New Concepts in Cerebral Ischemia (Frontiers in Neuroscience)

NEW CONCEPTS IN CEREBRAL ISCHEMIA Edited by Rick C.S. Lin

CRC PR E S S Boca Raton London New York Washington, D.C.

Library of Congress Cataloging-in-Publication Data New concepts in cerebral ischemia / Rick C. S. Lin, editor. p. ; cm. — (Methods & new frontiers in neuroscience) Includes bibliographical references and index. ISBN 0-8493-0119-X (alk. paper) 1. Cerebral ischemia—Pathophysiology. 2. Cerebral ischemia—Molecular aspects. I. Lin, Rick C. S., 1945- II. Methods & new frontiers in neuroscience series. [DNLM: 1. Cerebrovascular Accident. WL 355 N5318 2001] RC388.5 .N464 2001 616.8′1—dc21 2001043360

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Methods & New Frontiers in Neuroscience Series Editors Sidney A. Simon, Ph.D. Miguel A.L. Nicolelis, M.D., Ph.D. Our goal in creating the Methods & New Frontiers in Neuroscience Series is to present the insights of experts on emerging experimental techniques and theoretical concepts that are, or will be, at the vanguard of Neuroscience. Books in the series cover topics ranging from methods to investigate apoptosis, to modern techniques for neural ensemble recordings in behaving animals. The series also covers new and exciting multidisciplinary areas of brain research, such as computational neuroscience and neuroengineering, and describes breakthroughs in classical fields like behavioral neuroscience. We want these books to be the books every neuroscientist will use in order to get acquainted with new methodologies in brain research. These books can be given to graduate students and postdoctoral fellows when they are looking for guidance to start a new line of research. Each book is edited by an expert and consists of chapters written by the leaders in a particular field. Books are richly illustrated and contain comprehensive bibliographies. Chapters provide substantial background material relevant to the particular subject. Hence, they are not only “methods books.” They also contain detailed “tricks of the trade” and information as to where these methods can be safely applied. In addition, they include information about where to buy equipment and Web sites helpful about solving both practical and theoretical problems. We hope that as the volumes become available, the effort put in by us, by the publisher, by the book editors, and by individual authors will contribute to the further development of brain research. The extent that we achieve this goal will be determined by the utility of these books.

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Preface Every year more than a half million American suffer from stroke. At present, stroke is the third leading cause of death in the U.S. and is the number one cause of disability. With the rapid growth of the elderly population, brain attack constitutes a major healthcare concern, even in our highly competitive and developed modernday society. In the United State alone, the estimated annual cost for healthcare may exceed an alarming fifty billion dollars. Despite our extensive research efforts to better understand the biological mechanisms of stroke-induced neuronal injury in animal models, a promising drug which protects neurons from various types of insult remains unavailable for clinical application. However, our endeavors have not gone unrewarded. In fact, multi-disciplinary approaches in search of a neuroprotective therapy have lead to many novel findings, and have provided investigators with valuable insight. With continuing progress, it is foreseeable that in the near future effective treatments for neuron survival will be a reality. The main goal of this book is to provide a detailed description of the various mechanisms involved in neuronal degeneration and the glial response to stroke and/or traumatic brain injury. There is an additional emphasis on potential strategies which may prove beneficial for neuroprotection following ischemic insult or a damage-inducing event. Eleven chapters are included in this book. Over the years, gender-dependent differences have been observed in response to neurological insult. Chapter 1 deals specifically with this issue, and provides a current view as well as experimental data for illustration purposes. Birth asphyxia can cause severe cerebral ischemic injury, and result in a lifetime of reduced mental as well as physical capacity. Chapter 2 introduces this topic and considers processes which elicit neonatal neuron death. The transplantation of stem cells for various types of CNS disease has been examined recently as a candidate approach for function restoration after brain injury. To address this exciting area of research, chapter 3 provides an overview of new findings and elaborates on the future implementation of this application after stroke. Chapter 4 presents issues related to excitotoxicity, oxidative stress, and apoptosis. Chapters 5 and 6 introduce new data and current ideas on the role of calcium and zinc in neuronal death. Adenosine related compounds have been utilized extensively for cardiac ischemia, but have not yet been adopted as a means for limiting the development and severity of neurological lesions. Chapter 7 provides a rationale for the inability of adenosine and its corresponding receptors to produce a beneficial neuroprotective effect in human stroke victims. Chapter 8 summarizes the cellular and molecular events triggered by calpain and caspase in ischemic as well as traumatic brain injury. The brain immune response has attracted a lot of attention. Chapter 9 provides the reader with a new, detailed view which favors the future usage of anti-inflammatory agents to improve neurological outcome following episodes of ischemia. Chapter 10 addresses the fundamentals of hyperbaric oxygen therapy as a potential means to

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prevent neuronal damage. Lastly, one of the most frustrating aspects associated with hypoxia is the lack of consensus regarding transient ischemic attack. Chapter 11 describes experimental models and recent advances in an area that has received little attention. Looking back over 40 years upon the painful memory of a child who lost his grandfather to stroke, I am privileged to have had the opportunity to further understand this devastating disease and contribute to a scientific community whose talented members are rapidly advancing along many fronts to provide hope for many. At this time, I would like to express my sincere appreciation to all of my colleagues who have shaped my professional career and influenced my research interests. First, I would like to thank my previous Duke University colleagues, Drs. Jim Davis, Barbara Crain, and Vic Nadler for introducing me to this gratifying field of study. Second, I would like to thank my good friends and colleagues, Drs. Miguel Nicolelis and Sidney Simon, also at Duke, for their endorsement of this publication and support throughout its assembly. Finally, I extend my gratitude to Barbara Norwitz, a senior editor from CRC, for her patience and commitment to this project.

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Preface The Editor Allelochemical Rick C.S. Lin interactions, was born in during Chung-King, the last three China, decades, in 1945. have He evolved was brought as anupimin portant Taipei, Taiwan, branch ofand plant came ecology. to theInU.S. this as book, a marine in general, biologist the effects in 1969. of chemical He received com-a pounds Master degree released in from marine plants biology (including at Dukemicroorganisms), University in 1971 onand other a Ph.D. plantsdegree in their in vicinity neuroanatomy are considered from Vanderbilt under the University term “allelopathy.” in 1976. From The1976 termto“allelochemical” 1979, Dr. Lin was is aused postdoctoral in a widerfellow context in in neurophysiology the field of ecology at University where itofincludes, Virginia.but In is 1979, not limited he was appointed to, plant and as an microorganism assistant professor interactions. at the Department Allelochemicals of Cell released Biology, from Southwestern plants (inMedical cluding microorganisms) School. He then have returned multifaceted to the Department influencesofon Anatomy, ecosystems; Duke these University, also inas fluence an assistant soil microbial professor ecology, in 1981. soil Innutrients, 1988, he jointed and physical, the Department chemical and of Physiology biological and soil factors. Biophysics, We believe Hahnemann that it is University, extraordinarily as andifficult associate to professor separate the and influence then proof moted allelochemicals to full professor on each ofin these 1992. components Dr. Lin of is an currently ecosystem. a full Effects professor on any one at the of Department these components, of Anatomy, due toUniversity allelochemicals, of Mississippi may influence School growth, of Medicine. distribution, and survival Dr. Lin’s of plant research species. interests are quite diverse, ranging from development, plasticity,The andaim normal of thisfunction, book is totoprovide neurological insightdiseases and recent such progress as stroke. on allelochemical His work has consistently research from been thisaimed multifaceted at the correlation standpoint. between Research structure articles—reporting and function at results the celof lular substantially level, especially completed in the work, visual and and review somatosensory articles—presenting systems. novel He hasand published critical exaptensively, praisals of including specific topics articles of interest, in majorare journals included. such Yetasit may Science, not be Nature, a comprehenand the sive treatise of onthe theNational subject.Academy The sequence of chapters in past the twenty book starts an Proceedings of Science. Over the years,with he has overview continuous followed byawards 34 chapters by scientists around the world, thus received from contributed National Institute of Health, National Science presenting aas global perspective onCompanies, allelochemical Section I— Foundation, well as Pharmaceutical for hisresearch. research activities. He Methodologies 2 –8), discusses important of methodology the also has served (Chapters as a regular member of grant reviewaspects committees for both theinNIH studyVA. of allelopathy, shortcomings of bioassays for allelopathy, bioassays for differand ent plant groups, extraction of allelochemicals from soil, sampling procedures, and an outline of analytical methods for different classes of allelochemicals. Section II— Interactions Among Plant and Microbial Systems (Chapter 9 –15), presents allelochemical research in aquatic and terrestrial ecosystems, and includes other important subjects like pollen allelopathy. Section III—Ecological Aspects (Chapters 16 –22), illustrates the significance of ecological studies in allelochemical research, and discusses the important role that the soil environment plays in the functioning of allelochemicals. Section IV—Biochemical, Chemical and Physiological Aspects (Chapters 23 –30), discusses biochemical, molecular, and physiological aspects of allelopathy, including information on modes of action of allelochemicals in allelopathy. Allelochemicals have been successfully used in biocontrol of plant pathogens and weeds. This important applied aspect of allelochemistry is discussed under Section V—Biological Control of Plant Disease and Weeds: Applied Aspects (Chapters 31 –34). Thus, in totality, the book illustrates the processes, procedures, and applications related to allelochemicals.

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Contributors Frank C. Barone, Ph.D. Department of Cardiovascular Pharmacology GlaxoSmithKline King of Prussia, Pennsylvania John A. Connor, Ph.D. Department of Neuroscience University of New Mexico School of Medicine Albuquerque, New Mexico Joseph A. Erhardt, Ph.D. Department of Cardiovascular Pharmacology GlaxoSmithKline King of Prussia, Pennsylvania Byoung Joo Gwag, Ph.D. Center for the Interventional Therapy of Stroke and Alzheimer’s Disease Department of Neuroscience and Pharmacology Ajou University School of Medicine Kyungkido, South Korea Edward D. Hall, Ph.D. CNS Pharmacology Pfizer Global Research and Development Ann Arbor, Michigan Michael V. Johnston, M.D. Department of Neurology and Developmental Medicine Kennedy Krieger Institute Johns Hopkins University School of Medicine Baltimore, Maryland

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Doo Yeon Kim, Ph.D. Center for the Interventional Therapy of Stroke and Alzheimer’s Disease Departments of Neuroscience and Pharmacology Ajou University School of Medicine Kyungkido, South Korea Yang-Hee Kim, Ph.D. National Creative Research Initiative Center for the Study of CNS Zinc Department of Neurology University of Ulsan College of Medicine Seoul, South Korea Jae-Young Koh, M.D., Ph.D. National Creative Research Initiative Center for the Study of CNS Zinc Department of Neurology University of Ulsan College of Medicine Seoul, South Korea Joo-Yong Lee, Ph.D. National Creative Research Initiative Center for the Study of CNS Zinc Department of Neurology University of Ulsan College of Medicine Seoul, South Korea Jeffrey J. Legos, Ph.D. Department of Cardiovascular Pharmacology GlaxoSmithKline King of Prussia, Pennsylvania

Rick C. S. Lin, Ph.D. Department of Anatomy University of Mississippi Medical Center Jackson, Mississippi Kook In Park, M.D., D.M.Sc. Department of Pediatrics and Pharmacology Yonsei University College of Medicine Seoul, South Korea Jeong Ae Park, Ph.D. National Creative Research Initiative Center for the Study of CNS Zinc Department of Neurology University of Ulsan College of Medicine Seoul, South Korea Andrew A. Parsons, Ph.D. Department of Cardiovascular Pharmacology GlaxoSmithKline King of Prussia, Pennsylvania Robin L. Roof, Ph.D. CNS Pharmacology Pfizer Global Research and Development Ann Arbor, Michigan C. William R. Shuttleworth, Ph.D. Department of Neuroscience University of New Mexico School of Medicine Albuquerque, New Mexico Kimberly L. Simpson, Ph.D. Department of Anatomy University of Mississippi Medical Center Jackson, Mississippi

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Evan Y. Snyder, M.D., Ph.D. Departments of Pediatrics, Neurosurgery, and Neurology Children’s Hospital, Harvard Medical School Boston, Massachusetts Ronald F. Tuma, Ph.D. Department of Physiology Temple University School of Medicine Philadelphia, Pennsylvania Dag K.J.E. von Lubitz, Ph.D. MedSMART, Inc. Ann Arbor, Michigan Kevin K.W. Wang, Ph.D. Department of CNS Molecular Sciences Pfizer Global Research and Development Ann Arbor, Michigan Seok Joon Won, Ph.D. Center for the Interventional Therapy of Stroke and Alzheimer’s Disease Departments of Neuroscience and Pharmacology Ajou University School fo Medicine Kyungkido, South Korea Eric J. Zoog, M.D. Emergency Department Baptist Medical Center Jackson, Mississippi

Preface Contents Allelochemical interactions, during the last three decades, have evolved as an important branch of plant ecology. In this book, in general, the effects of chemical compounds released from plants (including microorganisms), on other plants in their CHAPTER 1 vicinity are considered under the term “allelopathy.” The term “allelochemical” is Gender Differences in Acute Neuroprotective of used in a wider context in theCerebral field of Ischemia: ecology where it includes, Mechanisms but is not limited Estrogen to, plant a Robin Roof and Edward D.multifaceted Hall cludingL.microorganisms) have influences on ecosystems; these also influence soil microbial ecology, soil nutrients, and physical, chemical and biological soil factors. We believe that it is extraordinarily difficult to separate the influence o

CHAPTER 2

these components, due to allelochemicals, mayTheir influence growth, distribution, and Neonatal Hypoxic-Ischemic Brain Insults and Mechanisms survival of plant species. Michael V. Johnston The aim of this book is to provide insight and recent progress on allelochemical research from this multifaceted standpoint. Research articles—reporting results of o substantially completed work, and review articles—presenting novel and critical apCHAPTER 3 topics of interest, are included. Yet it may not be a comprehenpraisals of specific Neural Stem Cell Biology Provides Insights into New Therapeutic Strategies for Hypoxic-Ischemic Brain Injury Kook presenting In Park a and global Evanperspective Y. Snyder on allelochemical research. Section I— Methodologies (Chapters 2 –8), discusses important aspects of methodology in the study of allelopathy, shortcomings of bioassays for allelopathy, bioassays for different plant groups, extraction of allelochemicals from soil, sampling procedures, and CHAPTER 4 an outline of analytical methods for different classes of allelochemicals. Section II— Excitotoxicity, Oxidative and Apoptosis in Ischemic Death alleloInteractions Among PlantStress and Microbial Systems (ChapterNeuronal 9 –15), presents Byoung Joo Gwag, Seok Joon Won and Doo Yeon Kim chemical research in aquatic and terrestrial ecosystems, and includes other important subjects like pollen allelopathy. Section III—Ecological Aspects (Chapters 16 –22), illustrates the significance of ecological studies in allelochemical research, and dis-

CHAPTER 5

lochemicals. Ca Section IV—Biochemical, Chemical and 2+ Signals Intracellular Underlying Rapid and Delayed Excitotoxicity in Physiological Aspects (Chapters 23 –30), discusses biochemical, molecular, and Mature CNS Neurons physiological aspects of allelopathy, including information on modes of action of alJohn A. Connor and C. William R. Shuttleworth lelochemicals in allelopathy. Allelochemicals have been successfully used in biocontrol of plant pathogens and weeds. This important applied aspect of allelochemistry is discussed under Section V—Biological Control of Plant Disease CHAPTER 6 Aspects (Chapters 31 –34). Thus, in totality, the book illustrates and Weeds: Applied Mechanism of Zinc-Induced Neuronal Death Jae-Young Koh, Yang-Hee Kim, Jeong Ae Park and Joo-Yong Lee

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CHAPTER 7 Cerebral Ischemia and Adenosine: “Spheres of Action” Dag K.J.E. von Lubitz

CHAPTER 8 Calpain and Caspase in Ischemic and Traumatic Brain Injury Kevin K.W. Wang

CHAPTER 9 Brain Inflammation, Cytokines, and p38 MAPkinase Signaling in Stroke Frank C. Barone, Ronald F. Tuma, Jeffery J. Legos, Joseph A. Erhardt, and Andrew A. Parsons

CHAPTER 10 The Potential Role of Hyperbaric Oxygen in the Treatment of Stroke Eric J. Zoog

CHAPTER 11 Cellular and Molecular Mechanisms of Ischemic Tolerance Kimberly L. Simpson and Rick C.S. Lin

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1

Gender Differences in Acute Cerebral Ischemia: Neuroprotective Mechanisms of Estrogen Robin L. Roof and Edward D. Hall

CONTENTS 1.1 1.2 1.3

Preface Introduction Gender Differences in Outcome after Ischemic Stroke, Hypoxia, and Subarachnoid Hemorrhage 1.3.1 Ischemic Stroke 1.3.2 Hypoxia 1.3.3 Subarachnoid Hemorrhage 1.4 Role of Circulating Estrogen in Gender Differences in Outcome 1.4.1 Effects of Ovariectomy 1.4.2 Effects of Estrogen Replacement 1.4.3 Estrogen Neuroprotection in Males 1.5 Potential Mechanisms for Estrogen Neuroprotective Effect 1.5.1 Cerebral Blood Flow Changes 1.5.2 Vascular Nitric Oxide Formation 1.5.3 Stimulation of Vascular Maxi-K Channels 1.5.4 Reduction of Leukocyte Adhesion 1.5.5 Antioxidant Effects 1.5.6 Decreased β Amyloid Production and Neurotoxicity 1.5.7 Protection against Glutamate-Induced Excitotoxicity 1.5.8 Activation of MAP Kinase Pathways 1.5.9 Upregulation of bcl-2 Expression 1.6 Genomic vs. Nongenomic Mechanisms of Estrogen Neuroprotection 1.7 Summary References

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1.1 PREFACE It is becoming increasingly clear that striking sex differences exist in the pathophysiology of and outcome after acute neurological injury. Females show a lesser susceptibility to postischemic brain injury in experimental models of stroke. Additional evidence suggests this sex difference extends to humans as well. The greater neuroprotection afforded to females is likely due, in large part, to the effects of circulating estrogens. In fact, exogenous administration of estrogen has been shown to improve outcome in experimental stroke models. The neuroprotection provided by peri-injury administration of estrogen extends to males as well. Multifactorial mechanisms most likely underlie this neuroprotective effect and probably depend on the type and severity of injury as well as the type and concentration of hormone present. Evidence suggests that genomic and nongenomic mechanisms may be involved. Estrogen’s putative effects include preservation of microvascular autoregulatory function, an antioxidant effect, reduction of Aβ production and neurotoxicity, reduced excitotoxicity, increased expression of the antiapoptotic factor bcl-2, and mitogen-activated protein kinase pathways. It is hypothesized that several of these neuroprotective mechanisms can be linked to estrogen’s ability to act as a potent chemical (i.e., electron-donating) antioxidant. The following chapter will discuss experimental and clinical evidence for sex differences in outcome after acute cerebral ischemia, review the evidence implicating estrogens as mediators of this neuroprotective effect, and, finally, address the specific mechanisms underlying estrogen’s neuroprotective effect.

I.2

INTRODUCTION

In spite of our increased understanding of the neuropathological events associated with cerebral ischemia, the roles of gender in the injury and recovery processes have not, until recently, been well studied. Both animal and clinical studies have traditionally employed predominantly male subjects; in those studies in which females are included, gender comparisons are only rarely analyzed. A general assumption has been made in the past that results from studies of brain injury in males would apply to females as well. Additionally, inclusion of females in animal studies adds a degree of complication as it becomes necessary to control for the hormonal fluctuations associated with the reproductive cycle. In human drug trials, there are limitations placed on the inclusion of women of childbearing capacity,1 although recently these have been reduced.2 As a result of these complicating factors, much of our current knowledge of (CNS) response to injury and potential treatments for such injury are based primarily on studies of male subjects. The last decade has seen increasing interest in the role of gender and gender-related hormones in CNS injury processes. The evidence emerging suggests that male and female nervous systems respond differently to injury caused by cerebral ischemia. A number of studies demonstrate gender differences in the pathophysiology of and outcome after CNS insult, with the brains of females consistently exhibiting less damage compared to their male counterparts. While some of these findings may

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be due to differences in the female vasculature, the relative neuroprotection observed in females appears to be largely due to the effects of estrogens. The efficacy of potential treatments for CNS injury may differ between the sexes as well. For example, a clinical trial for the use of aspirin for threatened stroke showed that while aspirin reduced the risk of continuing ischemic attacks, stroke or death, the effect was sex-dependent. The risk reduction for men was 48%, whereas no significant effect for women was observed.3 In a phase III clinical trial of the 21aminosteroid-antioxidant tirilazad conducted in Europe, Australia, and New Zealand, differing efficacy was also found for men and women.4 The drug was found to significantly decrease mortality and improve neurologic recovery 3 months after subarachnoid hemorrhage. Once again, however, the beneficial effects occurred primarily in men. Since a drug development effort may target a mechanism that only works in one gender, it is imperative that we assess possible gender-related differences in response to CNS injury in order to develop effective treatments for both men and women. Furthermore, an understanding of these differences may suggest new mechanistic approaches to acute neuroprotection.

1.3 GENDER DIFFERENCES IN OUTCOME AFTER ISCHEMIC STROKE, HYPOXIA, AND SUBARACHNOID HEMORRHAGE 1.3.1 ISCHEMIC STROKE A lower incidence of ischemic stroke occurs in premenopausal women compared to men.5-9 Explanations for this difference include lifestyle, vascular differences,10 direct and indirect effects of estrogen on the blood vessel wall,11-13 and other endocrine influences.14 Similar sex differences in the incidence of stroke have also been documented in animal studies. For example, in one such study, a greater proportion of male gerbils showed clinical signs of stroke during a 3-hour unilateral carotid occlusion than did the females.15 The relative vulnerability of males and females to CNS tissue damage once a stroke has occurred has also been examined. Prior to the 1990s, only a few studies had addressed this issue.10,16,17 With the increased interest over the past decade, there are now numerous studies indicating that the magnitude of injury after experimental ischemia is gender-linked.15,18-20 The early studies focused primarily on survival rate and incidence of lesions following permanent carotid-artery occlusion. Berry et al.,10 for example, found that more male gerbils developed cerebral infarctions after permanent carotid-artery occlusion than did females. Following the same procedure, Payan and Conrad16 found that mortality, as well as the number of brain lesions, was significantly higher in male gerbils compared to females. Sadoshima et al.17 found that length of survival was greater for females than males after permanent bilateral carotid-artery occlusion. In addition, severe ischemic changes in the brain were seen in 50% of the males but only 15% of the females. This group also examined gender

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FIGURE 1.1 Comparison of the extent of neuronal preservation at 24 hours after ischemia (3 hours of unilateral carotid occlusion) in hippocampal CA1 and lateral cerebral cortex of male vs. female gerbils. P values were obtained using student’s t test (two-tailed). (Originally published in Hall et al., J. Cereb. Blood Flow Metab., 11:292, 1991. Reproduced with permission.)

differences after temporary bilateral carotid-artery occlusion and found a higher survival rate among females. More recent studies have shown that, in addition to sex differences in survival after ischemia and occurrence of infarcts, infarct size and degree of neuronal loss also tend to be less in females than in males after ischemic injury. Hall et al.15 examined a subpopulation of stroke-prone gerbils and found that, 24 hours after an equivalent degree of severe incomplete ischemia produced by temporary unilateral carotid occlusion, males demonstrated significantly more neuronal loss in cerebral cortex and the CA1 of the hippocampus than did females. This finding is shown in Figure 1.1. Alkayed et al.,1,8 using a model of temporary (2-hour) middle-cerebral artery occlusion (MCAO), found that both Wistar and hypertensive female rats had smaller infarcts in cortex and caudate putamen than males of the same strain. Zhang et al.,19 using a similar model, also found larger infarct areas in male rats than in females. Moreover, Hall and Sutter20 reported sex differences in ischemic infarct size after permanent MCAO in mice. Once again, brain infarcts were significantly larger in males than in females.

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1.3.2 HYPOXIA Studies of hypoxia support sex differences in vulnerability to loss of oxygen delivery to the brain. Britton and Kline,21 for example, produced hypoxia in rats by placing the animals in a barometric chamber and reducing ventilation. They found a striking sex difference in survival duration, with females surviving 40% longer than males of the same age. Stupfel et al.22 measured sex differences in mortality after repeated (29 times over 40 months) 20-min periods of inhalation of 5.5% oxygen in nitrogen. Mortality rate was again found higher in male mice (57%) than in females (41%). In another study, Stupfel et al.23 compared mortality in male and female mice after progressively lowering normoxic PO2 over several hours along with nitrogen flushing and found a higher survival rate among females. Saiyed and Riker,24 on the other hand, found no sex difference in survival after acute severe hypoxia in mice. However, it appears, in general, that females experience less CNS damage than males during hypoxia.

1.3.3 SUBARACHNOID HEMORRHAGE Gender differences in another form of stroke, subarachnoid hemorrhage (SAH), may exist as well. In a recent clinical trial, Kassell et al.4 showed that 3-month mortality after severe SAH was lower in females (37%) than in males (53%). In addition, incidence of favorable outcome after severe SAH was higher in females (42%) than in males (26%). The pathophysiology of SAH includes three key features. These are: (1) opening of the blood–brain barrier (BBB), protein extravasation and vasogenic edema; (2) loss of microvascular autoregulation; and (3) delayed cerebral vasospasm. Concerning the first, Smith et al.25 found no differences between male and female rats in the extent of abnormal opening of the BBB following SAH. Possible gender differences in post-SAH microvascular autoregulatory failure and vasospasm have not been explored.

1.4 ROLE OF CIRCULATING ESTROGEN IN GENDER DIFFERENCES IN OUTCOME The most obvious explanation for the observed sex differences in neuroprotection is that circulating gonadal hormones provide this benefit. This hypothesis has been examined quite thoroughly in the area of cerebral ischemia research and to a lesser degree in association with traumatic brain injury. The majority of available reports suggest that females may be protected due to higher levels of circulating estrogen, although progesterone has been shown to exert neuroprotective effects as well.

1.4.1 EFFECTS OF OVARIECTOMY One way to test the hypothesis that circulating gonadal hormones are responsible for the gender differences in outcome from ischemic or traumatic injury to the brain is

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to remove the primary source of these hormones by ovariectomizing females and comparing outcome with that of intact females or with males. Alkayed et al.18 used this strategy to assess the role of hormones in outcome after temporary MCAO. As hypothesized, the degree of damage sustained by the brains of the ovariectomized females was more similar to that of males than to intact females. In other words, loss of the normally circulating female sex hormones eliminated the neuroprotection associated with females in this model. Similarly, Zhang et al.19 found that ovariectomy prior to temporary MCAO increased mortality levels compared to that of intact females and, interestingly, above that seen in intact males. Lesion size was also larger in ovariectomized females compared to intact females. Pelligrino et al.26 also reported that ovariectomized female rats showed greater neurological dysfunction after transient forebrain ischemia (i.e., global ischemia) relative to intact females. In a follow-up study, the same group replicated this finding and reported that ovariectomized rats had more severe histopathology than did intact females.27 The role of the female gonadal hormones in the gender difference in neuroprotection against brain injury is further evidenced by Alkayed et al.,28 who report that unlike normally cycling female rats, infarct size after ischemia in reproductively senescent females does not differ from that of males. Thus, the removal of the female sex hormones, by ovariectomy or with age, eliminates the observed gender difference in outcome after brain injury.

1.4.2 EFFECTS OF ESTROGEN REPLACEMENT The above studies suggest that the cause of poorer outcome in the ovariectomized females was lack of circulating estrogen. Support for this possibility comes from studies in which outcome is examined in ovariectomized females given estrogen injections or implants to replace that normally provided by the ovaries. In the first of such studies, Simpkins et al.29 found that pretreatment of ovariectomized rats with 17β-estradiol decreased mortality following temporary MCAO compared to nontreated ovariectomized rats. In addition, the size of the ischemic area was smaller in the estrogen-treated ovariectomized rats compared to ovariectomy alone. Dubal et al.30 showed an estrogen-related decrease in infarct volume after permanent cerebral ischemia. Several other groups, including Shi et al.,31 Rusa et al.,32 and Stubley et al.,33 have reported similar reductions in the volume of ischemic damage when ovariectomized animals are given estrogen replacement. Pelligrino et al.26 demonstrated not only improved histopathology but also improved neurologic outcome after transient forebrain ischemia with estrogen replacement. Kondo et al.34 also found that estrogen replacement improved behavioral outcome after transient forebrain ischemia. In their study, ovariectomized rats implanted with an estradiol benzoate capsule one week prior to temporary bilateral occlusion of the common carotid performed better after the injury in a water tank task than nontreated ovariectomized rats. These findings support the hypothesis that the loss of neuroprotection associated with ovariectomy is due to an attenuation of circulating estrogen.

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1.4.3 ESTROGEN NEUROPROTECTION IN MALES Estrogen may also provide neuroprotection to males, even with dosing shortly before delivery of the insult. Sudo et al.35 found that the water-soluble form of 17βestradiol, when infused into the brains of gerbils beginning 2 hours prior to transient forebrain ischemia, prevented the deficits in a passive avoidance task seen in nontreated gerbils. Estrogen-treated gerbils also showed less delayed pyramidal neuronal death in the CA1 region of the hippocampus. Chen et al.36 similarly found 17β-estradiol treatment to reduce cell loss in the hippocampus CA1 after transient forebrain ischemia. Toung et al.37 examined the effects of both acute and chronic 17β-estradiol treatments on outcome after temporary MCAO in male rats. They found that in both cases, infarct volume was reduced compared to nonestrogentreated males. A 17β-estradiol-induced reduction of infarct size in male rats after temporary MCAO was also found by Hawk et al.38 Finally, Culmsee et al.39 recently reported that male mice given 17β-estradiol or 2-OH-estradiol 24 hours before induction of permanent focal ischemia had smaller infarcts than those given the vehicle only.

1.5 POTENTIAL MECHANISMS FOR ESTROGEN NEUROPROTECTIVE EFFECT The evidence discussed above clearly suggests that gonadal hormones are responsible for the greater neuroprotection observed in females following cerebral ischemia. The more thoroughly studied of these hormones is estrogen. The following is a discussion of the possible mechanisms by which estrogen may act to provide neuroprotection.

1.5.1 CEREBRAL BLOOD FLOW CHANGES A lower incidence of stroke and vascular events in premenopausal women has been well documented, as has an increase in these events in women after menopause.7 Although indirect effects of estrogens on cardiac risk factors — such as estrogen’s lipid-lowering effect — are partially responsible, direct effects of estrogen on the blood vessel wall may play an important role. These direct vascular effects of estrogens may also underlie much of the hormone’s neuroprotection after brain injury. A number of published studies have shown an effect of estrogen on microvascular vasomotor tone and production of vasoactive substances. Mendelsohn and Karas12 describe four mechanisms by which estrogen can produce rapid vasomotor effects. These include estrogen-induced, endothelium-derived relaxing factor (EDRF) release, estrogen antagonism of vasoconstrictor responses to endothelin, direct hyperpolarizing effects of estrogen on resting vascular smooth muscle, and rapid estrogen antagonism of vascular smooth-muscle calcium channels. Estrogen may also upregulate genes for rate-limiting enzymes in the biosynthesis of two important vasodilators, prostacyclin (PGI2)40-43 and EDRF,44 the latter now identified as nitric oxide (NO•). All of these actions would serve to counteract vasoconstriction or cause vasodilation. ©2002 CRC Press LLC

Such effects on the microvasculature by estrogen could become important because of the hypoperfusion that often immediately follows stroke. A reduction in adequate blood supply to the brain is among the secondary intracranial events that result from the brain’s physiologic response to severe traumatic injury. If sufficient blood flow to the brain is not maintained, ischemic damage to brain tissue will rapidly follow. An improvement in blood flow by estrogen would serve to reduce the extent of secondary damage that results. Several studies provide evidence for such a role of estrogen after acute brain ischemia. Hurn et al.,45 for example, examined postischemic cerebral blood flow (CBF) in male, female, and estrogen-treated female rabbits subjected to a brief episode of four-vessel occlusion-induced forebrain ischemia followed by 3 hours of reperfusion. They found that while baseline blood flow did not differ among the groups, postreperfusion hyperemia was greater in females than in males after a fourvessel occlusion and 3-hour reperfusion. Chronic 17β-estradiol treatment of females caused higher CBF during ischemia and reduced postreperfusion hyperemia in several brain regions compared to non-treated females. In another study by the same group, the effect of ovariectomy on the gender difference in postischemic CBF was examined.18 Age-matched male, female, and ovariectomized female rats from two strains — normotensive Wistar and strokeprone spontaneously hypertensive rats — were subjected to 2 hours of intraluminal MCAO, followed by 22 hours of reperfusion. CBF was monitored by laser-Doppler flowmetry. Female rats of both strains maintained a higher relative flow during ischemia compared to males and ovariectomized females. Volume of tissue with endischemic CBF <10 ml/100 g/min was smaller in females compared to males (but not different from ovariectomized females). Female rats also sustained smaller cortical and striatal infarcts after occlusion compared to age-matched males of both strains. Ovariectomy did not affect the volume of severely ischemic tissue. However, in a later study,46 the same group found that 17β-estradiol treatment of ovariectomized rats did not improve CBF during ischemia, although it did result in infarct volume reduction. These combined findings support a flow-preserving effect of endogenous estrogen during cerebral ischemia but suggest that other mechanisms are involved in estrogen’s neuroprotection as well. Further evidence for estrogen’s CBF-preserving effect has been provided by Stubley et al.,33 who compared post-MCAO CBF in ovariectomized rats with that of ovariectomized rats implanted with 17α-estradiol pellets. During reperfusion, nontreated ovariectomized rats showed an average recovery in CBF of approximately 50% of the pre-MCAO levels, where it remained for 24 hours. The CBF of estrogentreated ovariectomized rats, on the other hand, promptly returned to pre-MCAO levels during reperfusion. The observed differences in CBF were associated with corresponding differences in infarct size. Pelligrino et al.,26 reported that ovariectomy resulted in an exacerbation of the CBF drop experienced by intact female rats subjected to 30 minutes of forebrain ischemia (right common carotid-artery occlusion + hemorrhagic hypotension to 30 mmHg). This effect was reversed with low-dose chronic estrogen treatment.

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FIGURE 1.2 CBF following closed head injury in (A) intact and ovariectomized female rats treated with estrogen or vehicle and (B) male rats treated with estrogen or the vehicle. Asterisk (*) indicates significant difference between the groups at that time point at the p < 0.05 confidence level.

Ovariectomized females injected with 0.1 mg/kg of 17β-estradiol showed CBF recovery equivalent to normal females. In a recent study of blood flow change after traumatic brain injury, Roof and Hall,47 using laser-Doppler flowmetry, demonstrated better recovery of CBF after impact-acceleration closed-head injury in female rats than in males. Ovariectomy partially eliminated the sex difference. Daily injections of 17β-estradiol for two weeks prior to injury resulted in improved postinjury CBF recovery in both males and females, as shown in Figure 1.2.

1.5.2 VASCULAR NITRIC OXIDE FORMATION The results of many of the studies described above suggest that estrogen provides neuroprotection after ischemic injury, at least in some instances, by improving CBF. Hypoperfusion after brain injury can result from a combination of several factors,

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including hypotension, increased intracranial pressure, damage to the microvasculature, or loss of microvascular autoregulation. Microvascular autoregulation is a protective mechanism by which CBF and capillary perfusion pressure are kept relatively constant during changes in systemic blood pressure.48 One component of autoregulation is the myogenic response, which is the constriction or dilation of vascular smooth-muscle cells in response to increases or decreases in transmural pressure. An important modulator of the myogenic response is EDRF or nitric oxide (NO•). Interestingly, the myogenic tone of rat cerebral arteries has been shown to differ between males and females; and this difference appears to result from estrogen enhancement of NO• production.44, 49-51 It has been shown that the vascular endothelium of female animals and humans produces more NO• than that of males; that ovariectomy reduces basal NO• levels; and that estrogen treatment of ovariectomized animals and postmenopausal women increases NO• generation.52 Estrogen has been linked to increased expression and activity of both of the Ca2+-dependent isoforms of NO• synthase (NOS), the endothelial isoform (eNOS), and neuronal isoform (nNOS). Although most of the evidence linking estrogen to increased NOS expression and activity comes from studies of peripheral tissue, there is evidence that the same occurs in the brain.26,27,53,54 Thus, it may be that the NO•-stimulating effect of estrogen is a key mechanism underlying gender differences in blood flow change after ischemic injury. One group recently directly addressed this possibility. Pelligrino et al.55,56 examined whether NOS activity is affected by ovariectomy and estrogen replacement and whether NOS-derived NO• supports vasodilation during ischemia. They measured both CBF changes and NOS levels after transient forebrain ischemia in intact, ovariectomized, and 17β-estradiol-treated ovariectomized female rats and found a direct correspondence between the two measures. Ovariectomized females showed greater reductions in CBF than intact females. Estrogen treatment eliminated the difference. Likewise, NOS levels in brain tissue from the ovariectomized females were lower than from intact females. This difference was eliminated by estrogen treatment. These findings support the hypothesis that estrogen improves postinjury blood flow recovery by enhancing NOS expression. Others have suggested that estrogen increases NO• in the absence of changes in endothelial NO• synthase gene expression.57,58 Barbacanne et al.58 found that exposure of bovine aortic endothelial cells to physiological doses of estrogens did not alter NOS gene expression, but rather induced an antioxidant effect (vide infra) that enhanced the biological activity of NO•. They also reported that in vivo estradiol treatment decreased lucigenin-enhanced chemiluminescence of thoracic aorta from ovariectomized rats, demonstrating an estrogen-related decrease in O2– production. Since O2– is known to react with NO• to form peroxynitrite, with O2– as the rate-limiting factor, decreased O2– results in more available NO•. This estrogen-induced increase in NO• availability would then lead to vascular relaxation and increased blood flow.

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1.5.3 STIMULATION OF VASCULAR MAXI-K CHANNELS A recent report suggests that estradiol may also produce vascular relaxation in an endothelium-independent manner by stimulating smooth muscle Maxi-K channel gates directly.59 Maxi-K channels are key modulators of vascular smooth-muscle tone. Estrogen appears to activate the Maxi-K channel through binding to this membrane receptor’s β subunit. Opening of these channels would cause K+ efflux, membrane repolarization, and closure of voltage-dependent Ca2+ channels, leading to a decrease in intracellular Ca2+ and relaxation of blood vessels.

1.5.4 REDUCTION OF LEUKOCYTE ADHESION Santizo and Pelligrino60 have recently shown that estrogen may improve blood flow after cerebral injury by reducing leukocyte adhesion. Leukocyte infiltration and adhesion occur after cerebral ischemia and can exacerbate neuropathology.61-65 Santizo and Pelligrino60 found increased leukocyte adhesion in the cerebral circulation of ovariectomized female rats, an effect that was reversed with estrogen treatment. The mechanism by which estrogen acts to repress leukocyte adhesion is not known, but one strong possibility is that it occurs secondarily to an estrogen-related increase in NO release. NO• has been shown to reduce leukocyte adhesion66 by diminishing the expression of adhesion molecules such as E-selectin, ICAM-1, and VCAM-1.67,68 Whether via increased NOS synthase expression, increased NO• availability or a nonendothelium-dependent mechanism such as interaction with Maxi-K channels, these data provide multiple mechanisms by which estrogen can improve CBF and exert subsequent neuroprotective effects.

1.5.5 ANTIOXIDANT EFFECTS The studies discussed above show a clear role of CBF enhancement in explaining the relative neuroprotection seen in females in some ischemic injury models. On the other hand, Hall et al.15 failed to find a difference in pre-, intra-, or postischemic cortical CBF between male and female gerbils subjected to a 3-h unilateral carotid occlusion and reperfusion. Females showed significantly less cortical neuronal loss at 24 h postreperfusion compared to males. However, CBF before, during, and for the first 2 h after ischemia in females was not different from that measured in male animals, as shown in Figure 1.3. Thus, improved CBF cannot be the sole explanation for female-associated neuroprotection. Another important mechanism by which estrogen may provide neuroprotection is by limiting an important component of the secondary injury cascade, free radicalinduced lipid peroxidation. Lipid peroxidation is a destructive process initiated by free radicals that has been shown to produce significant damage following acute brain ischemia or traumatic injury (see references 70 and 71 for reviews). Lipid peroxidation of cell membranes is a geometrically progressing process that spreads over the surface of the membrane and on to surrounding cells. Free-radical scavengers such as superoxide dismutase69 and lipid antioxidants, such as the glucocorticoid steroid methylprednisolone,70 U-72099E,71 the 21-aminosteroid tirilazad mesylate,72

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FIGURE 1.3 Comparison of cortical blood flow in male vs. female gerbils before, during, and for 2 h after a 3-h period of unilateral carotid occlusion as measured by hydrogen clearance. (Originally published in Hall et al., J. Cereb. Blood Flow Metab., 11:292-298, 1991. Reproduced with permission.)

and the nonsteroidal 2-methylaminochroman U-78517F,73 have been reported to attenuate posttraumatic pathophysiology and/or to promote survival and recovery in experimental head injury. Tirilazad and U-78517F have also been found protective in models of focal cerebral ischemia.74 Thus, there is extensive evidence that antioxidant compounds are neuroprotective. Estrogens have been shown to be powerful antioxidants15,75-78 and more potent inhibitors of lipid peroxidation than β-carotene, superoxide dimutase,76 and vitamin E.15,76 The chemical structure of estrogen allows for the donation of an electron in the form of a hydrogen atom from the hydroxyl in the 3 position of the aromatic A ring to a lipid peroxyl radical, thus neutralizing its ability to react with neighboring polyunsaturated fatty acids. A schematic of estrogen’s antioxidant activity is shown in Figure 1.4. This antioxidant property of estrogen allows for inhibition of the propagation of lipid peroxidation reactions.76,79,80 Indeed, efforts have been undertaken to improve upon the antioxidant properties of 17β-estradiol by introduction of additional phenolic hydroxyl moieties into the estrogen structure.81

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FIGURE 1.4 Chemical basis for 17β-estradiol’s lipid peroxyl radical scavenging chemical antioxidant action.

Lipid peroxidation is initiated when a free radical species such as an hydroxyl radical (•OH) steals an electron from an allylic carbon of a polyunsaturated fatty acid (LH) esterified to a membrane phospholipid. The resulting alkyl radical (L•) then reacts with molecular oxygen (O2) to form a lipid peroxyl radical (LOO•). The peroxyl can react with another LH creating a second L• representing propagation of the lipid peroxidation chain reactions. Alternatively, 17β-estradiol can step in and donate an electron from the phenolic hydroxyl moiety (3-OH) to the lipid peroxyl radical. As a result, the membrane chain reaction is stopped. The resulting lipid hydroperoxide (LOOH) can be decomposed by the antioxidant enzyme glutathione peroxidase (GSHPx) to an innocuous lipid alcohol (LOH). While uncertain, the resulting 17βestradiol radical can be reduced by acceptance of an electron from ascorbate in the same way that ascorbate regenerates vitamin E from vitamin E radical. The antioxidant effects of estrogen have been mainly demonstrated in neural tissues. Hall et al.15 showed that 17β-estradiol potently inhibits iron-catalyzed lipid peroxidation in rat brain homogenates and is more potent in that regard than vitamin E. Behl et al.82 assessed oxidative cell death caused by β amyloid (Aβ), H2O2, and glutamate in mouse clonal hippocampal cells and found that preincubation of the cells with 17β-estradiol prevented oxidative stress-induced cell damage and cell death (measured as cell viability and cell lysis). Goodman et al.,83 using primary rat hippocampal cultures, likewise found a 17β-estradiol attenuation of the oxidative neurotoxicity produced by Aβ. Culmsee et al.39 found that 17β-estradiol and 2-OHestradiol reduced the percentage of damaged chick embryonic neurons when treated with FeSO4, an initiator of lipid peroxidation. In this primary neuron culture, reactive

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oxygen species were also found to be diminished after treatment with 17β-estradiol or 2-OH-estradiol, suggesting that protection was due to an antioxidant effect of the estrogens used. Vedder et al.84 found 17β-estradiol reduced iron-induced lipid peroxidation in four cell systems: rat brain homogenates, hippocampal HT22 cells, rat primary neocortical cultures, and human brain homogenates. The incubation of primary neuronal cultures with 17β-estradiol showed that the inhibitory effect on lipid peroxidation was paralleled by an increase in survival of cultured cells. Several in vivo studies of ischemic brain injury have addressed the question of estrogen’s ability to act as an antioxidant and reduce lipid peroxidative damage. Hall et al.15 measured brain vitamin E levels before and after unilateral carotid occlusion in male and female gerbils. While they found no difference in baseline levels in brain vitamin E, a difference in vitamin E loss was seen after injury — with much more depletion in males than in females, as shown in Figure 1.5. This suggests that a greater level of oxygen radical-induced lipid peroxidative damage was experienced by the male, resulting in lowered levels of vitamin E. Similarly, Ferris et al.85 examined ascorbate levels and loss after decapitation ischemia in male and female rats.

FIGURE 1.5 Comparison of vitamin E levels in the ischemic hemisphere of male vs. female gerbils 2 hours after a 3-hour period of unilateral carotid occlusion. P values were obtained using student’s t tests.

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They found significant ascorbate loss in the males, but not the females, again suggesting higher levels of oxidative damage in the males. In a follow-up study, KumeKick et al.86 demonstrated that gonadectomy of the female eliminated this sex difference, suggesting that female gonadal hormones are responsible for the effect. Gonadectomy of the male, on the other hand, did not alter ascorbate loss after ischemia. The same group more recently demonstrated that, in some brain regions at least, chronic estrogen replacement (17β-estradiol) prevented the ovariectomy-induced enhanced ascorbate loss associated with ischemia.87 Additional evidence for estrogen’s in vivo antioxidant neuroprotective effect comes from Hall and Sutter,20 who compared infarct size after unilateral carotid occlusion in male and female transgenic mice that overexpress the antioxidant enzyme Cn, Zn superoxide dismutase (Cu, Zn SOD) with wild type males and females. In the nontransgenics, lesion size was greater in males than females, hypothesized to be due to an antioxidant neuroprotective effect of estrogen in the females. In male transgenics, lesion size was smaller than in nontransgenic males, presumably because of the overexpressed Cu, Zn SOD. Female transgenic mice, however, had lesions the same size as females of the wild type. Thus, while overexpression of Cu, Zn SOD provided protection to the males, it did not provide any additional protection in the females, likely because the females already have the benefit of the antioxidant effects of endogenous estrogen. Estrogen may also exert an indirect antioxidant action secondary to its ability to increase NO• (vide supra). Increased NO• levels decrease lipid peroxidative damage because NO• can act as an inhibitor of the lipid peroxidation chain reactions by scavenging lipid peroxyl radicals by the reaction LOO + NO• → LOONO.88-90 Finally, Wang et al.91 demonstrated that estrogen limits 3-nitrotyrosine (3-NT) levels after transient forebrain ischemia in rats. 3-NT is a marker of peroxynitrite, which is now thought to play a critical role in postischemic oxidative neuronal damage. Greater levels of 3-NT immunoreactivity were found in the hippocampus and intermediate cortical layers in ovariectomized compared to intact female rats after unilateral carotid occlusion plus hemorrhagic hypotension, suggesting endogenous estrogen reduces peroxynitrite production in vulnerable brain regions. As noted earlier, peroxynitrite is formed by the reaction of NO• with O2–. Whether NO• reacts with O2– to form peroxynitrite or with peroxyl radicals to halt lipid peroxidation depends on the balance of O2– and NO• concentrations.89 When NO• concentration is lower or equal to O2– concentration, NO• reacts with O2–. When NO• concentration is higher than O2–, NO• remains free to react with and neutralize lipid peroxyl radicals. Pathological conditions such as stroke tend to enhance local release of O2–, leading to increased peroxynitrite and oxidative tissue injury. Enhancement of NO• production, however, allows NO• to play a protective role and interrupt the propagation of the lipid peroxidation reactions down the membrane. Thus, an estrogen-associated increase in NO• levels is neuroprotective.

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1.5.6 DECREASED β AMYLOID PRODUCTION AND NEUROTOXICITY Evidence for yet another possible estrogenic neuroprotective mechanism is derived from studies linking ischemic and traumatic brain injury and susceptibility to the development of Alzheimer’s disease (AD). There exists a strong relationship between cerebral insults and consequent dementia.92 For example, the incidence of dementia during the first year after a cerebral infarct is nine times greater than expected in agematched controls. Amyloid plaques are a characteristic feature of AD that account for more than 90% of the dementias.93 Aβ, the major component of the plaque, accumulates in the brain due to an overproduction of insoluble amyloid precursor protein (APP) cleavage fragments. It is cytotoxic, as is the Aβ25–35 fragment.94 Increased APP expression and Aβ deposition have been demonstrated to occur in the brain after traumatic brain injury,95 focal ischemia,93,96,97 and global cerebral ischemia.98,99 That Aβ deposition plays a role in secondary brain injury is strongly supported by the fact that transgenic mice which overexpress APP have been shown to be more susceptible to ischemic brain damage.100 Estrogen, which has been suggested to be beneficial in the prevention of AD,101,102 is known to protect neurons from the cytotoxic effects of Aβ. For instance, it has been shown that both high83 and physiological103,104 doses of 17β-estradiol protect hippocampal neurons in vitro against Aβ-induced toxicity. In another in vitro study, Gridley et al.105 examined human SK-N-SH neuroblastoma cells exposed to Aβ25–35 with or without the addition of 17β-estradiol. They reported improvements of up to 71% in cell survival 72 hours after exposure to the toxic Aβ fragment when estrogen was added. Behl et al.106 showed that rat primary hippocampal neurons and mouse clonal hippocampal HT22 cells pretreated with either 17α-estradiol or 17β-estradiol were protected against a 24-h challenge by Aβ.25–35 Estrogen may also lessen production of the Aβ precursor APP. An in vivo study93 found that a single injection of 17β-estradiol given two hours before unilateral MCAO to ovariectomized female rats reduced the overexpression of APP by more than 60% in both the penumbra and core of the ischemic area. Therefore, estrogen may protect against Aβ toxicity and decrease its production as well. A number of reports94,107-111 have demonstrated that Aβ toxicity is in large part due to the formation of oxygen radicals and the subsequent initiation of membrane lipid peroxidation. Behl et al.94 determined that hydrogen peroxide (H2O2) mediates Aβ toxicity, since Aβ causes the intracellular accumulation of H2O2. It has been suggested that high doses of 17β-estradiol attenuate Aβ toxicity by reducing the oxidative stress caused by it.82,83,105 Thus, it may be that the neuroprotective effects of estrogen against Aβ in stroke and AD are due in part to the antioxidant action discussed above. In addition to a direct toxic effect, Aβ increases the susceptibility of neurons to excitotoxins,107,112 possibly another avenue through which estrogen may provide neuroprotection against Aβ and brain injury in general.

1.5.7 PROTECTION AGAINST GLUTAMATE-INDUCED EXCITOTOXICITY Several studies have shown that estrogen may protect against neuronal death due to excitotoxicity. Behl et al.82 reported that preincubation with 17β-estradiol attenuated

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the toxicity of glutamate exposure to a hippocampal cell line culture, as measured by cell viability and lysis. Goodman et al.,83 using primary rat hippocampal cultures, likewise found a 17β-estradiol attenuation of the neurotoxicity produced by glutamate exposure. Twenty-four hour pretreatment with 17β-estradiol significantly reduced cell death in primary rat cortical neurons after exposure to glutamate in vitro.113 Regan and Guo114 reported that 17β-estradiol reduced neuronal loss in murine cell cultures due to 24-hour kainate or NMDA exposure, and Weaver et al.115 reported the same in a rat hippocampal culture. The mechanism by which estrogen reduces excitotoxicity has not been determined. There are, however, a number of hypotheses. Regan and Guo114 and Moosmann and Behl116 hypothesized that much of estrogen’s beneficial effect against glutamate toxicity is mediated by its direct antioxidant activity. Indeed, the concentrations of estrogen needed to protect against glutamate-induced excitotoxicity in a cell culture are several orders of magnitude higher than those reported to saturate CNS estrogen receptors,117 making it likely that a non-genomic mechanism is involved. Moosmann and Behl116 also found significant cytoprotection against glutamate toxicity in mouse HT22 cells only when 17β-estradiol concentrations were five orders of magnitude above that needed to induce transcriptional alterations. The antioxidant hypothesis is also supported by the fact that concomitant treatment with the protein synthesis inhibitor, cycloheximide, or the antiestrogen, ICI 182,780, had little114 or no effect on estrogen’s neuroprotection116 in these studies. Weaver et al.,115 on the other hand, suggest that estrogen directly inhibits the NMDA receptor. Their argument is based primarily on their finding that 17α-estradiol, unlike 17β-estradiol, did not reduce NMDA-induced neuronal death in their study, even though both are antioxidants. However, the antioxidant capacities of the two estrogens differ, with 17α-estradiol being slightly weaker.75 Without examining a range of doses, therefore, this argument is problematic. Weaver et al.115 agree with Regan and Guo114 that the mechanism does not likely involve the cytosolic estrogen receptor since they, too, found that an antiestrogen (tamoxifen) did not interfere with the neuroprotective effect of 17β-estradiol. In addition, Behl et al.82 demonstrated estrogen protection against oxidative stress in murine hippocampal HT-22 cells, a cell line that lacks estrogen receptors. These combined data suggest that estrogen’s neuroprotective effects against glutamate-mediated excitotoxicity are due to steroid’s chemical antioxidant action rather than its ability to produce a genomic effect via the estrogen receptor that is critical for neuroprotection against excitotoxicity.

1.5.8 ACTIVATION OF MAP KINASE PATHWAYS Singer et al.118 have very recently suggested that, in addition to an antioxidant effect, estrogen protection against glutamate toxicity may be mediated by activation of signaling pathways similar to those used by growth factors. Mitogen-activated protein kinase (MAPK) pathways are known to play an important role in growth factor signaling and stress signaling.119 One such signaling pathway leads to expression of ERK-MAPK, which may be involved in blocking apoptosis.120

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Estrogen has been shown to rapidly activate MAPK in neuroblastoma cells,121 and in nonneuronal cells.122 In a recent experiment, Singer et al.118 found a rapid and sustained increase in MAPK activity in primary cortical neurons within 30 min of estrogen exposure. In addition, they report that inhibition of MAPK signaling with the MAPK kinase inhibitor PD98059 blocked both the increased MAPK activity and the estrogen-induced neuroprotection of these cells against glutamate toxicity. This effect appears to be estrogen-receptor dependent since it was blocked by the antiestrogen ICI 182,780.

1.5.9 UPREGULATION OF BCL-2 EXPRESSION The antiapoptotic protein bcl-2 is a product of a proto-oncogene originally identified in human B-cell lymphomas.123 Bcl-2 is a survival factor that can block both necrotic and apoptotic cell death124 and promotes cell survival in a variety of tissues including brain. For example, overexpression of bcl-2 in neurons inhibits death in response to deprivation of serum, glucose or growth factors, and exposure to the calcium ionophore A23187.125–127 Bcl-2 overexpression protects neurons from glutamate toxicity as well.127,128 Bcl-2 has multiple neuroprotective actions including prevention of the activation of capases, inhibition of free radical formation, and regulation of calcium sequestration.129 Estrogen upregulates bcl-2 expression in various tissues including brain. Garcia-Segura et al.,130 for example, showed that bcl-2 was increased in the arcuate nucleus of rats on the day of estrus (i.e., peak estrogen levels) compared to other days, was decreased by ovariectomy, and was increased with 17β-estradiol treatment of ovariectomized rats. Bcl-2 induction may therefore be a mediator of estrogen’s neuroprotection. Consistent with this concept, Singer et al.128 showed that 24 h of 17β-estradiol pretreatment significantly increased bcl-2 levels and enhanced survival of NT neurons in vitro after exposure to H2O2 or glutamate. This effect was blocked by the antiestrogen, ICI 182,780, suggesting a mechanistic role of the estrogen receptor. Two in vivo studies further support the hypothesis that bcl-2 is a mediator of estrogen’s neuroprotection. Alkayed et al.18 compared bcl-2 expression and infarct size in male, female, ovariectomized, and estrogen-treated ovariectomized female rats after a 2-h MCAO/22-h reperfusion ischemic injury. The bcl-2 signal was concentrated primarily in the ischemic penumbra and was higher in female and estrogentreated ovariectomized rats compared to male and nontreated ovariectomized rats. The bcl-2 levels correlated negatively with infarct size. In a similar study, Dubal et al.30 found that after unilateral MCAO, the brains of female ovariectomized rats treated with 17β-estradiol showed more bcl-2 expression on the injured side of the brain and smaller infarct size compared to nontreated, ovariectomized female rats. Figure 1.6 summarizes the various neuroprotective actions of 17β-estradiol and their interactions.

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FIGURE 1.6 Mechanisms by which 17β-estradiol may exert its neuroprotective effects. PCS = prostacyclin synthetase.

1.6 GENOMIC VS. NONGENOMIC MECHANISMS OF ESTROGEN NEUROPROTECTION Some of the neuroprotective actions of estrogen are independent of an action on the classic cytosolic estrogen receptor (ERβ). This is no doubt the case for 17β-estradiol’s chemical antioxidant effect. However, for many of the other mechanisms, the issue of the role of estrogen receptor activation is unresolved. In the vast majority of studies examining neuroprotection by estrogen, the 17β-estradiol form was used. 17β-estradiol is the “traditional” estrogen and is responsible for many of the reproductive effects associated with estrogen. The classical mechanism by which 17βestradiol exerts its effects is by binding to the cytosolic receptor, causing either stimulation or repression of gene transcription. Therefore, any effects associated with it, including neuroprotection, theoretically could be mediated via this mechanism. This is not the only mechanism by which 17β-estradiol can act, however.131–136 For example, nonclassic membrane “receptor” actions of estrogen also exist,135 such as a reduction of calcium currents in rat neostriatal neurons,137 in addition to the aforementioned antioxidant action. Evidence that estrogen provides neuroprotection via a mechanism other than the classical ERα action comes from several sources. Hurn et al.,138 for example, compared infarction size in ERα knockout (KO) vs. wild-type mice in a model of MCAO previously found sensitive to the effects of 17β-estradiol. They found that infarct size was unchanged by ERα deficiency in female mice. This suggests that the ERα is not

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necessary for estrogen’s neuroprotection. Another way to determine the involvement of ERα when 17β-estradiol is used is to co-administer an antiestrogen such as tamoxifen or ICI 182,780. Both are ERα antagonists and will block the effects of 17βestradiol that occur through the classic genomic receptor mechanism. Culmsee et al.39 found that neither the neuroprotective effect nor the free radical scavenging properties of estrogens against oxidative stress in a cell culture were influenced by the addition of tamoxifen. Several of the studies looking at 17β-estradiol neuroprotection from excitotoxic injury also used antiestrogens to assess the involvement of estrogen receptors in estrogen’s neuroprotective effects. Unfortunately, the results are mixed. Tamoxifen113 and ICI 182,780118 blocked the protective effects that 17βestradiol provided against glutamate exposure to primary cortical neurons in vitro. On the other hand, Regan and Guo114 reported that ICI 182,780 did not block neuroprotection provided by 17β-estradiol from cell death caused by glutamate exposure to a murine cortical cell culture. Weaver et al.115 also found that tamoxifen did not block 17β-estradiol’s neuroprotection from NMDA and kainate-induced cell death in a rat hippocampal cell culture. The conflict between the results of these studies leaves the issue of ERα involvement, at least in excitotoxic injury, unresolved. The only other relevant studies in which antiestrogens were used were two that demonstrated 17β-estradiol can upregulate NOS via a receptor mechanism. Hayashi et al.139 found that exposure of endothelial cells in vitro to 17β-estradiol upregulates NOS, an effect blocked by both tamoxifen and ICI 182,780. Weiner et al.140 found a pregnancy-induced increase in NOS activity in several guinea pig tissues including cerebellum. This effect was blocked by tamoxifen as well. This limited information leaves open the possibility that estrogen’s effects on NOS, and subsequently on blood flow, are mediated by a receptor mechanism. Another method of determining whether estrogen’s neuroprotection is mediated by a classical receptor is use of the epimer of 17β-estradiol, 17α-estradiol. This form of estrogen binds only very weakly to ERα. Thus, neuroprotective effects produced by 17α-estradiol are not likely to involve ERα activation. There are but a few examples of this strategy available. Green et al.141 found that 17α-estradiol was effective in reducing cell death caused by serum deprivation of human neuroblastoma SK-N-SH cells. Weaver et al.115 assessed the effects of 17α-estradiol against NMDA and kainate-induced cell death in a rat hippocampal cell culture. They found that, unlike 17β-estradiol, it provided no protection — a finding consistent with an ER-mediated action of estrogen. Ironically, in this same study, tamoxifen did not block 17β-estradiol’s effect, suggesting the opposite. On the other hand, Behl et al.106 found that rat primary hippocampal neurons and mouse clonal hippocampal HT22 cells were protected from damage caused by Aβ 25–35 by 17α-estradiol. Because Aβ 25–35 causes oxidative stress and 17α-estradiol’s neuroprotection against it may be mediated via its antioxidant effect, this latter finding is not surprising. A new class of estrogen receptors, ERβ, has been recently described.142 Although some domains of ERα and ERβ are homologous and share functional similarities, their localization and mechanisms related to transcription may differ.143-145 Lindner et al.146 reported the presence of ERβ in rat aorta, suggesting a possible role in mediating estrogen’s effects on blood flow. In addition, upregulation of these receptors

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occurred after injury to the aorta. There is very little information concerning the distribution of this receptor type in brain or of its involvement in neuroprotection. Dubal et al.,30 however, demonstrated that not only is the ERβ present in rat cortex, but that ERα and ERβ are differentially regulated by ischemic injury. In nonestrogen-treated rats, ERα was dramatically upregulated by injury, while ERβ was downregulated. 17β-estradiol treatment blocked the downregulation of ERβ, resulting in a higher ratio of ERβ/ERα. While these data do not tell us which, if either, of the two receptor types mediates a receptor-based mechanism, the fact that their numbers are altered by injury suggests there may indeed be a role for the classical hormone receptor in estrogen’s neuroprotective effects.

1.7 SUMMARY Striking differences in susceptibility to postischemic brain damage between male and female animals have been demonstrated in many experimental models with females having a distinct neuroprotective advantage. Evidence also supports a similar gender difference in vulnerability in humans, at least regarding the outcome after SAH. It is likely that the lesser vulnerability of the female brain is based upon the intrinsic multimechanistic neuroprotective effects of estrogen. The list of mechanisms includes a preservation of microvascular autoregulatory function, a prominent chemical (i.e., electron-donating) antioxidant effect, an attenuation of Aβ production and neurotoxicity, an antagonism of glutamate excitotoxicity, an increase in expression of the antiapoptotic factor bcl-2, and activation of MAPK pathways. To what extent these actions involve estrogen receptor-based vs. nongenomic mechanisms remains to be defined. The specific mechanism(s) that come into play probably depend on a variety of factors including severity and type of injury, the endogenous level or exogenous dose administered, and the type of estrogen employed in the case of pharmacological studies. In any event, the unraveling of the mechanisms of gender-based differential vulnerability to ischemic brain injury and estrogen-mediated neuroprotective effects may lead us to an improved understanding of the mechanisms of secondary brain injury and to the identification of novel pharmacological neuroprotective strategies.

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124. Bredesen, D.E., Neural apoptosis, Ann. Neurol., 38, 839, 1995. 125. Allsop, D. and Williams, C.H., Amyloidosis in Alzheimer’s disease, Biochem. Soc. Trans., 22, 171, 1994. 126. Mah, S.P., Zhong, L. T., Liu, Y., Roghani, A., Edwards, R.H., and Bredesen, D.E., The protooncogene bcl-2 inhibits apoptosis in PC12 cells, J. Neurochem., 60, 1183, 1993. 127. Zhong, L.T., Kane, D.J., and Bredesen, D.E., BCL-2 blocks glutamate toxicity in neural cell lines, Brain Res: Mol. Brain Res., 19, 353, 1993. 128. Singer, C.A., Rogers, K.L., and Dorsa, D.M., Modulation of Bcl-2 expression: a potential component of estrogen protection in NT2 neurons, Neuroreport, 9, 2565, 1998. 129. MacManus, J.P. and Linnik, M.D., Gene expression induced by cerebral ischemia: an apoptotic perspective, J. Cereb. Blood Flow Metab., 17, 815, 1997. 130. Garcia-Segura, L.M., Cardona-Gomez, P., Naftolin, F., and Chowen, J.A., Estradiol upregulates Bcl-2 expression in adult brain neurons, Neuroreport, 9, 593, 1998. 131. Teyler, T.J., Vardaris, R.M., Lewis, D., and Rawitch, A.B., Gonadal steroids: effects on excitability of hippocampal pyramidal cells, Science, 209, 1017, 1980. 132. Wong, M. and Moss, R.L., Electrophysiological evidence for a rapid membrane action of the gonadal steroid, 17 beta-estradiol, on CA1 pyramidal neurons of the rat hippocampus, Brain Res., 543, 148, 1991. 133. Wong, M. and Moss, R.L., Long-term and short-term electrophysiological effects of estrogen on the synaptic properties of hippocampal CA1 neurons, J. Neurosci., 12, 3217, 1992. 134. Morley, P., Whitfield, J.F., Vanderhyden, B.C., Tsang, B.K., and Schwartz, J.L. A new, nongenomic estrogen action: the rapid release of intracellular calcium, Endocrinol., 131, 1305, 1992. 135. McEwen, B.S., Alves, S.E., Estrogen actions in the central nervous system, Endocrinol. Rev., 20, 279, 1999. 136. McEwen, B.S., The molecular and neuroanatomical basis for estrogen effects in the central nervous system, J. Clin. Endocrinol. Metab., 84, 1790, 1999. 137. Mermelstein, P.G., Becker, J.B., and Surmeier, D.J., Estradiol reduces calcium currents in rat neostriatal neurons via a membrane receptor., J. Neurosci., 16, 595, 1996. 138. Hurn, P.D., Sampei, K., Sawada, M., Goto, S., Crain, B.J., Alkayed, N.J., Korach, K. S., Traystman, R.J., Demas, G.E., and Nelson, R.J., Stroke in mice deficient in classical estrogen receptors, Conference of the Society for Neuroscience, Los Angeles, 1998. 139. Hayashi, T., Yamada, K., Esaki, T., Kuzuya, M., Satake, S., Ishikawa, T., Hidaka, H., and Iguchi, A., Estrogen increases endothelial nitric oxide by a receptor-mediated system, Biochem. Biophys. Res. Commun., 214, 847, 1995. 140. Weiner, C.P., Lizasoain, I., Baylis, S.A., Knowles, R.G., Charles, I.G., and Moncada, S., Induction of calcium-dependent nitric oxide synthases by sex hormones, Proc. Nat. Acad. Sci. U.S.A., 91, 5212, 1994. 141. Green, P.S., Bishop, J., and Simpkins, J.W., 17 alpha-estradiol exerts neuroprotective effects on SK-N-SH cells, J. Neurosci., 17, 511, 1997. 142. Mosselman, S., Polman, J., and Dijkema, R., ER beta: identification and characterization of a novel human estrogen receptor, FEBS Lett., 392, 49, 1996. 143. Kuiper, G.G., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J.A., Cloning of a novel receptor expressed in rat prostate and ovary, Proc. Nat. Acad. Sci. U.S.A., 93, 5925, 1996. 144. Tremblay, G.B., Tremblay, A., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Labrie, F., and Giguere, V., Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor beta, Mol. Endocrinol., 11, 353, 1997.

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145. Kuiper, G.G. and Gustafsson, J.A., The novel estrogen receptor-beta subtype: potential role in the cell- and promoter-specific actions of estrogens and anti-estrogens, FEBS Lett., 410, 87, 1997. 146. Lindner, V., Kim, S.K., Karas, R.H., Kuiper, G.G.J.M., Gustafson, J.A., and Mendelsohn, M. E., Increased expression of estrogen receptor-b mRNA in male blood vessels after vascular injury, Circ. Res., 83, 224, 1998.

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2

Neonatal HypoxicIschemic Brain Insults and Their Mechanisms Michael V. Johnston

CONTENTS 2.1 2.2

Introduction Major Syndromes of Neonatal Hypoxic-Ischemic Injury 2.2.1 Syndromes Caused by Asphyxia 2.2.1.1 Near-total asphyxia 2.2.1.1.1 Pattern of Injury in Human Infants 2.2.1.1.2 Pathogenesis of the Near-Total Asphyxia Pattern 2.2.1.1.3 Models of Near-Total Asphyxia 2.2.1.1.3.1 Acute Total Asphyxia in Monkeys 2.2.1.1.3.2 Acute Asphyxia in Piglets 2.2.1.2 Partial Prolonged Asphyxia 2.2.1.2.1 Injury in Human Infants 2.2.1.2.2 Experimental Models 2.2.1.2.2.1 Partial Prolonged Asphyxia in Monkeys 2.2.1.2.2.2 Partial Projonged Asphyxia in Piglets and Rodents 2.2.1.3 Parasagittal Watershed Infarction 2.2.1.3.1 Injury in Human Infants 2.2.1.3.2 Experimental Models of Parasagittal Watershed Infarction 2.2.1.4 Syndrome of Hypoxic-Ischemic Encephalopathy 2.2.2 Syndromes Caused Primarily by Ischemia 2.2.2.1 Focal Ischemic Infarction 2.2.2.1.1 Injury in Human Neonates 2.2.2.1.2 Focal Ischemia Models 2.2.2.2 Periventricular Leukomalacia (PVL) 2.2.2.3 Hypothermic Circulatory Arrest with Heart Surgery

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2.2.2.3.1 2.2.2.3.2

Insult in Human Neonates Models of Brain Injury from Hypothermic Circulatory Arrest 2.3 Mechanisms for Deplayed Neuronal Injury 2.3.1 Excitotoxic Mechanism 2.3.1.1 Disruption of Glutamate Synapse Function 2.3.1.2 Selective Vulnerability Due to Connectivity of Neuronal Circuits 2.3.2 The Neurotoxic Cascase and Delayed Energy Failure 2.3.3 The Apoptosis-Necrosis Continuum 2.3.4 Experimental Interventions to Modify Injury 2.3.4.1 Hypothermia 2.3.4.2 Oxygen Free Radical Scavengers 2.3.4.3 Carbon Dioxide and Acidosis 2.3.4.4 Glutamate Antagonists 2.3.4.5 Nitric Oxide Synthesis Inhibition 2.3.4.6 Caspase Inhibition 2.3.4.7 Neuronal Growth Factors 2.3.4.8 Glucocorticoids 2.3.4.9 Hypoxic Preconditioning 2.4 Conclusion References

2.1 INTRODUCTION Cerebral ischemia is an important cause of brain injury and permanent neurologic disability in fetuses and neonates, and considerable progress is being made to determine underlying causes and to develop neuroprotective interventions. In contrast to adults — where focal single-vessel occlusions from emboli or thrombi are the most common causes of cerebral ischemia — in the neonate, ischemia superimposed on severe hypoxemia secondary to disruption of ventilation or oxygen delivery is more common.1 For example, in the syndrome of near-total asphyxia due to nearly complete disruption of delivery of oxygen to the infant through the umbilical cord, severe hypoxemia leads to reduced cardiac output, which in turn causes relatively symmetric reduction in cerebral blood flow to the brain.2 When focal occlusion of a cerebral vessel occurs in the infant, it is more likely to be caused by infection associated with intravascular clotting, emboli of cardiac origin, or genetic clotting disorders than in adults.3 Major differences in the selective vulnerability of brain regions also distinguish the infant from the adult. For example, selective vulnerabiltiy of the periventricular white matter in premature infants,4 or of the basal ganglia and thalamus in term infants subjected to severe asphyxia,5,6 are syndromes that are far less frequent in older individuals. Another factor that distinguishes the infant from the adult is the enhanced role of glutamate-mediated excitotoxicity in the infant.7 Although the infant brain is protected from hypoxia from an energetic standpoint because of its

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modest energy consumption,8 failure to supply the neonate with a critical threshold of oxygen and glucose causes severe excitotoxic injury.9 This injury is associated with prominent signs of heightened excitatory activity including seizures and epileptic changes on EEG and encephalopathy.10 Developmental changes in excitatory neurotransmitter circuits, especially in the subunit composition of NMDA type glutamate receptors, may make them more likely to mediate injury in the neonate.11 Therefore, hypoxic-ischemic insults in the neonatal brain can be distinguished from ischemic disorders in adults by their causation, age-dependent differences in regional vulnerability, and molecular factors involved in their pathogenesis.

2.2 MAJOR SYNDROMES OF NEONATAL HYPOXICISCHEMIC INJURY 2.2.1 SYNDROMES CAUSED BY ASPHYXIA 2.2.1.1

Near-Total Asphyxia

2.2.1.1.1 Pattern of Injury in Human Infants The fetus is vulnerable to complete disruption of oxygen delivery from the placenta if the umbilical cord becomes kinked or compressed. This type of insult or absence of ventilation in neonates can result in a severe impairment of oxygen delivery together with a buildup of carbon dioxide, a condition referred to as asphyxia.9 The risk of brain injury from asphyxia is most closely related to the degree of hypoxemia causing metabolic acidosis from accumulation of lactic acid. Clinical studies suggest that the risk of brain injury can reach 50% if the cord pH after an asphyxial episode falls to less than 7.0 with a concurrent metabolic acidosis associated with a base deficit of more than 20.5,9,12,14 A recent study in asphyxiated infants showed a strong link between an elevation in the ratio of lactic acid to creatinine in urine and severity of brain dysfunction (encephalopathy).13 Infants exposed to severe asphyxia at the end of a term gestation cannot endure the insult for longer than approximately 30 min because the heart fails to maintain cardiac output and arrests after longer intervals.14 Infants with a brain insult associated with the syndrome of near-total asphyxia have undergone severe hypoxemia, acidosis, and reduced cardiac output nearly to the point of cardiac arrest but are able to be resuscitated.2 Neonates who have survived such an intense but relatively brief insult have a characteristic pattern of brain injury which can be visualized on MRI (magnetic resonance imaging) scanning, and sometimes on computerized tomographic (CT) radiologic scanning.5,6,15 Within several days to weeks after the insult, an MR image weighted for T1 (time for longitudinal magnetization relaxation) exhibits a symmetric increased signal in the peri-Rolandic sensorimotor cortex, the putamena, the thalami bilaterally, and sometimes in the brainstem.16 Careful analysis of brain density with CT scans has demonstrated reduced density of the thalamus and basal ganglia within several days after the injury in some infants.6 At an interval of several months to many years later, the T1-weighted signal disappears and there is enhanced signal in the same regions on images weighted for T2 (time for transverse magnetization

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relaxation), suggesting gliosis.5 Although very severe injury can extend beyond these selectively vulnerable regions into the central and subcortical white matter, in most infants damage from the near-total asphyxia syndrome spares completely the periventricular white matter and cerebral cortex.2,5 This pattern of injury is virtually always associated with severe motor impairment fitting a pattern called extrapyramidal cerebral palsy. 2.2.1.1.2 Pathogenesis of the Near-Total Asphyxia Pattern The pattern of selective vulnerability in the putamen, thalamus, and peri-Rolandic cerebral cortex corresponds to neuronal areas that are relatively more active than others in the neonatal brain but not to recognized patterns of arterial distribution.17,18 This suggests that the pattern of injury is related more to the intrinsic vulnerability of these areas to energy failure than to regional patterns of ischemia. One potentially important link among these areas is that they are interconnected by excitatory neuronal circuits.19 Glutamate-containing projections from the cortex innervate both the thalamus and putamen, which is the primary motor component of the basal ganglia. The thalamus in turn sends excitatory projections to the sensorimotor cortex. Overactivity in these excitatory pathways could contribute to excitotoxic injury in these regions, and clinical evidence of excessive excitatory activity after an asphyxial insult is consistent with this hypothesis.20 Functional brain imaging using positron emission tomography (PET) with 18Fluro-deoxyglucose (FD-glucose) in human infants indicates that these selectively vulnerable regions are also relatively more active than other regions in the period after an asphyxial insult.17,18 Experimental evidence indicates that the regional cerebral metabolism of glucose (rCMRgl) in the brain is tightly linked to the turnover of glutamate at synapses, suggesting that the relative increase in rCMRgl observed after asphyxia may correlate with increased activity at excitatory synapses.21 Therefore, the selective pattern of injury seen after near-total asphyxia in human neonates may reflect the location of vulnerable regions within maturing excitatory circuits.22 2.2.1.1.3 Models of Near-Total Asphyxia Several models of near-total asphyxial brain injury in human neonates have been developed in laboratory animals, including subhuman primates and piglets.14,23 Although there are important differences between the models and the human disorder, there are also remarkable similarities, especially with respect to the pattern of selective neuronal vulnerability. 2.2.1.1.3.1

Acute Total Asphyxia in Monkeys

Myers’ model of acute total asphyxia in monkeys remains one of the best replications of the insult seen in human neonates. In this model, term monkey fetuses were exposed to timed intervals of 10 to 25 min of asphyxia by occluding the umbilical cord and slipping a thin, saline-filled rubber sac over the fetal head at surgical delivery.14 The neonates were resuscitated with ventilation, cardiac massage, and intra-arterial epinephrine and maintained for intervals of several days to weeks after the insult. The initial insult was associated with a marked drop in systemic blood pressure to below 10 mm Hg, severe metabolic acidosis with a pH as low as 6.7, and a base

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deficit approaching 20 mEQ. In this model the first evidence of brain injury one week later was observed after 10 minutes of asphyxia, while animals asphyxiated for longer than 25 minutes generally died of heart failure shortly after the insult. Neuropathologic examination of the brains in these animals demonstrated widespread injury in the thalamus and brainstem when the insult lasted longer than 16 to 18 min.14 Myers noted that the most vulnerable structures, such as the posterior and lateral ventral thalamic nuclei, also had the highest regional cerebral blood flow in the newborn period in the baseline state when studied with 14C-antipyrine autoradiography.14 This is consistent with the hypothesis that vulnerable structures have a relatively high baseline level of neuronal activity. Interestingly, Myers commented that the injury pattern produced in the monkey fetus bore no relation to human perinatal damage recognized at the time. He speculated that this was based in part on the fact that the newborn monkey is more mature than the human term neonate. However, modern MR imaging does reveal that the thalamus is a focus of injury in most infants with the syndrome of near-total asphyxia.5,6 2.2.1.1.3.2

Acute Asphyxia in Piglets

Asphyxia in one-week-old piglets has provided one of the best models for the neuropathologic and neuroimaging pattern of injury observed in human neonates with the syndrome of near-total asphyxia.23 In the model described by Martin et al.,23 oneweek-old piglets are intubated under anesthesia with pentobarbital, paralyzed, and ventilated, and physiologic variables and temperature are controlled. Hypoxemia is initiated by reducing the arterial oxygen saturation to 30% for 30 min, followed by 5 min of ventilation with room air, followed by 7 min of airway occlusion during which arterial oxygen saturation is reduced to less than 5%. At the end of asphyxia, the animals are resuscitated with 100% oxygen and chest compressions, and epinephrine and bicarbonate are infused to maintain arterial pH at 7.4. Animals that survive 4 d are perfusion-fixed for pathologic examination. Analysis of six piglets prepared in this way revealed a selective pattern of injury in the somatosensory cortex, basal ganglia (particularly in putamen, subthalamic nucleus, and substantia nigra), and ventral thalamus that strikingly resembles the pattern seen in neuroimaging in human neonates with near-total asphyxia.5,6 It is noteworthy that in animals with less severe injuries, there were lesions in the thalamus, putamen, and small areas of somatosensory cortex, while more severely injured animals had involvement of the caudate and more extensive involvement of the cerebral cortex. The preferential involvement of the thalamus and putamen parallels lesions seen on MR images in human neonates. Thoresen et al. have also developed a useful model of asphyxial injury in piglets in which the degree of hypoxemia is controlled by monitoring the amplitude of EEG activity.24 This model has been extensively studied from the standpoint of the relationship between encephalopathy and neuropathology. In this model, one-day-old piglets are intubated and ventilated under halothane anesthesia, and physiologic and EEG monitoring are initiated. After a period of stabilization, the concentration of inspired oxygen (FiO2) is abruptly reduced to 6% and maintained at the highest value at which the EEG amplitude is reduced to 7 µV or less. Blood pressure in these

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animals ranged from 23–40 mm Hg, and animals that arrested were resuscitated with 100% oxygen and cardiac compressions. Groups of animals were compared with sham, short (mean of 27 min) and long (mean of 40 min) insults. In an analysis of 19 animals prepared in this way, Thoresen et al.24 recorded postinsult seizures that resembled those in human neonates with asphyxia in 39% of the animals. The insult produced extensive neuropathologic damage that preferentially affected the cortex, hippocampus, and cerebellum, more than the thalamus and basal ganglia, suggesting many of the animals prepared in this way sustain more of a partial-prolonged insult, as described below, than the human syndrome of near-total asphyxia. This model has been very useful to study the pathogenesis of hypoxic-ischemic encephalopathy and postasphyxial seizures seen in human infants. 2.2.1.2

Partial Prolonged Asphyxia

2.2.1.2.1 Injury in Human Infants In many infants, the asphyxial insult is not abrupt and extremely severe as in the near-total syndrome. Rather, it is sustained over a period of several hours.14, 25 The major physiologic difference between the near-total and partial prolonged insults is that cardiac pumping is maintained in the latter, while cardiac arrest terminates the near-total insult after 30 min to 1 h.14 Although there are instances of overlap between these two types of insult in clinical practice, in many cases the patterns seen on neuroimaging and neuropathology are remarkably distinct. In contrast to the triad of putamenal, thalamic, and somatosensory cortical pathology seen after near-total asphyxia, partial prolonged insults generally produce predominantly cortical involvement.12 In the period of several days immediately following the insult, there is generally extensive cortical edema and blurring of the gray-white matter junction seen on neuroimages. After several months, the cortex has often been replaced by multiple cysts, the pattern of multicystic encephalomalacia. 2.2.1.2.2 Experimental Models 2.2.1.2.2.1

Partial Prolonged Asphyxia in Monkeys

Myers produced partial asphyxia in monkey fetuses by constriction of the maternal aorta, which lowers the fetal arterial partial pressure of oxygen and pH in a stepwise fashion depending on the aortic blood pressure.14 At levels of blood pressure that produced a pH of 7.10 or 7.15, no brain injury occurred despite a duration of several hours. However, fetal brain injury did occur with acidosis below pH 7.0, with partial asphyxia lasting from a half hour to several hours. Myers was able to produce a spectrum of neuropathology in these animals ranging from virtually complete cortical hemispheric necrosis with little basal ganglia injury with partial asphyxia lasting several hours to combinations of basal ganglia and severe cortical injury when episodes of total asphyxia were superimposed on milder more prolonged episodes.14 These changes resemble those seen in human neonates with similar insults.

