. Doc Ophthalmol 10:91–131 11. Snyder WB (1967) Laser coagulation of the anterior segment: I. Experimental laser iridotomy. Arch Ophthalmol 77:93–98 12. Perkins ES (1970) Laser iridotomy. Br Med J 2:580–581 13. Khuri CH (1973) Argon laser iridectomies. Am J Ophthalmol 76:490–493 14. Del Priore LV, Robin AL, Pollack IP (1988) Neodymium:YAG and argon laser iridotomy: long term follow-up in a prospective, randomized clinical trial. Ophthalmology 95;1207–1211 15. Thomas JV, Simmons RJ, Belcher CD III (1982) Argon laser trabeculoplasty in the presurgical glaucoma patient. Ophthalmology 89:187–197

Index

A Aberrations of cornea, 40–41 of eye, 39–40 ABT. See Aldehyde-Bisulfite-Toluidine Blue Accessory lachrymal gland, 306–307 ACE inhibitors, role in IOP in elderly patient, 298 Acetazolamide, 376 role in IOP in elderly patient, 298 Acidic proteoglycans, 136. See also Proteoglycans Actin, 10 Advanced glycation end products (AGEs), 53, 283, 313 Advanced non-exudative AMD. See Geographic atrophy Aged people, visual impairment in cataract, 375–378 degenerative myopia, 376 Age-related changes, 133, 134 in apraxia of eyelid, 332 in blepharospasm, 334–335 blink rate, 324 and eyeblink conditions, 336 eyelid movement, 325–328 in lachrymal gland, 309–310 animal studies, 312–313 human studies, 310–312 in subnuclear system, 360 cranial nerves, 361–363 distal insertions and associated structures, 368–369 extraocular muscle, 363–365 muscle fiber population, 365–367 in supranuclear system the cerebellum, 346–349 the cerebrocerebellum and pontocerebellar pathway, 356–360

the spinocerebellar pathway, 351–356 the vestibulocerebellar pathway, 349–351 in tear fluid, 309 Age-related eye disease, 7, 291, 293 Age-related Eye Disease Study, 291 Age-related Eye Disease Study II, 293 Age-related macular degeneration (AMD), 239, 240, 273–274, 287, 376, 381–383 abundance of peroxisomes in RPE in, 282 anisotropy in, 283 basal lamina deposits, 241 basement membrane of RPE in, 278 Bruch’s membrane (BM), 241 bruch’s membrane changes in, 278 drusen in ocular fundus, 241–242 eyes structure, 279–280 histochemical composition of bruch’s membrane in, 280 methods for detection of, 275–277 morphological alterations of mitochondria in, 281 neovascular AMD, 244–246 anti-angiogenic therapies, 249–250 laser photocoagulation, 248 photodynamic treatment, 248 retinal rotation techniques, 251 surgical removal of CNV, 250–251 transplantation of the autologous RPE, 251 transpupillary thermotherapy, 248–249 triamcinolone and cortison in, 250 non-exudative, 287 management of, 290–293 stages of, 289–290 non-neovascular abnormalities in, 288–289

399

400 Age-related macular degeneration (AMD) (cont.) non-neovascular AMD, 247–248 lutein and zeaxanthin effects, 252 rheopheresis and laser application, 252 peroxisomal contribution to basement membrane thickening of RPE, 278–279 photoreceptor (PR) cells, damage of, 240 risk factors for, 242–244 RPE alteration in, 277–278 RPE role, 240 statistical analysis of anisotropy in, 279–280 AGEs. See Advanced glycation end products Aging, 133 eye, optics of, 41–43 Alcian blue, 136 ALCIAN BLUE-CEC Method, 136. See also Trabecular meshwork, stining methods Aldehyde-Bisulfite-Toluidine Blue, 276–277 Alpha-actin (ACTA1) gene, 13 Alpha-tropomyosin, 15 AMD. See Age-related macular degeneration AMD and normal eyes structure, statistical analysis of, 280 Amyloidosis, 164–166 Anecortave (Retaane), cortisene, 250 Anterior mosaic crocodile shagreen, 48 Antidesmin labeling, 10 Antihypertensive drugs, role in IOP in elderly patient, 298–299 Antioxidative vitamins, 252 Apoptosis, 71 Apraxia, 332 Arcus senilis, 45 AREDS. See Age-related Eye Disease Study AREDS II. See Age-related Eye Disease Study II Asteroid hyalosis, 166–167 Astigmatism, 49 Astrocytes, 148–152, 173, 266

