Neuroprotective Agents and Cerebral Ischaemia

International Review of NEUROBIOLOGY VOLUME 40 Neuroprotective Agents and Cerebral Ischaemia International Review o...

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International Review of

NEUROBIOLOGY VOLUME 40

Neuroprotective Agents and Cerebral Ischaemia

International Review of

NEUROBIOLOGY VOLUME 40 Series editors RONALDJ. BRADLEY Department of Psychiatry, School of Medicine, Louisiana State University Medical Center, Shreveport, Louisiana, USA

R. ADRONHARRIS Department of Pharmacology, University of Colorado, Health Sciences Center, Denver, Colorado, USA

PETERJENNER Biomedical Sciences Division, King’s College, London, UK

Editorial Board Ross J. BALDESSARINI

~ N Y KURIYAMA A

TAMAS BARTFAI

BRUCE S. MCEWEN

COLINBLAKEMORE

HERBERT Y. MELTZER

FLOYD E. BLOOM

NOBORU MIZLJNO

PHILIPBRADLEY

SALVADOR MONCADA

DAVIDA. BROWN

TREVOR W. ROBBINS

MATTHEWJ. DURING

SOLOMON H. SNYDER

KJELLG. FUXE

STEPHEN G. WAXMAN

PAULGREENCARD

CHIEN-PING Wu

SUSAND. IVERSEN

RICHARD J. WYATT

PAUL JANSSEN

Neuroprotective Agents and Cerebral Ischaemia Editors

A. RICHARD GREEN Astra Arcus, Loughborough, UK

and

ALAN J. CROSS Astra Arcus USA, Rochester, New York, USA

ACADEMIC PRESS San Diego London Boston New York

Sydney Tokyo Toronto

This book is printed on acid-free paper. Copyright 0 1997 by ACADEMIC PRESS

All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press, Inc. 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://w.apnet.com Academic Press Limited 24-28 Oval Road, London NWl 7DX, UK http://www.hbuk,co.uk/ap ISBN 0- 12-366840-9 0- 12-197880-X @bk) A catalogue record for this book is available from the British Library Typeset by Servis FilmsettingLtd. Printed in Great Britain by HartnoUs Ltd, Bodmin, Cornwall

CONTENTS Contributors............................................................................................................. Preface .....................................................................................................................

xi ... xiii

I . Mechanisms of nerve cell death: apoptosis or necrosis after cerebral ischaemia

R.M.E. CHALMERS-REDMAN, A.D. FRASER, W.Y.H.Ju. J. WADIA.N.A. TATTON AND W.G. TATTON 1.1

1.2 I .3 1.4 I .5 I .6 1.7 1.8

Stroke and neuronal necrosis ................................................................................... Neuronal apoptosis caused by trophic factor insufficiency ...................................... Neuronal apoptosis due to a wide variety of causes................................................. Neuronal apoptosis in ischaemia-hypoxia ............................................................... Genes and proteins that promote or retard apoptosis ............................................. Evidence that mitochondria contribute to the initiation of apoptosis ..................... Possible reduction of apoptosis caused by ischaemia-hypoxia with trophic factors . Possible new anti-apoptotic functions for two old drugs ..........................................

1 2 3

7 10

12 14 15

2. Changes in ionic fluxes during cerebral ischaemia TIBOR KRISTIAN AND Bo K . SIESJO 2.1 2.2 2.3 2.4 2.5 2.6

Introduction ............................................................................................................. Changes in ionic fluxes ............................................................................................ Disturbances in ionic fluxes at restricted energy production ................................... Bioenergetic failure and ionic fluxes ........................................................................ Ion fluxes during focal ischaemia............................................................................. Ionic fluxes in the postinsult period .........................................................................

27 31 32 33 40 40

3 . Techniques for examining neuroprotective drugs in vivo A . RICHARD GREEN AND ALAN J. CROSS 3.1 3.2 3.3 3.4

3.5 3.6

General introduction ............................................................................................... Global models of acute ischaemic stroke ................................................................. Focal models of acute ischaemic stroke.................................................................... The design of studies using animal models to discover clinically useful neuroprotective drugs .......................................................................................... Protocols required when using animal models to discover new therapeutic entities General discussion ...................................................................................................

47 49 53

58 63 63

4. Techniques for assessing neuroprotective drugs in v i m MARK €! GOLDBERG. UTASTRASSER AND LAURAL. DUGAN 4.1 4.2 4.3 4.4

Introduction ............................................................................................................. Simulatingischaemic conditions in Uitro...................................................................... Combined oxygen-glucose deprivation in dissociated cortical neuronal cultures ... Combined oxygen-glucose deprivation in organotypic hippocampal cultures........ V

70 73 78 84

vi

CONTENTS

4.5

Comparison of dissociated cell and organotypic slice models of oxygen-glucose deprivation injury ................................................................................................

90

5. Calcium antagonists: their role in neuroprotection A.JACQUELINE HUNTER 5.1 5.2 5.3 5.4 5.5 5.6

Introduction ............................................................................................................. The role of calcium in ischaemic stroke................................................................... Classification of voltage-operated calcium channels................................................ In vitm studies with calcium antagonists ................................................................... In Uivo studies with calcium antagonists.................................................................... Clinical studies .........................................................................................................

95 95 96 99 100 105

.

6 Sodium and potassium channel modulators: their role in neuroprotection

TIHOMIR €! OBRENOVITCH Introduction........................................................................................................... Down-modulation of voltage Na' channels during ischaemia: an inherent adaptive mechanism for neuronal survival ........................................................ 6.3 Na' channel blockade protects neurones against ischaemia: experimental evidence............................................................................................................. Neuroprotective agents acting on Na' channels .................................................... 6.4 6.5 Clinical relevance and suitability........................................................................... K' channel openers: introduction ......................................................................... 6.6 Effect of ischaemia on K' channels ....................................................................... 6.7 Rationale for opening K' channels to protect neurones against ischaemia........... 6.8 6.9 K' channel openers and neuroprotection in ischaemia: experimental evidence ... 6.10 Concluding remarks .............................................................................................. 6.1 6.2

110 110 111

114 118 120 121 124 126 127

7. NMDA antagonists: their role in neuroprotection DANIEL L. SMALLAND ALISTAIR M . BUCHAN 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9

Introduction ............................................................................................................. Excitotoxicity hypothesis .......................................................................................... NMDA receptor molecular biology ......................................................................... NMDA receptor biophysics ..................................................................................... NMDA receptor pharmacology .............................................................................. NMDA antagonist neuroprotection in in Uitro models of ischaemia......................... NMDA antagonist neuroprotection in in Uivo models of ischaemia ......................... How to determine what goes to trial ....................................................................... Conclusion ...............................................................................................................

137 138 140 142 145 150 153 157 158

8. Development of the NMDA ion-channel blocker. aptiganel hydrochloride. as a neuroprotective agent for acute CNS injury ROBERT N. MCBURNEY 8.1 8.2 8.3

Introduction ............................................................................................................. Difficulty of developingdrugs for acute CNS injury ............................................... Development of NMDA antagonists for acute CNS injury .....................................

173 176 177

CONTENTS

8.4 8.5 8.6 8.7

Aptiganel hydrochloride: from laboratory to clinic ................................................. Clinical experience................................................................................................... Comments on the progress of aptiganel hydrochloride ........................................... The future ................................................................................................................

vii 179 182 192 193

9. Pharmacology of AMPA antagonists and their role in neuroprotection RAMMY GILLAND DAVID LODGE 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9 9.10

Discovery of glutamate receptor subtypes ............................................................... 197 AMPA agonists......................................................................................................... 198 Elucidation of more potent and selective AMPA antagonists.................................. 200 Molecular biology of AMPA receptors .................................................................... 202 Antagonist pharmacology of recombinant AMPA receptors .................................. 203 AMPA antagonists and cerebral ischaemia ............................................................. 203 Role ofAMPA/kainate antagonists in focal ischaemia models ............................... 206 Mechanism of protection following focal ischaemia ................................................ 213 Role of AMPA/kainate antagonists in transient forebrain ischaemia models ......... 214 Side-effect profile of AMPAIkainate antagonists and relevance to clinical testing... 220

I 0. GABA and neuroprotection PATRICK D. LYDEN 10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.9 10.10

Introduction ............................................................................................................. Excitotoxicity........................................................................................................... The GABA strategy ................................................................................................. The excitotoxic index............................................................................................... Anatomy ofthe GABA receptor .............................................................................. Response of GABA to ischaemia ............................................................................. GABA, agonists are neuroprotective....................................................................... Pharmacology of GABA mimetics........................................................................... Combinatorial strategies .......................................................................................... Future directions ......................................................................................................

233 233 235 236 236 238 240 250 252 253

.

I I Adenosine and neuroprotection

BERTIL B. FREDHOLM 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8

Introduction ............................................................................................................. Formation and levels of adenosine........................................................................... Adenosine receptors................................................................................................. Effects of drugs that affect adenosine levels ............................................................. Acute effects of adenosine receptor agonists and antagonists.................................. Mechanism of actiop of adenosine .......................................................................... Long-term actions of adenosine receptor agonists and antagonists ........................ Summary .................................................................................................................

259 260 262 267 268 269 273 273

.

I 2 lnterleukins and cerebral ischaernia

NANCY J. ROTHWELL. SARAH A. LODDICK AND PAULSTROEMER 12.1 12.2

Introduction ............................................................................................................. Interleukin-1 ............................................................................................................

281 282

...

CONTENTS

VlU

12.3 12.4 12.5 12.6 12.7 12.8 12.9

Interleukins in the brain ........................................................................................... Role of interleukins in ischaemic brain damage ...................................................... Other interleukins in stroke ..................................................................................... Effects of rIL- Ira on other forms of neurodegeneration ......................................... Mechanisms of action of IL-1 and r I L l r a .............................................................. Pharmacological approaches to cytokine modulation ............................................. Therapeutic considerations......................................................................................