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2.2.1.2.2.2

Partial Prolonged Asphyxia in Piglets and Rodents

Several other animal models replicate some features of partial prolonged asphyxia as seen in human infants. As mentioned above, Thoresen’s piglet model produces a predominantly cortical and hippocampal neuropathology that resembles partial asphyxia in monkeys and human infants.24 Other piglet models have produced partial asphyxia by combining hypoxia with periods of carotid occlusion and hypotension or by inflating a cuff around the neck at pressures above systemic blood pressure.26-28 These preparations are different from models described above because they do not expose the heart and other organs to asphyxia as occurs in human neonates. In another acute, nonsurvival model partial prolonged asphyxia is induced by ventilating piglets with 5–7% oxygen for 60 min.29 Vannucci’s laboratory pioneered the combination of unilateral carotid ligation and exposure to 8% oxygen to produce hypoxic-ischemic injury in 7-day-old rats, a model that has been used extensively in physiologic, neurochemical, and neuroprotection studies.30 The model replicates certain features of injury in human neonates, especially the relatively selective vulnerability of the basal ganglia (corpus striatum in the rodent), cerebral cortex, and thalamus. It also appears to replicate the fact that brain injury is dependent on reduction in cardiac output. Unlike the adult Levine preparation, which it resembles, studies of regional cerebral blood flow showed that cerebral perfusion on the side of carotid ligation is normal in the baseline state but falls by 70% or more during the period of hypoxia.1 Therefore, the insult resembles asphyxia in the human infant, in which ischemia is superimposed on severe hypoxemia. After recovery, perfusion on the ligated side is restored, in contrast to focal occlusion models. Neuronal injury in this model is strongly dependent on NMDA-mediated glutamate neurotoxicity.7 This is a convenient model with many strengths, and one which combines several features of both partial prolonged and near-total asphyxia. Weaknesses in the model include the difficulty in controlling physiologic variables, as in larger models, and a relatively high rate of interanimal variability in brain injury.31 A similar model of common carotid artery ligation and exposure to hypoxia in the rabbit has been reported by D’Arceuil et al.32 2.2.1.3

Parasagittal Watershed Infarction

2.2.1.3.1 Injury in Human Infants The parasagittal watershed infarction syndrome in human neonates following asphyxia was described by Volpe and Pasternak using clinical criteria and technetiumenhanced brain scanning in an infant after asphyxia associated with severe systemic hypotension.33 Infants with this syndrome typically have weakness in the upper arms and shoulders compared to the hands and lower extremities due to injury to cerebral cortex and subcortical white matter in the vascular watershed between the anterior and middle cerebral arteries. Severe loss of blood pressure causes diminished perfusion and infarctions in zones that are midway between end-vessels served by these two major cerebral vessels. This syndrome has been documented by postitron emission tomography (PET) as well as by conventional neuroimaging.34

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2.2.1.3.2 Experimental Models of Parasagittal Watershed Infarction Myers produced parasagittal watershed infarctions in newborn monkeys with severe hypotension superimposed on partial prolonged asphyxia14 and Pasternak and Groothuis produced similar lesions with hypotension in newborn puppies.35 More recently, Williams and colleagues36 produced a model of parasagittal infarction in fetal sheep. In this preparation the head, neck, and forelimbs of fetal sheep of 119–126 d gestation are externalized from the uterus and the vertebro-occipital anastomoses between the carotid arteries and veretebral arteries are ligated bilaterally. This procedure restricts the vertebral vascular supply to the carotid arteries. Inflatable occluder cuffs are placed around the carotid arteries and shielded electrodes are inserted via parasagittal burr holes overlying the parietal lobe of the brain. To produce cerebral ischemia, the carotid artery cuffs are inflated for 10–40 min and after which animals survive for 3 d. In this model, the greatest damage occurs in the parasagittal cortex and CA1 and CA3 regions of the hippocampus, with much less injury in the lateral cortex, striatum and thalamus. In contrast to the human, in which carotid occlusion would allow blood flow to be redistributed through the circle of Willis from the posterior circulation, in this model in the sheep, the pressure in the entire anterior circulation of the brain can be controlled by flow in the carotid arteries. The model has been used to advantage to study the evolution of seizures following neonatal asphyxia. Intrauterine electrocortigraphic monitoring demonstrated that onset of seizures in the model is delayed approximately 8 h after asphyxia, similar to the time period for appearance of seizures in humans following asphyxia. 2.2.1.4

Syndrome of Hypoxic-Ischemic Encephalopathy (HIE)

Each of these patterns of asphyxial injury in human full-term neonates is associated with the clinical syndrome of hypoxic-ischemic encephalopathy (HIE).10 HIE describes clinical manifestations of brain dysfunction including seizures, decreased level of consciousness, and hypotonia as well as electroencephalograhic abnormalities such as electrographic seizures, a burst-suppression pattern and/or severe slowing or voltage suppression.9,37 Signs of HIE in human neonates usually evolve after a delay of 8–24 h after the asphyxial insult, then worsen and improve over the next week to 10 d.9,38 Clinical studies suggest that the stage and severity of clinical HIE based on clinical and EEG manifestations is directly related to the severity of brain injury.39 Signs of mild encephalopathy on the Sarnat scale, which include autonomic hyperactivity and hyperalertness and mild EEG changes, generally are not associated with permanent brain injury while severe encephalopathy usually is associated with permanent injury as described above. Infants who have had an asphyxial insult, but do not manifest clinical signs of HIE during the following week of observation in the nursery, have virtually no chance of sustaining a permanent brain injury.40 This makes assessment of the clinical and EEG signs of HIE important for predicting which infants are at greatest risk for permanent brain injury.

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There appears to be an important physiologic principle underlying the observation that evolving injury from asphyxia is reflected in the severity of clinical signs of HIE in neonates. The infant is generally more likely than the more mature brain to respond to a variety of insults and injuries with excitatory signs such as seizures and epileptic EEG patterns than the older child or adult.41 For example, approximately 10% of young children experience seizures with high fever without any underlying neuropathology but this is very uncommon at later ages. Seizures of all types are more common in infants and young children than in older individuals as are the EEG abnormalities associated with them.41 This may be related in part to the fact that excitatory synapses are rapidly proliferating in the immature brain, reaching levels approximately twice as high as the adult at age 2 years in humans.42 Glutamate-mediated excitatory neurotransmission probably contributes to the enhanced excitatory activity in the neonate.7 For example, the physiologic activity of the N-methyl-D-aspartate (NMDA)-type glutamate receptor appears to be enhanced in the immature brain and may contribute to seizures as well as neuronal damage.41 Excitatory signs of brain dysfunction in HIE probably correlate strongly with the degree of brain injury from asphyxia because the neonatal brain is intrinsically more excitable than the adult and because, as described in more detail below, dysfunction of excitatory synapses contributes directly to the evolution of the injury.

2.2.2 SYNDROMES CAUSED PRIMARILY BY ISCHEMIA 2.2.2.1

Focal Ischemic Infarction

2.2.2.1.1 Injury in Human Neonates Brain injury caused by focal ischemic infarctions is less common than asphyxial syndromes in neonates and they appear to be caused by fundamentally different mechanisms.3 Except for rare situations, such as in cases of carotid cannulation of the carotid artery used for extracorporeal membrane oxygenation (ECMO) therapy in very hypoxic and hypotensive infants, it is unusual for asphyxia to be associated with single vessel strokes.43 Cerebral infarcts in fetuses and neonates are most likely to be caused by arterial emboli, and the most common cause of these emboli is infection related to sepsis and disseminated intravascular coagulation.3 Emboli from intracardiac sources are becoming less common as early repair of cyanotic congenital heart disease has become commonplace. Inherited disorders of coagulation including Leiden Factor V mutation, antiphospholipid antibodies, homocystinuria and protein C, and protein S deficiencies are gaining wider recognition as causes of neonatal strokes.44,45 It is also noteworthy that many of these causes of increased coagulability are also also associated with an increased risk of complications of pregnancy in the mother such as pre-eclampsia and toxemia.46 Other genetic syndromes, such as carbohydrate-deficient glycoprotein disorder, have also been recognized relatively recently as carrying a higher risk of stroke.47 This information suggests that focal ischemic infarctions in neonates are generally caused by different mechanisms than the asphyxia syndromes discussed above. Although many focal infarctions are recognized at birth by the onset of focal seizures, epidemiologic information suggests

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that more strokes associated with permanent hemiparetic cerebral palsy probably originate prenatally than in the immediate intrapartum period.48 Much less is known about the mechanisms for infarctions on the venous side of the circulation which are responsible for two additional neonatal disorders: venous sinus thrombosis and periventricular hemorrhagic infarction. Venous sinus thrombosis is being recognized more frequently with modern neuroimaging including color Doppler ultrasound of the brain and MR imaging and MR angiography.9,49 Although no large series have been reported, infection, disseminated intravascular coagulation, dehydration, and thrombocytosis secondary to acquired disorders such as polycythemia and inherited clotting disorders are potential causes.9 Thrombosis of the internal cerebral vein with rupture and intracranial hemorrhage, called periventricular hemorrhagic infarction, has also been implicated in the pathogenesis of grade IIIIV intracranial hemorrhage in premature infants.4 2.2.2.1.2 Focal Ischemia Models Focal cerebral ischemia has been studied less commonly in neonatal experimental models of focal infarction than asphyxia models. Ashwal and colleagues have used an 18-day-old rat model in which the middle cerebral artery is occluded transiently by passing a 0.07-mm nylon filament into it via the carotid artery.50 This preparation produces a relatively reproducible area of injury, making it possible to distinguish core from penumbral regions, and has been used to study the neuroprotective effects of nitric oxide synthase inhibition.51 Renolleau et al. also developed a focal occlusion model in 7-day-old rat pups by permanently occluding the left middle cerebral artery in association with a one-hour period of reversible occlusion of the left common carotid artery.52 A similar model has also been reported in 7-day-old rats by Derugin, et al.53 2.2.2.2

Periventricular Leukomalacia (PVL)

Periventricular leukomalacia or PVL is one of the most important causes of disability in premature infants and is the most common cause of permanent motor dysfunction or cerebral palsy in this group.4 PVL is generally considered as a developmentally determined ischemic syndrome of premature infants with a peak incidence between27–30 weeks gestation.4 However, as clinical study of the disorder has intensified, etiologies such as maternal and neonatal infection have also emerged as important causes.54 Nevertheless, it is appropriate to condsider PVL here as an important cause of ischemic injury in the developing brain. PVL was originally described in postmortem brain by Banker and Larroche as symmetric bilateral necrosis of periventricular white matter that is most severe around the frontal horns and in the occipital-parietal region around the ventricular trigone.55 As neuroimaging with head ultrasound and then MRI scanning developed, PVL could be studied in more detail in living infants and a greater variety of lesions was apparent.56 PVL associated with cystic lesions in the white matter can often be observed to develop in premature infants with serial imaging in the postnatal period.56 In this type, there may be little evidence of pathology initially after birth, but

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serial ultrasound images first reveal enhanced echogenicity around the ventricles and then formation of cysts.57 These cysts eventually collapse after several weeks, as the surrounding white matter reorganizes, simultaneously enlarging the ventricular space filled by cerebrospinal fluid. In many cases, the formation of these periventricular cysts is probably caused by ischemia in the periventricular white matter, which may be more vulnerable because of the anatomy of its arterial supply.58 Although the role of systemic hypotension has been proposed as a cause of PVL, this theory currently appears to have less support than the hypothesis of impaired intracerebral arterial autoregulation.59 Premature infants cannot regulate the diameter of intracerebal arterioles to maintain constant perfusion pressure in the face of variations in systemic pressure as well as older babies. Careful studies with Doppler ultrasound suggest that there is a strong correlation between fluctuations in cerebral perfusion in sick premature infants and development of PVL.60 Ischemia may cause white matter necrosis through excitotoxic mechanims that parallel those involved in neuronal damage.61 Immature oligodendroglia that synthesize and maintain myelin are vulnerable to high concentrations of glutamate that may be released in white matter during ischemia, in part because they are more vulnerable to damage from oxidative stress.62 The enhanced vulnerability of immature oligodendroglia during a critical point in their development has been proposed as one of the factors that makes premature infants most vulnerable to PVL during a relatively narrow time window of gestational age.63 Excitotoxic mechanisms may also be a link to death of oligodendroglia from inflammatory cytokines stimulated by infection.54 Both agents such as endotoxin and overstimulation of excitatory amino acids may stimulate proinflammatory cascades that kill oligodendroglia by apoptotic mechanism. This mechanistic interaction between glutamate-mediated excitotoxicty stimulated by ischemia and inflammatory cascades stimulated by infectious agents, as well as the association between infection and alterations in blood pressure, probably contribute to the interactions between these two mechanisms to cause PVL.54 2.2.2.3

Hypothermic Circulatory Arrest With Heart Surgery

2.2.2.3.1 Insult in Human Neonates Hypothermic circulatory arrest (HCA), in which total circulatory arrest is combined with reduction in body temperature of 18°C or lower, is commonly used to support surgery for complex congenital heart disorders such as transposition of the great vessels. This procedure is associated with brain injury in a small group of childern, who may have severe permanent impairment.64 One of the most severe disorders associated with HCA is the syndrome of postpump choreoathetosis related to injury to the basal ganglia.65 This syndrome typically begins after a latent period between the time when the infant awakes from anesthesia and onset of reduced level of consciousness, agitation, and choreoathetotic movements. Kupsky and colleagues found that this syndrome was associated with damage to the globus pallidus in postmortem analysis of 2 patients.66 Holden et al. identified basal ganglia lesions on neuroimaging in 6 of 11 patients with postpump choreoathetosis and identified cyanosis as a risk

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factor.65 Curless et al. reported that severe respiratory alkalosis and hypocapnia during rewarming might be a risk factor in 3 patients they reported with postpump choreoathetosis.67 Respiratory alkalosis occurs in patients maintained by the socalled “alpha-stat” method of acid base management during heart surgery. Du Plessis et al. reported that use of the more acidotic “pH-stat” strategy was associated with a better neurologic outcome in a study of 182 infants undergoing deep hypothermic cardiopulmonary bypass.68 This suggests that severe respiratory alkalosis may worsen ischemia during reperfusion. HCA may also cause seizures and cognitive impairments in some infants. Newburger et al. compared HCA with low-flow cardiopulmonary bypass in infants undergoing heart surgery for D-transposition of the great vessels and found that HCA increased the odds ratio for postoperative seizures to 11.4 and lengthened the recovery time to the first appearance of EEG activity.69 Bellinger et al. analyzed the developmental and neurologic status on the same group of infants and found that the HCA group had a higher percentage of low developmental scores (27% vs. 12%).70 Developmental scores in the HCA group were inversely related to the duration of circulatory arrest and the incidence of neurologic abnormalities were positively related to the length of arrest. These studies comparing two methods of support for cardiac surgery, rather than simply comparing HCA with control children, are important because the rate of neurobehavioral disorders in infants with congenital heart disease prior to surgery is elevated.71 A prolonged period of severe cyanosis prior to surgery has also been identified as a risk factor for cognitive decline in children with transposition of the great arteries by Newburger et al.72 2.2.2.3.2 Models of Brain Injury from Hypothermic Circulatory Arrest Several groups have developed experimental models of hypothermic circulatory arrest in dogs and piglets. Vannucci’s group developed a model of hypothermic circulatory arrest for 1 to 1.75 hours in newborn dogs induced by intravenous potassium chloride followed by resuscitation with closed chest compression, epinephrine, and bicarbonate administration with a recovery period up to 72 hours.73 They found neuropathological evidence of damage in cortex and basal ganglia that was related to the duration of arrest and a relationship between the severity of cortical injury and neurobehavioral abnormalities. Nomura et al. developed a model of hypothermic circulatory arrest and cardiopulmonary bypass in one-week-old pigs that replicates more closely the clinical approach used in heart surgery.74 They used near-infrared spectroscopy (NIRS) to examine intravascular hemoglobin and mitochondrial (cytochrome aa3) oxygenation and found changes similar to that reported in human infants by du Plessis et al.75 Although cooling was associated with an increase in hemoglobin oxygenation despite the reduction in cerebral blood flow, cytochrome aa3 continued to fall during cooling, consistent with a net cellular oxygen deficit. In human infants, reperfusion following circulatory arrest caused a rapid return of intravascular hemoglobin oxygenation but the level of oxidized cytochrome aa3 was delayed, especially in infants over 2 weeks of age.75 Recent studies in the piglet model indicate that measurement

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of a reduction in cytochrome signal on NIRS correlates well with concurrent reductions in high-energy phosphates and evidence of histologic brain injury.76 Therefore, work in this piglet model suggests that NIRS might be a useful clinical tool for monitoring brain oxygenation during HCA. Another model of hypothermic circulatory arrest and cardiopulmonary bypass in older dogs has allowed extensive studies of mechanisms of glutamate-mediated excitotoxcity and neuronal death that are relevant to injury in neonates.77 In this model, 10-month-old hound dogs are placed on cardiopulmonary bypass under anesthesia and cooled to 18°C over 30 minutes. Then the pump is turned off and venous blood is drained and circulatory arrest is maintained for 2 hours. After circulation is restored, animals are rewarmed and maintained for intervals up to 72 hours. Circulatory arrest results in selective neuronal necrosis and apoptosis in layers 3 and 5 of entorhinal cortex and neocortex, and in the basal ganglia, Purkinje cells of the cerebellum, and CA-1 of the hippocampus. Pretreatment with the NMDA antagonist MK-80177 and the neuronal nitric oxide synthase (nNOS) inhibitors 7-nitroindazole and AR17477 (Astra Arcus) are strongly protective against histopathologic and neurologic manifestations of injury.78 Studies utilizing microdialysis indicate that extracellular levels of glutamate and citrulline, which reflect the production of nitric oxide from arginine, are markedly elevated in the period immediately after restoration of circulation.78 Immunocytochemical studies indicate that immunoreactivity for nNOS containing neuronal fibers is also induced over 24 hours after the insult.79 These studies suggest that the syndrome of delayed onset of encephalopathy, seizures, and movement disorders after hypothermic circulatory arrest reflects a prolonged elevation in extracellular glutamate and activation of excitatory amino acid receptors as well as induction of nNOS and prolonged elevation in nitric oxide (NO) during the reperfusion period.

2.3 MECHANISMS FOR DELAYED NEURONAL INJURY The neonatal brain has very low energy requirements,8 and reactive hyperemia in response to even severe hypoxemia can usually maintain levels of ATP (adenosine triphosphate) through anaerobic glycolysis. However, failure of cerebral perfusion superimposed on severe hypoxemia allows ATP to fall to critical levels,1 triggering the first steps in a cascade of biochemical events that can result in death of brain tissue.

2.3.1 EXCITOTOXIC MECHANISMS One of the earliest events triggered by the initial phase of energy failure is depolarization of neuronal membranes and disruption in synaptic function. The prominent role that excitatory neuronal activity plays in the clinical signs of encephalopathy that evolve after hypoxic ischemia10 appears to reflect the relatively selective impact of the insult on excitatory synapses and excitatory neuronal circuits. Disruption in pre- and postsynaptic components of synapses and persistent, enhanced firing in these neuronal circuits probably contribute to selective patterns of brain injury.

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2.3.1.1

Disruption of Glutamate Synapse Function

Hypoxic ischemia severe enough to damage the neonatal brain produces a prominent disruption in the function of glutamate synapses by reducing the reuptake of glutamate into glia by its energy and sodium-dependent transporter.80–82 This leads to accumulation of neurotransmitter within synapses and the extracellular space.83,84 In human infants after asphyxia, levels of glutamate, aspartate, and glycine are elevated in cerebrospinal fluid in proportion to the severity of HIE.85,86 Disruption of glucose delivery to ischemic tissue may be responsible for early reduction in the activity of glutamate transporters because, in the face of hypoxemia, increased glucose is needed to produce ATP anaerobically to power Na+/K+ ATPase linked to the transporter.21 Postsynaptic glutamate receptors are activated through combinations of elevated neurotransmitter levels and depolarization of postsynaptic membranes.87 NMDA (Nmethyl-D-aspartate) glutamate receptor-operated channels and voltage-sensitive calcium channels open when mitochondrial energy levels fall.88 Experimental evidence suggests that NMDA receptors are strongly activated during hypoxic ischemia, through a combination of elevated levels of glutamate and glycine in the synapse and depolarization of neuronal membranes from energy failure with passive opening of NMDA receptors.87,89,95 This allows potentially toxic levels of intracellular calcium and sodium to enter neurons and some glia, including oligodendroglia.61 NMDA receptor antagonist drugs are strongly neuroprotective in neonatal models of hypoxic ischemia, reflecting the fact that damage mediated by the NMDA receptor is markedly enhanced in the immature brain compared to the adult.90–94 Therefore, combinations of hypoxemia and ischemia have a profound effect on pre- and postsynaptic elements of excitatory synapses. 2.3.1.2

Selective Vulnerabilty Due to Connectivity of Neuronal Circuits

As described in the section on near-total asphyxia pattern, certain groups of neurons, such as those in the thalamus, putamen, and sensorimotor cortex, may be predisposed to injury by virtue of their location within excitatory neuronal networks.22 The elevated rCMRgl in vulnerable regions in the period after the insult may reflect elevated levels of glutamate release and reuptake in excitatory projections from the sensorimotor cortex to the putamen and reciprocal connections between the cortex and the thalamus that could mediate excitotoxic injury.22 These regions may be selectively vulnerable to near-total asphyxia because of their connectivity through excitatory pathways.22

2.3.2 THE NEUROTOXIC CASCADE AND DELAYED ENERGY FAILURE Hypoxic ischemia and disruption of synaptic function trigger the opening of membrane channels that allow calcium and sodium to flood into neurons and some glia, such as oligodendroglia, and these ionic changes trigger a cascade of intracellular events that can result in cell death (Figure 2.1). One of the early events in this cascade

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is the accumulation of free radicals of oxygen including superoxide (·O–) and hydroxyl ions (·OH–), as well as the free radical gas, nitric oxide (NO).1,96 Free radicals of oxygen are generated after hypoxic ischemia due to defects in the electron transport chain caused by calcium overload of mitochondrial and inadequate saturation of cytochrome oxidase with oxygen, as well as by enhanced synthesis of prostaglandins and the conversion of hypoxanthine to xanthine and uric acid.98 Nitric oxide is produced in the postinsult period by neuronal nitric oxide synthase (nNOS) or immunologic NOS (iNOS). Transgenic mice lacking the gene for nNOS (NOS I) are protected from hypoxic ischemic injury compared with controls who express the gene, suggesting that nitric oxide stimulated by NMDA-mediated activation of nNOS contributes to brain injury.99 Enhanced production of NO• has been documented in several models of HIE.78,100,101 Individual oxygen free radicals or combinations with nitric oxide, such as the combination of nitric oxide with superoxide to form peroxynitrite, can attack attack lipid membranes.98,102 Nitric oxide can also impair metabolic steps involved in energy metabolism including glycolysis, the tricarboxylic acid cycle, and the mitochondrial electron transport chain.98 Attack on DNA by oxygen free radicals or NO can also trigger the DNA repair enzyme poly(ADPribose) polymerasae (PARP), with polyadenylates nuclear proteins as part of the repair process.102,103 Activation of PARP consumes nicotinamide adenine dinucleotide (NAD+), an intermediate that is needed for mitochondrial production of ATP, further impairing mitochondrial function. Activation of PARP has been linked to overstimulation of NMDA, but not non-NMDA glutamate receptors.104 DNA fragmentation produced by cysteine proteases (caspases) also contributes to activation of PARP. These steps play an important role in delayed energy failure after hypoxic ischemia. Neuroimaging studies of infants after hypoxic-ichemic insults as well as histologic study of experimental models of ischemic injury indicate that damage evolves over a period of several days.105 Magnetic resonance spectroscopy has been used to measure high-energy phosphorus intermediates as well as lactate in human infants after hypoxic ischemic insults.106 These studies revealed that full-term infants had normal levels of high-energy phosphates soon after resuscitation from asphyxia, but levels dropped in infants with severe injury after 24 hours.106 Studies with proton spectroscopy showed that infants with elevated levels of lactate/N-acetylaspartate had poor neurologic outcome.107 Similar spectroscopic changes showing a delay in energy failure that corresponds with poor outcome have been reproduced in a piglet model of asphyxia.108 In the 7-day-old rat pup model of hypoxic ischemia, impairment of mitochondrial function following the insult has been shown to be related in part to overstimulation of NMDA recetpors.109 These results are consistent with the concept of hypoxic ischemia as a cascade of delayed biochemical events that impair mitochondrial oxidative metabolism over a period of hours to days after the initial insult.

2.3.3 THE APOPTOSIS-NECROSIS CONTINUUM Delayed or secondary energy failure causes cell death either by the genetically programmed process of apoptosis or by the explosive destruction of cellular membranes

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Excitotoxic Cascade Elevated Glutamate NMDA Channel

Open Apoptosis

Ca+ Flood O. Radicals & NO.

Mitochondria

Nucleus

PARP NAD+

Caspase

Necrosis

FIGURE 2.1 Schematic of the cascade of major events that lead to cell death in the neonatal brain after an hypoxic-ischemic insult.

called necrosis (Figure 2.1)110,111. Although a great deal of work remains to be done in specific models of ischemia in neonates, a general conceptual outline is available based on both animal and cell culture experiments.112 Apoptosis appears to be far more prominent in neonatal animal models of hypoxic ischemia than in the adult, and cell death is expressed in a continuum from apoptosis to necrosis at this age.113–117 In neonates it is common to observe “hybrid” cells that have morphologic features of both apoptosis and necrosis.118,119 The same continuum is observed in the immature brain when excitotoxic amino acids are injected directly into the brain.118 Evidence that a pan-caspase inhibitor is strongly protective against hypoxic-ischemic damage in the 7-day-old rat model suggests that apoptosis plays a major role in cell death at this age.120,121 In the neonatal rat model of hypoxic ischemia, NMDA antagonists prevent activation of caspase 3 in the brain.122 In the same model, apoptosis persists for more than a week after the insult, suggesting that new cells continue to commit to apoptosis over that time.119 Postmortem neuropathology from neonates who have died after hypoxic ischemia also demonstrates prominent apoptosis.113 This may be related to the fact that cellular programs for apoptosis are more active in the immature brain because they are used normally to remove redundant neurons. Regarding Figure 2.1, synaptic dysfunction involving accumulation of glutamate and other amino acids and depolarization of neuronal membranes leads to excessive calcium entry into neurons and some glia. Production of oxygen-free radicals leads to mitochondrial dysfunction and secondary energy failure over hours to days after

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the insult. Consumption of NAD+ by activation of PARP poly(ADP-ribose) polymerase may contribute to mitochondrial dysfunction by reducing energy intermediates. Activation of caspases by mitochondrial dysfunction can lead to apoptosis. The nature and intensity of mitochondrial dysfunction may help to determine whether neurons die by necrosis, with total destruction of neuronal membranes, or apoptosis, which involves nuclear condensation and cytoplasmic shrinkage with membranes left intact until phagocytosis. Experiments in cell culture suggests that the severity of mitochondrial energy failure may have a connection to the decision that cells make for apoptosis or necrosis.110,111,123 Ankarcrona et al. found that the expression of apoptosis or necrosis in cultured neurons was related to the severity of energy failure produced by NMDA, with necrosis associated with more intense excitotoxic insults.110 Less severe insults may impair metabolism and stimulate mitochondria to release cytochrome C and other proteins to activate caspase 3 and apoptosis by the intrinsic pathway.124 Recent evidence suggests that hypoxic-ischemic injury in neonatal rats is also associated with activation of the extrinsic Fas death receptor pathway associated with cleavage of procaspase 8 and downstream activation of caspase 3.125 Activation of other proteases such as the interleukin 1 β converting enzyme (ICE) family also appear to be important in triggering apoptosis after hypoxic ischemia injury in neonates, providing a connection to cytokine-mediated inflammatory pathways.126,127 The calpain protease system is also activated by hypoxia-ischemia.126 This information suggests ways in which the nature and severity of energy disorders in the neonatal brain influence the decision for apoptosis or necrosis.

2.3.4 EXPERIMENTAL INTERVENTIONS TO MODIFY INJURY Numerous interventions have been shown to reduce brain injury in experimental neonatal models although most are effective only when given before the insult or within a few hours afterward.96,129 So far it has been difficult to translate these observations into useful clinical therapies, in part because of toxic side effects and in part because of the difficulty of chosing comparable groups of patients for clinical trials. 2.3.4.1

Hypothermia

Hypothermia is clearly protective against brain injury from hypoxic ischemia when started before the insult, as demonstrated by its use since the 1950s to protect the brain during infant cardiac surgery.130 Several studies in the 1950s also suggested that hypothermia might be neuroprotective for asphyxiated infants, but the practice was abandoned a short time later when it was reported that premature infants had a higher mortality if exposed to hypothermia.130 Recently there has been a resurgence of interest in this form of therapy, following on reports from several animal models that mild hypothermia reduces falls in ATP, rises in brain lactate, elevations in nitric oxide production, and brain injury associated with asphyxia.131,101,132 Results from these experimental models suggest that the duration of postinsult hypothermia is important since short periods can delay but ultimately not prevent brain injury.133,134 Mild

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selective cooling of the head is being examined in human infants and experimental models with potentially positive results.135 In the near-term fetal sheep model of transient cerebral ischemia, delayed selective head cooling begun before the onset of postischemic seizures and continued for 3 days was associated with improvement in EEG activity and a reduction in neuronal loss in the cortex.136 There remain questions about whether head cooling can penetrate into deep brain regions, which are vulnerable to ischemic injury.137 Although the method was effective in cooling deep regions of the piglet brain, numerical modeling of temperature distributions within the neonatal human head suggest that deep cooling may require a combination of systemic and head cooling.137,138 No adverse effects were noted in a safety study in 22 term infants with asphyxia exposed to combinations of mild systemic and head cooling.135 Even if it is not effective alone, mild hypothermia may prolong the therapeutic window in which other interventions, such as glutamate antagonists, could be effective. 2.3.4.2

Oxygen Free Radical Scavengers

Scavengers of cytotoxic oxygen free radicals include the xanthine oxidase inhibitor allopurinol, the cyclooxygenase inhibitor indomethacin, and the iron chelator desferoxamine which can prevent iron from reacting with oxygen free radicals to form more toxic intermediates.96 Several studies in the neonatal rat model of hypoxic ischemia have demonstrated that high doses of allopurinol prevent brain injury, probably by a direct free radical scavenging mechanism rather than by inhibiting xanthine oxidase.140 Shadid et al. showed that allopurinol, indomethacin, or desferoxamine improved cerebral metabolism and electrocortical brain activity after asphyxia in newborn lambs, but a combination of the three had no additional effect.141 In another study, Van Bel et al. showed that allopurinol reduced free radical formation, cerebral perfusion, and electrical brain activity in a group of 11 severely affected infants compared to a comparable control group without significant side effects. 142 2.3.4.3

Carbon Dioxide and Acidosis

The arterial partial pressure of carbon dioxide (PaCO2) has a potent effect on cerebral perfusion and hypocapnia, which causes vasoconstriction, has been associated with an increased risk of periventricular leukomalacia in ventilated premature infants.143 Vannucci et al. found that 7-day-old rats with mild hypercapnia (PaCO2 = 54 mm Hg) had less hypoxic-ischemic injury than pups with normocapnia, while hypocapnia was associated with greater injury.144 This suggests that hypercapnia could be a clinically useful intervention, although this has not been established by clinical trials. However, the observation that respiratory acidosis is not harmful is consistent with other data indicating that acidosis, whether respiratory or metabolic, is not itself directly harmful to the neonatal brain.145 Although severe lactic acidosis is a marker for hypoxic ischemia severe enough to damage the brain, evidence from Vannucci’s laboratory suggests that it is the degree of energy deficiency rather than the lactic acid that causes injury.145

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2.3.4.4

Glutamate Antagonists

When administered before or within 1–2 hours after experimental hypoxic ischemia, NMDA type glutamate receptor-channel antagonists such as dizocilpine (MK-801), ketamine, or dextromethorphan, are the most potent neuroprotective agents in the neonatal brain.90–93 In rodents this protection is related to the enhanced toxicity of NMDA receptor overstimulation at 6–7 days from ischemia or trauma, probably related to a special role that these receptors play in development at this age.94 NonNMDA glutamate receptor antagonists also have some protective effects in neonatal rodent models of hypoxic ischemia, though less than for NMDA antagonists.92 NonNMDA antagonists have recently been reported to protect against white matter injury in a neonatal rodent model, suggesting a potential intervention for periventricular leukomalacia.61 Although NMDA antagonists are less effective against global ischemia in adult animals, they are strongly protective against hypothermic circulatory arrest in young dogs.77 Magnesium, which normally blocks the NMDA channel is also a potent protective agent in the immature rodent model, although it was not effective in a model of umbilical cord occlusion in sheep.145,147 Magnesium therapy for human infants with asphyxia may be limited by its tendency to produce systemic hypotension.96,148 2.3.4.5

Nitric Oxide Synthesis Inhibition

Inhibition of nitric oxide synthesis shows promise in animal models of hypoxic ischemia. In the 7-day-old rat model, we found that 7-nitroindazole (7-NI), an inhibitor of the neuronal isoform of NOS, is protective in a dose of 100 mg/kg that is sufficient to suppress nNOS catalytic activity in the brain for 6–9 hours after the insult, but not at a lower dose of 50 mg/kg that reduces nNOS for only an hour. This is consistent with previous reports that nNOS containing neurons are less vulnerable to ischemic injury than other neurons and respond to injury with an increase in immunoreactivity in injured regions of the brain after the insult.99,149,150 Recent experiments also showed that inhibition of immunologic NOS (iNOS or NOS II) is also protective in the 7-dayold rodent model. Tsuji et al.151 found that the iNOS inhibitor aminguanidine administered before the insult produced by carotid ligation and exposure to hypoxia and for 3 days afterward markedly reduced the delayed rise in NO metabolites in the ischemic hemisphere, and markedly reduced brain injury. Selective inhibition of NOS isoforms may have promise for protecting the brain, in contrast to some nonspecific inhibitors of NOS which may produce mixed results because of interference with endothelial NOS (NOS III) that regulates cerebral blood flow.98 2.3.4.6

Caspase Inhibition

Inhibition of cysteine-containing, aspartate-preferring proteases (caspases) is potentially the most potent neuroprotective pharmacologic strategy in the neonatal brain yet discovered, aside from inhibition of NMDA channels.152,153 Caspase 3, the main downstream executioner caspase, and others are strongly activated after hypoxic ischemia in the neonatal brain, probably related in part to the prominent role that the

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apoptosis-necrosis continuum plays at this age.152 Caspase inhibition can produce synergism with NMDA antagonism because caspases are activated downstream in the excitatory cascade. Cheng et al. showed that pan-caspase inhibition provides strong neuroprotection in the neonatal rat model of hypoxic ischemia when given more than 2.5 hours after completion of hypoxia.120 Inhibition of another family of cysteine proteases, the calpains, also has some neuroprotective effect, and these protease families interact with each other following an ischemic insult.128 Although current caspase-inhibiting peptides have difficulty penetrating the blood brain barrier, this is a potentially exciting new area for therapeutic intervention. 2.3.4.7

Neuronal Growth Factors

Neuronal growth factors such as nerve growth factor, basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I), and brain-derived neuronal growth factor (BDNF) have all been demonstrated to have a neuroprotective effect in neonatal models of hypoxic-ischemic brain injury.154–160 For example, Hossain et al. showed that implantation of endothelial cells genetically engineered to secrete human FGF (FGF-1) protected against quinolinate, an endogenous NMDA receptor angonist in neonatal rats.155 The neuroprotective effect of BDNF against injury in hypoxic ischemic neonatal rats is correlated with inhibition of caspase-3 activity, acting through the Ras-MAP kinase signaling pathway.156,160 This suggests that one important component of the neuroprotective action of growth factors is the ability to activate intracellular signaling cascades that block pathways involved in apoptosis and caspase activation. 2.3.4.8

Glucocorticoids

Glucocorticoids such as dexamethasone have a potent effect on the outcome of brain injury, but this effect is markedly dependent on the timing of administration in relation to the insult. Although administration of corticosteroids immediately before or after the insult has little effect on injury and may increase mortality in rat pups,96,161 Barks et al. showed that administration 24 hours before was strongly protective. Adminstration 6 hours before also offered some protection.162 Although the effects of administration at 24 hours before the insult suggest the possibility of an induction of gene expression, the mechanism for this effect remains to be discovered. Recent clinical reports indicate that dexamethasone may have an adverse effect on brain development in premature infants when given after birth to prevent lung disease.163,164 2.3.4.9

Hypoxic Preconditioning

Hypoxic or ischemic preconditioning is the reduction in injury to an organ that occurs when it has been exposed to an earlier insult, often at an interval of 24 hours. Gidday et al. reported a preconditioning effect in neonatal rat pups exposed to an 8% oxygen environment for 3 hours at 24 hours prior to an hypoxic ischemic insult.165 This group also reported that this effect may be mediated by nitric oxide derived

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from endothelial NOS.166 An understanding of the mechanism for this effect could help to uncover new steps in the pathways to mediate hypoxic-ischemic injury.

2.4 CONCLUSION Ischemic injury in the neonatal brain differs from the adult in several ways, including mechanisms of injury, patterns of selective vulnerability, and the contribution of apoptosis vs. necrosis as a mode of cell death. Many of the molecular mechanisms of injury appear to be similar to those activated in the adult brain, but major variations occur in large part related to the developmental importance of these pathways in brain development at the time of injury. Another important age-related difference is the greater plasticity of the immature brain, which contributes to different patterns of recovery after fetal or neonatal injuries.

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107. Penrice, J., Cady, E.B., Lorek, A., et al. Proton magnetic resonance spectroscopy of the brain in normal preterm and term infants, and early changes after perinatal hypoxia-ischemia, Ped. Res., 40, 6, 1996. 108. Penrice, J., Lorek, A., Cady, E.B., et al. Proton magnetic resonance spectroscopy of the brain during acute hypoxia-ischemia and delayed energy failure in the newborn piglet, Ped. Res., 41, 795, 1997. 109. Gilland, E., Puka-Sundvall, M., Hillered, L., et al. Mitochondrial function and energy metabolism after hypoxia-ischemia in the immature brain: involvement of NMDA receptors. J. Cereb. Blood Flow Metab., 18, 297, 1998. 110. Ankarcrona, M., Dypbukt, J.M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S.A., and Nicotera, P., Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function, Neuron., 15, 961, 1995. 111. Abe, K,, Aoki, M., Kawagoe, J., Yosida, T., Hattori, A., Kogure, K., and Itoyan, Y., Ischemic delayed neuronal death, a mitochondrial hypothesis, Stroke, 26, 1478, 1995. 112. Banasiak, K.J., Xia, Y., and Haddad, G.G., Mechanisms underlying hypoxia induced neuronal apoptosis, Prog. Neurobiol., 62, 215, 2000. 113. Edwards, A.D., Yue, X., Cox, P., Hope, P.L., Azzopardi, D.V. Squier, M.V., and Mehmet, H., Apoptosis in the brains of infants suffering intrauterine cerebral injury, Pediatr. Res., 42, 684, 1997. 114. Beilharz, E.J., Williams, C.E., Dragunow, M., Sirimanne, E.S., and Gluckman, P.D., Mechanisms of delayed cell death following hypoxic-ischemic injury in the immature rat: evidence for apoptosis during selective neuronal loss, Brain Res. Mol. Brain Res., 29, 1, 1995. 115. Pulera, M.R., Adams, L.M., Liu, H., Santos, D.G., Nishimura, R.N.. Yang, F., Cole, GM, and Wasterlain, C.G., Apoptosis in a neonatal rat model of cerebral hypoxia-ischemia, Stroke, 29, 2622, 1998. 116. Yue, X., Mehmet, H., Penrice, J., Cooper, C., Cady, E., Wyatt, J.S., Reynolds, E.O., Edwards, A.D., and Squier, M.V., Apoptosis and necrosis in the newborn piglet brain following transient cerebral hypoxia- ischemia, Neuropathol. Appl. Neurobiol. 23, 16, 1997. 117. McDonald, H.W., Behrens, M.I., Chung, C., Bhattacharyya, T., and Choi, D.W., Susceptibility to apoptosis is enhanced in immature cortical neurons, Brain Res., 759, 228, 1997. 118. Martin, L.J., Al-Abdulla, N.A., Brambrink, A.M., Kirsch, J.R., Sieber, F.E., and Portera-Cailliau, C., Neurodegeneration in excititoxicity, global cerebral ischemia, and target deprivation: A perspective on the contributions of apoptosis and necrosis, Brain Res. Bull., 46, 281, 1998. 119. Nakajima, W., Ishida, A., Lange, M.S., Gabrielson, K.L., Wilson, M.A., Martin, L.J., Blue, M.E., and Johnston, M.V., Apoptosis has a prolonged role in the neurodegeneration after hypoxic ischemia in the newborn rat, J. Neurosci., 20, 7994, 2000. 120. Cheng, Y., Deshmukh, M., D’Costa, A., et al. Capase inhibitor affords neuroprotection after delayed administration in a rat model of neontal hypoxic-ischemic brain injury, J Clin. Invest., 101, 1992, 1998. 121. Hu, B.R., Liu, C.L., Ouyang, Y., Blomgren, K., and Siesjo, B.K., Involvement of caspase-3 in cell death after hypoxia-ischemia declines during brain maturation, J. Cereb. Blood Flow Metab., 20, 1294, 2000. 122. Puka-Sundvall, M., Hallin, U., Zhu, C., et al. NMDA blockade attenuates caspase-3 activation and DNA fragmentation after neonatal hypoxia-ischemia, Neuroreport, 11, 2833, 2000.

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123. Puka-Sundvall, M., Wallin, C., Gilland, E., et al. Impairment of mitochondrial respiration after cerebral hypoxia-ischemia in immature rats: relationship to activation of caspase-3 and neuronal injury, Brain Res. Dev. Brain Res., 125, 43, 2000. 124. Budd, S.L., Tenneti, L., Lishnak, T., et al. Mitochondrial and extramitochondrial apoptotic signaling pathways in cerebrocortical neurons, Proc. Nat. Acad. Sci. (USA), 97, 6161, 2000. 125. Northington, F.J., Ferriero, D.M., Flock, D.L., et al. Delayed neurodegeneration in neonatal rat thalamus after hypoxia-ischemia is apoptosis, J Neurosci., 21, 1931, 2001. 126. Liu, X.H., Kwon, D., Schielke, G.P., Yang, G.Y., Silverstein, F.S. and Barks, J.D., Mice deficient in interleukin-1 converting enxyme are resistant to neonatal hypoxicischemic brain damage, J. Cereb. Blood Flow Metab., 19, 1099, 1999. 127. Hagberg, H., Gilland, E., Bona, E., et al, Enhanced expression of interleukin-1 (IL-1) and IL-6 messenger RNA and bioactive protein after hypoxia-ischemia in neonatal rats, Pediatr. Res., 40, 603, 1996. 128. Blomgren, K., McRae, A., Elmered, A., et al, The calpain proteolytic system in neonatal hypoxic-ischemia. Ann. NY Acad. Sci., 825, 104, 1997. 129. Johnston, M.V., Trescher, W.H., Ishida, A. and Nakajima, W., Novel treatments after experimental brain injury, Semin. Neonatol. 5, 75, 2000. 130. Wyatt, J.S. and Thoresen, M., Hypothermia treatment and the newborn, Pediatrics, 100, 1028, 1997. 131. Yager, J.Y. and Asselin, J., Effect of mild hypothermia on cerebral energy metabolism during the evolution of hypoxic-ischemic brain damage in the immature rat, Stroke, 27, 919, 1996. 132. Amess, P.N., Penrice, J., Cady, E.B., et al. Mild hypothermia after severe transient hypoxia-ischemia reduces the delayed rise in cerebral lactate in the newborn piglet, Pediatr. Res., 41, 803, 1997. 133. Trescher, W.H., Ishiwa, S., and Johnston, M.V., Brief post-hypoxic-ischemic hypothermia markedly delays neonatal brain injury, Brain Dev., 19, 326, 1997. 134. Bona, E., Hagberg, H., Loberg, E.M., et al. Protective effects of moderate hypothermia after neonatal hypoxia-ischemia: short- and long-term outcome, Pediatr. Res., 43, 738, 1998. 135. Gunn, A.J., Gluckman, P.D., and Gunn, T.R., Selective head cooling in newborn infants after perinatal asphyxia: a safety study, Pediatrics, 102, 885, 1998. 136. Gunn, A.J., Gunn, T.R., Gunning, M.I., et al. Neuroprotection with prolonged head cooling started before postischemic seizures in fetal sheep, Pediatrics, 102, 1098, 1998. 137. Thoresen, M., Simmonds, M., Satas, S., et al. Effective selective head cooling during posthypoxic hypothermia in newborn piglets, Pediatr. Res., 49, 594, 2001. 138. Van Leeuwen, G.M.J., Hand, J.W., Lagendijk, J.J.W., et al. Numerical modeling of temperature distributions within the neonatal head, Pediatr. Res., 48, 351, 2000. 139. Dietrich, W.D., Lin, B., and Globus, M.Y.T., Effect of delayed MK-801 (dizocilpine) treatment with or without immediate postischemic hypothermia or chronic neuronal suvival after global forebrain ischemia in rats, J Cereb, Blood Flow Metab., 15, 960, 1995. 140. Palmer, C., Vannucci, R.C., and Towfighi, J., Reduction of perinatal hypoxic-ischemic brain damaage with allopurinol, Pediatr. Res., 27, 332, 1990. 141. Shadid, M., Moison, R., Steendijk, P., et al. The effect of antioxidative combination therapy on post hypoxic ischemic perfusion, metabolism, and electrical activity in the newborn brain, Pediatr. Res. 44, 119, 1998.

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142. Van Bel, F., Shadid, M., Moison, R.M., et al. Effect of allopurinol on post asphyxial free radical formation, cerebral hemodynamics, and electrical brain activity, Pediatrics, 101, 185, 1998. 143. Greisen, G., Munck, H., and Lou, H., Severe hypocarbia in preterm infants and neurodevelopmental deficit, Acta Paediatr. Scand., 76, 401, 1987. 144. Vannucci, R.C., Towfighi, J., Heitjan, D.F., et al. Carbon dioxide protects the perinatal brain from hypoxic-ischemic damage: an experimental study in the immature rat, Pediatrics, 95, 868, 1995. 145. Vannucci, R.C., Brucklacher, R.M., and Vannucci, S.J., The effect of hyperglycemia on cerebral metabolism during hypoxia-ischemia in the immature rat, J. Cerebral Blood Flow Metab., 16, 1026, 1996. 146. McDonald, J.W., Silverstein, F.S., and Johnston, M.V., Magnesium reduces Nmethyl-D-aspartate (NMDA)-mediated brain injury in perinatal rats, Neurosci. Lett., 109, 234, 1990. 147. Thordstein, M., Bagenholm, R., Thiringer, K., et al. Scavengers of free oxygen radicals in combination with magnesium ameliorate perinatal hypoxic-ischemic brain damage in the rat, Pediatr. Res., 34, 23, 1993. 148. Levene, M., Blennow, M., Whitelaw, A., et al. Acute effects of two different doses of magnesium sulphate on infants with birth asphyxia, Arch. Dis. Child Fetal Neonatl. Ed., 73, F174, 1995. 149. Trifiletti, R., Neuroprotective effects of NG-nitro-L-arginine in focal stroke in the 7 day old rat, Eur. J. Pharmacol., 218, 197, 1992. 150. Hamada, Y., Hayakawa, T., Hattori, H., et al., Inhibitor of nitric oxide synthesis reduces hypoxic-ischemic brain damage in the neonatal rat, Pediatr. Res., 35, 10, 1994. 151. Tsuji, M., Higuchi, Y., Shiraishi, K., et al. Protective effect of aminoguanidine on hypoxic-ischemic brain damage and temporal profile of brain nitric oxide in neonatal rat, Pediatr. Res., 47, 79, 2000. 152. Ma, J., Endres, M., and Moskowitz, M.A., Synergistic effects of caspase inhibitors and MK-801 in brain injury after transient focal cerebral ischemia in mice, Brit. J. Pharmacol., 124, 756, 1998. 153. Schulz, J.B., Weller, M., and Moskowitz, M.A., Caspases as treatment targets in stroke and neurodegenerative diseases, Ann. Neurol., 45, 421, 1999. 154. Nozaki, K., Findlestein, S.P., and Beal, M.F., Basic fibroblast growth factor protects against hypoxia- ischemia and NMDA neurotoxicity in neonatal rats, J. Cereb. Blood Flow Metabol., 13, 221, 1993. 155. Hossain, M.A., Fielding, K.E., Trescher, W.H., Ho, T., Wilson, M.A., and Laterra, J., Human FGF-1 gene delivery protects against quinolinate-induced striatal and hippocampal injury in neonatal rats, Eur. J. Neurosci. ,10, 2490, 1998. 156. Cheng, Y., Gidday, J.M., Yan, Q., Shah, A.R., and Holtzman, D.M., Marked age-dependent neuroprotection by BDNF against neonatal hypoxic-ischemic brain injury. Ann. Neurol., 41, 521, 1997. 157. Johnston, B.M., Mallard, E.C., Williams, C.E., and Gluckman, P.D., Insulin-like growth factor-1 is a potent neuronal rescue agent after hypoxic-ischemic injury in fetal lamb, J. Clin. Invest.,97, 300, 1996. 158. Gustafson, K., Hagberg, H., Bengtsson, B.A., Brantsing, C., and Isgaard, J., Possible protective role of growth hormone in hypoxia-ischemia, Pediatr. Res., 45,318, 1999. 159. Holtzman, D.M., Sheldon, R.A., Jaffe, W., Cheng, Y., and Ferriero, D.M., Nerve growth factor protects the neonatal brain against hypoxic-ischemic injury, Ann. Neurol., 39,114, 1996.

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160. Han, B.H. and Holtzman, D.M, BDNF protects the neonatal brain from hypoxic-ischemic injury via the ERK pathway, J. Neurosci., 20, 5775, 2000. 161. Supko, D.E. and Johnston, M.V., Dexamethasone potentiates NMDA receptor-mediated neuronal damage in the postnatal rat, Eur. J. Pharmacol., 270, 105, 1994. 162. Barks, J.D.E., Post, M., and Tuor, U.I., Dexamethasone prevents hypoxic-ischemic brain damage in the neonatal rat, Pediatr. Res., 29, 558, 1991. 163. Shinwell, E.S., Karplus, M., Reich, D., et al. Early postnatal dexamethasone treatment and increased incidence of cerebral palsy, Arch. Dis. Child Fetal Neonatal Ed., 83, F177, 2000. 164. Murphy, B.P., Inder, T.E., Huppi, P.S., et al. Impaired cerebral cortical gray matter growth after treatment with dexamethasone for neonatal chronic lung disease, Pediatrics, 107, 217, 2001. 165. Gidday, J.M., Fitzgibbons, J.C., Shah, A.R., and Park, T.S., Neuroprotection from ischemic brain injury by hypoxic preconditioning in the neonatal rat, Neurosci. Lett., 168, 221, 1994. 166. Gidday, J.M., Shah, A.R., Maceren, R.G., et al. Nitric oxide mediates cerebral ischemic tolerance in a neonatal rat model of hypoxic preconditoning, J. Cerebral Blood Flow Metab., 19, 331, 1999.

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3

Neural Stem Cell Biology Provides Insights into New Therapeutic Strategies for Hypoxic-Ischemic Brain Injury Kook In Park and Evan Y. Snyder

CONTENTS 3.1 Hypoxic-Ischemic Brain Injury as a Major CNS Disorder 3.2 Therapeutic Potentials of Neural Stem Cells 3.3 The Neural Stem Cell Response of Hypoxic-Ischemic Injury 3.4 Combining Cell Replacement with Gene Therapy via Neural Stem Cells 3.5 The Attempt of Self-Repair in the Injured Mammalian Brain 3.6 Translating Stem Cell biology into Therapy 3.7 Application of Biodegradable Synthetic Polymer to Neural Stem Cells 3.8 Summary 3.9 Acknowledgments References

3.1 HYPOXIC-ISCHEMIC BRAIN INJURY AS A MAJOR CNS DISORDER Stroke is one of the most common causes of death and severe disability in adults of developed countries,1 accounting for a large proportion of health care costs. About 200 per 100,000 adults per year will have their first stroke. Because the incidence of stroke increases with age, the absolute number of patients with stroke is likely to increase even further, given that the population of aged adults is also increasing.2,3 However, ischemic brain injury does not only affect the adult population, it is a major cause of mortality and severe neurodevelopmental disability (cerebral palsy, mental retardation, epilepsy, neurological handicap, and learning disability) in the pediatric, especially the newborn, population.4,5 Although the etiologies of hypoxic-ischemic (HI) brain injury in adults and children may differ, much of the pathophysiology

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underlying neural cell death and dysfunction is quite similar. In the case of newborn infants, despite advances in perinatal monitoring, obstetric and neonatal care, and a deeper understanding of the pathophysiology of perinatal asphyxia, the incidence of hypoxic-ischemic encephalopathy (HIE) in neonates has remained essentially unchanged over the last few decades. Except for thrombolysis therapy for acute stroke in the adult, current clinical management of both adult stroke and perinatal HIE has been limited to supportive measures; it is not directed toward preventing or interrupting the processes underlying brain injury or promoting regeneration.1,4,5 Given the absence of effective therapies for stroke and perinatal HIE, it is important to derive new strategies. There has been an intense search recently for new approaches that might be rooted in the growing knowledge of the molecular mechanisms that mediate neural cell death and degeneration. Unfortunately, despite recent substantial research in neuroprotection, to date, no neuroprotective agents have been shown conclusively to be clinically effective.1,5-8

3.2 THERAPEUTIC POTENTIALS OF NEURAL STEM CELLS Recently, there has been a growing interest in the therapeutic potential of neural stem cells (NSCs) and progenitors for therapy in stroke, HIE, and other central nervous system (CNS) dysfunctions. NSCs are the primordial, multipotent, self-renewing cells that, during the earliest stages of development, are believed to give rise to the vast array of specialized cells of the nervous system. They are thought to persist all throughout life, not only in a few discrete regions but probably throughout the brain, serving homeostatic and perhaps self-repair functions. The growing interest in NSC biology, as it might apply to HIE and stroke, represents a somewhat different focus on CNS injury. While most strategies under investigation seek to short-circuit cell death and/or promote neuroprotection — i.e., to combat progression of neuropathological processes, stem cell biology shines the spotlight instead on a nonpathological process — i.e., on reinvoking developmental processes for purposes of regeneration. In other words, a putative stem cell-mediated strategy would be rooted not so much in “combating” pathology as in abetting natural self-repair processes postulated — at least based on data emerging from our lab — to exist in the CNS in response to a wide range of injury and degenerative processes. In this context, therefore, the interest in NSCs derives from the realization that these cells are not simply a substitute for fetal tissue in transplantation paradigms or simply a “better” vehicle for gene delivery. We in the field of developmental neuroscience believe that the basic biology of these cells endows them with a potential that other vehicles for gene therapy and repair may not possess.9-11 This biologic potential endows them with the ability actually to integrate into the neural circuitry after transplantation. This property, in turn, may allow for the regulated release of various gene products. It may also allow for literal neural cell replacement. While presently available gene transfer vectors usually depend on relaying new genetic information through established neural circuits, which may, in

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fact, have already degenerated and become dysfunctional, NSCs may actually participate in the reconstitution of these pathways. The replacement of enzymes and of cells may be targeted not only to specific, anatomically circumscribed regions of the CNS,12-16 but also, if desired, by simple modifications in technique, to actually large areas of the CNS in a widespread manner.17-23 This ability is important because most neurologic diseases are not localized to specific sites, as is Parkinson’s disease. Rather, their neuropathologies are often extensive, multifocal, or even global; stroke and HIE provide ideal examples of just how broad the regions of degeneration may be. Intriguingly, NSCs may actually be uniquely responsive to neurodegenerative environments.24-26 This type of responsiveness of NSCs may optimize cell replacement and therapeutic gene expression within the damaged CNS.

3.3 THE NEURAL STEM CELL RESPONSE TO HYPOXICISCHEMIC INJURY Little is actually known about the response of NSCs to CNS injury in general, let alone HI brain injury in particular. Is it possible to repopulate an “ablated” CNS with NSCs in the way hematopoietic stem cells reconstitute lethally irradiated bone marrow? HI brain injury was initially viewed as an injury that is not only of importance in its own right but also might serve as a prototype for other large, acquired brain injuries.27 It occurred to us that to help answer this question we might be able to use one of our prototypical NSC clones, clone C17.2,28–30 as “reporter cells.” This wellcharacterized clone is just one of several with stem cell features that exist in the literature — multipotent, self-renewing, self-maintaining, nestin-positive, responsive to various stem cell trophins. As one would demand of a putative stem cell, NSCs from clone C17.2 are able to participate in the development of the CNS throughout the neuraxis and across developmental periods, from fetus to adult.17,18,23,25,26,28,29 Engrafted and integrated NSCs are visible because they have been transduced also with a reporter gene, lacZ, that allows the cells to stain blue when processed with Xgal histochemistry, or to appear brown of fluorescent following reaction with an antibody against E. coli β-galactosidase (βgal) in immunoperoxidase and immunofluorescence protocols, respectively.26,28 This ability to identify progeny of a donor NSC is important because, by their nature, NSCs integrate and intermingle seamlessly into the host following transplantation, do not form a discernible graft-host border, and actually come to resemble host neural cells of the same phenotype. When we talk about using clone C17.2 NSCs as “reporter” cells, we mean using well-characterized, indelibly marked cells with known ancestry, potential, and clonal relationships that are traceable, abundant, and homogenous, that intermingle imperceptibly with host cells in vivo and that can, therefore, be used as a tool for mirroring, probing, and tracking — i.e., “reporting” on — the behaviors of neighboring endogenous progenitors that are otherwise invisible to such monitoring and whose own clonal relationships and degree of homogeneity are much less certain. Such cells would also allow well-controlled experiments to proceed with minimal variability in cell population under study from experiment to experiment, animal to animal,

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condition to condition. The type of injury in which NSCs would be investigated in these preliminary experiments would be focal HIE engendered by permanent ligation of the right common carotid artery of a week-old mouse followed by exposure of the animal to 8% ambient oxygen. This combination of ischemia and hypoxia results in extensive injury to the hemisphere ipsilateral to the carotid ligation while leaving the contralateral hemisphere as an intact control. In the first set of pilot experiments,27 we wondered what might be observed if we took a normal animal in which “reporter” NSCs had become stably integrated throughout the brain during a critical period of its development (creating virtually a chimeric brain of host and reporter cells) and then exposed that animal to unilateral HI injury. The experimental paradigm, therefore, was as follows: clone C17.2 NSCs were transplanted into the cerebral ventricles of mice on the day of birth (P0), allowing the NSCs access to the subventricular germinal zone (SVZ) that lines the ventricular system running the length of the neuraxis; this results in widespread migration, stable integration, and intermixture of donor NSCs with host cells throughout the parenchyma.17 The right hemisphere was then subjected to HI injury at 1 week of age (P7). The brains were analyzed 2–5 weeks later. The resulting picture in these preliminary studies was complex but intriguing. In contrast to the intact side, where the reporter NSCs remained widely and evenly interspersed throughout the intact parenchyma, the reporter NSCs in the HI-injured hemisphere appeared to be densely and preferentially clustered around the infarction cavity. The heavy accumulation and number of cells in that location suggested either that many NSCs had migrated to that particular area, or that the cells near there had proliferated, or both. In addition, in the penumbra of the infarction, an increased number of donor-derived cells were identified immunocytochemically as oligodendrocytes and neurons. Neurons and oligodendrocytes are the two neural cell types that are most susceptible to HI injury and that are least likely to regenerate spontaneously in the “post-developmental” mammalian cortex. Furthermore, in the intact hemisphere, as might be expected from NSCs implanted after the completion of embryonic cortical neurogenesis, no donor-derived neurons and many fewer oligodendrocytes were noted. Therefore, following HI brain injury, NSCs appeared to evidence components of altered proliferation, migration, and differentiation. This is precisely the type of behavior one might expect of a stem cell. We decided to start examining each of these components in a systematic fashion.27 First we asked whether there was new transient proliferation by quiescent NSCs, both reporter and host cells. To answer this question, a transplant of reporter NSCs was performed at P0 into the cerebral ventricles; unilateral HI injury was induced at P7 (after the cells had stably integrated, differentiated, and become quiescent); the mice were then pulsed with bromodeoxyuridine (BrdU), a nucleotide analogue, at various post-HI injury time points. The preliminary analysis revealed that, before injury, donor-derived cells were completely quiescent; however, after HI injury, the percentage of reporter (lacZ+) cells that became mitotic (i.e., incorporated BrdU) increased rapidly, peaked at about 3 days after induction of HI, and then fell back to 0 by 1 week after HI. Host cells did precisely the same thing; their pattern of proliferation was virtually superimposable upon that of donor cells, peaking at ~3

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days after HI and then returning to 0, also suggesting an induction of transient proliferation. That there are so many changes peaking at 3 days following injury is intriguing. The literature on stroke — and, in fact, on other injuries — has suggested that the interval of 3–7 days after insult is a very metabolically, biochemically, and molecularly active temporal “window” during which a variety of mitogens, trophins, extracellular matrix molecules, and other factors are uniquely elaborated. We shall return to this “window” and its impact on neural stem cell biology later in the review. Next, we began to approach the question whether reporter NSCs (and, by extension, host NSCs), in fact migrated to the areas of neurodegeneration. NSCs (clone C17.2) were transplanted into only the left intracerebroventricular space at P0. At 1 week of age, unilateral HI injury was induced in the contralateral right hemisphere in some animals, while in others the right hemisphere was left intact. In animals with an intact right hemisphere, engrafted stem cells simply remained stably distributed and densely integrated throughout the parenchyma of only the transplanted left hemisphere. But in animals in which the right hemisphere had been infarcted, cells at multiple levels throughout the cerebrum dramatically appeared to migrate across the corpus callosum and any available interhemispheric commissure to the infarcted region (Figure 3.1). With high magnification under light and electron microscopy, one could appreciate leading processes of NSCs migrating along interhemispheric connections toward the damaged areas. Even within the infarct, one could see reporter cells migrating into the heart of the necrotic area.

FIGURE 3.1 Migration by transplanted “reporter” stem cells to the ischemic area of a mouse brain subjected to unilateral, focal hypoxic-ischemic brain injury. Clone C17.2 neural stem cells were injected into the left cerebral ventricle of a mouse on the day of birth (postnatal day 0 [P0]). At 1 week of age (P7), the animal was subjected to contralateral right-sided hypoxic-ischemic injury. The animal was analyzed at maturity with Xgal histochemistry to identify lacZ-expressing donor-derived cells (which stain blue). Some cells appeared to migrate along the corpus callosum (arrowhead) throughout the cerebrum toward the highly ischemic area (arrow).

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Therefore, there seems to be evidence that NSCs already integrated into the CNS will migrate to an area of subsequent infarction. Will reporter NSCs implanted after HI injury also be drawn to areas of damage? To investigate this question, the following paradigm is followed: Unilateral (right) HI injury is induced at P7, and reporter NSCs are transplanted into the contralateral (left) cerebral ventricle 3 days later (at P10). As a control, some animals not subjected to right HI are also transplanted on the left at P10. As before, in intact animals, the NSCs remain nicely but stably integrated on the transplanted left side. However, in animals that have been infarcted on the right before transplantation on the left, reporter NSCs migrate avidly across the corpus callosum and other interhemispheric commissures to the area of infarction throughout the length of the cerebrum. Furthermore, they integrate into those infarcted areas as if drawn or directed by a tropism for the region. When reporter NSCs are injected directly into the infarcted area on the right, they never migrate in the other direction to the contralateral intact side, in these pilot studies. This last manipulation, that of injecting NSCs directly into the infarct, suggests what our next set of experiments entailed. NSCs (clone C17.2) are transplanted directly into the degenerating infarcted region at various time points following the induction of unilateral HI. When implantation is performed shortly after an HI (e.g., the following day), robust engraftment is seen throughout the infarcted area. If

FIGURE 3.2 Robust engraftment by transplanted neural stem cells within the ischemic region of a mouse brain subjected to unilateral focal hypoxic-ischemic injury (HI). This mouse was subjected to right hypoxic-ischemic injury on postnatal day 7 (P7). Three days later (P10), the animal received a transplant of clone C17.2 neural stem cells within the region of infarction. The animal was analyzed at maturity with Xgal histochemistry. A representative coronal section is shown. Robust engraftment was evident within the ischemic are (arrow). Similar engraftment was evident throughout the hemisphere. Even cells that implanted outside the region of infarction appeared to migrate along the corpus callosum toward the ischemic area. The most exuberant engraftment was evident 3–7 days after HI. Immunocytochemical and ultrastructural analysis revealed that a subpopulation of donor-derived cells, especially those in the penumbra, differentiated into neurons and oligodendroglia, the two neural cell types most characteristically damaged by HI and the cell types least likely to regenerate spontaneously in the postnatal brain.

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transplantation is postponed until 5 weeks after HI, virtually no, if any, engraftment is achieved. Engraftment is most exuberant 3–7 days after HI (Figure 3.2). Is there, indeed, a change in differentiation fate by these reporter NSCs in these areas of degeneration compared with what might be seen in intact brain? Immunocytochemical and ultrastructural examination of the engrafted regions, particularly in the penumbra of the infarct, suggests that indeed there is. Donor-derived cells (recognized by an anti-βgal antibody) are assessed for the expression of neural cell type-specific antibodies (e.g., NeuN, neurofilament, MAP-2 for neurons, CNPase for oligodendrocytes, GFAP for astrocytes, nestin for immature, undifferentiated progenitors). A subpopulation of donor NSCs in the injured postnatal neocortex differentiate into neurons (~5%) and oligodendrocytes (~4%). Other cell types are astroglial — though no scarring seems apparent — and undifferentiated progenitors. As noted below, these numbers contrast significantly with what one finds in an intact age-matched recipient neocortex. The presence of donor-derived neurons can be detected as much as 1 mm away from the heart of the infarction cavity on the side of the lesion, suggesting a relatively large “sphere of influence” exerted by the injured tissue. (Interestingly, occasionally we would note host-derived neurons in an otherwise severely destroyed cortex; this type of finding is consistent with our belief that some host NSCs, just as the reporter NSCs do, try to shift their differentiation toward compensation for neuronal cell death, a phenomenon that we are perhaps augmenting with our transplants; more on this phenomenon later.) Examination of the penumbra under the electron microscope in these preliminary studies supports the immunocytochemical assessments. A significant number of donor-derived oligodendrocytes and neurons are appreciated. Some donor-derived pyramidal neurons receive synaptic input from the host. Quantification of the differentiation pattern by transplanted reporters NSCs in the injured neocortex compared with that in the intact neocortex is dramatic and illuminating. Whereas 5% of engrafted NSCs on the injured side differentiate into neurons, no neuronal differentiation by NSCs is seen at all in the intact neocortex, consistent with both the normal absence of neurogenesis in the postnatal mammalian cortex and with our own prior findings.25,31 There is a 5-fold increase in the number of donor-derived oligodendrocytes in the injured neocortex compared with the intact neocortex. The number of astrocytes does not significantly differ between the two sides. Also, there is an upregulation of nestin in donor NSCs in response to injury (almost three times as many donor cells are nestin-positive in the injured cortex compared with the intact cortex, suggesting that they may become activated or primed to make a differentiation choice). These preliminary quantitative data are presented to make a qualitative point. On the intact side of the infarcted animal, there is no neuronal differentiation at all; on the injured side, 5% of donor-derived cells are now neurons. The magnitude of that number is less significant than the phenomenon of qualitatively going from consistently no neurons to neurons of any number at a stage in development when no cortical neurons should normally be born. As mentioned previously, oligodendrocytes and neurons are the two neural cell types most damaged by HI injury. It appears from these preliminary data that NSCs may be attempting to repopulate and reconstitute

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that area of injury — particularly within a certain temporal window — by “shifting” their normal differentiation fate to compensate for the loss of those particular cell types, especially neurons. It seems indeed likely that, as a consequence this type of neurodegeneration, signals are elaborated to which NSCs (donor and probably host) are able to respond in a reparative fashion. Precisely what those signals are is an area of ongoing active investigation. They no doubt are a complex mix of various mitogens, neurotrophins, adhesion molecules, cytokines, and so forth. Although the preliminary numerical data cited above are presented principally to illustrate the “shift” toward neuronal differentiation by NSCs in response to injury, it is instructive to note that, given the vast number of NSCs that engraft into the infarcted region, a differentiation of even 5% of such cells into neurons translates into tens of thousands of replacement neurons supplied to that degenerating region. We don’t actually know how many neurons and how much circuitry is required to functionally reconstruct a damaged mammalian system. We do know that, fortunately, 100% restoration is not needed; older lesion data would suggest that as little as 10% may be sufficient.

3.4 COMBINING CELL REPLACEMENT WITH GENE THERAPY VIA NEURAL STEM CELLS Despite the fact that neuronal differentiation of 5% of transplanted NSCs may be sufficient to repair an HI injured region of brain, we nevertheless wondered whether that percentage could be increased. Neurotrophin-3 (NT-3) is known to play a role in inducing neuronal differentiation.32,33 It appeared feasible that neuronal differentiation of both host and donor NSCs might be enhanced if the latter were engineered before transplantation to (over)express NT-3. A subclone of NSCs was transduced with a retrovirus encoding rat NT-3.34 The engineered NSCs successfully produce large amounts of NT-3 in vitro and in vivo. We have determined that both the parent NSCs and the NT-3-overexpressing NSC subclones, express trkC receptors (the receptor for NT-3).35 These receptors are appropriately tyrosine-phosphorylated in response to exogenous NT-3; this phosphorylation can be blocked by K252a, an inhibitor of neurotrophin-induced tyrosine kinase activity. Therefore, it appeared that these engineered NSC clones not only could secrete excess amounts of NT-3 but also could likely respond to NT-3 in an autocrine or paracrine fashion — a very appealing scenario. In tissue culture, these NT-3-overexpressing NSCs, like the parent NSC clone, still differentiate into all three neural cell types (neurons, astrocytes, and oligodendrocytes). However, unlike the parent clone, whose percentage of neurons falls in serum-containing medium as new cells are born, the proportion of this NT-3-expressing subclone that continues to express neuronal markers in culture for prolonged periods (>3 weeks) remains quite high (~90%).35 In an experimental paradigm identical with that described previously, cells from the NT-3-expressing NSC subclone are implanted into the infarct of a unilaterally asphyxiated postnatal mouse brain 3 days after induction of HI injury.35 The brains are

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analyzed 2–4 weeks later as described above. Indeed, on preliminary analysis, the percentage of donor-derived neurons is dramatically increased, to 20% in the infarction cavity and to as high as 80% in the penumbra. Many of the neurons are calbindin-positive; they are also variously GABAergic, glutamatergic, or cholinergic. Donor-derived glia are rare. It appears, therefore, that when NSCs are transplanted within regions of HI injury, a greater percentage of them engineered ex vivo to express NT-3, differentiate into neurons. The NT-3 likely does act on donor cells (as well as host cells) in an autocrine/paracrine fashion to enhance that neuronal differentiation. Interestingly, this pilot experiment enunciates the feasibility of using NSCs for simultaneous, combined gene therapy and cell replacement in the same transplant procedure using the same clone of cells in the same transplant recipient — an appealing stem cell property with implications for therapies in other degenerative conditions.

3.5 THE ATTEMPT OF SELF-REPAIR IN THE INJURED MAMMALIAN BRAIN In the transplant studies described above, the grafted and stably integrated NSC clones, whose response to focal HI cerebral degeneration was tracked, were viewed as “reporter cells,” mirroring the behavior of the brain’s own NSCs which putatively alter their fate — their proliferation, migration, and differentiation — in an effort to repopulate damaged areas. The thinking would be, of course, that if the brain’s inclinations are toward self-repair via the NSCs, then that response might be augmented. Is this truly what endogenous progenitors “attempt” to do? We launched a series of nontransplant-based experiments to explore whether the “reporter cells” were indeed reporting on a true phenomenon. It has been recognized for decades36–38 that two highly circumscribed regions of the mammalian cerebrum continue to generate neurons throughout life. These “privileged” areas are designated “neurogenic regions” and exist lifelong in the olfactory bulb (OB) by way of the SVZ and in the hippocampal dentate gyrus (DG),39–43 including in humans.44,45 The remainder of the CNS is termed “nonneurogenic”; in other words, neuronal generation does not take place beyond fetal life, their normal period of neuron birth. Consequently, neuronal regeneration does not occur in the vast majority of the “postdevelopmental” CNS after injury or disease.46 However, the fact that cerebrum does retain a capacity for neurogenesis from proliferating cells in the SVZ and DG throughout life, suggests that these neural progenitor cells may provide an endogenous population with significant neuroregenerative potential (either constitutively or following manipulation). The findings in the previous sections postulated that, following brain injury and during phases of neurodegeneration, signals might be transiently elaborated, even in “nonneurogenic” regions, to which progenitor and stem cells can respond in a reparative fashion. Were an intrinsic capacity for producing new neural cells (including neurons) in classically nonneurogenic regions to be apparent — even at low, ostensibly clinically silent levels — this might attest to a degree of inherent CNS plasticity not previously appreciated, might explain certain observed levels of unanticipated

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recovery often seen by clinicians following adult and pediatric stroke, and might lend insight into the teleological significance of persistent neurogenic zones while offering a substrate from which better strategies for brain repair might be launched. Following unilateral HI brain injury, in preliminary studies, the migration and differentiation of mitotic neural progenitor cells (NPCs) in the SVZ of both hemispheres was assessed using two methods.47 First, we tracked the behavior of newly proliferative endogenous NPCs by injecting intraperitoneally the proliferation marker BrdU which is selectively and permanently incorporated into the nuclear genomic material of all cells entering S-phase, hence labeling dividing cells. Starting 2 hours after induction of unilateral HI, mice were pulsed with BrdU every 4 hours for the subsequent 12 hours. As an additional independent marker of newly mitotic cells, in parallel experiments, a replication-incompetent, help virus-free retroviral vector encoding the lacZ reporter transgene48 was also employed to label such cells directly. A retroviral provirus becomes permanently integrated into the genome and passed stably to the progeny of only those cells progressing through S-phase. Successful infection (as indicated by lacZ expression) is, therefore, another unambiguous marker of mitotic cells. In order to label proliferating SVZ cells, the lacZencoding vector was injected into both lateral ventricles of mice being subjected to unilateral HI. HI brain injury induced a significantly increased proliferation of the SVZ progenitor population ipsilateral to the lesioned right side compared to the grossly intact contralateral left side and uninjured control group. Expansion of BrdU-positive cells was most pronounced in the dorsolateral wall of the lateral ventricles adjacent to the infarction cavity, and a relatively dense stream of “newly born” cells oriented toward and into the injured cerebral cortex was apparent. The normal fate of most of the cells born in the SVZ (particularly the anterior portion) is to migrate rostrally along the rostral migratory stream (RMS) into the OB, where they differentiate into neurons.39–42 Certainly that typical developmental program is evident in the intact left hemisphere. Intriguingly, although more cells were actually born in the right SVZ ipsilateral to the lesion in response to HI, significantly fewer BrdU-positive cells were present in the RMS and the number of newly born cells that actually reached the right OB was reduced significantly — as if the newly born cells on the damaged side were “shunted” or “drawn” away from their normal migratory route toward the site of injury. Interestingly, the number of newborn cells that reached the RMS and OB from the SVZ contralateral to the lesion, though certainly much greater than that ipsilateral, was also significantly reduced compared to the noninjured control group, suggesting that injury has a broad effect throughout the brain and may draw cells from even distant regions. In other words, the CNS environment appears to change radically after injury, particularly that induced by HI. To help determine the differentiation fate in vivo of injury-generated BrdU-labeled cells, particularly in nonneurogenic regions, they were analyzed for their co-expression of neural cell type-specific antigens. Over a 3-week period following the final BrdU pulse, many of the cells induced to proliferate yielded new oligodendrocytes, astrocytes, and intriguingly, neurons (4.0%, 1.2%, and 1.2% at 1, 2, and 3 weeks, respectively). Interestingly, these new neurons (likely an underrepresentation

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given the time course of the BrdU pulses) were evident not only in the compromised hemisphere, but in the contralateral hemisphere as well, suggesting again the widespread “ripple” effect of signals emanating from even an ostensibly localized lesion. (No BrdU+ neurons were seen in the cortices of uninjured control mice.) That these newly born neurons in nonneurogenic regions might persist permanently was suggested by their continued detection essentially undiminished for at least 2 months after injury. As a complement to BrdU labeling and to track more rigorously the fate of these newly proliferative injury-responsive periventricular NPCs, a retroviral vector encoding lacZ was injected into both lateral ventricles of mice being subjected to unilateral HI. In response to HI, lacZ-expressing (i.e., βgal+) periventricular cells migrated into the adjacent striatum and hippocampus, into the cortex ipsilateral to the lesion, and into the cortical penumbra. Confirming the observation noted previously, a subpopulation of the newly proliferative and migratory βgal+ cells now expressed the mature neuronal marker, NeuN, in all of these “nonneurogenic” regions, suggesting de novo neurogenesis. (Interestingly, even in the grossly intact contralateral hemisphere, some βgal+ periventricular cells (often in groups) also migrated into the cortex and overlying hippocampal CA1 area, becoming neurons.)