B Balacco-Gabrieli hypothesis, 386–387 B cells, 153, 176, 179 Bell’s palsy. See Hemifacial paralysis Bevacicumab (Avastin), drug, 220, 250 B-galactosidosis enzyme, 382 Blepharoptosis, 11, 327–328 Blepharospasm, 334–335

Index Blessig-Iwanoff cysts, 198 Blessig-Iwanoff holes, 194 Blindness AMD cause, 274, 287 cataract cause, 394–395 corneal diseases and, 395 degenerative myopia, 386–387 diabetic retinopathy cause, 383, 385, 395–396 eye problems and, 394 permanent, 378 prevention of, 376 primary open angle glaucoma, 379 and reduced vision causes of, 4 estimated prevalence of eye diseases, 4 world prevalence of, 1–3 retinal detachment, 396 retinal disorders cause, 395 Blink definition of, 319 neuroanatomical and neurophysiological circuit, 320–324 rate in human, 324 recovery in Bell’s palsy, 329 reflex, 321 reflex circuit, 321 reflex excitability, 12 β–Blockers, role in IOP in elderly patient, 298, 301 Blood pressure β–blocker reduces systemic, 298 correlation of IOP and, 299, 302 reduction of, 300 risk factor for glaucoma, 297 SBP and DBP values of, 301 systolic, 296 values of arterial, 298 Botulinum toxin, 331, 334 Bovine vitreous, 158 Brophenol blue (BPB), 269 Brown atrophy, 134 Bruch’s membrane, 219, 225, 230, 241, 274, 278, 283, 288 alteration of, 274–275 changes, 278 empty vacuoles accumulation in, 281 histochemical composition of, 279 inner collagenous layer of, 278 lipid deposits in, 274 lipid peroxides in, 282 polarization microscopy of, 283 thickening of, 278–279 Bursa, 160

Index C Ca2+ channels, 11 Calcitonin gene-related peptide, 307 Calcium channel blocker, role in IOP, 298, 300 Calreticulin protein, 25 Calsequestrin, 22 cAMP. See 3′, 5′–cyclic adenosine monophosphate CAPT. See Complications of Age-Related Macular Degeneration Prevention Trial Captopril drug, role in IOP in elderly patient, 302 Catalase, 11 Cataract diabetic retinopathy and, 384 secondary glaucoma and, 379 symptoms of, 394–395 types of, 376–378 visual impairment and, 375 CCN. See Central caudal nucleus Cell-mediated, immune response, 153 Cellular atrophy, 134 Central caudal nucleus, 320 Central core disease, 16 Central nervous system (CNS), 147 Cerebellum functional role of, 336, 346 PEF and, 358 purkinje cells in, 347 spinocerebellar pathway and, 351–352 Cerebrocerebellum definition of, 347 and pontocerebellar pathway FEF, 356–358 PEF, 358 PPRF, 358–359 SC, 359–360 CGRP. See Calcitonin gene-related peptide Cholesterol granulomas, 170 Cholesterolosis bulbi, 168 Chondroitin sulphates, 134, 158, 161 Choriodal neovascularization (CNV), 240 Choroid, 217 acute ischemic lesions, 232 age-related changes, 225–226 age-related macular degeneration (AMD), 229–231 anterior ciliary arteries, 219 areas supplied by, 222 choriocapillaris, 218–219 hypertensive choroidopathy, 233–234 ICG-angiographic study, 226–229 lamina vitrea, 219

401 multiple functions of, 224 posterior ciliary arteries (PCAs), 220 circulation disorder, 231–232 long posterior ciliary arteries, areas supplied by, 223 occlusions, 232 vascular patterns of, 220–222 watershed zone, 223 stroma, 218 suprachoroidal layer or lamina fusca, 218 venous drainage of, 224 Choroidal neovascularization, 290, 381 Choroidal Neovascularization Prevention Trial, 292–293 Choroidal neovascular membranes, 381–382 Chronic lachrymation, 10 Ciliary neurotrophic factor (CNTF), 240 Cistern, 160 Cloquet’s canal, 160 CNV. See Choroidal neovascularization CNVM. See Choroidal neovascular membranes CNVPT. See Choroidal Neovascularization Prevention Trial Collagen fibrils, 28, 53, 158–160, 277 Collagen level, in normal and AMD eyes structure, 279 Collagen of vitreous, 158 Collagen vascular disease, 16 Complicated cataract, 377 Complications of Age-Related Macular Degeneration Prevention Trial, 291, 293 Conditioned blinks, 325–326 Congenital cataract, 377 Congenital glaucoma, 379 Conjunctival inflammation, 10 Connective tissue changes, 28. See also Eyelid, aging of intramuscular connective tissue, 28–29 metabolic exchange and neurotransmission, 30 Contrast sensitivity function (CSF), 35 Cornea, aging of, 45 Cornea, coupling of, 41, 43 Corneal aberrations, 41, 51 Corneal Arcus, 45–47 deposited lipid molecules, 47 lucid interval of Vogt, 46 prevalence with age, 46 Corneal coma, 42, 51 Corneal coma-like aberrations, 105 Corneal hysteresis (CH), 54