282 284 288 288 289 292 293

I3 . Nitrone-based free radical traps as neuroprotective agents in cerebral ischaemia and other pathologies

KENNETH HENSLEY. JOHN M . CAIWEY.CHARLES A . STEWART. TAHERA TABATABAIE. QUENTIN PYEAND ROBERT A. FLOYD 13.1 Introduction ............................................................................................................. 299 1 3.2 Solution chemistry and neuroprotective potential of nitrone-based free radical 300 spin traps .............................................................................................................. 13.3 PBN mitigates postischaemic brain free radical production. protein oxidation. metabolic impairment. and infarction when administered prior to or following 304 the ischaemic event .............................................................................................. 13.4 PBN suppresses postischaemic gene induction: implications for apoptosis ............. 305 13.5 Other pharmacological action of nitrones............................................................... 306 13.6 Possible mechanisms of nitrone action: moving beyond the ‘simple’ free radical 309 scavenging hypothesis .......................................................................................... 312 13.7 Summary .................................................................................................................

14. Neurotoxic and neuroprotective roles of nitric oxide in cerebral ischaemia

TURGAY DALKARA AND MICHAEL A. MOSKOWITZ 14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8

Introduction ............................................................................................................. Synthesis and metabolism of NO ............................................................................ Molecular mechanisms of NO action ...................................................................... NOS inhibition ........................................................................................................ Functions of NO in the brain .................................................................................. NO-mediated neurotoxicity..................................................................................... NO and the NMDA receptor .................................................................................. NO and cerebral ischaemia.....................................................................................

319 320 322 323 324 325 327 327

I 5. A review of earlier clinical studies on neuroprotective agents and current approaches NILSGUNNAR WAHLGREN 15.1 15.2 15.3 15.4 15.5 15.6 15.7

Introduction ............................................................................................................. Calcium antagonists................................................................................................. Monogangliosides.................................................................................................... Naloxone .................................................................................................................. Piracetam ................................................................................................................. GABA agonists/clomethiazole ................................................................................ NMDA antagonists..................................................................................................

337 339 342 345 345 345 347

15.8 15.9 15.10 15.11

CONTENTS

ix

Inhibition of glutamate release ................................................................................ Free radical scavengers............................................................................................. Inhibition of leucocyte adhesion .............................................................................. General discussion ...................................................................................................

351 352 353 353

EX ........................................................................................................................ CONTENTS OF RECENT VOLUMES ...............................................................................

365 375

This Page Intentionally Left Blank

CONTRIBUTORS A.M. Buchan Clinical Neurosciences, University of Calgary, Foothills Hospital, 1403-29 Street W ,Calgary, Alberta, 2TN 2T9 Canada J.M. Carney Centaur Pharmaceuticals Inc., 484 Oakmead Parkway, Sunnyvale, CA 94086, USA R.M.E. Chalmers-Redman Department of Physiology and Biophysics, Institute of Neuroscience, Dalhousie University, Halifax, Nova Scotia, Canada A.J. Cross Astra Arcus USA, 755Jefferson Road, Rochester, New York, NY 14623, USA T. Dalkara Stroke and Neurovascular Regulation Laboratory, Department of Neurology and Neurosurgical Service, Massachusetts General Hospital and Harvard Medical School, 149 13th Street, CNY 6403, Charlestown, MA 02 129, USA and Department of Neurology, Hacettepe University, Ankara 06 100, Turkey L.L. Dugan Center for the Study of Nervous System Injury, and Department of Neurology, Washington University School of Medicine, St Louis, Missouri 63 110, USA R.A. Floyd Oklahoma Medical Research Foundation Department of Free Radical Biology and Aging, Oklahoma City, Oklahoma USA and Centaur Pharmaceuticals Inc., 484 Oakmead Parkway, Sunnyvale, CA 94086, USA A.D. Fraser Department of Physiology and Biophysics, Institute of Neuroscience, Dalhousie University Halifax, Nova Scotia, Canada B.B. Fredholm Department of Physiology and Pharmacology, Section of Molecular Neuropharmacology, Karolinska Institutet, S-177 77 Stockholm, Sweden R. Gill Hoffmann La Roche, Pharma Division, PRPN, BAU 68/410, Grenzacher Strasse, 4002 Basel, Switzerland M.P. Goldberg Center for the Study of Nervous System Injury, and Department of Neurology, Washington University School of Medicine, St Louis, Missouri 63 1 10, USA A.R. Green Astra Arcus, Bakewell Road, Loughborough, Leicestershire L E l l 5 R H , UK K. Hensley Oklahoma Medical Foundation Department of Free Radical Biology and Aging, Oklahoma City, Oklahoma, USA AJ. Hunter SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM 19 5AW, UK W.Y.H. Ju Department of Physiology and Biophysics, Institute of Neuroscience, Dalhousie University, Halifax, Nova Scotia, Canada T. Kristih Laboratory for Experimental Brain Research, Lund University, University Hospital, S-22 1 85 Lund, Sweden S.A. Loddick School of Biological Sciences, 1.124 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9pT, UK xi

Xii

D. Lodge

CONTRIBUTORS

Lilly Research Centre Limited, Erl Wood Manor, Windlesham, Surrey, GU20 6PH, UK RD. Lyden UCSD Stroke Center, Department of Neurosciences, University of California, 200 W. Arbor Drive 8466, San Diego, CA 92 103-8466 and Veteran’s Administration Medical Center, Department of Neurology, 3350 La Jolla Village Drive, San Diego, CA 92 161, USA R.N. McBurney Cambridge Neuroscience Inc., One Kendall Square, Building 700, Cambridge, MA 02 139, USA M.A. M o s k o ~ t z Stroke and Neurovascular Regulation Laboratory, Department of Neurology and Neurosurgical Service, Massachusetts General Hospital and Harvard Medical School, 149 13th Street, CNY 6403, Charlestown, MA 02 129, USA T.R Obrendtch Department of Neurochemistry, Institute of Neurology, Queen Square, London WC 1N 3BG, UK Q, Pye Oklahoma Medical Research Foundation Department of Free Radical Biology and Aging, Oklahoma City, Oklahoma, USA NJ. Rothwell School of Biological Sciences, 1.124 Stopford Building, University of Manchester, Oxford Road, Manchester M 13 9PT, UK B.K. SiesjZI Laboratory for Experimental Brain Research, Lund University, University Hospital, S-22 1 85 Lund, Sweden D.L. Small National Research Council Canada, Institute for Biological Sciences, Building M-54, 1200 Montreal Road, Ottawa, Canada K1A OR6 C.A. Stewart Oklahoma Medical Research Foundation Department of Free Radical Biology and Aging, Oklahoma City Oklahoma, USA U. Strasser Center for the Study of Nervous System Injury, and Department of Neurology, Washington University School of Medicine, St Louis, Missouri 63 1 10 USA R Stroemer School of Biological Sciences, 1.124 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK T. Tabatabaie Oklahoma Medical Research Foundation Department of Free Radical Biology and Aging, Oklahoma City Oklahoma, USA N.A. Tatton Department of Physiology and Biophysics, Institute of Neuroscience, Dalhousie University, Halifax, Nova Scotia, Canada W.G. Tatton Department of Physiology and Biophysics and Department of Psychology, Institute of Neuroscience, Dalhousie University, Halifax, Nova Scotia, Canada J. Wadia Playfair Unit, Toronto Hospital, Toronto, Ontario, Canada N.G. Wahlgren Karolinska Stroke Research, The Stroke Research Unit, Department of Neurology, Karolinska Hospital, S-17 1 76 Stockholm, Sweden

PREFACE Stroke is the third leading cause of death in major industrialized countries and a major cause of long-lasting disability. It has profound effects not only on the survivor, but also the family and society as a whole. There are now, however, significant grounds for believing that treatments may soon be available which, if given soon after a cerebrovascular accident, will substantially lessen the long-term neuronal damage that normally occurs, that is, a neuroprotective drug. In this book an international group of experts reviews the biochemical mechanisms which accompany as acute ischaemic episode and discuss ways that this knowledge may be applied to develop therapeutic compounds. Initial chapters by Tatton and colleagues and Kristian and Siesjo examine the way that cells die. The following chapters by Green and Cross and Goldberg and colleagues reviews techniques for examining the activity of putative neuroprotective drugs in uivo and in uitro. Since there are currently no drugs in use with proven clinical eficacy, a ‘battery’ of animal models of stroke and in vitro studies are required at present in order to have any confidence that a compound should proceed into clinical study. Subsequent chapters examine how knowledge of the biochemical changes occurring during an ischaemic episode may be applied to developing novel therapeutic compounds. Thus,Jackie Hunter and Tiho Obrenovich review the rationale for altering the function of ion channels following an ischaemic insult. This is followedby two chapters on NMDA antagonists since, reasonably, it can be claimed that the current enthusiasm for research on neuroprotective agents was initiated by studies on these compounds. Small and Buchan examine the current experimental and clinical status of NMDA antagonists, while Robert McBurney describes how one such compound was discovered and the work required to progress this drug to the clinic. Further chapters review the involvement of the AMPA receptor (Gill and Lodge), GABA (Lyden), adrenosine (Fredholm),interleukins (Rothwell and colleagues), free radicals (Floyd, Carney and colleagues) and nitric oxide (Dalkara and Moskowitz) in the neurodegenerative process; each chapter also describes how selective compounds acting on these Werent neurochemical processes might lead to novel neuroprotective agents. Finally, Nils-Gunnar Wahlgren reviews the many clinical trials that have been, and are being, conducted on potential neuroprotective drugs. Reading this chapter gives an insight into the challenges faced in producing a successful drug. However, these trials have taught us much and we feel hopeful that efficacious treatments are now ‘around the corner’. We also hope that researchers reading this book will gain ideas which will ensure that this hope becomes a reality,

A. Richard Green and Alan j! Cross Astra Arcus

...