3.6 TRANSLATING STEM CELL BIOLOGY INTO THERAPY The findings in the previous section (as did the transplant studies described previously) suggest that, following CNS injury and during acute phases of resultant neurodegeneration, factors are elaborated to which donor-derived and endogenous neural progenitor and stem cells may respond in a reparative fashion and which can promote the establishment of new neurons even within nonneurogenic regions of the “postdevelopmental” CNS. Neural stem and progenitor cells appear to be capable of responding to neurogenic signals not only during their normal developmental expression, but also when induced in later stages during critical periods following injury. Stem cells seem to have a tropism for and a trophism within degenerating CNS regions. They seem to be able to “shift” their differentiation fate. This phenomenon seems to be magnified at the peak of active neurodegeneration. Given these observations, we further speculate that the CNS may “attempt” to repair itself with its own endogenous pool of progenitors and stem cells, but that supply may simply be insufficient either in number or in factors regulating mobilization, recruitment, migration, differentiation, survival, neurite extension, and synaptogenesis in the context of HI injury. Therefore, the net impact of the production of new nerve cells may be limited. If this is the case, perhaps we can augment that stem cell population with exogenous stem cells and/or exogenous trophic factors to enable more significant recovery. Such a strategy would certainly benefit from identifying those transiently expressed signals. Such identification may permit them to be supplied exogenously in order to recruit the host’s own internal stem cell reservoir more effectively. In fact, donor stem cells genetically engineered ex vivo (as we did with the NT-3-expressing

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stem cells) may be one method for supplying some of those tropic and trophic factors. Under certain circumstances, in fact, one clone of transplanted stem cells may be able to serve multiple therapeutic functions: both gene delivery and cell replacement. Therefore, one strategy, which can take its place in the repertoire with other valuable repair strategies, may be stem cell-based: using the host’s own appropriately activated reserve of stem cells augmented by an exogenous supply of stem cells introduced during or shortly after injury or neurodegeneration (apparent “windows of opportunity”). It may, in fact, be possible to treat chronic lesions by re-expressing certain “signals” (e.g., certain cytokines) that emulate the more acute phase, to which stem cells may then respond in a reparative fashion. All these speculations are absolutely predicated on exploring the dynamic processes by which multipotent stem cells make their phenotypic choices in developing and degenerating CNS. The abiding faith in “translational neuroscience” is, of course, that the biology that endows rodent neural stem cells with their therapeutic potential is conserved in the human CNS. Progress in this regard is, gratifyingly, being made. Several neural stem cell clones and populations have been isolated from human fetal brains and these cells appear to emulate many of the appealing properties of their rodent counterparts;22,45,49–53 they differentiate, in vitro and in vivo, into all three neural cell types; they vouchsafe conservation of neurodevelopmental principles following engraftment into developing mouse brain; they express foreign genes in vivo in a widely disseminated manner; and they can replace missing neural cell types when grafted into various mutant mice. In order to determine whether findings with rodent NSCs in response to injury might extend to cells from the human CNS and to explore their therapeutic potential in the treatment of HI in infants, human NSCs (in pilot studies) were injected into the infarction cavity of mice, employing the same experimental paradigm described above.54 Human NSCs show robust engraftment within the ischemic region and its penumbra, migrate extensively and preferentially toward the site of injury, and differentiate into all three neural cell types. A subpopulation of donor-derived neurons express glutamate, GABA, tyrosine hydroxylase, and choline acetyltransferase in various CNS regions. Preliminary data suggest that human NSCs grafted into the HI-injured brain sites in mice partially restore some motor and cognitive functions, as demonstrated by rotarod performance, the step-through type passive avoidance test, and the habituation of exploratory behavior test. These findings suggest that human NSCs might be capable of replacing some neural cell populations lost to experimental HI injury in mice and could provide a rationale for ultimate stem cell-based therapy for human ischemic and other degenerative CNS diseases.

3.7 APPLICATION OF BIODEGRADABLE SYNTHETIC POLYMER TO NEURAL STEM CELLS Although neural stem cells appear to have the capacity to repopulate HI-injured brain, particularly in the penumbra, their ability to reform structural and functional neural connections may be limited by the vast amount of brain parenchymal loss.

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The core of the infarct changes rapidly to a cystic cavity. Even the most capable stem cell may need intrinsic organization and a template to guide restructuring. Furthermore, large volumes of cells will not survive if located greater than a few 100 µm from the nearest capillary. To address this need, a pilot experiment was performed.55 Polyglycolic acid (PGA) is a synthetic biodegradable polymer used widely in clinical medicine.56 Highly hydrophilic, PGA loses its mechanical strength rapidly over 2–4 weeks in the body. We hypothesized that three-dimensional highly porous “scaffolds” composed of PGA, if co-transplanted with neural stem cells into the infarction cavity might facilitate reformation of structural and functional circuits, particularly if the cells had been engineered ex vivo to also express factors that might attract ingrowth of host fibers. The scaffold might initially provide a matrix to guide cellular organization and growth, allow diffusion of nutrients to the transplanted cells, become vascularized, and then disappear, obviating concerns over long-term biocompatibility. To test this hypothesis, clone C17.2 neural stem cells were seeded onto PGA scaffolds in culture. The cells grew robustly, migrated readily throughout the structure, and differentiated spontaneously into neurons and glias, adhering to the polymer fibers. Immunostaining showed that most cells robustly differentiated into neurons, sending out long neurofilament (NF)+ processes. They extended long axonlike processes along the fibers and developed small, complex dendrite-like processes. After 4–6 days, in vitro, the cell-polymer unit was implanted into the evolving cystic infarct cavity of mice subjected 1 week prior to unilateral HI brain injury. After 2–6 weeks, the cells had, indeed, completely impregnated the polymer, and the polymer/stem cell unit refilled the infarction cavity, becoming incorporated into animal’s cerebrum and even becoming vascularized by the host (Figure 3.3). The NSCs seeded on polymers displayed robust engraftment, foreign gene (lacZ)

FIGURE 3.3 Implantation of PGA (polyglycolic acid) polymers-neural stem cells complex into the infracted region of a mouse brain subjected to unilateral focal hypoxic-ischemic (HI) injury. This mouse was subjected to right hypoxic-ischemic injury on postnatal day 7 (P7). Seven days later (P14), the animal received a transplant of clone C17.2 neural stem cells-PGA matrix complex, initially generated in vitro, into HI-generated infarction cavity. The animal was analyzed at maturity. A representative coronal section is shown. NSCs-polymer complex can fill that cavity (arrow in A), appear to be incorporated into the infracted cerebrum (A), and even support neovascularization (arrows in B point to host blood vessels in the stem cellpolymer unit) (arrowheads in A and B point to dark fibers from the polymer).

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expression and differentiation into neurons and glia within the region of HI injury, virtually repopulating the injured brains. Many long neuronal processes of host and donor-derived neurons enwrapped the polymer fibers and ran along the length of the fibers, often interconnecting with each other. Donor-derived neurons appeared to extend many exceedingly long, complex processes along the length of the disappearing matrix, extending ultimately into host parenchyma apparently as far as the opposite intact hemisphere. Host neuronal processes, in a reciprocal manner, appeared to enter the matrix, possibly making contact with donor-derived neurons. In order to confirm the ability of engrafted NSCs-polymer complex to establish longdistance neuronal connections, both the anterograde and retrograde label DiI, and the anterograde label “biotinylated dextran amine conjugated with fluorescein” (BDAFITC) was stereotactically injected into NSCs-polymer complex transplant site or contralateral intact cortex 8 weeks after transplantation. Indeed, neuronal tracing studies showed that long-distance neuronal circuitry between donor-derived and host neurons in both cerebral hemispheres may have been reformed through the corpus callosum in some instances55. Although these findings are still preliminary, it appears feasible to implant NSCs-polymer complexes in order to augment reparative responses, to facilitate even further the differentiation of host and donor neurons, and to enhance the ingrowth/outgrowth of such cells in an effort literally to facilitate reformation of structural/functional cortical tissue and promote neuronal connectivity.

3.8 SUMMARY Together, the preliminary and published studies in this review suggest that NSCs may play a role in a wide range of repair strategies for the CNS injured by ischemia. Indeed, NSCs seem to serve as the glue that holds these various repair strategies together: cell replacement, gene therapy, and biomaterial tissue engineering.

3.9 ACKNOWLEDGMENTS: K.I. Park was partly supported by a special grant of the Dean of Yonsei University College of Medicine Research Fund of 1998. EYS was partly supported by grants from the March of Dimes and from NINDS (NS34247 and NS33852).

REFERENCES 1. Davenport, R. and Dennis, M., Neurological emergencies: acute stroke, J. Neurol. Neurosurg. Psych., 68, 277, 2000. 2. Sudlow, C. and Warlow, C., Comparing stroke incidence worldwide. What makes studies comparable?, Stroke, 27, 550, 1996. 3. Warlow, C. P., et al., The organization of stroke services, in Stroke. A Practical Guide to Management, Warlow, C.P. et al. (eds.), Blackwell, Oxford, 1996, 598. 4. Volpe, J.J., Neurology of the Newborn, 3rd ed., WB Saunders Company, Philadelphia, 1995. 5. Vannucci, R.C. and Perlman, J.M., Interventions for perinatal hypoxic-ischemic encephalopathy, Pediatrics, 100, 1004, 1997.

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6. du Plessis, A.J. and Johnston, M.V., Hypoxic-ischemic brain injury in the newborn. Cellular mechanisms and potential strategies for neuroprotection, Clin. Perinatol., 24, 627, 1997. 7. del Zoppo, G., et al., Inflammation and stroke: putative role for cytokines, adhesion molecules and iNOS in brain response to ischemia, Brain Pathol., 10, 95, 2000. 8. Tan, S. and Parks, D. A., Preserving brain function during neonatal asphyxia, Clin. Perinatol., 26, 733, 1999. 9. Snyder, E.Y. and Wolfe, J.H., CNS cell transplantation: a novel therapy for storage diseases?, Curr. Opin. Neurol., 9, 126, 1996. 10. Snyder, E.Y. and Fisher, L.J., Gene therapy for neurologic diseases, Curr. Opin. Pediatr., 8, 558, 1996. 11. Snyder, E.Y. and Senut, M.C., Use of non-neuronal cells for gene delivery, Neurobiol. Dis., 4, 69, 1997. 12. Martinez-Serrano, A., et al., CNS-derived neural progenitor cells for gene transfer to nerve growth factor to the adult rat brain: complete rescue of axotomized cholinergic neurons after implantation into the septum, J. Neurosci., 15, 5668, 1995. 13. Martinez-Serrano, A., Fischer, W., and Bj?rklund, A., Reversal of age-dependent cognitive impairments and cholinergic neuronal atrophy by NGF-secreting neural progenitors grafted to the basal forebrain, Neuron, 15, 473, 1995. 14. Martinez-Serrano, A., et al., Longterm functional recovery from age-induced spatial memory impaiments by nerve growth factor gene transfer to the rat basal forebrain, Proc. Natl. Acad. Sci. USA, 93, 6355, 1996. 15. Martinez-Serrano, A. and Bjklund, A., Protection of the neostriatum against excitotoxic damage by neurotrophin-producing, genetically modified neural stem cells, J. Neurosci., 16, 4604, 1996. 16. Martinez-Serrano, A. and Snyder, E.Y., Neural stem cell lines for CNS regeneration: Basic Science and Clinical Applications, in CNS Regeneration, Tuszynski, M. and Kordower, J. (eds.), Academic Press, San Diego, 1999, 203. 17. Snyder, E.Y., Taylor, R.M., and Wolfe, J.H., Neural progenitor cell engraftment corrects lysosomal storage throughout the MPS VII mouse brain, Nature, 374, 367, 1995. 18. Lacorazza, H.D., et al., Expression of human β-hexosaminidase α-subunit gene (the gene defect of Tay-Sachs disease) in mouse brains upon engraftment of transduced progenitor cells, Nat. Med., 2, 424, 1996. 19. Lynch, W.P., et al., Late virus replication events in microglia are required for neurovirulent retrovirus-induced spongiform neurodegeneration: evidence from neural progenitor-derived cheimeric mouse brains, J. Virol., 70, 8896, 1996. 20. Lynch, W.P., Shapre, A.H., and Snyder, E.Y., Neural stem cells as engraftable packaging lines optimize viral vector-mediated gene delivery to the CNS evidence from studying retroviral env-related neurodegeneration, J. Virol., 73, 6481, 1999. 21. Billinghurst, L.L., Taylor, R.M., and Snyder, E.Y., Remyelination: cellular and gene therapy, Sem. Pediatr. Neurol., 5, 211, 1998. 22. Flax, J.D., et al., Engraftable human neural stem cells respond to developmental cues, replace neurons and express foreign genes, Nat. Biotech., 16, 1033, 1998. 23. Yandava, B.D., Billinghurst, L.L., and Snyder, E.Y., “Global” cell replacement is feasible via neural stem cell transplantation: evidence from the dysmyelinated shiverer mouse brain, Proc. Natl. Acad. Sci. USA, 96, 7029, 1999. 24. Snyder, E.Y. and Macklis, J.D., Multipotent neural progenitor or stem-like cells may be uniquely suited for therapy for some neurodegenerative conditions, Clin. Neurosci., 3, 310, 1996.

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25. Snyder, E.Y. et al., Multipotent neural precursors can differentiate toward replacements of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex, Proc. Natl. Acad. Sci. USA, 94, 11663, 1997. 26. Rosario, C.M. et al., Differentiation of engrafted multipotent neural progenitors towards replacement of missing granule neurons in Meander tail cerebellum may help determine the locus of mutant gene action, Development, 124, 4213, 1997. 27. Park, K.I., Jensen, F.E., and Snyder, E.Y., Nerual progenitor transplantation for hypoxic-ischemic brain injury in immature mice, Soc. Neurosci. Abs., 21, 2027, 1995. 28. Snyder, E.Y. et al., Multipotent neural cell lines can engraft and participate in development of mouse cerebellum, Cell, 68, 33, 1992. 29. Snyder, E.Y. et al., Transplantation and differentiation of neural “stem-like” cells: possible insights into development and therapeutic potential, in Research and Perspectives in Neurosciences: Isolation, Characterization, and Utilization of CNS stem cells, Gage, F.H. and Christen, Y. (eds.), Springer-Verlag, New York, 1997, 173. 30. Snyder, E.Y., Neural stem-like cells: developmental lessons with therapeutic potential, Neurosci., 4, 408, 1998. 31. Gage, F.H. et al., Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain, Proc. Natl. Acad. Sci. USA, 92, 11879, 1995. 32. Ghosh, A. and Greenberg, M.E., Distinct roles for bFGF and NT-3 in the regulation of cortical neurogenesis, Neuron, 15, 89, 1995. 33. Johe, K.K. et al., Single factors direct the differentiation of stem cells from the fetal and adult central nervous system, Genes Dev., 10, 3129, 1996. 34. Liu, Y. et al., Intraspinal delivery of neurotrophin-3 using neural stem cells genetically modified by recombinant retrovirus, Exp. Neurol., 158, 9, 1999. 35. Park, K.I. et al., Transplantation of neurotrophin-3 (NT-3) expressing neural stem-like cells into hypoxic-ischemic brain injury, Soc. Neurosci. Abs., 23, 346, 1997. 36. Sidman, R.L., Miale, I.L., and Feder, N., Cell proliferation and migration in the primitive ependymal zone: an autoradiographic study of histogenesis in the nervous system, Exp. Neurol., 1, 322, 1959. 37. Altman, J. and Das, G.D., Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats, J. Comp. Neurol., 124, 319, 1965. 38. Altman, J., Autoradiographic and histological studies of postnatal neurogenesis: IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb, J. Comp. Neurol., 137, 433, 1969. 39. Lois, C. and Alvarez-Buylla, A., Long distance neuronal migration in the adult mammalian brain, Science, 264, 1145, 1994. 40. Lois, C., Garcia-Verdugo, J. M., and Alvarez-Buylla, A., Chain migration of neuronal precursors, Science, 271, 978, 1996. 41. Goldman, S.A. and Luskin, M.B., Strategies utilized by migrating neurons of the postnatal vertebrate forebrain, Trend Neurosci., 21, 107, 1998. 42. Kakita, A. and Goldman, J.E., Patterns and dynamics of SVZ cell migration in the postnatal forebrain: monitoring living progenitors in slice preparation, Neuron, 23, 461, 1999. 43. Wei, W., et al., Directional guidance of neuronal migration in the olfactory system by protein slit, Nature, 400, 331, 1999. 44. Eriksson, P.S., et al., Neurogenesis in the adult human hippocampus, Nat. Med., 4, 1313, 1998. 45. Pincus, D.W., et al., FGF2/BDNF-associated maturation of new neurons generated from adult human subependymal cells, Ann. Neurol., 43, 576, 1998.

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46. Ramon y Cajal, S., Degeneration and Regeneration of the Nervous System, Oxford University Press, London, 1928. 47. Park, K.I. and Snyder, E.Y., Transplantation of human neural stem cells, propagated by either genetic or epigenetic means, into hypoxic-ischemic (HI) brain injury, Soc. Neurosci. Abs., 25, 212, 1999. 48. Price, J., Turner, D., and Cepko, C., Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer, Proc. Natl. Acad. Sci. USA, 84, 156, 1987. 49. Svendsen, C. N., et al., Long-term survival of human central nervous system progenitor cells transplanted into a rat model of Parkinson’s disease, Exp. Neurol., 148, 135, 1997. 50. Vescovi, A.L., et al., Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation, Exp. Neurol., 156, 71, 1999. 51. Fricker, R.A., et al., Site-specific migration and neuronal differentiation of human neural progenitor cells after transplantation in the adult rat brain, J. Neurosci., 19, 5990, 1999. 52. Villa, A., et al., Establishment and properties of a growth factor-dependent, perpetual neural stem cell line from the human CNS, Exp. Neurol., 161, 67, 2000. 53. Javier Rubio, F. et al., Genetically perpetuated human neural stem cells engraft and differentiate into the adult mammalian brain, Mol. Cell. Neurosci., 16, 1, 2000. 54. Park, K.I. and Snyder, E.Y., Transplantation of human neural stem cells, propagated by either genetic or epigenetic means, into hypoxic-ischemic (HI) brain injury, Soc. Neurosci. Abs., 25, 212, 1999. 55. Park, K.I., et al., Transplantation of neural stem cells (NSCs) seeded in biodegradable polyglycolic acid (PGA) into hypoxic-ischemic (HI) brain injury, Soc. Neurosci. Abs., 26, 868, 2000. 56. Shalaby, S.W. and Johnson, R.A., Synthetic absorbable polyesters, in Biomedical Polymers, Shalaby, S.W. (ed.), Carl Hanser, 1994, 2.

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4

Excitotoxicity, Oxidative Stress, and Apoptosis in Ischemic Neuronal Death Byoung Joo Gwag, Seok Joon Won, and Doo Yeon Kim

CONTENTS 4.1. Introduction 4.2. Excitotoxicity 4.2.1 Background 4.2.2 Ca2+-Mediated Fast Excitotoxicity 4.2.3 Ca2+ Overload-Induced Neuronal Death following Hypoxic Ischemia 4.2.3.1 Calpains 4.2.3.2 Cytosolic Phospholipase A2 (cPLA2) 4.2.3.3 Ca2+-Dependent Protein Kinases 4.2.3.4 Endonucleases 4.2.3.5 Nuclear Factor Kappa B 4.2.3.6 Mitochondria 4.2.3.7 Oncosis vs. Apoptosis 4.2.3.8 Limitation of Anti-Excitotoxicity Therapy for Hypoxic-Ischemic Injury 4.3. Oxidative Stress 4.3.1 Background 4.3.2 Free Radical Production During Hypoxic Ischemia and Reperfusion 4.3.2.1 Free Radical Production in Mitochondria 4.3.2.2 Activation of Xanthine Oxidase by Calcium-Activated Proteases 4.3.2.3 ROS Production by Transition Metals 4.3.2.4 Reactive Nitrogen Radicals 4.3.2.5 Metabolism of Arachidonic Acid 4.3.2.6 Apoptosis vs. Oncosis 4.3.2.7 Limitation of Antioxidant Therapy for Hypoxic Ischemic Injury 4.4. Apoptosis

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4.4.1 4.4.2

Background Initiation of Ischemic Apoptosis 4.4.2.1 pH 4.4.2.2 Calcium Imbalance 4.4.2.3 Fas Receptor 4.4.3 Propagation of Ischemic Neuronal Apoptosis 4.4.3.1 MAPKs 4.4.3.2 The Proapoptotic Family of bcl-2 4.4.3.3 Caspases 4.4.3.4 Reevaluation of Ischemic Neuronal Apoptosis 4.5. Maximization for Prevention of Hypoxic-Ischemic Neuronal Death 4.6. Conclusion 4.7 Acknowledgments References

4.1 INTRODUCTION Neuronal function and survivorship require appropriate supply of blood to the nervous system. Neuronal activity disappears immediately after blood flow drops below one fourth of the normal values. If the ischemic condition persists for a prolonged time, primary neuronal death appears rapidly in the core areas and is accompanied by the secondary death in the ischemic penumbra that slowly evolves subsequent to activation of multiple death pathways and thus has been targeted for therapeutic intervention. The first line of interventional therapy stems from findings that excitotoxicity underlies a leading cause of neuronal death following hypoxic-ischemic insults. Accordingly, antagonists of ionotropic glutamate receptors have been developed, shown to reduce hypoxic-ischemic brain injury in various animal models, and applied for clinical trial of ischemic stroke with little therapeutic efficacy to date. Free radicals are produced primarily during a period of reperfusion and believed to contribute to delayed neuronal death. Finally, several lines of evidence suggest that apoptosis or programmed cell death comprises a portion of ischemic neuronal death. It is conceivable to postulate that the therapeutic intervention of ischemic neuronal death likely involves appropriate combination of neuroprotective drugs designed to prevent excitotoxicity, oxidative stress, and apoptosis.

4.2 EXCITOTOXICITY 4.2.1 BACKGROUND Glutamate mediates excitatory synaptic transmission through activation of ionotropic glutamate receptors sensitive to NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) or kainate. While this excitatory transmission is indispensable for normal information processing and neuronal plasticity, excess and sustained activation of the glutamate receptors results in fulminant neuronal death, namely glutamate neurotoxicity or excitotoxicity.

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4.2.2 CA2+-MEDIATED FAST EXCITOTOXICITY The pentameric NMDA receptors consist of the fundamental subunit NMDAR1 and modulatory subunits of NMDAR2A-2D.94,230 The heteromeric NMDA receptors are highly permeable to Ca2+ as well as Na+ and K+. NMDA receptors mediate slowly evolving and desensitizing component of excitatory postsynaptic currents. NMDA receptors can be fully activated under depolarization of plasma membrane that abolishes the inhibition of NMDA receptors by Mg2+. As a brief (>3 min) activation of NMDA receptors is sufficient to trigger neuronal death, activation of NMDA receptors has been proposed as a primary cause of neuronal death after focal cerebral ischemia that is accompanied by transient (~30–60 min) elevation of extracellular glutamate.11,236 Ca2+ influx through NMDA receptors mediates the rapidly triggered NMDA neurotoxicity while Na+ influx contributes to swelling of neuronal cell body (Figure 4.1).31 AMPA receptors consist of a combination of GluR1–GluR4 subunits.94,180 Most AMPA receptors are highly permeable to Na+ and K+ and mediate rapidly evolving and desensitizing component of excitatory postsynaptic currents. In contrast to NMDA, a prolonged (>60 min) activation of AMPA receptors is required to trigger neuronal death.121 While most neurons express the GluR2 subunit that renders AMPA receptors impermeable to Ca2+, expression and function of GluR2 appear to be reduced in neurons vulnerable to hypoxic-ischemic insults. A transient forebrain ischemia produces delayed neuronal death in the CA1 hippocampal area that is

FIGURE 4.1 Scheme of excitotoxic neuronal death in hypoxic-ischemic brain injury.

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preceded with downregulation of GluR2 mRNA expression.72,181 NADPH diaphorase-containing neurons in the cortex and striatum express Ca2+-permeable AMPA receptors and can undergo degeneration following a brief (~10 min) exposure to AMPA.249 Single-cell RT-PCR analysis has demonstrated that striatal diaphorasecontaining neurons reveal reduced ratio of GluR2/GluR1 and unedited expression of GluR2 mRNA.113 This altered expression of GluR2 results in Ca2+-dependent AMPA neurotoxicity that may underlie potential mechanisms for selective neuronal death following hypoxic-ischemic brain injury. The GluR5-7 and KA1-2 proteins comprise the subunits for functional KA receptors permeable to Na+ and K+. Administration of kainate produces nondesensitizing currents at AMPA receptors and fast desensitizing currents at kainate receptors. Like AMPA-mediated slow excitotoxicity, a prolonged (1 h>) exposure to kainate is needed to trigger neuronal death. Whereas it has been well documented that Ca2+ entry through NMDA or AMPA receptors mediates the fast excitotoxicity, it remains to be delineated how slow excitotoxicity is triggered and propagated. One possibility is that Na+ entry through AMPA or kainate receptors likely contributes to AMPAor kainate-mediated slow excitotoxicity.

4.2.3 CA2+ OVERLOAD-INDUCED NEURONAL DEATH FOLLOWING HYPOXIC ISCHEMIA The concentration of free Ca2+ in the cytoplasm of a resting neuron is extremely low (approximately 100 nM) whereas its extracellular concentration is estimated to 1–2 mM. The intraneuronal levels of Ca2+ are maintained through (1) the entry of extracellular Ca2+ through ligand-operated receptor or voltage-gated Ca2+ channels, (2) the release of Ca2+ from the endoplasmic reticulum through stimulation of IP3 receptors or from the mitochondria through Na+-Ca2+ exchanger, (3) the extrusion of Ca2+ through Ca2+-ATPase or Na+-Ca2+ exchanger in the plasma membrane, (4) the binding of Ca2+ to target proteins, and (5) the sequestration of Ca2+ into the endoplasmic reticulum through Ca2+-ATPase or mitochondria through an electrophoretic (uniport) mechanism.22,68,80 Thus, energy failure in hypoxic ischemia will cause accumulation of intraneuronal free Ca2+ (Ca2+ overload) by enhancing the entry and release of Ca2+ and interfering with the ATP-dependent extrusion and sequestration of Ca2+. The entry of Ca2+ through NMDA receptors appears to underlie a major portion of Ca2+ overload following hypoxic ischemia as NMDA antagonists block the entry and accumulation of Ca2+ in central neurons deprived of oxygen and glucose (a hypoxic-ischemic condition in vitro).70,75 Prolonged elevation of intracellular Ca2+ leads to catabolic process of vital molecules and irreversible death of neuronal cells through multiple mechanisms involving activation of Ca2+-dependent effector proteins. 4.2.3.1

Calpains

Calpains are a family of Ca2+-dependent cysteine proteases that consist of an 80-kD catalytic subunit and a 30-kD subunit.225 Active calpain cleaves vital proteins such as spectrin, fodrin, Ca2+-ATPase, protein kinase C, and nuclear factor kappa B,21,144

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which contributes to dendritic remodeling, interruption of membrane and cytoplasmic transportation, modulation of gene expression, and neuronal degeneration.56,245,246 Activation of calpain I has been reported following activation of ionotropic glutamate receptors or hypoxic-ischemic insults.176,196,214 Selective inhibitors of calpain I can reduce NMDA-, kainate-, or AMPA-mediated excitotoxicity to some extent and attenuate brain damage after hypoxia, focal cerebral ischemia, or transient global cerebral ischemia.95,139,191 However, causative activation of calpain I for excitotoxicity has been challenged with findings that blockade of calpain I by several inhibitors does not protect neurons from glutamate neurotoxicity.56,148 In addition to calpains, other proteases such as cathepsin D are activated in cortical neurons exposed to NMDA or kainate (Yoon and Gwag, unpublished data) and thus likely involved in the process of excitotoxicity. 4.2.3.2

Cytosolic Phospholipase A2 (cPLA2)

Accumulated Ca2+ in neurons results in translocation of cPLA2 into plasma membrane. The Ca2+-activated cPLA2 produces free fatty acid (e.g., arachidonic acid) and lysophospholipids by catalyzing the cleavage of the free fatty acid from glycerophospholipids. Arachidonic acid is further utilized for production of prostaglandins and leukotrienes with concomitant production of superoxide.129,207 Activation of cPLA2 may contribute to neuronal death in excitotoxicity and hypoxic ischemia. Activity of cPLA2 is increased following focal cerebral ischemia or in cultured neurons following activation of ionotropic glutamate receptors.50,112,136,204,206 Pharmacological inhibitors of cPLA2 reduce excitotoxic and hypoxic-ischemic neuronal death.13,201 Moreover, knockout mice of cPLA2 (cPLA2-/-) shows substantially reduced cerebral infarct, brain edema, and neurological deficits following occlusion of the middle cerebral artery.12 Direct application of cPLA2 or melittin, a cPLA2 activator, produces neuronal death in cultured neurons and rats.33 Thus, Ca2+ overload through glutamate receptors induces activation of cPLA2 that produces neurotoxic metabolites such as prostaglandins, leukotrienes, reactive oxygen species, and platelet activating factor through metabolism of arachidonic acid and lysophospholipids. While cPLA2 mediates Ca2+-dependent excitotoxicity and hypoxic-ischemic injury in part, further study will be needed to delineate how the neurotoxic metabolites are coupled to the propagation and execution of neuronal death after hypoxicischemic injury. 4.2.3.3

Ca2+-Dependent Protein Kinases

Administration of glutamate results in Ca2+-dependent inhibition or activation of calcium/calmodulin protein kinase II (CaMK II) in the cortical and hippocampal neurons through activation of NMDA glutamate receptors.32,64,166 Activity of CaMK II is decreased in a way sensitive to NMDA antagonists rapidly following transient focal or global cerebral ischemia.88,216 While homozygous knock-out mice lacking the alpha subunit of CaMK II enhances sensitivity to hypoxic-ischemic insults in vivo,248 a selective cell-permeable inhibitor of CaMK II, KN62, attenuates neuronal death

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following exposure to NMDA or deprivation of oxygen-glucose in vitro.87 Thus, the exact role of CaMK II should be unveiled for understanding and therapeutic prevention of excitotoxic and hypoxic-ischemic neuronal death. Administration of excitotoxins can activate the major members of the mitogenactivated protein kinase (MAPK) family: p42/p44 extracellular signal-regulated kinases (ERK1 and 2), c-Jun N-terminal protein kinase (JNK)/stress-activated protein kinases (SAPKs) and p38.107,119,209,241,259 It has been well established that SAPK and p38 act as a downstream mediator for execution of neuronal and nonneuronal cell apoptosis.39 While activated p38 MAPK appears to mediate NMDA-induced neuronal apoptosis in cerebellar granule cells,107 inhibitors of p38 did not reduce NMDA-induced neuronal cell necrosis in cortical cell cultures.118 In a recent report, PD98059, a selective ERK inhibitor, was shown to reduce infarct volume up to 55% by 1 d and 36% by 3 d following occlusion of the middle cerebral artery.1 In addition to p38, ERKs and SAPKs likely participate in the process of excitotoxicity as well as hypoxic-ischemic neuronal death. 4.2.3.4

Endonucleases

Intraneuronal Ca2+ overload or acidification can activate Ca2+/Mg2+-dependent endonucleases or DNAse II, respectively, that cleave a region of linker DNA between nucleosomes, resulting in internucleosomal DNA fragments of multiples of 200 base pairs (or DNA ladders).197 Administration of glutamate, NMDA, AMPA, or kainate produces DNA ladders in cultured neurons and rat brain.3,83,185 Although activation of caspase-3 mediates liberation of caspase-activated DNase (CAD)/DNA-dependent protein kinase-40 (DFF-40) by cleaving inhibitor of CAD (ICAD)/DFF-45, and then produces DNA ladders, excitotoxins appear to produce DNA ladders through activation of CAD-independent endonucleases as excitotoxins-induced DNA ladders are produced in the absence of caspase-3 activation.83,118 The process of DNA fragmentation has been implicated in chromatin condensation and morphological changes of nucleus in nonneuronal cells,54,255 but its role in excitotoxic neuronal death needs to be resolved. 4.2.3.5

Nuclear Factor Kappa B

The transcription factor nuclear factor kappa B (NF-κB) plays a dynamic role in survival and death of neuronal and nonneuronal cells under physiological and pathological conditions.7 It has been well documented that activation of NF-κB enhances neuronal survival by preventing apoptosis. The activation of NF-κB by cytokines appears to be required for preventing apoptosis of sensory neurons as evidenced by findings that the antiapoptosis action of cytokines disappears in neurons treated with a super-repressor IkappaB-alpha protein or lacking the RelA (p65) subunit of NFκB.163 Inhibition of NF-κB renders various types of cells highly vulnerable to apoptosis.234 In contrast to the antiapoptosis action of NF-κB, controversial results have appeared regarding the causative role of NF-κB to excitotoxicity. Treatment with TNF or C2-ceramide was shown to reduce excitotoxicity and free radical

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neurotoxicity through mechanisms involving activation of NF-κB.71,158 Administration of excitotoxins induces activation of NF-κB in neurons in vitro and in vivo.77,254 A causative activation of NF-κB has been proposed from findings that aspirin or salicylate prevents NMDA neurotoxicity by blocking the activation of NF-κB.77 However, this possibility has been challenged with the inhibitory action of aspirin or salicylate against NMDA-induced activation of SAPK.119 Thus, the exact role of NFκB in excitotoxicity needs to be reexamined turning to pharmacological and genetargeted knockout of NF-κB. 4.2.3.6

Mitochondria

Mitochondria constitute approximately 25% of the cytoplasmic volume and produce cellular energy in the form of ATP via the electron transport and oxidative phosphorylation in most eukaryotic cells. Mitochondria have been recognized as target organelles for regulation and execution of cell death under pathological conditions.18,132 Role of mitochondria in excitotoxicity stems from various observations. First, entry and accumulation of cytoplasmic Ca2+ through ionotropic glutamate receptors result in subsequent accumulation of Ca2+ in mitochondria ([Ca2+]m).182,251 The cytoplasmic Ca2+ is transported into mitochondria through an electrophoretic uniporter whose driving force is generated by the negative membrane potential, ∆ψm.79 Second, mitochondria become depolarized due to the transport of Ca2+ into the matrix and the inhibition of the oxidative phosphorylation.194,247,250 Third, inhibiting mitochondrial Ca2+ uptake reduces Ca2+-mediated glutamate neurotoxicity.228 Finally, increasing mitochondrial membrane and redox potential blocks accumulation in [Ca2+]i and neuronal death following activation of NMDA receptors.213 Excess Ca2+ in mitochondria results in mitochondrial dysfunction and neuronal death in various ways. With accumulation of [Ca2+]m, ATP synthesis in mitochondria is impaired due to the collapse of the mitochondrial oxidative phosphorylation.111,243 ATP depletion will interfere with actions of ATP-dependent Ca2+ pumps, amplify accumulation of [Ca2+]i, and thus enhance the process of excitotoxic neuronal death. Activation of NMDA receptors produces reactive oxygen species in mitochondria primarily through the inhibition of the oxidative phosphorylation and membrane potential.194,213 Ca2+-induced mitochondrial damage causes increased mitochondrial membrane permeability,48 which can result in mitochondrial release of cytotoxic substances such as cytochrome c.145 4.2.3.7

Oncosis vs. Apoptosis

Activation of ionotropic glutamate receptors allows entry of Ca2+ and Na+ through plasma membrane. The massive entry and accumulation of the cations induce secondary influx of Cl– and H2O, resulting in marked swelling of neuronal cell body within a few hours.31 Transmission electron microscopy reveals swelling of cytoplasmic organelles including mitochondria and scattering condensation of nuclear chromatin early in the process of excitotoxicity.83 Plasma membrane and cytoplasmic organelles are disrupted but nuclear membrane remains intact. In contrast to

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apoptosis (shrinkage necrosis), membrane blebbing is not observed in the process of excitotoxicity. The morphological features suggest that neurons undergo oncosis* (swelling necrosis) following superfluous activation of NMDA, AMPA, or kainate receptors. Although DNA ladders, TUNEL-positive neurons, and chromatin condensation visible by DNA-binding fluorescence dyes have been used as evidence of apoptosis in excitotoxicity,3,185 they are all observed in the process of necrotic cell death or oncosis.27,74,84,224 Excitotoxic neuronal oncosis appears to propagate through signal transduction different from oxidative stress and apoptosis as neither antioxidants nor antiapoptosis agents (e.g., inhibitors of macromolecule synthesis or growth factors) prevent excitotoxicity.83,85 4.2.3.8

Limitation of Anti-Excitotoxicity Therapy for Hypoxic-Ischemic Injury

It has been reasoned that blockade of excitotoxicity can be applied to treat fulminant neuronal death appearing in hypoxic-ischemic patients. Therefore, a number of NMDA and AMPA/kainate receptor antagonists have been developed for the prevention of hypoxic-ischemic neuronal death. Several NMDA receptor antagonists, including MK-801 and dextrophan, provide substantial protection against ischemic injuries induced by oxygen-glucose deprivation in vitro or occlusion of middle cerebral artery.70,221 Although NMDA receptor antagonists should be promptly delivered to ischemic patients for appropriate efficacy, most patients are hospitalized after 3 hours from onset of symptoms. The therapeutic potential of NMDA receptor antagonists is further limited by behavioral effects such as psychosis and hyperlocomotion,53,177 less protective effects against global ischemic injuries,17,200 and direct neurotoxicity in several areas of the brain.175 While antagonists (such as NBQX) acting on AMPA and kainate receptors mitigate selective neuronal loss following transient global ischemia in the rat,218 they are partially neuroprotective against focal ischemic insults.16 Co-administration of NMDA and AMPA/kainate antagonists blocks excitotoxic neuronal oncosis but unveils slowly evolving apoptosis following hypoxic-ischemic insults.84 Thus, applying glutamate antagonists to treat hypoxic-ischemic injury should be compromised with the deleterious effects of antagonist itself and appearance of glutamate-independent death pathways (e.g., apoptosis and oxidative stress).

4.3 OXIDATIVE STRESS 4.3.1 BACKGROUND Oxidative stress, the excess accumulation of prooxidants over antioxidants, can damage essential components for cell function and survival. Oxidative stress increases lipid peroxidation, oxidizes DNA and proteins, and can result in dysfunction of Apoptosis was named to mean “shrinkage cell necrosis.” As necrosis has been used often to describe “swelling cell necrosis,” it seems to be more meaningful to use oncosis (derived from onkos, meaning swelling) instead of necrosis. *

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mitochondrial oxidative metabolism,189,237,266 ion homeostasis,131 various enzyme activity6,215 and maintenance of plasma membrane integrity.24 Brain consumes as much as about 20% of O2 in the body. Neurons contain high content of polyunsaturated fatty acids (target molecules of lipid peroxidation) in the plasma membrane, low levels of catalase (an antioxidant enzyme that catalyzes decomposition of H2O2 to O2), and can thus be prone to oxidative stress under certain pathological conditions that disturb the prooxidants-antioxidants balance.

4.3.2 FREE RADICAL PRODUCTION DURING HYPOXIC-ISCHEMIA AND REPERFUSION Energy failure during hypoxic ischemia induces depolarization of plasma membrane that results in activation of voltage-dependent Ca2+ channels and Ca2+-permeable glutamate receptors. Entry and accumulation of Ca2+ in neurons can produce free radicals through activation of prooxidant pathways including phospholipases,6,215 nitric oxide synthase,43 xanthine oxidase,159 and loss of mitochondrial potential.189,266 In addition to Ca2+, transition metals such as Fe2+, Cu2+, and Zn2+ may contribute to generation of free radicals in hypoxic-ischemic injury. Reintroduction of excess oxygen into the ischemic area provides a major source of ROS and thus causes reperfusion injury (Figure 4.2). 4.3.2.1

Free Radical Production in Mitochondria

Mitochondria produce ATP by utilizing about 90% of O2 that is taken up by neurons. During the electron transfer in the inner mitochondrial membrane, electrons

FIGURE 4.2 Routes of ROS production following hypoxic-ischemic brain injury.

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spontaneously leak from the electron transport chain and react with available O2 to produce superoxide. While this superoxide is normally cleared to H2O by superoxide dismutases (e.g., MnSOD in the mitochondria) and glutathione peroxidase in neurons, Ca2+ is accumulated in the cytosol during hypoxic-ischemic injury (see above) and enters into mitochondria, which results in production of mitochondrial free radicals and oxidation of mitochondrial lipid and DNA.49,51,69,89,183,190 Excess Ca2+ in the mitochondria interrupts the electron transport chain and collapses the mitochondrial membrane potential.194 Therefore, free electrons are accumulated in the mitochondria, which react with oxygen supplied after reperfusion and cause production of superoxide. The superoxide is further processed to produce the hydroxyl radical by a Fenton reaction or peroxynitrite by reacting with nitric oxide. Reactive oxygen and nitrogen species (ROS and RNS) also inhibit the electron transport chain in the mitochondria and amplify generation of mitochondrial free radicals.23,189,210,237,266 4.3.2.2

Activation of Xanthine Oxidase by CalciumActivated Proteases

Administration of glutamate to central neurons activates xanthine oxidase through activation of NMDA receptors and Ca2+-dependent proteases.5 Accumulated [Ca2+]i induces activation of the neutral protease calpain that results in conversion of xanthine dehydrogenase into xanthine oxidase. Xanthine oxidase catalyzes oxidation of xanthine and hypoxanthine into uric acid, producing superoxide as a by-product.159 4.3.2.3

ROS Production by Transition Metals

Copper and iron are abundant (~0.1–0.5 mM) in brain and have been implicated in generation of ROS in various neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease.45,262 These transition metals mediate formation of hydroxyl radical through the iron-catalyzed or copper-catalyzed Haber-Weiss reactions.86 During transient focal ischemia, Fe2+ is released from iron-binding proteins and can convert hydrogen peroxide to hydroxyl radical.124,264 The transition metal Zn2+ mediates death of neuronal and nonneuronal cells in hypoxic ischemia, epilepsy, and trauma.36,123,138,233 Zn2+ is stored in the presynaptic vescicles of glutamatergic neurons, released with glutamate in an activity-dependent manner, and translocated into adjacent neurons.60 The Zn2+ translocation was observed in degenerating neurons after transient forebrain ischemia.123,233 This ischemic neuronal death was prevented by blockade of Zn2+ translocation with Ca-EDTA or overexpression of metallothionein-1, a Zn2+-binding protein.240 Zn2+ ions enter into target cells through voltage-gated calcium channels (VGCC), NMDA or AMPA/kainate glutamate receptors permeable to Ca2+, Na+/Ca2+ exchanger, or Zn2+ transporter.38,61,120,211,212 Entry and accumulation of Zn2+ into neurons result in transient generation of reactive oxygen species that mediate latent neuronal death.114,116,212 Zn2+ produces ROS possibly through activation of cyclooxygenases and PKC.115,174

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4.3.2.4

Reactive Nitrogen Radicals

Besides roles of nitric oxide (NO or nitrogen monoxide) as endothelial-derived relaxing factor in blood vessels, NO plays a role in the pathogenesis of hypoxic-ischemic injury. Three isoforms of nitric oxide synthase (NOS) catalyze the conversion of arginine to NO and citrulline. NO may exert neurotoxicity or neuroprotection depending upon the isoforms and localization of activated NOS. Ca2+/calmodulin-dependent NOSs such as neuronal NOS (nNOS) and endothelial NOS (eNOS) are activated by raised [Ca2+]i.117 Expression of inducible NOS (iNOS) is increased in astrocytes and microglia by various cytokines including interleukin1, interleukin-2, interferon-γ, or tumor necrosis factor.160,167 nNOS is expressed primarily in NADPH-diaphorase-positive neurons and activated by raised Ca2+ during hypoxic-ischemic injury.143,205 Inhibiting nNOS reduced NMDA-mediated excitotoxicity and focal cerebral ischemia.20,44,154,168 NO derived from neurons can diffuse freely across membranes and cause degeneration of surrounding neurons. NO may react with superoxide to produce perxoynitrite (ONOO–), another highly reactive free radical, that appears to mediate toxicity associated with ischemia and reperfusion.126,187-189 Peroxinitrite is ultimately converted to hydroxyl radical and nitrogen dioxide. These free radicals cause tissue damage by nitration of DNA and proteins as well as oxidization of lipids, DNA, and proteins.10,261 In addition, NO may produce neuronal death by activating poly (ADP-ribose) synthetase and thus depleting beta-nicotinamide adenine dinucleotide265 and inhibiting mitochondrial ATP synthesis as an electron acceptor.15 NO can be produced in the endothelial cells of blood vessels through activation of eNOS, released, and influences hypoxic-ischemic brain injury. Endothelial NO mediates vasodilation and prevents thrombosis. Thus, endothelial NO appears to act as a neuroprotective signal against hypoxic ischemia. This has been supported by recent findings that inhibiting eNOS worsens neuronal death after hypoxic ischemia.40,217,227 Finally, the expression of iNOS is increased in glial cells and neutrophils over several days after reperfusion, which provides a substantial amount of NO in the ischemic area.100 Induction of iNOS potentiates neuronal death following deprivation of oxygen and glucose while inhibition of iNOS reduces the infarct volume after transient cerebral ischemia.99 Increased levels and activation of iNOS appear to contribute to somewhat delayed neurotoxicity following hypoxic-ischemic injury. 4.3.2.5

Metabolism of Arachidonic Acid

Glutamate or hypoxic ischemia produces arachidonic acid through activation of PLA2 (see above). Arachidonic acid is converted into the eicosanoids with concomitant production of superoxide by cyclooxygenases and 5-lipooxygenase.125,130 Increased expression and activation of COX-2 was observed in neurons and glial cells in rodent and human ischemic brain37,184,203 and shown to depend upon activation of NMDA receptors in neurons.165 COX-2 inhibition attenuates NMDA-mediated excitotoxicity and hypoxic-ischemic injury.92,98 Recently, activation of constitutive

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COX-2 was shown to mediate Zn2+-induced free radical production and neurotoxicity in cortical cell cultures.115 The exact role of activated COX-2 in neurons and nonneuronal cells needs to be determined to correlate with free radical production and neuronal death following hypoxic ischemia. 4.3.2.6

Apoptosis vs. Oncosis

Accumulated ROS in cells are expected to randomly attack DNA, lipid, and proteins and therefore to produce cell death in a way different from apoptosis that is propagated through a cascade of well-orchestrated molecular events. However, several lines of evidence suggest that ROS may act as mediators of apoptosis in nonneuronal cells. First, ROS are produced in the process of apoptotic cell death.14,93,133,186 Second, ROS scavengers prevent apoptosis.76,93,134,239 Third, exposure to prooxidants can induce apoptosis.193,252 The causative role of ROS for apoptosis has been reported in various populations of neurons,193,252 mostly turning to DNA ladders, DNA-binding fluorescence dyes, and TUNEL method that do not differentiate apoptosis from oncosis (see above). We also observed that cortical cell cultures exposed to prooxidants such as Fe2+ or buthionine sulfoximine revealed DNA ladders, condensation of nuclear chromatin, and TUNEL-positive neurons.202 However, ultrastructural analysis of free radical neurotoxicity demonstrated the occurrence of typical oncosis in most neurons evident by swelling of cell body and mitochondria, scattering condensation of nuclear chromatin, and fenestration of plasma membrane prior to nuclear membrane.202 4.3.2.7

Limitation of Antioxidant Therapy for HypoxicIschemic Injury

It has been well documented that free radicals (e.g., ROS and RNS) contribute to hypoxic-ischemic neuronal death. Pharmacological or genetic intervention of ROS and RNS has been neuroprotective against hypoxic-ischemic injury as discussed above. However, neither tirilazad mesylate, a lipid peroxidation inhibitor, nor ebselen, a seleno-organic compound with antioxidant activity, showed therapeutic efficacy in primary outcome measure of stroke patients. This lack of efficacy may be attributed to inappropriate administration of drugs that is insufficient to block ROS production following hypoxic ischemia. In addition, blockade of ROS neurotoxicity may result in appearance of the other death pathways, excitotoxicity, and apoptosis.

4.4 APOPTOSIS 4.4.1 BACKGROUND Kerr et al. reported electron microscopic features of shrinkage necrosis or apoptosis that would play a role in the regulation of cell number under physiological and pathological conditions.108 Apoptotic cells were accompanied by condensation of nucleus and cytoplasm, nuclear fragmentation, and aggregated condensation of nuclear chromatin. Interestingly, apoptosis is prevented by inhibitors of protein and mRNA

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synthesis and thus appears to require the expression and activation of death-regulating proteins in neurons and nonneuronal cells.152,257 Current understanding of proapoptosis and antiapoptosis proteins stems to a great extent from genetic study of developmentally occurring cell death in C. elegans that is executed by activation of the caspase homologue Ced-3, the Apaf-1 homologue Ced-4, and the BH3 domaincontaining proapoptotic member of the Bcl-2 family Egl-1 and prevented by the structural and functional homologue of Bcl-2, Ced-9.19 Morphological and molecular features of apoptosis have been reported in the nervous system during development and various neurological diseases. Apoptosis has been recognized as additional pattern of hypoxic-ischemic neuronal death. Various proapoptosis proteins are activated in ischemic brain areas and inhibitors of protein synthesis attenuate hypoxicischemic neuronal death (Figure 4.3). Inhibitors of caspases or overexpression of Bcl-2 attenuates neuronal death following focal and global ischemia.

4.4.2 INITIATION OF ISCHEMIC APOPTOSIS Although evidence has been accumulated demonstrating occurrence of neuronal apoptosis after hypoxic-ischemic insults, it remains to be determined how interruption of blood supply to brain triggers neuronal cell apoptosis. 4.4.2.1

pH

Extracellular and intracellular acidosis occurs following hypoxic ischemia and has been considered as a primary cause of cell death.220 The possibility has been raised

FIGURE 4.3 Multiple pathways for hypoxic-ischemic neuronal apoptosis.

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that intracellular acidification comprises an upstream event in the process of apoptosis. The proton gradient across the mitochondrial inner membrane is collapsed in neurons deficient in oxygen and glucose, which can result in a complete loss of the mitochondrial membrane potential and render the outer mitochondrial membrane permeable to cytochrome c. The released cytochrome c in the cytosol binds to Apaf1, activates procaspase-9, and leads to activation of downstream caspases such as caspase-3.42 Hypoxic ischemia or the apoptosis-inducing protein kinase inhibitor staurosporine causes acidification in the cytosol that promotes activation of cytochrome c-dependent caspases and acidic endonucleases.156 The intracellular acidification was shown to cause apoptosis in cultured neurons and haematopoietic cells.47,65 Growth factors and the antiapoptosis members of Bcl-2 prevent the cytosolic acidification, which likely results in attenuation of caspase activation and apoptosis.65,109,195 It is possible that acidosis may mediate neuronal apoptosis following hypoxic-ischemic insults. 4.4.2.2

Calcium Imbalance

Hypoxic-ischemic insults to brain can cause excess accumulation of calcium in neurons by enhancing the entry and release of Ca2+ and interfering with the ATP-dependent extrusion and sequestration of Ca2+ (see above). While disrupted ion homeostasis produces neuronal cell oncosis as shown in excitotoxicity, selective accumulation of intracellular Ca2+ can cause neuronal apoptosis. Raised [Ca2+]i by calcium ionophores causes neurite damages, depriving cell bodies of target-derived neurotrophic factors, and then produces hallmarks of apoptosis such as DNA ladders, shriveled cell body, aggregated and condensed nuclear chromatin, and sensitivity to various antiapoptosis agents.81 When neurons are exposed to NMDA in low levels of extracellular Na+ and thus become more permeable to Ca2+, they reveal apoptotic features.263 Raised [Ca2+]i can trigger apoptosis through mechanisms involving activation of calpain, caspases, PLA2, and endonucleases that is observed following focal and global cerebral ischemia.170,171,198,199 4.4.2.3

Fas Receptor

The death receptor Fas (CD95 or APO-1) belongs to the tumor necrosis factor (TNF) receptor superfamily and plays a role in death and survival as well as proliferation and differentiation.169,229 Fas ligand (Fas-L) activates Fas in autocrine or paracrine fashion, which causes trimerization of Fas with Fas-associating protein with death domain (FADD) and procaspase-8. Activation of Fas has been demonstrated as a necessary step for apoptosis of neuronal cells deprived of trophic factors.28,137,192 Expression of Fas and Fas-L is increased in ischemic brain area following hypoxicischemic injury.57,91,151,155,157 The recruitment of FADD and procaspase-8 to the Fas receptor is observed in the cerebral cortex and hippocampal formation subjected to focal cerebral ischemia and global forebrain ischemia, respectively.106,253 This implies that the Fas-mediated death pathway may underlie hypoxic-ischemic neuronal apoptosis. In support of this, the infarct size following occlusion of middle cerebral artery

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is reduced in lpr mice deficient in Fas.151 Further study will be needed to understand mechanisms for expression of Fas and Fas-L and to correlate the Fas pathway and neuronal apoptosis following hypoxic-ischemic injury.

4.4.3 PROPAGATION OF ISCHEMIC NEURONAL APOPTOSIS The intracellular signaling pathway leading to apoptotic cell death has been extensively studied in various types of cell. In particular, a family of mitogen-activated protein kinases (MAPKs), caspases, and the proapoptotic family of Bcl-2 appear to mediate the execution of neuronal apoptosis. 4.4.3.1

MAPKs

MAPKs, p42/p44 extracellular signal-regulated kinases (ERK 1 and 2), c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinase, play a role in development and survival of neurons and nonneuronal cells.102 Among these, JNK and p38 have been established as common mediators of cell death.164 In brain, JNK and p38 are activated to execute apoptotic death of neuronal cells following trophic factor deprivation or exposure to calyculin A, haloperidol or NO donors.67,119,135,150,173,258 Activation of JNK and p38 is also observed in the vulnerable brain areas after hypoxic-ischemic injury.91,103,178 Administration of SB203580, a p38 inhibitor, reduces the infarct size after transient focal cerebral ischemia and the delayed neuronal death in the CA1 sector after global forebrain ischemia,8,232 suggesting that p38 and possibly JNK may mediate the execution of hypoxic-ischemic neuronal apoptosis. Activated JNK and p38 may execute apoptosis by preventing the antiapoptosis actions of Bcl-2 and Bcl-XL, activating the translocation of the cytosolic Bax into mitochondria, and thus activating the mitochondrial apoptosis signals such as cytochrome c release.67,110,226,238 4.4.3.2

The Proapoptotic Family of Bcl-2

The physiological and pathological roles of the Bcl-2 family of proteins have been extensively reviewed.26,161 In general, physical balance between antiapoptotic and proapoptotic members of the Bcl-2 family appears to determine death and survival of developing and mature cells. The proapoptotic Bcl-2 family includes Bax, Bcl-Xs, Bak, Bad, and Bid and participates in the process of hypoxic-ischemic neuronal death. The expression of Bax is increased selectively in neurons undergoing apoptosis after global forebrain ischemia and focal cerebral ischemia.104,128 The cerebral infarct and the delayed neuronal death in the hippocampus are significantly reduced in mice and gerbil overexpressing Bcl-2, respectively.4,153,222 Hypoxic ischemia can activate the proapoptotic Bcl-2 family possibly through two separate routes. The intracellular acidosis can induce pH-sensitive conformational change of Bax and then the translocation of Bax into mitochondria.109 Bax is oligomerized, inserted into the outer membrane of mitochondria, and in turn shown to induce cytochrome c release, caspase activation, and apoptosis.78,96 Alternatively, raised [Ca2+]i induces BAD dephosphorylation and dissociation from 14-3-3 in the cytosol through activation of the

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protein phosphatase calcineurin, which results in BAD translocation to mitochondria and heterodimerization with Bcl-XL.244 4.4.3.3

Caspases

The caspases, a family of cysteine-dependent aspartate-directed proteases, mediate the propagation and execution of apoptosis and can be classified into “initiator caspases” and “effector caspases.”52 The initiator caspase, caspase-9, is proteolytically activated by apaf-1, a cytoplasmic protein homologous to ced-4, and cytochrome c. The latter is located in the intermembrane space of the mitochondria and released into the cytoplasm by the proapoptotic family of Bcl-2 (e.g., Bax) that is transported from the cytoplasm into the mitochondria in the early phase of apoptosis. The mitochondria Bax-like proteins may rupture the outer mitochondrial membrane or induce the formation of a channel complex for cytochrome c release.46 Another initiator caspase, caspase-8, is activated through the interaction with the Fas receptor and the FADD adapter. Activation of caspase-9 and caspase-8 is observed in the vulnerable brain areas prior to appearance of neuronal apoptosis following global and focal hypoxic-ischemic injury in brain.127,242,253 Activated caspase-8 and caspase-9 can induce activation of downstream caspases such as caspase-3, 6, and 7 that can cleave a number of proteins essential for structure, signal transduction, and cell cycle and result in the termination of the overall apoptosis process. Caspase-3-mediated neuronal death has been reported following hypoxic ischemia in vitro and in vivo.29,63,73,170,231 4.4.3.4

Reevaluation of Ischemic Neuronal Apoptosis

Wyllie reported that the genomic DNA was cleaved into oligonucleosomal size in the course of apoptosis.256 This internucleosomal DNA fragmentation (or DNA ladders) has been shown using the agarose gel electrophoresis of DNA and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method and widely accepted to define apoptosis.66,256 In addition, apoptotic condensation of the nuclear chromatin has been demonstrated using membrane-permeable fluorochromes (e.g., Hoechst dye and propidium iodide) binding to DNA.41,172 As DNA ladders, TUNEL-positive neurons, and the chromatin condensation were observed in the process of neuronal death in the hypoxic-ischemic brain areas, apoptosis as well as oncosis has been considered an additional type of hypoxic-ischemic neuronal death.140,142,147 However, similar patterns of DNA damage are observed in the process of necrotic cell, suggesting that analysis of DNA damage is not sufficient to define apoptosis.55,74,224 The electron microscopic examination reveals that neurons primarily undergo oncosis evident by swelling of cell body and mitochondria, translucent cytoplasm, scattering condensation of the nuclear chromatin, and fenestration of the plasma membrane with the preserved nuclear membrane following occlusion of the middle cerebral artery (Figure 4.4). The similar morphological patterns of degenerating neurons were reported in the hippocampal region following global forebrain ischemia.35,141 Taken together, hypoxic-ischemic insults to brain appear to produce neuronal cell oncosis possibly through mechanisms involving

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FIGURE 4.4 Transmission electron micrographs of a normal neuron (A), a typical apoptotic neuron in the dentate gyrus 1 d after the intraventricular injection of colchicines (B), and neurons showing necrotic features 1 day after occlusion of middle cerebral artery for 60 min (C) and (D). Abbreviations: N = nucleus; PM = plasma membrane; Mi = mitochondria. Scale bar, 2.5 mm.

excitotoxicity and oxidative stress (Table 4.1). While some biochemical and molecular events underlying apoptosis have been observed following focal cerebral ischemia and global forebrain ischemia, these may be overridden by fulminant necrotic pathway. The apoptosis component of hypoxic-ischemic neuronal death may be unveiled with blockade of excitotoxicity and oxidative stress.84

4.5 MAXIMIZATION FOR PREVENTION OF HYPOXICISCHEMIC NEURONAL DEATH While antagonizing excitotoxicity does reduce neuronal death after hypoxic-ischemic injury, its beneficial effect is confronted by unwanted observations. Systemic injections of NMDA antagonists alone produce neuronal vacuolization and death in adult rats.59 Neuronal death by NMDA antagonists reveals hallmarks of apoptosis such as shrinkage of cell body, aggregated condensation of nuclear chromatin, and sensitivity to inhibitors of protein synthesis.59,97,101,235 In addition, prolonged deprivation of oxygen and glucose undergoes slowly evolving apoptosis through activation of caspases under blockade of excitotoxicity.73,84 Accordingly, combined treatment

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with an NMDA antagonist and a caspase inhibitor results in synergetic neuroprotection against hypoxic-ischemic injury in vitro and in vivo.2,146,208 Neurotrophins enhance neuronal survival by preventing programmed cell death or apoptosis of developing neurons.42,223 The neuroprotective effects of neurotrophins have been demonstrated under various pathological conditions. For example, neurotrophins protect various populations of neurons from axotomy.30,34,62,90,149,162,260 Neurotrophins can attenuate neuronal death following global or focal cerebral ischemia.9,25,219 Besides the beneficial effects, neurotrophins appear to exacerbate neuronal injury under certain circumstances. BDNF, NT-3, or NT-4/5 renders neurons highly vulnerable to deprivation of oxygen and glucose, possibly by enhancing Ca2+ influx through NMDA glutamate receptors and thus oncosis.58,122 BDNF or NGF potentiates neuronal cell oncosis by reactive oxygen species or nitric oxide.82,105,116,179 Taken together, the neuroprotective action of neurotrophins appears to be limited to apoptosis and should be compromised with the potentiation effects of NMDA or free radical neurotoxicity. Evidence is being accumulated demonstrating that excitotoxicity, oxidative stress, and apoptosis contribute to hypoxic-ischemic neuronal death through mutually exclusive pathways. To date, glutamate antagonists, antioxidants, or antiapoptosis agents such as growth factors have been examined to treat hypoxic-ischemic brain injury. However, therapeutic efficacy of neuroprotective drugs against either excitotoxicity, oxidative stress, or apoptosis can be confronted with deleterious effects on the other death pathways that also participate in the process of hypoxic-ischemic neuronal death. In the future, appropriate combination of glutamate antagonists, antioxidants, and antiapoptosis agents should be applied for maximal neuroprotection against hypoxic-ischemic injury.

4.6 CONCLUSION Excitotoxicity, oxidative stress, and apoptosis comprise major routes of hypoxic-ischemic neuronal death. Each route is likely activated and propagated through selective transmembrane and intracellular signaling systems. Further understanding of distinct and integrated mechanisms leading to three death routes is warranted for efficient and secure treatment of hypoxic-ischemic brain injury.

4.7 ACKNOWLEDGMENTS This work was supported by a National Research Laboratory grant from the Korean Ministry of Science and Technology (BJG) and in part by the Korea Science and Engineering Foundation (KOSEF) through the Brain Disease Research Center at Ajou University (BJG).

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TABLE 4.1 Comparison of Apoptosis, Oncosis, and Hypoxic-Ischemic Neuronal Death Apoptosis

Oncosis

Ischemia

Cell body swelling

+

+

Scattered condensation of nuclear chromatin

+

+

Dilation of mitochondria

+

+

Early loss of plasma membrane

+

+

Potentiation by trophic factors

+

+

Prevention by antioxidants

+

+

Prevention by glutamate antagonists

+

+

Criteria for Oncosis

Criteria for Apoptosis Membrane blebbing

+

Cell body shrinkage

+

Aggregation and condensation of nuclear chromatin

+

Early loss of nuclear membrane

+

DNA ladder

+

+

+

TUNEL staining

+

+

+

Cytochrome c release

+

+

Prevention by caspase inhibitors

+

+

Prevention by trophic factors

+

+

Prevention by protein synthesis inhibitors

+

+

Prevention by bcl-2

+

+

+

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5

Intracellular Ca2+ Signals Underlying Rapid and Delayed Excitotoxicity in Mature CNS John A. Connor and C. William R. Shuttleworth

CONTENTS 5.1 5.2 5.3

Introduction Rapid Neuronal Death and Secondary Ca2+ Responses Delayed Neuronal Death Hypotheses 5.3.1 Delayed Increases in Intracellular Ca2+ 5.3.2 Apoptosis 5.3.3 Chronic Depression of Intracellular Ca2+ Signaling 5.4 Conclusion 5.5 Acknowledgments References

5.1 INTRODUCTION At the outset it is acknowledged that this review accepts the common view that many forms of extraordinary CNS neuronal death, from transient ischemia, mild mechanical or pharmacological trauma, result from variants of excitatory amino acid (EAA) toxic causality. Specifically these conditions may arise from local and transient ischemic events or more widespread occurrences such as moderate head trauma or brief cardiac arrest. This set of conditions comprises a group that inflicts great social cost; and, in cases where there is an extended survival time course of neurons, the possibility of effective rescue exists. It has often been assumed, or implied, that a “one size fits all” model applies to all neuronal death ascribed to EAA toxicity. However, the present review emphasizes the view that immediate (within a few hours) and delayed (1–4 days) neuronal death should be viewed as having different, but perhaps, sequential etiologies. Olney and colleagues55,68 are primarily responsible for formulating the initial model of cell damage following excessive EAA

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stimulation, and the work has spawned a great diversity of detailed models of the cascades leading to cell death. Cellular mechanisms mediating rapid neuron death have been most extensively studied in dispersed monolayer, or in organotypic tissue culture preparations and to a more limited extent in acute slices. In all these experimental models, a large increase in intracellular Ca2+ is a primary event initiating EAA toxicity. The findings from these studies have been ably and exhaustively reviewed in a number of places,12,45,46 and only those findings considered relevant to the reviewers’ thesis will be incorporated here.

5.2 RAPID NEURONAL DEATH AND SECONDARY CA2+ RESPONSES Is the initial Ca2+ transient that EEAs elicit sufficient to activate death cascades, or are there intervening mechanisms, possibly blockable or reversible, that are normally interposed? It is tempting to suppose that the former is the case since this early Ca2+ increase has been thoroughly documented over many years;47 however, there may be much more to the story. Primary Ca2+ responses to a short (1–10 sec) burst of EAA stimulation last no longer than a few seconds after the EAA is removed. Protocols in which glutamate is applied for many minutes will not be considered in detail here. In addition to these well-described primary Ca2+ responses, it has become apparent that brief EAA exposure can also lead to Ca2+ rises that persist for many minutes following agonist removal. This was first demonstrated in 1988 with a report from the author’s lab using acutely isolated neurons from CA1 hippocampus. Initial responses to the applied EAA could be either recovery completely with no longer term effect on intracellular Ca2+, or a short-lasting recovery interrupted by an EEA-independent rise in Ca2+ that persisted for many minutes. This increase often resulted in rapid cell death.16,84 The rise in Ca2+ that occurred after washout of the EAA was termed a secondary response. The isolation procedures for fully differentiated neurons, pioneered by Kay and Wong,39 produced specimens with a considerable portion of the apical dendritic tree intact. This feature was exploited to demonstrated that the secondary response was most easily initiated by repeated 1-sec applications of glutamate (from a microelectrode) to a portion of the apical dendrite 10s of µm from the soma. The immediate (primary) response to the applications was a Ca2+ elevation throughout the neurons, due to voltage and receptor gated Ca2+ influx and possibly intracellular release. Responses to a first glutamate application, or to multiple applications in sphingosine pretreated neurons (see below) recovered with a t1/2 of ~10 sec. In non-sphingosine-treated neurons, a second or third application produced primary responses that recovered everywhere in the cell except near the site of the glutamate application. Under these conditions, Ca2+ levels only partially recovered at this location, and then slowly increased (secondary response). These high Ca2+ levels were locally maintained for periods of minutes without significant Ca2+ increases in the soma or proximal part of the dendrite (see Figure 5.1). The region of high Ca2+ then slowly spread toward the soma until the whole cell was involved, a process that

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FIGURE 5.1 Slow propagation of the secondary Ca2+ response in an acutely isolated CA1 neuron. A: Glutamate was delivered via iontophoresis from a microelectrode (seen at lower right). The neuron was exposed to three glutamate stimuli (1-s duration) at 3-min intervals. During the first two stimuli, Ca2+ levels rose throughout the cell but recovered to resting levels within 30 to 40 sec. Following the third stimulus, Ca2+ levels in the distal dendrite showed a partial, transient recovery, but then slowly increased throughout the neuron. B: Spatial profiles illustrating Ca2+ concentrations at different distances from the soma (regions indicated by boxes in A) showing the slow spread of the secondary response along the apical dendrite and into the soma. Indicated times on the profile are measured following the third glutamate stimulus.

required from 5 to 20 minutes. Pretreatment of the neurons with sphingosine, a protein kinase C and Ca2+-calmodulin kinase inhibitor, prevented the induction of the secondary response. The primary Ca2+ responses to EAA applications were unaffected, as determined by fura-2 ratio measurements. Other studies were subsequently reported from different laboratories showing that strong or repeated glutamate stimulation triggered secondary Ca2+ increases or Ca2+ current that persisted beyond the actual exposure to agonist.51,66 In one study done in tissue culture, the induction of secondary responses was prevented by induction of the response by sphingosine.85 The question of whether the secondary response was only generated in neurons previously stressed by isolation, or in tissue culture, was addressed more recently by

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FIGURE 5.2 Comparison of Ca2+ responses for two sequential glutamate stimulus episodes in a single CA1 neuron microinjected with fura-2. Left: Ca2+ transients recorded at five locations (see inset) in a previously unstimulated neuron. Small horizontal bars mark iontophoretic glutamate applications. Proximal locations (1, 2, and 3) show more rapid recovery than distal regions (4 and 5). Recovery was complete (>90%) in all regions 1 min after stimulus termination. Right: Responses to a second identical set of stimuli show a greatly extended time course as compared to the first. Ca2+ levels in the most distal dendrite (5) now remain elevated for 6 to 7 min after stimulus termination. Recovery in the soma is also slowed, possibly reflecting diffusion of Ca2+ from the dendrite and persisting membrane depolarization (see text). (Reprinted with permission from Connor, J.A. and Cormier, R.J. (2000), Cumulative effects of glutamate microstimulation on Ca2+ responses of CA1 hippocampal pyramidal neurons in slice, J. Neurophysiol., 83:90-98.)

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studying responses of mature CA1 neurons in brain slice.14 Here glutamate was delivered to the apical dendritic tree by iontophoreses from a microelectrode. One cycle of stimulation consisted of five10-sec applications repeated at 1-minute intervals. In this protocol glutamate could be delivered to only a restricted portion of the slice, ~100 µm radius, around the microelectrode. Surprisingly this protocol produced responses that were very similar to those in the acutely isolated neurons, indicating that the stress of isolation was not a major factor in response initiation. After an initial glutamate stimulus cycle, Ca2+ levels were restored within 20–30 sec, but following 2 or 3 identical stimuli, Ca2+ levels in the dendrites nearest the site of glutamate ejection remained at elevated Ca2+ levels. This behavior is illustrated in Figure 5.2 where a secondary response was initiated during the second stimulus cycle. Although there are significant differences in the time course of Ca2+ responses in the soma and proximal apical dendrite between the 2 episodes (increased maximum amplitude and slowing of recovery, especially after the fifth pulse), the greatest difference is seen in the more distal apical dendrites. Instead of following the soma/proximal dendrite response, the more distal dendrite Ca2+ levels plateau or “hang” for several minutes after termination of the last stimulus pulse, before recovering to prestimulus levels in a relatively rapid fashion. Further stimulation with glutamate resulted in the high Ca2+ front invading the proximal dendrite and then the soma followed by cell deterioration. Thus the response was very similar to events observed in isolated neurons. A striking example of the involvement of the secondary Ca2+ response in EAA triggered neuronal death has recently been demonstrated in a mouse model of kainic acid (KA) toxicity.73 Experiments exploited the very different sensitivity of two inbred strains to KA excitotoxicity. Some inbred strains (e.g., C57Bl/10, FVB/N), are well suited for studies of excitotoxicity, and behave in many respects like rat, in that systemic kainate exposure results in extensive pyramidal neuron death in the hippocampus. Unlike rat, in which CA3 neurons are most strongly affected, both CA3 and CA1 pyramidal neurons are killed in vulnerable mice. In contrast, other strains (eg C57Bl/6) are remarkably resistant to these same excitotoxic insults, and despite experiencing similar seizure activity, show virtually no pyramidal neuron death.70,78 Experiments employing administration of KA to acute slices revealed major differences in dendritic Ca2+ responses elicited by KA (Figure 5.3). (See color insert following page 114.)During the exposure period to KA there were large Ca2+ increases in the distal dendrites of neurons from the vulnerable strain but much smaller increases in the analogous regions in neurons from the resistant strain. Soma and proximal dendritic Ca2+ changes were similar in neurons of the two strains. This difference was also reflected in the Ca2+ changes evoked by Schaffer collateral stimulation.73 The greatest difference was expressed after washout of KA as illustrated in the rightmost panels of Figure 5.3A. Complete Ca2+ recovery was exhibited in the resistant strain while one of the dendrites from the vulnerable strain remained “hanging” at a high level (~1500 nM measured by bis-fura-2 ratio). This Ca2+ distribution was not stationary. Figure 5.3B follows the development of the secondary Ca2+ response in the same neuron as it propagated and engulfed the entire neuron over a 25-min period following KA washout. It can be seen that the propagation occurred both toward the soma and away from the soma, as branch-points with uninvolved dendrites were reached. The hallmark of this

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FIGURE 5.3 (See color Figure 5.3.) Initial and secondary Ca2+ responses to KA superfusion (10 µM, 10 min) in CA1 pyramidal neurons from Bl/6J (KA resistant) and Bl/10J (vulnerable) mice. Black and white images (upper left panels), show bisFura-2 fluorescence excited at 380 nm. Panels a,a’–h,h’ show color-coded intracellular Ca2+ levels (scale bar at right) with uniform resting Ca2+ in panels a,a’. Peak of the response (panel c,c’) occurred ~ 4.5 min after onset of KA exposure with strong increases in Ca2+ in the proximal and distal apical dendrites. Panels c,c’ show Ca2+ levels 15 s after the peak response. After reexposure to normal saline (panel d,d’), Ca2+ returned to resting levels throughout the Bl/6J neuron and in the Bl/10J neuron except in a portion of the apical dendritic tree (arrow in panel d’). After KA washout, Ca2+ levels remained very high in this restricted dendritic region (panel e’). Part B shows the slow propagation of the secondary response throughout the neuron. Note calibration of the color bar is different in A & B to optimize display contrast. Scale bar 50 µm. (Reprinted with permission from Shuttleworth, C.W.R. and Connor, J.A,. Strain dependent differences in calcium signaling predict excitotoxicity in murine hippocampal neurons, J. Neuroscience, 21, 4225–4236, 2001.)

slowly propagating secondary Ca2+ response in the vulnerable strain neurons was its initiation in the smaller dendrites with subsequent, very slow propagation throughout the neuron. During the initial and propagating phases of the response, very steep Ca2+ gradients existed within the dendritic tree (Figure 5.4). The midpoint of pooled data was approximately 1.5 µM, and it has been proposed that this is near the critical level of Ca2+ for the response to be initiated. Although other sources of Ca2+ may be contributing to the response, the primary factor supporting the elevated levels is Ca2+ influx across the plasma membrane. Figure 5.5 illustrates the rapid decrease of intracellular Ca2+ levels when the dendritic tree at the advancing front was superfused with Ca2+-free ACSF (containing 10 mM BAPTA), delivered from a micropipette positioned adjacent to the neuron. After a brief exposure to Ca2+-free solution, the superfusion flow was stopped and normal extracellular Ca2+ was restored by diffusion from the surrounding medium, the

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intracellular Ca2+ levels climbed to their preceding values. The rapidity of the recovery during the zero-Ca2+ superfusion suggests that energy-dependent Ca2+ transport is still competent near the advancing Ca2+ front. If this is the case, then the advancing front of high Ca2+ is produced by increases in Ca2+ permeability of the plasmalemma that allow Ca2+ to enter at a high enough rate to overwhelm the normal uptake and export mechanisms. Sooner or later, though, one would expect ATP levels to be depleted; and indeed, if the response is allowed to invade the whole neuron, as in Figure 5.3, it requires many minutes for Ca2+ levels to be restored in Ca-free external saline. It has become clear from work in a number of laboratories that Ca2+ levels in small spiny dendrites reach very high levels during normal electrical stimulation, e.g., spiny dendrite levels have been shown to reach 40–60 mM during 1 sec tetani in rat.62 More recent work using 2-photon excitation of low affinity indicators in small dendrites and dendritic spines is also consistent with micromolar levels of Ca2+ being reached.29,86 These rapidly decaying events are reproducible, and in general do not lead to secondary responses or overt cell damage. Taken together, these observations suggest that the time course of Ca2+ transients may be a critical determinant in separating normal Ca2+-dependent physiology from pathological cascades. Since recovering dendritic secondary responses as long as 300–400 sec were encountered, the safety factor for this separation could be well over 100. Sustained Ca2+ elevations could activate a number of targets that lead to spreading secondary responses. These cytoplasmic Ca2+ concentrations are in the range that could activate calpain, a Ca2+-dependent protease that has been shown to contribute to KA-stimulated excitotoxicity in cultured neurons4,5 and may contribute to dendritic remodeling after excitotoxic injury.23 A progressive modification of

FIGURE 5.4 Pooled data illustrating the Ca2+ concentration gradient near the front of the propagating secondary response. Data were derived from regions 50–100 µm from the cell soma after secondary responses were initiated in distal dendrites (n = 9 neurons). (Reprinted with permission from Shuttleworth, C.W.R. and Connor, J.A., Strain dependent differences in calcium signaling predict excitotoxicity in murine hippocampal neurons, J. Neurosci., 21, 4225–4236, 2001.)