402 Corneal structure Bowman’s membrane, 52 Descemet’s membrane, 55–56 endothelium, 56–57 epithelium, 51–52 stroma, 53–55 subbasal nerve plexus, 52–53 Corneal thickness, 50–51 Corneal ulceration, 10 Cornea shape, 49 Cryopexy, 175 Cuticular Drusen, 288 3′,5′-cyclic adenosine monophosphate, 308 Cyclodiathermy, 381 Cystoid macular edema (CME), 257 Cytokines, 63, 153, 240, 310

D Dacryology, 9 Darglitazone role, in decorin gene, 283 DBP. See Diastolic blood pressure Deep crocodile shagreen, 47–48 anterior and posterior, 48 Degenerative myopia, 376 Balacco-Gabrieli hypothesis, 387 definition of, 386 nature of disease, 386 Del Rio Hortega cells, 148 Denervation atrophy, 16 Dermatan, 134, 141, 143 Dermatochalasis, 11 Desmin, 25 Diabetic retinopathy, 4, 6, 180, 182, 376, 383, 395–396. See also Vitreous hemorrhage cause of blindness in, 385 groups of, 383 major types of, 384–385 visual reduction, 385–386 Diacylglycerol (DAG), 308 Diastolic blood pressure, 296, 299 Dislipidemia, 6 Diuretic drug, role in IOP in elderly patient, 298 Drusen increased risk of CNV, 290–291 increased risk of visual loss, 289 large, 289–292 laser treatment of, 293 types of, 288 Drusenoid PED, 288 Dry AMD, 376, 382

Index Dry eye, 395 conjunctivitis and, 312 definition of, 305–306 frequency in elderly women, 310 Parkinson’s patients and, 328 syndrome, 327 tear glands and, 395

E ECCE. See Extracapsular cataract extraction ECM. See Extracellular matrix Ectropion, 11 Eepiphora, 10 Effectors-armed T cells, 153 EGF. See Endothelial growth factor Electron microscope, role in RPE detection, 276 Electroretinogram, 383 Elschnig’s spots, 232 Endoplasmic reticulum (ER), 23 Endothelial choroidal cells, 151 Endothelial growth factor, 381 Entropion, 11 EOM. See Extraocular muscle Epidermal growth factors (EGFs), 63, 309, 381 Epinucleus, 65 Epiretinal membranes (ERM), 167, 170, 173, 257, 265, 267 clinical examination of, 266 diagnostic characteristics using OCT, 266–267 dyes, application of, 269 enhancement of visualization, 267 ERM surgery, indication for, 268 histological evaluations, 269 ILM peeling, 269 macular function and morphology, assessment of, 267 phacoemulsification and IOL implantation, 269 surgical complications, 269 sutureless 25-gauge and 23-gauge vitrectomy systems, 270 three-dimensional UHR-OCT system, evaluation, 267–268 vitreoretinal surgery, 268–269 ERG. See Electroretinogram Ethics Committees, 135 Extracapsular cataract extraction, 377–378 Extracellular matrix, 28, 275, 282 Extraocular muscle, 363–365

Index Eye blink generator, 321, 323 diseases, 4 diseases and disorders, 394 AMD, 395 diabetic retinopathy, 395–396 low vision, 396 retinal detachment, 396 movement during blinking reticular formation and cervical spinal cord, 321, 323 reticular formation in, 321 problems, 394 Eyelid aging of, 9 changes in kinematics of blinking, 12 cutis laxa, 12 epidermal innervation, 13 horizontal eyelid fissure, 11 hyperexcitable blink reflex, 12 laxity of lower eyelid, 12 orbicular muscle motoneuron activity, 12 raised eyebrows and skin creases, 12 trigeminal reflex blink, 13 kinematics, 325–328 effect of aging, 328 investigated by electromagnetic recordings and OO-EMG, 326 movement, 319, 326 problems, 394 Eyesight protection, steps for, 393–394