XU1


Chapter I

MECHANISMS OF NERVE CELL DEATH: APOPTOSIS OR NECROSIS AFTER CEREBRAL ISCHAEMIA R.M.E. Chalmers-Redman*-*.A.D. Fraser***,W.Y.H. Ju***,J.Wadias, N.A. Tatton*" and W.G. Tatton**t*o Departments of *Physiology/Biophysics, +Psychology,and the 'Institute of Neuroscience. Dalhousie University, Halifax, Nova Scotia, and the *Playfair Unit, Toronto Hospital, Toronto, Ontario, Canada

1.1 I .2 I .3 I .4 I .5 I .6 I .7 I .8

Stroke and neuronal necrosis Neuronal apoptosis caused by trophic factor insufficiency Neuronal apoptosis due to a wide variety of causes Neuronal apoptosis in ischaemic-hypoxia Genes and proteins that promote or retard apoptosis Evidence that mitochondria contribute to the initiation of apoptosis Possible reduction of apoptosis caused by ischaemia-hypoxia with trophic factors Possible new anti-apoptotic functions for two old drugs References

I 2

3 7 10 12 14 15 16

I. I Stroke and neuronal necrosis Nerve cell necrosis was thought to be mainly, ifnot entirely responsible for the neurological deficits found in animal models of nervous system ischaemia or hypoxia (see Degirolami et al., 1984 for example). Accordingly, nerve cell death in human stroke was believed to be necrotic. A progressive and marked reduction of neuronal membrane potential, caused in part due to changes in K+ conductance, has been shown in the first 3-10 minutes after the onset of ischaemia or anoxia (see Martin et al., 1994). That loss of membrane potential causes massive synaptic glutamate release which activates glutamatergic receptors and thereby opens Ca2+channels on nearby nerve cells (Choi and Rotham, 1990).The resulting calcium overload sets in motion a series of events which rapidly lead to the swelling of organelles like mitochondria and the fracture of external cellular membranes. The high cytoplasmic calcium was proposed to have a number of effects: (1) a duablmg of mitochondrial function causing ATP NEUROPROTECTIVE AGENTS AND CEREBRAL ISCHAEMIA, IRN 40 ISBN 0-12-366840-9; 0-12-197880-X @bk)

Academic Press Limited Copyright 0 1997 All rights ofreproduction in any form resmved

1

2

R.M.E. CHALMERS-REDMAN et al.

levels to fall precipitouslywith a loss of ATP-dependent processes like Ca2+pumping; (2) rapid increases in the concentrations of cytoplasmic reactive oxygen species (ROS) with hdespread peroxidation of membrane lipids; and (3) the activation of Ca2+ dependent proteolytic enzymes. Together these changes caused swelling of the nerve cells, dissolution of intracellular organelles, fracture of external membranes, and a local inflammatory reaction to components of the extruded cytoplasmic contents. Treatment strategies for human stroke were proposed to counter the Ca2+induced cascades of events leading to neuronal necrosis (see Choi, 1995). Those strategies included the use of N-, P- or L-type Ca" channel blockers, non-activating Na' channel blockers, N-methyl-D-aspartate(NMDA) or a-amino-3-hydroxy-5-methyl-4isoxazoleproprionic acid (AMPA)receptor blockers, intracellular Ca2+chelators, and the reduction of ROS levels using agents like the 2 1 aminosteroids (Hall and McCall, 1994) or iron chelators (Connor and Menzies, 1995).

I.2 Neuronal apoptosis caused by trophic factor insufficiency In the 1970s and early 1980s, Kerr, Wyllie and coworkers (Kerr et al., 1972; Wyllie, 1987)provided evidence for a different form of cell death termed apoptosis. One clear example involved lymphocytes that were exposed to glucocorticoids (Wyllie, 1980). Characteristically,the apoptotic cells showed nuclear chromatin clumping followed by DNA condensation and the fractionation of the nucleus into nuclear bodies. DNA electrophoresisgels provided evidence for internucleosomal DNA fragmentation due to digestion by endonucleases. There was a definite delay between glucocorticoid exposure and the onset of the nuclear changes. The cells shrank rather than swelled; the plasma membrane remained intact but showed characteristic blebs. Finally the cellular remains appeared to be engulfed by macrophages without an apparent inflammatory reaction. Apoptosis seemed particularly important as a counterbalance to overexuberant cell replication but seemed to have little to do with nerve cells that were postmitotic and unable to replicate. About the same time, it was recognized that the massive death of neurones that occurred as part of vertebrate prenatal and postnatal brain development depended on competition for trophic factors (see Oppenheim, 1989, 1991). The neurotrophic hypothesis proposed that nerve cells depended on trophic support from their targets (i.e. other neurones or muscles which received their synapses)for survival and that the targets could supply only limited amounts of trophic molecules (see Johnson and Oppenheim, 1994).Hence developing neurones compete for trophic support. Elegant experimentsshowed that the neurones with 'the best connections' survived and maintained their connections while weakly connected neurones died (see Oppenheim, 1991). Death due to neurotrophic insufficiencywas termed programmed cell death as it was thought that it depended on the activation of an intrinsic programme leading to self-destruction. Most particularly it was established that developmentalprogrammed

MECHANISMS OF NERVE CELL DEATH

3

neuronal death involved the de nouo expression of 'death genes' and therefore required new protein synthesis (Oppenheim et al., 1990).The dependence on new protein synthesis was similar to that found in the death of cultured sympathetoblasts deprived of nerve growth factor (NGF') which showed many of the characteristics of apoptosis (Martin et al., 1988). Subsequently, it has become clear that a requirement for new protein synthesis is a hallmark of programmed neuronal death but not necessarily of neuronal apoptosis (Dragunow and Preston, 1995;Johnson et al., 1995). Some forms of neuronal apoptosis can proceed without new protein synthesis despite displaying the characteristic findings of cell and nuclear shrinkage, chromatin condensation, DNA fragmentation and membrane blebbing. For example, PC 12 cells that have been exposed to serum but not NGF undergo apoptosis with serum withdrawal that is unaffected by treatment with transcriptional/translational blockers that greatly reduce new protein synthesis (Rukenstein et al., 1991). Similarly, PC 12 cells that have been exposed to NGF for 6 days and have initiated process growth, but are not fully differentiated, undergo apoptosis after serum and NGF withdrawal that is similarly independent of new protein synthesis (Tatton et al., 1994). In contrast, PC 12 cell apoptosis caused by trophic withdrawal after 12 days of NGF exposure requires new protein synthesis (Mesner et al., 1992). It seems that macromolecules necessary for the progression of apoptosis are constitutive in some cells but must be induced in others (Eastman, 1993; Raff et al., 1993).Therefore, neuronal apoptosis can be dependent on new protein synthesis (programmed), independent of new protein synthesis (unprogrammed), or can even be facilitated by the inhibition of new protein synthesis (see Koh and Cotman,1992).

I .3 Neuronal apoptosis due to a wide variety of causes

It has rapidly become apparent that neuronal apoptosis is not only a result of trophic withdrawal but that a wide range of different insults can induce the process, even those that were thought to represent prototypic models of neuronal necrosis (Enokido and Hatanaka, 1994). For example, neuronal apoptosis can be induced by exposure to excitatory amino acids (Bonfoco et al., 1995b; Mitchell et al., 1994; Portera-Cailliau et al., 1995b), the Parkinsonian toxin 1-methyl-4-phenyl- 172,3,6-tetrahydropyridine (MPTP) (Seniuk-Tatton and Kish, personal communication), its metabolite, 1methyl-4-phenyl-pryridinium ion (MPP') (Dipasquale et al., 1991; Mochizuki et al., 1994; Mutoh et al., 1994) and 6-hydroxydopamine (Walkinshaw and Waters, 1994), all of which were once thought to cause only necrosis. Similarly, p-amyloid protein (the 25-35 fragment), mitochondrial complex I inhibitors (Hartley et al., 1994), calcium channel blockers (Koh and Cotman, 1992), pro-oxidants like H 2 0 2(Slater et al., 1995a), methamphetamine (Finnegan and Karler, 1992) excessive iron levels (Farinelli and Greene, 1996; Zsnagy et al., 1995); colchicine (Bonfoco et al., 1995a), ceramide (Brugg et al., 1996), agents found in cycad flour (Gobe, 1994), the AIDS protein, gp120 (Muller et al., 1992), some sialoglycoproteins (Kobayashi et al., 1994),

4


DNA synthesis blockers (Dessi et al., 1995),high levels of dopamine (Ziv et al., 1994), and axonal transection or crush (Ju and Tatton, unpublished observations) have been proposed to induce apoptosis in experimental models. Lower doses/concentrations of the agents, particularly if delivered over a more protracted time course, induce apoptosis, while high levels induce necrosis (Bonfoco et al., 199510). For example, higher concentrations of P-amyloid protein fragment induce necrosis (Behl et al., 1994) while smaller concentrations cause apoptosis (Forloni et al., 1993; Gschwind and Huber, 1995; Loo et al., 1993; Watt et al., 1994). Intermediate doses/concentrations often induce a mixture of necrosis and apoptosis, with the necrosis occurring in the first few hours after exposure and apoptosis appearing after a number of hours or even days. For example, we have found that 1-2 p~ MPP' induces apoptosis in embryonic mesencephalic dopaminergic neurones cultured in serum-free media, while 10 p~ causes rapid necrosis, and 5 mM causes a mixture of necrosis and apoptosis (Fraser,Leopold and Tatton, unpublished observations). Similarly, exposure to H 2 0 2(0.1 and 0.25 mM) largely induces apoptosis in NGF-differentiated PC 12 cells beginning 6 hours and more after the onset of exposure, while 1 .O mM induces necrosis within 3 hours. Exposure to 0.5 mM H202initially induces necrosis which is followed by apoptosis (Chalmers-Redrnan,Ju and Tatton, unpublished observations). Three methods have been used to identify neuronal apoptosis: (i)DNA fragmentation; (ii)cell shrinkage and the condensation of chromatin, and (iii) a requirement for de nouo protein synthesis. In most studies, changes in nuclear DNA or chromatin have been examined to differentiate neuronal apoptosis and necrosis: (i) DNA electrophoresis gels showing the 'ladder' pattern typical of oligonucleosomal DNA digestion (e.g. Batistatou and Greene, 1993); (ii) flow cytometry techniques sensitive to nuclear DNA condensation (e.g. Darzynluewicz et al., 1992); (iii) in situ staining with fluorescent dyes showing chromatin condensation and apoptotic bodies (e.g. Deckwerth and Johnson, 1993); and (iv) in situ 3' linker techniques (usually the ApopTag or terminal deoxynucleotidyl-transferase-mediateddUPT-x nick end labelling (TUNEL) methods) which attach a chromagen to the 3' cut ends of DNA (e.g. Tatton et al., 1994). Some authors have challenged the utility of 3' end labelling as a reliable in situ marker of apoptosis if used by itself (Charriaut-Mariangue and BenAri, 1995). They have argued that necrosis can cause light nuclear or cytoplasmic DNA labelling, possibly by labelling single-strand DNA breaks, which is not associated with the DNA condensation characteristic of apoptotic nuclei viewed with EM. We have used computer deconvolution techniques (Agard and Sedat, 1983; Shaw, 1994) to examine nuclei in mesencephalic embryonal dopaminergic neurones (MEDNs) treated with varying concentrations of MPP' and stained using the ApopTag peroxidase method (Fraser, Leopold and Tatton, unpublished observations). Figure 1A shows untreated control neurones raised in serum-free media and immunoreacted with an antibody against tyrosine hydroxylase (TH). Figure 1B shows identically raised and immunostained neurones at 72 hours after exposure to 2 p~ MPP'. Note the typical MPP' damage to the T H immunopositive neuronal processes in Figure 1B. DNA gel electrophoresis strips are superimposed on Figures 1A and 1B