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FIGURE 5.5 Reduction of extracellular Ca2+ collapses the Ca2+ gradient of the secondary response. Three successive applications of BAPTA/zero Ca2+ solution (arrows, pressure ejection from a small pipette, 15-µm tip diameter) were applied during the propagation of the secondary response toward the soma. Dendrite Ca2+, measured approximately 30 µm from the soma (open circles), dropped rapidly during the application period, reaching almost prestimulation levels. Upon termination of the zero Ca2+ solution flow, normal extracellular Ca2+ was restored from the bath and intracellular Ca2+ levels rapidly assumed their previous values. As a consequence of the proximity of the secondary response, the soma Ca2+ level (filled circles) was also elevated and showed reductions during the zero Ca2+ application. (Reprinted with permission from Shuttleworth, C.W.R. and Connor, J.A., Strain dependent differences in calcium signaling predict excitotoxicity in murine hippocampal neurons, J. Neuroscience., 21, 4225–4236, 2001.)

voltage-dependent Ca2+ channels following calpain activation28 could provide substantially increased Ca2+ influx, as could expression of a nonselective inward current as has previously been described in isolated neurons following prolonged exposure to NMDA or glutamate.9 The effects of secondary Ca2+ responses on dendritic mitochondrial function are not yet known, but because of its extended duration it is probable that secondary Ca2+ responses would overload mitochondria in the affected areas and mitochondrial dysfunction that is associated with excitotoxic cell death would result.79,67,83 It is proposed here that the secondary or reactive Ca2+ responses are a critical element in neuronal damage following short-term exposure to EAAs. Of course if the exposure period to EEAs is extended, it becomes impossible to separate the primary response to the EAA from the reactive response since they run together in time. Thus, depending upon the injury model used, say 5–10 minute transient ischemia vs. more permanent occlusions, the relative importance of each would be expected to change.

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5.3 DELAYED NEURONAL DEATH HYPOTHESES Although it is attractive to ignore it, the neuronal degeneration generally studied in culture or in acute in situ preparations such as brain slice occurs within a period of a few hours or less. As originally pointed out by Pulsinelli et al. and Kirino et al.,41,42,61 there is a very significant component of cell death in CA1 hippocampus that does not occur until a few days after a well-defined global ischemic insult. In addition to this model, the widely used kainic acid (KA) model of excitotoxicity produces significant cell death in the mouse hippocampus only after a significant delay of 2–3 days, similar to the loss in rat and gerbil after brief global ischemia.24 Ion probe and morphological analysis of postischemic hippocampus have shown that mitochondria in most brain regions take on heavy Ca loads even during brief insults,21,33,76 but that after the maelstrom, in cells that survive the initial shock, mitochondrial Ca content appears largely restored to normal levels in 1–2 hours.21 In vivo measurements with ion-sensing electrodes in the rat indicated that intracellular Ca2+ increases to 30 µM during an 8-minute ischemic episode, but falls to ~3 µM by 10 minutes reperfusion before returning to preischemic levels by 20 minutes.74,75 These latter measurements also give some indication that secondary responses akin to those described in the preceding section also occur.74,75 Thus the straightforward excitotoxic hypothesis may serve well to account for rapidly ensuing cell death (within hours after reperfusion) but cannot directly explain the death that is delayed by days, long after the presumed causative parameters have returned to basal levels. Hypotheses by which the initial disruption in Ca2+ homeostasis may lead to delayed cell death fit into four broad groups: (1) things happen that cause delayed increases in intracellular Ca2+ loads, activating the same cell death mechanisms prevalent in acute death; (2) apoptosis is the operative mechanism, involving the induction of cascades leading to programmed cell death which may require many hours to complete; (3) chronic depression of cytosol/organellar Ca2+ levels which, over time, leads to cell death through disruption of Ca2+-dependent protein processing or trafficking; and (4) other factors such as extraordinary release of zinc are involved.44

5.3.1 DELAYED INCREASES IN INTRACELLULAR CA2+ A long-standing hypothesis is that excitability of neurons marked for delayed death exhibit increased excitability following ischemia, causing accumulating overload of Ca2+ due to the added influx during firing. Conceptually this condition might arise from a loss of inhibitory interneurons after the initial insult or a modification of intrinsic properties of the neurons themselves. Experimental findings regarding postischemic excitability have been mixed. Decreased or unchanged excitability has been reported following brief ischemia in gerbils and hypertensive rats.7,36,80 Increased excitability of CA1 neurons has also been reported in some studies of both gerbil81 and rat.8 In slices that were cut within 2 days of a 5-minute ischemic episode in awake gerbils, one study applied intracellular-recording and whole-cell techniques to CA1 cells43 and often observed depolarized resting potentials (– 40 to –50

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mV) and phenomena that were consistent with failing Ca2+ homeostasis. A similar study of later postischemic times (2.5 to 3.5 days) reported more severe indications of failing Ca2+ homeostasis.82 The view is taken here that the examples of depressed resting potentials and failing Ca2+ homeostasis represent the last stages of deterioration, and not the perhaps treatable stages that occur at intermediate times. This view is also supported by autoradiographic and indicator dye evidence of Ca2+ accumulation in postischemic CA1 neurons.3,19 These findings show that the timing of Ca2+ accumulation correlates so closely with morphological death markers as to imply that Ca2+ accumulation did not appreciably precede death in area CA1.3,19 A second hypothesis for delayed Ca2+ overload arose from the discovery that mRNA for a specific glutamate receptor subunit (GluR2) was downregulated, rather specifically in area CA1 24 hours after 10 min of 4-vessel occlusion in rat.60 Incorporation of this subunit into the AMPA-KA-type glutamate receptor renders the ion pore impermeable to Ca2+. Without this subunit present in the AMPA-KA receptor complex, this normally monovalent pore allows the passage of Ca2+.31,35 Consequently, without mRNA for GluR2 present, the normal Ca2+-impermeable AMPA-KA receptors in the pyramidal cells would be turned, over time, to Ca2+-permeable receptors. These modified receptors could impose Ca2+ overburden to the CA1 neurons during periods of excitation and also in response to ambient, interstitial glutamate.60 A similar, although possibly slower disappearance of mRNA for this subunit has been confirmed in the gerbil for 5-min occlusion where an increased KAstimulated Ca2+ accumulation has been demonstrated in neurons 3 days after the insult,25 but no significant changes were found earlier than this. In slice preparations, where the Ca2+ signals were studied, there was no significant elevation of ambient Ca2+ levels, but it is quite probable that near the surface of a brain slice, where the analyzed neurons lay, ambient glutamate levels are smaller than in vivo. Thus it remains possible that increased Ca2+ influx in response both to synaptic input and to ambient glutamate levels in vivo could result in sustained calcium elevations and death. It is also possible that the GluR2 downregulation is a compensatory mechanism for the loss of voltage-gated Ca2+ influx (see below). However, it would appear to be inadequate to effect a reversal of degeneration.

5.3.2 APOPTOSIS The hypothesis that apoptosis plays a significant role in delayed, postischemic neuronal death has become very prominent in the past 4 to 5 years, and only a small part of the literature most closely related to proposed studies can be included here. Mitochondrial dysfunction and release of cytochrome C is thought to trigger cleavage and activation of a cascade of cysteine proteases (caspases), which in turn lead to organized cellular degradation.48 Support for this model has come from finding suggestive morphological markers in postischemic neurons such as DNA laddering on agarose gels and TUNEL labeling of DNA that are associated with apoptotic death.37,40,49 The apoptosis gene homologs of C. elegans (ced-3 and ced-4)22 are expressed in cortical and hippocampal neurons following in vivo ischemic insults.32,48 The activation and cleavage of caspase 3, the family member with highest homology

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to CED-3, has been shown to precede the DNA laddering and TUNEL staining in a 2 hr cerebral artery occlusion model in mouse.54 Moreover, benzyloxycarbonyl inhibitors of cysteine proteases (Z-DEVD-FMK and z-VAD, zD) have proved effective in reducing postischemia neuron loss in rat, mouse, and gerbil models.10,11,27,30 Despite these suggestive findings, there have been reservations all along as to whether apoptosis actually occurs after in vivo ischemia in adult animals (as opposed to excitotoxic models in tissue culture). Evidence has been provided that the hallmark DNA fragmentation indeed occurs after cell death.61 A thorough electron microscopic study of postischemia cell death in the gerbil 5-min occlusion model failed to find evidence that classical apoptosis occurred in area CA1.13 The authors concluded: “The results show that untreated global ischemic injury has necrotic, not apoptotic, morphology but do not rule out programmed biochemical events of the apoptotic pathway occurring before neuronal necrosis.” With regard to ischemic cell death, it must be emphasized that findings from one model system are not necessarily applicable to another. Much of the direct evidence on apoptotic death has come from neurons in culture or from early stages of development, in vivo. Thus, much of the uncertainty around the issue of necrosis vs. apoptosis may arise from actual differences due to developmental stage. There is evidence of developmental differences in the behavior of at least one element of the apoptotic cascade.38 Nevertheless, the finding that cysteine protease inhibitors are effective in preventing CA1 neuron death in the rodent transient ischemia models is a strong lead regardless of whether they are acting exactly as proposed or otherwise.

5.3.3 CHRONIC DEPRESSION OF INTRACELLULAR CA2+ SIGNALING Despite the above hypotheses regarding heavy Ca2+ loading and neuronal death, when one looks at conditions in CA1 neurons that are going to die at some point in the near future, one finds quite the opposite conditions. For several years the author’s lab has been engaged in electrophysiological and Ca2+ measurements using CA1 pyramidal neurons that had been subjected to a lethal ischemic insult in vivo. In marked contrast to initial expectations, the neurons examined 3 days after global ischemia showed normal, or possibly subnormal, resting Ca2+ levels and greatly depressed Ca2+ increases during current injection stimulation.15,72 Figure 5.6 shows trains of action potentials elicited from a control neuron (upper) and a neuron examined 3 days after an ischemic insult. Action potential firing was superficially similar in both neurons, but the maximum Ca2+ increase in the postischemic neuron was much smaller than in the control neuron. The numerical increases given in the figure were measured in the apical dendrite in the region of largest change. Spatial maps comparing typical Ca2+ increases in control and postischemic neurons are shown in Figure 5.7 (see color insert following page 114), illustrating that the postischemic reduction occurred throughout the neuron. This change in Ca2+ signaling occurred over several days, but did not begin immediately. At 1 day after the insult there was no significant loss of Ca2+ signal, but over the next 2 to 3 days the loss was progressive. Over this same time period, the Ca2+ influx-dependent, tetrodotoxin-sensitive action potential (Ca-spike) disappeared (Figure 5.8). This suggests that the depressed

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FIGURE 5.6 Spike trains and associated maximum dendritic Ca2+ increases in a CA1 pyramidal neuron from a control gerbil and a neuron taken from an animal that had undergone a 5-min ischemic insult 48 hours before. Trains were elicited by 0 3 nA depolarizing current pulses via the recording/indicator-injecting microelectrode. The spike number is similar in both neurons, but the Ca2+ increase is far less in the postischemic neuron (amplitudes of corresponding Ca2+ transients indicated under traces).

FIGURE 5.7 (See color figure 5.7.) Spatial maps of depolarization-driven Ca2+ increases (c–e) and recoveries (f–h) comparing a normal CA1 pyramidal neuron (upper panels) with a CA1 pyramidal neuron in a slice cut 48 hours after transient ischemic insult (lower panels). Depression of the Ca2+ signal (accumulation) after ischemia was severe and occurred in all parts of the neuron. Estimated values of Ca2 > 1 µM, near the limit of fura-2 measurements, have been coded as grey levels.

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FIGURE 5.8 Disappearance of TTX-insensitive Ca2+ spike after ischemia. Upper traces show spiking patterns in a control CA1 neuron in normal and TTX containing saline. Lower traces, spiking patterns 2 days after bilateral ischemia. After 10 min in 1 µM TTX, 1.6 nA (depolarizing current injected via recording electrode) evoked Ca2+ spikes in the control cell (upper right), but even much larger stimuli (2.5 nA) failed to evoke Ca2+ spikes in the postischemic cell (lower right). Standard KAc recording electrodes were used in this series. (Reprinted with permission from Connor, J.A., Razani-Boroujerdi, S., Greenwood, A.C., Cormier, R.J., Petrozzino, J.J., and Lin, R.C. [1999], reduced voltage-dependent Ca2+ signaling in CA1 neurons after brief ischemia in gerbils, J. Neurophysiol., 81:299-306.)

Ca2+ signal from the intracellular indicators is due in large part to a loss of voltagegated Ca2+ channel (VGCC) activity. Other factors such as intracellular Ca2+ release or increased rapid buffering could also account for a part of the depression but it is unlikely that these factors contribute much. First, Ca2+ release appears to provide only a minor contribution to the Ca2+-indicator signal in CA1 dendrites.69 Second the recovery phase from amplitude-adjusted loads is unchanged between control and postischemic neurons (Figure 5.9). The comparative loss of Ca2+ signaling is more severe when measured by action potential train stimulus than by steady depolarizations. Figure 5.10 shows population averaged responses from 6 neurons in each group where the decrease was approximately 80%, compared to the ~60% decreases observed for maintained depolarization. This may imply that slowly- inactivating entry routes are less affected after ischemic insult than certain channels that show more rapid inactivation. By contrast, pyramidal neurons from the CA3 area, which survive standard ischemic insults in vivo, show none of the above changes in Ca2+ homeostasis. Indicator signals and Ca2+ spikes of 2-day postischemic neurons are identical to neurons from control animals.72

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FIGURE 5.9 Ca2+ measurements emphasizing similar recovery time-courses for Ca2+ transients in control and postischemic neurons Ca2+ load. 1-sec depolarizing current injections were given to each group, control and post-ischemic. Depolarizing current for the control neurons was less than for the post-ischemic neurons in order to produce matched peak Ca2+ excursions (n = 6). (Reprinted with permission from Connor, J.A., Razani-Boroujerdi, S., Greenwood, A.C., Cormier, R.J., Petrozzino, J.J., Lin, R.C. [1999], reduced voltage-dependent Ca2+ signaling in CA1 neurons after brief ischemia in gerbils, J. Neurophysiol. 81:299306.)

The above findings have led to a hypothesis that the reduced Ca2+ influx after ischemic insult might ultimately lead to a depletion of Ca2+ in the endoplasmic reticulum (ER), Golgi apparatus, and other intracellular compartments such as mitochondria. Based upon the known dependence of protein synthesis, processing, and transport in the ER and Golgi on sufficiently high intraluminal Ca2+,6,53 it has been proposed15,72 that depletion of this intraluminal Ca2+ would cause degeneration of the neurons that might be expected to occur over a fairly extended time course. Early morphological studies of postischemic CA1 neurons showed marked proliferation of the ER,41,42 and also a disaggregation of polyribosomes18,41 in this population but not in other, surviving, types of neurons. These features suggest disruption of ER function. In addition to effects on protein synthesis, calcium depletion of the ER and nuclear envelope (a continuation of the ER) has been shown to inhibit nuclear signaling by closing nuclear pores26,77 and may also contribute to delayed cell death.

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FIGURE 5.10 Comparison showing the strong decrement of maximum dendritic Ca2+ transients elicited by action potential trains fired by 1-sec current injections in normal CA1 neurons (control) and neurons studied 2 days after bilateral ischemia. Measurements were made in a small region of the apical dendrite showing maximal excursions (see Fig. 5.7) Mean resting Ca2+ levels were not significantly different between the populations. Action potential trains were similar (see Fig. 5.6). For each time point, the mean [Ca2+] ± SEM is plotted for each group (n = 6). Horizontal bar denotes time of stimulus. “R” denotes recovery level, measured at ~ 30 sec. (Reprinted with permission from Connor J.A., Razani-Boroujerdi, S., Greenwood, A.C., Cormier, R.J., Petrozzino, J.J., Lin, R.C. [1999], reduced voltage-dependent Ca2+ signaling in CA1 neurons after brief ischemia in gerbils. J. Neurophysiol. 81:299-306.)

The hypothesis that low ER calcium is upstream from delayed, postischemic death has also been developed independently, based upon different types of evidence, by Paschen and colleagues.56,57,58 Their hypothesis follows from observations that depletion of intracellular Ca2+ pools (using thapsigargin, caffeine, or exogenous buffering) induced metabolic changes resembling those produced by transient cerebral ischemia in vivo. Among these changes were phosphorylation of the eucaryotic initiation factor eIF-2 alpha, disaggregation of polyribosomes and depressed protein synthesis, and increased expression of growth arrest genes gadd34 and gadd153.20,59 Importantly, these effects were more tightly linked to Ca2+ levels in intracellular pools than to cytosolic Ca2+ levels, raising the possibility that these stores are key elements in the relationship between disrupted calcium homeostasis and delayed cell death. This work places emphasis on a possible breakdown of Ca2+ transport into ER as a cause of decreased ER levels. RTPCR studies of multiple brain regions indicates a selective loss of endoplasmic reticulum calcium-ATPase (SERCA) mRNA;58 however, it is not yet known whether these changes are expressed selectively in neurons or glia, and it remains to be determined how rapidly the drop in mRNA expression is reflected in loss of functional SERCA protein. A reduction in activity of this pump could contribute to decreased ER Ca2+ levels, but our finding of depressed Ca2+ influx strongly suggests that decreased ER levels would result even if SERCA expression is

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unaffected, or the reduction in activity occurs at a later time point than reduction in voltage-dependent Ca2+ entry. This is because voltage-dependent influx of extracellular Ca2+ appears to be the key determinant of ER filling in hippocampal CA1 neurons. Unlike many types of somatic cells which display a mechanism for opening specialized plasmalemma Ca2+ channels following the depletion of intracellular Ca2+ stores (capacitative Ca2+ entry)65 hippocampal pyramidal neurons do not. The same is probably true for other CNS neurons but data are unavailable. CA1 neurons exposed to thapsigargin do not show the persisting elevation of cytosolic Ca2+ 63 that exocrine gland or mast cells2,34 do, for example. Also, strong evidence has been developed implicating depletion of Ca2+ from intracellular stores as a key trigger of apoptosis in cell lines and in tissue, cultured neurons.1,17,52,71 As outlined above, the importance of apoptotic cell death after in vivo ischemia in adult models is controversial, and it is quite likely that not all of the subtleties of apoptotic gene expression in mature neurons have been defined. The next few years should clarify the actions of shared elements of apoptosis in the different models of delayed cell death.

5.4 CONCLUSION Obviously the above observations raise more questions than they answer, but then both sets are relatively new and unexplored as applied to neuronal degeneration. It is hoped that a summary of what seem to be first-order questions might serve to kindle interest in expanded experimentation at the cellular function level in the important and challenging area of delayed neuronal loss. 1. What are the Ca-carrying channels that produce the secondary response? Are they ordinary channels that have undergone modification that alters their kinetics so that open probability is increased at more negative voltage, or is a set of new channels inserted into the membrane. This second possibility is not so remote as it seemed a few years ago, before the elegant demonstrations of rapid trafficking of channels between internal and plasma membranes.50 2. What are the factors that limit the primary, agonist-generated Ca2+ increases in EAA resistant strains. This has possibly important implications for therapies. That is, both resistant and nonresistant strains perform their basic functions well enough to survive. Therefore neuroprotection might be accomplished without compromising vital functions such as is experienced with glutamate receptor blocker approaches. 3. Are there strategies that can selectively block generation or propagation of secondary responses? Previous work suggests that Ca2+-dependent phosphorylation may be a useful line of investigation, as are studies of sequestration and extrusion mechanisms that must come into play with the sustained very high Ca2+ levels experienced in secondary responses.

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4. Are there factors present in vivo that prevent the secondary response from causing early toxicity? There is good evidence from in vivo Ca2+ electrode recordings that a secondary response occurs in CA1 neurons during and after ischemic insult.74,75 Since these neurons do not die immediately right away (as most neurons do in vitro), what factors are most important in this temporary rescue? 5. Which Ca2+ channels are disappearing or becoming inoperable after the in vivo ischemic insult. Can strategies be developed to maintain VDCC function in the days following transient insults, and assess impact on neuronal survival? 6. Does the loss of activity-driven Ca2+ influx observed after in vivo ischemia really produce a decrease in organellar Ca? 7. Can selective Ca channel blockers be found that when administered in vivo produce delayed neuronal death?

5.5 ACKNOWLEDGMENTS Supported by NIH grant NS 35644. We thank Dr. S. Razani-Boroujerdi for help in constructing Figure 5.7.

REFERENCES 1. Bian, X., Hughes, F.M., Huang, Y., Cidlowski, J.A. and Putney, J.W. Roles of cytoplasmic Ca2+ and intracellular Ca2+ stores in induction and suppression of apoptosis in S49 cells, Am. J. Physiol., 272, C1241, 1997. 2. Bird, G.S., Louzan, M.C., Ribeiro, C.M. and Putney, J.W. Calcium signalling in exocrine glands, Eur. J. Morphol., 36, Suppl. 153., 1998. 3. Bonnekoh, P., Kurolwa, T., et al. Time profile of calcium accumulation in hippocampus, striatum and frontoparietal cortex after transient forebrain ischemia in the gerbil, Acta Neuropath., 84, 400, 1992. 4. Brorson J.R., Manzolillo P.A., and Miller R.J. Ca2+ entry via AMPA/KA receptors and excitotoxicity in cultured cerebellar Purkinje cells, J. Neurosci., 14, 187, 1994. 5. Brorson J.R., Marcuccilli C.J., and Miller R.J. Delayed antagonism of calpain reduces excitotoxicity in cultured neurons, Stroke, 26, 1259, 1995. 6. Brostrom M.A. and Brostrom, C.O. Calcium dependent regulation of protein synthesis in intact mammalian cells, Ann. Rev. Physiol., 52, 557, 1990. 7. Buzsaki, G., Freund, T. F., et al. Ischemia-induced changes in the electrical activity of the hippocampus, Exp. Brain Res., 78, 268, 1989. 8. Chang, H.S., Sasaki, T. and Kassel, N.F. Hippocampal unit activity after transient cerebral ischemia in rats, Stroke, 20, 1051, 1989. 9. Chen, Q.X., Perkins, K.L., Choi, D.W., and Wong, R.K.S., Secondary activation of a cation conductance is responsible for NMDA toxicity in acutely isolated hippocampal neurons, J. Neurosci., 17, 4032, 1997. 10. Chen, J., Nagayama, T., Jin, K., Stetler, R. A., Zhu, R. L., Graham, S. H. and Simon, R. P., Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient crebral ischemia, J. Neurosci., 18, 4914,1998.

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11. Cheng, Y., Deshmukh, M., D’Costa, A., Demaro, J.A., Gidday, J.M., Shah, A., Sun, Y., Jacquin, M.F., Johnson, E.M. Jr., Holtzman, D.M., Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury, J. Clin. Invest., 101, 1992, 1998. 12. Choi D.W., Calcium: still center-stage in hypoxic-ischemic neuronal death, Trends Neurosci., 18, 58, 1995. 13. Colbourne, F., Sutherland, G.R., and Auer, R.N., Electron microscopic evidence against apoptosis as the mechanism of neuronal death in global ischemia, J. Neurosci., 19, 4200, 1999. 14. Connor, J.A., Cormier, R.J., Cumulative effects of glutamate microstimulation on Ca2+ responses of CA1 hippocampal pyramidal neurons in slice, J. Neurophysiol., 83, 90, 2000. 15. Connor J.A., Razani-Boroujerdi S., Greenwood A.C., Cormier R.J., Petrozzino J.J., and Lin R.C., Reduced voltage-dependent Ca2+ signaling in CA1 neurons after brief ischemia in gerbils, J Neurophysiol., 81, 299, 1999 16. Connor J.A., Wadman W.J., Hockberger, P.E., and Wong, R.K., Sustained dendritic gradients of Ca2+ induced by excitatory amino acids in CA1 hippocampal neurons, Science, 240, 649, 1988. 17. Deckwerth, T.L., Easton, R.M., Knudson, C.M., Korsmeyer, S.J., and Johnson, E.M.Jr., Placement of the BCL2 family member BAX in the death pathway of sympathetic neurons activated by trophic factor deprivation, Exp. Neurology, 152, 150, 1998. 18. Deshpande, J.K., Siesjo, B.K. and Wieloch, T., Calcium accumulation and neuronal damage in the rat hippocampus following cerebral ischemia, J. Cereb. Blood Flow Metab., 7, 89, 1987. 19. Dienel, G.A., Regional accumulation of calcium in postischemic rat brain, J. Neurochem., 43, 913, 1984. 20. Doutheil, J., Althausen, S., Gissel, C. and Paschen. W., Activation of MYD116 (gadd34) expression following transient forebrain ischemia of rat: implications for a role of disturbances of endoplasmic reticulum calcium homeostasis, Brain Res. Mol. Brain Res., 63, 225, 1999. 21. Dux, E., Mies, G., et al., Calcium in the mitochondria following brief ischemia of gerbil brain, Neurosci. Letts., 78, 295, 1987. 22. Ellis, H.M. and Horvitz, H.R., Genetic control of programmed cell death in the nematode C. elegans, Cell, 44, 817, 1986. 23. Faddis, B.T., Hasbani, M.J., and Goldberg, M.P., Calpain activation contributes to dendritic remodeling after brief excitotoxic injury in vitro. J. Neurosci., 17, 951, 1997. 24. Faherty, C.J., Xanthoudakis, S., and Smeyne, R.J., Caspase-3-dependent neuronal death in the hippocampus following kainic acid treatment, Brain Res. Mol. Brain Res., 70, 159, 1999. 25. Gorter, J.A., Petrozzino, J.J., Aronica, E.M., Rosenbaum, D.M., Opitz, T., Bennett, M.V.L., Connor, J.A., and Zukin R.S., Global ischemia induces downregulation of GluR2 mRNA and increases AMPA receptor-mediated Ca2+ influx in hippocampal CA1 neurons of gerbil, J. Neurosci., 17, 6179, 1997. 26. Greber, U.F. and Gerace, L., Depletion of calcium from the lumen of endoplasmic reticulum reversibly inhibits passive diffusion and signal-mediated transport into the nucleus, J. Cell Biol., 128, 5, 1995.

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27. Hara, H., Friedlander, R.M., Gagliardini, V., Ayatam, C., Fink, K., Huang, Z., Shimizu-Sasamata, M., Yuan, J., and Moskowitz, M.A., Inhibition of interleukin 1 converting enzyme family proteases reduces ischemic and excitotoxic damage, Proc. Natl. Acad. Sci. USA, 94, 2007, 1997. 28. Hell, J.W., Westenbroek, R.E., Breeze, L.J., Wang, K.K., Chavkin, C., and Catterall, W.A., N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons, Proc. Natl. Acad. Sci. USA, 93, 3362, 1996. 29. Helmchen, F., Svoboda, K., Denk, W., and Tank, D.W., In vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons, Nat. Neurosci. 2, 989, 1999. 30. Himi, T., Ishizaki, Y., and Murota, S., A caspase inhibitor blocks ischaemia-induced delayed neuronal death in the gerbil, Eur. J. Neurosci., 10, 277, 1998. 31. Hollmann, M., Hartley, M., and Heinemann, S., Ca2+ permeability of KA-AMPAgated glutamate receptor channels depends on subunit composition, Science 252, 851, 1991. 32. Honkaniemi, J., Massa, S.M., Breckinridge, M. and Sharp F.R., Global ischemia induces apoptosis-associated genes in hippocampus, Mol. Brain Res., 42, 79, 1996. 33. Hossmann, K.-A., Ophoff, B. G., et al., Mitochondrial calcium sequestration in cortical and hippocampal neurons after prolonged ischemia of the cat brain, Acta Neuropath., 68, 230, 1985. 34. Huang, Y. and Putney, J.W., Relationship between intracellular calcium store depletion and calcium release-activated calcium current in a mast cell line (RBL-1), J. Biol. Chem., 273, 19554, 1998. 35. Hume, R.I., Dingledine, R., and Heinemann, S., Identification of a site in glutamate receptor subunits that controls calcium permeability, Science, 253, 1028, 1991. 36. Imon, H., and Mitani, A., et al., Delayed neuronal death is induced without postischemic hyperexcitability: Continuous multiple-unit recording from ischemic CA1 neurons, J. Cereb. Blood Flow Metab., 11, 819, 1991. 37. Iwai, T., Hara, A., Niwa, M., Nozaki, M., Uematsu, T., Sakai, N., and Yamada, H., Temporal profile of nuclear DNA fragmentation in situ in gerbil hippocampus following transient forebrain ischemia, Brain Res., 671, 305, 1995. 38. Johnson, M.D., Kinoshita, Y., Xiang, H., Ghatan, S., and Morrison, R.S., Contribution of p53-dependent caspase activation to neuronal cell death declines with neuronal maturation, J. Neurosci., 19, 2996, 1999. 39. Kay, A.R. and Wong, R.K.S., Isolation of neurons suitable for patch-clamping from adult mammalian central nervous system, J.Neurosci.Meth., 6, 227, 1986 40. Kihara, S., Shiraishi, T., Nakagawa, S., Toda, K. and Tabuchi, K., Visualization of DNA double strand breaks in the gerbil hippocampal CA1 following transient ischemia, Neurosci. Letts., 175, 133, 1994. 41. Kirino, T. and Sano, K., Selective vulnerability in the gerbil hippocampus following transient ischemia, Acta. Neuropath., 62, 201, 1984. 42. Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239, 57, 1982. 43. Kirino, T., Robinson, H. P. C., et al., Disturbance of membrane function preceding ischemic delayed neuronal death in the gerbil hippocampus, J. Cereb. Blood Flow Metab., 12, 408, 1992. 44. Koh, J.Y., Suh, S.W., Gwag, B.J., He, Y.Y., Hsu, C.Y. and Choi, D.W., The role of zinc in selective neuronal death after transient global cerebral ischemia, Science, 272, 1013, 1996.

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45. Kristian, T. and Siesjo, B.K., Calcium in ischemic cell death. Stroke 27, 1592, 1998. 46. Lipton P., Ischemic cell death in brain neurons, Physiol. Rev., 79, 1431, 1999 47. MacDermott, A.B., Mayer, M.L., Westbrook, G.L., Smith, S.J., and Barker, J.L., NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones, Nature, 321, 519, 1986. 48. MacManus, J.P. and Linnik, M.D., Gene expression induced by cerebral ischemia: an apoptotic perspective, J. Cereb. Blood Flow Metab., 17, 815, 1997. 49. MacManus, J.P., Buchan, A.M., Hill, I.E., Rasquinha, I. and Preston E., Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain, Neurosci. Letts., 164, 89, 1993. 50. Man, H.Y., Ju, W., Ahmadian, G., and Wang, Y.T. Intracellular trafficking of AMPA receptors in synaptic plasticity, Cell Mol. Life Sci., 57, 1526, 2000 51. Manev, H. Favaron, M. Guidotti, A., and Costa, E. Delayed increase of Ca2+ influx elicited by glutamate: Role in neuronal death, Mol. Pharmacol., 36:106, 1989. 52. Marks, N., Berg, M.J., Guidotti, A., and Saito, M., Activation of caspase-3 and apoptosis in cerebellar granular cells, J. Neurosci. Res., 52, 334, 1998. 53. Mizuno, K., Nakamura, T., and Matsuo, H., A unique membane, calcium-dependent endopeptidase with specificity toward paired based residues in rat liver Golgi fractions, Biochem. Biophys. Res. Commun., 164, 780, 1989. 54. Namura, S., Zhu, J., Fink, K., Endres, M., Srinivasan, A., Tomaselli, K.J., Yuan, J., and Moskowitz, M.A., Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia, J. Neurosci., 18, 3659, 1998. 55. Olney, J. W. and Sharpe, L. G., Brain lesions in an infant rhesus monkey treated with monosodium glutamate, Science, 166, 386, 1969. 56. Paschen, W. and Doutheil, J., Disturbances of the functioning of endoplasmic reticulum: a key mechanism underlying neuronal cell injury? J. Cereb. Blood Flow Metab., 19, 1, 1999. 57. Paschen, W., Doutheil, J., Gissel, C,, and Treiman, M., Depletion of neuronal endoplasmic reticulum calcium stores by thapsigargin: effect on protein synthesis, J. Neurochem., 67, 1735, 1996. 58. Paschen, W., Doutheil, J., Uto, A. and Gissel, C., Changes in endoplasmic reticulum Ca2+-ATPase mRNA levels in transient cerebral ischemia of rat: a quantitative polymerase chain reaction study, Neurosci. Letts., 217, 41, 1996. 59. Paschen, W., Gissel, C., Linden, T., Althausen, S. and Doutheil, J., Activation of gadd153 expression through transient cerebral ischemia: evidence that ischemia causes endoplasmic reticulum dysfunction, Brain Res. Mol. Brain Res., 60, 115, 1996. 60. Pellegrini-Giampietro, D.E., Zukin, R.S., Bennett, M.V., Cho, S. and Pulsinelli, W.A., Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats, Proc. Nat.l Acad. Sci. USA, 89, 10499, 1992 61. Petito, C.K., Torres-Munoz, J., Roberts, B., Olarte, J.P., Nowak, T.S., and Pulsinelli, W.A., DNA fragmentation follows delayed neuronal death in CA1 neurons exposed to transient global ischemia in the rat, J. Cereb. Blood Flow Metab., 17, 967, 1997. 62. Petrozzino, J.J., Pozzo-Miller, L.D., and Connor, J.A., Micromolar Ca2+ transients in dendritic spines of hippocampal pyramidal neurons in brain slice, Neuron, 14, 1223, 1995. 63. Pozzo-Miller, L.D., Petrozzino, J.J., Golari, G., and Connor, J.A., Ca2+ 2+ release from intracellular stores induced by afferent stimulation of CA3 pyramidal neurons in hippocampal slices, J. Neurophysiol., 76, 554, 1996.

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64. Pulsinelli, W.A., Brierley, J.B., and Plum, F., Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11, 491, 1982. 65. Putney, J.W. and McKay, R.R., Capacitative calcium entry channels, Bioessays 1, 38, 1999. 66. Randall, R.D. and Thayer, S.A., Glutamate-induced calcium transient triggers delayed calcium overload and neurotoxicity in rat hippocampal neurons, J. Neurosci., 12, 1882, 1992. 67. Reynolds, I.J., Mitochondrial membrane potential and the permeability transition in excitotoxicity, Ann. N.Y. Acad. Sci., 893, 33, 1999. 68. Rothman, S.M., and Olney, J.W., Glutamate and the pathophysiology of hypoxic—ischemic brain damage, Ann. Neurol., 19, 105, 1986. 69. Sandler, V.M. and Barbara, J.G., Calcium-induced calcium release contributes to action potential-evoked calcium transients in hippocampal CA1 pyramidal neurons, J. Neurosci., 19, 4325, 1999. 70. Schauwecker, P.E. and Steward, O., Genetic determinants of susceptibility to excitotoxic cell death: implications for gene targeting approaches, Proc. Natl. Acad. Sci. USA, 94, 4103, 1997. 71. Schultz, J.B., Weller, M., and Klockgether, T., Potassium deprivation-induced apoptosis of cerebellar granule neurons: a sequential requirement for new mRNA and protein synthesis, ICE-like protease activity, and reactive oxygen species, J. Neurosci., 16, 4696, 1996. 72. Shuttleworth, C.W.R., Greenwood, A.C., and Connor, J.A., Ca2+ 2+ signaling in gerbil CA3 hippocampal neurons following transient in vivo ischemia, Neurosci. Letts., 286, 75, 2000. 73. Shuttleworth, C.W.R. and Connor, J.A., Strain dependent differences in calcium signaling predict excitotoxicity in murine hippocampal neurons, J. Neurosci. (in press). 74. Silver, I. A. and Erecinska, M., Intracellular and extracellular changes of [Ca2+] in hypoxia and ischemia in rat brain in vivo, J. Gen. Pysiol., 95, 827, 1990. 75. Silver, I. A. and Erecinska, M., Ion homeostasis in rat brain in vivo: intra- and extracellular [Ca2+] and [H+] in the hippocampus during recovery from short-term, transient ischemia, J. Cereb. Blood Flow Metab., 12, 759, 1992. 76. Simon, R. P., Griffith, T., et al., Calcium overload in selectively vulnerable neurons of the hippocampus during and after ischemia: an electron microscopy study in the rat, J. Cereb. Blood Flow Metab., 4, 350, 1984. 77. Stehno-Bittel, L., Perez-Terzic, C., and Clapham, D.E., Diffusion across the nuclear envelope inhibited by depletion of the nuclear Ca2+ store, Science, 270, 1835, 1995. 78. Steward, O., Schauwecker, P.E., Guth, L., Zhang, Z., Fujiki, M., Inman, D, Wrathall, J., Kempermann, G., Gage, F.H., Saatman, K.E., Raghupathi, R., and McIntosh, T., Genetic approaches to neurotrauma research: opportunities and potential pitfalls of murine models, Exp. Neurol., 157, 19, 1999. 79. Stout, A.K., Raphael, H.M., Kanterewicz BI, Klann, E., and Reynolds, I.J., Glutamate-induced neuron death requires mitochondrial calcium uptake, Nat. Neurosci., 1, 366, 1998. 80. Suyama, K., Changes of neuronal transmission in the hippocampus after transient ischemia in spontaneously hypertensive rats and the protective effects of MK-801. Stroke 23, 260, 1992. 81. Suzuki, R., Yamaguchi, T., et al., The effects of 5-minute ischemia in mongolian gerbils: II. Changes of spontaneous neuronal activity in cerebral cortex and CA1 sector of hippopcampus, Acta Neuropath., 62, 217, 1983.

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82. Tsubokawa, H., Oguro, K., et al., Abnormal Ca2+ homeostasis before cell death revealed by whole cell recording of ischemic CA1 hippocampal neurons, Neuroscience, 49, 807, 1992 83. Vergun, O., Keelan, J., Khodorov, B.I., and Duchen, M.R., Glutamate-induced mitochondrial depolarisation and perturbation of calcium homeostasis in cultured rat hippocampal neurones, J. Physiol. (Lond.), 519.2, 451, 1999. 84. Wadman, W.J. and Connor, J.A., Persisting modification of dendritic calcium influx by excitatory amino acid stimulation in isolated CA1 neurons, Neuroscience, 48, 293, 1992. 85. Weiss, S., Hochman, D., and MacVicar, B.A. Repeated NMDA receptor activation induces distinct intracellular calcium changes in subpopulations of striatal neurons in vitro, Brain Res., 627, 63, 1993. 86. Yuste, R., Majewska, A., Cash, S.S., and Denk, W., Mechanisms of calcium influx into hippocampal spines: heterogeneity among spines, coincidence detection by NMDA receptors, and optical quantal analysis, J. Neurosci., 19, 1976, 1999.

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2+ A FIGURE 5.3 Initial and secondary Ca

responses to KA superfusion (10 M, 10 min) in CA1 pyramidal neurons from Bl/6J (KA resistant) and Bl/10J (vulnerable) mice. Black-and-White images (upper left panels), show bisFura-2 fluorescence excited at 380 nm. Panels a,a’–h,h’ show color-coded intracellular Ca2+ levels (scale bar at right) with uniform resting Ca2+ in panels a,a’. Peak of the response (panel c,c’) occurred ~ 4.5 min after onset of KA exposure with 2+ B strong increases in Ca in the proximal and distal apical dendrites. Panels c,c’ show Ca2+ levels 15 s after the peak response. After reexposure to normal saline (panel d,d’), Ca2+ returned to resting levels throughout the Bl/6J neuron and in the Bl/10J neuron except in a portion of the apical dendritic tree (arrow in panel d’). After KA washout, Ca2+ levels remained very high in this restricted dendritic region (panel e’). Part B shows the slow propagation of the secondary response throughout the neuron. Note calibration of the color bar is different in A & B to optimize display contrast. Scale bar 50 m. (Reprinted with permission from Shuttleworth, C.W.R. and Connor, J.A., Strain dependent differences in calcium signaling predict excitotoxicity in murine hippocampal neurons, J. Neuroscience, 21, 4225–4236, 2001.)

Ca2+ responses to injected current is greatly reduced in 2 day post-ischemic CA1 neuron measured by injected fura-2 8 sec

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FIGURE 5.7 Spatial maps of depolarization-driven Ca increases (c–e) and recoveries (f–h) comparing a normal CA1 pyramidal neuron (upper panels) with a CA1 pyramidal neuron in a slice cut 48 hours after transient ischemic insult (lower panels). Depression of the Ca2+ signal (accumulation) after ischemia was severe and occurred in all parts of the neuron. Estimated values of Ca2+ > 1 M, near the limit of fura-2 measurements, have been coded as grey levels. 2+

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6

Mechanism of ZincInduced Neuronal Death Jae-Young Koh, Yang-Hee Kim, Jeong Ae Park, and Joo-Yong Lee

CONTENTS 6.1 6.2 6.3

Introduction Zinc, a Novel Ionic Mediator of Neuronal Cell Death In Vivo Sources for Toxic Zinc Accumulation: Vesicular and Nonvesicular 6.4 Routes of Zinc Entry 6.5 Intracellular Signaling Events Mediating Zinc Toxicity 6.6 An Effector Mechanism of Zinc Toxicity: Oxidative Stress 6.7 Induction and Activation of NADPH Oxidase by Zinc 6.8 Another Effector Mechanism of Zinc Toxicity: p75NTR/NADE-Mediated Apoptosis 6.9 Protective Measures Against Zinc Toxicity 6.10 Summary 6.11 Acknowledgment References

6.1 INTRODUCTION Zinc, a transitional metal, is required for the normal function of a number of proteins. For instance, zinc is an essential component of many enzymes (zinc metalloenzymes) including carbonic anhydrase,1 superoxide dismutase (SOD)-1, and metalloproteases.2 In addition, a large number of transcription factors3 and various signaling proteins contain zinc-binding motifs such as zinc fingers, RING fingers, and zinc clusters;4 the binding of zinc to these sequences provide proteins with correct protein conformations for their normal activity. Hence, it is not surprising that our body contains a substantial amount of zinc in every cell. In fact, zinc is the second most abundant trace element in the body. The central nervous system is not an exception, and contains even higher levels of zinc than the rest of the body with a possible exception of pancreatic islets; a gram of wet brain tissue contains about 10 µg of zinc, which translates into about 150 µM concentration.5,6 In particular, the forebrain contains higher levels of zinc than does the brain stem, cerebellum, or spinal cord. In the brain, zinc is more enriched in gray

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matter than white matter, and the highest amount of zinc is found in hippocampal mossy fibers. Most of the zinc in the brain, like in the rest of the body, is tightly bound to proteins, and thus inaccessible to simple histochemical detection methods.7 However, interestingly, a substantial fraction of the forebrain zinc (10–15%) is present in chelatable (free or loosely bound) forms,8 and hence can be stained with simple histochemical methods such as neo-Timm9 and N-(6-methoxy-8-quinolyl)-p-toluene sulphonamide (TSQ) fluorescence stainings.10 With these methods, zinc is visible in neocortical layers I-III and V, hippocampus, subiculum, amygdala, thalamus, and striatum.6,11 Ultrastructurally, most of the chelatable zinc in the brain is localized within vesicles of excitatory synaptic boutons.12-14 Whereas anatomical information about synaptic vesicle zinc has been available for some time, serious investigations into its functional roles, physiologic or pathologic, were only recently initiated. Here we are going to discuss the cytotoxic role of endogenous zinc and its mechanisms.

6.2 ZINC, A NOVEL IONIC MEDIATOR OF NEURONAL CELL DEATH While calcium is still taking center stage in the field of neuronal death,15,16 an increasing body of evidence suggests that endogenous zinc is another important ionic mediator of ischemic and epileptic neuronal death.17,18 The brain contains quite high levels of zinc, especially inside vesicles of excitatory synaptic boutons. Moreover, like glutamate, zinc inside synaptic vesicles is released into the extracellular space in acute injurious conditions;19–21 during the intense release, the extracellular peak concentration of zinc may reach 300 µM.20 In cortical cultures, brief exposure to several hundred µM zinc leads to extensive neuronal and sometimes glial cell death.22,23 More direct support for zinc’s role in neuronal cell death has come from in vivo experiments. Following transient global ischemia or repeated seizures in the rat, zinc is found accumulated in the cell body of most degenerating neurons.24–27 In fact, the correlation between zinc accumulation and neuronal cell death has been reported to be nearly perfect.24,25 In both cases, blockade of zinc accumulation using a zinc chelator, CaEDTA, results in remarkable protection against neuronal death; CaEDTA completely blocks zinc toxicity but has no effect on calcium overload excitotoxicity in cortical culture.25 Further supporting zinc’s role in neuronal death, the expression of metal-regulating proteins is altered in brain injury. Zinc transporter 1 (ZnT-1) that pumps out zinc from cells, and metallothionein-III (MT-III), a high-capacity neuronal zinc buffer, are induced in hippocampal CA1 neurons following ischemia.28–30 Considering all these evidences, it is highly likely that endogenous zinc plays a critical role in ischemia- or seizure-induced neuronal death.

6.3 IN VIVO SOURCES FOR TOXIC ZINC ACCUMULATION: VESICULAR AND NONVESICULAR In animal models of ischemia, seizures, and trauma, almost all the degenerating neurons exhibit zinc accumulation in their cell bodies. As discussed above, synaptic

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vesicle zinc is likely the major source for the toxic zinc accumulation in most cases;24 it is situated specifically in injury-vulnerable brain areas and released with injury. However, in certain situations, sources other than synaptic vesicles seem to contribute to toxic zinc accumulation. Of known zinc transporters, zinc transporter3 (ZnT-3) is found to be responsible for recruiting zinc into synaptic vesicles in the brain,8 because knocking out ZnT-3 gene in mice results in complete disappearance of synaptic vesicle zinc.31 However, even in ZnT-3-null mice, following seizures, zinc accumulation still occurs in the cell body of degenerating neurons.32 Hence, in ZnT-3-null mice, cytotoxic zinc accumulation must have come from sources other than synaptic vesicles. It is possible that functions of synaptic zinc are somehow compensated,33 for example, by another mechanism of zinc release independent of the synaptic vesicle mechanism. Alternatively, zinc accumulation inside neurons may come from internal zinc sources such as metallothioneins.34,35 To be able to answer the question where zinc comes from, further studies on zinc homeostasis in the brain seem warranted.

6.4 ROUTES OF ZINC ENTRY How does released zinc gain access to the inside of neurons? Zinc ion, as a positively charged molecule, cannot easily permeate intact lipid bilayer membrane. Hence specific routes such as ion channels seem to be required for zinc to enter cells. The fact that depolarization enhances zinc neurotoxicity36 suggests that voltage-gated channels may be involved. Zinc exchanges its hydration shell at a rate intermediate between that of Ca2+ and that of cationic calcium channel blockers such as Mg2+ or Co2+..37 Consistently, often zinc attenuates current through voltage-gated calcium channels.38 The channel blocking effect, however, is not incompatible with slow permeation.39,40 In fact, depolarization enhances zinc influx estimated by TSQ fluorescence in an L-type calcium channel blocker-dependent manner.36 Another route of zinc entry is Ca2+-permeable AMPA/kainate channels. These channels may lack GluR2 subunit that limits Ca2+ permeability.41 In a small subset of neurons such as ischemia-vulnerable hippocampal hilar neurons, these channels may provide the major route for toxic zinc entry.18 In addition, dynamic downregulation of GluR2 subunit in CA1 neurons after global ischemia42 may render these neurons more vulnerable to subsequent zinc entry. NMDA receptor is also a calcium-permeable channel which is blocked by zinc. The flickering voltage-dependent block of NMDA channels by zinc may reflect channel block, followed by slow permeation.43 Suggesting that this route is important in zinc-induced neuronal death, particularly under nondepolarizing conditions, NMDA antagonists reduced TSQ fluorescence and zinc neurotoxicity in cortical culture.23 In addition to these channels, zinc also serves as a substrate for the Na+/Ca2+ exchanger or related molecules.44 Na+/Ca2+ exchanger may facilitate zinc entry into neurons in normal situations and zinc extrusion after intraneuronal zinc accumulation. The precise role of Na+/Ca2+ exchanger in zinc neurotoxicity has not yet been determined.

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6.5 INTRACELLULAR SIGNALING EVENTS MEDIATING ZINC TOXICITY Once zinc enters neurons in large enough quantity, it seems that neurons are doomed to demise. Then, which toxic mechanisms are invoked by raised intracellular zinc? Are the signaling cascades involved in zinc neurotoxicity? Zinc can activate diverse signaling enzymes such as protein kinase C (PKC).45,46 The members of PKC are highly enriched in the central nervous system (CNS) and serve diverse physiological functions.46 For example, it regulates neurotransmitter synthesis, ionic fluxes, exocytosis, gene regulation and modulation of various receptor-mediated signals.48,49 Besides these physiological regulatory functions, alterations in PKC activity are thought to be involved in excitotoxicity,50 ischemic neuronal death,51 and brain trauma.52 Therefore, it seems possible that activation of PKC by zinc may be one of the signaling mechanisms involved in zinc-induced neuronal death. In cortical cultures, PKC activity in the membrane fraction is markedly increased by zinc exposure, to a similar degree in sister cultures exposed to a potent PKC activator phorbol-12-myristate-13-acetate (PMA) (Figure 6.1A).53 A selective PKC inhibitor GF109203X not only blocked the increase in the membrane PKC activity but also neuronal death induced by zinc (Figure 6.1AC). The increase of membrane PKC activity by zinc may not require intermediary cytosolic events, because direct addition of zinc to a reaction mixture containing the membrane isolates in test tube also increases the PKC activity.53 Extracellular signal-regulated kinase (Erk), a member of the mitogen-activated protein kinase (MAPK) family, is thought to have survival-promoting effect on neurons.54,55 Erk is normally activated by the upstream kinase MEK-1, and transmits the signal to the downstream kinase Elk.56 In cortical culture, a brief intense zinc exposure markedly increases Erk activity for a prolonged period of time (Figure 6.2A).57 Indicating that this pathway contributes to zinc neurotoxicity, a specific inhibitor of MEK-1 (PD98056) substantially reduces zinc-induced neuronal death (Figure 6.2B). Whereas Erk conveys survival-promoting signals in many cases, under certain circumstances, it seems to contribute to cell death.58,59 In cortical culture, Erk activation leads to the induction of the transcription factor egr-1 (Figure 6.2C). Experiments with antisense oligonucleotides suggest that egr-1 induction is also a critical event for zincinduced neuronal death (Figure 6.2D).57 Hence, activation of Erk may cause neuronal death via an egr-1-dependent mechanism. In this regard, it is noteworthy that egr-1 was found to play a critical role in ischemic and oxidative injury to brain cells.60,61 Although PKC and Erk/egr-1 pathways seem important for zinc neurotoxicity in our cortical culture, it is likely that the list of involved signaling cascades is much larger. For instance, zinc has been shown to activate Src kinase,62 which upregulates currents through NMDA receptors.63 This type of indirect toxicity may be important in certain pathological situations.

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A Membrane PKC activity (fold increase of radioactivity)

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+ trolox + H89 + OA

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FIGURE 6.1A (A) Forty min after the onset of exposure to indicated drugs (300 µM zinc for 15 min alone or with addition of indicated agents, or 100 nM PMA for 40 min), cells were lysed and membrane fractions were prepared for the evaluation of PKC activity. Bars denote folds of membrane PKC activity (mean + SEM, n = 3) normalized to the mean value in sham wash controls. The increase induced by the 15-min zinc exposure was completely blocked by the addition of 1.2 mM CaEDTA, 1 µM GF109203X, or 50 µM PKC peptide inhibitor 19-36 (PI). The increase in membrane PKC activity by zinc exposure was close to the level increased by 100 nM PMA. (B) Upper panel: Phase-contrast photomicrographs of cortical cultures 24 h after 15 min exposure to 300 µM zinc alone (left) or with addition of the selective PKC inhibitor GF 109203X (1 µM) (right). Lower panel: Fluorescent photomicrographs of the same fields after staining with propidium iodide (bright spots represent nuclei of dead neurons). Scale bar, 100 µm. (C) Bars represent LDH release (mean + SEM, n = 6–9) in sister cultures 24 h after 15 min exposure to 300 µM zinc alone or in the presence of GF109203X (0.1 and 1 µM), a vitamin E analog antioxidant trolox (100 µM), a PKA inhibitor H-89 (1 µM), a phosphatase inhibitor okadaic acid (OA; 100 nM), or a CaM kinase II inibitor KN62 (1 µM). Asterisks denote difference from zinc alone (p < 0.05, two-tail t test with Bonferroni correction for 6 comparisons). (Adapted from Noh, K.M., Kim, Y.H., and Koh, J.Y., Mediation by membrane protein kinase C of zinc-induced oxidative neuronal injury in mouse cortical cultures, J. Neurochem., 72, 1609, 1999, with permission.)

A Zinc (300 µM)

Se

CTL 0.08 0.5 1 2 4 8

0.08 0.5 1 2 4 8

h Erk1/2-p

160 120

PD

CTL

C Zinc

LDH release (%)

B

Zinc+PD

Erk1/2

Erk1/2-p

80

Erk1/2

40

Egr-1

0

+ -

+ +

+

Zinc PD

60 50 40 30 20 10 0

Zi

(A

eg r-1

+

S)

r-1 eg

+

nc

nc

nc

Zi

(N S)

*

Zi

LDH Release (%)

D

FIGURE 6.2 (A) Time course of ERK activation by zinc: Western blots for ERK 1/2 (low) and phosphorylated ERK 1/2 (upper) at indicated hours after sham wash (CTL) or 20 min exposure to 300 µM zinc. Addition of serum was used as a positive control. (B) Bars denote LDH release (mean + SEM, n = 3) in cortical cultures 12 h after 20 min exposure to 300 µM zinc in the absence or presence of PD098059 (75 µM). (C) Western blots revealed that the specific MEK1 inhibitor PD098059 (PD) blocked phosphorylation of ERK 1/2 (ERK 1/2-p) as well as induction of egr-1 in cortical cultures, 4 h after 20 min exposure to 300 µM zinc. (D) Bars denote LDH release (mean + SEM, n = 3), 6 h after 30 min exposure to 300 µM zinc, in cortical cultures with treatment with 40 µM egr-1 antisense Zinc + egr-1 (AS) or non-sense oligonucleotides Zinc + egr-1 (NS). Asterisks denote difference from zinc (p < 0.01, two-tail t test with Bonferroni correction for two comparisons). (Adapted from Park, J.A. and Koh, J.Y., Induction of an immediate early gene egr-1 by zinc through extracellular signal-regulated kinase activation in cortical culture: its role in zinc-induced neuronal death, J. Neurochem., 73, 450, 1999, with permission.)

6.6 AN EFFECTOR MECHANISM OF ZINC TOXICITY: OXIDATIVE STRESS Oxidative stress has been proposed as a critical contributory mechanism in both necrosis and apoptosis.64,65 As a direct death effector, massively generated ROS can rapidly damage cell and mitochondrial membranes causing energy failure, cell swelling, and necrosis. On the other hand, in various models of apoptosis, transient

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ROS generation precedes cytochrome C release and caspase activation, suggesting the role as a signaling event. Zinc-induced neuronal death, at least in our hands, seems to involve oxidative stress to a significant extent. First, increases in ROS and lipid peroxidation levels in neurons accompany zinc neurotoxicity (Figure 6.3A).66 Second, zinc-induced neuronal death is attenuated by various antioxidative measures such as trolox, SOD, and catalase (Figure 6.3B). Consistent with this idea, ROS generation is increased following zinc exposure in neurons that are doomed to die.67,68 Our finding that PKC is involved in zinc toxicity seems consistent with zinc toxicity occurring mainly via oxidative injury. PKC activators such as phorbol esters, also induce oxidative injury in cortical neurons,53 indicating that intense PKC activation is sufficient in causing oxidative injury in these cells.

6.7 INDUCTION AND ACTIVATION OF NADPH OXIDASE BY ZINC Whereas activation of PKC may be a critical step for oxidative injury by zinc,53 the identity of the ROS-generating steps of this toxic reaction were unknown. Thus, we attempted to identify the responsible ROS-generating enzymes in zinc toxicity. NADPH oxidase is a superoxide-producing enzyme consisting of the membrane and the cytosolic components.69 Additionally, small G-proteins regulate its activity.70 Although NADPH oxidase is mainly expressed in phagocytic cells, it is also expressed in nonphagocytic cells.71,72 Furthermore, NADPH oxidase may contribute to ROS generation in NGF deprivation apoptosis of sympathetic neurons.73 Because NADPH oxidase is an enzyme regulated by PKC74,75 it seems possible that NADPH oxidase is an enzyme mediating the PKC-dependent oxidative injury in the setting of zinc neurotoxicity in cortical culture. In fact, zinc exposure markedly increases the levels of NADPH oxidase subunits, particularly p47PHOX and p67PHOX (Figure 6.3C,D), and translocates cytosolic subunits of NADPH oxidase to the membrane (Figure 6.3C), which is a signature event of NADPH oxidase activation.76 Both the induction and activation of NADPH oxidase are dependent on PKC activity (Figure 6.3D). Increased ROS production and neuronal cell death after zinc exposure are attenuated by NADPH oxidase inhibitors (Figure 6.3A,B). All these results suggest that NADPH oxidase is indeed one of the effector enzymes mediating oxidative injury in zinc toxicity. Of course, this result does not imply that NADPH oxidase is the only ROS-generating enzyme in zinc toxicity, and thus further studies should examine the role of other known ROS-generating enzymes such as xanthine oxidase and cyclooxygenase that are upregulated in ischemic brain.77,78

6.8 ANOTHER EFFECTOR MECHANISM OF ZINC TOXICITY: P75NTR/NADE-MEDIATED APOPTOSIS In addition to features of necrosis, those of classical apoptosis are also induced by zinc exposure. For example, zinc exposure induces DNA fragmentation in cortical culture,66 preferentially at low-intensity exposure.79 Also in cerebellar granule neuron

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A

B

100 80 60 40

LDH release (%)

*

20

C

*

* *

0

zinc

trolox catalase SOD AEBSF

cytosol

membrane

C2 Z2 C4 Z4 C2 Z2 C4 Z4 p67KDa p47KDa

gp91

p91KDa

D

+D PI +G FX

p21KDa

CT RL Zi nc

rac1

P67PHOX

cytosol

membrane

CT RL Zi nc +G FX CT RL Zi nc +G FX

E

P67PHOX

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FIGURE 6.3 (A) DCF fluorescence in cortical cultures 3 h after sham wash (left) or 15-min exposure to 400 µM zinc without (middle) or with addition of 50 µM AEBSF, a specific inhibitor of NADPH oxidase (right) during and after the zinc exposure.(B) Bars denote LDH release in cortical cultures 24 h after 15 min exposure to 400 µM zinc without or with addition of trolox (100 µM), catalase (300 mU/ml), Cu/ZnSOD (50 U/ml), or AEBSF (50 µM) after the exposure. Asterisks denote difference from zinc (p < 0.01). (C) Translocation of NADPH oxidase by zinc: Western blots for indicated subunits of NADPH oxidase in the cytosolic fraction and the membrane fraction of cortical cultures, 2 or 4 h after sham wash or 15 min zinc exposure. Whereas levels of rac1 (p21) in each fraction did not change much with zinc exposure, levels of p67PHOX appeared to be decreased in the cytosolic fraction and to be increased in the membrane fraction. Also the membrane levels of p47PHOX markedly increased upon zinc exposure. The level of the membrane-anchored subunit gp91PHOX did not change much with zinc exposure. (D) Western blots show levels of p67PHOX in cortical cultures 4 h after sham wash (CTRL) or 15-min exposure to 400 µM zinc without (Zinc) or with addition of an NADPH oxidase inhibitor, DPI (100 nM), or a PKC inhibitor, GF109203X (3 µM) during and continuously after the zinc exposure. GF109203X markedly attenuated the induction of p67PHOX by zinc. (E) Western blots of the membrane fractions with anti-p67PHOX antibody after 4 h exposure to 400 µM zinc. Addition of 3 µM GF109203X during and after the zinc exposure decreased membrane translocation of p67PHOX by zinc. (Adapted from Noh, K.M. and Koh, J.Y., Induction and activation by zinc of NADPH oxidase in cultured cortical neurons and astrocytes, J. Neurosci., 20, RC111, 2000, with permission.)

cultures, zinc induces mitochondrial injury and subsequent apoptosis.80 As to the mechanism, oxidative stress and mitochondrial injury may lead to secondary apoptosis. In addition to these more or less nonspecific stimuli, p75NTR-dependent apoptosis may specifically mediate zinc-induced apoptosis in cortical neurons.81 p75NTR is a neurotrophin receptor, which binds all neurotrophins with more or less equal affinity.82 Interestingly, activation of p75NTR by neurotrophins elicits not only survival effect but also apoptogenic effect, depending upon the downstream signaling cascades. For the survival effect, p75NTR may facilitate Trk signaling.83–85 By contrast, especially in the absence of Trk, p75NTR can induce apoptotic cell death through caspase activation.82,86,87 p75NTR is a type I transmembrane receptor, which lacks kinase activity but contains death domain motif in its cytoplasmic terminus. Therefore, its cytoplasmic death domain motif may play a critical role in transducing the apoptogenic effect of p75NTR. Several proteins — TRAFs, SC-1, NRIF, NRAGE, and NADE — were discovered to interact with the cytoplasmic domain of p75NTR.88-92 However, although the interaction of TRAF-2, NRIF, and NRAGE with p75NTR has been linked to its apotogenic signaling, no definite evidence is available that any of these mediate p75NTRmediated apoptosis. On the other hand, Mukai and colleagues have shown that NADE may be such a death effector in certain cell types including a neuronal cell line.92 Consistent with the idea that this apoptogenic system is activated in cortical culture by zinc, zinc exposure induces NGF, p75NTR and NADE in neurons (Figure 6.4A). Further indicating that the p75NTR contributes to zinc toxicity, blockade of its signaling with REX (a p75NTR function-blocking antibody) as well as inhibition of NADE induction with antisense oligonucleotides, attenuate zinc-induced neuronal death (Figure 6.4B). In a rat model of transient global ischemia, p75NTR and NADE are co-induced in hippocampal CA1 neurons.81 Chelation of zinc with CaEDTA blocks co-induction of p75NTR and NADE as well as neuronal death, which finding is consistent with zinc being the trigger for p75NTR, NADE, and cell death. As zinc neurotoxicity is likely relevant in other injury models, the role of p75NTR/NADE system in these conditions needs to be investigated.

6.9 PROTECTIVE MEASURES AGAINST ZINC TOXICITY As discussed above, inhibition of the involved signaling events or antioxidative measures might be useful against zinc neurotoxicity. More straightforwardly, application of the zinc chelator CaEDTA that is markedly protective against ischemic and epileptic neuronal death in rats, might be helpful, although the passage through blood brain barrier might pose a practical problem. In addition to these, we have recently reported that a thrombolytic agent — tissue plasminogen activator (tPA) — has remarkable protective effect against zinc toxicity in cortical culture, but independent of its well-known proteolytic effect.93 Whereas the mechanism involved is yet unclear, tPA seems to enhance the removal of zinc from zinc-overloaded neurons, perhaps via Na+/Ca2+ exchangers or related molecules.94

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A Membrane PKC activity (fold increase of radioactivity)

3

FIGURE 6.4 (A) Western blots are for p75NTR and NADE (22kD) at indicated hours after 15-min exposure to 300 µM zinc. Photomicrographs show immunocytochemical staining with anti-NGF antibody of a sham washed control culture (CTRL) and a sister culture 8 h after 15min exposure to 300 µM zinc (Zinc). Scale bar, 200 µm. (B) Left bars denote LDH release in cortical cultures (mean + SEM, n = 4), 18 h after 15-min exposure to 300 µM zinc without (Zinc) or with addition of REX (1/400) (+REX). Right bars represent LDH release in cortical cultures after 20 h exposure to 25 µM zinc without (Zinc) or with 10 µM of NADE anti-sense (+AS #1 or +AS #2) or 10 µM non-sense (+NS) oligonucleotides. Asterisks denote difference from Zinc (p < 0.05, two-tail t test with Bonferroni correction for three comparisons). (Reprinted with permission from Park, J.A., Lee, J.Y., Sato, T.A., and Koh, J.Y., Co-induction of p75NTR and p75NTR-associated death executor in neurons after zinc exposure in cortical culture or transient ischemia in the rat, J. Neurosci., 20, 9096, 2000.)

2

*

*

1

*

0 CNTL

PMA

Zn

+CaEDTA

+GFX

+PI

B

C

100

LDH release (%)

80

60

40

* * *

20

0

Zn alone

0.1

1 + GFX

100

1

0.1

+ trolox + H89 + OA

1 + KN62

Another molecule exhibiting a remarkable protective effect against zinc toxicity is pyruvate.95 Pyruvate is an endogenous triose produced in glycolysis. Although its cytoprotective mechanism has not yet been fully elucidated, it blocks the zinc-induced depletion of nicotinamide ademine dinucleotide (NAD)+ and ATP. As pyruvate is an endogenous molecule derived from normal glucose metabolism, if it keeps its beneficial effect in vivo, it may be used without much concern about side effects.

6.10 SUMMARY Neuronal death induced by endogenous zinc may be a key mechanism of neuronal death in a variety of pathological conditions. Although exceptions may exist, it is

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likely that chelatable zinc in the synaptic vesicles of glutamatergic neurons or other releasable pools of zinc may be the main source of toxic zinc accumulation. Upon accumulating in postsynaptic neurons, zinc can exert neurotoxicity by at least two parallel mechanisms. First, zinc activates signaling molecules such as PKC, Erk, and egr-1, which as a result increase oxidative stress in neurons. Of these, PKC may induce and activate NADPH oxidase, an enzyme-generating toxic ROS. Overall, the oxidative injury in zinc toxicity seems to result mainly in necrosis. Second, zinc induces and activates the apoptogenic NGF/p75NTR/NADE system in cortical neurons, which leads to caspase activation and classical apoptosis. Considering the relevancy of zinc-induced neuronal death in various brain injury conditions, the thorough understanding of the associated toxic mechanisms may help us to find effective neuroprotective measures in those cases.

6.11 ACKNOWLEDGMENTS This work was supported by the Creative Research Initiative Program of the Korean Ministry of Science and Technology.

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52. Joo, F.A., Tosari, Z.O., and Koltai, M., Inhibition by H-7 of the protein kinase C prevents formation of brain edema in Sprague-Dawley DFY rats, Brain Res., 490, 141, 1989. 53. Noh, K.M., Kim, Y.H., and Koh, J.Y., Mediation by membrane protein kinase C of zinc-induced oxidative neuronal injury in mouse cortical cultures, J. Neurochem., 72, 1609, 1999. 54. Boulton, T.G., Nye, S.H., Robbins, D.J., Ip, N.Y., Radziejewska, E., Morgenbesser, S.D., DePinho, R.A., Panayotatos, N., Cobb, M.H., and Yancopoulos, G.D., ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF, Cell, 65, 663, 1991. 55. Segal, R.A. and Greenberg, M.E., Intracellular signaling pathways activated by neurotrophic factors, Annu. Rev. Neurosci., 19, 463, 1996. 56. Karin, M. and Hunter, T., Transcriptional control by protein phosphorylation: signal transmission form the cell surface to the nucleus, Curr. Biol., 5, 747, 1995. 57. Park, J.A. and Koh, J.Y., Induction of an immediate early gene egr-1 by zinc through extracellular signal-regulated kinase activation in cortical culture: its role in zinc-induced neuronal death, J. Neurochem., 73, 450, 1999. 58. Runden, E., Seglen, P. O., Haug, F.M., Ottersen, O.P., Wieloch, T., Shamloo, M., and Laake, J.H., Regional selective neuronal degeneration after protein phosphatase inhibition in hippocampal slice cultures: evidence for a MAP kinase-dependent mechanism, J. Neurosci., 18, 7296, 1998. 59. Murray, B., Alessandrini, A., Cole, A.J., Yee, A. G., and Furshpan, E.J., Inhibition of the p44/42 MAP kinase pathway protects hippocampal neurons in a cell-culture model of seizure activity, Proc. Natl. Acad. Sci. USA, 95, 11975, 1998. 60. Hirata, H, Asanuma, M., and Cadet, J.L., Superoxide radicals are mediators of the effects of methamphetamine on Zif268 (Egr-1, NGFI-A) in the brain: evidence from using CuZn superoxide dismutase transgenic mice, Brain Res. Mol. Brain Res., 58, 209, 1998. 61. Yan, S.F., Fujita, T., Lu, J., Okada, K., Shan Zou, Y., Mackman, N., Pinsky, D.J., and Stern, D.M., Egr-1, a master switch coordinating upregulation of divergent gene families underlying ischemic stress, Nat. Med., 6, 1355, 2000. 62. Manzerra, P., Behrens, M.M., Heidinger, V., Ichinose, T., Yu, S.P., and Choi D.W., Zinc exposure results in the activation of src kinase and the phosphorylation of NMDA receptor subunits (NR2A/2B), Abstr. Soc. Neurosci., 26, 2145, 2000. 63. Yu, X.M., Askalan, R., Keil, G.J., and Salter, M.W., NMDA channel regulation by channel-associated protein tyrosine kinase Src, Science, 31, 674, 1997. 64. Cai, J. and Jones, D.P., Mitochondrial redox signaling during apoptosis, J. Bioenerg. Biomembr., 31, 327, 1999. 65. Kruman, I.I. and Mattson, M.P., Pivotal role of mitochondrial calcium uptake in neural cell apoptosis and necrosis, J. Neurochem., 72, 529, 1999. 66. Kim, Y.H., Kim, E.Y., Gwag, B.J., Sohn, S., and Koh, J.Y., Zinc-induced cortical neuronal death with features of apoptosis and necrosis: mediation by free-radicals, Neuroscience, 89, 175, 1999. 67. Sensi, S.L., Yin, H.Z., Carriedo, S.G., Rao, S.S., and Weiss, J.H., Preferential Zn2+ influx through Ca2+-permeable AMPA/kainate channels triggers prolonged mitochondrial superoxide production, Proc. Natl. Acad. Sci. USA, 96, 2414, 1999. 68. Sensi, S.L., Yin, H.Z., Weiss, J.H., AMPA/kainate receptor-triggered Zn2+ entry into cortical neurons induces mitochondrial Zn2+ uptake and persistent mitochondrial dysfunction, Eur. J. Neurosci., 12, 3813, 2000.

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69. DeLeo, F.R. and Quinn, M.T., Assembly of the phagocytic NADPH oxidase: molecular interaction of oxidase proteins, J. Leukoc. Biol., 60, 677, 1996. 70. Knaus, U.G., Heyworth, P.G., Evans, T., Curnutte, J.T., and Bokoch, G.M., Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2, Science, 254, 1512, 1991. 71. Fukui, T., Lassegue, B., Kai, H., Alexander, R.W., and Griendling, K.K., Cytochrome b-558 alpha-subunit cloning and expression in rat aortic smooth muscle cells, Biochim. Biophys. Acta, 1231, 215, 1995. 72. Jones, S.A., O’Donnell, V.B., Wood, J.D., Broughton, J.P., Hughes, E.J., and Jones, O.T., Expression of phagocytic NADPH oxidase components in human endothelial cells, Am. J. Physiol., 271, H1626, 1996. 73. Tammariello, S.P., Quinn, M.T., and Estus, S., NADPH oxidase contributes directly to oxidative stress apoptosis in nerve growth factor deprived sympathetic neurons, J. Neurosci., 20, RC53, 2000. 74. Heinecke, J.W., Meier, K.E., Lorenzen, J.A., and Shapiro, B.M., A specific requirement for protein kinase C in activation of the respiratory burst oxidase of fertilization, J. Biol. Chem., 265, 7717, 1990. 75. Benna, J.E., Dang, P.M., Gaudry, M., Fay, M., Morel, F., Hakim, J., and GougeritPocialdo, M.A., Phosphorylation of the respiratory burst oxidase subunit p67phox during human neutrophil activation. Regulation by protein kinase C-dependent and -independent pathways, J. Biol. Chem., 272, 17204, 1997. 76. Noh, K.M. and Koh, J.Y., Induction and activation by zinc of NADPH oxidase in cultured cortical neurons and astrocytes, J. Neurosci., 20, RC111, 2000. 77. Kinuta, Y., Kimura, M., Itokawa, Y., Ishikawa, M., and Kikuchi, H., Changes in xanthine oxidase in ischemic rat brain, J. Neurosurg., 71, 417, 1989. 78. Petroni, A., Bertazzo, A., Sarti, S., and Galli, C., Accumulation of arachidonic acid cyclo- and lipoxygenase products in rat brain during ischemia and reperfusion: effects of treatment with GM1-lactone, J. Neurochem., 53, 747, 1989. 79. Lobner, D., Canzoniero, L.M., Manzerra, P., Gottron, F., Ying, H., Knudson, M., Tian, M., Dugan, L.L., Kerchner, G.A., Sheline, C.T., Korsmeyer, S.J., and Choi, D.W., Zinc-induced neuronal death in cortical neurons, Cell Mol. Biol., 46, 797, 2000. 80. Manev, H., Kharlamov, E., Uz, T., Mason, R.P., and Cagnoli, C.M., Characterization of zinc-induced neuronal death in primary cultures of rat cerebellar granule cells, Exp. Neurol., 146, 171, 1997. 81. Park, J.A., Lee, J.Y., Sato, T.A., and Koh, J.Y., Co-induction of p75NTR and p75NTR-associated death executor in neurons after zinc exposure in cortical culture or transient ischemia in the rat, J. Neurosci., 20, 9096, 2000. 82. Casaccia-Bonnefil, P., Gu, C., Khursigara, G., and Chao, M.V., p75 neurotrophin receptor as a modulator of survival and death decisions, Microsc. Res. Tech., 45, 217, 1999. 83. Hempstead, B.L., Martin-Zanca, D., Kaplan, D.R., and Chao, M.V., High affinity NGF binding requires co-expression of the trk proto-oncogene and the low affinity NGF receptor, Nature, 350, 678, 1991. 84. Barker, P.A. and Shooter, E.M., Disruption of NGF binding to the low affinity neurotrophin receptor p75LNTR reduces NGF binding to trkA on PC12 cells, Neuron, 13, 203, 1994. 85. Rydén, M., Hempstead, B., and Ibáñez, C.F., Differential modulation of neuron survival during development by nerve growth factor binding to the p75 neurotrophin receptor, J. Biol. Chem., 272, 16322, 1997.

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86. Frade, J.M. and Barde, Y.A., Nerve growth factor: two receptors, multiple functions, Bioessays, 20, 137, 1998. 87. Gu, C., Casaccia-Bonnefil, P., Srinivasan, A., and Chao, M.V., Oligodendrocyte apoptosis mediated by caspase activation, J. Neurosci., 19, 3043, 1999. 88. Khursigara, G., Orlinick, J.R., and Chao, M.V., Association of the p75 neurotrophin receptor with TRAF6, J. Biol. Chem., 274, 2597, 1999. 89. Ye, X., Mehlen, P., Rabizadeh, S., VanArsdale, T., Zhang, H., Shin, H., Wang, J.J., Leo, E., Zapata, J., Hauser, C.A., Reed, J.C., and Bredesen, D. E., TRAF family proteins interact with the common neurotrophin receptor and modulate apoptosis induction, J. Biol. Chem., 274, 30202, 1999. 90. Chittka, A. and Chao, M.V., Identification of a zinc finger protein whose subcellular distribution is regulated by serum and nerve growth factor, Proc. Natl. Acad. Sci. USA, 96, 10705, 1999. 91. Casademunt, E., Carter, B.D., Benzel, I., Frade, J.M., Dechant, G., and Barde, Y.A., The zinc finger protein NRIF interacts with the neurotrophin receptor p75NTR and participates in programmed cell death, EMBO J, 18, 6050, 1999. 92. Mukai, J., Hachiya, T., Shoji-Hoshino, S., Kimura, M., Nadano, D., Suvanto, P., Hanaoka, T., Li, Y., Irie, S., Greene, L.A., and Sato, T.A., NADE, a p75NTR-associated cell death executor, is involved in signal transduction mediated by the common neurotrophin receptor p75NTR. J. Biol. Chem., 275, 17566, 2000. 93. Kim, Y.H., Park, J.H., Hong, S.H., and Koh, J.Y., Nonproteolytic neuroprotection by human recombinant tissue plasminogen activator, Science, 284, 647, 1999. 94. Kim, Y.H. and Koh, J.Y., EGF receptor-dependent cytoprotection by tPA and HGF against zinc toxicity in cortical culture, Abstr. Soc. Neurosci., 26, 775, 2000. 95. Sheline, C.T., Behrens, M.M., and Choi, D.W., Zinc-induced cortical neuronal death: contribution of energy failure attributable to loss of NAD+ and inhibition of glycolysis, J. Neurosci., 20, 3139, 2000.

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7

Cerebral Ischemia and Adenosine: “Spheres of Action”* Dag K.J.E. von Lubitz

CONTENTS 7.1 Spheres of Action 7.2 Introduction 7.3 Pathology of Stroke 7.4 Adenosine Release During Ischemia 7.5 Adenosine Receptors 7.6 Disease and Drug-Induced Adenosine Receptor Fluctuations 7.7 Adenosine Actions During Cerebral Ischemia 7.8 Indirect Effects of Adenosine and Stroke 7.9 Adenosine and the Treatment of Ischemic Stroke 7.10 Conclusion References

7.1 SPHERES OF ACTION Until recently, the main focus of studies of adenosine and its receptors in the context of stroke concentrated on A1 receptors whose stimulation has been consistently shown to result in the reduction of brain damage following experimentally induced global and focal brain ischemia in animals. The promising results of these experiments led many authors to propose the development of therapeutics targeted specifically at A1 receptors and their clinical testing as therapeutics not only against stroke-induced damage but in the context of other neurodegenerative diseases as well. The most recent discoveries indicate that adenosine-mediated actions might be far more complex than originally anticipated, and they may range from purely protective to lethal. The systemic complexity of adenosine-induced effects appears to cast doubts about the rapid development of stroke treatment based on the direct activation of the adenosine receptors, although improving the understanding of these effects may lead to entirely new notions about the treatment of neurodegenerative * Letter from Lord Manville to Count von Münster.

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disorders through the manipulation of secondary processes triggered by the adenosine receptor stimulation. On the other hand, it is equally likely that the current exceptions may set the final rule. Thus, the treatment of vascular dementia based on the broad concept of adenosine receptor stimulation is the subject of current clinical tests, and the clinical trials of aspirin and dipyridamole (adenosine uptake inhibitor) have been also concluded very recently. In both cases, the results are highly promising and warrant a larger scale effort. There is no doubt that the present explosive growth of interest in adenosine will have a significant impact on disciplines other than cardiology, where adenosine has been accepted and used for well over a decade.1 It is particularly in the context of the nervous system and its disorders that the attitudes undergo a very rapid change. Until recently, studies of neural effects of adenosine were the domain of the “selected few,” with the majority considering such work as the fringe of the “en vogue” neuroscience. The persistence of those who continued their work was followed by the growing recognition of the paramount importance of adenosine in the modulation of a wide range of phenomena extending well beyond the control of neurotransmitter release. It is, therefore, increasingly likely that the improved understanding of the significance of adenosine in normal and pathological functions of a living organism may ultimately result either in the prophylaxis of stroke or in the therapies aimed at the reduction of poststroke brain damage.