F Facial motor nucleus, premotor neurons, 321 Facial motor system in, eyelid movement, 320 Facial movement disorders apraxia, 332 blepharospasm, 334–335 hemifacial paralysis, 328–332 hemifacial spasm, 332–334 FAG. See Fluorangiography Familial amyloidotic polyneuropathy (FAP), 164 FEF. See Frontal Eye Field Felderstruktur fiber conventional innervation of, 367 cranial nerves and, 361 micrograph of, 364–365 nerve terminals of, 355 Fibrillenstruktur fiber micrograph of, 364, 366–367 ratio of, 362 Fibroblast growth factors (FGFs), 63

403 Filamentary sarcoplasmic inclusion, 26 Final common pathway, the, 345–346 Fingerprint inclusion, 26 Flocculonodular lobe, 347, 349 Flouresceine angiography, 244–246, 258. See also Age-related macular degeneration (AMD) Flow cytometry, diagnosis systemic lymphoma, 179 Fluorangiography, 383 Frontal Eye Field, 356–358 Full-thickness macular hole (FTMH), 257–259. See also Macular holes

G GAD. See Glutamic acid decarboxylase GAG. See Glucosaminoglycans; Glycosaminoglycan Ganglioside composition, age-related changes in, 89 Geographic atrophy, 288–289, 376 Gerontoxon, 45 Ginkgo biloba (Egb761), 149 Glaucoma, 5, 378–386 AMD and CNVM prevalence in, 382 congenital, 379 diabetic retinopathy, 383–384 IOP fluctuations and, 378–379 medical treatment of, 380 ophthalmology examination, 379–380 pharmacological treatment of CNV in AMD, 383 photodynamic therapy, 383 primary angle closure, 379 primary open angle, 379 secondary, 379 surgical treatment of, 381 symptoms and progresses of, 378 visual reduction, 385–386 Glial cells, 147–150, 169, 172, 199, 266 Glial fibrillar acidic protein (GFAP), 150 Glicans level, in normal and AMD eyes structure, 279 Glucosaminoglycans, 133 Glutamic acid decarboxylase, 322 Glutathione, 11, 16, 91, 149, 150, 214 Glycation end products (AGEs), 29 Glycosaminoglycan, 29, 143, 158, 159 Goldmann applanation tonometer, 54 Goldmann perimetry, in ERM diagnosis, 267 Golgi tendon organ, 354 GTO. See Golgi tendon organ

404 H Hassall-Henle warts, 48 Hemifacial paralysis, 328–332 Hemifacial spasm, 332–334 Hemosiderin-laden macrophages, 170 Henle’s warts. See Hassall-Henle warts Heparan sulphate, 141, 143 Herpes simplex virus, 328 High-order aberrations (HOAs), 98 Homeostasis, 24, 89, 134, 147, 149, 159, 280 Horizontal eyelid movement, 326 Horizontal eye movement, 345 HSV-1. See Herpes simplex virus Human accessory lachrymal gland, 306 Human crystalline lens, optical changes of. See also Human lens aging modulation transfer function, 102–104 wavefront analysis, 104–105 Human extraocular muscles, 344 Human lachrymal gland, 306 Human lens aging biometric, optical and physical changes, 82–84 crystalline lens position, 84 lens equatorial and pole-pole dimensions, 82 refractive index and lens paradox, 83–84 embryology of lens, 62–63 lens modifications with aging, 98–105 lens morphology, 64–66 lens capsule, 66–69 lens fiber cells, 72–76 ocular lens epithelium, 69–72 lens optical quality, 79–80 metabolic changes and external agents, 86–93 calcium, sodium, potassium, 87 ganglioside composition, 89 hormonal influence, 92–93 ibiquitin conjugation, 91–92 lens phospholipid, 87–89 oxidative stress, 90–91 water content, 90 nuclear fiber compaction, 80–82 physical agents and medication, effect of, 93–98 allopurinol, 97 amiodarone, 98 antimalarials and phenothiazines, 97–98 aspirin and nonsteroidal anti-inflammatory drugs, 95–96 cholesterol-lowering medications, 97 diuretics and antihypertensives, 96 ultraviolet radiation (UVR), 93–95

Index transparency of the crystalline lens, 77–79 zonular apparatus, 84–86 Human oculomotor system, 343 age-related changes in subnuclear system, 360 cranial nerves, 361–363 distal insertions and associated structures, 368–369 extraocular muscles, 363–365 muscle fiber population, 365–367 age-related changes in supranuclear system the cerebellum, 346–349 the cerebrocerebellum and the pontocerebellar pathway, 356–360 the spinocerebelar pathway, 351–356 the vestibulocerebellar pathway, 349–351 somatic motor system and, 344 structural and functional system of, 345–346 Hyaluronic acid, 133, 141, 158, 160, 198 Hyperlipoproteinemia, 45 Hypertensive choroidopathy, 234 Hypertrophic astrocytes, 151. See also Astrocytes Hypocellular vitreous, 159 Hypothyroid myopathy, 16