5

FIGUREI Apoptosis in mesencephalic embryoha1 dopaminergic neurones exposed to MPP’ mesencephalic dopaminergic neurones taken from E 15 rat embryos and immunostained for tyrosine hydroxylase after 1 1 days in vitro. (A) Vehicle-treated controls. (B) Neurones treated with 2 mM MPP’ for 3 days. The insets show the DNA pattern obtained from gel electrophoresis with each treatment; note the ‘laddering’ with MPP’ treatment. (C) Examples of ApopTag-positive nuclei in MPP+-treated culture. (D) Deconvolution of ApopTag-positive nuclei demonstrating different stages of DNA strand breakage and changes in the conformation of nuclear DNA characteristic of apoptosis.

showing that 200 base pair (BP) ‘laddering’ was detectable in cells treated with the 2 p~ MPP’ concentrations but not in control cells (DNA was taken at 24 hours after MPP’ or control vehicle addition). ApopTag-positive nuclei were evident in MEDNs treated with 2 p~ MPP’ (an example is presented in Figure 1C , see Tatton et al. (1994) for detailed methods) but were not evident in untreated control neurones. Figure 3D shows examples of the ApopTag-positive nuclei after deconvolution ‘slicing’. The deconvolutions revealed changes in nuclear DNA conformation including so-called ‘nuclear capping’ and ‘buttoning’ which are identical to the nuclear changes shown with DNA-binding fluorescent dyes in apoptosis (Bonfoco et al., 1995b). In effect, the deconvolution methods allow the ApopTag method to simultaneously provide information on DNA strand breaks, nuclear DNA conformational changes, and DNA condensation. Similar deconvolution imaging may serve to obviate the need for EM images in determinations of neuronal apoptosis and thereby serve to strengthen or deny evidence for apoptosis which presently rests solely on ApopTag or TUNEL staining.

6

R.M.E. CHALMERS-REDh4ANet al.

The TUNEL and ApopTag in Situ methods have been used to examine apparently intact nerve cells in human postmortem material for evidence of DNA strand breaks consistent with apoptosis (Migheli et al., 1994).TUNEL or ApopTag-positive nuclei have been found in neurones examined in brain tissue from patients with Parkinson’s disease (Anglade et ab, 1995), Alzheimer’s disease (Cotman and Anderson, 1995; Cotman et al., 1994; Dragnow et al., 1995;Johnson, 1994; Lassmann et al., 1995; Smale et al., 1995; Su et al., 1994),glaucoma and several hereditary retinal dystrophies (Steinberg, 1994; Tso et al., 1994), Huntington’s disease (Dragunow et al., 1995; Portera-Cailliau et ab, 1995b), amyotrophic lateral sclerosis (Thomas et al., 1995; Yoshiyama et al., 1994), spinal muscular atrophy, AIDS encephalitis (Petito and Roberts, 1995b),and status epilepticus (Pollard et al., 1994).Other investigators have failed to find evidence of nuclear DNA strand breaks and therefore of apoptosis in some of the above conditions (Migheli et al., 1994). Relatively high percentages of nuclei with in situ detected DNA strand breaks have been reported in Parkinson’s (1-2% of substantia nigra compacta neurones in Anglade et al., 1995)and Alzheimer’s disease brains (as high as 85% in some parts of the entorhinnal cortex in Anderson et al., 1996). Given the likely short life of nuclei with strand breaks in nervous system apoptosis (Raff et al., 1993) and the relatively slow progression of nerve cell death in Parkinson’s and Alzheimer’s diseases, the percentages seem far too high, thus raising the possibility that the in situ end labelling methods are detecting nuclear events other than those associated with apoptosis. Maintenance of human brain tissue in some fixatives or drying of tissue can induce false positives (Petito and Roberts, 1995a)while maintenance in formalin beyond 3-5 weeks can reduce the capacity of the techniques to detect DNA strand breaks. The recent finding of co-localization of c-JUN immunoreactivity, known to represent an early event in neuronal apoptosis (see below), in the same nerve cells as those showing in situ evidence of nuclear DNA strand breaks in Alzheimer’s brains, lends support to the view that the in situ methods were in fact marking nerve cells which have entered the apoptotic process (Anderson et al., 1996).It therefore has been suggested that increased c-JUN levels mark nerve cells that have entered apoptosis and that the relatively high percentages of nuclei that are positive for nuclear DNA strand breaks reflects an immediately pre- or post-agonal acceleration in DNA cleavage in nerve cells that were already committed to apoptosis (Petito and Roberts, 1995a). Recent studies of dopaminergic nerve cells exposed to MF’TP have shown increased Bax expression in cells that are apparently entering apoptosis (Hassouna et al., 1996). In order to avoid future controversy about the utility of in situ DNA end labelling in detecting apoptosis in experimental models or human brain tissue, three steps should be taken: (i) the ApopTag or TUNEL methods should be combined with fluorescent strainingof nuclear DNA in order to provide evidence of two independent changes in nuclear DNA, (ii) deconvolution methods should be used with light or fluorescence microscopy to define subnuclear structural changes in DNA strand breaks or chromatin condensation: and (iii) immunocytochemistxyfor death-promotingproteins like BAX or c-JUN should be combined with the DNA end labelling techniques or fluorescent straining of nuclear DNA.


7

The apparent profusion of experimental models of nerve cell death and neurological conditions that appear to involve apoptosis have been interpreted by some as evidence that the methods used to identift apoptosis may be nonspecific. However, it may not be surprising that neuronal apoptosis is common in neurological conditions, if one takes the view that apoptosis is the product of low level insults which are insufficient to kill nerve cells but are sufficient to activate a ‘suicide’ system.

I .4 Neuronal apoptosis in ischaemia-hypoxia Numerous studies have now reported apoptosis as contributing to the neuronal death found in experimental models of ischaemia. As shown in Table 1, the ischaemic models have differed widely in terms of species, site of arterial occlusion, duration of occlusion, the time after the onset of occlusion when the tissue was studied, and the brain region or regions that were studied. Similar to the variability of the models employed, the studies have varied in the criteria used to establish apoptosis or necrosis. Table 1 attempts to demonstrate ‘ladders’ on DNA electrophoresis under 14 experimental ischaemic conditions, and under 11 of those conditions DNA ‘laddering’was evident in tissue taken from the cortex, hippocampus, striatum or other forebrain areas. The DNA ‘laddering’ presents strong evidence for the endonuclease activation thought to be a major characteristic of apoptosis. Eighteen of 22 studies using ApopTag or TUNEL staining found in situ evidence of nuclear DNA strand breaks consistent with apoptosis. Eight studies combined DNA electrophoresiswith ApopTag or TUNEL staining: all found both DNA ‘ladders’and in situ evidence of nuclear DNA strand breaks. Another five studies combined either DNA electrophoresis with fluorescent staining of nuclear DNA or ApopTag or TUNEL with fluorescent staining of nuclear DNA as a means of merentiating apoptotic and necrotic neuronal death. All of these studies found agreement between the different methods in terms of identifying apoptosis. To date, only two studies have used all three methods - DNA electrophoresis, in situ methods to detect nuclear DNA strand breaks, and fluorescent staining of nuclear DNA. In each case, the three methods were found to agree on the presence of apoptotic nerve cell death after ischaemia (MacManus et al., 1993; 00et al., 1995).One study (Linnik et al., 1993)provided evidence for neuronal apoptosis after ischaemia using DNA electrophoresis with flow cytometry methods. The study also found that the neural death was reduced by treatment with a protein synthesis inhibitor, suggesting that at least some forms of neuronal apoptosis caused by ischaemia are programmed in nature. Two studies have found increased c-JUN immunoreactivity in hippocampal or other forebrain neurones after ischaemia (Dragunow et al., 1993, 1994) and have suggested that increased c-JUN expression is an early marker of commitment to apoptosis (see below). In situ methods to detect nuclear DNA strand breaks were not employed in those studies to determine if nerve cells with evidence of increased c-JUN levels also

TABLE 1 NECROSIS O R APOITOSIS AFTER ISCHAEMIA

Species

Apoptosis Necrosis

Reference

Rat

X

p k i l h m d ol., 1995)

Rat Rat

x

(Beilharz d d.,1995) (Charriatut-Mariangue

x

Damage oragent

Type

Ischacmia/ uCAO hypoxia uCAO kchem./ hypoxia Ischaemia MCAO

APOP

Duration ischaemia orhypoda

Survival

NeweccU

time

'ype

I5 m

5 h-5 d

Cortex

Wm

5 h-5 d

Cortex

Ih

W

Forebrain

60m I20 m decr. O2

Id Id 48h

C5 m

4d

COrtCX, S U . cortex cerebellar

&I8 h

I8 h

Forchain

Ih

1W20m

C O R ~hippo ,

h

DNA Ladders

Somal size

Tag Flow Chrom.con- T d . Transc. NudearEM TUNEL cytom. densation blockade blodrade findine P

-

dol., 1995)

Rat Rat

we

(Chen d d.,1995) (Chen ad.,1995) (Emuardsdd.,1995) (Hara rt d.,1995)

x x x

H u m

Rat

X

(Hiuttd.,1995)

Rat

x

(Islam el d.,1995)

Ischaemia

Ischacmia Ischaemia Hchaemia

MCAO MCAO hCA0 dccr. BP

hypoxia Ischaemia/ UCAO hypoxia lschaemia hCCAO

su. -

P

-

P

-

?