7.2. INTRODUCTION Stroke is the second most common cause of death and only heart disease causes higher mortality.2 Paradoxically, while the rate of strokes decreases, their absolute number, due to the advancing average age of the population, begins to increase. Moreover, the ready acceptance of the traditional Western style of life with its dietary excesses, sedentary life, and the exposure to a wide variety of environmental toxins results in the increased number of strokes in the societies where cerebrovascular disorders were an exception rather than the rule as seen in Europe and in the U.S. There is no doubt that stroke, even in the mildest of forms, is a devastating experience to the patient and close relatives. Even in the absence of physical deficits, the psychological impact may affect the entirety of future life and reduce its subsequent quality. For the society as such, the emotional impact of a person’s experience of stroke is, sadly enough, of no essential interest. Even royal and presidential strokes are matters of only a passing curiosity. However, the price of the treatment of stroke has a paramount impact. It affects the entire society, it has its own component within the entire societal tax burden, it diverts resources from other diseases, it demands creation of highly specialized units, raising the cost of hospital operations. And the treatment of stroke is expensive. It belongs among the most costly of disorders, not only in terms of the immediate clinical interventions, but also as a continuing fiscal burden during the subsequent, and frequently protracted, rehabilitation period3 followed by often lifelong disability that may severely affect the continuation of one’s previous professional life.4 In its most severe form, stroke-related deficits may pre-

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vent execution of even the simplest daily functions, requiring continuous advanced nursing care and specialized facilities. Unsurprisingly, a lot of effort and resources are spent to develop the efficient means of treating this disorder.5 The notion of stroke treatment based on adenosine and its receptors has been advocated for over two decades.6–9 Theoretically at least, and the theory was supported by a significant amount of bench-derived evidence, stimulation of adenosine receptors (primarily A1) by their agonists resulted in a wide range of neuroprotective effects that were of paramount interest for the clinical world dealing with the treatment of stroke. The release of toxic neurotransmitters was reduced, synaptic transmission depressed, activation of N-methyl-D-aspartate (NMDA) receptors was markedly attenuated, while Ca2+ influx into the postsynaptic neurons was quite impressively diminished. Moreover, there was the reduction of neuronal activity, blood vessels were relaxing, and the reduction of cerebral metabolism causing hypothermia — a side effect of quite a significance in stroke — constituted the additional blessing and a background of a colossal debate whether neuroprotection by A1 receptor agonists was real or a figment of imagination. Still, there were also molecular effects with beneficial genes and proteins expressed. But most importantly, at least from the clinical (and neuropathological, as it were) point of view, in animal models at least, there was a very striking reduction of postischemic mortality accompanied by an equally striking reduction of neuronal damage that hardly any other drugs could or can surpass. Unhappily, there was also a profound hypotension and bradycardia, unwanted excitation in the hippocampus resulting from the activation of A2A receptors, apoptosis caused by the stimulation of A3 sites, and — as of late — the discovery of lethal effects of adenosine itself.10–12 Moreover, prolonged exposure to adenosine receptoracting agents results in desensitization whose importance in terms of practical treatment is, at best, very poorly understood.

7.3. PATHOLOGY OF STROKE The events that result from the occlusion of a cerebral vessel, particularly a major one, have been the subject of numerous reviews to which the reader is referred for further, detailed information13–22 (see also other chapters in this volume). To provide the context for the discussion that follows, suffice to say that all cellular constituents of the brain are susceptible to ischemic damage. While cerebral ischemia may be global (e.g., resulting from heart attack, strangulation, or drowning) or focal (stroke, and as a constituent of mechanical brain damage), the arrest of cerebral blood flow invariably results in a rapid depletion of ATP, failure of ionic pumps and collapse of ion homeostasis, release of excitotoxic neurotransmitters, and the subsequent neuronal calcium overload that triggers a series of highly destructive enzymatic cascades. Cumulative consequences of the ensuing free radical damage, degeneration of cytoskeletal proteins, loss of cellular membrane integrity, and degradation of DNA lead ultimately to either apoptotic or necrotic death. Reperfusion damage with its own contribution of cerebral destruction amplifies the severity of the original ischemic event.

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Until recently, it has been uniformly thought that endogenous adenosine is a benevolent yet powerful agent whose release during stroke antagonizes many of the damaging processes involved in its evolution.6,8,23 Recent studies of this cell-protecting “retaliatory metabolite”24 show that the relationship is not as straightforward as originally thought, and the excess of extracellular adenosine may be lethal.10

7.4 ADENOSINE RELEASE DURING ISCHEMIA Neurons, glia, endothelium, and even blood provide the source of extracellular adenosine in the brain, although some studies25 indicate that glia may be its primary source.26,27 Under normal conditions, adenosine produced through catabolic activity6,8 is released into the extracellular space along its concentration gradients using a system of bidirectional nucleoside transporters.6 In addition, extracellular cleavage of ATP by ecto-5′-nucleotidases provides another significant source of interstitial adenosine.28 Metabolic stress induced by elevated electrical activity, hypoxia, or ischemia result in a very rapid degradation of ATP which, in turn, leads to significantly increased liberation of adenosine6,8 whose concentration may be elevated even further by leakage from the damaged or necrotic cells.26 The true concentration of adenosine at rest is difficult to determine due to its very rapid metabolism, and even the most accurate measurements by means of indwelling microdialytic probes result in a range of 30 to 300 nM.29 There is, however, little doubt that ischemia, head injury, and seizures rapidly increase basal concentration of extracellular adenosine to a 30- to 100-fold higher level.30–32 It appears that the process is not uniform and substantial regional differences have been observed31 which, in turn, may be related to the variations in the distribution of adenosine deaminase. The activity of the latter enzyme, together with that of adenosine kinase, decreases during ischemia while the contribution of ecto-5′-nucleotidases increase. Since the efficiency of the equilibrative nucleoside transporter (rENT1) decreases as well, the overall result of these changes is a rapid shift toward the generation of extracellular adenosine.33 The process of hypoxic/ischemic generation of adenosine appears to be closely and sequentially tied to the release of excitotoxic neurotransmitters since in experimental focal ischemia (and very likely in human stroke as well) the reduction of cerebral blood flow (CBF) to 25 ml/100 g/min is sufficient to elevate intracerebral concentration of adenosine.34 It thus precedes the release of excitatory amino acids that occurs at CBF levels of approximately 20 ml/mg/min.34 It seems that the brain is capable of detecting the “level of criticality;” and by turning the endoprotective mechanism of adenosine-mediated effects, it attempts to either prevent or at least delay flooding of the extracellular space with the cytotoxic neurotransmitters. From the clinical point of view, while there is a wide variety of stroke-characteristic processes that may result in adenosine release,35 the characteristic shifts of its concentration may be used as a very sensitive indicator of cerebral ischemia.36,37

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7.5. ADENOSINE RECEPTORS Four adenosine receptor subtypes have been identified and characterized (A1, A2A, A2B, and A3. See Ref. 38). Although all subtypes belong to the G-protein-coupled superfamily of receptors, differences in the specifics of the transducer system within the adenosine group have been noted, with A1 and A3 interacting with Gi/Go proteins, and A2A and A2B with Gs.39 The affinity of individual receptor types for adenosine varies. Thus, the affinity of A2 receptors is lower than that of A1, while that of A2B is lower still. A3 receptors appear to have the lowest affinity. The further point of distinction is the effect of adenosine receptor stimulation on cyclic AMP, where activation of A2 subtype elevates, while activation of both adenosine A1 and A3 receptors reduces the intracellular concentration of cAMP.40,41 Finally, and contrary to A2 receptors, stimulation of both A1 and A3 subtypes leads to the increased activity of phospholipase C,42 while A3 receptors appear to be involved in the regulation of phospholipase D as well.43 Release of internal calcium stores and phospholipase C activation are among the injurious mechanisms characteristic of stroke.17 Hence, the ability of adenosine receptors to stimulate phosphoinositide hydrolysis, thereby influencing the subsequent release of internal Ca2+ stores,44–46 may need further experimental attention in the context of cerebral ischemia and cerebroprotective therapies. Studies using radioautographic and functional assays in both animal and human brains have demonstrated the presence of neuronal and astrocytic adenosine A1 receptors in the hippocampus, I, IV, and VI laminae of the cortex, superior colliculus, and cerebellum.47–52 Both A2A and A2B receptors are found on the smooth muscle fibers and on the endothelial cells of cerebral blood vessels.53 However, the densest population of high affinity neuronal adenosine A2A sites is present in the striatum.47,54 In addition, functional assays indicate the presence of A2A receptors on astrocytes and microglia, and low affinity A2B receptors on astrocytes.55–58 Receptor binding studies show that adenosine A3 receptors are distributed throughout the entire brain, although at a density that is significantly lower than that of the other subtypes.59 However, Rivkees et al.60 studied the distribution of A3 receptors using specific A3 mRNA probes and radioligands, and they failed to confirm the presence of cerebral A3 receptors. This is a very surprising finding in view of specific cerebral effects elicited by the exposure to A3 receptor agonists and antagonists administered either prior or following both focal and cerebral ischemia.8,61–64 Moreover, electrophysiological experiments confirm the presence of neuronal A3 receptors as well.65 While the 3I-AB-MECA used by Rivkees et al.60 is not fully selective for A3 receptors (although, at the moment, it is the only radioligand available for the studies of A3 receptors), the lack of mRNA-based detection is unquestionably an important finding that needs further exploration.

7.6 DISEASE AND DRUG-INDUCED ADENOSINE RECEPTOR FLUCTUATIONS The density of at least A1 receptors is subject to circadian variation.66 Hence, measurably decreased density of A1 receptors at night may be a potentially important

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contributor to the damage caused by the ischaemic stroke whose circadian rhythms have been described as well.67,68 Significantly, A1 receptors are also affected by hypertension that results in their loss69 or desensitization.70 Since hypertension is frequently associated with stroke,71,72 reduced efficacy of adenosine A1 receptor-mediated neuromodulation may be one of contributory elements to the onset and the subsequent exacerbated cerebral damage seen in stroke patients with prior history of hypertension.73,74 Age, another factor predisposing for stroke, is also associated with the adenosine receptor changes: the density of A1 receptors decreases75–77 although the affinity remains unaltered.76,78 Persistent, age-related increase in the adenosine concentration in the extracellular space of the aged brain may be related to the reduced number of A1 receptors.78 Both non-Alzheimer and Alzheimer disease-related dementias are also associated with the loss of A1 receptors,79,80 but it is unclear whether the loss is the consequence of the ongoing neuronal destruction or one of its causes. Nonetheless, just as with hypertension, dementias of various etiologies are associated with increased risk of stroke81–83 and elucidation of the possible influence of the functional loss of A1 receptor (either through the decrease of their number or aberrations of binding properties) on the incidence and outcome of stroke in patients with dementia may be of therapeutic significance. Importantly, aging seems to have opposite effects on A2A receptors, resulting in the increase of their absolute number, coupling to G-proteins, and efficiency.84,85 There are no studies that address the significance of these discrepancies. However, at a purely speculative level, the enhanced presence of A2A receptors in older animals and in aging humans may represent an adaptive change of the endogenous set of cerebroprotective mechanisms, where age-related inflammatory processes pose a greater level of risk than other forms of cerebrovascular pathology. Hence, it is quite possible that the enhanced level of interstitial adenosine, increased receptor density, and the well-documented anti-inflammatory consequences of adenosine A2A receptor stimulation,86 are simply the result of a gradual, age-dependent shift of the protective tasks of the “adenosine complex” whose target is a different, but biologically most likely, form of injury. Under normal circumstances, the process of downregulation and desensitization of G-protein coupled receptors plays an important role in the coordination of their biological effects.87 However, since downregulation of adenosine receptors is also induced by the prolonged ligand exposure,88–90 the influence of this phenomenon on the potential therapeutic outcomes must be evaluated when adenosine-based therapies are considered therapeutic importance. The issue is emphasized by several studies of experimental ischemia showing reversal of therapeutic effect induced by a long-term preischemic exposure to A1 agonists and A2 antagonists.8,35,88,91,92 Finally, although all adenosine receptors desensitize, the process is much faster at A3 receptors than at the other subtypes.93–95 There is thus a possibility that despite the similarity of the second messenger systems, specific “downstream” effects elicited by the stimulation of individual receptor subtypes are both distinct and independent of each other,96 providing another target for eventual therapeutic utilization. Although rapid desensitization of A3 receptor may play a significant role in stroke pathology and in the neuroprotective complex provided by adenosine and its

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receptors,97,98 much more is known about ischemia-induced fluctuations of A1 receptors. Cerebral ischemia and stroke reduce both their density99–101 and the expression of A1 as well as A2 receptor mRNA.101 In focal ischemia, the initial decrease in A1 receptor density is slow during the first 24 hrs of reperfusion.100 There is thus a possibility that functional A1 sites present in the penumbra98 may offer a very tempting target for therapeutic interventions.19,20,35,102 The loss of A1 receptors is much faster following global ischemia, particularly in the selectively vulnerable regions.99,103 Since the reduction of adenosine binding sites does not correspond to the loss of neurons, and since the interaction of A1 receptors with their associated G-proteins is preserved,104 it is most likely that sequestration rather than full receptor degradation takes place during the initial 24 hr of postischemic reflow. The definitive loss of adenosine receptors is the consequence of the subsequent degeneration of adenosine receptor mRNA,101 followed by the physical disappearance of irretrievably damaged neurons. From the therapeutic point of view, the postischemic reduction in the number of functional adenosine A1 receptors may have a substantial impact on the neuronal capacity to withstand ischemic stress due to the long-lasting disinhibition of excitatory inputs and the ensuing protracted low-level activation of NMDA sites.35

7.7 ADENOSINE ACTIONS DURING CEREBRAL ISCHEMIA Together with γ-aminobutyric acid (GABA), adenosine acting at A1 receptors serves as the principal inhibitory neuromodulator in the brain.105 A2 receptors support excitatory and anti-inflammatory events,106–110 while the role of adenosine A3 receptors in neuronal functions continues to be ill-defined. The inhibitory effects of adenosine A1 receptor stimulation are both pre- and postsynaptic, and their details have been discussed in several recent and extensive reviews.6,7,35 Here, suffice to say that among the chief consequences of presynaptic A1 receptor activation that are relevant to cerebral ischemia and stroke is the inhibition of presynaptic calcium currents,111 and the concomitant reduction in the liberation of glutamate, acetylcholine, dopamine, noradrenaline, and serotonin.112–114 Release of GABA remains essentially unaffected.115–117 Postsynaptic consequences of A1 receptor stimulation8,35,118 are reflected in the stabilized postsynaptic membrane potential and depressed excitability of NMDA receptors. As a consequence of these actions, the postsynaptic influx of Ca2+ — one of the major causative factors in the development of the subsequent neurodestructive cascades — is attenuated.119 Recently, Brundege and Dunwiddie120,121 showed that adenosine generated through the activity of postsynaptic pyramidal neuron in the hippocampus is transported to the extracellular space. It then interacts with A1 receptors located on the very same neuron and inhibits its electrophysiological activity. Since the process of egotropic inhibition35 may account for approximately 80% reduction in excitatory responses,120 ischemiainduced internalization of A1 receptors has a devastating impact on their ability to reduce the intensity of the events that ultimately lead to the functional derangement of brain tissue.

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A number of studies demonstrated recently the involvement of adenosine A1 receptors in phosphoinositol metabolism mediated through the activation of phospholipase C.122–126 Pathologically modified metabolism of phosphoinositides is a potent contributor to the generation of postischemic injury, particularly through its effect on the inflammatory processes.127,128 Hence, the recent findings that adenosine A1 receptor agonists and bradykinin — a very powerful autacoid released during ischemia and promoting both blood–brain barrier opening and the development of brain edema129 — act synergistically in the release of inositol (1,4,5)-triphosphate (IP3) from renal artery smooth muscle cells in vitro are of significant interest.122 The possibility that such synergies are tissue-specific or even characteristic of individual species cannot be excluded since, for example, in the Chinese hamster ovary (CHO) cells A1 receptor agonist N6-cyclopentyladenosine (CPA) promotes IP3 release,130 while in the hippocampus the same agonist has a powerful inhibitory effect.131 In the presence of another proinflammatory mediator, histamine, selective adenosine A1 receptor agonists diminish IP3 release from cerebromicrovascular endothelium as well.132 Both A2A and A3 receptors are involved in the adenosine-histamine interplay, and the activation of A2A and A3 receptors located on mast cells produces opposite effects on histamine release and microvascular responses, with A2A inhibiting133 and A3 receptors stimulating degranulation of mast cells and the consequent vasoconstriction and increased vasopermeability.134–138 The existence of these complex and still incompletely elucidated effects indicates the clear need for further study of the interactions between adenosine and other receptor types located on both neurons and glia, particularly in the pathologically altered environment. Involvement of adenosine in the synthesis of nitric oxide represents another complex pattern of interactions between adenosine and other trophic systems of the brain.139–141 A1, A2A, and A3 receptors seem to participate in nitric oxide synthesis, release, and signaling,98,142 although receptor-independent role of adenosine alone has been described as well.143 In the context of cerebral ischemia and stroke, NO is considered as one of the major participants in the generation of the subsequent damage.144–150 On the other hand, it is also known that a variety of nitric oxide synthase isoforms exist149,151 — some clearly destructive (inducible, neuronal), others having neuroprotective functions (endothelial). However, the details of the relationship between adenosine, its receptors, and the specific aspects of ischemic NO synthesis and its release are practically unknown, despite their potential importance in the context of stroke therapies. Exposure to agents enhancing extracellular concentration of adenosine as the means of protecting the brain from ischemic damage represents one such approach.152,153 The contribution of adenosine and nitric oxide to vasorelaxation under both normal and pathological conditions has been the subject of several recent papers. 139–141,154–157 The fact that A2A receptors are the main participants in the regulation of both normoxic and hypoxic cerebral blood flow has been known for several years.158,159 However, their roles during postischemic reperfusion are far less certain,160 although a significant improvement of postischemic cortical blood flow has been described following either inhibition of adenosine transport by 4-nitrobenzylthioinosine (NBTI) in pigs,153 or acute treatment with A2A receptor agonist

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2-[(2-aminoethylamino)-carbonylethylphenylethylamino]-5′-N-ethylcarboxoamidoadenosine (APEC) in gerbils.161 While the importance of A1 receptors in experimentally induced ischemia and stroke has been established in a number of studies,6–8,23,35 the available data underline a contradictory role of A2 receptors in the pathology of cerebrovascular disease. There is now no doubt A2A receptors play essential functions in the normal and pathologically altered brain.90,106,107 Moreover, experiments in which adenosine A2A receptors were inhibited by the selective antagonists immediately prior to the arterial occlusion showed significant reduction of cerebral damage induced by both global 161–164 and focal ischemia.165 Decreased infarct volume and improvement of neurological recovery have been also described in A2A receptor deficient mice exposed to experimental stroke.166 Reduction in the release of excitatory amino acids consequent to A2A antagonist administration167,168 is undoubtedly among the mechanisms involved in the demonstrated protection of the ischemic brain,161–165 especially since inhibition of the excitatory actions mediated by A2A receptors would enhance the attenuating impact of A1 receptors. Blockade of microglial vascular A2A receptors may confer additional protection as has been recently suggested by Ongini et al.,164 while Stone and Behan169 indicated the possibility of both glial and vascular components as well. Furthermore, an intriguing mechanism involving interplay of A1 and A2A receptors has been described by Phillis,170 who showed that exposure to the A2 agonist CGS 21680 inhibits spontaneous firing of cortical neurons due to the possible enhancement of A2 receptor-mediated GABA release. The persistent release of GABA in the striatum of rats exposed to experimental middle cerebral artery occlusion171 may be the direct consequence of the interactions described by Phillis. If such assumption were true, then, despite consistently demonstrated neuroprotective effects of A2 antagonists, it is likely that the effects observed by Phillis may play an important part not only in the normal functions of cerebral neurons, but also as a very important constituent of the protective actions sustained by A2A receptors during the postischemic reflow stage. A2A receptor agonists have been consistently shown to prevent platelet aggregation, neutrophil adhesion to the vascular endothelium, and the subsequent phagocytosis,172 all of which are intimately involved in postischemic reperfusion damage.173–175 Since neutrophil invasion begins approximately 15 hours after ischemia,176 it is possible that the protective effects resulting from A2A receptor activation can be best obtained by time-specific exposure to appropriate agents, i.e., antagonists to reduce the impact of excitotoxicity in the immediately postischemic period, followed by agonists at a stage where their protective impact on neutrophil activation would be most pronounced and have the greatest chance of interrupting inflammatory cascades.177–179 Apart from acute brain pathologies, A2A receptors are also intimately involved in chronic diseases of the CNS, e.g., Parkinson’s, Alzheimer’s, and Huntington’s. A series of recent reviews has been devoted to this subject and to the possible use of A2A receptor-acting agents as therapeutics.180–182 Cerebral functions of adenosine A2B receptors are very poorly known. It has been shown that they contribute to the accumulation of cAMP in primary astrocyte culture183 and mediate elevation of interleukin-6 mRNA in human astroglioma cells.56

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There is a possibility that the involvement of A2B receptors in the activity of mast cells184 may have an indirect effect on the brain either through vascular phenomena, e.g. regulation of leukocyte adhesion,185,186 or through mast cell participation in brain pathology, e.g., allergic encephalomyelitis.187,188 However, specific studies are needed in order to determine A2B receptor participation in the pathophysiology of stroke. Very recently it has been reported that A2B receptors serve as the receptors of netrin1,189 a critical protein involved in the control of axonal elongation and pathfinding.190 Although there is no information of the role of netrin-1 in postischemic repair of brain damage, at least one report demonstrates aberrations in neuronal circuitry and activity in netrin-1 deficient mice. Taken together, the limited data on A2B receptors indicate both their potential importance in cerebral functions and the clear need for more extensive studies of this adenosine receptor subtype. Stimulation of A3 receptors causes degranulation of mast cells and hypotension.98 Thus, in similarity to adenosine A2B receptor, the A3 receptor subtype may be involved in some aspects of cerebral blood flow regulation, although, due to its low affinity for adenosine (1.0M as opposed to 10–30 nM for either A1 and A2 receptor respectively), its impact would be felt, most likely, only during stroke, seizures, or other forms of extreme metabolic stress, i.e., under conditions that markedly elevate extracellular adenosine concentration. The success of A3 receptor agonists in experimental treatment of cardiac ischemia191,192 shifted attention away from the brain. However, the available results indicate a very high complexity of A3 receptor involvement both in the normal activities of the brain as well as in cerebral pathology.35,97,98,193 Electrophysiological studies have shown that activation of hippocampal adenosine A3 receptors results in desensitization of the adenosine A1 receptor-mediated inhibition of excitatory synaptic transmission,65 while in CA3 neurons stimulation of the adenosine A3 receptors potentiate calcium currents.194 In addition, activation of A3 receptors results in a sustained activation of phospholipase D195 necessary for neutrophil phagocytosis and generation of the oxidative bursts.196–199 Whether the A3 receptors are involved in postischemic inflammatory processes is unclear,177 especially that their stimulation has been shown to inhibit both the induction of TNF gene and the liberation of this cytokine in murine macrophages.199,200 Yet, treatment with N6-(3-iodobenzyl)-5′-(N-methylcarbamoyl) adenosine (IB-MECA) immediately prior to focal ischemia in mice results in the enhanced activation of microglia201 known to be the primary source of inflammatory cytokines.202–204 However, the enhanced activation of microglia observed in the latter study may be the result of prolonged hypoxia caused by a significant delay in the return of normal cerebral perfusion97,98 rather than the direct consequence of microglial A3 receptor activation. Adenosine A3 receptors have been suggested to act as the regulators of cellular differentiation and death,10 with low (nM) concentrations of A3 agonists promoting differentiation, and high (M) inducing apoptosis. Studies of neurotoxicity in vitro,205 and cerebral ischemia in vivo64,97,98,201 confirm the observations of the destructive nature of A3 receptor activation. In vitro, only high (>10 M) concentrations of the selective adenosine A3 receptor agonist Cl-IB-MECA were capable of inducing necrotic death of cultured rat cerebellar granule cells. However, exposure to nontoxic levels of

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glutamate (50 M) followed by the addition of only 1 M Cl-IB-MECA induced very swift necrosis.205 In vivo, pretreatment with IB-MECA prior to global ischemia or focal ischemia resulted in a very significant increase of postischemic morbidity and mortality.61,97,98 Chronic treatment with IB-MECA, and acute preischemic administration of the selective A3 receptor antagonist 3-ethyl-5-benzyl 2 methyl-6phenyl-4phenylethynyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS 1191) reversed these effects, indicating that the effects are, indeed, receptor mediated. Moreover, chronic treatment with A3 agonist or acute exposure to the antagonist results in a longlasting depression of NOS. Upregulation accompanies acute treatment with the agonist IB-MECA.97,98 As with all other forms of adenosine and NO interaction (see above), the type of nitric oxide synthase that is affected remains to be determined, preliminary observations indicate that the chronic treatment with IB-MECA depresses neuronal rather than epithelial synthase. (von Lubitz and Lin, unpubl.) Rapid induction of astrocyte apoptosis64 by micromolar concentration of adenosine A3 receptors indicates that increased concentration of extracellular adenosine will have an adverse, maybe even destructive, impact also on these cells.64,193,206 Nonetheless, it has been suggested recently35,98 that, despite their seemingly highly destructive role, A3 receptors constitute an important element of the cerebral endogenous protection complex. Accordingly, in focal ischemia the main function of A3 receptors is to promote isolation of the irreparably damaged volume from the still surviving cerebral tissue by a rapid induction of astrocyte proliferation.10,193 The rapidly expanding astrocytic processes create both a physical and functional “wall” surrounding the irreparably damaged ischemic core that may provide an extension of the time available for marshalling other endogenous repair mechanisms (e.g., redirection of blood flow to still salvageable parts of the brain, activation of A1 receptors in more distal regions, and so forth). Experimental support for this hypothesis has been recently provided.61,201

7.8 INDIRECT EFFECTS OF ADENOSINE AND STROKE Even moderate hyperthermia has a profoundly adverse effect on the outcome of stroke, and elevation of body temperature by as little as 10C increases the risk of poor outcome 2.2-fold.207 Induced hypothermia has been frequently suggested as a therapeutic intervention in stroke,208–210 and the experimental and clinical studies of its effects on the outcome of traumatic brain injury and stroke indicate the beneficial effect of lowered brain temperature.211–218 Both peripheral and central exposure to adenosine receptor A1 agonists results in a significant hypothermia209 consequent to the depression of energy metabolism.219–221 Under normal conditions, and at concentrations of extracellular adenosine close to the receptor affinity constant, the depressant effect of adenosine on cerebral energy metabolism is insignificant.222 However, its modulatory effect on energy demand/supply becomes much more pronounced in ischemia or seizures, i.e., when the concentration of extracellular adenosine is substantially elevated.223

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Quantitative studies of brain metabolism established a direct relationship between brain temperature and metabolism,215 while mapping cerebral metabolism indicates that functional activity elevates metabolic rate predominantly within the terminal projection zone of the activated pathway,224 i.e., within the area topographically corresponding to the location of adenosine A1 receptors.225 It is thus more than likely that the hypothermic effects of adenosine A1 receptor activation are related to their depressant impact on the electrical activity, the latter responsible for at least 40% of the cerebral metabolism.226 Moreover, since hypothermia induces supersensitivity of adenosine receptors,227 it is conceivable that the temperature, lowering these effects of A1 receptor agonists, may play a part in the robustly neuroprotective effects following stimulation of these receptors6,7,8,35 not only as a result of reduced metabolism but also due to the modification of receptor binding properties. It is also of significant interest that a substantial reduction of cerebral metabolism and neuroprotection induced by the commonly used anesthetic isoflurane228–230 may involve adenosine A1 receptors.231 On the other hand, the report of the predictive value of the effects of isoflurane on the extent of cerebral damage following ischemia232 indicates the need for particular caution when determining the true nature of the involved receptors. This is particularly true in the studies that combine the use of this common anesthetic with the goal of assessing the protective value of specific receptor ligands. Reduced blood pressure is frequently associated with less than satisfactory outcome of the ischemic stroke,233–235 and several studies have shown that systemic administration of adenosine (and many of its analogues) causes hypotension.236–238 Whether this serious side effect can be eliminated by careful dosing and administration rate of adenosine and its related compounds,239 or by employment of new drugs with reduced cardiovascular profile240 remains to be conclusively demonstrated. Successful simulation of the former approach has been recently performed using computer modeling and a human patient simulator. (von Lubitz et al., unpubl.)

7.9 ADENOSINE AND THE TREATMENT OF ISCHEMIC STROKE Since elevation of the extracellular adenosine concentration in stroke is both a siteand event-specific phenomenon,241 the use of adenosine itself, either through drugs enhancing its production or arresting its breakdown, would appear to be the most obvious therapeutic approach in the context of stroke. After all, adenosine has long-standing FDA approval for clinical use in supraventricular tachycardia, and it has been used with very positive results during angioplasty in acute myocardial infarction.242 Moreover, the half-time of the (typically) bolus-injected adenosine is very short, and any side effects that may be present when adenosine concentration is elevated for a protracted period are irrelevant in this context. Preconditioning may offer an alternative approach and its potential benefits in cardiovascular disorders have been recognized for a long time.243 There is a distinct probability that transient ischemic attacks (TIAs) may result in attenuation of the subsequent stroke in humans.244 Whether TIAs “prepare” the brain for a major event remains to be determined. There

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are, however, several experimental indicators (discussed in this volume) indicating such probability. The involvement of K+-ATP ion channels and protein kinase C in preconditioning has been demonstrated by several authors.245–248 Since adenosine A1 receptors have been shown to regulate both PKC and K-ATP channels, it is not surprising that the major preconditioning role of both adenosine and A1 receptors has been described.249–251 Moreover, it has been shown that preconditioning upregulates the density of adenosine receptors.252,253 The prospect of using endogenous adenosine as a treatment of stroke (and other neurological disorders) either through increasing the number of available receptors, or through the exposure to adenosine uptake blockers,254,255 raises the possibility of side effects, as clearly evidenced by the results of Abbracchio and her colleagues.10,193,256 The studies have shown that high concentration of adenosine analogue (2-chloro-adenosine; CADO) promotes apoptotic and necrotic death of several cell types10,193,257 Moreover, preexposure of proliferating myoblasts and differentiated myotubes to CADO sensitizes them to glutamate, and results both in a large increase of intracellular Ca2+ concentration and destruction of cytoskeletal integrity. Based on these results the authors proposed a novel and fascinating hypothesis of a completely novel pathology for muscle dystrophies.10,257 It is unknown whether these results apply directly to the ischemic conditions in the brain, and what effect preexposure to adenosine has on neurons during clinical stroke. However, our own data quoted in the preceding section of this review61,97,98,205 indicate that pretreatment with A3 receptor agonist is, indeed, deleterious. Furthermore, chronically elevated extracellular concentration of adenosine may result in severe systemic disturbances as indicated by Blackburn et al.258 who described pulmonary pathology followed by death within 3 weeks in adenosine deaminase-deficient mice. There are significant advantages to the prophylactic stroke treatment based on antiplatelet therapy.259 The results of the recent European Stroke Prevention Study showed that both salicylic acid and dipyridamole were effective in reducing the incidence of ischemic stroke and transient ischemic attacks.260 Piccano and Abbracchio261 presented a tempting explanation based, in part, on “adenosine theory,” suggesting that adenosine-related cerebrovascular and neuroprotective effects of the administered dipyridamole (adenosine uptake blocker and a clinically used antiplatelet aggregation agent) played a significant role in the beneficent outcome of the platelet antiaggregation trials. However, animal studies have shown that adenosine receptors desensitize rapidly71,88 and the therapeutic benefits seen during acute treatment are lost in a chronic regimen. Thus, one must consider other possible explanations for the mechanisms involved in the prevention of stroke through combined dipyridamole and aspirin therapy.35 Such considerations should not preclude further work exploring the value of currently available agents, e.g., carbamazepine,262–264 dipyridamole, or propentophylline (see below). Adenosine kinase inhibitors offer another promising approach265 but the experimental data are conflicting, with some investigators showing reduction of postischemic damage266–268 and others269 reporting failure. It is worth noting, however, that in studies of analgesic properties of GP 3966, CNS hemorrhages were observed in both rats and dogs.270 Studies of postischemic administration need to be made before

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any conclusions can be made. Finally, despite a promising start, insufficient data prohibit even preliminary conclusions on the usefulness of agents that enhance adenosine A1 receptor binding properties.271,272 The “classical” approaches based on treatment with the adenosine A1 receptor agonists, most of which demonstrate robust attenuation of cerebral damage following both global and focal ischemia, have been the subject of a number of exhaustive recent reviews.6–8,35 The major advantage of using selective A1 agonists is their indicated efficacy even when the treatment is initiated several hours after ischemia. However, the study of Bischofberger et al.19 is presently the only one that describes successful administration of A1 agonist as late as 18 h after global ischemia in gerbils. Hence, further confirmation, particularly in the context of focal ischemia, is badly needed. The problem of side effects elicited by A1 agonists (bradycardia, hypotension) still awaits definitive solution. The recent emergence of new selective adenosine A1 receptor agonists with a low cardiovascular profile239 is highly promising in this context. While exposure to A2A receptor-acting agents proved experimental merits, the question of “treat early or late” needs an answer — both agonists and antagonist appear to be protective, depending on the time of administration and the targeted process (see above). Utilization of A3 receptors as therapeutic targets in stroke is probably the most enigmatic. Dependence on the nature of the treatment regimen,35,97 its timing in relation to ischemia (von Lubitz, in press), and the pronounced dependence of the curative vs. lethal effect on the concentration of the drug at the target10,195,206 make this approach of high scientific but little practical value in the context of stroke treatment. Although impressive cardioprotection by A3 receptor agonists has been demonstrated, the data originate from ex vivo systems. Critical trials in which animal models of cardiac resuscitation are used (and where global brain ischemia is an invariable component of the ensuing pathology) must be conducted before definitive conclusions on the clinical usefulness of A3 receptor agonists in heart ischemia can be made. Propentofylline HWA 285; 1-(5’-oxyhexyl)-3-methyl-7-propylxanthine, a moderately efficient inhibitor of adenosine uptake and weak adenosine A1 receptor antagonist35 has been the subject of clinical trials against vascular- and Alzheimer-type dementia. The results of dementia studies were encouraging,273,274 but the trials of pentoxifylline (another methylxanthine) and a limited trial of propentofylline were inconclusive in the context of ischemic stroke.275

7.10 CONCLUSION Despite fervent wishes to the contrary expressed by almost all authors writing on the experimental results of adenosine-based treatment of global and focal ischemia, the clinical reality of such treatment is as remote as it has always been. Several reasons conspire against considering adenosine-acting drugs as clinical candidates. The stigma of “side effects,” of which hypothermia is among the most persistent, still plagues the concept, despite the fact that hypothermia is actually among the very

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useful therapeutic components of such treatment. Hypotension and bradycardia appear to be very substantially reduced by the recently introduced, novel drugs.240 Far more disturbing are inconsistencies that characterize many of the currently available data. While neuroprotection by A1 receptor agonists has been accepted, the use of different models, different dosing regimens, different evaluation criteria, and outcome cut-offs convert the results into a series of interesting but rather loosely connected facts. Once elementary dilemmas such as the efficacy of clinically relevant postischemic treatment, the nature of the lethal effects of the intense stimulation of adenosine receptors,10 the efficacy of new side-effect-free drugs, long-term effect of treatment, etc. are settled, the question of adenosine-based therapy in stroke will be answered by “very likely” instead of a vague “maybe.” Adenosine receptors represent a very alluring therapeutic target because of the very potent effects that are elicited by their stimulation. Yet, because of their ubiquitous nature, and because of the very broad involvement of adenosine in almost every function of the organism, the potential for either very unspecific or for intense side effects is very real. The emerging data10 on the role of A3 receptors is highly indicative of this problem. However, new approaches to the design of adenosine receptor-acting drugs,276 vigorous further experimentation concentrating on clinically relevant issues, and adherence to the guidelines on standards regarding preclinical neuroprotective and restorative drug development277 may promote adenosine-based therapy up the ranks of competing ideas on how to treat stroke. However, before this happens, the funding agencies and the industry must realize the significant therapeutic potential of adenosine. Presently, cardiologists “own” the only convincing body of data that incorporates both basic and clinical elements. In the realm of neurology and neurological disease, the data are widely scattered, the studies executed with minimal coherence, and only a perfunctory reference to their clinical usefulness is made by the authors who often give the impression of the clinical relevance of their work as a strictly peripheral issue. There is no doubt that adenosine and its receptors, due to their range of direct and indirect influences on the functions of a living organism, represent a splendid target for therapeutic interventions. Equally, there is no doubt that due to the complexity of adenosine-mediated actions, the task is also exceedingly complex, and there are no instantaneous rewards that the industry needs in order to show interest. Moreover, the fashionable “mechanistic” approach of NIH led to a plethora of papers describing a wide range of fascinating phenomena that are, essentially, of no use to the neurosurgeon, neurologist, or emergency medicine physician who still view adenosine as hypothermic, bradycardiac, and of limited interest in anything else but treatment of a special form of tachycardia. Thus, in similarity to many other agents, the future of adenosine as a therapeutic in stroke or any other neurological disaster is not a matter of good science of which there is already a lot, but of politics that dictate the science. While exploratory work on the regulatory role of adenosine must clearly continue, a well-coordinated effort aimed at the exploration of its therapeutic potential that will be based on well-defined guidelines recently provided by AHA needs to be instituted as well.

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REFERENCES 1. Sommersschild, H.T. and Kirkeboen, K.A., Adenosine and cardioprotection during ischemia and reperfusion, Acta Aenesthesiol. Scand., 44, 1038, 200. 2. Murray, C.J.L. and Lopez, A.D., Mortality by cause for eight regions of the world: global burden of disease study, Lancet, 349, 1269, 1997. 3. Gubitz, G. and Sandercock, P., Acute ischaemic stroke, Brit. Med. J. 320, 692, 2000. 4. Hankey, G.J. and Warlow, C.P., Treatment and secondary prevention of stroke: evidence, costs, and effects on individuals and populations, Lancet, 354, 1457, 1998. 5. Barnett, H.J.M., Eliasziw, M., and Meldrum, H.E., Evidence based cardiology: prevention of ischaemic stroke, Brit. Med. J., 318, 1539, 1999. 6. Rudolphi, K.A. et al., Adenosine and brain ischemia, Cerebrovasc. Brain Metab., 4, 346, 1992. 7. Sweeney, M.I., Neuroprotective effects of adenosine in cerebral ischemia: window of opportunity, Neurosci. Biobehav. Rev., 21, 207, 1997. 8. von Lubitz, D.K.J.E., Acute treatment of cerebral ischemia and stroke: put out more flags, in Purinergic Approaches in Experimental Therapeutics, Jacobson, K.A., Jarvis, M.E. (eds.), Wiley-Liss, New York, 1997, 449-70. 9. Deckert, J. and Gleiter, C.H., Adenosine — an endogenous neuroprotective metabolite and neuromodulator, J. Neural Transm. Suppl., 43, 23, 1994. 10. Jacobson, K.A. et al., Adenosine-induced cell death: evidence for receptor-mediated signaling, Apoptosis, 4, 197, 1999. 11. Satoh, A. et al., Activation of adenosine A1 receptor pathway induces edema formation in the pancreas of rats, Gastroenterol., 119, 829, 2000. 12. Imura, T. and Shimohama, S., Opposing effects of adenosine on the survival of glial cells exposed to chemical ischemia, J. Neurosci. Res., 62, 539, 2000. 13. Back T, Pathophysiology of the ischemic penumbra — a revision of a concept, Cell Mol. Neurobiol., 18, 621, 1998. 14. Dirnagl, U., Iadecola, C., and Moskowitz, M.A., Pathobiology of ischemic stroke: an integrated view, Trends Neurosci., 22, 391, 1999. 15. Del Zoppo, G.J. and Hallenbeck, J.M., Advances in vascular pathophysiology of ischemic stroke, Thromb. Res., 98, 73, 2000. 16. Wolozin, B. and Behl, C., Mechanisms of Neurodegenerative disorders: Part 2: Control of cell death, Arch. Neurol., 57, 801, 2000. 17. Lee, J.-M. et al., Brain tissue responses to ischemia, J. Clin. Invest. 106, 723, 2000. 18. Zipfel G.J., Lee, J-M., and Choi, D.W., Reducing calcium overload in the ischemic brain, New Engl. J. Med., 341, 1543, 1999. 19. Heiss W.-D. et al, Which targets are relevant for therapy of acute ischemic stroke?, Stroke, 30, 1486, 1999. 20. Fisher, M. and Baron J.-C., Which targets are relevant for therapy of acute ischemic stroke?, Stroke, 31, 984, 2000. 21. Vaughan C.J. and Delanty, N., Neuroprotective properties of statins in cerebral ischemia and stroke, Stroke, 30, 1969, 1999. 22. Rubattu, S., Giliberti, R., and Volpe M., Etiology and pathophysiology of stroke as a complex trait, Am. J. Hypertens., 13, 1139, 2000. 23. Schubert, P. et al., Modulation of nerve and glial function by adenosine — role in the development of ischemic damage, Int. J. Biochem., 26, 1227, 1994. 24. Newby, A.C., Adenosine as a retaliatory metabolite, Trends Biol. Sci., 9, 42, 1984. 25. Ciccarelli, R. et al., Rat cultured astrocytes release guanine-based purines in basal conditions and after hypoxia/hypoglycemia, Glia, 25, 93, 1999.

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194. Fleming, K.M. and Mogul, D.J., Adenosine A3 receptors potentiate hippocampal calcium current by a PKA-dependent/PKC-independent pathway, Neuropharmacology, 36, 353, 1997. 195. Ali, H. et al., Sustained activation of phospholipase D via adenosine A3 receptors is associated with enhancement of antigen- and Ca2+-ionophore-induced secretion in a rat mast cell line, J. Pharmacol. Exp. Ther., 276, 837, 1996. 196. Serrander, L., Fallman, M., and Stendahl, O., Activation of phospholipase D is an early event in integrin-mediate signaling leading to phagocytosis in human neutrophils, Inflammation, 20, 39, 1996. 197. Bonser, R.W. et al., Phospholipase D activation is functionally linked to superoxide generation in the human neutrophil., Biochem. J., 264, 617, 1990. 198. Watson, F. and Edwards, S.W., Stimulation of primed neutrophils by soluble immune complexes priming leads to enhanced intracellular Ca2+ elevations, activation of phospholipase D, and activation of the NADPH oxidase, Biochem. Biophys. Res. Comm., 247, 819, 1998. 199. McWhinney, C.D. et al., Activation of adenosine A3 receptors on macrophages inhibits necrosis factor-alpha, Eur. J. Pharmacol., 310, 209, 1996. 200. Bowlin, T. et al., Adenosine A3 receptor agonists inhibit macrophage necrosis factoralpha production in vitro and in vivo, Cell Mol. Biol., 43, 345, 1997. 201. von Lubitz, D.K.J.E., Simpson, K.L., and Lin, R.C.S., Right thing at a wrong time? Adenosine A3 receptors and cerebroprotection in stroke, Proc. N.Y. Acad. Sci., in press 202. Mabuchi, T. et al., Contribution of microglia/macrophages to expansion of infarction and response of oligodendrocytes after focal cerebral ischemia in rats, Stroke, 31, 1735, 2000. 203. Stoll, G. and Jander, S., The role of microglia and macrophages in the pathophysiology of the CNS, Prog. Neurobiol., 58, 233, 1999. 204. Stoll, G., Jander, S., and Schroeter, M., Inflammation and glial responses in ischemic brain lesions, Prog. Neurobiol., 56, 149, 1998. 205. Sei, Y. et al., Adenosine A3 receptor agonist-induced neurotoxicity in rat cerebellar granule neurons, Drug Dev. Res., 40, 267, 1997. 206. Glowinski, J. et al., Glial receptors and their interaction in astrocyto-astrocytic and astrocyto-neuronal interactions, Glia, 11, 201, 1994. 207. Reith, J. et al., Body temperature in acute stroke: relation to stroke severity, infarct size, mortality, and outcome, Lancet, 347, 1415, 1996. 208. Ginsberg, M. and Busto, R., Combating hyperthermia in acute stroke: a significant clinical concern, Stroke, 29, 529, 1998. 209. Meden, P., Kammersgaard, L., and Overgaard, K., Can acute stroke be treated with hypothermia? Nordic. Med., 113, 3, 1998. 210. Lanier, W.l., Cerebral metabolic rate and hypothermia: their relationship with ischemic neurologic injury, J. Neurosurg. Anesthesiol., 7, 216, 1995. 211. Corbett, D. and Thornhill, J., Temperature modulation (hypothermic and hyperthermic conditions) and its influence on histological and behavioral outcomes following cerebral ischemia, Brain Pathol., 10, 145, 2000. 212. Young-Su, P. and Ishikawa, J., Analysis of mild barbiturate-moderate hypothermia therapy on the authors-152 cases, No Shinkei Geka, 25, 529, 1997. 213. Kammersgaard, L.P. et al., Feasibility and safety of inducing hypothermia in awake patients with acute stroke through surface cooling: a case-control study: the Copenhagen Stroke Study. Stroke, 31, 2251, 2000. 214. Correia, M., Silva, M., and Veloso, M., Cooling therapy for acute stroke, Cochrane Database Syst. Rev., CD001247, 2000.

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215. Laptook, A.R. et al., Neonatal ischemic neuroprotection by modest hypothermia is associated with attenuated brain acidosis, Stroke, 26, 1240, 1995. 216. Zeevalk, G.D. and Nicklas, W.J., Hypothermia and metabolic stress: narrowing the cellular site of early neuroprotection, J. Pharmacol. Exp. Ther., 279, 332, 1996. 217. Colbourne, F. et al., Prolonged but delayed postischemic hypothermia: a long-term outcome study in the rat middle cerebral artery occlusion model, J. Cereb. Blood Flow Metab., 20, 1702, 2000. 218. Maier, C.M. et al., Delayed induction and long-term effects of mild hypothermia in a focal model of transient cerebral ischemia: neurological outcome and infarct size, J. Neurosurg., 94, 90, 2001. 219. Anderson, R., Sheehan, M.J., and Strong, P., Characterization of the adenosine receptors mediating hypothermia in the conscious mouse, Br. J. Pharmacol., 113, 1386, 1994. 220. Kulinski, V.I., Olkhovski, I.A., and Kovalevski, A.N., Biochemico-pharmacological mechanisms and the relation between anticalorigenic, hypothermic, and antihypoxic effects of adenosine, Vopr. Med. Khim., 33, 107, 1987. 221. Zhong, W. et al., The effects of adenosine on the energy metabolism of the reperfused intestine in rats, Jpn. J. Surg., 28, 178, 1998. 222. Daval, J-L. and Nicolas, F., Non-selective effects of adenosine A1 receptor ligands on energy metabolism and macromolecular biosynthesis in cultured central neurons, Biochem. Pharmacol., 55, 141, 1998. 223. Bruns, R.F., Role of adenosine in energy supply /demand balance, Nucleos. Nuclet., 10, 941, 1991. 224. Sokoloff, L., Sites and mechanisms of function-related changes in energy metabolism in the nervous system, Dev. Neurosci., 15, 194, 1993. 225. Deckert, J. and Jorgensen, M.B., Evidence for pre- and postsynaptic localization of adenosine A1 receptors in the CA1 region of rat hippocampus: a quantitative autoradiographic study, Brain Res., 446, 161, 1988. 226. Astrup, J., Sorensen, P.M., and Sorensen, H.R., Oxygen and glucose consumption related to Na+-K+ transport in canine brain, Stroke, 12, 726, 1981. 227. Broadley, K.J., Broome, S., and Paton, D.M., Hypothermia-induced supersensitivity to adenosine for responses mediated via A1 receptors but not A2 receptors, Br. J. Pharmacol., 84, 407, 1985. 228. Newberg, L.A., Milde, J.H., and Michenfelder, J.D., The cerebral metabolic effect of isoflurane at and above concentrations that suppress cortical electrical activity, Anesthesiology, 59, 23, 1983. 229. Popovic, R., Liniger, R., and Bickler, P.E., Anesthetics and mild hypothermia similarly prevent hippocampal neuron death in an in vitro model of cerebral ischemia, Anesthesiology, 92, 1343, 2000. 230. Kawaguch, M. et al., Isoflurane delays but does not prevent cerebral infarction in rats subjected to focal ischemia, Anesthesiology, 92, 1226, 2000. 231. Roscoe, A.K. et al., Isoflurane, but not Halothane, induces protection of human myocardium via adenosine A1 receptors and adenosine triphosphate-sensitive potassium channels, Anesthesiology, 92, 1692, 2000. 232. Nellgård, B. et al., Anesthetic effects on cerebral metabolic rate predict histologic outcome from near-complete forebrain ischemia in the rat, Anesthesiology, 93, 431, 2000. 233. McCulloch, J., Neuroprotective drug development in stroke: blood pressure and its impact, J. Hypertension, 5, S131, 1996.

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234. Dietrich, W.D. et al., Delayed hypovolemic hypotension exacerbates the hemodynamic and histopathologic consequences of thromboembolic stroke in rats, J. Cereb. Blood Flow Metab., 19, 918, 1999. 235. Ahmed, N., Nasman, P., Wahlgren, N.G., Effect of intravenous nimodipine on blood pressure and outcome after acute stroke, Stroke, 31, 1250, 2000. 236. Kassell, N. et al., Cerebral systemic circulatory effect of arterial hypotension induced by adenosine. J. Neurosurg., 58, 69, 1983. 237. Stange, K., Lagerkranser, M., and Sollevi, A., Effect of adenosine-induced hypotension on the cerebral autoregulation in the anesthetized pig, Acta Anesthesiol.Scand., 33, 450, 1989. 238. White, P.J., Rose-Myer, R.B., and Hope, J.W., Functional characterization of adenosine receptors in the nucleus tractus solitarius mediating hypotensive responses in the rat, Br. J. Pharmacol., 117, 305, 1996. 239. Bulley, S.R. and Wittnich, C., Adenosine infusion: a rational approach towards induced hypotension, Can. J. Cardiol., 11, 327, 1995. 240. Knutsen, L.J., Lau ,K., and Oetersen, H., N-substituted adenosines as novel neuroprotective A1 agonists with diminished hypotensive effects, J. Med. Chem., 42, 3463, 1999. 241. Marangos, P.J. et al., Adenosine: its relevance to the treatment of brain ischemia and trauma, Prog. Clin. Biol. Res. 361, 331, 1990. 242. Marzili, M. et al., Beneficial effect of intracoronary adenosine as an adjunct to primary angioplasty in acute myocardial infarction, Circulation, 101, 2154, 2000. 243. Tomai, F. et al., Ischemic preconditioning in humans: models, mediators, and clinical relevance, Circulation, 100, 559, 1999. 244. Weih, M. et al., Attenuated stroke severity after prodromal TIA: a role for ischemic tolerance in the brain? Stroke, 30, 1851, 1999. 245. Beauchamp, P. et al., Protective effects of preconditioning in cultured rat endothelial cells: effects on neutrophil adhesion and expression of ICAM-1 after anoxia and reoxygenation, Circulation 100, 541, 1999. 246. Geshi, E. et al., The role of ATP-sensitive potassium channels in mechanism of ischemic preconditioning, J. Cardiovasc. Pharmacol., 34, 446, 1999. 247. Perez-Pinzon, M.A. and Born, J.G., Rapid preconditioning neuroprotection following anoxia in hippocampal slices: role of the K+ATP channel and protein kinase C, Neuroscience, 89, 453, 1999. 248. Dana, A. et al., Adenosine A1 receptor induced delayed preconditioning in rabbits: induction of Hsp27 phosphorylation via tyrosine kinase- and protein kinase C-dependent mechanisms, Circ. Res. 86, 989, 2000. 249. Perez-Pinzon, M.A. et al., Anoxic preconditioning in hippocampal slices: role of adenosine, Neuroscience, 75, 687, 1996. 250. Blondeau, N. et al., K(ATP) channel openers, adenosine agonists and epileptic preconditioning are stress signals inducing hippocampal neuroprotection, Neuroscience, 100, 465, 2000. 251. Sato, T. et al., Adenosine primes the opening of mitochondrial ATP-sensitive potassium channels: a key step in ischemic preconditioning?, Circulation, 102, 800, 2000. 252. Zhang, W.L. and Lu, G.W., Changes of adenosine and its A1 receptor innhypoxic preconditioning, Biol. Signals Receptors, 8, 275, 1999. 253. von Arnim, C.A. et al., Adenosine receptor up-regulation: initiated upon preconditioning but not upheld, Neuroreport 11, 1223, 2000. 254. Tian, Y.H. et al., Adenosine deaminase inhibition attenuates reperfusion, low flow, and improves graft survival after rat liver transplantation, Transplantation, 69, 2277, 2000.

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255. Parkinson, F.E. et al., Effects of nitrobenzylthioinosine on neuronal injury, adenosine levels, and adenosine receptor activity in rat forebrain ischemia, J. Neurochem., 75, 795, 2000. 256. Kulkarni, J.S. et al., Adenosine induces apoptosis by inhibiting mRNA and protein synthesis in chick embryonic sympathetic neurons. Neurosci. Lett., 248, 187, 1998. 257. Ceruti, S. et al., Adenosine- and 2-chloro-adenosine-induced cytopathic effects on myoblastic cells and myotubes: involvement of different intracellular mechanisms. Neuromuscul. Disorders, 10, 436, 2000. 258. Blackburn, M.R. et al., Metabolic consequences of adenosine deaminase deficiency in mice are associated with defects in alveogenesis, pulmonary inflammation, and airway obstruction, J. Exp. Med., 192, 159, 2000. 259. Bednar, M.M. and Gross, C.E., Antiplatelet therapy in acute cerebral ischemia, Stroke, 30, 887, 1999. 260. Forbes, C.D., European stroke prevention study 2: dipyridamole and acetylsalicylic acid in the secondary prevention of stroke, Int. J. Clin. Pract., 51, 205, 1997. 261. Picano, E. and Abbracchio, M.P., European Stroke Prevention Study-2: serendipitous demonstration of neuroprotection induced by endogenous adenosine accumulation? Trends Pharmacol. Sci., 1, 14, 1998. 262. Murakami, A. and Furui, T., Effects of the conventional anticonvulsants, phenytoin, carbamazepine, and valproic acid, on sodium-potassium-adenosine triphosphate in acute ischemic brain, Neurosurgery, 34, 1047, 1994. 263. Vivancos Mora J., Treatment of neurological complications: cerebral edema and epileptic seizures, Neurologia 10 (Suppl.2), 23, 1995. 264. Schierhout, G. and Roberts, I., Anti-epileptic drugs for preventing seizures following acute traumatic brain injury, Cochrane Database Syst. Rev., CD000173, 2000. 265. Kowaluk, E.A. and Jarvis, M.F., Therapeutic potential of adenosine kinase inhibitors, Exp. Opin. Invest. Drugs, 9, 551, 2000. 266. Miller, L.P. et al., Pre- and peristroke treatment with the adenosine kinase inhibitor, 5’-deoxyiodotuberacin, significantly reduces infarct volume after temporary occlusion of the middle cerebral artery in rats, Neurosci. Lett. 220, 73, 1996. 267. Jiang, N., Adenosine kinase inhibition protects brain against transient focal ischemia in rats, Eur. J. Pharmacol. 320, 131, 1997. 268. Tatlisumak, T., Delayed treatment with an adenosine kinase inhibitor, GP683, attenuates infarct size in rats with temporary middle cerebral artery occlusion, Stroke, 29, 1952, 1998. 269. Phillis, J.W. and Smith-Barbour, M., The adenosine kinase inhibitor, 5-iodotuberacidin, is not protective against cerebral ischemic injury in the gerbil, Life Sci., 53, 497, 1993. 270. Erion, M.D. et al., Therapeutic potential of adenosine kinase inhibitors as analgesic agents, Drug Dev. Res., 1, 22 (S14-06), 2000. 271. Cao, X. and Phillis, J.W., Adenosine A1 receptor enhancer, PD81,723, and cerebral ischemia/reperfusion injury in the gerbil, Gen. Pharmacol. ,26, 1545, 1995. 272. Halle, J.N. et al., Enhancing adenosine A1 receptor binding reduces hypoxic-ischemic brain injury in newborn rats, Brain Res., 759, 309, 1997. 273. Kittner, B., Clinical trials of propentofylline in vascular dementia. European/Canadian Propentofylline Study Group, Alzheimer Dis. Assoc. Disorders, 13 (Suppl.3), S166-71, 1999.

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274. Bachynsky, J. et al., Propentofylline treatment for Alzheimer disease and vascular dementia: an economic evaluation based on functional abilities, Alzheimer Dis. Assoc. Disorders, 14, 102, 2000. 275. Bath, P.M., Bath, F.J., and Asplund, K., Pentoxifylline, propentofylline and pentifylline for acute ischaemic stroke, Cochrane Database Syst. Rev., CD000162, 2000. 276. Jacobson, K.A. and Knutsen L.J.S., P1 and P2 purine and pyrimidine receptor ligands, in Handbook of Experimental Pharmacology, Vol. 151/I Abbracchio, M.P., Williams, M. (eds.), Springer-Verlag, Berlin, in press. 277. Recommendations for standards regarding preclinical neuroprotective and restorative drug development (special report), Stroke, 30, 2752, 1999.

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8

Calpain and Caspase in Ischemic and Traumatic Brain Injury Kevin K.W. Wang

CONTENTS 8.1 8.2 8.3 8.4 8.5 8.6

Two Different Forms of Neuronal Cell Death: Apoptosis and Oncosis Apoptosis and Oncosis in Neurodegeneration Activation of Calpain 1 and 2 in Oncotic Death Caspase Cascade in Apoptosis Calpain Activation in Certain Apoptosis Systems Caspase-3 and Calpain 1 and 2: What They Have in Common 8.6.1 Caspase-3 and Calpain 1 and 2 Share Many Common or Related Substrates 8.6.2 Turning Bax-Things Worse 8.7 Calpain-Caspase Crosstalk 8.8 Perspective 8.9 Acknowledgments References

8.1 TWO DIFFERENT FORMS OF NEURONAL CELL DEATH: APOPTOSIS AND ONCOSIS Programmed cell death or apoptosis is cell death generally characterized by the presence of DNA fragmentation at the nucleasome linkage regions and DNA condensation, as well as cell shrinkage giving way to the formation of apoptotic bodies.1 Apoptosis occurs physiologically during development and other stages of its lifetime to eliminate unwanted cells. Unscheduled apoptosis also occurs in a large number of pathological or injurious conditions.2 Oncosis (or oncotic necrosis) occurs when cells have been acutely and severely injured to a point that is beyond repair.1 It is characterized by the presence of massive ion (Ca2+ and Na+) influx, mitochondria and cell swelling, massive multisite DNA breakage and plasma membrane bursting. Both forms of cell death have been well documented in acutely injured neurons. Necrosis is a term that means “deadness,” which occurs in the end-stage of both apoptosis and oncosis, thus is confusing and should be avoided.3

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8.2 APOPTOSIS AND ONCOSIS IN NEURODEGENERATION Not very long ago it was commonly believed that acute neurological cell death is oncotic in nature. That changed when newer evidence for apoptosis such as the presence of DNA laddering and nuclear DNA condensation in dying/dead neurons were identified in models of cerebral ischemia and excitotoxicity.4-10 It was followed by the evidence for apoptotic neuronal death in experimental TBI as well as spinal cord injury (SCI).11,12 In fact, apoptotic neuronal death has been reported in a large number of chronic neurodegenerative conditions, such as Parkinsonism, Huntington’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis (ALS).13-15 More recently, various researcher groups have pointed out that apoptosis is likely a less dominant form of neuronal death.16-19 Thus it is more likely both apoptotic and oncotic neuronal death are manifested in these disorders.

8.3 ACTIVATION OF CALPAIN 1 AND 2 IN ONCOTIC DEATH In ischemic and traumatic brain injury, it is now well established that sustained intracellular free calcium revelation results from calcium in flux via the ionotrophic glutamate receptors (NMDA, AMPA, and kainate receptors) as well as through voltage-gated calcium channels. This leads to calpain-mediated proteolysis of various cellular proteins and contributes to oncotic neuronal death.20 Calpain overactivation was also found in myocardial infarct and ischemic renal injury.21-23 At least in neuronal culture models, calpain activation can be mimicked by treatment with glutamate, hypoxia, A23187, and calcium channel opener maitotoxin.24,25 Calpain is generally in an inactive proenzyme form in a resting cell but becomes overactivated under extreme conditions that result in sustained [Ca2+]cytosol elevation, which is generally associated with necrotic oncosis (Table 8.1). Instances include calcium-ionophore-treated cells (such as A23187-Molt-4 cells), or glutamate-treated glutamatergic central neurons. In the first case, calcium rushes in through the poreforming A23187. In the latter case, calcium comes in through the ionotropic glutamate-receptors, which also function as ligand gated calcium/sodium channels.20,25 Similarly, maitotoxin, a potent neurotoxin that opens both voltage- and ligand-gated calcium channels also induces rapid and massive calcium influx and subsequent calpain activation.24 We investigated the form of cell death with MTX treatment and found no DNA laddering or cell swelling. General DNA degradation and dissolution rather than condensation were observed. These are properties that are associated with necrotic oncosis.26,27 More recently, we found that by elevating the extracellular calcium concentration from 0.8 mM to 5.8 mM alone is sufficient to create calcium influx and calpain activation in the calcium-treated cells.28 For evidence of calpain activation, we found the specific fragmentation pattern for alpha-spectrin (280 kDa), forming immunoreactive fragments of 150 kDa and 145 kDa,20,25,27 identical to those forms by in vitro digestion of control cell lysate with purified calpain. Furthermore, calpain inhibitors, such as calpain inhibitor I and PD150606, inhibit the formation of these fragments. Calpain1 and 2 activities are also regulated by endogenous protein inhibitor calpastatin.29

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TABLE 8.1 Compare and Contrast: Caspase-3 and Calpain 1 and 2 Caspase-3

Calpain 1 and 2

Protease class

Cysteine protease

Cysteine protease

Endogenous inhibitors

XIAP, cIAP1, cIAP2, NAIP

Calpastatin

Resting mode

Inactive pro-enzyme (32 kDa)

Inactive pro-enzyme (80 kDa + 29 kDa)

Activation mode

Proteolytic processing (to 17 kDa + 12 kDa)

Ca2+, then autolytic processing (to 78 kDa + 18 kDa)

Preferred cleavage site

DxxD*x

(L,V,I)x*x

Endogenous substrates

Subset of cytoskeletal, cytosolic, Subset of cytoskeletal, and nuclear proteins or enzymes cytosolic, and nuclear proteins or enzymes

Consequence of substrate proteolysis

Produces limited fragments, Produces limited fragments, sometimes with gain of function sometimes with gain of function

Cell death involvement

Apoptosis

Most forms of oncosis and mixed oncosis-apoptosis,

Inhibitors as neuroprotectants

Yes

Yes

Calpain 1 and 2 are ubiquitous mammalain proteins and are enriched in CNS neurons. They do not have strict recognition sequence in substrate cleavage site but prefer Val, Ile, or Leu in the P2 position (second residue N-terminal to cleavage site) (Table 8.1). Only a small subset of cellular proteins are susceptible to calpain attack while the majority of cellular proteins are resistant. Calpains tend to cleave substrates to large “limited fragments” without further degradation.30 Calpain 1 and 2 substrates include cytoskeletal proteins (e.g., alpha-spectrin, beta-spectrin, MAP2, neurofilaments H and M),25 membrane receptors (EGF receptor, PDGF receptor) and G-proteins (alpha subunit),31,32 signal transduction enzymes, protein kinase C isoforms, and many calmodulin-dependent enzymes (e.g., CaMPK-II, plasma membrane calcium pump, calcineurin, and neuronal nitric oxide synthase), and PEST-containing proteins.30,33 A number of transcription factors have been shown to be calpain-regulated (c-fos, c-Jun, c-myc, c-mos and NF-kappaB).34-36 When calpain 1 and 2 are uncontrollably activated the destruction and/or modification of these cellular proteins could prove to be detrimental to the host cells.

8.4 CASPASE CASCADE IN APOPTOSIS Caspases are a family of related cysteine proteases discovered based on the initial discovery that a C. elegans apoptosis-linked protein CED-3 is homologous to the mammalian caspase-1 (then called interleukin-1beta-converting or ICE). This led to the discovery of a large number of ICE-like proteases (now renamed caspases) and

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their roles as mediator of apoptosis in a wide range of cells.37,38 Caspase-3 and caspase-7 appear to be common downstream apoptosis effectors. First, caspase-3/7 can be activated intrinsically or extrinsically. The intrinsic pathway is mitochondria-dependent. Upon an apoptotic trigger, Bax or Bax-like proteins translocate to the mitochondrial outer membrane, dimerise with Bcl-2, which allows the release of cytochrome C into the cytosol. There, cytochrome C complex with Apaf-1 and caspase-9 leads to autolytic activation and the processing/activation of caspase-3 and 7. The extrinsic pathway involves ligand binding to the so-called death-domain containing receptors (such as TNF-aR-1, Fas).39 The ligand-bound receptor recruits adapter protein(s), which also contains death domains (FADD for Fas, TRADDFADD pair for TNFa-R1). FADD then induces caspase-9 molecules to colocalized and thus the autolytic activation of the associated caspase-8 or caspase-10. Like caspase-9, caspase-8/10 processes and activates caspase-3. Caspase-7 is highly homologous to caspase-3 and shares identical substrate cleavage site requirement Asp-Xaa-Xaa-Asp. Thus it might substitute for caspase-3 in most cell types and tissues. However, in the CNS, no mRNA for caspase-7 was detected.40 Consistent with that, caspase-3-/- knockout mice showed normal development except the CNS, in which too many neurons were found.41 Therefore, it seems that caspase-3 is more important in neurodegeneration and we will focus our discussion on caspase-3. Caspase activity can also be directly suppressed by endogenous inhibitors (cIAP1, 2, XIAP, NAIP)42 (Table 8.1). Caspase-3 also has a finite number of cellular protein substrates.37,38 The key specificity determinant is the Asp (D) in the P1 and P4 positions, identified using synthetic peptide as substrates (Table 8.1). Like calpain, caspase-3 tends to produce limited fragments of its substrates, leaving them as fingerprint for caspase-3 activation. The list of substrates has grown rapidly. They can be classified into the following classes: (i) cytoskeletal proteins (such as actin, a and b-spectrin, GAS-2), (ii) signal transduction enzymes (protein kinase C delta and theta isoforms CaMPK-II and IV, FAK (focal adhesion kinase), phospholipase C, P21-activated kinase, MEKK1), (iii) cell cycle proteins (PITSLRE kinase; Rb), and (iv) nuclear substrates, such as DNA-PKcs, PARP, U1-70K and NuMA and caspase-activated DNAase which is responsible for DNA fragmentation in the internecleasome regions.2 Caspase-3 also activates caspase-6, which degrades lamin A, B1, B2, and C. Three biochemical markers have been used extensively to detect caspase-3 activation in neurological or neurodegenerative disorders: (i) processing of intact 32 kDa proenzyme form of caspase-3 to the 17 kDa and 12 kDa dimeric form,43,44 (ii) fragmentation of the 113 kDa poly (ADP-ribose) polymerase (PARP) to a 89 kDa form,45 and (iii) processing of nonerythroid alpha-spectrin (280 kDa) into a 150 kDa and 120 kDa spectrin breakdown product (SBDP120).46 In fact, caspase-3, PARP, and alphaspectrin processing have been reported in several neuronal apoptosis models, such as cultured neuroblastoma or neuronal cells subjected to staurosporine as well as cerebellar granule neurons subjected to potassium deprivation.27,44,47-49 Furthermore, similar evidence has been observed in in vitro models of hypoxia-hypoglycemia, excitotoxicity, and MPTP toxicity (in cultured primary neurons). Antibodies that specifically detect the activated caspase-3 (17 kDa), the 89-kDa PARP fragment, and

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the SBDP120 have recently been reported,48,50,51 making immunohistochemical studies possible. More recently, there is a large body of literature identifying the presence of caspase-3 activation and proteolytic activity in in vivo models for cerebral ischemia, excitotoxicity, TBI, and SCI.52-54 Caspases appear to be centrally involved in any apoptotic cascade in various cell types, as caspase inhibitors such as Z-D-DCB, Z-VAD(oMe)-fmk, and Ac-DEVD-CHO almost universally protect against any forms of apoptosis. Similarly these agents are found to be excellent neuronal apoptosis inhibitors.26,55-57 Also they were ineffective against necrotic oncosis.48 Recently several inhibitors of this class have been used in suppressing the “apoptotic” component in a cerebral ischemia and TBI.58-62

8.5 CALPAIN ACTIVATION IN CERTAIN APOPTOSIS SYSTEMS Calpain activation was first identified in thymocyte and T-cell apoptosis,63,64 as measured by calpain autolysis, calpain substrate proteolysis and that calpain inhibitors protect against apoptosis in immune cells.65,66 We then showed that calpain is also activated in staurosporine-treated neuroblastoma SH-SY5Y cells, in NGF-deprived rat PC-12 cells, and in low potassium-treated rat cerebellar granule neurons (CGN),26,27 based on detection of calpain autolysis and specific spectrin fragments generated by calpain (SBDP150 and SBDP145) (see below). Caspases appears to be centrally involved in any apoptotic cascade as caspase inhibitors ( Z-D-DCB, Z-VAD-fmk, and Ac-DEVD-CHO) almost universally protect against any forms of apoptosis. Unlikely caspases, calpain inhibitors protect against a subset of apoptotic conditions.26,64,67-69 We found that in potassium-induced CGN apoptosis can block calpain inhibitors partially. Furthermore, the combined use of both calpain and caspase inhibitors provided an additive protective effect.26 Since then several laboratories have demonstrated the antiapoptotic effects of calpain inhibitors.68,70,71

8.6 CASPASE-3 AND CALPAIN 1 AND 2: WHAT THEY HAVE IN COMMON 8.6.1 CASPASE-3 AND CALPAIN 1 AND 2 SHARE MANY COMMON OR RELATED SUBSTRATES Table 8.1 illustrates the many aspects in which calpain 1 and 2 are similar to caspase3, including that they are both cysteine proteases, have endogenous protein inhibitors, produce limited fragments of selected protein substrates, and are involved in cell death. Here we illustrate that they even share many identical or related endogenous substrates (Table 8.2). Alpha II-spectrin (alpha-fodrin) has long been recognized as a preferred substrate for calpain 1 and 2.2 Calpain mediates alpha II-spectrin breakdown to 150 kDa and 145 kDa doublet (SBDP150 and SBDP145). Several groups also found that alpha II-spectrin was degraded to a 120 kDa fragment

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TABLE 8.2 Common or Related Substrates for Calpain and Caspase-3 Substrate Protein

Major Fragments Produced by Caspase-3

Major Fragments Produced by Calpain

αII-Spectrin (280 kDa)

150 kDa; 120 kDa

150 kDa; 145 kDa

βII-Spectrin (260 kDa)

110 kDa; 85 kDa

110 kDa

Actin (43 kDa)

35 kDa

40-42 kDa

Vimentin (54 kDa)

52–64 kDa

44 kDa and others

Tau (55kDa)

45 kDa

42 kDa and others

CaMPK-IV (55 kDa)

38 kDa

40 kDa

CaMPK-II (50 kDa)

35 kDa

35 kDa

Calcineurin A (60 kDa)

45 kDa

45 kDa

FAK (125 kDa)

85 kDa

90 kDa

PARP (113 kDa)

89 kDa + 24 kDa

70 kDa + 40 kDa

DNA polymerase ε (261 kDa)

140 kDa

140 kDa

Calpastatin (105 kDa)

75 kDa

Multiple

Caspase-3 (32 kDa)

Autolysis

29 kDa

Calpain I and II (80 kDa)

Not degraded

Autolysis

PKCα, β, γ (75 kDa)

Not degraded

46 kDa + 36 kDa

PKCδ, θ (75 kDa)

40 kDa

Not degraded

Transglutaminase II (80 kDa)

? kDa

Degraded

Not degraded

100 kDa

70 kDa

Not degraded

Multiple

Multiple

AMPA receptor (110 kDa)

Multiple

90 kDa

IP3R1, 2

Degraded

Not degraded

IP3R3

Not degraded

Degraded

Bcl2 (24 kDa)

22 kDa

Not degraded

Bax (21 kDa)

Not degraded

18 kDa

Bid (20 kDa)

18 kDa

Not degraded

Bcl-XL (24 kDa)

20 kDa

20 kDa

Phospholipase C-beta3 (155 kDa; membrane) Phospholipase A2 (100 kDa; cytosol) Amyloid precursor protein (80 kDa)

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(SBDP120) by caspase-3 during apoptosis in T-cells, neurons, and other cell types.27,46,72 We also found that the SBDP120 was observed only in apoptotic neurons but not in oncotic neurons,27,50 which is consistent with the fact that caspase is only activated in apoptosis but not in oncosis.48 We also observed that calpain-mediated alpha II-spectrin breakdown to 150 kDa and 145 kDa doublets were also present in apoptotic neurons (Fig. 8.1). The SBDP145 and SBDP120 can be easily distinguished on SDS-PAGE. Anti-calpain-produced SBDP150-specific antibodies have been produced by various groups.50,73,74 Taking advantage of the new N-terminal of SBDP120, we recently developed an anti-SBDP120-specific antibody.50 In fact, using SBDP120 as a marker for apoptosis is convenient as several commercial antialphaII-spectrin antibodies react with rat (e.g., cerebellar granule neurons) and human alpha II-spectrin (e.g., SH-SY5Y cells).27,46,72 This antibody detects apoptotic neurons in culture as well as in vivo models of TBI.53,75,76 Thus the usage of these spectrin and SBDP antibodies are powerful tools in detecting the apoptotic and oncotic neurons. Pro-apoptotic Signal

Caspase 9 Caspase 8

Membrane Permeability

Cytoskeletal Ca2+ influx substrates α,, β -spectrin 80K Actin Tau 30K Pro-Calpain

32K

76K

Degradation Calpastatin

Pro-caspase 3

Activated Calpain 18K

17K Activated Caspase 3

12K

Degradation Degradation Cytoplasmic substrates PKC Phospholipase CAMPKII Calpastatin

Nuclear substrates PARP CaMPKIV

Caspase 6

Cell Death

Lamins A, B1, B2, C

Nucleus

FIGURE 8.1 Calpain 1 and 2 and caspase-3 in mixed oncosis and apoptosis. Upon activation, caspase-3 can activate calpain 1 and 2 in two ways: (1) by degrading cytoskeletal and membrane proteins and thus compromising cell membrane permeability to Ca2+; and (2) partial inactivation of calpastatin by proteolysis. Once activated, calpains join caspase3 in degrading a whole range of cytoplasmic, cytoskeletal, and nuclear substrates, resulting in mixed oncotic/apoptotic cell death.

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Besides alpha II-spectrin, we found that the beta II-spectrin is also simultaneously degraded by calpain and caspase-3 in apoptosis and by calpain only in oncosis.27 Adjacent calpain and caspase-3 cleavage sites lead to 110 kDa beta-SBDP’s while caspase-cleavages at another major site produces an 85 kDa. Vimentin is an intermediate filament protein of the cytoskeleton network. It was found to be a calpain in lens cells.77,78 Recently, vimentin degradation by caspase is again observed in apoptotic skin fibroblasts and prostate epithelial cells.79,80 Actin, a major spectrin-binding protein, has been shown to be degraded by caspase-3 and calpain.77,81 However, actin is not a particularly good substrate to calpain or caspase and is only partially fragmented, which leads to the failure of some researchers to consistently observe its cleavage in cell death. Overall, we speculate that the simultaneous degradation of various cytoskeletal proteins in apoptosis (and oncosis) significantly compromise the cytoskeletal network and cell structural integrity and possibly membrane permeability. Tau, a microtubule (MT) binding protein that stabilizes the assembly of MT, which is of extreme importance in neurons since protein and other biomolecules transport in neurites (axons and dendrites), is supported by MT. Tau is composed of up to 6 isoforms in adult human neurons. Tau aggregates and hyperphosphorylated Tau have been linked to the formation of neurofibrillary tangles in Alzheimer’s disease.82,83 Sensitivity of Tau to calpain proteolysis has been suggested as playing a role in neuronal degeneration.84 In fact, multiple fragments are generated with the major fragment of about 30-40 kDa. Canu et al.85 further showed that in CGN apoptosis, tau is also degraded and is sensitive partially to caspase and calpain inhibitors. In fact, the combination of inhibitors of both proteases provided the best protection. This prompted the authors to examine and subsequently confirm the direct susceptibility of tau to caspase and calpain. More recently, the degradation of tau by caspases in mixed cerebrocortical neurons was confirmed.86,87 Amyloid precursor protein (APP) is another key protein associated with AD.82 Apparently, when APP was inappropriately cleaved, it produced the b-amyloid peptides 1–40 and 1–42, which form extracellular aggregates (amyloid plaques). Although the responsible proteases have been recently identified as beta-secretase and presenilins, other proteases might contribute to producing truncated forms of APP that are more readily cleaved by BACE and presenilins (PS).88 Calpain can cleave (APP) at three different sites, all located in the extracellular N-terminal domain.89 It is therefore likely that internalized APP might be cleaved. The C-terminal cleavage of APP could indeed produce a fragment that contains the whole of the b-amyloid peptides 1–40 and 1–42 (amyloidogenic), which can be further processed by BACE and PS. APP is also cleaved by caspase 3 at two sites in the extracellular N-terminal domain and at a third site near the C terminus. Again, the cleavage in the C-terminal region might help facilitate the production of b-amyloid peptides indirectly.90,91 A subgroup of signal transduction enzymes are sensitive to calpain and/or caspase-3 degradation. We established that CaM-PKIV is fragmented in caspase-3 and calpain in staurosporine-induced apoptosis but cleaved by calpain only in MTX-induced necrotic oncosis.92 Here caspase produces an immunoreactive 38 kDa N-terminal fragment while calpain produces a 35 kDa N-terminal fragment. In parallel with its proteolysis during apoptosis, we observed a corresponding decrease in

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CAMPK activity in these cells. Previously, others and we have shown that CaMPKII is sensitive to calpain proteolysis.93,94 In our recent study, we found that CaMPKII is also sensitive to caspase-3 proteolysis in apoptotic cells 92 (Table 8.1). The anti-apoptotic properties of CaMPK-IV activity were recently confirmed in cerebellar granule neurons by See et al.95 The calcium-dependent alpha-, beta- and gammaisoforms of protein kinase C have been found to be degraded by calpain in vitro and in cell culture under a variety of conditions.96,97 In fact, calpain is suspected of playing a role in the downregulation of PKC. Interestingly, two calcium-independent PKC isoforms (delta and theta) have now been shown to be degraded by caspase-3 but not the calcium-dependent forms.98,99 PI-specific phospholipase C-beta3 is proteolytic activated by calpain,100 whereas phospholipase A2 and phospholipase Cgamma1 were found to be caspase-3 substrates.101 Both phospholipid hydrolases are involved in transmembrane signal transduction. In platelets, focal adhesion kinase (FAK, pp125FAK) is a 125 kDa nonreceptor tyrosine kinase implicated in integrinmediated signal transduction. It is degraded by calpain during platelet activation to 90 kDa and 45 kDa, 40 kDa fragments.102,103 Recently, FAK was also found to be degraded by caspase-3/7 during apoptosis in Jurkat cells to an 85 kDa fragment.104 IP3 receptors (IP3R) are involved in IP3 signaling. Recent reports found that IP3R1 and IP3R2 are degraded by caspase-3 while insensitive to calpain. Conversely, IP3R3 is degraded only by calpain.105 PARP is the most well-known nuclear substrate for caspase-3. During apoptosis, the 113 kDa PARP is degraded by caspase to a distinct 89 kDa fragment and 24 kDa fragment.44,45 But recently, PARP has been found to be cleaved at alternative site(s), generating fragments from 70 kDa to 40 kDa during necrotic oncosis.106,107 Another recent study described the purification of a non-caspase-protease that cleaves bovine PARP.108 They identified the protease as bovine m-calpain. The fragmentation of PARP by calpain in vitro108 again yields fragments ranging from 70–40 kDa, similar to the PARP cleavage pattern obtained in oncotic cells.106 Using maitotoxin (MTX) as a necrotic challenge, we indeed found that PARP is cleaved into 70 kDa and 40 kDa fragments. This process is sensitive to calpain inhibitor I, but the extent of calpain-mediated proteolysis in oncosis is much less than that observed by caspase-3mediated apoptosis.109 DNA polymerase e (catalytic subunit) is another nuclear protein that is degraded by both calpain and caspase-3.110 In apoptosis, proteolysis is likely to be an important event in shutting down cell function by disabling a number of signal transduction enzymes, in disabling the cell’s ability to repair its DNA or going through cell cycle and in degrading the cytoskeleton network, therefore allowing membrane blebbing and the subsequent phagocytosis by macrophage. In oncotic necrosis, although not necessarily by design, calpain-mediated proteolysis undoubtedly plays a similar role in disabling the cells in the signal transduction, membrane and cytoskeleton integrity front, and nuclear function. Furthermore, calpain might facilitate the apoptotic cell death by aiding caspase in proteolysis of cellular proteins.