I ICCE. See Intracapsular cataract extraction ICG. See Indocyanine green angiography Idiopathic epiretinal membrane (ERM), 168, 171, 173 Idiopathic FTMHs, 259 Idiopathic macular holes, 257, 262, 266 IIV. See Italian Institute for Vision Increasing intraocular pressure, 296–297, 378–380 treatment of, 297–298 Indocyanine green angiography, 383 Indocyanine green (ICG), 261, 269 Insulin-like growth factor (IGF-1), 240 Insulin-like growth factors (IGFs), 63 Internal limiting membrane (ILM), 257, 261, 262, 266 Internal optics aberrations, 41 Intracapsular cataract extraction, 377 Intraocular hemorrhage, 168 Intraocular lenses, optics of, 41–43 Intraocular Pressure (IOP), 5 Ischemia, 232 Italian Institute for Vision, 393

Index K Keratan, 143 Keratoconjunctivitis, 10 Keratocyte density, 54, 55 Keratopathy, 48, 175 Krause gland, 306 Kupffer cells, 153

L Lachrymal gland, 305 age-related changes in, 309–310 animal studies, 312–313 human studies, 310–312 anatomy of gross anatomy of, 306 histology of, 306–307 innervation of, 307 secretory function of protein secretion, 308 water secretion, 308–309 Lamellar macular holes (LMH), 257 Laser iridotomy and trabeculoplasty treatment, of glaucoma, 380 Laser photocoagulation, 385 Lateral canthus, 12 LDL. See Low-density lipoprotein Lens bio-densitometric changes. See also Human lens aging scheimpflug photography features, 98–101 Lens capsule aging of, 69 anatomy of, 66–67 mechanical properties, 68 ultrastructure of growth and thickness, 67–68 Lens, development of apoptosis, 62 polypeptide growth factors and cytokines in, 63 tunica vasculosa lentis, 63 Lens fiber cells fiber proteins role of, 73 organization and lens sutures, 72–73 suture formation, 75–76 Lens sutural anatomy, 73–75. See also Lens fiber cells Levator palpebrae superior muscle, 321–322 Lewy bodies, 25 Lightgreen SF yellowish (LGSF), 269 Lipids level, in normal and AMD eyes structure, 279 Lipofuscin, 10, 11 Lipofuscin-laden RPE cells, role in AMD, 288

405 Localized hemosiderosis, 168. See also Vitreous hemorrhage Low-density lipoprotein, 280 Low vision cataract and, 376–378 causes of, 375–376 degenerative myopia, 386–387 degenerative myopia in, 386–387 diabetic retinopathy, 383 open angle glaucoma in, 375 symptoms of, 396 Lymphocytes, 152–153, 174, 178, 307, 313

M Macroglia, 148, 149 Macrophage-migration inhibitory factor (MIF), 63 Macrophages, 148, 149, 153–154, 174, 382 Macula, diabetic tractional retinal detachment of, 171 Macular choroid, ischemic lesions, 232–233. See also Choroid Macular cysts, 257 Macular holes, 257 hole closure rate, improvement for, 261 ILM peeling, 261, 262 intraocular tamponade, 261–262 OCT images, 258–259 SLO and fundus autofluorescence, lesion evaluation, 258 sutureless 25-gauge and 23-gauge vitrectomy systems, 262 ultrahigh resolution OCT (UHR-OCT) for, 259 vital dyes for staining, 261 vitreomacular traction, 259 vitreoretinal surgery, 261 vitreoretinal surgery for FTMHs, complications, 262 Macular pseudoholes (MPH), 257 Mallory bodies, 25 MALT. See Mucosa-associated lymphoid tissue MAP. See Mean arterial pressure Mean arterial pressure, 299 Medial longitudinal fasciculus, 345, 350 MEP. See Motor endplate Microglia, 148, 149, 152, 153 Microglial cells, 152. See also Glial cells Mieloperoxidase, 153 MIRA I. See Multi-center Investigation of Rheophoresis I