P

Y

P

-Pus Cerhil Rat

(Iwaidd., 1995)

x X

x

( L d d . , 199%)

Rat

x

Rat MOUW Mouse Rat

X

X

(Iid d.,1995d) (Iicf d.,1995a) ( L i e f & , l995b) (Iid d.,l995b) (LinniLdd, 1993)

Rat

X

(Linnikdd., 1995)

Mouse Gerbil Rat

x

(Matsuyama d d.,1995)

X

(Nitatond d,1995)

X

(00d d,1995)

X

X

bCCA0 uMCA0 lschaemia MCAO Hchaemia MCAO Ischaemia MCAO MCAO lXhacmia Ischaemia "CC+ MCAO Ischaemia "CC+ MCAO Ischaemia bCA0 lschacmia bCCA0 Ischaemia/ UCAO hypoxia Ischaemia

Lpchaemia

5m 2h 2h 1&120m 2h 2h 24 h

2-7 d

Hrppo -pus

.2-28 d 22 h 48h 22 h 22 h 0

Cortex, str. Cortex, SU. cortex, str. cortex, l r . cortex, su. Cortex

24 h

24 h

Cortex

30 m 5m H h

€&24 h

Hippacampus, etc %pacampus SN

12h-7 d &7 d

shrink

P P

P Shrink P P -

-

Y

P -

-

P

-

P -

P

P

-

Y

Shrink NC

P -

-

P

Apoptosi bodies Dense patches

Gerbil

x

Rat Rat Rat

x x x

Rat

X

Rat

x

Gerbil Rat Rglet

x

Gerbil

x

Rat

x

(Kihara d d., 1994) (MacManur d a[., 1994) (Mehmetdd., 1994)

x

x

x Rat x Gerbil x Gerbil/rat x Rat

Rat

Gerbil x Monkey Monkey x

(Tohitadd., 1995) (Volpe ctd., 1995) (Volpecfd., 1995) (Volpe d d.,1995) (Volpe d ol., 1995) (Dragunow d d.,1994)

(Seidd., 1994) (Dragunow d d.,1993) X

Garcia tf d.,1993) (MacManusdnL, 1993) ( O h o t o dd.,1993) (Roberts-Lewiscld.,

X

x x

1993) (Deshpande dol., 1992) (Shigcno d d.,1990) (Degbolamidd., 1984) (Garcia and Kimijyo, 1974)

bCA0 MCAO MCAO MCAO MCAO lschaemia/ CAO hypoxia Ischaemia K C A O lscharmia MCAO lschaemia/ h C A 0 hypoxia lschaemia b C A 0 khaemia/ CAO hypoxia Ischaemia MCAO khaemia CA+MCAO lschaemia bCCAO Ischaemia/ M C A O hypoxia Ischaemia bCCAO Irhaemia hCAO khaemia M C A O lschaemia M C A O

lschaemia lschaemia lschaemia Ischaemia Ischaemia

15m 20m 20m 20 m 20 m 15m

M h 72 h 24h 1-120 h 1-120 h 24h

2m 2h 61 m

I-7d 22 h 48 h

IOm 15 m

24 h

0.5 k 7 d

12-96 h

Hippocampus Hippocampus, LC. Str.

Thalamus Cortex forebrain hippocampus cortex

IOm

0h 48 h 2+%h 24

10 m 5m 15m-15 d 2.5 h-7 d

Hippocampus 6 7 2h Hippocampus 7d 24 h-28 d Forebrain 0h Forebrain

8-16 m

5m

Y Y N N

~

Y

cortex

Hippocampus Forebrain Forehain Hippocampus, st^. Forebrain

>

~

Y

-

-

P P

-

N N

-

P P P

~

~

Y

Swd -

Y

-

-

P

~

P

-

-

-

N

-

P

-

Cortex

-

-

-

N Shrink Both

~

-

-

N

NC

Y

-

-

Key: b - bilaterak BP - blood pressure; CA - cerebral artery; CAO - cerebral artery occulsion; CCAO - common carotid artery occlusion; cerebell.- cerebellum;Chrom. - chromatin, concen. - concentration; Cytom. - Cytomeq; d days;durn. - duration; decr. - decreased; E M - electron microscopic; h - hours; inhibit. - inhibition;m - minutes; MCAO -middle cerebral artery occlusion; N - no;/negative; N C - no change; P - positive. SN substantia n i p ; str striaturn; Transc. - transcriptional,T d . - transktional, uCAO - unilateral cerebral artery occlusion; uCC unilateral common carotid; uMCAO - unilateral middle cerebral ancry occlusion; Y - yes ~

~

~

10


showed evidence of nuclear DNA digestion or chromatin condensation. Similarly, neurones were found with increased BCL-2 immunoreactivity after middle cerebral artery occlusion in the rat (Chen et al., 1995).It was not determined whether neurones with or without evidence of nuclear DNA changes showed the increased BCL-2 immunoreactivity. If neurones without increased BCL-2 levels were those with evidence of nuclear DNA changes, then BCL-2 might be taken as a marker for surviving nerve cells after ischaemia (see below). Taken together, the results of the studies presented in Table 1 strongly support a role for apoptosis in neuronal death after ischaemia. The extent of apoptosis versus that of necrosis under specific conditions and in relation to regional changes in arterial perfiusion remains uncertain. Table 2 presents similar information to Table 1 but summarizes findings from in viva and in uitro experimental models that have examined neuronal death caused by factors such as excitotoxin exposure, pro-oxidant exposure, mitochondrial poisoning and hypoxia, which are similar to the events that are thought to play a role in the cascades initiated by ischaemia. Similar to the ischaemic studies shown in Table 1, DNA electrophoresis revealed ‘laddering’ in most studies in which it was utilized, and the finding of DNA ‘laddering’ was mirrored by in situ findings of nuclear DNA strand breaks or chromatin condensation when multiple techniques were employed. Some authors ruled against apoptosisbased on an inability of transcriptional or translational blockers to reduce neuronal death, even when DNA ‘ladders’ or in situ nuclear changes were present (see examples in Table 2). The absence of a dependence on new protein synthesis showed that the neuronal death was not programmed but should not have been interpreted as evidence against the presence of apoptosis (see above).

I.5 Genes and proteins that promote or retard apoptosis Apoptosis has been divided into four stages: (i) cell cycle arrest; (ii) capacitation for apoptosis and proliferation; (iii) irreversible commitment to death or pre-apoptosis, and (iv)nucleolysis, chromatolysis and proteolysis (see Kromer etal., 1995for a current review of apoptotic mechanisms and pertinent general references for apoptosis). In recent years, a number of genes and their protein products have been identified which have the capacity to influence the progression of apoptosis. The gene products that can influence neural apoptosis have been extensively reviewed recently (Bredesen, 1995)and will not be detailed here, but pertinent references can be gleaned from that source. (In the following material, upper case letters usually indicate the protein product and lower case letters usually indicate the gene.) From the aspect of the potential treatment of neuronal apoptosis, the genes that promote the transition from stage 2 to stage 3, or those that reverse the stage 1 to stage 2 transition, are most interesting (see Kroemer et al., 1995).The genes bax, bcl-K, bad, bak, ICE (interleukin 1b converting enzyme), prICE and ICE- 1L promote the transition from stage 2 to stage 3, while bcl-2, bcl-xL, Bcl-x,, A 1, Mcl- 1, BAG- 1, abl, raf- 1 and ICH- 1, decrease entry into that transition. In nerve cells, the bax/bcl family @ax,

TABLE 2 NECROSISO R APOprOSIS CAUSED BY C O M P O N E N T S OF THE ISCHAEMIC CASCADE ~~

~~

~

~~~

~~~

I n i d Species inoiho

Apop- Ncctosir rosb Rererence

Damage or agent

Excitotoxic Rat mfro

x

Lawrencc etnl., 1996

Rat Rat Rat

mtra

mim

x

x x

Rat

~ino

x

x

Rat Rat Rat Rat

Lifm

x

uifm

x

vifm

?

UiDO

x

uiha

Diho

x

Rat mfm Rat viho Rat dm Rat vim Rat V*o uiw Rat Rat vim PrWxidant Rat mfm Rat mfm

x

Rat Mouse

mfm

x

vim

x

Rat

x

x

x

x x

x x

x

x

Mouse V*o x Rat mfro x Mitochondrialtoxin Mouse uirm x Mouse vifro x

x

Bonloco d nl., 1995 Bndixo d aL, 1993 Portera-Cailliall r t d . , 1995 Portera-Cailliallrtd., 1995 ReganctoL, 1995 Regancfd., 1995 Pollard cf oL, 1994 Prehn eta/., 1994 Bchlrtd, 1993 Behlcfd, 1993 DerJi ct d. 1993 Kure d al., 1991 Kurc cf aL, 1991 LeppinefaL, 1992 Leppin ct ol., 1992 Leppin rf 01.. 1992

Duration/ exposure

Survival time

Doseor concentration

Nervecell

Delivery

"Ipe

DNA Somal ladders size

Glutamate/ hypoglycacmia NMDA NMDA Quinolmicacid

-

30m

-

25200pM

Hippocampus

-

IOm

10 h

300 pM 2 mhl

COrtCX Cortex

240 nhV0.5 phl

Quinolinicadd

IC

-

12-16 h

10m SI2h

20m 12h 12h 15m W24h

Glutamate Kainatc,AMPA Kainatc NMDA Glutamate Glutamate Glutamate Glutamate Glutamate NMDA AMPA Kainatc

IOm IC

~

IC

IC 1c IC IC

~

-

su.

N Y

NC Swell -

P N P

240 nM/0.5 pM

str.

Y

-

P

&72 h

500 pM 35, 10 pM 1.2 pgO.3 pI

Y Y

100 PM

NC Shrink SweU

-

-

Cortex Cortex amyg.,hippo. Hippocampus PC-12,etc. PC-12, etc. Cerebellum cortex Hippocampus

-

0-24h 7-IOd 7-IOd 7-10d

C6.25mM 12.5 mM IWpM ImM 20mM/ZpM 100-2OOmM 50mM

Y -

P -

N Y

Y

SV.