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8.6.2 TURNING BAX-THINGS WORSE Another way that caspase and calpain can work together is at the Bcl-2/Bax protein family level (Figure 8.2). Bcl-2, which is a well-studied antiapoptotic protein associated with the mitochondria, is cleaved by caspase-3 in a positive feedback loop to an N-terminally truncated form (tBcl-2) which become proapoptotic.111 Similarly Woods et al.112 and we113 reported that Bax are also truncated N-terminally by calpain, but not caspase. tBax is in fact more proapoptotic114 (Figure 8.2). Bid is a neutral molecule but once it is cleaved by caspase-8, it will translocate to mitochondria and induce cytochrome C and Smac release.115 This has been viewed as a crosstalk from the receptor pathway to the mitochondria-pathway of apoptosis. Lastly, Bcl-XL, another antiapoptotic protein, can be truncated by either calpain or caspase to a proapoptotic form116,117 (Figure 8.2). Thus, both calpains and caspases are involved in a positive feedback mechanism to further protein degradation and apoptotic cell death.

Bax

+

-

BclXL

+

tBclXL

Calpain 1, 2

tBax

Caspase-3 / Calpain

++

Bid

Cyto. C. & Smac Release

-

tBid

+

Bcl-2 Caspase-3

Caspase- 8

+

tBcl-2

Activation of Caspases

Apoptosis

FIGURE 8.2 Cysteine protease regulation of Bcl-2/Bax family proteins. Proapoptotic Bax, when truncated by calpain, becomes more active. Bid, a normally neutral molecule, becomes proapoptotic when truncated by caspase-8. Likewise, antiapoptotic molecules Bcl-2 and BclXL truncated by caspase-3 and/or calpain become apoptotic. Truncated Bax and Bid exert their effects by translocating to the nuclei and therefore causing the release of cytochrome C and Smac, both of which activate caspase-9 and caspase-3, respectively.

8.7 CALPAIN-CASPASE CROSSTALK It has been recently reported that calpastatin is degraded by calpain in A23187treated cells118 (Table 8.2). We suspect that the caspase pathway might be linked to calpain pathway through calpastatin (Figure 8.1). We indeed found that the 105 kDa HMW form of CAST is in fact very sensitive to caspase proteolysis in apoptosis in staurosporine-treated neuroblastoma SY5Y cells. A major fragment of 75 kDa is formed in SY5Y. We concluded that caspase-1 and 3 are the likely proteases

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involved. However, there is only a two-fold drop of inhibitory activity of CAST upon fragmentation since the major cleavage sites lie outside of the inhibitory sequence in each repeat unit.119 We speculate that the degradation of CAST might have yet other functions in unleashing calpain activity or other calpain-independent roles in apoptosis. Again, the susceptibility of CAST to caspase is confirmed by Porn-Ares et al.120 Recently, it was shown by others and us that pro-caspase-3 is a substrate of calpains.109,121,122 As it turns out, pro-caspase-3 (32 kDa) is truncated by calpain at the Nterminal to a 30 kDa form, which appeared to be less vulnerable to being activated by caspase 8 or caspase 9. Chua further showed that caspase-7, 8 and 9 are also truncated at the N-terminal.122 The truncation of the upstream caspase–9 (and caspase-8) is even more significant as it abolishes their ability to interact with the mitochondriareleased Apaf-1 or with the “death domain” receptors, respectively. We interpret this as a mechanism in which during oncosis, when there is high level of calpain activity, the caspase activation cascade and thus apoptotic phenotype would be shut down (Figure 8.3). On the other hand, Nakagawa et al. recently showed that caspase-12 is in fact processed and activated by calpain.117 Since caspase-12 is the ER-associated caspase that is turned on during A-beta toxicity, it has additional meaning. Lee et al.123 have shown that A-beta treatment of hippocampal cultures can in fact activate calpain. Interestingly, calpain also processes a cdk5 activator protein p35 (to a truncated p25 form) thereby activating cdk5. They further showed that calpain and cdk5 inhibitors in fact could protect against A-beta toxicity, which was also confirmed by others.124,125 Together, this would suggest that calpain plays a key role in A-beta induced toxicity.

Caspases

Calpains

-

+

Ca2+

+

Calpastatin

Caspases

+

+

Calpains

+

Apoptosis

+

Mixed Apoptosis-Oncosis

Oncotic Necrosis

FIGURE 8.3 Calpains, caspase and neuronal apoptosis and oncosis. Under specific conditions, strict caspase-dependent and calpain-independent apoptosis can occur. However, in most neuronal injury, calcium influx invariably causes calpain activation as well, which can lead to a mixed oncosis/apoptosis phenotype. Lastly, in severe excitotoxicity and ischemic injury, strict oncosis with calpain activation is the most likely phenotype.

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8.8 PERSPECTIVE In this chapter, we described the presence of three phenotypic forms of cell death: Caspase-dependent apoptosis, mixed oncosis-apoptosis with calpain and caspase activation and caspase-independent oncosis. Indeed, apoptosis might or might not be associated with Ca2+ overload. In antiFAS-treated Jurkat T cells, there is little calpain activation (Figure 8.3). The difference could be the rapid onset of apoptosis and the resultant cell death (which takes 3–4 h). Thus, in rare conditions that no intracellular calcium elevations occur, a strict calpainindependent but caspase-dependent apoptosis would occur. In fact, artificially, we can demonstrate that using long-term treatment (24–48 h) with calcium chelator (EGTA) (Figure 8.3). However, more than likely, in delayed neuronal injury induced by hypoxia, excitotoxicity etc., calcium level invariably rises and thus, you have a mixed apoptotic-oncotic phenotype with the activation of both calpains and caspases (Figure 8.1). This is initiated by caspase-mediated calcium entry and calpastatin fragmentation (Figure 8.1). Calpain and Caspase-3 go on and degrade or modify the large arrays of similar or identical cellular proteins (Table 8.2), ultimately resulting in neuronal death. It therefore follows that calpain and caspase inhibitors have neuroprotective effects, which have been demonstrated by various laboratories. In fact both calpain and caspase inhibition have been shown to have an extended therapeutic window (3–4 delay treatment is still efficacious).58,59,126 Furthermore, Rami et al.71 recently showed that the combination treatment of a calpain inhibitor with a caspase inhibition provide additive neuroprotection in a 4-vo global ischemia model. A third scenario is when neurons are injured acutely with challenges such as severe ischemia (in core of infract), large and rapid rises of intracellular free calcium levels result in high calpain activation which most likely shut down caspase activation cascade by truncation of caspases and Apaf-1, producing a strict oncotic phenotype (Figure 8.3).

8.9 ACKNOWLEDGMENTS I would like to thank my present and former associates Rathna Nath, Satavisha Dutta, Albert Probert Jr., Dr. Kim McGinnis, Dr. Rand Posmantur, and my collaborators, Dr. Ronald Hayes, Dr. Po-wai Yuen, and Dr. Margaret Gnegy for contributing to the work.

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5. MacManus, J.P., Buchan, A.M., Hill, I.E., Rasquinha, I., and Preston, E., Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain, Neurosci. Lett., 164 (1-2), 89, 1993. 6. Heron, A., Pollard, H., Dessi, F., Moreau, J., Lasbennes, F., Ben-Ari, Y., and Charriaut-Marlangue, C., Regional variability in DNA fragmentation after global ischemia evidenced by combined histological and gel electrophoresis observations in the rat brain, J. Neurochem., 61 (5), 1973, 1993. 7. Filipkowski, R.K., Hetman, M., Kaminska, B., and Kaczmarek, L., DNA fragmentation in rat brain after intraperitoneal administration of kainate, Neuroreport, 5 (12), 1538, 1994. 8. Li, Y., Chopp, M., Jiang, N., and Zaloga, C., In situ detection of DNA fragmentation after focal cerebral ischemia in mice, Brain Res. Mol. Brain Res., 28 (1), 164, 1995. 9. Bonfoco, E., Krainc, D., Ankarcrona, M., Nicotera, P., and Lipton, S.A., Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures, Proc. Natl. Acad. Sci. USA, 92 (16), 7162, 1995. 10. Chen, J., Jin, K., Chen, M., Pei, W., Kawaguchi, K., Greenberg, D.A., and Simon, R.P., Early detection of DNA strand breaks in the brain after transient focal ischemia: implications for the role of DNA damage in apoptosis and neuronal cell death, J. Neurochem., 69 (1), 232, 1997. 11. Emery, E., Aldana, P., Bunge, M.B., Puckett, W., Srinivasan, A., Keane, R.W., Bethea, J., and Levi, A.D., Apoptosis after traumatic human spinal cord injury, J. Neurosurg., 89 (6), 911, 1998. 12. Rink, A., Fung, K.M., Trojanowski, J.Q., Lee, V.M., Neugebauer, E., and McIntosh, T.K., Evidence of apoptotic cell death after experimental traumatic brain injury in the rat, Am. J. Pathol.. 147 (6), 1575, 1995. 13. Anglade, P., Vyas, S., Javoy-Agid, F., Herrero, M.T., Michel, P.P., Marquez, J., Mouatt-Prigent, A., Ruberg, M., Hirsch, E.C., and Agid, Y., Apoptosis and autophagy in nigral neurons of patients with Parkinson’s disease, Histol. Histopathol., 12 (1), 25, 1997. 14. Cotman, C.W., Apoptosis decision cascades and neuronal degeneration in Alzheimer’s disease, Neurobiol. Aging, 19, S29, 1998. 15. Troost, D., Aten, J., Morsink, F., and de Jong, J.M., Apoptosis in amyotrophic lateral sclerosis is not restricted to motor neurons. Bcl-2 expression is increased in unaffected post-central gyrus, Neuropathol. Appl. Neurobiol., 21 (6), 498, 1995. 16. Dessi, F., Charriaut-Marlangue, C., Khrestchatisky, M., and Ben-Ari, Y., Glutamateinduced neuronal death is not a programmed cell death in cerebellar culture, J. Neurochem., 60 (5), 1953, 1993. 17. Scott, R.J. and Hegyi, L., Cell death in perinatal hypoxic-ischaemic brain injury, Neuropathol. Appl. Neurobiol., 23 (4), 307, 1997. 18. MacManus, J.P., Rasquinha, I., Black, M.A., Laferriere, N.B., Monette, R., Walker, T., and Morley, P., Glutamate-treated rat cortical neuronal cultures die in a way different from the classical apoptosis induced by staurosporine, Exp. Cell Res., 233 (2), 310, 1997. 19. Portera-Cailliau, C., Price, D.L., and Martin, L.J., Excitotoxic neuronal death in the immature brain is an apoptosis-necrosis morphological continuum, J. Comp. Neurol., 378 (1), 70, 1997. 20. Wang, K.K. and Yuen, P.W., Calpain inhibition: an overview of its therapeutic potential, Trends Pharmacol. Sci., 15 (11), 412, 1994.

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102. Carragher, N.O., Fincham, V.J., Riley, D., and Frame, M.C., Cleavage of Focal Adhesion Kinase by Different Proteases during Src-Regulated Transformation and Apoptosis: Distinct Roles for Calpain and Caspases, J. Biol. Chem., 2000. 103. Cooray, P., Yuan, Y., Schoenwaelder, S.M., Mitchell, C.A., Salem, H.H., and Jackson, S.P., Focal adhesion kinase (pp125FAK) cleavage and regulation by calpain, Biochem. J., 318 (Pt 1), 41, 1996. 104. Wen, L.P., Fahrni, J.A., Troie, S., Guan, J.L., Orth, K., and Rosen, G.D., Cleavage of focal adhesion kinase by caspases during apoptosis, J. Biol. Chem., 272 (41), 26056, 1997. 105. Diaz, F. and Bourguignon, L.Y., Selective down-regulation of IP(3)receptor subtypes by caspases and calpain during TNF alpha -induced apoptosis of human T-lymphoma cells [In Process Citation], Cell Calcium, 27 (6), 315, 2000. 106. Shah, G.M., Shah, R.G., and Poirier, G.G., Different cleavage pattern for poly(ADPribose) polymerase during necrosis and apoptosis in HL-60 cells, Biochem. Biophys. Res. Commun., 229 (3), 838, 1996. 107. Sallmann, F.R., Plancke, Y.D., and Poirier, G.G., Rapid detection of poly(ADP-ribose) polymerase by enzyme-linked immunosorbent assay during its purification and improvement of its purification, Mo. Cell. Biochem., 185 (1-2), 199, 1998. 108. Buki, K.G., Bauer, P.I., and Kun, E., Isolation and identification of a proteinase from calf thymus that cleaves poly(ADP-ribose) polymerase and histone H1, Biochim. Biophys. Acta., 1338 (1), 100, 1997. 109. McGinnis, K.M., Gnegy, M.E., Park, Y.H., Mukerjee, N., and Wang, K.K., Procaspase-3 and poly(ADP)ribose polymerase (PARP) are calpain substrates, Biochem. Biophys. Res. Commun., 263 (1), 94, 1999. 110. Liu, W. and Linn, S., Proteolysis of the human DNA polymerase varepsilon catalytic subunit by caspase-3 and calpain specifically during apoptosis, Nucleic Acids Res., 28 (21), 4180, 2000. 111. Cheng, E.H., Kirsch, D.G., Clem, R.J., Ravi, R., Kastan, M.B., Bedi, A., Ueno, K., and Hardwick, J.M., Conversion of Bcl-2 to a Bax-like death effector by caspases, Science, 278 (5345), 1966, 1997. 112. Wood, D.E., Thomas, A., Devi, L.A., Berman, Y., Beavis, R.C., Reed, J.C., and Newcomb, E.W., Bax cleavage is mediated by calpain during drug-induced apoptosis, Oncogene, 17 (9), 1069, 1998. 113. McGinnis, K.M., Gnegy, M.E., and Wang, K.K., Endogenous bax translocation in SH-SY5Y human neuroblastoma cells and cerebellar granule neurons undergoing apoptosis, J. Neurochem., 72 (5), 1899, 1999. 114. Gao, G. and Dou, Q.P., N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome C release and apoptotic cell death [In Process Citation], J. Cell. Biochem., 80 (1), 53, 2000. 115. Li, H., Zhu, H., Xu, C. J., and Yuan, J., Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis, Cell, 94 (4), 491, 1998. 116. Clem, R.J., Cheng, E.H., Karp, C.L., Kirsch, D.G., Ueno, K., Takahashi, A., Kastan, M.B., Griffin, D.E., Earnshaw, W.C., Veliuona, M.A., and Hardwick, J.M., Modulation of cell death by Bcl-XL through caspase interaction, Proc. Natl. Acad. Sci. USA 95 (2), 554, 1998. 117. Nakagawa, T. and Yuan, J., Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis, J. Cell Biol., 150 (4), 887, 2000.

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9

Brain Inflammation, Cytokines, and p38 MAP Kinase Signaling in Stroke Frank C. Barone, Ronald F. Tuma, Jeffrey J. Legos, Joseph A. Erhardt, and Andrew A. Parsons

CONTENTS 9.1 9.2

Abstract Introduction 9.2.1 Stroke: How Important Is It? 9.2.2 Stroke: Its Pathophysiology 9.3 The Brain Inflammatory Response to Injury 9.4 TNFα in Stroke and Brain Injury 9.5 IL-1β in Stroke and Brain Injury 9.6 Leukocyte Adhesion Molecules in Stroke and Brain Injury 9.7 Neurodestructive and Neuroprotective Gene Expression Following Stroke 9.8 Strategies to Target Stroke: Brain Inflammation and Its Complexities 9.9 Strategies for Treatment of Brain Inflammation/Injury 9.9.1 Blocking Cytokine Actions 9.9.2 Anti-Adhesion Molecule Antibodies 9.9.3 Anti-Leukocyte Treatments 9.10 Other Approaches: Blocking Cytokine Production 9.10.1 cAMP and PKC 9.10.2 p38 Mitogen-Activated Protein Kinase Activation and Inhibition in Stroke 9.10.3 CSAIDs p38 Inhibitors 9.10.4 p38 MAPK Activation Following Focal Stroke 9.10.5 p38 MAPK Inhibition on Brain Injury, Downstream Signaling, and Inflammatory Cytokine Expression Post-Stroke 9.10.6 SB 239063 Effects Development and Resolution of Infarct Post-Stroke 9.10.7 SB 239063 Neuroprotection Following In Vitro Ischemia 9.11 Summary and Conclusions References

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9.1 ABSTRACT The importance of cytokines, especially TNFα and IL-1β, is emphasized in the propagation and maintenance of the brain inflammatory response to injury. Much data supports the case that ischemia and trauma elicit an inflammatory response in the injured brain. This inflammatory response to brain injury consists of mediators (cytokines, chemokines, and adhesion molecules) followed by cells (neutrophils early after the onset of brain injury and then a later monocyte infiltration). De novo upregulation of proinflammatory cytokines, chemokines, and endothelial-leukocyte adhesion molecules occurs soon following focal ischemia and trauma during a time when the tissue injury is evolving. Presented here is a discussion of the role of TNFα and IL-1β in brain injury and its associated inflammation, and the cooperative actions of cytokines, chemokines, and adhesion molecules in driving this brain inflammatory process. We also address novel approaches to target cytokines and reduce the brain inflammatory response, and thus brain injury, in stroke. The mitogen-activated protein kinase (MAPK), p38, has been linked to inflammatory cytokine production and cell death following stress. Stroke-induced p38 enzyme activation in the brain has been demonstrated, and treatment with a second-generation p38 MAPK inhibitor, SB 239063, provides a significant reduction in infarct size, neurological deficits, and increased inflammatory cytokine expression produced by focal stroke. SB 239063 also provides direct protection of cultured brain tissue to ischemia in vitro. The most effective dose was further evaluated in detail, and SB 239063 significantly (p < 0.05) reduced neurologic deficit and infarct size for at least 1 week. Also, early reductions in MRI DWI intensity following treatment with SB 239063 correlated highly with neuroprotection seen up to 7 days poststroke. Since increased protein levels for various proinflammatory cytokines cannot be detected prior to 2 h in this stroke model, the early improvements due to p38 inhibition, observed using DWI, demonstrate that p38 inhibition can be neuroprotective through earlier direct effects on ischemic brain cells, in addition to effects on inflammatory cytokines and brain inflammation. This was also demonstrated by direct effects of calcium influx on p38 early activation on neurons in vitro. The robust SB 239063-induced neuroprotection emphasizes a significant opportunity for targeting MAPK pathways in ischemic stroke injury, and also suggests that p38 inhibition be evaluated for protective effects in other experimental models of nervous system injury and neurodegeneration.

9.2 INTRODUCTION 9.2.1 STROKE: HOW IMPORTANT IS IT? Stroke is the third largest cause of death in the United States (first in Japan), ranking only behind heart disease and all forms of cancer. It is the leading cause of disability in the United States (i.e., highest “disease-burden cost”). No medical treatment is approved for the treatment of stroke beyond a thrombolytic (e.g., tPA has to be administered within 3 hours after stroke and, thus, is only available for 1–2% of stroke

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patients). Aspirin and anticoagulants (where embolic phenomena are documented) are utilized as preventative therapy. Thus, stroke represents a large, unmet medical need. Estimates indicate that there are about 775,000 stroke cases per year in the United States, with about 4 million surviving, but at increased risk of a secondary cardiovascular event. It has an estimated market of $1 billion with an annual health care cost in excess of $50 billion (US). In the UK, 100,000 cases of stroke per year account for 5% of total government expense. Estimates indicate that stroke is responsible for half of all patients hospitalized for acute neurological disease. Twentyeight percent of annual stroke victims are under age 65. Although the incidence of stroke previously appeared to be on the decline, recent analyses indicate that this is not the case, and that we might even expect increased stroke numbers with an everincreasing morbidity and mortality as the human population increases in longevity. Predictions are that the US (only) stroke rate will increase by the year 2005 to over 1 million per year.1,2 Stroke risk factors include both genetic (predisposing) and environmental factors. These factors are a function of natural processes or result from a person’s lifestyle. Stroke risk factors that can be treated include (i) high blood pressure (considered the most important controllable risk factor for stroke), (ii) heart disease, (iii) cigarette smoking (use of oral contraceptives combined with cigarette smoking greatly increases stroke risk), (iv) transient ischemic attacks (e.g., these are the first sign of cerebrovascular disease and occurrence increases risk for major stroke by 10fold), and (v) high red blood cell count (e.g., a moderate increase in the number of red blood cells thickens the blood and makes clots more likely). Risk factors for stroke that cannot be changed include (i) increased age (chances of having stroke more than doubles for each decade of life after age 55), (ii) gender (men have about a 19 percent greater chance of stroke than women), (iii) race (blacks have a much higher risk of death and disability from stroke, in part because they have a greater incidence of high blood pressure), (iv) diabetes mellitus, (v) prior stroke, and (vi) family history of strokes. Other controllable risk factors are secondary risk factors for stroke that contribute to heart disease including high blood cholesterol and lipids, physical inactivity and obesity.3

9.2.2 STROKE: ITS PATHOPHYSIOLOGY Stroke is most commonly the result of an obstruction of blood flow in a major cerebral vessel (usually the middle cerebral artery), which, if not resolved within a short period of time (minutes), will lead to a core of severely ischemic brain tissue that may not be salvaged. However, the ultimate size of the brain infarct also depends on the “penumbra,” a zone of tissue around the core of the infarct where blood flow is still maintained above a neuronal disabling level or the critical 20–25% of normal blood flow. If blood flow in the penumbral zone further decreases below the critical level of flow, the infarct zone will inevitably expand. The magnitude of blood flow (i.e., by providing the conditions essential to maintenance of cellular energy hemostasis), therefore, is the principal factor in determining the size of the core infarct and penumbral zones. Figure 9.1A (See color insert following page 114.) provides an

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Embolus/Thrombus in Cerebral Vessel

A

Cerebral Blood Flow ATP/PCr Depletion

Free Radicals (NO, SO-)

Ion permeability Cai+2 Cli-

Nai+

Neurotransmitter Release

H2Oi

Ke+

[Ca+2]i

Cell Swelling

Cellular Activation of Enzymes Cytotoxic (ionic) Edema FFA/PUFA

Lipases

Lipid/Protein/Nuclear Damage

Proteases

Endonucleases

Cellular Damage/Death

Inflammation

B

Progression of Brain Injury and Recovery Following Stroke

Necrotic and Apoptotic Cellular Damage

Neurodistructive Gene Expression IL-1, TNF Early ICAM-1, ELAM-1 IL-8, MCP-1 iNOS, MMPs BBB Break

Injury Neutrophil

Neuroprotective Gene Expression

Healing and Recovery

ICAM-1

Circulating Monocytes

bFGF NGF, BDNF TGF, GDNF (Neuroprotection)

EC Metalloproteinases Cytokines Chemokines Microglia

O2

-

NO

Neuron/Astrocyte Cytotoxicity

Cavitation of Infarct Microglia/Macrophages Astrogliosis/Glial Scar

é Plasticity of Remaining Neurons

FIGURE 9.1 (See color Figure 9.1.) (A) Schematic box diagram illustrating major changes that occur in thromboembolic ischemic stroke. (B) Ongoing neuroprotective and neurodestructive gene expression and their roles poststroke.

outline of some of the events known to be involved in the initiation of brain injury/neuronal death following ischemic stroke. The events resulting from “embolus/thrombus” are also relevant to brain trauma. Decreased blood flow leads to a reduction in phosphocreatinine and ATP (especially the latter), and if ischemia is prolonged, the energy source depletion will be sufficient to lead to severe impairment of cellular function by disruption of ATP-dependent processes (e.g., Na+/K+ ATPase) (see Refs. 4–6 for reviews). Regarding Figure 9.1A, ischemic stroke reduces energy availability and therefore membrane ionic pumps fail rapidly. The rise in extracellular potassium can reach levels sufficient to release excitotoxic neurotransmitters (e.g., glutamate and aspartate) to stimulate sodium/calcium channels coupled to glutamate receptors that can facilitate developing cytotoxic edema. The significant influx of calcium through calcium channels increases free cytosolic calcium that causes mitochondrial calcium

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overload, cessation of already compromised ATP production, and extensive breakdown of cellular phospholipids, proteins, and nucleic acids owing to Ca2+ activation of phospholipases, proteases, and endonucleases. Free radicals (nitric oxide, superoxide, etc.) are produced in the process and contribute to membrane lipid peroxidation, protein and nuclear DNA toxic changes, and cellular injury (i.e., necrosis and/or apoptosis). These processes initiate neurodestructive and neuroprotective gene expression responses in the injured brain. The inflammatory response to tissue injury occurs after these initial changes but contributes to the ongoing evolution of tissue injury. Regarding Figure 9.1B, the early depletion in energy-rich phosphates that occurred in part A has immediate effects on membrane transport processes resulting in increased extracellular potassium, increased intracellular sodium and calcium, and glutamate release. All these events are over within 1 to 2 hours. Immediate early response genes are expressed within 2 hours, and then neurodestructive gene expression occurs consisting of inflammatory cytokine (e.g., IL-1β and TNFα), chemokine and adhesion molecule (e.g., ICAM-1) expression that drives the brain inflammatory response to injury. Leukocytes accumulate within vessels, altering normal flow, and within tissues (e.g., initially neutrophils). Resulting rheologic, cytokine, and proteinase cytotoxic effects contribute to ongoing brain injury. In this same time period, neuroprotective gene expression (e.g., protective growth factors including NGF, BDNF, GDNF) is observed in defense of ongoing neurodestructive processes. Tissue remodeling factors (TGFβ) are expressed later and is associated with resolution/healing of infarcted/injured brain tissue. Generally following initiation of brain injury (as depicted in part A and outlined in the text), neurodestructive gene expression (primarily those inflammatory cytokines, adhesion molecules, chemokines, and inflammatory proteins such as inducible NOS and metalloproteinases) can drive brain inflammation and necrotic/apoptotic cell death. Also, neuroprotective gene expression includes neurotrophic and growth factors from circulating mononuclear cells that have infiltrated into damaged tissue or resident activated glial cells that can protect cells/tissue, can facilitate repair and remodeling and/or increase neuronal plasticity, and can facilitate the recovery of function for remaining viable neurons/tissue. Under reduced blood flow, ATP depletion occurs and results in the disruption of ionic gradients across excitable (neuronal) and nonexcitable (glial) membranes that is marked by efflux of K+ from the cells and influx of Na+, Cl-, and Ca2+ into the cells. Of these events, the increase in extracellular K+, along with a fall in pH, precedes the other ionic changes. In this phase, ATP stores are rapidly depleting, but still maintain sufficient energy to prevent the second phase, which is marked by sudden and dramatic ion conductance changes. It is postulated that the rise in K+ can reach levels sufficient to release neurotransmitters such as glutamate, which in turn will stimulate Na+/Ca2+ channels coupled to the NMDA receptor; these events will further lead to Na+, Cl-, and H2O accumulation, cell swelling, and cytotoxic edema. Parallel to these events, extracellular Ca2+ enters into cells through both voltage-operated calcium channels (VOCC) and receptor-operated calcium channels (ROCC), leading to elevated free cytosolic Ca2+, which causes mitochondrial calcium overload and

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cessation of ATP production. Transient extracellular K+-induced depolarizations can contribute to the expansion of the neuronal lesion. Peri-infarct depolarization produces disruption of ionic gradients and transmitter release, with the associated accumulation of free Ca2+ in the cells resulting in a rapid and extensive breakdown of phospholipids and proteins and even nucleic acids by activation of calcium-dependent phospholipases, proteases, and endonucleases. Depolarization causes an increase in intracellular calcium and an increase in extracellular glutamate. Glutamate, an excitatory neurotransmitter implicated in ischemic neuronal damage, results in excitotoxicity in which excessive extracellular glutamate kills neurons through an increase in intracellular calcium. Glutamate-mediated excitotoxicity is thought to occur because of the overactivation of AMPA and NMDA synaptic glutamate receptors. The NMDA receptor channel conducts calcium as do some AMPA receptor channels. AMPA receptors also control the initial membrane depolarization caused by glutamate, and affect the opening of the NMDA receptors. AMPA, NMDA and glycine receptors are targets that have been pursued pharmacologically (see Refs. 6-9 for reviews). Accumulation of products such as free fatty acids (especially the unsaturated types) that are transformed to lipid peroxides and their metabolites (via lipid peroxidation) further contribute to structural and functional perturbations of the membrane and cell function. Free radicals (a group of highly reactive oxygen species) are generated during ischemia, and cause considerable damage to lipids, DNA and proteins and contribute to the process of neuronal death. Free radicals also contribute to the breakdown of the blood-brain barrier and brain edema. Levels of free radical scavenging enzymes (e.g., superoxide dismutase) fall during ischemia and nitric oxide levels are elevated. Nitric oxide generated primarily by neuronal and inducible nitric oxide synthases promote neuronal damage following ischemia. For example, activation of cytosolic proteases by the increased [Ca2+]i can result in the direct disassembly of the cellular microtubule system and proteolytic degradation of structural and functional protein. Another specific event associated with protease activation, the conversion of xanthine dehydrogenase to xanthine oxidase, bears directly on cellular formation of toxic oxygen free radicals which further break down membrane, cytoskeletal, and nuclear structures. Therefore, to summarize those factors that contribute to initial brain injury, the reduction in cerebral blood flow depletes energy to sufficiently low levels to significantly perturb cellular ionic homeostasis. A key consequence is an increase in intracellular calcium. This has been the main, consistent event focused upon by scientists over the past three decades of stroke research.10 Increased intracellular calcium is responsible for the release of neurotransmitters and the activation of many enzymes. Glutamate has been focused upon as the neurotransmitter released by the ischemic/depolarizing events in stroke because of its excitotoxic effect on neurons. Also, another consequence of increased intercellular calcium is the activation of lipases, proteases, and endonucleases. The consequences of endonuclease activation might trigger debates over the importance of apoptosis or programmed cell death in focal stroke, but there is no doubt that DNA damage is a consequence of ischemia. Free radicals contribute to these effects. Overall, cell destruction in ischemic stroke

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is associated with multiple events including depletion of energy, ion perturbations, neurotransmitter release, and enzyme activation. In terms of the pharmaceutical industry, basic research in this area has provided for the discovery and development of calcium channel blockers as an important potential therapeutic approach to the treatment of stroke. Also, the development of a variety of glutamate receptor antagonists (i.e., targeting various glutamate and glutamate-associated receptors) are major development areas as well. Approaches targeting oxygen free radicals have also been pursued.6,7 Although the research on these approaches has been and still is quite extensive, a calcium channel blocker has not yet demonstrated efficacy in the clinic. Many phase III clinical studies on a variety of glutamate receptor antagonists have been ongoing, but the data is still inconclusive and there have been many failures. There might be a reason for the difficulties in providing therapeutic benefit associated with these targets, since changes associated with them occur very soon following stroke. Calcium concentration changes happen within seconds to minutes after the ischemia insult. In fact, calcium level maximizes around two minutes. Glutamate release has been demonstrated to be at maximum after 20–40 minutes, and most of the damage associated with excitoxicity may occur within the first 20 minutes, and certainly within an hour. Oxygen free radicals are very short-lived. Perhaps the difficulties in obtaining clinical efficacy is that these events, while clearly valid targets to intervene upon, may not be much of an opportunity by the time the patient gets into the emergency room or other facility for therapeutic intervention. The thrombolytic tissue plasminogen activator (tPA) which degrades fibrin clots can now be used as a therapeutic for stroke, but only if administered within 3 hours.2 Typically, patients are available for therapeutic intervention at earliest 3 hours poststroke. There are other opportunities for intervention that can impact on the evolution of brain injury. These will be discussed below.

9.3 THE BRAIN INFLAMMATORY RESPONSE TO INJURY Direct blows (e.g., blunt or closed impact), deprivation of oxygen and nutrients, transplantation, neurotoxic injury (e.g., excitotoxic damage), viral attack, and immunological challenge produce the well-defined brain response of “gliosis.”11 The activation, proliferation, and hypertrophy of cells derived from the mononuclear phagocytic system (e.g., macrophages and microglia) characterize this reaction. The significance of this response was originally thought to trigger processes that mediate the repair of brain injury by restoration of blood supply, reestablishing the integrity of the blood brain barrier and promoting general homeostasis at the site of injury.12-17 Although activated astrocytes have been shown to secrete growth factors that stimulate axonal growth,18 their accumulation at sites of injury has also been shown to suppress axonal regrowth and damaged neural tissue16,19 This data suggests a potential of the brain to regenerate in an orderly manner following an insult. Since cytokines activate glial cells in vivo,15 and glial cells produce cytokines in response to stimulation in vitro,20-22 a close relationship would appear to exist

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between inflammation, cytokine production, and gliosis. Indeed, elegant experiments have shown that gliosis can be induced by TNFα, IL-1β, and IFNα.23,24 Further implications for the potential central involvement of cytokines in brain inflammation have been derived from studies on the localization of TNFα and IL-1β following surgical injury to the hippocampus in rats.25 in multiple sclerotic plaques in human brain26 and human head injury, in which colocalization of cytokines with β-amyloid precursor expression, a marker for Alzheimer’s disease, was reported.27 A key issue concerning the involvement of cytokines in brain inflammation is the identification of those key cellular elements responsible for their production. Indeed, there is still much debate as to whether inflammatory mediators are of central origin or whether local production is as a result of recruitment of activated immune cells from the peripheral circulation. Although several cell types are able to secrete cytokines including microglia, astrocytes, and neurons, there is also evidence to support the involvement of peripherally derived cells in contributing to brain inflammation and injury. Furthermore, the notion that the blood-brain barrier is impermeable to cells of the immune lineage is no longer valid, and it is clear now that it becomes leaky following injury to the brain,28,29 but the actual extent of the bloodbrain barrier breakdown in stroke is not well understood. Indeed, peripherally derived mononuclear phagocytes, T-lymphocytes, Natural Killer cells, and polymorphonuclear cells (PMNs) that produce and secrete cytokines, may all contribute to brain inflammation or gliosis. In support of this, irradiation of the bone marrow or treatment in vivo with colchicine, attenuate gliosis, wound repair, neovascularization, and generalized inflammation.29 The inflammatory response to brain injury has been studied systematically following focal stroke by our laboratory and also by many other investigators.30-41 Unlike normal vessels that are empty and collapsed upon histopathological evaluation, in the focal ischemic cortex, vessels become filled with leukocytes, and many have a significant zone of edema around them. Many of the leukocytes, primarily neutrophils, in the ischemic tissue vessels are adherent to the postcapillary venules and arteriole microvascular walls. Although they do not adhere directly to capillary endothelium, they can impede flow within microvessels and capillaries. This is not normally observed in brain vessels, or in most other vessels for that matter. Some of these neutrophils find their way outside the vascular walls in the focal ischemic cortex. Evolving brain injury is associated with the expression of inflammatory mediators (e.g., inflammatory cytokines) and an inflammatory response, that includes adhesion of leukocytes to the wall of blood vessels, infiltration of these cells into ischemic brain tissue, and the activation of microglia resident in the brain (for review see Ref. 42). This inflammatory reaction not only contributes to lipid-membrane peroxidation, but also exacerbates the degree of tissue injury due to the rheologic effects of “sticky” leukocytes in the microvessels (i.e., an interference with normal microvascular perfusion) and also due to the release of cytotoxic enzymes from these activated leukocytes (i.e., cytodestructive for an already compromised tissue bed).43-47 Therefore, the brain is capable of mounting a pronounced inflammatory response to injury manifested in part by gliosis and driven by cytokines and both centrally and peripherally derived immune cells. The exact nature of the signaling

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mechanisms still remains to be elucidated, but undoubtedly involves TNFα22 and IL-1β, chemotactic cytokines (e.g., IL-8) as well as the expression of adhesion molecules that together promote both recruited cell adherence and infiltration, and enhanced permeability of brain endothelium. For example, for a neutrophil to adhere to the endothelium and then migrate unidirectionally into the tissue, adhesion molecules have to be upregulated. In order to upregulate adhesion molecule expression, injury must upregulate specific cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). This has to happen very rapidly, and proteins also have to be rapidly translated. In addition, when those cytokines are expressed and translated, they have to upregulate adhesion molecules and produce in parallel chemokines that will produce a chemoattraction to drive neutrophils into the brain tissue from the vasculature. To summarize, in addition to gliosis, the classical hallmarks of brain inflammation are neuronal loss, edema formation, and the presence of recruited PMNs (initially and primarily neutrophils) and mononuclear (later monocytes and macrophages; M∅) leukocytes. The recruitment process must involve the liberation of chemotactic factors that promote margination and migration of leukocytes to the damaged area. Cytokines that predispose or “prime” endothelium for cellular adherence include TNFα and IL-1β. Additionally, adhesion molecules such as CD11/CD18 integrins, ICAM-1, ELAM-1, and P-selectin are also thought to be pivotal in this inflammatory process.48 The early accumulation of neutrophils in ischemic brain damage has been clearly demonstrated based upon histopathological,30,32–37 biochemical38-40 and 111In-labelled leukocyte studies.31,41 The importance of leukocyte infiltration in the pathogenesis of brain injury has been reviewed previously.42–44 It is postulated that PMNs induce tissue damage due to their vascular plugging/rheologic effects, and by generation and release of oxygen radicals and cytotoxic products as they are activated in ischemic tissue.37,42–47 This primary inflammatory response occurs later than the initial “damaging” events discussed above, is driven by cytokines, and contributes to the evolution/maturation or progression of tissue injury. There is also even a later, more chronic inflammatory stage34,35 that is involved in repair, recovery, and plasticity brain processes that occurs following brain injury that will be discussed in more detail below (Figure 9.1B).

9.4 TNFα IN STROKE AND BRAIN INJURY TNFα is a pleiotrophic cytokine released by many cell types upon appropriate stimulation. TNFα exerts diverse array of biological activities, among them including its ability to stimulate of acute phase protein secretion and increased vascular permeability.49 TNFα and its receptors have been identified in the CNS.50 Numerous clinical studies have shown a distinct relationship between elevated levels of several cytokines, including TNFα, neurodegenerative disorders, and brain injury (see Refs. 42 and 51 for review). Elevated TNFα levels occur in various experimental models of brain injury. Systemic kainic acid administration induces TNFα mRNA levels in cerebral cortex,

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hippocampus, and hypothalamus 2–4 hrs later.52 Systemic or intracerebroventricular administration of lipopolysaccharide (LPS) has also been shown to increase brain TNFα levels as determined by bioassay,53 while injection of the excitotoxin ibotenic acid into the rat posterior hypothalamic region increased hippocampal TNFα two weeks later.54 In a model of closed-head injury, Shohami et al.55 reported an early increase in TNFα peptide at the site of the focal insult. Also, in rat traumatic head injury, TNFα mRNA and protein levels are rapidly elevated.56,57 Furthermore, in mice challenged with particles of charcoal injected into the hippocampus, an increase in striatal levels of TNFα mRNA was observed.55 Elevated serum TNFα was also observed following severe head injury in man.58 Elevated expression of TNFα mRNA and protein occurs soon following middle cerebral artery occlusion (MCAO) in rats. In these studies, ischemic cortex levels of TNFα mRNA were elevated as early as 1 hr postocclusion (i.e., prior to significant early influx of inflammatory neutrophils), peaked at 12 hr and persisted for about 5 days. The early expression of TNFα mRNA followed by leukocyte infiltration suggest that TNFα may be involved in this response. Double-label immunofluorescence studies localized the de novo synthesized TNFα to neurons but not astroglia. At 5 days following the ischemic insult, neuronal-associated TNFα was diminished, and TNFα immunoreactivity was localized in the inflammatory cells. The significance of TNFα expression in the brain was studied by microinjection of TNFα into the rat cortex; TNFα induced leukocyte adhesion to microvessel endothelium, but no evidence for neurotoxicity at the site of injection was found. Buttini et al.61 also identified a rapid upregulation of TNFα mRNA and protein in activated microglia and macrophages following focal stroke, again suggesting that TNFα is part of an intrinsic inflammatory reaction of the brain following ischemia. TNFα may exert a primary effect on microvascular inflammatory response as reflected by TNFα-induced neutrophil adhesion to brain microvessel endothelium.59 Furthermore, very recent data from our laboratory has demonstrated that intracerebroventricular injection of TNFα 24 hr prior to MCAO, exacerbates tissue injury.62 This effect was reversed by intracerebroventricular administration of anti-TNFα monoclonal antibody in the contralateral ventricle. Further evidence for the involvement of TNFα in stroke-induced injury is supported by findings that spontaneously hypertensive rats have higher levels of TNFα production in the brain as compared with normotensive rats.53 This would further suggest that TNF (and other cytokines) may predispose brain endothelium to subsequent brain injury. These data suggest that TNFα may prime the brain for subsequent damage by activating vascular endothelium to a “partial,” proadhesive state possibly through the upregulation of surface endothelial adhesion molecules (see below).

9.5 IL-1β IN STROKE AND BRAIN INJURY Accumulating evidence shows that IL-1β can be produced in the brain from various cellular elements including microglia, astrocytes, neurons and endothelium.63 Like TNFα, IL-1β has many proinflammatory properties,64 and receptors for this cytokine

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have been demonstrated in the central nervous system.65 Increase in IL-1β mRNA expression has been shown to occur following several types of injury to the brain including kainate excitotoxicity52 and LPS.66,67 Furthermore, mechanical damage following implantation of a microdialysis probe has been shown to induce expression of IL-1β.68 Following fluid percussion brain trauma procedure in the rat, a rapid increase in IL-1β mRNA expression has been reported.69 Microglial IL-1α expression has been observed in human head injury.27 IL-β mRNA expression has been shown to increase following transient brain ischemia in the rat.70,71 The exacerbation of ischemic brain injury due to exogenous IL-1β administered into the brain has been observed.72 A rapid (3–6 hr postischemia) increase in IL-1β mRNA following MCAO occurs which peaks at 12 hr and returned to basal values at 5 days,73 mimicking the profile of TNFα.59,60 Early IL-1β expression following focal stroke has also been demonstrated using in situ hybridization.74 The recent development of tools such as specific antibodies to rat IL-1β will now permit a more detailed (i.e., immunohistochemical) evaluation of increased IL-1β peptide and its cellular location. Interleukin-1 receptor antagonist (IL-1ra), a 23-25 KDa glycosylated protein, is a naturally occurring inhibitor of IL-1 activity that competes with IL-1 for occupancy of IL-1RI without inducing a signal of its own. IL-1ra has a higher binding affinity for IL-1RI than IL-1α and IL-1β.75 IL-1ra is produced by many different cellular sources including monocytes/macrophages, endothelial cells, fibroblasts, neurons and glial cells.75 A large number of studies indicate that IL-1ra can block IL-1 activity in vitro and in vivo. Accumulating evidence demonstrates the protective effects of IL-1ra in brain injury. Thus, intracerebroventricular administration of recombinant IL-1ra produced a marked reduction in brain damage induced by focal stroke,76,77 brain hypoxia/ischemia78 or fluid percussion injury in the rat.79 This neuronal protective effect of IL-1ra in focal stroke was further supported by a recent study using an adenoviral vector that overexpressed IL-1ra in the brain.80 Of interest is the fact that the peripheral administration of IL-1ra has been shown to reduce brain injury,81 which suggests a potential use of IL-1ra as a neuroprotective therapeutic in human stroke and/or neurotrauma. However, data to date indicate that peripheral administration must be immediately after stroke in order to provide efficacy.81 The biological responses to IL-1 are mediated by specific surface receptors. Two primary receptors for IL-1 have been identified that belong to the immunoglobulin (Ig) super-family.75 The type I IL-1 receptor (IL-1RI), an 80 kDa glycoprotein, is prevalent in T-cells, endothelial cells, smooth muscle cells, and fibroblasts, whereas the type II receptor (IL-1RII), a 68 kDa protein, is found in B-cells and macrophages.75 The cDNA encoding both receptors has been cloned from human, mouse, and rat.82–85 The major difference between the two receptors exists in their cytoplasmic domain, i.e., the IL-1RI contains a larger cytoplasmic domain than IL1RII. This difference allows IL-1RI but not IL-1RII to engage in intracellular signal transduction,86 while IL-1RII may act as a “sink” for IL-1β. The expression of IL-1RI mRNA in brain has been localized in specific regions by means of in situ hybridization in mouse and rat.87,88 Recently, the upregulation of IL-1RI mRNA in brain was observed in mouse after peripheral administration of bacterial endotoxin.89 In contrast, no previous study has evaluated the IL-1RII

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expression in the normal or diseased brain. Very recently, the expression of IL-1ra and IL-1R mRNA following focal stroke has been evaluated.90 The level of IL-1ra mRNA was markedly increased in the ischemic cortex at 6 hr (6-fold increase compared to the sham-operated samples), then reached a significantly elevated level from 12 hr to 5 days following MCAO. The temporal mRNA expression for IL-1RI and IL-1RII was investigated using the same samples as applied for IL-1ra.90 Overall, there was a relatively high basal expression of IL-1RI mRNA in the cortical sample, and upregulation of IL-1RI mRNA was clearly observed at 5 days after MCAO. In contrast, IL-1RII mRNA expression was rapid and robust, and maintained high expression levels for several days post MCAO. The presence of IL-1ra in the normal brain and the upregulation of IL-1ra mRNA after ischemic injury suggest that IL-1ra may serve as a defense system to attenuate the IL-1-mediated brain injury. It is interesting to observe that the temporal induction profile of IL-1ra following MCAO virtually parallels that of IL-1β as demonstrated previously,73 except that IL-1ra mRNA exhibited prolonged elevation beyond that of IL-1β. Thus, the balance between the message levels of IL-1β and IL-1ra expressed postischemia may be more critical to the degree of tissue injury than IL-1 levels per se. However, actual protein expression levels can be even more important and will be discussed later in this review. The mediators responsible for IL-1ra induction after focal stroke are not known. However, previous studies indicate that some cytokines such as IL-1, TNF, IL-6, and TGFβ are inducers of IL-1ra.75 Ischemia-induced expression of IL-1ra mRNA could originate from monocytes/macrophages, endothelial cells, fibroblasts, neurons, and glial cells as observed previously under normal conditions.75 The same cellular sources may be responsible for IL-1β and IL-1ra production based upon the close temporal, and perhaps functional coupling, of these two genes after focal stroke. Differences in the expression of the two IL-1 receptors after focal stroke may reflect their distinct roles in ischemic injury. For example, IL-1RI, but not IL-1RII, stimulates IL-1-mediated signal transduction. Also, IL-1RI has the highest binding affinity for IL-1ra, whereas IL-1RII more readily binds IL-1β.75 The remarkable parallelism in the temporal expression of IL-1RI mRNA and leukocyte infiltration following MCAO in this stroke model35,36,38–40 suggests that the upregulation of this signal transducing receptor may contribute to the IL-1β-mediated leukocyte recruitment after ischemic insult. It is also interesting that the expression pattern for IL1RII mRNA is remarkably parallel with that of IL-1β mRNA following MCAO.65 Since (soluble or membrane bound) IL-1RII binds IL-1β with a higher affinity but without transducing a signal, the upregulation of IL-1RII after focal stroke might provide an action similar to IL-1ra, i.e., it could provide a natural compensatory mechanism to counter the activity of IL-1β. These data do suggest that the modification of IL-1β, either via an agonist to increase IL-1ra and/or IL-RII production, or a specific antagonist against IL-1RI signal transduction, may be of therapeutic benefit in focal stroke. In addition, these data also emphasize the importance of studying all the components of a given cytokine system (e.g., cytokine, endogenous

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antagonist, and cytokine receptor subtypes) for better understanding of its potential functional significance in brain injury.

9.6 LEUKOCYTE ADHESION MOLECULES IN STROKE AND BRAIN INJURY The presence of recruited leukocytes at the site of inflammation is critically dependent upon the coordinated expression of adhesion molecules (ligands and receptors) on inflammatory cells and the activated vascular endothelium, respectively. These timed responses facilitate the efficient “docking” of activated immune cells to their respective receptors which is the primary step in the process of transendothelial migration and diapedeis. Some of the candidate molecules include ICAM-1, ELAM-1, and P-selectin on the endothelial side and CD11/CD18, MAC-1, LFA-1 on the leukocyte side.91,92 Following MCAO in rats, we have demonstrated that ICAM-193 and ELAM-194 mRNA expression is increased within 1 to 3 hr. ICAM-1 protein was localized to the microvascular endothelium in the infarcted cortex. In an MCAO model in baboons,95 P-selectin and ICAM-1 were shown to be upregulated and similarly localized to the endothelium of the microvasculature in the ischemic penumbra. Consistent with these observations, several studies have demonstrated the beneficial effects of antiadhesion molecules in experimental models of stroke (see below). We have also demonstrated the upregulation of several other cytokines, chemokines, and adhesion molecules following focal stroke (see below).

9.7 NEURODESTRUCTIVE AND NEUROPROTECTIVE GENE EXPRESSION FOLLOWING STROKE As pointed out above (previous text and Figure 9.1A), focal ischemia is a very powerful stimulus to elicit genomic responses in the brain in the form of multiple gene expression. It is important that we do not create the impression that two cytokines, TNFα and IL-1β, are the only messages or proteins that are expressed in focal cerebral ischemia. We must point out that focal ischemia is a very powerful reformatting, reprogramming stimulus for the brain. There are very broad and robust genomic responses that occur following focal stroke, and the pattern of gene expression is exhibited as temporal episodes of different groups of gene expression. Transcription factors (immediate early genes; IEG) are the first “wave” as shown by c-fos and others (c-jun, zif268, Jun-B), which are very quickly upregulated, but are also quick to disappear.96–100 A second “wave” consists of the heat shock proteins (HSP). Heat shock protein mRNA is usually expressed within 1–2 hours and then downregulated by 1–2 days.97,100 Of great interest is the third “wave” that is largely comprised of increased cytokine gene expression as described earlier for TNF-α and IL-1β59,60,73 and including IL-6,99 and of course IL-1 ra.90 Chemokines such as IL-8,101 IP-10,102 and MCP-1103 are also increased, and are certain to play a role in neutrophil and mononuclear cell infiltration. The third “wave” also includes increased expression of the

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endothelial cell adhesion molecules such as ICAM-1, ELAM-1, and P-selectin93–95 that are important in establishing endothelial adhesion to the leukocyte prior to infiltration. In addition, growth factors96 (e.g., nerve growth factor; NGF; brain-derived nerve growth factor; BDNF) that might be expected to play neuroprotective role(s) following stroke, and the tumor suppressor gene p53104 also increase in this time frame. It is this third wave that is closely related to inflammation, and the occurrence of this wave of gene expression parallels leukocyte infiltration into the brain following injury. We have focused primarily on the cytokine, chemokine, and adhesion molecule expression associated with this wave of gene expression. It is this wave that drives the leukocyte infiltration (initially neutrophils; PMN, that are followed by monocytes/macrophages; Mono/M∅).35,38–40 The “third wave” starts after about one to three hours and comes up to a peak at about 12 hours. Usually by two or three days, it is almost completely downregulated, but for some genes, it lingers on for several days. This wave is really the complete gene expression package of inflammatory mediators, including inflammatory cytokines, chemokines, and the endothelial cell adhesion molecules (e.g., the expression of everything necessary to initiate and maintain the machinery of brain inflammation). More recently, we have identified a fourth “wave” of new gene expression that may well be associated with the acute inflammatory reaction to brain ischemia. This fourth “wave” includes proteolytic enzymes (metalloproteases; Col92) implicated in damage to extracellular matrix,105,106 and their endogenous protease inhibitors TIMP1;107 following focal stroke. The expression of these genes in stroke appears to be related to the influx of inflammatory cells, and is associated with secondary brain injury and repair processes following stroke. A fifth “wave” of new gene expression includes mediators such as transforming growth factor-β (TGF-b)108 and osteopontin (Osteo),109,110 which appears to be important in tissue remodeling (i.e., resolution of ischemic tissue injury),35,36 including the later glial changes and scarring that follow the inflammatory brain reaction that occurs in response to brain injury.

9.8 STRATEGIES TO TARGET STROKE: BRAIN INFLAMMATION AND ITS COMPLEXITIES Recent information emphasizes the complexity and our need to understand the basic mechanisms and timings of destructive, degenerative, and restorative functions of the brain inflammatory response to brain injury. For example, inflammatory activity in the brain extends from early initial leukocyte infiltration into later repair and remodeling and into neuronal plasticity and recovery of function periods.35,36,42 Although initial brain inflammation can contribute to the degree of brain damage following injury, longer-term antiinflammatory interventions to limit the degree of damage might interfere with nervous regeneration and recovery.111,112 Recent data113 indicate that autoimmune T cells can protect neurons from secondary degeneration after central axonomy. Apparently, a more restricted central nervous system (CNS) recruitment and activation of macrophages is linked to regeneration failure following injury in the CNS.114 Inflammation within the CNS is associated with facilitation

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Strategies to Target Dynamic Changes in Stroke Days

Hours 2

>50%patientss 8 hrs

7

Weeks/Months 14

Ca Na+ Glut Enzymes OR

I N J U R Y

Necrosis

Inflammation Repair Remodeling

Plasticity

Stroke Strategies Prevention/Protection

Apoptosis

Acute Intervention Regeneration/Functional Recovery

FIGURE 9.2 Schematic diagram depicting relative time sequence of the dynamic changes that occur following focal ischemic stroke in reference to potential Stroke intervention strategies. Of particular interest is the brain inflammation that occurs throughout all other phases of brain changes in response to initial ischemia/injury. Text describes the ying/yang involvement of brain inflammation in exacerbation and evolution of initial ischemic injury, and in the repair and recovery of function of neuron tissue following injury.

of neuronal plasticity/recovery (see Figure 9.2). This is likely a function of neurotrophic factor secretion by activated macrophages/microglia following injury. For example, improved regeneration occurs in the CNS associated with more marked inflammation114,115 and activated macrophage and microglia facilitation of neuronal plasticity/recovery following injury is associated with the secretion of neurotrophic factors.113 Therefore, the inflammation that does occur in response to CNS injury appears to serve multiple purposes. Clearly, more must be learned about these complex interactions, and the timing(s) of specific intervention(s) may be critical to development of significant neuroprotective antiinflammatory therapy.42

9.9 STRATEGIES FOR TREATMENT OF BRAIN INFLAMMATION/INJURY 9.9.1 BLOCKING CYTOKINE ACTIONS A very attractive therapeutic possibility is to aim directly for cytokine and chemokine suppressive agents. However, definitive proof for a role of TNFα and IL-1β in the development of ischemia has only been accomplished relatively recently

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as a result of the availability of selective and potent antagonists of its production or actions. For example, Betz et al.80 used an adenovirus vector constructed to overexpress IL-1ra. The excess of IL-1ra significantly reduced infarct size following focal stroke. While such modes of IL-1ra delivery are impractical in clinical terms, the studies point out a potential therapeutic remedy if delivery of IL-1ra can be achieved in a timely fashion. These data are consistent with the protective effects of the intracerebroventricular administration of recombinant IL-1ra which produced a marked reduction in experimental brain damage induced by focal stroke,6,7 brain hypoxia/ischemia,78 or fluid percussion injury.79 Again, the fact that the peripheral administration of IL-1ra has been shown to be neuroprotective is of interest from a therapeutic point of view.81 However, critical issues for the therapeutic potential of this agent will be the timing of administration and brain penetration in human stroke. TNFα is significantly involved in the pathophysiology of ischemic and traumatic brain injury.51 Following closed-head injury, agents that block TNFα activity provide neuroprotection.116 Pentoxifyline (a methylzanthine that reduces TNFα production at the transcriptional level) administered intravenously posttrauma blocked trauma-induced TNFα production, reduced maximum brain edema at 24 hr and facilitated recovery of motor function as late as 14 days postinjury. Soluble TNF receptor I (a physiological inhibitor of TNFα which acts by competing with the cell surface receptor) administered intravenously posttrauma provided similar protection and in addition reduced the disruption of the blood brain barrier and protected hippocampal cells from delayed cell death following trauma. TNFα administration into the brain (i.e., intracerebroventricularly) prior to focal stroke in hypertensive rats exacerbates ischemic brain injury. This increase in brain injury due to TNFα administration also occurs in transient focal ischemia with reperfusion.62 This effect was demonstrated to be specific, since TNFα mAb (intracerebroventricularly) completely reversed the exacerbation of infarct size and neurological deficit, while a nonimmune IgG antibody control treatment had no such effect.62 To test the hypothesis that endogenous TNFa is an important mediator of focal ischemic injury, two different but specific methods of blocking TNFα during ischemia: an anti-TNFα monoclonal antibody (mAb) and soluble TNF receptor I.62 TNFα was blocked by repeated intracerebroventricular administrations before and during focal stroke which reduced focal ischemic injury. A significant reduction in infarct size following anti-TNFα mAb administration compared to control (equivalent amount of nonimmune IgG) treatment was observed. Similar results on percent hemispheric infarct were observed for soluble TNF receptor I compared to vehicle treatments.99 Others have also demonstrated protective effects of blocking TNFα. In murine focal stroke, the topical administration of soluble TNF receptor I to the brain significantly reduced ischemic brain injury.117,118 In addition, in another study evaluating the effect of TNF blockade on focal stroke in hypertensive rats, soluble TNF receptor I administered intravenously pre- or post-MCAO significantly reduced the impairment in ischemic cortex microvascular perfusion and the degree of cortical infarction, strongly suggesting an inflammatory/vascular mechanism for TNFα in focal stroke.119

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Monoclonal antimurine TNFα effectively neutralizes the bioactivity of TNF. This has been shown to be specific for (i.e., effectively antagonizes) both mouse and rat TNFα,120,121 and has been demonstrated previously to prevent the transfer of experimental allergic encephalomyelitis.122 Based on these studies, the intracerebroventricular anti-TNFα mAb dose utilized was expected to provide a brain concentration of the antibody that would block TNFα-mediated effects. Indeed, repeated intracerebroventricular administrations of mAb just before and during focal ischemia produced a significant reduction in ischemic brain injury.62 Soluble TNF receptors are truncated forms of the extracellular domains of the receptors, and act as endogenous inhibitors of TNFα by competing with the cell-surface receptors for its binding.123,124 Soluble TNF receptor I efficacy was also demonstrated in other brain injury models which are associated with elevated TNFα levels, e.g., brain trauma,116 experimental autoimmune encephalomyelitis,125,126 and lethal endotoxemia.127 Repeated intracerebroventricular doses of soluble TNF receptor I were utilized62 to provide brain concentrations of the soluble receptor that were expected to block TNFα (i.e., similar to that achieved in other models where efficacy was demonstrated). Both brain62,117,118 and vascular119 administrations of soluble TNF receptor I administered up to 1 hr poststroke produced a significant decrease in focal stroke injury. In addition, the reduced microvascular perfusion that occurs following focal stroke was reduced by blocking TNF.119 Thus, these new data support previous reports on TNFα mRNA and peptide expression in the same stroke model, and provide a very strong case for a role of TNFα as a mediator of ischemic brain injury. It is important to note that persistent attempts to provide evidence for direct TNFα toxicity on neurons in relatively pure or mixed cultures were unsuccessful.62 Direct neurotoxicity of cytokines may depend on the absence of neurotrophic growth factors, which may explain some inconsistencies in available data. However, an indirect augmentation of neuronal damage by TNFα does seem likely. Astrocytes, microglia, and endothelial cells are the prime brain cell candidates likely to produce and respond to cytokine stimulation, and maintain an inflammatory response that will ultimately result in long-term neuronal loss after ischemic stroke. The direct injection of picomole amounts of TNFα into the brain of spontaneously hypertensive rats produced a dramatic increase in leukocyte adhesion to vascular walls and an infiltration of these inflammatory cells into tissue, but no direct neurotoxicity to neurons at the site of injection.59 Others have also shown that TNFα is not directly toxic to neurons,128,129 and some investigators even suggest a protective effect of TNFα on neurons.130-132 The broad scope of TNFα’s injurious and beneficial effects have been emphasized previously.133 Cytokines have also been suggested to provide beneficial effects in brain injury as inferred from studies with TNF-receptor knock-out mice (p55 and p75 knock-out), which display increased sensitivity to brain ischemia134,135 and the capacity of IL-1 to elicit a state of ischemic tolerance upon repeated administration.136 The later expression of TNFα in macrophages has been demonstrated and is involved in resolution of ischemic brain injury.35,36,59 However, the data to date demonstrate that increasing the acute effects of TNFα is not protective (and in fact increases ischemic injury), and that blocking the acute

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increased activity of TNFα that occurs following focal stroke is neuroprotective. In the future, an increased understanding of the specific TNF receptors involved in TNF protective and destructive effects should be pursued. The toxic effects of TNFα and its role as a mediator of focal ischemia may involve several mechanisms. For example, TNFα increases blood-brain barrier permeability and produces pial artery constriction that can contribute to focal ischemic brain injury, and there appears to be a direct toxic effect of TNFα on the capillary.137,138 TNFα increases microvascular permeability and opens the blood-brain barrier,138,139 apparently by increasing matrix-damaging metalloproteinase (gelatinase B) production, that is also expressed early following focal stroke.105,106,140 TNFα also causes damage to myelin and oligodendrocytes141,142 and increases astrocytic proliferation, thus potentially contributing to demyelination and reactive gliosis during brain injury. In addition, TNFα activates the endothelium for leukocyte adherence and procoagulation activity (i.e., increased tissue factor, von Willebrand factor, and platelet activating factor) that can exacerbate ischemic damage.48 Indeed, increased TNFα in the brain and blood in response to LPS appears to contribute to increased brain stem thrombosis and hemorrhage, and can contribute to increased stroke sensitivity/risk in hypertensive rats.42,53,143,144 Therefore, TNFα plays a pivotal role in inflammatory processes.145 It activates neutrophils,146 increases leukocyte-endothelial cell adhesion molecule expression,23 and increases leukocyte adherence to blood vessels and their subsequent infiltration into the brain (see Ref. 42 for review). Clearly, leukocyte transit via microvessels and capillaries is impaired after stroke which contributes to negative rheologic effects due to microvascular occlusion or plugging.37,46,47,147 Interference with either IL-1 or TNF has now proven to be protective in focal stroke and head trauma. antagonism of TNFα action by anti-TNFα mAbs or soluble TNFα receptors or IL-1β by recombinant IL-1ra could provide antiinflammatory effects against brain injury in man as demonstrated in laboratory animals. The utility of blocking inflammatory cytokines in conjunction with thrombolysis using tPA has been discussed.148

9.9.2 ANTI-ADHESION MOLECULE ANTIBODIES Another attractive approach is the inhibition of endothelial interactions with leukocytes. Chen et al.149 treated MCAO rats intravenously with an antibody against the leukocyte counterpart of ICAM-1 binding (i.e., MAC-1) and demonstrated reduction in infarct size by 45–50% in a rat transient MCAO model. Zang et al.150 used the intravenous administration of an anti-ICAM-1 antibody (i.e., blocking the endothelial side of the adhesion molecule interaction) to demonstrate a 40% reduction of infarct size in a similar model. Other studies verified these effects, but also illustrated that these antibodies could not reduce infarct size following permanent focal ischemia.151-155 However, the strategy may work if both leukocyte and endothelial adhesion proteins are blocked in permanent focal stroke. Furthermore, in a rabbit embolic model of stroke, anti-ICAM-1 antibody were shown to increase the amount of clot necessary to produce permanent damage.153 In addition, in a baboon model of transient focal ischemia, anti-CD18 mAb administered 25 min prior to the reperfusion led to an increase

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in reflow in microvessels of various sizes.156 Whether the experimentally demonstrated anti-ischemic effect of antiadhesion molecules will lead to a potential therapy is still an open issue, but the availability of recombinantly expressed soluble adhesion molecules (e.g., sICAM-1, sELAM-1) may provide superior therapeutic potential. The use of quantitative blood flow determinations in human stroke may also offer advantages in identifying effective therapeutic doses and serve as a surrogate marker of improved outcome.

9.9.3 ANTILEUKOCYTE TREATMENTS Matsuo et al.157 have used the RP-3 monoclonal antibody that selectively depletes leukocytes in the rat (about 90-95%) and reported a dramatic reduction in both neutrophil accumulation in focal ischemic brain tissue and infarct size (decreased by 4550%). Other studies have verified these effects in various experimental models,33,41,158–163 although some controversy exists.164 However, although these “depletion” experiments support the hypothesis that early inflammatory cell infiltration in stroke exacerbates ischemic injury, this strategy is unlikely to translate into clinical use.

9.10

OTHER APPROACHES: BLOCKING CYTOKINE PRODUCTION

9.10.1 CAMP AND PKC The evaluation of additional potent and specific anticytokine therapies in proper models of brain injury is clearly warranted. Much evidence has accumulated that indicates TNFα production is regulated at both transcriptional and translational levels.49 Thus, TNFα mRNA synthesis inhibitors such as rolipram,165 a phosphodiesterase IV inhibitor, could be of benefit in the treatment of brain inflammation. Other novel classes of drugs include highly specific protein kinase C (PKC) inhibitors of microbial origin such as calphostin C,166 which has been shown to potently inhibit LPS-stimulated TNFα production from human monocytes in vitro167 and LPS and Newcastle disease virus-stimulated TNFα production in astrocytic cell lines.21,168 Interestingly, PKC also serves as a “focal point” in the regulation of production of IL-1b169 and endothelial cell-derived adhesion molecules.48 The use of PKC inhibitors in vivo, however, may be hindered by widespread inactivation of PKC distant from the desired target within the CNS and therefore toxic consequences. However, the finding that liposome-entrapped staurosporine greatly enhanced survival in a rat model of endotoxemia possibly related to the suppression of serum TNFα levels,170 supports the feasibility of such a pharmacological paradigm to prevent brain inflammation. Further advances in this approach will be derived from the development of isozyme-specific PKC inhibitors.171

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9.10.2 P38 MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION AND INHIBITION IN STROKE Inflammatory mediator- and apoptosis-associated signaling pathways may provide novel neuroprotective targets in stroke. One such target might be selective inhibition of the mitogen-activated protein kinase (MAPK) intracellular signaling pathways (see Figure 9.3A). Three distinct but interlinked MAPK pathways have been characterized.172,173 Many neurotrophins/growth factors bind to tyrosine kinase receptors and signal through Ras via the Extracellular Signal-Regulated Kinase (ERK) MAPK pathway. Trophic/growth factors can mediate neuronal development, growth, survival, and protection that are linked biologically to cell growth and differentiation processes through this path. The Stress-Activated MAPKs (p38 and c-Jun N-terminal Kinase; JNK) comprise the other two pathways. p38 and JNK play important roles in transducing stress-related signals by phosphorylating intracellular enzymes, transcription factors, and cytosolic proteins174,175 involved in cell survival, apoptosis, and inflammatory cytokine production.176-179 Sustained activation of JNK and p38 MAPK have been shown to be associated with neuronal death/apoptosis,172,180–184 and selective p38

B

stimulus

growth factors

cytokines, stress

c

MEK (MAPKK)

MKK (SAPKK)

Infarct Volume (mm3)

Mitogen-Activated Protein Kinase Signaling

a s c a d e

ERK (MAPK)

p38/CSBP X X CSAID

c-Jun

MAPKAP-K1, Elk-1

MAPKAP-K2&3

125 100 75 50 25 0

VEHICLE

20

SB 239063

15

*

10

* *

*

*

*

* *

5

* *

-4

-3

-2

-1

0

1

2

3

4

5

Brain Sections (mm from Bregma)

6

7

8

5 15 30 60 Doses (mg/Kg, p.o.)

Oral SB 239063 Reduces Mean Deficit Grade

D

25

Infarct Areas (in mm2 )

150

0

Forebrain Infarct Area Profile

0 -5

Oral SB 239063 Reduces Infarct Volume

Regulation of Transcription and Translation

response

C

JNK

Mean Neurological Grade

A

2.5 2 1.5 1 0.5 0

0

5 15 30 60 Doses (mg/Kg, p.o.)

FIGURE 9.3 (A) General features of the MAP kinase family. (B) Treatment with SB 239063 produced marked reduction of infarct volume. (C) The marked decreases in infarct areas on individual braine slices throughout the forebrain at the most effective 15 mg/kg SB 239063 compared to vehicle treatment is presented graphically. (D) SB 239063 reductions in neurological deficits were similar to reductions in infarct volume shown in part B. * = p < 0.05 different from vehicle; ** =p < 0.01 different from vehicle.

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MAPK inhibitors can promote the survival of a variety of neurons in vitro.172,176,179–181 Nerve growth factor withdrawal causes neuronal apoptosis that is preceded by decreased ERK and increased JNK/p38 activities,179 suggesting a balance between ERK and stress-activated MAPKs under some conditions is required to mediate cell survival. In addition, insulin can promote the survival of neurons concomitant with inhibition of p38,185 and p38 MAPK activation is involved in glutamate toxicity-induced neuronal apoptosis.186 In global forebrain ischemia, p38 MAPK activation has been identified in microglial cells adjacent to dying, vulnerable neurons.187 Regarding Figure 9.3A, stimuli, typically acting at the cell surface, provide second messenger signaling via a cascade of protein phosphorylations to result in a response, typically at the level of protein transcription or translation. Of particular interest is the stress-activated MAP kinase pathway involving p38 mitogen-activated protein kinase (MAPK) which is selectively inhibited by Cytokine Suppressive AntiInflammatory Drugs (CSAIDs). p38 MAPK was originally referred to as the CSAID Binding Protein (CSBP). CSAIDs inhibit the production of the inflammatory cytokines TNF and IL-1. CSAIDs/p38 MAPK inhibitors act after p38 MAPK activation (via its phosphorylation from upstream kinases) to prevent p38 activation-induced phosphorylation of downstream kinases (e.g., see “XX CSAID” at arrow to MAPKAPK2). Thus, the cellular inflammatory cytokine production and apoptosis in response to various cellular stressors (e.g., ischemia or trauma) can be blocked. Regarding Figure 9.3B, in this study, SB 239063 was administered orally (0 or vehicle, 5, 15, 30, and 60 mg/kg) at 1-hour pre- and 5 hour poststroke in spontaneously hypertensive rats (SHR) (N = 6-30 per group).206 Production of moderate focal stroke by distal MCAO in SHR and measurements of brain injury and neurological deficits were as described previously.35,36,38-40 Briefly, SHR (280–340 g) were anesthetized and underwent right permanent middle cerebral artery occlusion (MCAO) by distal electrocautery of the MCAO at the level of the inferior cerebral vein. After 24 hours, animals were graded for neurological deficits, and brain forebrain sections were stained and analyzed for degree of injury using image analysis.

9.10.3 CSAIDS (P38 INHIBITORS) A class of “cytokine-suppressive antiinflammatory drugs” (CSAIDs) that inhibit TNFα and IL1β production have been developed.183,184 Interestingly, these pyridinyl imidazole drugs have been shown to hamper translation of both TNFα and IL-1β by mechanisms independent of cAMP involvement. Selected compounds from the pyridinyl imidazole chemical class have demonstrated therapeutic utility to inhibit TNFα and IL1β production,178,188-193 as well as the expression of several other inflammatory mediators/proteins, including inducible nitric oxide synthase,194–199 cyclooxgenase-2,199,200 IL-6,201,202 and IL-8,177,202-205 thus their use significantly impacts on the inflammatory process and the ultimate degree of tissue injury in animal models of disease/tissue injury. Initially utilizing a photoaffinity label, the isolation, purification, cloning, and expression of the CSAID target protein (i.e., CSAID binding protein) was achieved and discovered to be p38 MAPK.177,178,192 CSAIDs inhibit the

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catalytic activity of p38 (but do not interfere with its phosphorylation/activation). One physiological substrate of p38 is MAPKAP-K2 whose activation by p38 is inhibited by CSAIDs. MAPKAP-K2, upon activation, serves as a nuclear exporter of p38 (and itself) that allows the phosphorylation of their substrates in the cytoplasm.206 For example, Hsp27 is phosphorylated by MAPKAP-K2. Another physiological substrate of p38 is the transcription factor, ATF2. Therefore, through the phosphorylation of other proteins, p38 can upregulate message transcription, can stabilize message, and can increase protein translation.178 CSAIDs or p38 inhibitors can be expected to interfere with these effects (i.e., these effects have been primarily associated with increased cytokine production and cell stress/death). SB 203580 (4-[5-(4-Fluoro-phenyl)-2-(4-methanesulfinyl-phenyl)-3H-imidazol4-yl]-pyridine; see structure in Table 9.1) was the first described selective p38 MAPK inhibitor.207-209 It was soon realized that it inhibited other protein kinases, albeit in vitro, with IC50s in the low micromolar range; e.g., SB 203580 inhibited JNK 2β2 and c-Raf with IC50s of 5 uM and 0.4 uM, respectively. In addition, it was recognized that there are at least 4 homologs of p38 MAPK.192 These kinases have about 60-70% sequence homology. SB 203580 inhibits p38α and p38β2 but not p38γ and p38δ. This pattern of selective inhibition of a/b2 vs. g/d p38 paralogues is a general property of the pyridinylimidazoles. From a large series of these compounds, SB 239063 (4-[4-(4Fluoro-phenyl)-5-(2-methoxy-pyrimidin-4-yl)-imidazol-1-yl]-cyclohexanol; see structure in Table 9.1), a second-generation p38 MAPK inhibitor207,208 was identified to exhibit potent inhibition of p38 activity and improved selectivity, cellular and in vivo activity over first-line p38 inhibitors (e.g., such as SB 203580).209,210

9.10.4 P38 MAPK ACTIVATION FOLLOWING FOCAL STROKE Immunohistochemical studies employing a phospho-specific p38 antibody were conducted to evaluate the spatial and cellular distribution of p38 activation following stroke.211 The intensity of phospho-p38 immunostaining was dramatically increased in many astrocytes and some neurons in the evolving ischemic cortical infarction area for up to 6 hours after stroke. In addition, p38 enzymes isolated from the ischemic cortex exhibited increased phosphorylation of the target transcription factor ATF2 in vitro. No activation of p38 was detected in nonischemic cortex or in the cortex following sham surgery. In agreement with the immunohistochemical data, the time course of p38 MAPK activation was an early event where p38 activity was seen at 1, 3, and 6 hours poststroke, and continued to be elevated for at least 24 hours. MAPKAP-K2 phosphorylation of Hsp27 was also observed poststroke, indicating that activated p38 was activating/phosphorylating downstream substrates.

9.10.5 P38 MAPK INHIBITION ON BRAIN INJURY, DOWNSTREAM SIGNALING, AND INFLAMMATORY CYTOKINE EXPRESSION POST-STROKE The improved selectivity and in vivo activity of SB 239063 prompted our evaluation of its effects upon oral and intravenous administration in focal stroke. Significant

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TABLE 9.1 In Vitro and In Vivo Activities of SB 239063 vs. SB 203580 (i.e., a Second- vs. First-Generation p38 MAPK Inhibitor) OH

N

O

H N

O

N

S

N N

N

N

F F

Assay

SB 203580

SB 239063

Inhibition of isolated enzyme activity (IC50 in µM): p38a MEK ERK MAPKAP-K2 JNK-1 c-Raf

0.040 >10 >10 >10 5 0.4

0.044 >10 >10 >10 >10 >10

In vitro inhibition of LPS-induced TNFα production in human monocytes (IC50 in µM)

1.00

0.35

In vivo inhibition of LPS-induced TNFα production in rat plasma (IC50 in mg/kg, p.o.)**

25.0

2.6

40% at 60 23% at 30 0% at 10

60% at 30 51% at 10 28% at 3

In vivo inhibition of adjuvantinduced arthritis in the rat (% inhibition at mg/kg, p.o. dose treatment)

Both SB 203580 and SB 239063 were evaluated for their inhibitory activity and selectivity on a series of isolated MAPKs. p38 (four isoforms; α, β ,γ and δ, MEK, ERK, MAPKAP-K2, JNK-1 and c-Raf) were cloned, expressed and purified at SmithKline Beecham Pharmaceuticals and assayed under optimum conditions for IC50 µM determinations.177,178,191,192 In addition, the inhibitory (IC50 in µM) effects of both compounds on lipopolysaccharide (LPS)-stimulated human monocyte TNFα production was determined in vitro as described previously.178,210 Finally, the inhibitory activity (IC50 in mg/kg, p.o.) on plasma TNFα production in Lewis rats injected with LPS and in the adjuvant-induced arthritis model of peripheral inflammation was determined as described previously.209 a

Values listed are for p38α. Similar results were obtained for p38β. Neither compound had any inhibitory activity (i.e., no effects at 10µM) on p38γ or p38δ.

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protection from brain injury and neurological deficits were observed in the same in vivo oral dose activity range as that seen for other models of inflammation (see Figure 9.3B-D).208,212,213 Significant protection from brain injury and neurological deficits was also demonstrated due to intravenous poststroke treatment with SB 239063.208,213 In addition, SB 269063 was administered intravenously following stroke (as above) and biochemical and gene expression studies were conducted on the ischemic and nonischemic brain tissue. SB 239063 treatment administered at a neuroprotective plasma level blocked the phosphorylation of a downstream target (e.g., Hsp 27), demonstrating the ability of SB 239063 to inhibit activated p38 from phosphorylating its downstream targets at plasma levels that provided significant neuroprotection from stroke-induced brain injury. In addition, the normal stroke-induced increased IL-1β and TNFα mRNA expression was decreased by the neuroprotective dosing regimen of SB 239063.

9.10.6 SB 239063 EFFECTS DEVELOPMENT AND RESOLUTION OF INFARCT POST-STROKE To better evaluate the neuroprotective effects following p38 inhibition with the most effective oral dose of SB-239063 (15mg/kg), global neurologic deficit (GND), Diffusion Weighted Imaging (DWI), and T2 weighted MRI (see Figure 9.4A) were evaluated following stroke to monitor both the development and resolution of the infarct.212 DWI was used to measure the early effects of cytotoxic edema to reflect the areas ultimately at risk of irreversible injury. This has been shown previously to reflect ultimate degree of injury/protection in this model.214 We have previously demonstrated a very high correlation (r > .90, p < 0.01) between TTC histology and T2 weighted MRI at 24 hours post-MCAO. At 2 hours postinjury, there was a significant reduction in the area of cell stress (e.g., cell depolarization, diffusibility of water) for the SB 239063 compared to the vehicle treated rats (Figure 9.4B). At 24 hours post-stroke, there was approximately 30% reduction in infarct size between the SB 239063 and the vehicle-treated groups (Figure 9.4B). There was a good correlation (r = .74, p < 0.01) between the protection observed using early DWI (n = 9/group) and 24 hour T2 MR imaging in these same rats. At 1 day post-MCAO, SB 239063 provided dramatic neuroprotection which was associated with a significant reduction in neurologic deficits (Figure 9.4C). At 7 days poststroke, animals receiving SB 239063 maintained the significant 30% reduction in infarct (Figure 9.4B). The neuroprotection as assessed by infarct size was not attributable to any significant differences in amount of swelling within the injured brain between the treatment groups (data not shown). Identical results were obtained when the infarct volumes were corrected for swelling. At 7 days poststroke, SB 239063 also significantly improved neurologic outcome assessed by the GND compared to the vehicle treated group (Figure 9.4C).