406 Mitochondria, 10 beta oxidation in RPE, 281 by-products of, 284 disease in ECM, 275 POS turnover, 280 role in RPE, 274, 277–279 Mitochondrial changes. See also Eyelid, aging of crystalline mitochondrial inclusion, 19 crystals in intermembrane space, 18–20 decline in number and loss of cristae, 17–18 enlargement of mitochondria, 18 irregularly in cristae, 21 matrix density, 18–19 mitochondrial creatine kinase role, 20–21 mitochondrial volume density, 22 osmiophilic or paracrystalline inclusions, 21 oxidative enzymes and focal activity, 21 succino-dehydrogenase activity, 21 Mitochondrial diseases, 275 Mitochondria, type I and type II crystals, 20 Mittendorf’s dot, 63 MLF. See Medial longitudinal fasciculus Modulation transfer function (MTF), 35, 98 Motor endplate, 366 Mucosa-associated lymphoid tissue, 307 Müller cells, 148, 150, 152 Multi-center Investigation of Rheophoresis I, 293 Multifocal electroretinography (mfERG), 267 Muscle dystrophies, 16 Muscle spindle in human extraocular muscle, micrograph of, 353 Mutation, of transthyretin gene, 164 Myofiber abnormalities. See also Eyelid, aging of ACTA1 mutations, 14 depletion of glutathion, 16 duplication of Z line, 16 missense mutation in TPM3, 15 muscular hypotonia, 15 Nemaline bodies (rods), 13–14 Z-line streaming, 15–16 Myopic maculopathy, 387

N Nadolol drug, role in IOP in elderly patient, 298, 301 National Eye Institute, 393 Nd:YAG. See Neodymium: yttrium-aluminium-garnet Nd-YAG laser capsulotomy, 378 NEI. See National Eye Institute

Index Nemaline myopathy, 14 Neodymium:yttrium-aluminium-garnet, 380–381 Nephropathy, 6 Nerve fibers (nf), 52, 154, 205, 307, 354, 358, 361, 375 Neurofilaments (NFL), 150 Neuronal blink circuit, 321 Neutral proteases, 153 NF-κΒ. See Nuclear factor-κΒ Nitrendipine drug, role in IOP, 299 Non-exudative AMD, 287 management of, 290–293 stages of, 289–290 Non-neovascular abnormalities, in AMD, 288–289 Non-trigeminal blinks, 321 Normal aging changes, 288 Nuclear bag fiber, 353 Nuclear factor-κΒ, 313 Nuclear inclusions, 27

O OCT. See Ocular computerized tomography Ocular aberrations, 39 Ocular computerized tomography, 383 Ocular lens epithelium site of transport, metabolism and detoxification, 71 ultrastructure of, 69–71 Oculator motor nerves, 345 Oculomotor system in eyelid movement, 320 restitution of, 369–370 OO-EMG. See Orbicularis oculi electromyography Open angle glaucoma, 5, 375 Optical aberrations, 36 Optical coherence tomography (OCT), 244, 246, 257, 258, 267, 387 Ora serrata, 193, 194 Orbicularis oculi electromyography, 326, 329 Orbicularis oculi muscle blepharospasm and, 334 botulin toxin injections for the treatment of, 334 facial motor system and, 320–321 myofibrous composition of, 319 paresis of, 331 Orbicular muscle, 9, 13 Orbicular muscle aging, 10 Oxidative stress, 11, 87, 90, 149, 194, 206, 242

Index P Paracrystalline sarcoplasmic inclusion, 27 Paramedian pontine reticular formation, 345, 356, 358–359 Parietal Eye Field, 358 Pars plana vitrectomy, 182, 250, 268, 270 PAS. See Periodic acid-Schiff PAS staining, 136. See also Acidic proteoglycans Pearse method. See ALCIAN BLUE-CEC Method PED. See Pigment epithelial detachment PEF. See Parietal Eye Field Pegaptanib (Macugen), drug, 249 Perfluoropropane, 175 C3F8, tamponade, 261 Perfusion pressure (PP), 296 Periductal fibrosis, 310 Perifoveal vitreous detachment (PVD), 257 Periodic acid-Schiff, 306 Peripheral facial nerve palsy, 330 Peripheral nervous system (PNS), 147 Peroxisome activation of, 284 in bruch’s membrane, 278 definition and metabolism of hydrogen peroxide of, 281–282 and mitochondria role in RPE, 274–275 role in accumulation of lipids, 275 role in RPE, 274, 277–280 Peroxisome proliferator-activated receptors, 281–282 PG. See Proteoglycans Phagocytosis, 148, 149, 153, 230 Phospholipase, 153, 308 Photodynamic treatment (PDT), 248 Photopsia, 259, 266 Photoreceptors’ outer segment, 280 Pigment epithelial derived factor (PEDF), 240 Pigment epithelial detachment, 288 PKC. See Protein kinase C Platelet-derived growth factors (PDGFs), 63 POAG. See Systemic blood pressure and chronic simple glaucoma Point spread function (PSF), 36, 39, 102 Polarization microscopy, 28, 134, 137, 273, 275, 276, 282 Pontocerebellum, 347, 356 Pontomedullary reticular formation, 321–322 POS. See Photoreceptors’ outer segment Posterior vitreous detachment (PVD), 161 PPARs. See Peroxisome proliferator-activated receptors