-

(t24 ph4 10 PM

-

IW pM

2-20111

IlOmg/kg

Hippocampus Cortex Cortex Cortex

-

ICV

G18h -

Mukhejec rf d,1995 Prehn cf oL, 1994

I-butylhyd. FcNS

Icv

-

1248h

24h

-

22mg/lrg 0.1-IOphl

Cortex Hippocampus

Myen tt d.,1995 Bchrenr rt nl., 1994

KCN, aglycaemia Rotenone

-

&6Om 2H8h 2W8h

-

3P M 10 PM

Hypothalamic Cortex

-

-

3p M

24-48h

-

-

Shrink -

Cortex

Ic M

corlcx

-

1.5h

-

I-IWpM

Cortex

24h

2h

-

Sympath.

mfm

x

Mow

Virro

x

Gwagd nl., 1994

Rat

miro

x

Oligomycin FCCP

Hypoxia/ aglycamia Roscnbaum ct d.,1994 Hypoxia

-

NI/COI

-

P N P P

StT.

5mM

Patches

-

str.

Pcroxynitrate f-butylhyd.

B e h n s d el., 1994 Bchrens d d.,1994

-

N N

N N

Condensation Lucent

-

N N N N

Bonfoco *Id., 1995 htukhejee tf aL, 1995

x

P N

-

-

mfro

-

-

Adriamycin Peroxpimtc

0-18h

Flow Chrom.con- Transl. Trans. NuclearEM Cytom densation blockade blockade findings

-

Lawence d ol., I996 Bonfoco t f al., 1995

Mouse Mouse

24h

Y

APOP Tag TUNEL

-

-

N Y Y -

-

NC NC -

P N P

-

P P

-

P

-

-

P

-

P -

-

-

Envelope disluption -

-

Y

-

Y

-

Shrink -

Y

-

N

Shrink

N

-

Y

-

-

Y

shrink

-

-

-

P -

P

-

-

-

Key: p~ - micromolar;AhPA - a-amin~3-hydro-5-mcthyl-4-oxazolcpropnonic acid; amyg. - amygdala; CO1 - carbon dioxide; d. - days; FCCP carbonyl-zyanidcptritluoromctho~hcnylhydrazone; FeNS - ferrous ammonium rulphate; h -hours; IC - intracerebral; ICV - htracerebrovcnuicular; KCN - potassium cyanide, kg - Idlograms, m - minutes, mito. -mitochondria, mg - milligrams, ml - milliliters, m~ - millimolar, Nz - nitrogen gar, N - no/nega&e, NC - no change; NMDA-N-methyl-Barpate; P - positive; PC-12- rat pheochromocytomacell Line; Su.- striaturn;sympath. - sympatheticneuron; f-butylhyd. -tertiary butylhydropcroxide. ~

12

R.M.E. CHALMERS-REDMANel al.

bcl-2, bcl-xL)(see Oltvai and Korsmeyer, 1994)and the ICE family (ICE, ICE-1L and ICH- 1J (see Takahashi and Earnshaw, 1996) have received particular attention. increased expression of bax of ICE- 1 promotes neuronal apoptosis, while increased expression of bcl-2, bcl-xL or ICH-1, promotes survival. The mechanisms through which the protein products of those genes act on apoptosis are of intense interest but remain uncertain. The BAXIBCL family are found in the membranes of mitochondria, endoplasmic reticulum and the nucleus. The fact that a major portion of the BCL and BAX proteins reside in mitochondria is interesting in light of new findings that implicate mitochondria in the initiation of apoptosis (see below). Two other genedproteins have been clearly shown to influence neuronal apoptosis. First, the intermediate early protein c-JUN seems to be expressed in the early stages of neuronal apoptosis (see Dragunow and Preston, 1995), antisense oligonucleotides against c-jun reduce neuronal apoptosis (Schlingensiepen et al., 1994) and the overexpression of a negative c-jun mutant facilitates neuronal survival, while overexpression of c-jun increases apoptosis (Ham et al., 1995). Second, the gene for the scavenger protein, Cu/Zn superoxide dismutase (SOD 1) has been shown to decrease apoptosis when overexpressed and to increase apoptosis when underexpressed (Rothstein etal., 1994; Troy and Shelanski, 1994).

I .6 Evidence that mitochondria contribute to the initiation of apoptosis

Electron energy from the tricarboxylic acid cycle is converted into a transmembrane proton concentration gradient by mitochondria. The proton gradient serves to convert ADP to ATP through ATP synthase (see Nicholls and Ferguson, 1992 for an extensive review of mitochondrial function). Each of three mitochondrial complexes (I, 111 and IV) reduce the energy of electrons in the carrier molecules NADH, ubiquinone or cytochrome C, and use that energy to pump protons out of the mitochondrial matrix. Mitochondrial membrane potential (AY, normally about - 150 mv inside) and proton concentration difference (ApH)contributes to proton electromotive force (6p) (6p=AY-60 ApH), where ApH=(mitochondrial pH-cytosol pH) (Chacon et al., 1994). Since AY is by far the greater contributor to 6p, AY covaries almost linearly with the production of ATP or the ATP/ADP ratio. If cytosolic Ca2+levels are elevated, the uptake of Ca2+is driven by the AY - allowing mitochondria to store relatively large amounts of Ca2+(Richter and Kass, 1991). Factors which cause a marked increase in mitochondrial membrane permeability and the free distribution of mitochondrial ions and small solutes result in a loss of AY and a failure of energy production (Bernardes et al., 1994). Situations in which Ca2+and other ions can pass freely across mitochondrial membranes or in which high levels of Ca2+ accumulate in mitochondria due to high cytosolic levels (van de Water et al., 1994)will result in a compromise of AY and of mitochondrial energy production. Mitochondrial impairment may contribute to neuronal death in neurodegener-


13

ative diseases (Beal, 1992; Bed et al., 1993; Frim et al., 1993; Mattson et al., 1993b; Mutisya et al., 1994). Experimental apoptosis induced by tumour necrosis factor-a (Schulze-Osthoffet al., 1992),MPP’ (Tipton and Singer, 1993),complex I inhibition by rotenone (Hartley et al., 1994),manganese (Brouilletet al., 1993),complex I1 inhibition by 3-nitroproprionic acid (Brouillet et al., 1993),inhibition of mitochondrial DNA replication (Baixera et al., 1994) and pro-oxidants like H 2 0 2(Richter, 1993) have all been shown to involve mitochondrial impairment. Richter has recently argued that abrupt reductions of the ATP/ADP ratio to less than 0.2 result in necrosis, while smaller reductions result in apoptosis (Richter et al., 1995).He points out that apoptosis requires energy and a complete loss of available ATP may therefore be incompatible with apoptosis. Raff and coworkers (Jacobson et al., 1993)showed that mitochondrial DNA deficient fibroblasts could die by apoptosis and that increased bcl-2 expression reduced that apoptosis. Based upon those findings, it was postulated that mitochondria are not essential to the progress of apoptosis and that BCL-2 does not reduce apoptosis through a mitochondrial mechanism. It has been argued that this view may not be valid since the fibroblasts compensate for the loss of proteins derived from mitochondrial DNA by upregulating the glycolytic production of ATP and by maintaining B Y through ATP hydrolysis (see Richter et al., 1995, for details and references). Hence mitochondria deficient in DNA could still generate AY dependent ‘signals’ that are critical to the progression of apoptosis and BCL-2 could interfere with that signalling. Recently experiments in cell-free systems have shown that mitochondrial factors are essential to apoptosis (Newmeyer et al., 1994)which may be in accord with an involvement of mitochondria in apoptosis signalling. A number of studies in non-neural cells have shown that a progressive decrease in B Y can begin well before the onset of the nuclear stigmata of apoptosis like chromatin condensation or DNA strand breaks (Petitet al., 1995;Vayssiere et al., 1994; Zamzami et al., 1995a,b).Similarly we have shown that AY begins to decrease in pre-apoptotic PC 12 cells by 3 to 6 hours after trophic withdrawal, 2 to 4 hours before most cells show evidence of chromatin condensation or nuclear DNA strand breaks (Tatton et al., 1996). Both BCL-2 and BAX have been localized to mitochondrial membranes. There is disagreement as to whether they are located in the outer or the inner mitochondrial membranes (Hockenbery et al., 1990; Janiak et al., 1994; Lithgow et al., 1994; Monaghan et al., 1992; Nakai et al., 1993).BCL-2 also has been found in endoplasmic reticular and nuclear membranes (Hockenbery et al., 1990; Janiak et al., 1994). Truncated BCL-2 which cannot dock in mitochondrial membranes and remains in the cytosol was less effective than BCL-2 located in mitochondrial membrane in reducing apoptosis (Hockenbery et al., 1993).Expression of human bcl-2 in C. eleganc blocks developmental nerve cell death in the worms in a similar manner to the endogenous gene, ced-9, which shares sequence homology with bcl-2. Ced-9 is an element of a polycistronic locus that contains the cyt- 1 gene, which encodes a protein similar to cytochrome b560 of complex I1 of the mitochondrial respiratory chain in mammals (Hengartner and Horvitz, 1994).

14


Bcl-2 overexpression in a fibrosarcoid cell line was shown to prevent a decrease in

AY that was associated with apoptosis caused by tumour necrosis factor (Hennet et al., 1993). Conversely, we have shown that a decrease in bcl-2 levels in trophically deprived PC 12 cells is associated with a progressive decrease in AY in the cells (Ju, Wadia et al., unpublished observations).The capacity of bcl-2 overexpressionto block apoptosis is overridden by mitochondrial dysfunction caused by inhibitors of the mitochondrial respiratory complexes (Smets et al., 1994; Wolvetang et al., 1994),and those inhibitors can induce apoptosis in cells that express normal levels of bcl-2 (Wolvetang etal., 1994).It has also been shown that the reduction in apoptosis induced by bcl-2 overexpression is associated with a decrease in oxidative radical levels and reduced peroxidation of membrane lipids (Hockenbery et al., 1993; Reed, 1994). We have used confocal microscopy with oxidative radical sensitive fluorescent dyes to show that a decrease in BCL-2 levels in trophically deprived PC 12 cells entering apoptosis is associated with high cytosolic levels of oxidative radicals (Tatton et al., 1996).BCL-2 has been shown to alter the subcellular partitioning of Ca2+with an increase in cytosolic Ca", which would be expected to compromise mitochondrial membrane potential and therefore mitochondrial respiration (Bafi et al., 1993). Therefore, the increases in cytosolic oxidative radical levels could be secondary to a failure of mitochondrial function due to a loss of mitochondrial BCL-2, since a reduction of ATP production has been shown to induce high levels of mitochondrially derived superoxide radicals (Richter, 1993).It has been suggested that mitochondrial BCL-2 might stabilize AY and therefore decrease oxidative radical production and prevent the progression of apoptosis (Richter et al., 1995). Increased expression of the gene for the scavenger protein, Cu/Zn superoxide dismutase (SODl), reduces oxidative radical levels and blocks neuronal apoptosis (Greenlund et al., 1995).Oxidative radicals can cause neuronal necrosis by lipid peroxidation and fragmentationof external cellular membrane. However, it also has been suggested that lower levels of oxidative radicals may serve to signal apoptosis (Johnson et al., 1995; Slater et al. 1995b).Accordingly, the decrease in oxidative radicals found with bcl-2 overexpressionmay be responsible for an alteration in apoptosis signalling, which may derive from a stabilizationof mitochondrial function and decreased oxidative radical production.