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A Hemispheric Infarct Size (%)

30

SB239063 Provides Long Term Brain Protection Vehicle

B

SB 239063 (15m g/kg) #

25 20

**

*

15

*

10 5 0 2 Hour DWI

24 Hour T2

7day T2

Global Neurologic Deficit

SB239063 (15mg/kg) Provides Long Term Neurologic Protection 6.00 5.00

Vehicle

C

SB239063 (15mg/kg)

4.00 3.00 2.00

** **

1.00 0.00 24 hour

7 day

FIGURE 9.4 (A) Representative example of T2-MRI determination of 15 mg/kg SB 239063 treatment (top series of forebrain slices) producing a reduction in MCAO infarct compared to vehicle treatment (bottom series of forebrain slices). (B) Group data on SB 239063 (15 mg/Kg) for percent hemispheric infarct for DWI MRI 2 h post-MCAO, for T2-MRI 24 h postMCAO, and for T2-MRI 7 d post-MCAO. A long-term neuroprotection was identified using MRI. (C) Long-term protection from neurological deficits also observed due to SB 239063 treatment in the same study. All data are represented as mean ± standard error; n = 9/group. Differences were considered significant at # p < 0.05 compared to day 1, ** p < 0.01 compared to vehicle, and * p < 0.05 compared to vehicle.

9.10.7 SB 239063 NEUROPROTECTION FOLLOWING IN VITRO ISCHEMIA The rapid protection provided by SB 239063 as demonstrated by DWI-MRI in the above study suggested that other protective effects of p38 inhibition beyond only cytokine and inflammation reducing effects might contribute to brain protection. Also, a recent study demonstrated that brain tissue protein concentrations for IL-6 and IL-1ra did not significantly increase until 12 hours following distal electrocoagulation of the middle cerebral artery.215 In addition, the increased levels of IL-1β were biphasic, increasing at 4 hours, approaching baseline, and then significantly increasing again at 12 hours (see Figure 9.5). The timing of increased brain protein levels agree

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FIGURE 9.5 Brain protein levels of Interleukin-1β (A), Interleukin-1α (B), Interleukin-1ra (C), and Interleukin-6 (D) following focal stroke in the rat. The quantitative determination of tissue protein levels were normalized to original tissue weight and are expressed in ng/g tissue wet weight as described previously. * p < 0.05 vs. nonischemic tissue. # p < 0.05 vs. sham tissue.

with the infiltration of inflammatory cells as assessed by myeloperoxidase activity in this particular model. Since cytokine production and neutrophil infiltration can be delayed up to twelve hours or may fall below the lower limit of quantification at these time points, p38 MAPK inhibitors may still be beneficial at these later timepoints via additional/alternative mechanisms (e.g., cytokine inhibition). We have yet to be able to quantify brain tissue levels of TNFα to understand this relationship. Finally, an early poststroke phosphorylation of p38 following stroke can be observed,211-213 suggesting that the activation of this signaling cascade and the neuroprotection by CSAID/p38 inhibition might be, at least to some degree, independent of the brain inflammatory response. Therefore, the more direct effects of SB 239063 were evaluated in an in vitro model of oxygen-glucose deprivation-induced neuronal cell death.208 SB 239063 also significantly reduced hippocampal CA1 cell death produced by this in vitro ischemia under these conditions (Figure 9.6A), suggesting that this secondgeneration p38 inhibitor can protect neurons directly in addition to effects at blocking

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Figure 9.6 (A) Direct neuroprotective effects of SB 239063 on oxygen-glucose deprivation (OGD) in organotypic hippocampal brain slices. Percent damage to CA1 neurons in the hippocampus of brain slices 23 h following 45 min of OGD (in vitro ischemia) was significantly reduced by SB 239063. N = 9 per concentration bar. * = p < 0.05 different from zero concentration control group. (B) Representative Western blot showing direct effects of calcium influx produced by the calcium ionophore (5 µM A23187) to increase phosphorylation (i.e., activation) of p38 in cultured PC12 cells.

inflammatory cytokine/mediator production and subsequent brain inflammation. In addition, it was more recently demonstrated that p38 could be activated very early associated with calcium influx produced by the calcium ionophore, A23187, in PC12

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neuronal cells in vitro (Figure 9.6B). Thus, this additional data demonstrates clearly that p38 activation and its inhibition can act directly on cells and perhaps interfere with other cellular signaling mechanisms related to cell death in addition to inflammatory cytokine expression and brain inflammation. Therefore, p38 activation following stroke has been demonstrated, and effective dosing regimens of SB 239063 have been used to reduce p38 activity and protect the brain from injury, to reduce the loss of neurological functions due to stroke injury, and to decrease the message expression of TNFα and IL-1β (i.e., inflammatory cytokines known to mediate brain injury) that occur following stroke. In addition, SB 239063 can also provide direct neuroprotection to cultured brain tissue and can be associated with very early neuronal calcium signaling/influx.

9.11 SUMMARY AND CONCLUSIONS Evidence accumulating during the last decade has shown that the CNS can mount a well-defined inflammatory reaction to a variety of insults including trauma, ischemia, transplantation, viral infections as well as neurodegeneration. Many aspects of this centrally derived inflammatory response parallels this reaction in the periphery. Through the recent application of molecular genetic techniques including PCR, utilization of cDNA probes in conjuncture with the availability of highly specific antibodies, new concepts are rapidly emerging about the molecular mechanisms associated with the development of brain injury. In particular, the importance of cytokines, especially TNFα and IL-1β, are emphasized in the propagation and maintenance of a CNS inflammatory response. Certainly, much data supports the hypothesis that ischemia and trauma elicit an inflammatory response in the injured brain. This inflammatory response consists of mediators (cytokines, chemokines, and adhesion molecules) followed by cells (neutrophils early after the onset of brain injury and then a later monocyte infiltration). It is clear that de novo upregulation of proinflammatory cytokines, chemokines, and endothelial-leukocyte adhesion molecules in the brain occur soon after focal ischemia and trauma and at a time when the tissue injury is evolving. The significance of the inflammatory response and its contribution to brain injury is now becoming better understood. Evidence has emerged in support of the role of cytokines in driving the inflammatory response, and that this process is causally related to the degree of brain injury. Evidence reviewed includes: (i) the capacity of specific cytokines to exacerbate brain damage; (ii) the capacity of specific cytokine blockade to reduce ischemic brain damage; (iii) depletion of circulating neutrophils reduce ischemic brain injury; (iv) antagonists of the endothelialleukocyte adhesion interactions (e.g., anti-ICAM-1) reduce ischemic brain injury. Targeting the cytokines that drive the brain inflammatory response to injury provides opportunities to intervene with novel therapeutics in stroke and neurotrauma. A very significant amount of data now demonstrates that the brain inflammatory response is a contributing factor to evolving brain injury that occurs following focal stroke and neurotrauma. Furthermore, some other genes that are upregulated by brain injury have growth-promoting capacities, which raises the additional possibility that

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increasing these other “neuroprotective” gene products might be useful to counteract brain damage by enhancing repair and establishing compensatory mechanisms that enhance histological and functional recovery. Certainly, the capacity of ischemia and trauma to induce inflammation in the brain provides new areas for the discovery of novel therapeutic agents that could confine the neuronal damage that follows ischemia and trauma. Along these lines, data continues to accumulate substantiating the fact that inhibiting inflammatory cytokines (e.g., IL-1 and TNF) can be an effective, emerging therapeutic approach to inhibit the brain inflammatory response and reduce brain injury due to focal stroke and brain trauma. Additional studies will be required to fully understand and characterize the protective effects of SB 239063. For example, a determination of minimum time to posttreatment efficacy (i.e., provides information on the time window of opportunity in stroke), the tighter linking of neuroprotection to reduced brain inflammation and/or direct effects on apoptosis, and establishing longer-term brain and neurobehavioral protection due to SB 239063 administration needs to be evaluated. However, the present data demonstrate a significant early and relatively long-term activation of p38 MAPK in some models of focal stroke in the rat. In addition, the reduced p38 activity, the significant reduction of lesion volume and the reduced stroke-induced increase in brain IL-1β and TNFα expression (i.e., molecules that are known to contribute significantly to stroke injury), and the improved behavioral outcome that occurs following the poststroke administration of SB 239063 indicates that p38 activation is significantly involved in the progression of cell death in focal ischemia. The fact that it is neuroprotective in vitro suggests some direct mechanisms contribute to the protection of ischemic neurons. The potential direct and indirect effects of p38 inhibition to protect the brain is interesting. The convergence/importance of inflammatory cytokines and apoptotic pathways has been demonstrated previously.216,217 The advantages of reducing apoptosis and the production of inflammatory cytokines using CSAIDs to intervene in focal stroke might be significant (e.g., provides an inherent combination therapeutic approach). Therefore, targeting p38 may provide an opportunity for stroke intervention by multiple mechanisms (see Figure 9.7, parts A and B, for schematic illustrating protective mechanisms based on all available data; also see color insert following page 114). Overall, the data demonstrate that treatment with SB 239063 provides dramatic neuroprotection up to and including 7 days post-MCAO. Since the protection observed at 7 days was identical to that assessed by DWI at 2 hours, it appears that no additional cell death (e.g., via apoptosis) had occurred over one week. The data also suggest that the reduction in DWI hyperintensity observed at 2 hours following treatment with SB 239063 may be attributed to additional direct protective effect on brain cells. This is in agreement with additional data where SB 239063 (20uM) significantly reduced hippocampal CA1 cell death (up to 40%) produced by OGD in cultured organotypic brain slices. These early improvements in neuronal injury following p38 inhibition do reflect final outcome up to and including 1 week postinjury. As discussed, calcium signaling may also be associated with p38 signaling/activation. Multiple in vitro cell systems currently are being utilized to better understand how SB 239063 may be involved with pathophysiological events in

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FIGURE 9.7 (See color figure 9.7.) Schematic diagrams based on available data depicting p38 role in focal stroke and brain injury. Diagrams depict how inhibiting p38’s actions via CSAIDs (e.g., SB 239063) can reduce brain injury (A) via interference with the early, more immediate damage due to direct/intracellular effects by attenuating cytokine and calcium signaling associated with cytotoxicity/necrosis and apoptosis, and (B) via interference with the later, evolving damage that is due to more indirect/intercellular roles of inflammatory cytokines and other inflammatory mediators by attenuating the brain inflammatory response to injury and mediator-associated cytotoxicity/apoptosis.

end-organ ischemia. These detailed in vitro studies are necessary to fully understand the role of p38 following ischemia and the mechanism(s) by which it can be beneficial. In general, the results point out potential opportunities provided by targeting MAPK signaling pathways to protect the brain following stroke. This approach should be extended in the future by evaluating p38 inhibition in other models of CNS injury, including cerebral hemorrhage, traumatic brain and spinal injury,42,218 and neurodegenerative diseases.219

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190. Boehm, J.C., Smietana, J.M., Sorenson, M.E., Garigipati, R.S., Gallagher, T.F., Sheldrake, P.L., Bradbeer, J., Badger, A.M., Laydon, J.T., Lee, J.C., Hillegass, L.M., Griswold, D.E., Breton, J.J., Chabot-Fletcher, M.C., and Adams, J.L., 1Substituted 4-Aryl-5-pyridinylimidazoles: A new class of cytokine suppressive drugs with low 5-lipoxygenase and cyclooxygenase inhibitory potency, J. Med. Chem., 39, 3929, 1996. 191. Young, P.R., McLaughlin, M.M., Kumar, S., Kassis, S., Doyle, M.L., McNulty, D., Gallagher, T.F., Fisher, S., McDonnell, P.C., Carr, S.A., Huddleston, M.J., Seibel, G., Porter, T.G., Livi, G.P., Adams, J.L., and Lee, J.C., Pyridinyl imidazole inhibitors of p38 MAP kinase bind in the ATP site, J. Biol. Chem. 272, 12116, 1997. 192. Lee, J.C., Laydon, J.T., McDonnell, P.C., Gallagher, T.F., Green, D., McNulty, D., Blumenthal, M.J., Heys, R.J., Landvatter, S.W., Strickler, J.E., McLaughlin, M.M., Siemens, I., Fisher, S., Livi, G.P., White, J.R., Adams, J.L., and Young, P.R., Identification and characterization of a novel protein kinase involved in the regulation of inflammatory cytokine biosynthesis, Nature, 372, 739, 1994. 193. Kumar, S., McDonnell, P.C., Gum, R.J., Hand, A.T., Lee, J.C., and Young, P.R., Novel homologues of CSBP/p38 MAP kinase: Activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles, Biochem. Biophys. Res. Comm., 235, 533, 1997. 194. Ajizian, S.J., English, B.K., and Meals, E.A., Specific inhibitors of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulated with lipopolysacharide and interferon-gamma, J. Infect. Dis., 179, 939, 1999. 195. Badger, A.M., Cook, M.N., Lark, M.W., Newman-Tarr, T.M., Swift, B.A., Nelson, A.H., Barone, F.C., and Kumar, S., SB 203580 inhibits cytokine suppressive binding protein/p38 kinase, nitric oxide production and inducible nitric oxide synthase in bovine cartilage-derived chondrocytes, J. Immunol., 161, 467, 1998. 196. Bhat, N.R., Zhang, P., and Bhat, A.N., Cytokine induction of inducible nitric oxide synthase in an oligodendrocyte cell line: role of p38 mitogen-activated protein kinase activation, J. Neurochem., 72, 472, 1999. 197. Bhat, N.R., Zhang, P., Lee, J.C., and Hogan, E.L., Erk and p38 subgroups of MAP kinases regulate inducible nitric oxide synthase and TNFα gene expression in endotoxin-stimulated primary glial cultures, J. Neurosci., 18, 1633, 1998. 198. Chen, C.C. and Wang, J.K., p38 but not p44/42 mitogen-activated protein kinase is required for nitric oxide synthase induction mediated by lipopolysaccharide in RAW 264.7 macrophages, Mol. Pharmacol., 55, 481, 1999. 199. Subbaramaiah, K., Chung, W.J., and Dannenberg, A.J., Ceramide regulates the transcription of cyclooxygenase-2. Evidence for involvement of extracellular signal-regulated kinase/c-Jun N-terminal kinase and p38 mitogen-activated protein kinase pathways, J. Biol. Chem., 273, 32943, 1998. 200. Ridley, S.H., Dean, J.L., Sarsfield, S.J., Brook, M., Clark, A.R., and Saklatvala, J., A p38 MAP kinase inhibitor regulates stability of interleukin-1-induced cyclooxygenase-2 mRNA, FEBS Lett., 439, 75, 1998. 201. Beyaert, R., Cuenda, A., Berghe, W.V., Plaisance, S., Lee, J.C., Haegeman, G., Cohen, P., and Fiers, W., The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 systhesis in response to tumour necrosis factor, EMBO J., 15, 1914, 1996.

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202. Krause, A., Holtmann, H., Eickemeier, S., Winzen, R., Szamel, M., Resch, K., Saklatvala, J., and Kracht, M., Stress-activated protein kinase/Jun N-terminal kinase is required for interleukin (IL)-1-induced IL-6 and IL-8 gene expression in the human eprdermal carcinoma cell line KB, J. Biol. Chem., 273, 23681, 1998. 203. Gon, Y., Hashimoto, S., Matsumoto, K., Nakayama, T., Takeshita, I., and Horie, T., Cooling and rewarming-induced IL-8 expression in human bronchial epithelial cells through p38 MAP kinase-dependent pathway, Biochem. Biophys. Res. Commun., 249, 156, 1998. 204. Hashimoto, S., Matsumoto, K., Gon, Y., Nakayama, T., Takeshita, I., and Horie, T., Hyperosmolarity-induced interleukin-8 expression in human bronchial epithelial cells through p38 mitogen-activated protein kinase, Am. J. Respir. Crit. Care Med., 159, 634, 1999. 205. Marie, C., Roman-Roman, S., and Rawadi, G., Involvement of mitogen-activated protein kinase pathways in interleukin-8 production by human monocytes and polymorphonuclear cells stimulated with lipopolysaccharide or Mycoplasma fermentans membrane lipoproteins, Infect. Immun., 67, 688, 1999. 206. Ben-Levy, R., Hooper, S., Wilson, R., Paterson, H.F., and Marshall, C.J., Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase 2, Curr. Biol., 8, 1049, 1998. 207. Adams, J.L., Boehm, J.C., Kassis, S., Gorycki, P.D., Webb, E.F., Hall, R., Sorenson, M., Lee, J.C., Ayrton, A., Griswold, D.E., and Gallagher, T.F., Pyrimidinylimidazole inhibitors of CSBP/p38 kinase demonstrating decreased inhibition of hepatic cytochrome P450 enzymes, Bioorg. Med. Chem. Lett., 17, 3111, 1998. 208. Barone, F.C., Irving, E.A., Lee, J.C., Kassis, S., Kumar, S., Badger, A.M., White, R.F., McVey, M.J., Legos, J.J., Erhardt, J.A., Nelson, A.H., Ohlstein, E.H., Hunter, A.J., K., Ward, K., Smith, B.R., Adams, J.L., and Parsons, A.A., SB 239063, a second generation p38 mitogen-activated protein kinase inhibitor, reduces brain injury and neurological deficits in cerebral focal ischemia, J. Pharmacol. Exp. Ther., 296, 312, 2001. 209. Badger, A.M., Bradbeer, J.N., Votta, B., Lee, J.C., Adams, J.L., and Griswold, D.E., Pharmacological profile of SB 203580, a selective inhibitor of cytokine suppressive binding protein/p38 kinase, in animal models of arthritis, bone resorption, endotoxin shock and immune function, J. Pharmacol. Exp. Ther., 279, 1453, 1996. 210. Cuenda, A., Rouse, J., Doza, Y.N., Meier, R., Cohen, P., Gallagher, T.F., Young, P.R., and Lee, J.C., SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1, FEBS Lett., 364, 229, 1995. 211. Irving, E.A., Barone, F.C., Reith, A.D., Hadingham, S.J., and Parsons, A.A., Differential activation of MAPK/ERK and p38/SAPK in neurons and glia following focal cerebral ischemia in the rat, Mol. Brain Res., 77, 65, 2000. 212. Legos, J.J., Erhardt, J.A., White, R.F., Chandra, S., Parsons, A.A., Tuma, R.F., and Barone, F.C., SB 239063, a novel p38 inhibitor, attenuates early neuronal injury following ischemia, Brain Res., 892, 70, 2001. 213. Barone, F.C., Irving, E.A., Ray, A.M., Lee, J.C., Kassis, S., Kumar, S., Badger, A.M., White, R.F., Nelson, A.H., Legos, J.J., Erhardt, J.A., Ohlstein, E.H., Hunter, A.J., Harrison, D.C., Philpott, K., Ward, K., Smith, B.R., Adams, J.L., and Parsons, A.A., Inhibition of p38 mitogen-activated protein kinase provides neuroprotection in cerebral focal ischemia, Med. Res. Rev., 21, 129, 2001. 214. Chandra, S., White, R.F., Coatney, R.F., Sarkar, S.K., and Barone, F.C., Use of diffusion weighted-MRI and neurological deficit scores to demonstrate beneficial effects of isradipine in a rat model of focal ischemia, Pharmacology, 58, 294, 1999.

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215. Legos, J.J., Whitmore, R.G., Erhardt, J.A., Parsons, A.A., Tuma, R.F., and Barone, F.C., Differential brain expression of IL-1α, IL-1β, Il-1ra, and IL-6 following focal stroke in the rat: quantitative determination of tissue protein levels, Neurosci. Lett., 28, 189, 2000. 216. Hara, H., Fink, K., Endres, M., Friedlander, R.M., Gagliardini, V., Yuan, J.Y., and Moskowitz, M.A., Attenuation of transient focal cerebral ischemic injury in transgenic mice expressing a mutant ICE inhibitory protein, J. Cereb. Blood Flow Metab., 17, 370, 1997. 217. Sidotidefraisse, C., Rincheval, V., Risler, Y., Mignotte, B., and Vayssiere, J.L., TNFalpha activates at least two apoptotic signaling cascades, Oncogene, 17, 1639, 1998. 218. Nakahara, S., Yone, K., Sakou, T., Wada, S., Nagamine, T., Niiyama, T., and Ichijo, H., Induction of apoptosis signal regulating kinase 1 (ASK1) after spinal cord injury in rats: possible involvement of ASK1-JNK and p38 pathways in neuronal apoptosis, J. Neuropathol. Exp. Neurol., 58, 442, 1999. 219. Hensley, K., Floyd, R.A., Zheng, N.Y., Nael, R., Robinson, K.A., Nguyen, X., Pye, Q.N., Stewart, C.A., Geddes, J., Markesberg, W.R., Patel, E., Johnson, G.V., and Bing, G., p38 kinase is activated in the Alzheimer’s disease brain, J. Neurochem., 72, 2053, 1999.

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FIGURE 9.1 (A) Schematic box diagram illustrating major changes that occur in thromboembolic ischemic stroke. (B) Ongoing neuroprotective and neurodestructive gene expression and their roles post-stroke.

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FIGURE 9.7 Schematic diagrams based on available data depicting p38 role in focal stroke and brain injury. Diagrams depict how inhibiting p38’s actions via CSAIDs (e.g., SB 239063) can reduce brain injury (A) via interference with the early, more immediate damage due to direct/intracellular effects by attenuating cytokine and calcium signaling associated with cytotoxicity/necrosis and apoptosis, and (B) via interference with the later, evolving damage that is due to more indirect/intercellular roles of inflammatory cytokines and other inflammatory mediators by attenuating the brain inflammatory response to injury and mediator-associated cytotoxicity/apoptosis.

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10

The Potential Role of Hyperbaric Oxygen in the Treatment of Stroke Eric J. Zoog

CONTENTS 10.1 Pathophysiology of Cerebral Ischemia 10.1.1 Alterations in Cerebral Hemodynamics 10.1.1.1 No-Re-Flow Phenomenon 10.1.1.2 Post-ischemic Hypoperfusion 10.1.2 Hypoxia and Hypoglycemia 10.1.3 Reperfusion Injury 10.1.3.1 Free Radicals 10.1.3.2 Microcirculation 10.2 Hyperbaric Oxygen 10.2.1 The Physics of HBO Therapy 10.2.1.1 Boyle’s Law 10.2.1.2 Dalton’s Law 10.2.1.3 Henry’s Law 10.2.2 The Effect of HBO in Stroke 10.2.2.1 PMNL-Endothelial Adherence 10.2.2.2 Free Radicals 10.2.3 Human Studies and Conclusions References Hyperbaric oxygen (HBO), the delivery of oxygen at greater than atmospheric pressure, has been shown to be a safe and efficacious treatment for a number of disease processes. Controversy remains over its use for patients suffering from acute ischemic stroke, however. This chapter will attempt to review the existing literature and the theory behind its application to these patients.

10.1 PATHOPHYSIOLOGY OF CEREBRAL ISCHEMIA Ischemia is the lack of blood flow, while hypoxia is a decrease in the amount of available oxygen. The human brain requires more oxygen and glucose than any other

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tissue in the body on a gram-per-gram basis. The average human brain weighs approximately 1400 grams and therefore represents 2% of total body weight. However, it utilizes 25% of the total body oxygen and glucose per minute. The typical cerebral blood flow rate is approximately 50 ml/gram/minute. Electrical activity ceases at flow rates of 16 to 18 ml/gram/minute, and membrane failure occurs at rates of 10 to 12 ml/gram/minute or below. Because of the existence of collateral flow and the diffusion capacity of molecules, an interruption of blood flow starves a “core” area of neurons completely and produces a surrounding area of cells that receives a decreased (but not absent) supply of blood, termed the ischemic penumbra. The mechanism by which ischemia produces neuronal injury has not been fully delineated, but it seems to most likely be multifactorial. Factors that initiate repercussions include the presence of a blood clot that causes a lack of oxygen, a lack of glucose, and alterations in cerebral hemodynamics. When circulation of blood resumes, more complications occur that may perpetuate injury.

10.1.1 ALTERATIONS IN CEREBRAL HEMODYNAMICS Significant volumes of knowledge concerning the molecular mechanisms of cellular damage secondary to ischemia have been gained in the past decade. However, mainstream attention has only recently come to also recognize the existence of hemodynamic disturbances involved in ischemia/reperfusion. There seem to have been identified two major types of flow disturbances that occur upon reperfusion, noreflow, and postischemic hypoperfusion. 10.1.1.1 No-Reflow Phenomenon Ames et al. initially made an interesting observation — the ischemic damage incurred by the CNS is not the same in vivo as it is in vitro.1 They noticed that when isolated rabbit retina is deprived of oxygen and glucose in vitro, electrical response to light ceases as quickly as it does with in vivo cardiac arrest models. However, the in vitro prep recovers its response to light after up to 30 minutes of oxygen/glucose deprivation, whereas irreversible damage occurs to the in vivo retina after only 8 to 10 minutes of nutrient deprivation. Keeping in mind that the retina has a higher metabolic rate than the cerebral cortex, it was suspected that factors other than molecular injury mechanisms secondary to hypoxia/hypoglycemia involving only the neurons themselves were influencing the return of function. This led to investigation of the vascular system after ischemia. The circulating blood of a cat was labeled with FITC–albumin, and after 7.5 minutes of global ischemia, it was noted that distinct areas of the brain remained white.1 The labeled albumin obviously was never brought into these areas by the flow of blood, and the phenomenon was therefore termed no-reflow. It was further noted that the sizes of these areas increased with longer ischemic periods.2 However, if blood flow reduction is incomplete, the no-reflow phenomenon takes much longer

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to develop (onset after 7 minutes of complete ischemia vs. after 3 to 6 hours of incomplete flow reduction).3 The pathophysiology of the no-reflow state is a result of both vascular factors and intrinsic properties of the blood. Ischemia of sufficient duration produces fluid and electrolyte shifts that are most pronounced in the vicinity of the blood vessels.4 This accumulation of fluid extravascularly causes extrinsic compression of the vessel, narrowing the lumen.4,5 It has also been noted that the endothelial cells become edematous, and their increase in diameter would also narrow the vessel lumen.6 Many investigators believe that blood viscosity increases and that this is a major factor in the no-reflow phenomenon. The fluid shift mentioned above depletes the plasma of free water, increasing the plasma viscosity. Microthrombi may play a part in the pathogenesis; however, this is refuted by the inability of heparin to prevent noreflow.7 Erythrocyte sludging8 and the formation of platelet aggregates9 have also been shown to occur in cerebral vessels during ischemia. 10.1.1.2 Postischemic Hypoperfusion Postischemic hypoperfusion develops after a transient phase of reactive hyperemia.10 It is similar to no-reflow in that it develops only if the ischemic period is longer than a threshold duration of approximately 5 minutes,11,12 but its effect does not increase with longer periods of ischemia. Longer ischemic periods do, however, cause a delayed onset of hypoperfusion, because the necessary preceding hyperemic phase lasts longer with extended ischemic times.13 The time to onset of hypoperfusion is unchanged after incomplete flow interruption, in contrast to no-reflow.14,15 The decreased flow seems to be secondary to alterations in vascular tone. During the hyperemic phase, pial artery vasodilation has been noted,3 edema is at its maximum as is intracranial pressure, and electrophysiological function begins to recover (i.e., the ensuing hypoperfusion is not secondary to energy failure).15 There is also loss of autoregulation and of vascular tone changes in response to alterations in CO2 concentration.16 When the hyperemic phase ends, autoregulation returns; however, the vascular tone is still not sensitive to CO2 concentration.15 Vascular tone may stabilize at a higher level, particularly in the arterial and arteriolar vessels. Currently, the reasons behind both the dissociation between autoregulation and CO2 reactivity and the pathophysiological significance of the postischemic hypoperfusion state are unclear. Flow may also be impaired by adhesion of polymorphonuclear leukocytes (PMNLs) to the blood vessel walls, creating intravascular obstructions. This will be discussed further in later sections. Cerebrovascular hemodynamic disturbances certainly cannot explain the entirety of the pathology behind ischemia and must be considered in conjunction with the cellular and molecular responses to hypoxia and hypoglycemia resulting from the lack of blood flow.

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10.1.2 HYPOXIA AND HYPOGLYCEMIA The nonavailability of oxygen and glucose shuts down the oxidative production of energy for the neuron. The brain is unable to store any significant amount of molecular fuel and therefore is quickly affected by ischemia. The membrane potential developed and maintained by a neuron is very expensive, energetically. This, then, is the first to suffer the blow of ischemia. As the level of ATP falls, so does Na/K ATPase activity, and intracellular K+ leaks into the extracellular fluid as Na+ diffuses its way into the cell. This is logically followed by membrane depolarization and the influx of Ca2+ via voltage-dependent calcium channels. In presynaptic neurons, second messenger systems and calcium-dependent enzymes are activated and neurotransmitters are released from the terminal bouton. The neurotransmitters cause a Ca2+ influx into the postsynaptic cell, which adds to the already high intracellular concentration of calcium present secondary to the ischemia itself. Both the pre- and postsynaptic intracellular cytosolic calcium concentrations are further elevated by the IP3-mediated mobilization of calcium. Calcium levels of this concentration induce a nonspecific activation of phospholipase A2 and of other phospholipases that begins to decompose the plasmalemma through destruction of phosphotidylcholine and phosphotidylethanolamine.17,18 Cell membrane decomposition is perpetuated by other calcium-activated enzymes as well, and so the hallmark ischemia-mediated cell death begins because the cell is no longer able to isolate itself from the surrounding environment. This theory has been referred to as the excitotoxic amino acid neurotransmitter theory because the primary neurotransmitter released from the terminal bouton is glutamate, which has been found to accumulate extracellularly in the brain during ischemia. It has also been noted that excess levels of glutamate applied to cultured neurons result in cell death.18 The above sequence of events is collectively known as the primary ischemic injury, whereas the following resupply of blood, and hence glucose and oxygen, initiates the secondary, or reperfusion injury. The primary injury is difficult to target therapeutically and is time-dependent. Currently, the only available intervention for resolving the primary injury involves the use of thrombolytic agents to disrupt the culprit blood clot. Two large trials using the systemic administration of tissue plasminogen activator (t-PA) have been carried out recently. The mechanism of action of the drug is to cleave plasminogen into plasmin, which in turn degrades fibrin and dissolves the clot. The European Cooperative Acute Stroke Study (ECASS) was a multicenter, double-blinded, placebo-controlled study in which patients were administered t-PA if they presented within 6 hours of the onset of symptoms and did not meet certain high-risk criteria. A statistically significant improvement in neurologic recovery was noted at 90 days postinfarct in the t-PA-treated patient group. However, a statistically significant increase was also noted in intracerebral hemorrhage in the same treatment group, which correlated with an increased death rate as well.19 The U.S. National Institute of Neurological Disorder and Stroke (NINDS) ran a similar study in which patients received t-PA if they presented within 3 hours of their onset of symptoms. They reported that the treatment group was 30% more likely to

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have no or minimal disability at 90 days postinfarct. Intracerebral hemorrhage occurred at a much lower rate than in the European trial. Certain subsets of patients were identified as having an increased rate of hemorrhage with administration of the drug.20 Therefore, the FDA has endorsed the administration of t-PA to patients presenting within 3 hours of symptom onset and not displaying any of the criteria known to place individuals at a higher bleeding risk. A hypothesis might then be that earlier reperfusion and less ischemic time portends a better recovery. This has been borne out in several animal models. By its very nature, this intervention initiates secondary injury by allowing reperfusion. Several attempts at pharmacologic prevention or attenuation of reperfusion injury have been made, all with marginal or no success. Therefore, most current research attempts to delineate the molecular mechanisms of neuronal injury.

10.1.3 REPERFUSION INJURY The molecular mechanisms of reperfusion injury will be discussed individually; however, note that these individual processes are many, and most likely occur concomitantly to produce the end product of reperfusion injury. 10.1.3.1 Free Radicals A primary component of reperfusion is the generation of free radicals.21 The compounds formed are oxyradicals that are highly unstable, with an unpaired electron in the outer shell; they are also therefore highly toxic to the cell. The cellular toxicity is brought about by free radical-induced lipid peroxidation, which in turn generates more free radicals. Lipid peroxidation damages cell and organelle membranes and interferes with cellular homeostasis. These free radicals are produced in both the tissue damaged from ischemia and in polymorphonuclear leukocytes (PMNL) recruited during reperfusion. Because free radicals are difficult to isolate and work with directly, the above theory is supported by the observation that adding chemicals that act as free radical scavengers improves outcome and decreases tissue damage in models of ischemia–reperfusion (I/R).22-26 Tissue damage is also decreased after blocking PMNL adhesion, which will be discussed more fully later in this chapter. Studies in which intravascularly administered free radical scavengers prevented reperfusion injury have suggested that vascular endothelial cells may be the source of free radical production in I/R injury.21 Brain capillary endothelial cells contain a number of enzymes that produce superoxide (SO) and hydrogen peroxide (H2O2), including monoamine oxidase, xanthine oxidase, and nitric oxide synthase, among others. It must also be remembered that free radicals act as vascular intercellular messengers, with SO causing vasoconstriction and nitric oxide (NO) causing vasodilation. Cerebral capillary endothelial cells also contain antioxidant enzymes. These include superoxide dismutase (SOD), catalase, and glutathione peroxidase. SOD scavenges SO by converting it into H2O2 and oxygen. Catalase then scavenges the H2O2,

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changing it into oxygen and water. The efficacy of the enzymes as agents to decrease cerebral I/R injury is dependent on their ability to cross the blood–brain barrier, which they do very poorly. They must therefore be carried across actively in liposomes by the glia, or first packaged in liposomes and then given therapeutically (as is currently done with some antibiotics). However, it is important to note that these enzymes are not uniformly distributed throughout all tissues. Recently, cultured human brain capillary endothelial cells were subjected to I/R, and levels of LDH were measured as indictors of cellular injury.21 Three subgroups were treated with SOD, catalase, and allopurinol (an XO inhibitor) before reperfusion. It was noted that LDH levels were remarkably lower in the SOD- and catalase-treated groups as compared to controls. However, the cells treated with allopurinol contained LDH levels equal to control groups. This indicates that XO may not be an important contributor to free radical injury secondary to I/R in brain endothelium. Another potential source of free radical production is the interaction of SO and NO. These two species are known to react rapidly to form peroxynitrate (ONOO), which is stable; however, it rapidly decomposes into an intermediate species which oxidizes biological molecules in a manner similar to that of OH radicals but at a rate constant 10,000 times slower than OH. The rate of this reaction is driven by the concentration of SO and NO. Since both of the species are formed in endothelial tissue, these cells may be vulnerable to this mechanism of injury. However, a beneficial effect of NO may also be inferred, since it serves as a sink to reduce the population of the more potent oxidant SO and the damage it mediates. NO is a free radical which has been recognized as an endothelial-derived relaxation factor. There are three subtypes of nitric oxide synthase that produce NO by converting L-arginine to L-hydroxy arginine and then to L-citrulline with the consumption of NADPH, oxygen, and release of NO. Neuronal NOS (nNOS, NOS1) is a constitutive isoform localized in neurons, while inducible NOS (iNOS, NOS2) is found in microglia/macrophages and astrocytes. Endothelial NOS (eNOS, NOS3) is a constitutive form found in the endothelium and is responsible for vascular smooth muscle relaxation via directly activating guanyl cyclase and generating cGMP. It should be noted that nNOS and eNOS activity are calcium-dependent and that NO produced by nNOS and iNOS has been shown to be toxic both in vitro and in vivo.27-29 Endothelial NOS-produced NO has been shown to be neuroprotective via its vasodilating effect. These different forms of NO and their respective beneficial or detrimental effects become important in understanding the potential role of HBO in I/R. 10.1.3.2 Microcirculation The effect of I/R on microcirculation has been partially discussed previously under the sections explaining postischemic hypoperfusion and the no-reflow phenomenon. Another factor in the damage produced during reperfusion is the PMNL. These cells are recruited by chemoattractants and arrive via the bloodstream to produce free radicals and release enzymes that are destructive to the surrounding tissue.

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In I/R-subjected muscle it has been noted that an increase in PMNL adhesion is seen in the postcapillary venules. The increased adhesion progresses to a magnitude that obscures the venule wall microscopically.30 The mechanism of I/R-mediated PMNL–endothelial adhesion involves the CD11/18 cell membrane protein and its endothelial cell ligand, intercellular adhesion molecule-1 (ICAM1).31 Once bound to the lining, the PMNL then migrates through the endothelium and basement membrane of the venule and has been observed to disrupt this membrane on electron microscopy.31 The endothelial cells are also injured by the “rolling” of PMNLs through the microcirculation. The adhesion of PMNLs to the endothelium is dependent on two factors; the shear force of the blood flow and the “adhesiveness” of the two surfaces.32 In the early stages of this process, the adhesiveness is overcome by the shear forces, and some of the CD11/18-ICAM bonds break (or the ICAM molecules are dislodged from the endothelial cell membrane, causing microdisruptions), allowing the PMNL to roll along the surface. Once the adhesiveness overcomes the shear force pushing the PMNL along the endothelial surface, the PMNL can begin to migrate through the endothelium. This migration also produces vessel wall damage by disrupting the endothelial basement membrane. Venous washouts of ischemic rat hind limbs contain elevated levels of prostaglandin B2 (the metabolite of the vasoconstrictor, thromboxane A2) in limbs that went on to complete microcirculatory failure.33 Through direct tissue measurements, it was also noted that the arterioles surrounding the above venules underwent a severe vasoconstriction.34 It may therefore be that venules disrupted by a large amount of PMNL attachment and migration produce a vasoactive substance, resulting in vasoconstriction of nearby arterioles and the development of the no-reflow state. It also seems that PMNL endothelial adherence plays a major role in the pathogenesis of I/R injury.

10.2 HYPERBARIC OXYGEN Hyperbaric oxygen (HBO) therapy is simply the delivery of oxygen at greater than atmospheric pressure to treat disease. It has been used very effectively to treat several disease processes, including decompression sickness, arterial gas embolism, and gas gangrene, among others. The application of HBO is also under investigation as a treatment for several other entities, one of which is I/R injury. Despite the concerns that increased amounts of oxygen could only worsen the deleterious effect of oxygen-derived free radicals, results of work to date have been promising. In 1878, Paul Bert described caisson disease, or decompression sickness (“the bends”), the bubble theory, and oxygen toxicity in his book Barometric Pressure. In 1906, J.S. Haldane, a British physiologist at the Lister Institute, developed stage decompression tables for treating decompression sickness. In 1960 Boerema described keeping pigs alive using HBO and red blood cell-depleted plasma in his book Life Without Blood. Before the advent of cardiopulmonary bypass, drenching the tissues with oxygen enabled the surgical correction of various cardiac anomalies. The idea caught on, and multiple chambers were built in the U.S. and abroad. However, as

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improved cardiopulmonary bypass equipment was developed, the need for HBO declined. It was applied to several other conditions without scientific basis, and was even overused in this regard. Today several medical societies exist, and the use of HBO is reserved for scientifically supported indications.

10.2.1 THE PHYSICS OF HBO THERAPY The characteristics of gas under pressure are governed by straightforward and wellaccepted gas laws. The units of measure used to describe the amount of pressure can be confusing, however. Clinically, the unit most often used in the practice of hyperbaric medicine is atmospheres absolute (ATA). The barometric pressure at sea level is 14.7 pounds per square inch, or 1 ATA. When referring to pressures greater than that at sea level, always include the pressure at sea level. Descending to 33 feet of depth in sea water adds another 14.7 pounds per square inch. Therefore, at 33 feet of depth in sea water, the pressure is 2 ATA. The confusion arises because an instrument gauge does not include the initial 14.7 pounds per square inch at sea level when measuring pressure. Pressure as measured by an instrument gauge is labeled psig. Therefore, the psig at 33 feet of depth in sea water is 14.7 psig. The pressure at 66 feet of sea water can be referred to as 3 ATA or 29.4 psig. Another unit of pressure is referred to as Torr, or millimeters of mercury (mmHg). In this case the amount of pressure at sea level is 760 mmHg. It is customary, however, to only use mmHg to designate pressures of one atmosphere or less. 10.2.1.1 Boyle’s Law Robert Boyle observed that the volume is inversely proportional to the pressure for a body of ideal gas at constant temperature, or P1/ P2 = V2 / V1. Thus, compressing a given volume of gas to twice its original pressure will halve its volume. This is an important consideration in patient care because the greatest risk of middle ear barotrauma occurs at the beginning of pressurization, when the volume changes affecting the gas in the middle ear are maximal. 10.2.1.2 Dalton’s Law John Dalton, an English chemist and physicist, formulated the gas law which states that the total pressure exerted by a mixture of gases is equal to the sum of the pressure of each of the different gases making up the mixture — that is, each gas acts as if it were alone and occupying the total volume. Otherwise stated, Ptotal = pO2 + pCO2 + pN2 + p…. Therefore, at sea level (760 mmHg), air contains roughly 160 mmHg of oxygen and 600 mmHg of nitrogen pressure.

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10.2.1.3 Henry’s Law This law states that the amount of gas dissolved in a liquid is directly proportional to the partial pressure of the dissolved gas. Therefore as the partial pressure of oxygen dissolved in plasma at higher pressures increases, so does the arterial oxygen content and oxygen carrying capacity of the blood, regardless of the amount of hemoglobin present. The oxygen content of the blood is simply the amount of oxygen contained in a specified volume of blood. It is the sum of the oxygen bound to hemoglobin plus the amount of oxygen dissolved in the plasma. Hemoglobin, when 100% saturated (all four binding spots for oxygen on every hemoglobin molecule are occupied by oxygen), carries 1.3 grams of oxygen for every gram of hemoglobin. Healthy lungs are able to saturate hemoglobin to 100% at sea level; increasing the ambient pressure does not enable hemoglobin to carry any more oxygen. The normal amount of oxygen dissolved in the plasma at sea level is 0.31ml O2/100 ml plasma; but by Henry’s law, increasing the ambient pressure dissolves more gas in the liquid, and at 3.0 ATA the amount of oxygen dissolved in the plasma is 6.6 ml O2/100 ml plasma. Thus, raising the surrounding pressure increases the amount of oxygen carried in the blood by increasing the amount of oxygen carried in the plasma (normal whole blood arterial oxygen content is 18.1 ml; whole blood arterial oxygen content at 3.0 ATA is 24.7 ml). This increase represents roughly a 6 ml increase in oxygen-carrying capacity — which also represents the average oxygen requirement of the body. Therefore, the physiology behind Boerema’s experiment as discussed above is explained. The increased concentration of oxygen in the blood also increases the distance across which the oxygen can diffuse into the tissue (Figure 10.1). The entire oxygen requirement can now be met with the extra oxygen dissolved in the plasma under hyperbaric conditions.

10.2.2 THE EFFECT OF HBO IN STROKE Initially, application of excess oxygen to the I/R process seems counterintuitive given the significant contribution of free radicals to the pathogenesis of this injury pattern. The beneficial effects of HBO seen in various models are referred to as the oxygen paradox. The mechanisms underlying these effects are most likely multifactorial and therefore will be discussed separately below. 10.2.2.1 PMNL — Endothelial Adherence The contribution of the PMNL to tissue injury during the reperfusion phase of stroke was discussed above. Briefly, the microcirculation becomes inundated with activated PMNLs adhering to the venule walls. These cells then contribute to the formation of free radicals and release destructive enzymes. A single PMNL is capable of occluding some of the smallest capillaries. One of the vital steps in the contribution of the PMNL to injury is adherence to the endothelium. Intravital microscopy in a rodent gracilis muscle preparation has shown that HBO treatment significantly decreased the adhesion of the PMNL to the

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FIGURE 10.1 The increased distance through which oxygen is able to diffuse under normobaric and hyperbaric conditions. It then stands to reason that if the blood vessels in the adjacent penumbra are under hyperbaric condistions, a portion of the completely hypoxic “core” may receive some oxygen.

endothelium.34 The question of whether HBO interfered with activation of the PMNL or with the adhesion molecules was then raised. Since the primary PMNL expressed adhesion molecule is CD 11/18 (responsible for binding with the endothelial intercellular adhesion molecule-1, or ICAM-1), it was the logical first choice for study. The carbon monoxide (CO) poisoning model has been used for the study of PMNL adherence in ischemia. Although CO poisoning is not a classic form of I/R injury, the end result is similar. CO poisoning leads to a more global ischemia as opposed to ischemia along a specific vascular distribution. Ischemia is produced via the direct action of CO on the hemoglobin molecule and by CO’s interference with the electron transport chain at the cytochrome level. However, the transient cellular hypoxia and CNS hypotension followed by CO washout and restoration of oxygenation may produce tissue injury similar to that seen after transient vascular occlusion. Rats poisoned with CO demonstrate increased PMNL sequestration in the brain and an increase in lipid peroxidation. These effects were felt to be secondary to PMNL adhesion because PMNL depletion and use of an anti-CD18 antibody prevented lipid peroxidation.35 HBO treatment also prevented PMNL accumulation in the brain as well as lipid peroxidation.36 PMNL adhesion prevention via HBO is a dose-dependent phenomenon with 3 ATA providing the greatest suppression. CD18 adherence to nylon is prevented by the presence of an anti-CD18 antibody. PMNLs isolated from CO-poisoned rats after they received HBO demonstrated a decrease in adherence to nylon, suggesting HBO acts at the CD18 receptor.36 This action on the CD18 receptor may be mediated by a decrease in the synthesis of cyclic guanosine monophosphate (cGMP). PMNLs isolated from both rodents and humans treated with HBO were again able to bind with nylon after being exposed to cGMP analogues.37 The exact mechanism of the HBO effect on cGMP remains unclear; however, NO is suspected to play a significant role (see below).

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The effect of HBO on expression of CD18 molecules has also been studied with inconsistent results. The studies also examined the surface expression of CD18 and found no difference between HBO-treated and -untreated PMNLs. It has also been noted that the number of CD18 molecules is not altered.38 Current studies have focused on early outcome (<24 hours), and infiltration of PMNLs into the tissue occurs over the first 24 to 48 hours after injury. The principal ligand for CD11/18 is the endothelial membrane protein ICAM-1. Recently, human and bovine endothelial cells were subjected to an I/R stimulus (hypoxia and hypoglycemia for 4 hours followed by normoxia and normoglycemia for 20 hours).39 This process induced expression of ICAM-1 on the cell surface. Treatment of the cells with HBO (2.5 ATA for 90 minutes) upon reperfusion reduced ICAM-1 expression to control levels. This HBO-induced decrease in ICAM expression was reversed via the addition of a NOS inhibitor (L–N–arginine methyl ester), suggesting that HBO-mediated NO production may play a role in the mechanism of ICAM suppression. Another family of endothelial cell membrane proteins is involved in adhesion, the selectins (P- and E-selectin). E-selectin is a glycoprotein that is not stored in the cell and requires protein synthesis to be expressed. This usually requires 4 to 6 hours, based on in vitro stimulation with cytokines. The PMNL binding site for E-selectin appears to be the CD15 oligosaccharide.40 It also seems that E-selectin/CD15 binding initiates intracellular events which further activate the PMNL, and enhance the binding of the PMNL to the endothelium.41 Preliminary work indicates that HBO downregulates E-selectin expression in the model described immediately above.42 Decreasing PMNL — endothelial adherence via antibodies blocking intercellular binding reactions produces a decrease in tissue damage following I/R-induced injury. The above experimental evidence proposed an alternative method of accomplishing the same decrease in adherence utilizing HBO and attempted to begin to outline possible mechanisms for the results stated. Administering HBO instead of antibodies to produce this outcome in vivo would eliminate the difficulties of drug delivery, i.e., getting large molecules across an intact blood–brain barrier. 10.2.2.2 Free Radicals The presence of free radicals in the I/R injury process promotes lipid peroxidation, which in turn disrupts cell and organelle membranes, interfering in cellular homeostasis. Exposure to 100% oxygen during reperfusion after transient global ischemia in the gerbil increases both lipid peroxidation and mortality.43 One possible conclusion from this data is that, given the contribution of oxygen-derived free radicals to I/R injury propagation, the relative increase in tissue oxygenation produced by HBO would serve to worsen tissue damage and the outcome of I/R injury. Quite to the contrary however, HBO has been shown in multiple studies to decrease lipid peroxidation.36, 44–48 This has been observed despite the evidence that HBO may increase free radical production. In a recent rat focal ischemia model, cerebral blood flow in the ischemic periphery (as measured by laser doppler) was reduced to roughly 50% of normal via ligation of the right middle cerebral and right common carotid arteries.49

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HBO exposure (3 ATA for 2 hours) significantly increased cerebral arterial pO2 to 1571 mmHg. The calculated increase in oxygen supply to the ischemic periphery was 20%. Lipid peroxidation was measured at the laser doppler probe site and was found to be no different from controls, and yet infarct volume was reduced by 18% as compared to control animals. HBO treatment increased oxygen supply to the ischemic penumbra and reduced I/R injury without the expense of increasing lipid peroxidation. A possible mechanism for this has been suggested by examining the role of oxygen on lipid peroxidation in vitro. In a cell-free system, oxygen at concentrations achieved at usual therapeutic treatment pressures antagonized lipid peroxidation, possibly through oxygen-mediated termination reactions.50 HBO is also known to affect the production of another free radical, nitric oxide, as well as the expression of both iNOS and eNOS. Increases in the concentration of NO after HBO treatment have been seen in bovine cerebellum51 and zymosaninduced rat shock.52 It has been noted, however, that HBO affects the expression of iNOS and eNOS differentially.34 HBO has been shown to decrease the LPS-induced transcription of iNOS.53 Protein production studies in an in vitro model of I/R injury revealed an increase in eNOS but not iNOS after HBO treatment.34 The activity of eNOS has also been noted to be calcium-dependent,54 and one of the effects of I/R is an increase in intracellular calcium. If HBO can help decrease PMNL–endothelial adhesion (as described in the previous section) and produce local vasodilation which increases blood flow, then an increase of eNOS-produced NO may explain most of the mechanism underlying the beneficial effect of HBO on ischemia-reperfusion injury. It may, in effect, upregulate a natural cellular defense to I/R-mediated damage.

10.2.3 HUMAN STUDIES AND CONCLUSIONS So far, multiple attempts at therapeutic intervention for the prevention of reperfusion injury have been unsuccessful (Table 10.1). These trials have utilized compounds directed at stopping one part of the cascade of events that occurs during the reperfusion period. Several trials investigating the efficacy of the use of HBO therapy for acute stroke have yielded conflicting results. Human trials conducted in the 1970s failed to uncover any benefit from the use of HBO.55, 56 However, these studies were small (n < 35); and one studied the effect of HBO anywhere from 3 to 108 months after the onset of symptoms. Another small study by Kapp in 198157 began to characterize the possible adjunctive nature of HBO therapy. He used improvement in symptomatology under HBO conditions as a screening mechanism to identify patients who would benefit from surgical revascularization. Patients who improved with HBO and who were successfully revascularized had no recurrence of neurologic symptoms at 20 months postop. A study conducted in 1995 in Lyon, France58 utilizing HBO at 1.5 ATA detected a favorable outcome trend for HBO therapy, and the investigators did not observe any of the feared negative effects of therapy. Two trials are currently ongoing concerning the effect of HBO on stroke in human subjects at the University of Indiana. They are double-blinded, placebo-controlled studies examining the effectiveness of HBO therapy at 2.5 ATA, initiated within 24 hours of symptom onset. They will utilize the NIHSS, Barthel index, Glascow outcome

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TABLE 10.1 Neuroprotective Agents in Advanced Clinical Trials Drug Lubeluzole

Proposed Mechanism of Actiona Inhibits NO synthesis

Dosing Time and Routeb

Status of Phase III Trial

6–8 h/IV

U.S. trial encouraging, but European trial negative; third trial terminated prematurely

Enlimomab

Antibody neutralization of ICAM

6 h/IV

Negative

Citicoline

Phospholipid precursor or free radical scavenger

24 h/PO

First trial inconclusive and second trial negative

Fosphenytoin

Membrane stabilizer

6 h/IV

Terminated prematurely

Aptiganel

NMDA antagonist

6 h/IV

Terminated prematurely

Eliprodil

NMDA antagonist

6 h/IV

Terminated prematurely

Selfotel

NMDA antagonist

6 h/IV

Terminated prematurely

Nalmefene

Opiate antagonist

6 h/IV

Unknown

Tirilizad

Free radical scavenger

6 h/IV

Negative

Nimodipine

Calcium channel blocker

24–48 h/IV

Negative

GM ganglioside

Ganglioside

Unknown

Negative

Basic fibroblast growth factor

Promoter of neuronal growth and differentiation

6 h/IV

Terminated prematurely

Clomethiazole

GABA agonist

12 h/IV

Ongoing

GVS150526

Glycine antagonist 6 h/IV

International trial negative; enrollment in U.S. trial complete and data analysis under way

BMS-204352

Potassium channel modula6 h/IV tor

Ongoing

NXY059

Spin trap agent

Proposed

N/A

Note: NO = nitric oxide; ICAM = intercellular adhesion molecule; NMDA = N-methyl-D-aspartate; GABA = gamma-aminobutyric acid; N/A = not available. a Time and route of drug administration after the onset of stroke symptoms.

scale, and modified Rankin scale as assessment tools to evaluate patients at 0 and 24 hours as well as at 3 and 12 months. The expected completion date is in December 2001. These studies are being conducted at a large tertiary care center and the results are eagerly anticipated. Multiple investigations in animals, however, have continued to reveal promising conclusions about the role of HBO in stroke (Table 10.2). There is great difficulty in

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TABLE 10.2 Selected HBO Use in Ischemia–Reperfusion Injury Model Systems Study Species Organ

TI/R

THBO

IHBO

Outcome Measure

ATA

Effect

Buras et al.

Human/ Bovine

Endothelial 4 h/20 h cell

90 min

A

2.5

ICAM-1 expression + PMN adherence +

Chen et al.

Rat

Liver

1 h/2 h

90 min

B

2.5

WBC adherence Lipid peroxidation ATP, blood flow

+ + +, +

Mink and Rabbit Dutka

Brain

10 min/ 75 min

75 min

A

2.8

Lipid peroxidation

+

Rabbit

Brain

10 min/ 75 min

75 min

A

2.8

SEP

+

Rat

Skeletal muscle

1.5, 3 h/5 h

45 min

A

2.5

ATP, lactate phosphocreatine

+, + +

Sterling et Rabbit al.

Heart

30 min/ 3h

90 min

D, A, DA

2.5

Infarct size

+

Sirsjo et al.

Rat

Skeletal muscle

4 h/ variable

90 min

A

2.5

Blood flow

NS/+

Rat

Skin

4 h/ variable

90 min

A

2.5

Capillary density

NS/+

Rat

Brain

20 min/ 90 mina

45 min

B, A

2.3

WBC sequestration XO production Lipid peroxidation

+ + +

Tjarnstrom, Rat et al.

Small intestine

2 h/ 90 min

90 min

A

2.5

WBC sequestration PMN activation

+ +

Yamada et Rat al.

Small intestine

2 h/ 30, 120 min

90 min

D, A

2.0

15-day survival ATP Histology

+/NS NS/NS +/equiv

Zamboni

Rat

Skeletal muscle

4 h/3 h

60 min

D, A

2.5

WBC adherence Vessel diameter

+ +

Zamboni, Rat et al.

Skeletal muscle

4 h/ 90 min

90 min

D

2.5

WBC sequestration

+

Nylander et al.

Thom

Note: TI/R = ischemia/reperfusion times; THBO = duration of HBO treatment; IHBO = HBO treatment relative to ischemia; B = before ischemia; D = during ischemia; A = after ischemia; + = beneficial outcome; – = negative outcome; NS = no significant difference with treatment; SEP = somatosensory evoked potentials. a

indicates ischemia induced by CO poisoning.

reproducing the stringent control of experimental variables that is available with the use of animal models when human subjects are studied. The animal model is also not a perfect model of the human stroke. These problems have proven to be significant obstacles in the evaluation of various pharmacological therapies for stroke, and they must be considered and overcome when investigating HBO use in stroke. The presence of these obstacles should not discourage physicians and scientists but con-

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versely should push us to work even harder to overcome them and provide relief and hope to those who suffer from this debilitating disease. Reperfusion injury is a series of events, the side effect of which produces cellular damage. It therefore seems that the most effective strategy for minimizing or preventing cellular damage would be to intervene at multiple places in the process. Such combination therapy has been very effective in treating other examples of ischemia–reperfusion injury, such as in myocardial infarction; but it has never been studied in reference to cerebral ischemia. Hyperbaric oxygen therapy seems to have an effect on several aspects of reperfusion injury and will no doubt play a promising role in the combination therapy that is the future treatment of cerebral ischemia.

REFERENCES 1. Ames, A. et al., Cerebral ischemia, II, The no-reflow phenomenon, Am. J. Pathol., 52: 437-453, 1968. 2. Fischer, M. and Hossman, K-A., No-reflow after cardiac arrest, Intensive Care Med., 21: 132–141, 1995. 3. Hossman, K-A., Reperfusion of the brain after global ischemia: hemodynamic disturbances, Shock, 8(2): 95–101, 1997. 4. Chiang, J. et al., Cerebral ischemia, III, Vascular changes, Am. J. Pathol., 52: 455–476, 1968. 5. Arsenio-Nones, M.L., Hossman, K-A., and Farkas-Bargeton, E. Ultrastructural and histochemical investigation of the cerebral cortex of cat during and after complete ischemia, Acta. Neuropathol., 26: 329–344, 1973. 6. Jamison, R.L. The role of cellular swelling in the pathogenesis of organ ischemia, West J. Med., 120: 205–218, 1974. 7. Cantu, R.C. and Snyder, M. Effect of anticoagulants, vasodilators, and dipyridamole on postischemic cerebral vascular obstruction, J. Surg. Res., 2: 70–71, 1972. 8. Hekmatpariah, J., Cerebral blood flow dynamics in hypotension and cardiac arrest, Neurol., 23: 174–180, 1973. 9. Obrenovitch, T.P. and Hallenbeck, J.M. Platelet accumulation in regions of low blood flow during the post-ischemic period, Stroke, 16: 224–234, 1985. 10. Kuroiwa, T., Bonnekoh, P., and Hossman, K-A. Laser Doppler flowmetry in CA1 sector of hippocampus and cortex after transient forebrain ischemia in gerbils, Stroke,23: 1349–1354, 1992. 11. Lin, S-R., Angiographic studies of cerebral circulation following various periods of cardiac arrest: a preliminary study in the dog, Invest. Radiol., 9: 374–385, 1974. 12. Blomquist, P. and Wieloch, T., Ischemic brain damage in rats following cardiac arrest using long-term recovery model, J. Cereb. Blood Flow Metab., 5: 420–431, 1985. 13. Zimmer, R., Lang, R., and Oberdörster, G., Post-ischemic reactive hyperemia of the isolated perfused brain of the dog, Pflügers Arch., 328: 332–343, 1971. 14. Suzuki, R. et al., The effects of 5-minute ischemia in Mongolian gerbils. I. Bloodbrain barrier, cerebral blood flow, and local cerebral glucose utilization changes, Acta. Neuropathol., 60: 207–216, 1983. 15. Hossman, K-A., Lechtape-Grüter, H., and Hossman, V., The role of cerebral blood flow for the recovery of the brain after prolonged ischemia, Z. Neurol., 204: 281–299, 1973.

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16. Lassen, N.A., The luxury-perfusion syndrome and its possible relation to acute metabolic acidosis localized within the brain, Lancet, 2: 1113–1115, 1966. 17. Kogure, K. et al., Inflammation of the brain after ischemia, Acta. Neurochir., 66(S): 40–43, 1996. 18. White, B. et al., Global brain ischemia and reperfusion, Ann. Emerg. Med., 27: 588–594, 1996. 19. Hacke, W. et al., Intravenous thrombolysis with recombinant tissue plasminogen activator for acute hemispheric stroke, JAMA, 274: 1017–1025, 1995. 20. The National Institute of Neurological Disorder and Stroke rt-PA Stroke Study Group, Tissue plasminogen activator for acute ischemic stroke, New Engl. J. Med., 333: 1581–1587, 1995. 21. Nagoshima, T. et al., Reoxygenation injury of human brain capillary endothelial cells, Cell Mol. Neurobio., 19(1): 151–161, 1999. 22. Feller, A. et al: Experimental evaluation of oxygen free radical scavengers in prevention of reperfusion injury to skeletal muscle, Ann. Plast. Surg., 22:321, 1989. 23. Im, J. et al., Effects of superoxide dismutase and allopurinol on the survival of acute island skin flaps, Ann. Surg., 201:357, 1985. 24. Manson, P. et al., The role of oxygen free radicals in ischemic tissue injury in island skin flaps, Ann. Surg. ,198:87, 1983. 25. Sagi, A. et al., Improved survival of island skin flaps after prolonged ischemia by perfusion with superoxide dismutase, Plast. Reconstr. Surg., 77:639, 1986. 26. Chan, P. et al., Brain injury, edema and vascular permeability changes induced by oxygen derived free radicals, Neurology, 34(3): 315–320, 1984. 27. Dawson, V. et al., Mechanisms of nitric oxide-mediated neurotoxicity in primary cell cultures, J, Neurosci.. 13: 2651–2661, 1993. 28. Iadecda, C. et al., Inducible nitric oxide synthase gene expression in vascular cells after transient focal cerebral ischemia, Stroke, 27: 1373–1380, 1996. 29. Xu, J. et al., Oxygen-glucose deprivation induces inducible nitric oxide synthase and nitrotyrosine expression in cerebral endothelial cells, Stroke, 31: 1744–1751, 2000. 30. Zamboni, W. et al., Effect of hyperbaric oxygen on reperfusion of ischemic axial skin flaps: a laser doppler analysis, Ann. Plast. Surg., 28: 339, 1992. 31. Zamboni, W. et al., Ischemia-reperfusion injury in skeletal muscle: CE18 dependent neutrophil–endothelial adhesion and arteriolar vasoconstriction, Plast. Reconstr. Surg., 99:2002–2007, 1997. 32. Buras, J., Basic mechanisms of hyperbaric oxygen in the treatment of ischemia-reperfusion injury, Int. Anesthesiol. Clin., 38(1): 91–109, 2000. 33. Feng, L. et al., Vasoactive prostaglandins in the impending no–reflow state: evidence for a primary disturbance in microvascular tone, Plast. Reconstr. Surg., 81: 775, 1988. 34. Zamboni, W. et al., Morphological analysis of the microcirculation during reperfusion of ischemic skeletal muscle and the effect of hyperbaric oxygen, Plast. Reconstr. Surg., 91: 1110–1123, 1993. 35. Thom, S., Leukocytes in carbon monoxide-mediated brain oxidative injury, Toxicol. Appl. Pharmacol., 123: 234–247, 1993. 36. Thom, S., Functional inhibition of leukocyte β2 integrins by hyperbaric oxygen in carbon monoxide-mediated brain injury in rats, Toxicol. Appl. Pharmacol., 123: 248–256, 1993. 37. Chen, Q., Banick, P., and Thom, S., Functional inhibition of rat polymorphonuclear leukocyte β2 integrins by hyperbaric oxygen is associated with impaired cGMP synthesis, J. Pharmacol. Exp. Therapeutics, 276: 929–933, 1996.

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38. Larson, J., Stephenson, L., and Zamboni, W., The effects of HBO on PMN expression of CD18 in a rat model of ischemia–reperfusion, Undersea Hyperbaric. Med., 25S: 50, 1998. 39. Buras, J. et al., Hyperbaric oxygen downregulates ICAM-1 expression induced by hypoxia and hypoglycemia: the role of eNOS, Am. J. Physiol., in press. 40. Walz, G. et al., Recognition by ELAM-1 of the sialyl–Lex determinant on myeloid and tumor cells, Science, 250: 1132–1135, 1990. 41. Bochner, B. et al., Adhesion of human basophils, eosinophils, and neutrophils to interleukin-1 activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules, J. Exp. Med., 173: 1553–1557, 1991. 42. Buras, J. and Reenstra, W., Hyperbaric oxygen decreases endothelial cell E-selectin protein expression in an in vitro model of ischemia–reperfusion, Ann. Emerg. Med., 32: S17, 1998. 43. Mickel, H. et al., Breathing 100% oxygen after global ischemia in Mongolian gerbils results in increased lipid peroxidation and increased mortality, Stroke, 18: 426–430, 1987. 44. Mink, R. and Dutka, A., Hyperbaric oxygen after global cerebral ischemia does not promote brain lipid peroxidation, Crit. Care Med., 23: 1398–1404, 1995. 45. Chen, M.-F., et al., Hyperbaric oxygen pretreatment attenuates hepatic reperfusion injury, Liver, 18: 110–116, 1998. 46. Kawamura, S. et al., Therapeutic effects of hyperbaric oxygenation of acute focal cerebral ischemia in rats, Surg. Neurol., 34: 101–106, 1990. 47. Reitan, J. et al., Hyperbaric oxygen increases survival following carotid ligation in gerbils, Stroke, 21: 119–123, 1990. 48. Takahashi, M. et al., Hyperbaric oxygen therapy accelerates neurologic recovery after 15 minute complete global cerebral ischemia in dogs, Crit. Care Med., 20:1588–1594, 1992. 49. Sunami, K. et al., Hyperbaric oxygen reduces infarct volume in rats by increasing oxygen supply to the ischemic periphery, Crit. Care Med., 28(8): 2831–2836, 2000. 50. Thom, S. and Elbukin, M., Oxygen dependent antagonism of lipid peroxidation, Free Rad. Biol. Med., 10: 43, 1991. 51. Rebgasany, A. and Johns, R., Characterization of endothelium-derived relaxing factor/nitric oxide synthase from bovine cerebellum and mechanism of modulation by high and low oxygen tensions, J. Pharm. Exp. Ther., 259: 310–316, 1998. 52. Luongo, C. et al., Effects of hyperbaric oxygen exposure on a zymosan-induced shock model, Crit. Care Med., 26: 1972–1976, 1998. 53. Kurata, S., Yamashita, U., and Nakajima, H., Hyperbaric oxygen reduces the cytostatic activity and transcription of nitric oxide synthetase gene of mouse peritoneal macrophages, Biochemi. Biophys. Acta, 1263:35–38, 1995. 54. Lowenstein, C. and Snyder, S., Nitric oxide, a novel biologic messenger, Cell, 70: 705–707, 1992. 55. Sarno, M., Sarno, J., and Diller, L., The effect of hyperbaric oxygen on communication function in adults with aphasia secondary to stroke, J. Speech Hearing Res., 15: 42–48, 1972. 56. Sarno, J. et al., The effect of hyperbaric oxygen on mental and verbal ability of stroke patients, Stroke, 3: 10–15, 1972. 57. Kapp, J., Neurological response to hyperbaric oxygen—a criterion for cerebral revascularization, Surg. Neurol., 15: 43–46, 1981. 58. Nighoghossian, N. et al., Hyperbaric oxygen in the treatment of acute ischemic stroke. a double-blind pilot study, Stroke, 26(8): 1369–1372, 1995.

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59. Warriner, R., Pressure points, Bulletin: Southeast Texas Center for Wound Care and Hyperbaric Medicine, 2(2), 1994. 60. DeGraba, T. and Pettigrew, L., Why do neuroprotective drugs work in animals but not humans? Neurol. Clin., 19(2): 475–493, 2000.

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11

Cellular and Molecular Mechanisms of Ischemic Tolerance Kimberly L. Simpson and Rick C. S. Lin

CONTENTS 11.1 Introduction 11.2 Models of Ischemic Tolerance 11.2.1 In vivo Global and Focal Ischemia Models 11.2.2 The Cortical Spreading Depression Model 11.2.3 The 3-Nitroproprionic Acid Chemical Preconditioning Model 11.2.4 Other Models 11.3 Hemodynamics 11.4 Glutamate and Receptors 11.5 Stress Proteins 11.6 Protein Synthesis 11.7 Bcl-2 Related Gene Expression 11.8 Structural Protein Degradation 11.9 Adenosine and Receptors 11.10 The Immune/Inflammatory Response 11.10.1 Interleukin-1 11.10.2 Tumor Necrosis Factor-α 11.10.3 Glia 11.11 Conclusions References

11.1 INTRODUCTION It has been estimated that 20–40% of the patient population diagnosed with stroke have experienced a brief episode of antecedent transient ischemic attack (TIA). This brain malady has been described clinically as an ischemia-related event which produces a neurologic deficit without obvious structural damage.1,2 At present, very limited information is available concerning the etiology of TIA and its subsequent correlation as a risk factor for stroke. Some studies indicate that prior exposure to TIA increases the likelihood of sustaining a full-blown ischemic attack by 7–13-

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fold. The controversial nature of existing reports has contributed significantly to the lack of consensus regarding the potential outcome for patients who sustain TIA before stroke onset, and those who do not.3–6 In terms of postepisode neurological status, TIA prior to stroke has been documented to be beneficial, while in other instances it has been reported to result in no demonstrable difference, or a worsened condition. Apparently, the number, severity, and time-course between TIAs each play a role in influencing the overall degree of impairment. However, the wide range of effects observed in epidemiological studies may also be attributed to other unknown variables. Several in vivo and in vitro experimental models have emerged over recent years to elucidate the connection between TIA and stroke. The initial findings from these investigations have revealed that sublethal ischemia augments the survival capabilities of cells to make them less vulnerable to injury. This intriguing phenomenon has been termed “ischemic tolerance” and has been shown to protect the heart7 as well as the brain8 against a more severe, secondary insult. The interest surrounding this topic has since prompted many investigators to speculate upon the mechanisms which mediate this defensive response. The primary goal of this chapter is to describe models that are currently being employed to advance our understanding of ischemic tolerance and to discuss the progress which has been gained as a direct result of this work. In addition, this section also explores targets of potential therapeutic value and provides new perspectives relevant to neuronal preservation following various types of injury.

11.2 MODELS OF ISCHEMIC TOLERANCE Much of the earlier work on ischemic tolerance utilized cardiac muscle preparations.7,9–11 In addition to revealing several clues about the sequential changes associated with the development of ischemic tolerance, these studies provided a foundation upon which to relate findings from other parts of the body. Tissue from the kidney, liver, and intestine has been examined, but due to the overlapping interests of many cardiologists and neuroscientists, some of the most extensive comparisons have been drawn between the heart and the brain. As a result, three issues integral to the establishment of ischemic tolerance have emerged. The first considers the minimum length of time that a sublethal, transient ischemic insult must be present in order to promote a tolerant condition. The second focuses on the period of time necessary for the development of ischemic tolerance. This time frame constitutes the interval proceeding the cessation of the priming event and extending to the point of tolerance induction. The last involves the “window” or duration of time that the tolerant state persists. The heart requires approximately 3–10 minutes of preconditioning in order to attenuate the impact of a secondary, more severe ischemic episode. Almost immediately (less than one minute) after the initial insult, cardiac myocytes demonstrate a heightened ability to resist injury. The corresponding “window of tolerance” is shortlived and usually remains effective for a span of only one to three hours. The

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homolog of this process in the brain, however, moves along a more expanded time scale. In general, the time needed to evoke and develop a neurological tolerance ranges from minutes to days. Fortunately, once the tolerance response has been initiated, it can be sustained for nearly a week. The following section will introduce various experimental models and further detail current opinion on the events related to ischemic tolerance in the brain.

11.2.1 IN VIVO GLOBAL AND FOCAL ISCHEMIA MODELS The global ischemia model has been frequently used to study stroke-related degeneration throughout the forebrain. This approach has proven to be particularly beneficial for investigating the phenomenon of ischemic tolerance. Subjects in these experiments, typically Mongolian gerbils, receive a transient, bilateral common carotid occlusion. The incompletely formed circle of Willis, a common trait among these animals, provides for a thorough disruption of the ascending blood supply upon insult by preventing collateral flow. Strategies, which employ mice or rats, usually necessitate a more elaborate procedure in order to simulate a global ischemia. Technically, this translates into a 4-vessel occlusion (2 common carotids; 2 vertebral arteries) or a 2-vessel common carotid obstruction with hemorrhagic hypotension. One of the earliest studies to clarify the time course of events involved in ischemic tolerance was performed by Kitagawa et al.8 and utilized the global ischemia model. This investigation revealed that a brief two-minute, but not one-minute, global insult was sufficient to spare CA1 hippocampal neurons from a subsequent, more severe global assault. The global-global paradigm, as it has come to be known, also demonstrated that tolerance develops over a period of 24 hours and lasts for 1–2 days. These initial findings were later confirmed by Kirino et al.12 within the same model. However, another report13 suggested that the window of protection actually extends from 1–7 days. This study closely paralleled previous investigations, but differed with regard to endpoint. Apparently, longer survival times permitted the temporal boundaries, which define this interval, to be more accurately assessed. Since a majority of patients do not present in the clinic with global ischemia, additional in vivo models have been engineered that more closely mimic the human condition. The middle cerebral artery occlusion model (MCAO) is one such strategy which has been widely accepted for the study of ischemic tolerance. This approach takes into account the focal nature of many ischemic episodes and involves localized manipulation of the cerebral vasculature. As the name implies, the middle cerebral artery is clamped, sutured, or stapled for various lengths of time. Most studies have adopted a modified methodology, which employs basic techniques from the focal as well as the global approach. These modified experimental designs have reaffirmed the beneficial effects of ischemic preconditioning on neuronal survival and have further delineated the temporal characteristics associated with tolerance.14–19 For example, the focal-global ischemia model, which was utilized by Glazier et al.,15 involved the pairing of a brief MCAO with a secondary global ischemia. Although only 24 hours of reperfusion separated these two events, neuronal damage was significantly attenuated in the cortical region which received prior

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exposure. Forebrain nuclei outside of the optimized zone were severely impacted and demonstrated significant cell loss. However, according to the global-focal model,14 which combines a global priming ischemia with a subsequent MCAO, ischemic tolerance requires a period of two days to develop. Yet other studies have provided evidence which links the length of time between insults to the duration of tolerance expression. This time-dependent relationship has been particularly well demonstrated in the focal-focal model. As has been noted previously and has remained consistent with the dual MCAO strategy, an ischemic challenge, which precedes a severe insult, promotes a reduction in cerebral infarct volume.19 Interestingly, when ischemic insults were spaced 30 minutes apart, a window of protection was conferred, but was limited to three days instead of seven.18,20 Despite the modest, but appreciable model-to-model variability in temporal patterning, ischemic tolerance remains a vital process which permits the nervous system to adapt in the midst of ischemic adversity.

11.2.2 THE CORTICAL SPREADING DEPRESSION MODEL Cortical spreading depression (CSD) is a physiological term which denotes a state of neuronal hyperexcitability. Propagating waves of depolarization are accompanied by phases of depressed electrical activity. Ischemic events, such as MCAO, as well as stab wounds, raised extracellular potassium, and strong electrical stimulation can trigger the suppression that is associated with this heightened responsiveness.21–23 An experimental model which precipitates a CSD-like condition was initially proposed by Kawahara et al.24 to examine ischemic tolerance. In this study, KCl was unilaterally injected into the hippocampus and three days later a global ischemia was delivered. Unlike cells in the opposing nucleus, neurons located in the vicinity of treatment were able to resist damage from the ischemic insult. This strategy was later modified by the application of KCl to the cortical mantle. The investigations, which utilized this approach, stipulated the administration of KCl to one hemisphere. Bilateral global25 and focal26 ischemias were inflicted one or three days later, respectively. Using the noninjected side as an internal control, KCl kindling was found to promote neuronal survival. Based upon these findings, it can be inferred that neuronal depolarization contributes to the production of ischemic tolerance. However, inhibitory aspects of CSD may also be responsible for safeguarding neuronal integrity against TIA.

11.2.3 THE 3-NITROPROPRIONIC ACID CHEMICAL PRECONDITIONING MODEL 3-Nitroproprionic acid (3-NPA) is a mitochondrial neurotoxin and an irreversible inhibitor of succinic dehydrogenase. In a recent hippocampal slice study, this compound was found to induce tolerance by inhibiting oxidative phosphorylation.27 Animals, which received an intravenous injection of 20 mg/kg 3-NPA 1–24 hours prior to tissue preparation, exhibited an increased propensity for neuronal survival when subjected to in vitro hypoxia. This protective action has been coined “chemical preconditioning”

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and has been found to peak between 3 and 24 hours postadministration.28 Other research teams have also utilized 3-NPA pretreatments to evaluate ischemic tolerance. However, these studies employed in vivo models of transient global29 or focal30,31 ischemia. A major objective was to obtain additional information regarding optimal dosages and therapeutic time frames. Kuroiwa et al.31 found that exposure to 4 mg/kg of 3-NPA was effective for reducing ischemia-induced deterioration, but 80 mg/kg aggravated the damage. Furthermore, these reports suggested that chemical tolerance persists for a longer period of time in systemic models than in vitro preparations. The benefits of preconditioning were typically maintained over a course of one to four days, and day 3 (posttreatment) often marked the development of maximal protection.29 3-NPA has also been used to examine aspects of ischemic tolerance in smaller populations of neurons. Primary cortical cultures were generated for the studies of Weih et al.32 and the ability of neurons to withstand oxygen-glucose deprivation was assessed. It was determined that two hours of preconditioning with 3-NPA rendered considerable improvement in terms of neuronal viability. This strategy also indicated that chemically induced tolerance develops within 24–48 hours of 3-NPA priming.

11.2.4 OTHER MODELS Ischemic tolerance can also be triggered by other types of toxins as well as by factors which evoke a stress response. For instance, low doses of lipopolysaccharide (LPS), a bacterial endotoxin derived from E. coli., have been shown to reduce infarct size following permanent or transient MCAO.33,34 This substance, which has been suggested to activate inflammatory processes by stimulating the release of cytokines, has been shown to be effective when administered 2–4 days prior to ischemic insult. Diethyldithiocarbamate (1g/kg), an inhibitor of superoxide dismutase, is another agent which has been shown to increase the threshold for injury. This chemical has been shown to promote a state of oxidative stress through the generation of oxygenderived free radicals.38 Likewise, several in vitro studies have introduced either oxygen-glucose deprivation,40,41 hypoxia,42,41 or Na+/K+ ATPase inhibition41 as a means of taxing cells. These investigations utilized culture models to demonstrate that sublethal preconditioning provides significant protection against a subsequent insult of greater magnitude. Prolonged mild hypoperfusion was found to produce similar results.39 In this study a “stroke-resistant” strain of atypical Mongolian gerbils, known to possess a definite anastomosis between the anterior cerebral arteries, received a unilateral common carotid occlusion. Following 30 days of reduced blood flow, a five-minute insult, which was presented to the opposite side, was found to promote negligible damage. Bright light and heat are also among the stressors considered to impart ischemic tolerance. Retinal models, which demonstrate photoreceptor degeneration in response to constant visible light exposure, have been particularly useful for illustrating this point. In one paradigm, rodents were preconditioned with 12–48 hours of white fluorescent light (115–130 candelas). Following a 48-hour “rest phase” and a seven-day secondary exposure of the same intensity, resistance to subsequent light damage was observed.36 An additional study revealed that transient thermal stimuli

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provide a similar type of relief.35 Chopp et. al.37 extended this finding by testing the hypothesis that whole-body hyperthermia protects against cerebral ischemic insult. First, rats were placed on a heating pad and enclosed within a high-humidity container until rectal temperature was elevated to 42°C for fifteen minutes. Twenty-four hours after the shock, a forebrain ischemia was initiated by bilateral carotid artery clamping, bleeding, and trimethaphan camsylate (15 mg/kg) injection. It was observed that damage in the hippocampus, inferior frontal cortex, and dorsal-lateral striatum was significantly less severe in the heated group vs. the nonheated group. To summarize, there is general agreement that many factors influence the expression of ischemic tolerance. The following section will further detail the application of these approaches toward another issue, potential mechanisms of protection. Special emphasis is placed upon neuronal components, intracellular molecules, and signaling cascades which may render brain cells resistant to injury.