407 PPRF. See Paramedian pontine reticular formation Precortical vitreous syneresis, 162 Prednisolone drug, 329 Premotor neurons, facial motor nucleus, 321 Presbyopia, 394 Primary angle closure glaucoma, 379 Primary ocular lymphoma (POL), 176–180 clumps of pigment epithelium, 177 genotypic analysis for, 179 methotrexate, localized treatment, 180 ratio of interleukin-10 to interleukin-6, 179 RPE detachments in, 176–177 stem-cell transplantation, 180 Primary open angle glaucoma, 379 Proliferative vitreoretinopathy (PVR), 173–175 Prophylactic Treatment of Age-Related Macular Degeneration, 291–292 Prostaglandin, 376 Protein kinase C, 308 Proteoglycans, 28, 67, 133, 143, 275, 282 PTAMD. See Prophylactic Treatment of Age-Related Macular Degeneration Pulley muscle role, in eye movement, 368–369 Purkinje cells, 347, 349

Q Quantimet analyzer, 137, 209

R Ranibizumab (Lucentis), drug, 249 Reactive astrocytes, 150, 151. See also Astrocytes Reactive oxygen species (ROS), 11 Reflex blink assessment of internal networks and nuclei by, 320 characteristics of, 336 divided into, 321 eyelid movement and, 329 eye movement and, 330 Refractive index, 36, 41, 64, 80, 90 Reichert ultramicrotome, 136, 276 REP. See Retinal epithelial pigment Reticular degenerative retinoschisis, 198 Retina acetylcarnitine (ALCAR), treatment degenerative factors, 200 age-related changes of, 194 cobblestone degeneration, 199–200

408 Retina (cont.) cystic degeneration, 195–196 types, 198 latex degeneration, 199 pavement degeneration, 196 retinoschisis, 198–199 senile degeneration of vitreous, 196–197 senile diseases of senile cataracts and senile detachment, 195 senile lesions, 193–194 senile peripheral pigment degeneration, 197 vascular alterations, 197 Retinal epithelial pigment, 382 Retinal image quality average double-pass images, 37 double-pass images and optical performance, 37 senile miosis and tolerance of defocusing, 38 Strehl ratio, 37, 38 Retinal pigmentary epithelial, 287–288 Retinal pigment epithelium (RPE), 170, 177, 199, 273–274, 381–382 age-related changes in macular region, 278 aging of, 205 alteration of, 277–278 basement membrane of, 278 Bruch’s membrane changes by, 278 cellular density, changes in, 205 granules of lipofuscin, 206–207 histochemical composition of bruch’s membrane, 279 lesions of, 234 light microscopy of, 210, 212, 213 lipids staining, 209 lipofuscin in, 280–281 melanin granules and phospholipids, 207, 212 melanolipofuscin and melanolysosomes, 208 neutral lipids, 212, 213 peroxisomal contribution to basement membrane thickening, 278–279 QAI and statistical methods, 209 ststistical analysis of anisotropy in, 279–280 TEM analysis, 209 Retinal tears, 161, 163, 174, 180, 262. See also Vitreous hemorrhage 9-cis retinoic acid receptor, 281 Retraction eyelid movement, 326 RPE. See Retinal pigmentary epithelial; Retinal pigment epithelium