I.7 Possible reduction of apoptosis caused by ischaemia-hypoxiawith trophic factors Recombinant molecular methods have made it relatively easy to produce large amounts of the human forms of a variety of trophic factors and therefore have made their therapeutic use possible (Baringaga, 1994). Some trophic factors have been shown to reduce neuronal loss in experimental models of nervous system ischaemia while others are thought to contribute to ischaemic cascades (Boniece and Wagner, 1993; Cheng and Mattson, 1991; Mattson and Cheng, 1993; Mattson et al., 1993a;


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Mattson and Scheff, 1994; Pechan et al., 1995; Zhang et al., 1993) and therefore may have utility in reducing neurological deficits after stroke. Some trophic factors were shown to be effective in reducing neuronal apoptosis in experimental models of neurodegeneration, such as experimental models of motoneurone death similar to that in amyotrophic lateral sclerosis (ALS)(see Sendtner et al., 1991, 1992a,b). Several trophic factors have been examined recently as therapeutic agents in neurodegenerative diseases. For example, human recombinant ciliary neurotrophic factor (CNTE) was utilized to treat A L S in a double-blind placebo trial (Miller et al., 1996). Systemically delivered CNTF did not provide discernible improvement in any of the A L S indices examined. Furthermore, the CNTF treatment was found to induce numerous deleterious side-effects like fever and marked weight loss and was associated with increased deaths. The weight loss was similar to that previously found with systemic (Henderson et al., 1994)or intrathecal (Zhang et al., 1995) CNTF delivery in rodents. Although delivery of CNTF into the immediate vicinity of damaged neurones (Hagg and Varon, 1993)or directly to cut axon ends (Sendtner et al., 1991) has been shown to be effective in increasing neuronal survival, intrathecal CNTF doses just sufKcient to increase neuronal survival produced weight loss and death in experimental animals (Zhang et al., 1995). These findings suggest that powerful neurotrophic agents like CNTF may have to be delivered into the immediate area of the damaged neurones in neurological disorders. Proteinaceous trophic factors do not cross the blood-brain barrier easily, although some are transported retrogradely along the axons of neurones projecting to the periphery. Hopefdy, targeted carrier molecules will be developed that will transport the trophic factors specifically to the damaged neurones. The development of targeted delivery methods may determine ultimately the utility of treatment with trophic factors as a means of reducing neuronal apoptosis in humans after brain ischaemia.

I .8 Possible new anti-apoptotic functions for two old drugs Two agents, both previously thought to reduce the levels of oxidative radicals in neurones by oxidative radical scavenging or by altering monoamine metabolism, have been shown to selectively alter gene transcription and to reduce apoptosis. N-acetyl cysteine (NAC)was thought to reduce apoptosis through its conversion to glutathione which served to scavenge oxidative radicals. Recent work has shown that NAC reduces apoptosis independently of its conversion to glutathione and the reduction in apoptosis depends on the induction of new protein synthesis by NAC (Ferrari et al., 1995; Yan et al., 1995). Similarly, (-)-deprenil was thought to act by inhibiting MAOB with a consequent reduction in H 2 0 2 production from dopamine metabolism (Olanow et al., 1995; Parkinson, 1993). It has been shown that the metabolite of (-)deprenil, (-)-desmethyldeprenil,reduces apoptosis and induces new protein synthesis in pre-apoptotic nerve cells in a manner similar to NAC (Tatton et al., 1994). The

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metabolite of (-)-deprenilinduces increased synthesis of members of the BCL family, decreased synthesis of BAX, and increased synthesis of several scavenger proteins like SOD 1 (Tatton et al., 1996). Likely by means of these changes in gene expression, in particular the increased synthesis of BCL-2, (-)-desmethyldeprenilmaintains AY and reduces oxidative radical levels in the pre-apoptotic nerve cells (Tatton et al., 1996). NAC induces the expression of a number of the same genes as (-)-desmethyldeprenil (Ju and Tatton, unpublished observations) and also appears to reduce apoptosis through a mitochondrial action (Cossarizza et ab, 1995). Agents which selectively alter transcription and/or mitochondrial function relative to apoptosis may offer a practical means of reducing neuronal apoptosis after brain ischaemia. Furthermore, their investigation may lead us to a better understanding of the molecular and cellular mechanisms underlying nerve cell death caused by ischaemia.

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damage consistent with apoptosis in transient focal ischaemic neocortex. NeuroReport, 5 , 493496. Martin, D.P., Schmidt, R.E., DiStephano, P.S., Lowry, O.H., Carter, J.G. &Johnson, E.M. (1988) Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation.J. Cell Bwl., 106,824-843. Martin, R.L., Lloyd, H.G. & Cowan, A.I. (1994) The early events of oxygen and glucose deprivation: setting the scene for neuronal death? Zenh Neurosci., 17,251-257. Matsuyama, T., Hata, R., Yamamoto, Y, Tagaya, M., Akita, H. & Uno, H., et al. (1995) Localization of Fas antigen mRNA induced in postischaemic murine forebrain by in situ hybridization. Mol. Brain Res,, 34, 166-1 72. Mattson, M.P. & Cheng, B. (1993) Growth factors protect neurones against excitotoxichchaemic damage by stabilizing calcium homeostasis. Stroke, 24. Mattson, M.P., Rydel, R.E., Lieberburg,I. &Smith Swintosky, VL. (1993a)Altered calcium signaling and neuronal injury: stroke and Alzheimer’s disease as examples. Ann. MAcud. Sci., 679,l-21. Mattson, M.P. & Scheff, S.W. (1994)Endogenous neuroprotection factors and traumatic brain injury: mechanisms of action and implications for therapy3 Nmrotrauma, 11, 3-33. Mattson, M.P., Zhang, Y & Bose, S. (1993b) Growth factors prevent mitochondrial dysfunction, loss of Ca” homeostasis, and cell injury, but not ATP depletion in hippocampal neurones deprived ofglucose. Exp. Neurol., 121, 1-13. Mehmet, H., Yue, X., Squier, M.V, Lorek, A., Cady, E. & Penrice,J., et al. (1994) Increased apoptosis in the cingulate sulcus of newborn piglets following ,transienthypoxia-ischaemia is related to the degree of high energy phosphate depletion during the insult. Neurosci. Letts., 181, 121-125. Mesner, PW., Winters, T.R. & Green, S.H. (1992)Nerve growth factor withdrawal-inducedcell death in neuronal PC 12 cells resembles that in sympathetic neurones. J. Cell Bwl., 119, 1669- 1680. Migheli, A., Cavalla, P., Marino, S. & Schiffer, D. (1994)A study of apoptosis in normal and pathologic nervous tissue after in Situ end-labellingof DNA strand breaks.J. Neuropathol.hip. Neurol., 53, 606- 16. Miller, R.G., Petajan,J.H., Bryan, W.W., Armon, C., Barohn, RJ. & Goodpasture,J.C., ef al. ( 1996) A placebo-controlled trial of recombinant human ciliary neurotrophic (rhCNTF‘) factor in amylotrophic lateral sclerosis. Ann. Nmrol., 39, 256-260. Mitchell, IJ., Lawson, S., Moser, B., Laidlaw, S.M., Cooper, AJ. & Walkinshaw, G. et al. (1994) Glutamate-induced apoptosis results in a loss of striatal neurones in the parkinsonian rat. Neuroscience, 63, 1-5. Mochizuki, H., Nakamura, N., Nishi, K. & Mizuno, Y (1 994)Apoptosis is induced by 1-methyl4-phenylpyridinium ion (MPP+) in ventral mesencephalic-striatal co-culture in rat. Neurosci. Letts., 170, 191-194. Monaghan, €?,Robertson, D., Amos, AS., Dyer, MJ.S., Mason, D.Y & Greaves, M.E (1992) Ultrastructural localization of Bcl-2 protein. J. Hidochem. Cytochem., 40, 1814-1825. Mukherjee, S.K., Yasharel, R., Klaidman, L.K., Hutchin, T.P. & Adams,J.D. (1995)Apoptosis and DNA fragmentation as induced by tertiary butylhydroperoxide in the brain. Brain Res. Bul., 38,595404. Muller, W.E., Schroder, H.C., Ushijima, H., Dapper,J. & Bormann, J. (1992) gp120 of Hiv-1 induces apoptosis in rat cortical cell cultures: prevention by memantine. Euz J. Pharmcol., 226,209-1 4. Mutisya, E.M., Bowling, A.C. & Bed, M.E (1994) Cortical cytochrome oxidase activity is reduced in Alzheimer’s disease. J. Nmrochem., 63,2 179-2 184. Mutoh, T., Tokuda, A., Marini, A.M. & Fujiki, N. (1994) I-Methyl-4-phenylpyridinumkills differentiated PC12 cells with a concomitant change in protein phosphorylation. Brain Res., 661.51-55.