11.3.

HEMODYNAMICS

Several studies have examined the possibility that altered blood flow plays a role in the establishment of ischemic tolerance.14,16,17,19 These investigations evaluated whether changes in cortical perfusion occur as a result of initial preconditioning, and were based on the premise that increases in collateral blood flow may compensate for vascular distress. Laser-doppler flowmetry and quantitative [14C]iodoantipyrine autoradiographic analyses were performed on animals which had received focal preconditioning or a sham operation prior to a severe MCAO. No significant differences in regional cerebral blood supply were observed between the two groups following the secondary procedure that could account for priming-induced neuroprotection. Furthermore, these findings paralleled other reports which indicated plasma glucose, blood plasma, and blood pressure are not influenced by preconditioning and do not contribute to the precipitation of tolerance. Thus, local adjustments in circulation can be ruled out as conferring an adaptive advantage during ischemic challenge.

11.4.

GLUTAMATE AND RECEPTORS

It has been well documented that severe ischemic insult promotes excessive release of glutamate and modifies the expression of glutamate receptors.43–45 For instance, Zhang et. al.46 showed that both the levels of protein and mRNA for the NMDA receptor subunits, NR2A and NR2B, were decreased following transient forebrain ischemia. As a result of these and similar studies, a line of research grew that specifically addressed the involvement of glutamate in the generation of tolerance. Of particular interest was the potential for sublethal preconditioning to trigger alterations in secondary postischemia glutamate release. The time course of such changes relative to the period of neuroprotection was considered a matter of equal importance. Since many investigations relied upon the measurement of extracellular glutamate concentrations, microdialysis techniques were extensively utilized. One of the earliest examinations48 compared the effect of ischemic-priming vs. single trial

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manipulation on glutamate levels in gerbil hippocampus following a three-minute severe forebrain ischemia. Pretreatment with a two-minute ischemia 1 hour or 4 days before the secondary insult did not alter the amount of glutamate that was liberated. However, this group49 later conducted a study which called for the application of three two-minute forebrain ischemias by bilateral common carotid occlusion. This report revealed that a sublethal insult does, indeed, impact glutamate emission upon secondary and tertiary exposure. Glutamate concentrations were found to rise after the second and third ischemic insult, but the increases were smaller than those observed during the first procedure. Due to the fact that these repeated forebrain ischemias were only separated by one-hour intervals, i.e., not a sufficient amount of time to permit the development of tolerance, there was minimal opportunity to confer protection and cumulative neuronal damage was sustained. A recent investigation50 did succeed in demonstrating a correlation between extracellular glutamate levels, preconditioning, and the window of tolerance. With respect to sham-operated controls, CSD prior to focal cerebral ischemia elicited a 70% reduction in cortical glutamate output. This decrease also coincided with a downregulation of glial excitatory amino acid transporters, isoforms EAAT1 and EAAT2, at 1, 3, and 7 days postCSD, but not at 0 or 21 days. Thus, it appears that sublethal priming does influence subsequent glutamate efflux, and that conservative release following a secondary, severe insult may contribute to the protection rendered during ischemic tolerance. Glutamate receptor-mediated actions have also been considered in the genesis of tolerance. Although there are several glutamate receptor subtypes, the relationship of the NMDA receptor to neuroprotection has been the most thoroughly studied. As a result, many experimental models have demonstrated that blockade of this receptor subtype disrupts the onset of preconditioning-induced tolerance. This finding has been reproduced on several occasions with different pharmacological agents which antagonize the NMDA receptor. Among the in vivo studies, Kato et. al.47 were the first to report that protection afforded by a 2-minute preconditioning insult was inhibited. In this investigation, 3 mg/kg MK-801 ((+)-5-methyl-10,11-dihydro-5Hdibenzo [a, d] cyclohepten-5, 10-imine maleate), a noncompetitive NMDA receptor antagonist, was administered i.p. one hour prior to the priming insult. Three days later a three-minute global ischemia was inflicted that caused pronounced neuronal loss in the CA1 region of hippocampus. When ketamine was used to survey the role of NMDA receptors in CSD,51 bilateral carotid artery occlusion was found to elicit profound damage within the cortex, hippocampus, and striatum. Tolerance to this secondary forebrain ischemia was prevented 1, 3, and 7 days following the co-administration of ketamine and KCl. Likewise, in a 3-NPA model of chemical preconditioning, neuronal susceptibility to hypoxia was increased by the application of 2-D(-)-2amino-5-phosphonopentanoic acid (APV) to hippocampal slices.52 Animals were primed by in vivo 3-NPA treatment (i.p. injection) 1 and 24 hours prior to tissue preparation. Forty-five minutes before superfusion with 95% N2 and 5% O2, the NMDA receptor antagonist, APV, was introduced. Posthypoxic recovery was assessed by comparing population spike amplitudes against baseline measurements. The response to synaptic activation of Schaeffer collaterals usually exceeded 90%,

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however, after APV treatment neuronal output was significantly compromised. This decline in activity (42%), therefore, signified the absence of tolerance. In vitro studies also support the view that NMDA receptors participate in the installation of tolerance. Marini and Paul53 demonstrated this point using preconditioned cerebellar granule cell cultures. Initial incubation with low concentrations of NMDA or glutamate markedly reduced neuronal degeneration following exposure to toxic concentrations of 1-methyl-4-phenyl-pyridinium ion (MPP+), or glutamate. Administration of MK-801 or APV (a.k.a 2-amino-5-phosphovalerate) 5 minutes prior to priming, however, abolished the neuroprotective effect. Similar tests in a murine cortical cell culture model further reinforced the connection between NMDA receptors and resistance to injury.54 Neurons and glia received brief periods (5–30 minutes) of oxygen-glucose deprivation 24 hours before being subjected to a 45–55 minute severe insult. The neuroprotection, which resulted from preconditioning, was lost if the NMDA antagonist, 3-((D)-2-carboxypiperazin-4-yl)-propyl1-phosphonic acid, D-CPP, was applied during the priming treatment. The benefits of sublethal exposure were also diminished if the time between challenges extended outside of a 7–72 hour tolerance window. Conversely, the activation of non-NMDA receptors has been predominantly associated with detrimental effects.55,56 The in situ hybridization studies of PelligriniGiampietro et. al.57 have provided strong support for this position. These investigators demonstrated that a defining factor in postischemic cell death may be the altered expression of AMPA/kainate glutamate receptor subunits. Their work was based upon two main lines of evidence; first, increased Ca++ influx through glutamate receptor channels contributes to cell mortality and second, heteromeric channels assembled from GluR2 subunits are significantly less permeable to calcium ions than those formed from GluR1/R3 subunits. Therefore, it was postulated that an ischemic event might bias the receptor complement to exaggerate Ca++ entry and promote tissue damage. Following a severe, ten-minute forebrain ischemia, GluR2 mRNA was preferentially reduced in CA1 hippocampal neurons at a time point that preceded neuronal degeneration. This modified pattern of expression was found to coincide with intervals of elevated Ca++, that were later confirmed by individual intracellular recordings and fura-2 optical imaging techniques to be AMPA mediated increases in cytoplasmic free Ca++ levels.58 Interestingly, 2,3-dihydroxy-6-nitro-7-sulfmoylbenzo(f)quinoxaline (NBQX), a relatively selective AMPA receptor antagonist, was found to confer neuroprotection under these circumstances, presumably by directly blocking kainate receptors rather than by restoring GluR2 transcription.59 Additional steps have been taken in an attempt to elucidate a functional connection between AMPA receptors and ischemic tolerance. A 3-NPA model of hippocampal preconditioning was initially utilized to determine if the AMPA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), adversely impacted the formation of ischemic tolerance.52 As mentioned previously, population spike amplitudes were recorded from in vitro tissue samples before and after exposure to hypoxic conditions. Following treatment with 10 uM CNQX, activity levels recovered to 72 ± 15% of baseline measurements. Simply interpreted, these results suggested that non-NMDA receptors do not affect ischemic tolerance. Grabb and Choi54

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directly approached this issue by assessing the ability of kainate to precondition against subsequent oxygen-glucose deprivation in murine cortical cultures. Sublethal priming with concentrations of 10, 30, and 45 uM 24 hours prior to insult, not only failed to produce a protective effect, but actually enhanced the death induced by severe insult. As demonstrated in a complementary RT-PCR study,60 changes in the relative proportion of AMPA subunits may have contributed to increased neuronal vulnerability. Rat hippocampal neurons were challenged with sublethal oxygen-glucose deprivation and mRNA was extracted from individual cells 24–48 hours later. Following amplification, levels of GluR-D flop were found to be upregulated in preconditioned neurons. These cultures also exhibited augmented AMPA-/kainate-induced 45Ca++ accumulation, which could be blocked by NBQX and Joro spider toxin, as well as an increased propensity for kainate-triggered cell loss. In short, AMPA receptors do not appear to participate in the cellular processes which govern neuroprotection via ischemic tolerance. These gated channels are, however, highly correlated with excitotoxic events which aggravate neuronal homeostasis and render neurons susceptible to injury. Unlike their AMPA-related ionotropic counterparts, metabotropic glutamate receptors (mGluRs) possess neuroprotective capabilities.61,62 Of the three major subdivisions, groups II (mGluR2, mGluR3) and III (mGluR4, mGluR6, mGluR7, and mGluR8) are primarily responsible for imparting resistance against acute or chronic neurodegeneration. Group I subtypes (mGluR1, mGluR5), on the other hand, largely exert a facilitatory effect on pathological processes. Recently, both in vivo and in vitro models have been utilized to characterize the permissive or inhibitory nature of specific mGluR subtypes under excitotoxic conditions. For example, Bruno et al.64 used mixed cortical cultures from wild-type and subtype-deficient mice to demonstrate the importance of mGluR4 in maintaining neuronal viability. It was observed that the group III agonists, L-2-amino-4-phosphonobutyrate (L-AP-4), L-serine-Ophosphate (L-SOP), and (R,S)-4-phosphonophenylglycine[(R,S)]-PPG, were able to provide protection against a 100-uM pulse of NMDA in wild-type and heterozygous preparations. However, neurons from knock-outs tended to succumb to the effects of NMDA toxicity. Likewise, when low doses (10 nmol) of (R,S)-PPG were co-infused with NMDA into the caudate nucleus of wild-type mice, neuronal death was substantially reduced. Genetically manipulated animals did not exhibit limitations on lesion size with agonist administration; instead neuronal damage paralleled that produced by NMDA administration alone. Another study63 revealed that the mGluR5 receptor subtype might also represent a suitable target for therapeutic intervention. Based on the premise that endogenous activation of mGluR5 contributes to neuronal degeneration, this investigation differed from the former in the utilization of potent and selective noncompetitive antagonists to elicit neuroprotection. 2-methyl-6phenylethynylpyridine (MPEP), [6-phenyl-2-(phenylazo)-3-pyridinol] (SIB-1757), and [(E)-2-methyl-6-(2-phenylethenyl)pyridine] (SIB-1893), were all found to bestow a survival advantage upon NMDA insult. Due to the apparent life-sustaining properties of certain mGluRs, it is conceivable that these receptors fulfill an integral role in the development of ischemic tolerance. Currently, only a few reports are available which discuss this topic, and of those

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that do the functional implications of mGluR involvement remain unclear. Sommer et al.15 approached this issue by using gel electrophoresis and immunoblotting techniques to examine the postischemic protein expression of subtypes 1b, 2, 3, and 5. Three groups of gerbils with reperfusion intervals of 8, 24, and 48 hours and 4 days were examined: the first was subjected to a five-minute ischemia, the second was exposed to a 2.5-minute sublethal insult, and the third received a 2.5-minute priming challenge followed by a five-minute bilateral occlusion of the common carotid artery four days later. The major finding was that preconditioned animals demonstrated a transient reduction in the expression of mGluR1b and mGluR5 at 8 hours. These results correspond well with the notion that downregulation of harmful mGluRs lends to the survival of vulnerable CA1 neurons during ischemic tolerance. However, no significant changes were detected in the pattern or intensity of mGluR1b and mGluR5 immunoreactivity after a single 2.5-minute ischemic insult. As the authors indicate, this piece of evidence calls into question the likelihood that an altered supply of mGluRs contributes to the induction of ischemic tolerance. In fact, the similar, yet dramatic, attenuation of the mGluR1b and mGluR5 receptor subpopulations after a single 5-minute (death-producing) occlusion, further argued against a decline in mGluR availability as a mechanism for invoking ischemic tolerance. Moreover, when mGluRs were preconditioned with 100–200uM of the nontoxic, broad-spectrum agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) 24 hours prior to oxygen-glucose deprivation, murine cortical cultures failed to demonstrate any obvious signs of neuroprotection.54 Taken together, this limited amount of data suggests that mGluRs do not directly promote the establishment of a tolerant state. The potential ancillary involvement of these G-protein coupled receptors in fostering ischemic tolerance, nonetheless, warrants further study.

11.5.

STRESS PROTEINS

Stress proteins are a group of highly conserved proteins that are thought to play a role in facilitating neuronal survival during and after stress. They are considered to work in association with other proteins — controlling protein conformation, stabilization, or transport to specific loci in the cell. Of the several size classes, those referred to as the hsp70 family (~70,000 kD or 70 K) are the most well described.66,67 In most mammalian cells there are two prominent forms, an abundant constitutive member (hsp73) and a highly stress-inducible member (hsp72).68 These cytosolic proteins are known to be synthesized (or activated) in response to noxious agents or harsh environmental conditions, such as inhibition of energy metabolism, D-lysergic acid diethylamide (LSD),69,70 sodium arsenite,71 surgical cutting,72 and concussive injury.73 However, temperature elevation has been used most frequently to profile the characteristics of these “heat shock” proteins (HSPs). For example, mild hyperthermia has been shown to promote the rapid sequestration of hsp70 into the nucleus. Studies indicate that high levels of this protein accumulate near the nucleolus and assist in the assembly of small ribonucleoproteins as well as preribosomes.74,75 Perhaps the most intriguing concept to emerge from heat shock investigations is the phenomenon

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referred to as acquired thermotolerance; whereby exposure of cells to a mild heat challenge greatly enhances cell survival after a subsequent, and what would otherwise be a lethal, elevation in temperature.76 Since the discovery of acquired thermotolerance, a number of examinations have shown a strong correlation between the expression/decay of thermal resistance and the induction/degradation of HSPs.77–79 Several investigations have also revealed that the acquisition of thermotolerance is precluded by the inability to produce functional HSPs.80 One such study reinforced this point by demonstrating that intact hsp70 is required in order to promote cell survival against a thermal stress.81 Rat embryo fibroblasts were impregnated with affinity-purified monoclonal antibodies to hsp70 and subjected to a 30-minute heat shock. Immunotreatment was found to impair the heat-induced translocation of hsp70 proteins into the nucleus, and compromise the ability of injected cells to withstand a brief incubation of 45°C. In agreement with these results, Johnston and Kucey82 found that competitive inhibition of hsp70 gene expression caused thermosensitivity in the Chinese hamster ovary cell line. In vivo examinations within the nervous system have significantly broadened our knowledge of the relationship between thermotolerance and the recruitment of HSPs. These investigations have also indicated that exposure to a brief hyperthermia can fortify neurons against damage from stress-inducing factors other than heat shock. Barbe et al.35 hypothesized that a mild increase in body temperature would protect retinal photoreceptors from bright light triggered injury. To test this notion, rats were placed in a heat stress chamber, which circulated air at 41–42°C, until rectal temperatures had acclimated to surround conditions for 15 minutes. Following intervals of 0, 2, 4, 10, 18, 25, and 50 hours, animals were exposed to a light source of 2690 lux for 24 hours at 30°C. Rodents which received bright light stimulation within 18 hours of hypothermia were completely spared from damage, while the protein had significantly waned for those that were subjected to visual distress at 50 hours postheat shock. Concomitant with heat-induced resistance was the increased synthesis of 3 HSPs. This was revealed in part by intraocular injections of [35S]-methionine, which were administered shortly proceeding thermal treatment, and onedimensional gel electrophoresis. HSP content was found to peak 18 hours after heat stress, suggesting that these proteins are produced in order to promote tolerance against damage from intense illumination. Along similar lines, the principle of thermal preconditioning has been successfully applied to models of ischemia.37 Although levels of protein synthesis were not evaluated in this particular study, whole-body hyperthermia was found to considerably minimize the detrimental effects of carotid artery occlusion. Ischemia has also been implicated among the stress-evoking events that initiate the production of HSPs. The ischemia-induced changes that occur in the level of stress protein synthesis over time was first described by Nowak.83 In this study gerbils received a transient five-minute bilateral carotid artery clamp and were sacrificed thereafter at various intervals between 2 and 24 hours. The cerebral hemispheres, including the basal ganglia and hippocampi, were removed and prepared for two-dimensional gel electrophoresis. As determined by increased incorporation of radioactively labeled amino acids, an in vitro translation product of

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approximately 70K was upregulated within 2 hours of recirculation. Expression reached a maximum at 8 hours, and continued to be slightly elevated at 24 hours postinsult. Later it was revealed that the hsp70 response to ischemic intervention could be induced in glia and endothelial cells, as well as neurons.84–87 The factor that seems to govern the morphological localization of increased stress protein production is the cell’s proximity to an infarct. Inside a lesion, hsp70 is expressed mainly in blood vessels and sometimes in scattered microglia and astrocytes. Along the margins of injury, this protein is concentrated in glia, but becomes more prevalent within neurons outside the area of damage. Kirino et al.12 extended these findings and demonstrated that the induction of the stress response promotes ischemic tolerance. This study was based upon two major pieces of evidence, (i) brief ischemia can invoke the production of hsp70 and (ii) hippocampal CA1 neurons that are slated to die exhibit minimal hsp70 accumulation.88 It was surmised that ischemic damage is the result of a failed stress response and upregulation of stress proteins may make selectively vulnerable neurons more tolerant to an ischemic attack. To test this notion, male Mongolian gerbils received a twominute sublethal ischemia by bilateral carotid artery occlusion in order to precipitate the stress response. One, two, or four days later animals were subjected either to a five-minute severe insult or were sacrificed for histological processing. Immunostaining with a monoclonal antibody, which specifically recognizes stressinducible forms of the hsp70 family, revealed an increase in a 70-kDa heat shock protein within the CA1 sub-field following a two-minute ischemia. Coupled with higher neuronal density measurements in preconditioned animals (40–70% survival) than nonconditioned controls, these data suggested that brief ischemia elicits the synthesis of heat shock proteins and renders neurons more resistant to subsequent metabolic stress. However, some discrepancies were observed in the temporal pattern of hsp70 labeling that did not correlate well with the sequential progression of induced tolerance. For example, the limited amount of hsp70 immunoreactivity in CA1 one day after a two-minute brief insult did not appear to correspond with the moderate preservation that was detected following a second ischemic episode. In addition, neuronal protection, which was more evident 4 days subsequent to a conditioning challenge than at 2 days poststress, could not be explained on the basis of hsp70 staining, as equally intense levels were noted at each time point. A regional disparity in hsp70 expression was also discerned between CA3, dentate gyrus, and CA1, at different time intervals proceeding a two-minute sublethal challenge. Although the reason for this spatial variation is unclear, hsp70 was robustly upregulated in CA3 and dentate gyrus within 24 hours, while substantial increases in CA1 hsp70 content were not manifested until 2 through 4 days postinsult. Despite these incongruities, numerous reports have cited an enhanced production of hsp70 in association with ischemic tolerance.15,17,47,89,90,91 From these immunohistochemical, Western blot, and in situ hybridization studies, it is apparent that the time course of synthesis closely parallels the window of tolerance-evoked neuroprotection. The temporal profile of hsp70 induction was found to extend roughly seven days from an initial priming insult, with postischemia day 1 marking the earliest accumulations and day 3 reflecting maximal yields. By further tracking the expression of hsp70 beyond a secondary,

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severe ischemia, Liu et al.90 not only reinforced the argument in favor of the protective properties of hsp70, but underscored the correlation between advanced induction of hsp70 synthesis and neuronal survival after severe ischemic insult. Two groups of animals received common carotid artery occlusions; the first group was subjected to a three-minute preconditioning challenge three days prior to a sixminute insult, whereas the second series of animals was exposed exclusively to the latter treatment. Immunohistochemical comparisons between the two groups of animals revealed that the most noticeable difference in hsp70 staining occurred almost immediately (2 h) after severe ischemia. Tissue that was taken from prestressed animals exhibited appreciable levels of hsp70 expression, while samples through the CA1 region of non-conditioned specimens did not demonstrate a remarkable degree of hsp70 labeling until 1 and 3 days following lethal occlusion. The intense pattern of staining, which both sets of animals developed, persisted throughout the 24–72 hour time period following severe insult and decayed upon day 7 when hsp70 production in nonconditioned rats declined as a result of neuronal deterioration. Given the residual expression of hsp70 from a three-minute ischemic challenge and the delayed synthesis of hsp70 in ill-fated neurons following a six-minute severe insult, it is reasonable to suspect that neuronal death may not stem from a failed stress response, but from the absence of early compensatory increases in heat shock protein levels. The protective role of hsp70 in ischemic tolerance has also been examined with the use of other in vivo models. The canine aortic cross-clamp preparation is one such model that was introduced initially out of a need to understand ways of safeguarding the functional integrity of spinal cord neurons during operative repair of thoracoabdominal and thoracic aneurysms. Since the invasive procedures to correct this condition often lower perfusion pressure and lead to a 3.8%–17.6% incidence of paraplegia, an effort was directed toward evaluating the defensive capabilities of spinal cord neurons. Matsuyama et al.92 pursued this issue by determining whether the benefits of ischemic preconditioning apply to the spinal cord, and elucidating potential mechanisms that may impart resistance to ischemia-induced paraplegia. To meet these aims blood flow through the descending aorta was interrupted distal to the left subclavian artery in twelve adult beagles for 60 minutes. Forty-eight hours prior to this extended surgical procedure, half of the animals were preconditioned by cross-clamping the aorta in a similar manner for 20 minutes. Three of six control animals, which were not treated with a priming exposure of ischemia, received a Tarlov score of 5, i.e., they exhibited paralysis. All of the dogs in the experimental group, however, demonstrated either a complete recovery (n = 5) or an irregular gait (n = 1). Upon immunohistochemical analysis, hsp70 was found to be upregulated in each of the subjects that displayed signs of mobility. Additional assessments of hsp70 staining after a single episode of preconditioning confirmed that hsp70 expression 48 hours postinsult coincided with improved neurological outcome following severe ischemia. These findings indicated that HSPs are involved in the acquisition of ischemic tolerance, and in terms of the spinal cord suggested that HSPs may provide a target in the future for the rescue of endangered motoneurons or an avenue for ongoing investigations into the prevention of ischemia-induced injury. In agreement with this view are studies, which have utilized hsp70 overexpressing transgenic

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(hsp70tg) mice to examine the protective role of hsp70.93,94 By genetically manipulating the production of hsp70, researchers were able to more directly assess whether the beneficial effects of sublethal insult are attributable to hsp70 synthesis alone, or due to other metabolic changes associated with preconditioning. Briefly, ischemiainduced injury was compared between wild-type littermates and HSP70tg mice, which demonstrated high levels of hsp70 mRNA as well as protein under normal conditions.94 Damage to both groups was inflicted by unilateral, permanent MCAO via intraluminal blockade with 5-0 nylon monofilament suture. Cerebral infarction volume 6 and 24 hours postinsult was evaluated by Nissl staining and found to be significantly reduced in the population with selectively altered expression of hsp70. Taken together with previous findings, these data invite speculation about a novel, neuron-sustaining approach that could one day permit the regulation of hsp70 either during or in anticipation of a circulatory distress. In vitro cell culture studies have complemented in vivo examinations by offering strong evidence in support of HSP-mediated tolerance against glutamate toxicity95,96 and oxygen-glucose deprivation.97 Lowenstein et al.95 demonstrated that cerebellar granule cells preconditioned with mild heat shock exhibit a stress protein response and preferentially withstand glutamate exposure over a wide range of concentrations. Neuronal cultures were heated to 42.5°C for 1.5 hours and returned to 37°C for 14 hours after treatment. Preparations were bathed in 0–1000 uM solutions of glutamate for 6 hours and assessed for tissue viability 18 hours thereafter by trypan blue exclusion or lactate dehydrogenase (LDH) release. In contrast to nonheated controls, the induction of hsp72 was identified in preconditioned samples by immunoblot techniques and correlated with a substantial reduction in glutamate-evoked toxicity. Similarly, the heat shock response was also shown by Rordorf et al.96 to have a protective effect against excitotoxicity in cortical cultures. Colonies that were heated to 42.2°C for 20 minutes and returned to a 37°C environment displayed an increase in the synthesis of ~72 and ~85 kD proteins as well as levels of hsp70 mRNA. A corresponding resistance to excitotoxic death was clearly demonstrated by the exposure of these samples to 50–125 uM glutamate 3 and 24 hours subsequent to thermal stress. Specimens were subjected to 10 minutes of glutamate treatment and assayed 24 hours later by (i) comparing the number of nonpyknotic neurons in identical fields pre- and postadministration and (ii) measuring LDH leakage into the medium. When the interval between heat challenge and glutamate insult was shortened to 15 minutes or lengthened to 48 hours, the extent of damage exhibited by heat-primed plates paralleled that observed in nonheated sister cultures. The link between HSP induction and cell survival was further depicted in studies which utilized astrocyte cultures prepared from Swiss Webster mice.97 Upon infection with a retroviral vector encoding the human hsp70 gene, cells were found to overexpress hsp70 in quantities that were comparable to those induced by a priming heat treatment. Along with the predisposition for hsp70 synthesis came the ability for these cells to overcome 4–5.5 h incubations at 43.5°C. In fact, surveys conducted after 24 hours of normothermia revealed that hsp70-expressing cultures were twice as likely as controls to sustain heat stress. Likewise, hsp70 production was correlated with protection against 7 hours of oxygen-glucose deprivation. HSP70 overexpressing astrocytes demonstrated a 12%

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mortality rate, while normal and β-galactosidase positive controls suffered cell losses of 82% and 65%, respectively. Other HSPs, such as hsp27, have also been implicated in the process which promotes ischemic tolerance. A role for hsp27 in conferring resistance to injury has been justified primarily by the overlap between hsp27 expression and the window of protection that follows sublethal ischemia. Kato et al.98 found that by preconditioning with bilateral common carotid artery occlusion for 3 minutes, hsp27 content was elevated 1–7 days postinsult and peak levels of immunostaining were apparent on day 3. Labeling differed, however, in comparison to hsp70 on several counts. First, hsp70 was detected predominantly within neurons, while hsp27 tended to be sequestered in glia. Second, hsp70-positive cell bodies were selectively expressed in CA1, while hsp27-containing profiles were distributed throughout all regions of the hippocampus. Additional disparities became evident following treatment paradigms which adopted six-minute exposures to ischemia. Although both HSPs were upregulated 1 day postinsult and the territory of neuronal hsp70 expression expanded to include CA1, CA3 as well as the dentate hilus, hsp70 was only weakly identified in glia of nonconditioned Wistar rats at 7 days postinsult. HSP27 labeling, on the other hand, was exhibited by a large number of hippocampal neurons with some glial expression at day 1 postinsult, but dramatically shifted toward an astrocytic bias 3 and 7 days subsequent to severe ischemia. Cases preconditioned with a three-minute challenge 3 days prior to lethal insult did not reflect this latter pattern of intense hsp27 staining nor the associated pyramidal cell loss. Instead, the trailing phase of the tolerance window in these animals corresponded with an attenuated level of hsp27-immunoreactivity in scattered glia. The temporal correlation between the postischemia induction of hsp27, hsp70, and tolerance led to the conclusion that hsp27 may work in concert with hsp70 to enhance survival against secondary, severe ischemic injury. The discrepancies that were observed in the regional expression and cellular localization of these HSPs, however, indicated a dichotomy in the actions performed by each stress protein. As such, hsp27 has been suggested to serve a complementary role to that of hsp70, and exert an indirect influence on neuronal protection. A similar interpretation was drawn from the results of Currie et al.99 Male spontaneously hypertensive rats were preconditioned with a ten-minute focal occlusion by placing the bent tip of a platinum-irridium wire under the middle cerebral artery and elevating the vessel 0.5 mm from the cortical surface. Following recovery periods of 6 hours to 4 weeks animals were sacrificed for Northern analysis or immunohistochemical processing. Glial fibrillary acidic protein (GFAP) and hsp27 staining confirmed that a remarkable astrogliosis with increased expression of hsp27 developed 1–7 days after challenge and persisted in reduced quantities 4 weeks postinsult. Consistent with the broad distribution of hsp27 labeling that was observed previously in the hippocampus,98 hsp27-containing astrocytes were detected well beyond MCA perfusion territories. HSP70, on the contrary, tended to be found in neurons and was concentrated within the preconditioned target area 1–5 days postinsult. Short-term expression within the preconditioned zone of cortex suggested direct induction of hsp70 in response to a sublethal ischemic stimulus, while elevations in glial hsp27 content outside the domain of the blocked MCA indicated hsp27 regulation through

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an alternative means. The glial hsp27 stress response is likely to sustain the support functions of astrocytes during a severe ischemic episode and potentially influences the development of ischemic tolerance via the preservation of neuron-glia interactions. With this in mind, it is reasonable to suspect that the prolonged activation of hsp27-positive astrocytes contributes to the remodeling and plasticity which accompanies a gradual poststroke recovery. Although prior exposure to a stressful, benign ischemia has been demonstrated time and again to improve neuronal survival following severe circulatory compromise, tolerance-induced protection does not always correlate with an upregulation of HSPs. Using the CSD model, Kobayashi et. al.25 reported that preconditioning failed to elicit the expression of hsp72 mRNA in cortical areas primed to resist injury. Indeed, 6 days subsequent to a six-minute bilateral forebrain ischemia neuronal necrosis was found to be significantly reduced, despite the fact that hsp72 message was restricted to the site of KCl application. The two-hour administration of KCl was found, however, to elicit transient, negative deflections in DC potential and promote a rapid, widespread transcription of c-fos mRNA. The c-fos signal, which was prevalent upon termination of KCl-treatment as well as 2 hours postdelivery, had degraded within 24 hours (the time point designated for introduction of ischemic challenge). These findings suggested that transcription factors encoded by immediate early genes may mediate the expression of endogenous neuroprotective agents and lead to the induction of ischemic tolerance. At the very least, the results of Kobayashi et al.25 indicate that the CSD model is not appropriate for evaluating the role of stress proteins in providing resistance to secondary injury. On a similar note, studies which have examined the effects of ischemic preconditioning on brain edema and bloodbrain barrier permeability have also called into question the function of heat shock proteins and their capacity to provide protection against severe ischemic insult. Stress-induced disruptions of the cerebrovasculature by laser100 or ischemia-reperfusion injury101 have been reported to elevate the production of hsp70 in endothelial cells. However, Masada et al.102 found that cortical preconditioning with a transient, 15-minute MCAO did not evoke the production of hsp70 in blood vessels 3 days postinsult, and in comparison to sham-operated controls resulted in a significant reduction of hsp70-immunoreactive labeling following secondary permanent occlusion. Non-challenged animals demonstrated intense hsp70 staining 3, 6, and 24 hours subsequent to MCAO, and exhibited a greater degree of neurological deficit, infarction volume, edema formation, and blood–brain barrier disruption than their primed counterparts. These findings suggested that (i) prior exposure to ischemia can minimize the amount of damage sustained by the cerebrovasculature after MCA block and (ii) increased levels of hsp70 denote cell stress. While this study did not directly address the issue of hsp70-mediated protection, the lack of expression following a brief interruption of the ascending blood supply appears to speak against the involvement of hsp70 in ischemic tolerance. Clinically, however, the early onset stress response that was observed 3 hours after permanent MCAO does indicate the beginning of endothelial cell dysfunction. The utilization of tissue plasminogen activator (t-PA), a thrombolytic drug and currently the only medication available to

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treat stroke, under these circumstances should be limited so as to avoid compounding injury by triggering the development of brain hemorrhage.

11.6

PROTEIN SYNTHESIS

Although the biosynthetic activity of some neurons is putatively compromised subsequent to bilateral common carotid artery occlusion,103 increases in protein synthesis generally accompany ischemic insult. In fact, exposure to extreme ischemia often induces the production of several proteins prior to cell death. Kiessling et. al.,104 for instance, demonstrated the upregulation of ~27,000, ~50,000, ~65,000, ~70,000, and ~110,000 kD proteins following a 30–minute transient ischemia, while Lowenstein et al.105 described the enhanced expression of mRNA for calbindin-D28K, a 78 kD glucose-regulated protein (grp78), as well as grp94. Associated with this trend has been the correlation of de novo protein synthesis with ischemic priming and tolerance. In the heart, protein synthesis as early as 60 minutes after sublethal insult106 was found to coincide with a 24–48 hour delayed myocardial protection,107–109 and in the CNS the beneficial effects of neuronal preconditioning were shown to decline with the decreased production of new protein. Barone et al.19 first revealed the dependence of brain tolerance on increased protein assembly, and utilized cyclohexamide (CHX), a protein synthesis inhibitor, in conjunction with a regimen of focal ischemia to establish this relationship. Animals received a temporary (10-minute) MCAO which was followed one day later by a 24-hour permanent occlusion. These spontaneously hypertensive rats routinely demonstrated minimal neurological deficit prior to sacrifice, and modest infarct sizes postmortem. CHX, however, blocked the protective effects of preconditioning on injury and motor function when administered 30 minutes before priming, but when given 30 minutes prior to permanent MCAO did not compromise neuronal survival. Others have also concluded that neosynthesis of protein and RNA is required for the expression of tolerance. Wiegand et al.30 found that preconditioning with a single intraperitoneal injection of 3-NPA 3 days before transient (90-minute) or permanent occlusion of the common carotid and middle cerebral arteries decreased lesion size by 35% and 70%, respectively. Administration of CHX 30 minutes prior to 3-NPA treatment prevented the induction of neuroprotection and resulted in infarct volumes four days following focal ischemia that were identical to nonconditioned controls. Similarly, the irreversible RNA synthesis inhibitor, actinomyosin D, has been shown in vitro to inhibit heat shock- and NMDA-evoked resistance against glutamate toxicity.53,96 Cultured cerebellar and cortical neurons were incubated with actinomyosin D or CHX for a period which extended 30 minutes to 2 hours prior to the presentation of a conditioning stimulus 60 minutes to 3 hours postchallenge. The increased susceptibility of neuronal cultures to secondary insult with high concentrations of glutamate suggested that tolerance is mediated through the initiation of protein synthesis and that this neuroprotective mechanism can be disabled at the level of transcription or translation. Some evidence, however, does oppose the view that protein synthesis is involved with the development of ischemic tolerance. The findings of Kato et al.47 especially

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refute this notion. Male Mongolian gerbils were treated with 50 mg/kg anisomyosin, a protein synthesis inhibitor, 1 hour before and once daily for 2 days after receiving 2 minutes of preconditioning via bilateral common carotid artery occlusion. Three days following ischemic challenge a three-minute secondary insult was inflicted, and at 7 days of survival animals were sacrificed. Neuronal density, which was assessed by hematoxylin-eosin and cresyl violet staining, remained relatively unchanged across groups of normal controls, preconditioned subjects as well as anisomyosintreated individuals. Taken at face value, this data indicated that anisomyosin failed to inhibit the induction of ischemic tolerance. It was, therefore, concluded that ischemic priming enhances the survival capabilities of neurons through a means other than the production of new protein. However, as stated by the authors, the data set is always open to interpretation and often other factors must be considered. In this particular case, tolerance may have developed in the presence of a protein synthesis inhibitor for several reasons. One explanation includes the possibility that anisomyosin evoked a stress response of its own. Such a reaction could have provided protection against further metabolic stress. Second, the drug treatment protocol may have been insufficient to completely inhibit the production of protective proteins. Third, anisomyosin may not be an ideal compound for blocking the synthesis of proteins which provide the most protection against severe ischemic insult. Alternatively, anisomyosin may have inhibited the synthesis of putative killer proteins.110 This prospect is supported by the findings of Goto et al.111 Rats were exposed to a tenminute lethal global ischemia, which typically elicited massive neuronal degeneration in the CA1 subregion of hippocampus. Shortly following insult, animals were treated subcutaneously with CHX, either through consecutive administrations or in a single injection. At 72 hours postischemia when neuronal density was examined, the animals that received CHX exhibited more neuronal preservation than salinetreated controls. Those subjects given only one dose of CHX demonstrated peak effects when CHX was administered 12 hours postchallenge, as opposed to 24 hours or immediately following insult. Such benefits persisted as long as 168 hours after occlusion with some decrement in the overall percentage of surviving neurons. Taken together with previous findings, these studies suggest that ischemic insult can trigger the production of detrimental as well as protective proteins. This premise may help to account for discrepancies between different investigations which have utilized protein synthesis inhibitors to reveal the mechanism of ischemic tolerance after preconditioning.

11.7 BCL-2 RELATED GENE EXPRESSION The Bcl-2 family is a group of proteins which are thought to play an important role in determining cell survival after ischemia. It is their involvement in the regulation of apoptosis (programmed cell death), tissue homeostasis, as well as providing resistance against pathogenic insult that initially led investigators to examine the expression of these protooncogenes relative to ischemia induced neuronal injury. Thus far, at least 15 Bcl-2 family members have been identified in mammalian cells.

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Among them, bax, bad, and bcl-XS (short) have been shown to promote apoptosis, whereas, bcl-2 and bcl-XL (long) have been found to antagonize the death process. Bcl-2 has been demonstrated to sustain life by blocking the translocation of cytochrome c from the mitochondria, thereby preventing the activation of caspase.112–117 Protective effects presumably produced by this action have been observed in a number of different cell lines including, rat pheochromocytoma PC12 cells,118 sympathetic neurons from the rat superior cervical ganglia,119 and conditionally immortalized nigral cells.120 More specifically, microinjection of retroviral expression vectors containing bcl-2 cDNA into the nuclei of cultured neurons, inhibited cell death from glucose-deprivation, growth factor withdrawal, membrane peroxidation, Ca++ ionophore exposure, and in some cases free-radical formation. The ability of bcl-2 transformants to overcome such a wide range of adverse conditions without exhibiting signs of DNA degradation has suggested the potential for bcl-2 to interfere with a final common pathway which leads to necrotic as well as apoptotic cell death. In studies, which were undertaken to evaluate the bcl-2 response to ischemia, bcl-2 upregulation was shown to correlate with the acquisition of tolerance. Shimizaki et al.121 were among the first to demonstrate this relationship by utilizing a gerbil model of global ischemia. Following a two-minute bilateral carotid artery occlusion, which was capable of inducing tolerance to a subsequent five-minute ischemia and preventing delayed neuronal death, intense bcl-2 immunoreactivity was noted in the CA1 area of hippocampus. Increased expression was detected at 30 hours and persisted for 4 days post-insult. Enhanced bcl-2 labeling was not observed, however, after one-minute challenges, which were ineffective at evoking pyramidal neuron protection against secondary injury. Elevated levels of cytosolic cytochrome c were further found by Nakatsu et al.122 to accompany cell loss at day 7 in nontolerant animals following 5 minutes of forebrain ischemia. The release of cytochrome c could be suppressed by ischemic preconditioning, but as immunoblot and immunostain records indicated such reductions often corresponded with the augmented expression of bcl-2. A protective role for bcl-2 in ischemic tolerance is also supported indirectly by the studies of Chen et al.123 This investigation revealed that sublethally injured neurons preferentially express bcl-2. Twenty four hours prior to Western blot and immunohistochemical analysis, rats were exposed to a 60or 120-minute temporary focal ischemia. In response to the shorter interval of MCAO, bcl-2 was induced in a large number of neurons within the frontoparietal cortex, a region that was ischemic but spared from infarction. Longer durations of ischemia, however, elicited neuronal degeneration within this territory. Interestingly, bcl-2 expression was limited to a few surviving neurons, which were scattered along the border of the lesioned area. Within the core of the affected zone, where pyknotic changes were the most prevalent, the only elements to demonstrate bcl-2 immunoreactivity were endothelial cells and vessel walls. In accordance with previous reports, evidence from our laboratory points to the involvement of bcl-2 in ischemia-induced tolerance. We observed an enhanced expression of the bcl-2 oncoprotein in gerbil CA1 hippocampal neurons following two minutes of either unilateral or bilateral common carotid artery occlusion. Although increased labeling was detected within the first 24–48 hours, the most dramatic

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FIGURE 11.1 Photomicrographs show the expression of bcl-2 in gerbil CA1 pyramidal cells after a 2-min sublethal, bilateral forebrain ischemia. Sections were immunohistochemically processed using the ABC method or the fluorescent markers, FITC and Texas-Red. (A) No labeling was detected in normal controls. (B) Small arrows point to numerous CA1 neurons which are bcl-2 positive following 4 d of survival. (C) Bcl-2 staining, as demonstrated with FITC, is reduced in the CA1 region at 10 d postinsult. Note large arrows that depict the nearly smooth appearance of Texas-red linked MAP-2.

upregulation was noted 3–4 days postinsult (Figure 11.1). This response appeared to be transient in nature, since the signal for bcl-2 was not obvious 10–14 days after sublethal challenge. Taking into consideration the short term recruitment of bcl-2 and its temporal overlap with the window of tolerance, it seems likely that bcl-2 contributes to tolerance-related neuroprotection. Data obtained after 3-NPA preconditioning not only supports this view, but further suggests that a shift in the balance between anti- and proapoptotic proteins provides a possible means to resist ischemic injury.124 Using a sensitive real-time PCR technique to quantitate mRNA, and immunohistochemical methods to evaluate protein translation, levels of bcl-2 were found to be increased within hours after chemical priming. This upregulation reached statistical significance at 24 hours postadministration, and was also observed during early recovery from a 15-minute global ischemia that was imposed 1 day subsequent to 3-NPA exposure. A corresponding decrease in the transcription of bax following combined treatment with 3-NPA and ischemic insult resulted in the elevation of the bcl-2:bax ratio. The differential influence of these gene products on neuron survival became particularly evident when expression patterns were examined in drug-free rats after severe carotid artery occlusion. Hippocampal neurons that were able to maintain their viability preferentially demonstrated pronounced bcl-2 immunoreactivity at 4 days postinsult, while those that showed signs of morphological deterioration consistent with ischemic cell death were markedly bax-positive. Other models have also proven endogenous bcl-2 to be a major determinant of neuronal survival following ischemic insult. For instance, Martinou et al.125

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generated lines of transgenic mice that overexpressed the human bcl-2 gene. Increased production of bcl-2 was found to reduce neuronal loss during the period of naturally occurring cell death, as well as confer resistance against MCAO injury. Actually, the volume of brain infarction in genetically manipulated animals was 50% smaller than that observed in wild-type controls. In complementary studies, antisense oligodeoxynucleotides (ODNs) were infused into the lateral ventricle for 72 hours following 60 minutes of MCAO in order to block the protective effects of endogenous bcl-2.126 Compared to vehicle-treated controls, animals subjected to the antisense strategy displayed larger infarction areas and volumes after ischemia. The apparent increase in the distribution of shrunken, TUNEL (see Section 11.9) stained nuclei indicated neuronal degeneration by an apoptotic process. Interestingly, this territory in the ventral cortical mantle and medial striatum was devoid of bcl-2 labeling after antisense administration, but contained many bcl-2 positive neurons in control brains. Utilization of antisense technology in a similar manner after episodes of preconditioning, has permitted the issue of bcl-2 as an effector in ischemic tolerance to be further addressed.127 Normally in the rat, 60 minutes of transient ischemia to the middle cerebral artery results in infarction of the caudate-putamen. Priming with 20 minutes of focal ischemia, however, attenuated the amount of damage which was sustained when a secondary severe insult was inflicted within 3, 5, or 7 days of the first. The same brief occlusion was also able to elicit bcl-2 reactivity within the region that was spared from injury. However, infusion of antisense ODNs during the 72 hour interval between sublethal challenge and secondary ischemia resulted in reduced expression of bcl-2 in the striatum and the loss of tolerance induction. Infarction volumes were comparable to those found in nonconditioned controls, which had been subjected to sham-operation prior to ventricular perfusion. Conversely, animals which received treatment with CSF or bcl-2 sense ODNs continued to demonstrate the tolerance phenomenon. These data strongly favor the view that (i) bcl-2 actively promotes neuronal survival and (ii) preconditioning triggers ischemic tolerance through a bcl-2-mediated mechanism. Bcl-2 gene therapy may one day be available for clinical application and afford several who are at increased risk the opportunity to curb the detrimental effects of stroke.

11.8

STRUCTURAL PROTEIN DEGRADATION

Aside from the changes which have been observed in the expression of different proteins, certain morphological alterations have been shown to occur in response to ischemic events. Granted, most of the detrimental effects that have been mentioned, thus far, have concerned the terminal decline of the cell body and the compositional decay of its internal milieu. However, ischemia-induced injury has been shown to influence the microscopic appearance of other parts of the cell. These variations are manifested primarily in the dendrites and mark the structural breakdown of fine aspects of the neuronal architecture. In fact, in vivo as well as in vitro studies have shown that dendritic degeneration is indicative of early pathogenesis and that the

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selective vulnerability of these processes to ischemic insult is a hallmark of stroke related injury. Such damage, which is characterized by focal swelling, or beading, has been described previously in abstract form by Bateman and Goldberg.128 Neocortical cell cultures from chicks were exposed to 45–60 minutes of oxygen-glucose deprivation or 5 minutes of 500 uM NMDA. Using the lipid-soluble carbocyanine dye 1′,1-dioctadecyl-3, 3, 3′, 3′-tetramethylindocarbocyanine perchlorate (DiI) for fluorescent visualization, neurites were found with “beads” ranging in size from punctate varicosities between dendritic spines to dilations several times the diameter of the branch. A report by Stewart et al.129 concurs with these findings and indicates that the smooth contours of healthy dendrites become disturbed upon insult. In this particular study, the excitotoxic effects of excitatory amino acid agonists and plant-derived excitotoxic food poisons were evaluated in an in vitro preparation of chick spinal cord. After dissection, tissue was incubated for 90 minutes in NMDA, quisqualic acid, kainic acid, β-N-oxalylamino-L-alanine, β-N-methylamino-L-alanine, or domoic acid. A few of the treated cases also received a dorsal root ganglia (DRG) implant which consisted of a small crystal of DiI. Each compound induced the formation of large focal dendritic swellings in a concentration-dependent manner. These outpocketings were spread along the length of each dendritic radiation as it extended away from the cell body. EM analysis demonstrated that the acute edematous response of dendrosomal structures in the dorsal horn and intermediate zone also included clumping and condensation of nuclear chromatin. In addition, motor neurons assumed a dark and compressed appearance that was offset by the presence of many intracytoplasmic vacuoles (dark cell degeneration). At the present time very little is known about “why” dendritic profiles degrade after insult, but the examination of microtubule-associated protein-2 (MAP-2) has helped to clarify some of the “whens” and “wheres” of neuronal deterioration. MAP2 is localized mainly in dendrites as well as dendritic spines,130 and its presence in the cytoskeleton has permitted the evaluation of dendritic damage through the use of various immunostrategies. MAP-2 labeling has proven to be a particularly important tool for revealing the early stages of dendritic dysfunction following ischemic insult, primarily because disintegration of MAP-2 is evident before changes in general dendritic morphology are observed.131 We demonstrated this point in ischemic CA1 hippocampal neurons by combining intracellular injections of Lucifer Yellow with MAP-2 immunofluorescent staining. Animals received a five-minute bilateral global occlusion of the common carotid arteries and were sacrificed after 18–24 hours of reperfusion. The apical dendrites of dye-filled pyramidal neurons exhibited a normal appearance, while MAP-2 displayed a studded pattern consistent with dendritic beading. According to Western blot analysis, MAP-2 degradation, which was detectable as early as six hours postinsult, became pronounced at 24 hours and almost complete after four days, especially in the dorsolateral striatum. Early changes were found to be preferentially associated with the dendritic apparatus, since extensive loss of MAP-2 immunoreactivity was detected in the stratum radiatum and stratum oriens within a similar one-day time frame. Decreased somal labeling, however, was not detected until 2 days postinsult, suggesting that dendrites are more sensitive to adverse conditions than perikarya.132 Although, Aoki et al.133 did not report a MAP-2

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loss in the gerbil CA1 subregion until 2 days after a 15 minute bilateral common carotid occlusion, the findings of Kitagawa et al.134 support ours and further reveal that MAP-2 reactivity is decreased in the subiculum-CA1 region as early as three minutes after unilateral common carotid ligation. Park et al.135 have shown that dendritic beading also occurs in response to sublethal insult and that depending upon the intensity of the offense (lethal vs. sublethal), subtle differences in the pattern of breakdown can be detected. Mouse neocortical cultures were stained with DiI or against MAP-2 to determine the extent of injury sustained after O2-glucose deprivation or NMDA exposure. When neurons were incubated for 20–50 min in medium, which lacked oxygen and glucose, a majority of dendrites took on a varicose appearance. Focal swelling was mild, involved only the distal dendrites, and bore a strong resemblance to that observed after three minutes of 30uM NMDA treatment. With prolonged distress (60–70 minutes) dendritic spines were lost, and the entire arbor was replaced by periodic beads that were joined by thin segments of membrane. Likewise, application of NMDA for durations in excess of 10 minutes produced extensive swelling of the cell body as well as the proximal dendrite, and resulted in neuronal death by the following day. Upon further inspection, sublethal changes were found to (i) be significantly attenuated by glutamate receptor inactivation with 10 uM of the NMDA antagonist, MK-801, and (ii) display signs of reversibility. Two hours and 24 hours postinsult the morphology of dendritic appendages mirrored the smooth appearance of normal preparations. These data reinforce the notion that dendrites are more vulnerable to injury than the cell body, and also indicate that despite evidence of an intact perikarya, slight imbalances in the cellular microenvironment can have a noticeable impact on neuronal equilibrium. As suggested by Park et al.,135 it is possible that rapid dendritic responses contribute to alterations in neuronal function under certain physiological or pathological conditions. Thinking along these lines, the “next step” was to examine the in vivo occurrence of dendritic beading relative to sublethal ischemic injury and the relationship of ischemic tolerance to the structural collapse of dendrites. The possibility that dendritic beading may confer resistance to a secondary ischemic attack has been a point of particular interest. Park et al.135 proposed that structural alterations may contribute to plasticity following intense stimulation. In the case of ischemic damage, the decline in synaptic efficacy and transmission failure, which accompanies the breakdown of the dendritic array, may account for a certain degree of neuroprotection.136 This is not an unreasonable concept considering that focal constrictions between varicosities can promote the electrical isolation of dendrites from neuronal somata137 and the loss of dendritic spines could greatly limit excitatory synaptic contacts. However, it is also likely that dendritic beading is just a phenomenon that is observed in response to insult. We decided to approach this issue in the gerbil by determining the extent of CA1 dendritic beading after two minutes of bilateral common carotid artery occlusion and by tracking the expression of MAP-2 labeled varicosities with respect to the tolerance window. Extensive fragmentation was detected, but the onset of this reaction was delayed. Focal swelling was induced 2–3 days following insult, and was most obvious between days 4–7 (Figure 11.2; see color insert following page 114).

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FIGURE 11.2 (See color figure 11.2.) Photomicrographs illustrate MAP-2 immunohistochemical labeling in CA1 dendrites. The large arrows in (A) and (B) indicate the continuous extension of apical dendrites in samples taken from normal animals. This staining pattern was visualized by employing Texas-red and the ABC/DAB method, respectively. (C) Small arrows point to beaded dendritic profiles in tissue processed with Texas-red and collected 4 d after infliction of a 2-min sublethal ischemia. (D) At 10 d postinsult the dendritic field consisted of a mixture of smooth and swollen MAP-2 immunolabeled processes.

This change was found to be transient and to temporally coincide with the window of tolerance, since a majority of dendritic profiles had resumed a smooth appearance 10–14 days following challenge. Although these experiments did not directly support a specific role for dendritic beading in ischemic tolerance, they did provide evidence indicative of a common denominator. Perhaps the mechanisms responsible for ischemic tolerance lead to the breakdown of structural components in the dendritic field. MAP-2 has been shown to be the target of the degradative, Ca++-dependent protease, calpain.138 Given the variances in the onset, duration, and pattern of dendritic beading associated with sublethal vs. lethal insult, the heterogeneity which has been observed in the dendritic response may be explained on the basis of differential Ca++

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influx and subsequent accumulation. Several studies have been undertaken to evaluate regional fluctuations in Ca++ concentration after varying periods of ischemic insult. Most of these investigations have utilized intravenous injections of 45 Ca to autoradiographically gauge time-dependent alterations in calcium levels as well as cresyl violet and hematoxylin/eosin staining for correlations with neuronal damage. Alizarin red S and arsenazo III, a metallochromic indicator, have also been used to verify increases in Ca++ permeability. As a result, calcium loading was found within the selectively vulnerable CA1 region of the hippocampus and the dorsolateral striatum after transient (3–30 minutes) or repeated episodes of ischemia.139–143 Evidence of increased uptake was also detected in the choroid plexus, substantia nigra, medial geniculate, inferior colliculus, and layers III/V of neocortex. On average, calcium content was elevated 2–4 days following insult, although under some circumstances enhanced calcium gradients were observed before a lapse of 24 hours. The topical and temporal overlap of high densities of 45Ca radioactivity with lesion formation not only indicated a close association between rises in intracellular Ca++ and neuronal degeneration, but provided the grounds for linking disturbed Ca++ homeostasis to ischemic cell death. Abnormal accumulations of Ca++, however, have not been observed following sublethal insult by two-minute bilateral common carotid artery occlusion.139,143 Keeping in mind, the lowest level of Ca++ that can be detected by histochemical staining is approximately 1 mM,144 i.e., three to four orders of magnitude above the amount required to induce the pathological activation of proteases and phospholipases, as well as the gross nature of previous Ca++ evaluations from regional overviews in whole tissue sections, it seems plausible that discrete fluctuations in inward Ca++ flow may occur at the single-cell level to influence MAP-2 stability, but not threaten neuronal viability. Therefore, the expression pattern of dendritic beading under lethal and sublethal conditions may be dictated in part by differences in Ca++ intake and/or release from intracellular stores.

11.9.

ADENOSINE AND RECEPTORS

The purine nucleoside, adenosine, is an endogenous modulator that has been shown to exert a protective effect against ischemic injury. Chapter 7 by von Lubitz provides an overview of the work that has been done thus far on adenosine, and offers a current opinion on the use of adenosine-like compounds to combat ischemia-induced neurodegeneration. Some protective properties of adenosine and its associated receptors will be briefly covered at this time in order to expound upon their role in ischemic preconditioning and tolerance. Adenosine is a breakdown product of ATP. Its formation reflects the respiratory status of the cell, accumulating rapidly under conditions of energy depletion. Latini et al.145 have demonstrated that during periods of in vitro ischemia extracellular adenosine concentrations can rise from 240 nM to 30 uM. Enhanced levels of adenosine are believed to confer protection in a variety of ways. Van Wylen et al.146 suggested that the elevation in cerebral interstitial fluid adenosine content, which was observed after situations of inadequate oxygen supply for oxygen demand,

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was supportive of the adenosine hypothesis — whereby adenosine dilates the blood vessels supplying a hypoxic region, increasing oxygen delivery and compensating for the discrepancy between blood flow and metabolism. In a review article by Martin et al.,147 the prospect of adenosine upregulation to temper the release of large glutamate loads following an ischemic insult was proposed. Typically, 15–90 seconds after the commencement of an ischemic episode neurons experience an alteration in ionic flux, which promotes a rapid depolarization of the membrane potential within 3–5 minutes. This electrophysiological event is thought to facilitate the massive emission of excitatory amino acids, which is presumably responsible for postischemic excitotoxic damage. Adenosine, which in many instances is transported outside of the neuron terminal, interacts with presynaptic A1 receptors and through a Gi/o-protein linked signal tranduction pathway leads to the block of voltage-dependent Ca++ channels. Vesicle fusion is discouraged and exocytosis of excitatory neurotransmitters is depressed. Postsynaptically, action at the A1 receptor triggers an increase in the conductance of K+ and Clions.148 The accompanying hyperpolarization, consequently, antagonizes Ca++ dependent metabolic processes that precipitate nerve cell damage by reducing Ca++ influx through voltage-gated Ca++ channels upon synaptic activation. Adenosine further limits the likelihood of developing an extreme rise in intracellular Ca++ and an uncontrolled membrane depolarization by preventing the removal of the voltage sensitive Mg++ blockade at NMDA receptor-operated channels.149 A2 receptor stimulation may also contribute to neuroprotection through the maintenance of astrocyte functions, such as uptake of excitatory amino acids from the extracellular space or the enzymatic breakdown of glycogen.148 Studies which have focused on the heart have also implicated adenosine in the protection of cardiac tissue.150 The investigations that have addressed adenosine’s role in preconditioning have been particularly well received, because they have provided key insight toward our understanding of ischemic tolerance in the CNS. Most of these examinations have involved treating isolated organ preparations151,152 or cultured ventricular myocytes153 with selective adenosine agonists or antagonists. As a result, A1 and A3 receptor activation has been shown to evoke a tolerant condition similar to that induced by ischemic priming.154 Contractile function/rate-force analysis, creatine kinase measurements, tetrazolium and trypan blue staining, as well as sedimentation evaluation are among the assays which have been used to confirm the beneficial effect of adenosine on cardiac muscle. Surveys of total purine release and ATP content have also been conducted for correlation with ischemic injury. A report published by Lee et al.153 is representative of the work that has advanced our knowledge of the relationship between different adenosine receptor subtypes and preconditioning. Cultured ventricular myocytes were subjected to a 90–minute period of simulated ischemia by placing cells in a hypoxic incubator which contained N2 instead of O2. Such treatment elicited a significant drop in ATP content, a rise in the amount of creatine kinase release, and over a two-fold increase in the percentage of nonviable cells as compared to preconditioned controls, which received a fiveminute trial episode of the challenge just prior to full exposure. When introduced to myocytes ten minutes before the onset of a 90-minute hypoxia, the respective A1 and

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A3 receptor agonists, 2-chloro-N6-cyclopentyl-adenosine (CCPA) and 2-chloro-N6(3-iodobenzyl)adenosine-5′-N-methyluronamide (Cl-IB-MECA), were each able to reduce cell loss and mimic the effect of ischemic preconditioning. Incubation with the nonselective adenosine receptor antagonist, 8-sulphophenyltheophylline (8SPT) during ischemic preconditioning prevented adenosine receptor activation and abolished the protective effect of ischemic priming against subsequent insult. Separate blockade of each receptor subtype with the selective A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), or the selective A3 antagonist, 3ethyl 5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydro-pyridine-3,5dicarboxylate (MRS-1191), only partially attenuated cell survival, indicating that adenosine-induced protection is not exclusively mediated by a single receptor subpopulation. Liu et al.151 provided early support for this idea with the isolated rabbit heart model by using N6-[2-(4-aminophenyl)ethyl]adenosine (APNEA) instead of IB-MECA to discriminate the A3 component of ischemic preconditioning from the A1 contribution. Endogenous adenosine is thought to initiate ischemic tolerance through a signal transduction cascade which involves protein kinase C (PKC) and ATP-sensitive potassium (KATP) channels. Sato et al.155 have summarized the background evidence suggestive of this pathway and have provided a detailed account of findings which support a link between adenosine receptor activation and the opening of mitochondrial KATP (mitoKATP) channels. Intact ventricular myocytes and diazoxide, a selective mitoKATP channel opener in cardiac cells, were used to assess the extent of adenosine’s influence. Since fluorescence levels are a general indicator of redox state and net oxidation is typically detected with the increased opening of mitoKATP channels, the autofluorescence of mitochondrial flavin adenine dinucleotide-linked enzymes was evaluated to serve as an index of mitoKATP channel activity. Although adenosine alone did not alter fluorescence measurements, it was found to increase diazoxide-induced flavoprotein oxidation and decrease the latency for mitoKATP channel opening. In the presence of 8-SPT; the PKC inhibitor, polymixin B; or the mitoKATP channel blocker, 5-hydroxydecanoate (5HD), these potentiating effects were suppressed. Such findings support the notion that receptor-mediated actions of adenosine are routed through the PKC messenger system to facilitate the response of mitoKATP channels. To determine whether cardioprotective effects are conferred through this same cellular mechanism, cell suspensions were treated with adenosine, diazoxide, or the KATP channel opener, pinacidil153 prior to 60, 90, or 120 minutes of ischemia. Histochemical analysis of trypan blue permeability revealed that pharmacological preconditioning significantly decreased the percentage of cell death. As a matter of fact, the benefits of diazoxide on cell survival were found to be considerably augmented by simultaneous application of adenosine. 5HD and glibenclamide, a KATP channel antagonist,153 eliminated the capacity for cells to withstand an ischemic insult despite preexposure to adenosine or ischemic preconditioning. These results indicate that adenosine increases the resistance of cardiac myocytes to ischemic damage by priming the opening of mitoKATP channels. Since adenosine appears to lack the ability to directly initiate the channel opening process, its protective influence can be explained on the basis that mitoKATP channels exist in three distinct

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states: resting, primed, and open.155 According to this scheme, channels must traverse through several intermediate, nonconducting states before the channel pore can open. Adenosine is thought to optimize mitoKATP channels by automatically shifting them into a “ready” conformation. This mode increases the responsiveness of the channel and rapidly permits the flow of ion traffic should an endogenous stimulus directly permit passage through these membranous gateways. Studies in the CNS have confirmed that adenosine plays a significant role in ischemic preconditioning and demonstrated that ischemic tolerance is conveyed through receptor interactions similar to those defined in cardiac tissue. The activation of A1 receptors and K+ATP channels has been shown several times to be particularly important for precipitating a protective response to ischemic insult. For instance, Heurteaux et al.156 used an in vivo model of global ischemia to illustrate that treatment with the A1 receptor agonist, N6-cyclopentyladenosine (CPA), or the K+ channel opener, levcromakalim, prior to a 6-minute ischemia could preserve hippocampal neurons in a manner comparable to ischemic preconditioning. Exposure to DPCPX before the infliction of a three-minute priming ischemia, or glibenclamide before the administration of either CPA or levcromakalim markedly reduced the protective effect of conditioning strategies and hence, decreased the density of neurons that survived a subsequent ischemic insult. These findings support the position that ischemic preconditioning causes the release of adenosine and that the resultant activation of A1 receptors signals the opening of KATP channels, thereby imparting resistance to injury. Interestingly, the benefits of CPA were observed after a shorter period of time than those associated with classical models of ischemic preconditioning. Instead of requiring a time course of 1–3 days for tolerance to develop, only 15 minutes from the end of drug infusion to the beginning of the ischemic episode were necessary to pharmacologically enhance neuronal endurance. In this respect, these data parallel the in vitro evidence of Goldberg et al.157 and Pérez-Pinzón et al.158 Goldberg et al.157 found that addition of adenosine or the selective A1 agonist, N6-cyclohexyladenosine (CHA), to cortical cultures could substantially ameliorate the morphological appearance of cell loss and cause a concentration dependent reduction in LDH efflux when introduced during lethal insult. Cells were exposed to a sixhour period of hypoxia or glucose deprivation, and evaluated 24 hours after their return to normoxic, glucose-enriched conditions. Likewise, Pérez-Pinzón et al.158 reported that a 10-minute superfusion with adenosine or the adenosine A1 receptor agonist, 2-chloroadenosine (2-CADO), just 30 minutes prior to a 2-minute interval of anoxic depolarization was effective for improving the recovery of synaptically driven potentials in hippocampal slices. DPCPX, on the other hand, was able to prevent the full return of evoked activity (46%) when administered during an episode of preconditioning a half an hour before the onset of anoxia. Other studies have also suggested the involvement of adenosine in the development of ischemic tolerance. In studying the existence of bidirectional cross-conditioning between kainic acid excitotoxicity and global ischemia, Plamondon et al.159 found that A1 receptors and KATP channels mediate the neuroprotection conferred by sublethal ischemic preconditioning as well as epileptiform priming. Several analogs of adenosine and at least two major experimental designs were

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employed to demonstrate that injury to a secondary, severe insult can be minimized, if not prevented. In the first protocol rats received a three-minute sublethal ischemic challenge, which was followed three days later by the i.p. injection of 7.5 mg/kg kainic acid (KA7.5). The second paradigm entailed the administration of 5.0 mg/kg kainic acid (KA5) 72 hours prior to a 6-minute bilateral common carotid artery occlusion. Damage was assessed by cresyl violet staining, while the extent of necrotic and apoptotic cell death was determined by silver impregnation and the terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′ triphosphate-biotin nick-end labeling (TUNEL) method, respectively. An i.p. dose of DPCPX given two hours before either preconditioning challenge resulted in numerous argyrophilic neurons in CA1 and CA3. When evaluated seven days after final treatment, 33%–72% of pyramidal neurons were found to have sustained damage. In contrast, intravenous inoculation of the selective A1 agonist, Rphenylisopropyladenosine (R-PIA), immediately preceding exposure to KA7.5 was able to reduce neuronal degeneration (85% preservation of CA1 neurons) and inhibit the formation of apoptotic nuclei three days postinsult. Intracerebroventricular infusion of glibenclamide 30 minutes prior to each of the conditioning regimens, including R-PIA treatment, attenuated priming-induced tolerance and produced a 32%–65% enhancement of cell loss in response to ischemic conditions or seizure-like activity. Conversely, cromakalim, administered in a fashion similar to glibenclamide and once daily during recovery afforded a significant amount of protection to neurons subjected to KA7.5. Survival rates of 75% and 89% were observed in CA1 and CA3, respectively, relative to nonconditioned controls. Prompted by pharmacological evidence of adenosine’s role in preconditioning-induced tolerance, Kawahara et al.160 hypothesized that further stimulation of the adenosine receptor after priming may potentiate adenosine’s protective effect upon subsequent lethal ischemia. To examine this concept 20 mg/kg i.p. injections of propentofylline (PPF), an adenosine uptake blocker, were utilized to increase endogenous levels of adenosine 24 hours after a 2-minute sublethal, bilateral common carotid occlusion. Following a 5-minute ischemia, which was inflicted 48 hours after the first, CA1 neuronal density was found to be significantly higher in animals treated with uptake inhibitors than in gerbils given shots of vehicle. Co-administration of theophylline with PPF negated the potentiating effect of PPF on ischemic preconditioning and reduced the number of intact hematoxylin-eosin stained neurons to a value slightly below that calculated for vehicle-treated subjects. At the present time the role of central A3 receptors in ischemic preconditioning remains unclear. Some reviews indicate that there is little evidence to support a function for the A3 receptor in nervous tissue161 and that the A3 receptor largely impacts peripheral organs/systems, especially the testis and mast cells.148 Rathbone et al.,162 however, have offered information pertaining to the interaction of the A3 receptor with astroglia. Apparently, impingement upon this receptor population can lead to one of two paradoxical events: (i) apoptosis or (ii) enhanced cell survival. Studies using A3 receptor agonists in astrocyte cultures have demonstrated that in the nanomolar range A3 agonists are cryoprotective, but at higher concentrations such

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agents tend to be cytotoxic. Attempts to further clarify this differential response in the HL-60 cell line with selective A3 antagonists revealed that receptor blockade induces cell death, while treatment with low levels of A3 agonists can prevent A3 antagonist-mediated fatality. These findings suggest that the A3 receptor is activated to a limited degree by the constitutive release of adenosine and that a disruption in binding either by A3 antagonists or receptor desensitization can induce apoptosis. Taken together, it would appear that under certain circumstances both the A1 and A3 receptor subtypes contribute to life-sustaining processes, and as such, a therapeutic approach, which capitalizes upon the neuroprotective effects of specific adenosine agonists warrants serious consideration (see Chapter 7 by von Lubitz).

11.10 THE IMMUNE/INFLAMMATORY RESPONSE Pathological events which result in brain injury, whether from episodes of ischemia, trauma, or a disease process, have been shown to trigger an immune/inflammatory reaction. This response is characterized by the onset of edema, microglia activation, and the expression of cytokines, which include interleukin 1β and tumor necrosis factor-α. It is now known that several cell types (microglia, astrocytes, endothelial cells, and neurons) secrete cytokines and that the immune/inflammatory response occurs rapidly and persists for days to weeks163,164 (see Chapter 9 by Barone et al.). Despite extensive work, the precise nature (detrimental or beneficial) of inflammatory mediators remains to be fully understood. Throughout the final portion of this chapter issues related to interleukin-1, tumor necrosis factor, and microglia will be specifically addressed with regard to sublethal ischemic insult.

11.10.1 INTERLEUKIN-1 Interleukin-1 (IL-1) is a polypeptide growth factor that is released by monocytes and macrophages, as well as brain cells following insult. Currently, only a limited amount of information is available to infer a relationship between IL-1 and ischemic tolerance. The first line of evidence is based upon studies which utilized models of permanent unilateral MCAO.165,166 IL-1β mRNA was found to be noticeably upregulated in the cortical hemisphere opposite to the site of occlusion i.e., the viable, contralateral cortex. Although this increase represented a fraction of that observed on the ipsilateral, damaged side, it was detected within two hours of obstruction and peaked at six hours. These data suggest that severe ischemia significantly elevates the transcription of IL-1β mRNA in lesioned areas, and more importantly indicate that low levels of message can be manifested in regions devoid of neuronal death. A second clue which alludes to potential protective capabilities of IL-1 derives from the research of Ohtsuki et al.167 Enhanced measures of serum IL-1α and IL-1β were recorded in the gerbil arterial circulation 1–3 days after a two-minute sublethal global ischemia. While the authors did not describe alterations which may have occurred in the brain, it seems likely based upon IL-1 augmentation in surviving cortical neurons of aforementioned reports165,166 that a preconditioning challenge would

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induce the neuronal expression of IL-1. Certainly, this issue is deserving of further investigation. Recently, a number of studies have focused on the role of the naturally occurring IL-1 receptor antagonist, IL-1ra, a 23-25 kD glycosylated protein which blocks all known actions of IL-1. From the examination of several animal models, it has been ascertained that i.v., i.c.v., and s.c. treatment with recombinant human IL-1ra can protect neurons from ischemic insult as well as other types of brain injury.168,169 Adenoviral vectors which enhance the expression of IL-1ra were also found to reduce neuronal damage in the rat when administered i.c.v. five days prior to permanent MCAO.170 In an attempt to assess the involvement of IL-1ra in ischemic tolerance, Barone et al.19 evaluated the prevalence of IL-1ra mRNA in preconditioned cortex following double exposure to unilateral MCAO. The combination of temporary and permanent occlusion resulted in the marked amplification of IL-1ra message. Transcription peaked 24 hours after the second intervention and remained elevated for an additional day. Using immunohistochemical staining techniques with IL-1ra antiserum, the presence of IL-1ra(+) brain cells with neuron-like morphologies was confirmed. These profiles emerged 1–2 days after the final insult and preferentially appeared in the conditioned cortical hemisphere. Visual inspection of control material on the opposite side of the midline revealed no labeling. Since the expression of IL-1β was found to overlap considerably with the upregulation of IL-1ra mRNA and protein levels, the authors suggested that activation of IL-1ra may counteract any negative effects of IL-1β after an ischemic event.

11.10.2 TUMOR NECROSIS FACTOR-α Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine that is also released from many cell types, including brain cells, upon insult. The enhanced expression of TNF-α after ischemia has been reported on several occasions171,172,165 and apparently occurs quite rapidly. After severe MCAO, the TNF-α response can first be detected within an hour. Levels of TNF-α have been shown to peak at 3–12 hours postinsult and subside slowly over survival periods of 4–5 days. Low level increases of TNFα mRNA, which have been observed in nondamaged cortical hemispheres contralateral to the placement of a unilateral MCAO, have suggested the potential involvement of TNF-α in ischemia-induced neuronal preservation.165,172 A few articles have documented the time course of TNF-α expression after transient occlusion. Interestingly, changes in the relative levels of TNF-α after sublethal insult closely parallel TNF-α responses that are observed following longer exposures to ischemia. For example, Wang et al.173 reported that TNF-α mRNA can be detected in the ipsilateral cortex as early as one hour after 10 minutes of unilateral MCAO preconditioning. Using newly developed real-time RT-PCR techniques, the TNF-α signal was found to peak at 12 hours postinsult, persist for two days, and return to baseline measures by day five. This induction corresponds well with the tolerance window and suggests that TNF-α mRNA expression may facilitate the progression of ischemic tolerance.

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Findings from several other studies also support a role for TNF-α in neuronal protection. Nawashiro et al.174 demonstrated that TNF-α pretreatment 48 hours prior to permanent MCAO significantly reduced infarct size in mice. Protection was found to be dependent upon the mode of delivery. Intracisternal administration of 0.05 or 0.5 ug TNF-α into the cisterna magna was effective, while i.v. and i.p. injection were not. Given the differences in TNF-α application, an explanation, which accounts for the discrepancy in drug performance, is currently lacking. Additional evidence from hippocampal cell culture models has revealed that TNF-α and TNF-β can provide protection against the neurotoxin, amyloid-β-peptide.175 TNF-binding protein (TNFbp), however, has been shown to block the protective effects of TNF-α in spontaneously hypertensive rats.176 As illustrated in the lipopolysaccharide (LPS) model of preconditioning, pretreatment with TNF-bp negates the benefits of endotoxin exposure against focal cerebral ischemia, presumably by suppressing the actions of TNF-α after LPS-evoked release.

11.10.3 GLIA Gliosis, or the proliferation of glial cells, is a well-recognized phenomenon that occurs in association with brain injury. The main cell types to demonstrate this behavior are microglia and astrocytes. Microglia, in particular, exhibit many notable characteristics: (i) their activation closely relates to the immune/inflammatory response, (ii) they react very early following severe or minor pathological insult, (iii) their responses occur in a graded fashion, and (iv) they exert either a cytotoxic or protective influence. The following section will concentrate on microglia and their putative role in ischemic tolerance and protection. Morioka et al.177 have shown that after 25 minutes of 4-vessel occlusion, microglia in the CA1 and dentate hilus of rat hippocampus become activated or sustain a change in shape. Cells assumed an ameboid, round, or rod-like morphology and stained intensely with Griffonia simplicifolia B4-isolectin as early as 20 minutes postinsult. The response peaked 4–6 days after challenge and subsided four weeks later. At this point, near control conditions were observed: resting microglia were weakly stained and displayed thin, highly ramified processes. Using the 3-vessel occlusion model and kainic acid injections, Jorgensen et al.178 additionally reported that reactive microglia can be located in areas devoid of neuronal degeneration as well as in severely damaged areas. The authors further demonstrated that within unlesioned regions activated microglia take on a modified appearance. The altered morphology, which returned to normal within 4–7 days, was characterized by the presence of thickened processes. Appendages resembled those found under resting conditions, but were observed to extend shorter distances from the cell body. A study by Buttini et al.172 supports these findings and indicates that reactive microglia appear bilaterally in the cortex following permanent unilateral MCAO in the rat. Profiles immunohistochemically stained for TNF-α and exhibited a morphological heterogeneity which was location-dependent. Microglia in the contralateral, noninjured hemisphere demonstrated coarse, ramified processes, while those ipsilateral to MCAO in the severely damaged cortex appeared ameboid/round. Based upon avail-

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able information, it seems reasonable to speculate that microglia are transiently activated following sublethal ischemic insult or in response to ischemic preconditioning. The morphological disparity that has been observed between lesioned and viable cortical territories further indicates an intermediate stage in the glial response to insult. Perhaps, in this state between resting and full activation microglia are best equipped to perform a neuroprotective function. A handful of studies have utilized neuroanatomical techniques to confirm elevated glial activity following sublethal insult and establish that glial responses differ depending upon the severity of injury. Using a two-minute model of sublethal ischemia Kato et al.179 found a moderate increase in the hippocampal staining of reactive microglia. This response was evident 1–3 days postchallenge and subsided to control levels on day 7, the last day of survival. During this period of time, not only were the processes of microglial profiles ramified and thorny, but reactive astrocytes were observed which exhibited enhanced GFAP labeling. Unlike the hypertrophied condition, which has been described previously for reactive astrocytes following severe insult, astroglia in this preparation maintained a normal conformation. In a related report, Liu et al.180 employed 5-bromo-2′-deoxyuridine-5′-monophosphate (BrdU) as a marker to identify microglial proliferation after either a single sublethal ischemia or ischemia-induced tolerance in the gerbil. A 2.5-minute global ischemia was given alone, or in combination with a 5-minute severe global ischemia 3 days later. Results from these cases were then compared to data from a third group that was subjected exclusively to a 5-minute ischemic intervention. Although no morphological descriptions were provided, diversity in microglial responsiveness could be distinguished by regional differences in gliosis. Animals treated with a lethal dose of ischemia demonstrated widespread evidence of microglial reactivity, especially in the hippocampus. Microglial involvement following less damaging manipulation, however, was restricted more or less to the striatum and neocortex. Since the phagocytic properties of reactive microglia, which are typically observed following severe injury, are not evident after exposure to sublethal insult, i.e., synaptic terminals are not displaced by microglial processes,181,182 it is likely that another specialized role is fulfilled by the transient activation of newly identified microglia. The precise nature of this response remains to be elucidated, but could very well include neuroprotection.

11.11 CONCLUSIONS It is safe to say that many rather complex cellular/molecular events surround the development of cerebral ischemia-induced injury, and at this time our understanding of the protective mechanisms underlying ischemic tolerance remains in its infancy. As attention toward stroke-related issues continues to grow, the knowledge that we have acquired through studies of the heart will undoubtedly be applied to shape the investigations of tomorrow. Despite the multifactorial response to ischemic insult and the questionable benefits of several neuronal, glial, and endothelial intermediaries, we should not be distracted from our goal of one day unveiling a therapeutic approach that will provide a counter-measure against injury for those at risk of stroke.

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FIGURE 11.2 Photomicrographs illustrate MAP-2 immunohistochemical labeling in CA1 dendrites. The large arrows in (A) and (B) indicate the continuous extension of apical dendrites in samples taken from normal animals. This staining pattern was visualized by employing Texas-red and the ABC/DAB method, respectively. (C) Small arrows point to beaded dendritic profiles in tissue processed with Texas-red and collected 4 d after infliction of a 2-min sublethal ischemia. (D) At 10 d post-insult the dendritic field consisted of a mixture of smooth and swollen MAP-2 immunolabeled processes.