Index RXR. See 9-cis retinoic acid receptor Ryanodine receptor (RYR 1), 24

S Sarcalumenin, 24 Sarcolemma, 10 Sarcopenia, 10, 11 Sarcoplasma, 10, 22. See also Eyelid, aging of cytoplasmic body formation in, 17 Desmin abundance, 25–26 excitation relaxation coupling apparatus, 25 Lipid composition of TAs, 24 muscle-specific impairment in SR Ca2+ pump, 25 proteins, in calcium uptake, storage, and release, 24 sarcoplasmic inclusions, 25–27 tubular aggregation, 23–24 Sarcoplasmic reticulum (SR), 10, 22 SBP. See Systolic blood pressure SC. See Superior Colliculus SCA. See Spinocerebellar ataxia Scanning electron microscopy (SEM), 64 Scanning laser ophthalmoscope (SLO), 258 Scheimpflug camera, 49 Schlemm’s canal, 137, 379 Secondary glaucoma, 379 Senile cataract, 376 Senile entropion and ectropion, 10 Senile miosis, 38 Silicone oil, 175, 261, 384 Social Security, expenditure on glaucoma patients, 7 Soluble proteins, vitreous, 158–159 Spastic entropion or ectropion, 10 Spectrin, 10 Spinocerebellar ataxia, 336 Spinocerebellar pathway, of the cerebellum, 351 the muscle spindle, 352–354 tendon receptors, 354–356 Spinocerebellum, 347 Strehl ratio, 37, 38 Subfoveal choroidal neovascularization, photodynamic treatment, 376 Subretinal fibrosis, 175 Sulfur hexafluoride gas, 175 Sulphated GAGs, 134, 135 Superior colliculus, 359–360 Synchysis scintillans, 167–168, 182 Systemic blood pressure and chronic simple glaucoma, 297 Systolic blood pressure, 296, 299

Index T TC. See Trabecular cells T cells, 153 Tear fluid, 305 age-related changes to, 309 Tendon receptors, 354–356 Tetramethylbensidine, 276 Thixotropy, 331 Tissue necrosis factor α (TNF-α), 310 Tm. See Trabecular meshwork Trabecular cells, 137–140 Trabecular meshwork, 134 corneoscleral and uveoscleral components, 137–138 Duncan’s multiple range test, 137 elastic fibers and TC, 137 electron density, 137–140, 142 lipids staining for microscopy, 135–136 lumen of Schlemn’s canal, 138 microtome analysis, 136 mitochondrial abnormalities, 137, 140 morphological changes and extracellular matrix, 143 proteoglycans sulphate and hyaluronic acid, 140, 142 quantitative analysis of images (QAI), 137 Schlemm’s canal study, 141 staining methods, 136 stroma of, 143 Transforming growth factor-β (TGFβ), 63, 261 Transmission Electron Microscopy (TEM), 52, 64, 136 Transparency in lens, physical concept extinction of light in crystalline lens, 77 large particle scattering, 78–79 small particle scattering, 78 Transpupillary thermotherapy (TTT), 248–249 Transthyretin, transport protein, 164 Traumatic cataract, 377 Triadin, 24 Triamcinolone acetonide (TA), 261, 269, 383 Triangular syndromes, 231, 232 Trigeminal blinks system, 321 Trypan blue (TB), 261, 269 Tubular aggregate myopathy, 24 Tubular aggregates, 22 Tubulo-reticular aggregates, 24 Tumour necrosis factor-alpha (TNFα), 63 Tunica vasculosa lentis, 63 Type II and Type IX collagen, 158, 159, 161. See also Vitreous aging Typical peripheral cystoid degeneration (TPCD), 198

409 U Uncorrected refractive errors (URE), 6

V Varicella-zoster virus, 328 Vascular endothelial growth factor (VEGF-A), 240, 382 Vasoactive intestinal polypeptide, 307 VEGF. See Vascular endothelial growth factor Verapamil drug, role in IOP in elderly parient, 300 Vertical eyelid movement, 326 Vestibular nerve, 349 Vestibule-ocular reflex, 349, 350, 356 Vestibulocerebellar pathway, 349–351 Vestibulocerebellum, 347 VIP. See Vasoactive intestinal polypeptide VISION 2020 initiative, 5 Vision loss cataract cause, 394–395 corneal diseases and, 395 drusen and, 289–290 Visual acuity (VA), 259, 267, 268 Visual reduction, 385–386 Vitrectomy, 165, 385 Vitreous aging hyaluronic acid, photodegradation of, 161 liquefaction, 160–161 type IX collagen, loss of, 161 Vitreous amyloid deposits. See Amyloidosis Vitreous body, dissection, 160 Vitreous cells, disease of, 175 hemoglobin spherulosis, 182 hemorrhage, 180 lymphoma, 176–180 Vitreous composition, 158–159 Vitreous hemorrhage, 163, 174, 180–183. See also Primary ocular lymphoma (POL) treatment of, 182 Vitreous humor, 151, 152 Vitreous membranes, 168–173 Vitreous syneresis, 161, 182. See also Vitreous aging Vitronectin in Bruch’ membrane, 283 VOR. See Vestibule-ocular reflex VZV. See Varicella-zoster virus

410 W Wave-aberrations, 39, 41 Wet AMD, 376, 382 White limbal gridle of vogt, 48 Wolfring gland, 306 World population, distribution of, 4

Index X X-linked megalocornea, 48

Z Z-line, 13 duplication of, 17 Zonular fibers, 84–85