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Myers, K.M., Fiskum, G., Liu, YB., Simmens, SJ., Bredesen, D.E. &Murphy, A.N. (1995)Bcl2 protects neural cells from cyanide/aglycemia-induced lipid oxidation, mitochondrial injury, and loss of viability. j! Neurochem.,65, 2432-2440. Nakai, M., Takeda, A., Cleary, M.L. & Endo, T. (1993)The bcl-2 protein is inserted into the outer membrane but not into the inner membrane of rat liver mitochondria in Vitro. Biochem. Biophys. Res. Camnun., 196, 233-239. Newmeyer, D.D., Farschon, D.M. & Reed, J.C. (1994) Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria. Cell, 79,353-364. Nicholls, D.G. & Ferguson, SJ. (1992)Bioenergetics 2. London: Academic Press. Nitatori, T., Sato, N., Waguri, S., Karasawa, Y., Araki, H. & Shibanai, K. et al. (1995)Delayed neuronal death in the CAI pyramidal cell layer of the gerbil hippocampus following transient ischaemia is apoptosis.j! Neurosci., 15, 1001-101 1. Okamoto, M., Matsumoto, M., Ohtsuki, T., Taguchi, A., Mikoshiba, K. & Yanagihara, T. et al. (1 993) Internucleosomal DNA cleavage involved in ischaemia-induced neuronal death. Biochem. Bio@ys. Res. Commun.,196, 1356-1362. Olanow, C.W., Hauser, R.A., Gauger, L., Malapira, T., Koller, W. & Hubble,J. et al. (1995)The effect of deprenyl and levodopa on the progression of Parkinson’s disease. Ann. Neural., 38, 771-777. Oltvai, Z.N. & Korsmeyer,S.J. (1994)Checkpoints of dueling dimers foil death wishes. Cell, 79, 189-192. 00,T E , Henchcliffe,C. & Burke, R.E. (1 995)Apoptosis in substantia nigra following developmental hypoxic-ischaemicinjury. Jveuroscimce, 69, 893-90 1. Oppenheim, R.W. (1 989) The neurotrophic theory and naturally occurring motoneurone death. %endcNeurosci., 12, 252-255. Oppenheim, R.W. (1991)Cell death during the development of the nervous system. Ann. Rev. Jveurosci., 14, 1356-1362. Oppenheim, R.W., Prevette, D., Tytell, M. & Homma, S. (1990) Naturally occurring and induced neuronal death in the chick embryo in vivo requires protein and RNA synthesis: evidence for the role ofcell death genes. Dev. Biol., 138, 104-1 13. Parkinson, S.G. (1993) Effects of tocopherol and deprenyl on the progression of disability in early Parkinson’s disease. New Eql. j! Med., 328, 176-183. Pechan, PA., Yoshida, T., Panahian, N., Moskowitz, M.A. & Breakefield, X.O. (1995) Genetically modified fibroblasts producing NGF protect hippocampal neurones after ischaemia in the rat. NeuroReport,6,669472, Petit, EX., Lecoeur, H., Zorn, E., Dauguet, C., Mignotte, B. & Gougeon, M.L. (1995) Alterations in mitochondrial structure and function are early events of dexamethasoneinduced thymocyte apoptosis.j! Cell BWL, 130, 157-167. Petito, C.K. & Roberts, B. (1 995a) Effect ofpostmortem interval on in situ end-labellingof DNA oligonucleosomes. j! Neurofiathol. Ex-.Neural., 54, 76 1-765. Petito, C.K. & Roberts, B. (1995b)Evidence of apoptotic cell death in HIV encephalitis [see comments]. A m . 3 Pathol., 146, 1121-1 130. Pollard, H., Cantagrel, S., Charriaut-Mariangue, C., Moreau,J. &Ben Ari, Y. (1994)Apoptosis associated DNA fragmentation in epileptic brain damage. NeuroReport, 5, 1053-1055. Portera-Cailliau, C., Hedreen, J.C., Price, D.L. & Koliatsos, VE. (1995a) Evidence for apoptotic cell death in Huntington disease and excitotoxic animal models. j! Neurosci., 15, 3775-3787. Portera-Cailliau, C., Hedreen, J.C., Price, D.L. & Koliatsos, VE. (1995b) Evidence for apoptotic cell death in Huntington’s disease and excototic animal models. j! Neurosci., 15, 37 75-3 787. Prhen, J.H., Bindokas, VP, Marcuccilli, CJ., Krajewski, S., Reed, J.C. & Miller, RJ. (1994) Regulation of neuronal Bc12 protein expression and calcium homeostasis by transforming


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growth factor type beta confers wide-ranging protection on rat hippocampal neurones. h c . NatlAcad. Sci. U U , 91, 12599-12603. Raff, M.C., Barres, B.A., Burne, J.E, Coles, H.S., Ishizaki, Y &Jacobson, M.D. (1993) Programmed cell death and the control of cell survival: lessons from the nervous system. Science, 262,695-700. Reed,J.C. (1994)Bcl-2 and the Regulation of Programmed Cell Death.3 Cell Bwl., 124, 1 4 . Regan, R.E, Panter, S.S., Witz, A., Tdy,J.L. & Giffard, R.G. (1995) Ultrastructure of excitotoxic neuronal death in murine cortical culture. Bruin Res., 705, 188-198. Richter, C. (1 993) Pro-oxidants and mitochondrial Ca2+:their relationship to apoptosis and oncogenesis. FEESLetts., 325, 104-107. Richter, C., Gogvadze, V, Laffranchi, R., Schlapbach, R., Schweizer, M. & Suter, M. et al. (1995) Oxidants in mitochondria: from physiology to diseases. Ewchem. Eiophys Acfu, 1271, 67-74. Richter, C. & Kass, G.E.N. (1991) Oxidative stress in mitochondria: its relationship to cellular Intaract, 77, 1-23. calcium homeostasis, cell death, proliferation,and differentiation.Chem.-Eiol. Roberts-Lewis,J.M., Marc5 XR., Zhao, Y , Vaught, J.L., Siman, R. & Lewis, M.E. (1993). Aurintricarboxylic acid protects hippocampal neurones from NMDA- and ischaemiainduced toxicity in ziu0.J. Neurochm., 61, 378-381. Rosenbaum, D.M., Michaelson, M., Batter, D.K., Doshi, F! & Kessler,J.A. (1994)Evidence for hypoxia-induced,programmed cell death of cultured neurones. Ann. Neurol., 36,864470. Rothstein,J.D., Bristol, L.A., Hosler, B., Brown, R.H., Jr. & Kuncl, R.W. (1994)Chronic inhibition of superoxide dismutase produces apoptotic death of spinal neurones. h c . Natl Acad. Sci USA, 91,415554159. Rukenstein, A,, Rydel, R.E. & Greene, L.A. (1991) Multiple agents rescue PC12 cells from serum-free cell death by translation- and transcription-independent mechanisms.j.NeuroSCi., 11,2552-2563. Schlingensiepen, K.H., Wollnik, E, Kunst, M., Schlingensiepen,R., Herdegen, T.& Brysch, W. (1994)The role ofJun transcription factor expression and phosphorylation in neuronal differentiation, neuronal cell death, and plastic adaptations in zivo. Cell Mol. Neurobiol., 14, 487-505. Schulze-Osthoff, K., Bakker, A.C., Vanhaesebroeck, B., Beyaert, R., Jacob, W.A. & Fiers, W. (1 992) Cytotoxic activity of tumour necrosis factor is mediated by early damage of mitochondrial functions.J. Bwl. Chem.,267, 5317-5323. Sei, Y., Von Lubitz, K.J., Basile, AS., Borner, M.M., Lin, R.C. & Skolnick, P. et al. (1994) InternucleosomalDNA fragmentation in gerbil hippocampus following forebrain ischaemia. Neurosci. Letts., 171, 1 79- 182. Sendtner, M., Arakawa, Y., Stockli, K.A., Kreutzberg, G.W. & Thoenen, H. (1991) Effect of ciliary neurotrophic factor (CNTF) on motoneurone survival. [Review.]3 Cell Sci., Supjdmmt, 15, 103-109. Sendtner, M., Holtmann, B., Kolbeck, R., Thoenen, H. & Barde, Y-A. (1992a) Brain-derived neurotrophic factor prevents the death of motoneurones in newborn rats after nerve section. Nature, 360,757-759. Sendtner, M., Schmalbruch, H., Stockli, K.A., Carroll, F!, Kreutzberg, G.W. & Thoenen, H. (1992b) Ciliary neurotrophic factor prevents degeneration of motor neurones in mouse mutant progressive motor neuroneopathy.Nuture, 358,502-504. Shaw, F? (1 994) Deconvolution in 3D optical microscopy. Histochem.3, 26,687-694. Shigeno,T., Yamasaki, Y & Kato, G. (1990)Reduction of delayed neuronal death by inhibition of protein synthesis. Nmrosci. Letts., 120, 117-1 19. Slater, A.F., Nobel, C.S. & Orrenius, S. (1995a)The role of intracellular oxidants in apoptosis. Ewchim. BW$lys. Acta, 1271,5942. Slater,A.F.G., Stefan, C., Nobel, I., van den Dobbelsteen,DJ. & Orrenius. S. (1995b)Signalling mechanisms and oxidative stress in apoptosis. Ericol. Letts., 82-3, 14%153.

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Yoshiyama, Y, Yamada, T., Asanuma, K. & Asahi, T. (1 994) Apoptosis related antigen, L e o and nick-end labelling are positive in spinal motor neurones in amyotrophic lateral sclerosis. A ~ t Nb0Pafh01. u Berl.,88, 207-2 1I . Zamzami, N., Marchetti, F!, Castedo, M., Decaudin, D., Macho, A. & Hirsch, T., et al. (1 995a) Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death.3 Exp. Med., 182, 367-377. Zamzami, N., Marchetti, F!, Castedo, M., Zanin, C., Vayssiere,J.L. & Petit, EX. et al. (1995) Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in viUo.3 Exp. Med., 181, 1661-1672. Zhang, E, Richardson, PM., Holland, D.P., Guo, G. & Tatton, W.G. (1995) CNTF or (-)deprenyl in immature rats: survival of axotomized facial motoneurones and weight loss. J. Neurosci. Res., 40, 564-570. Zhang, Y, Tatsuno, T., Carney,J.M. & Mattson, M.P. (1993)Basic FGF, NGF, and IFGs protect hippocampal and cortical neurones against iron-induced degeneration. 3 Cmeb. Blood Flow Metub., 13,378-388. Ziv, I., Melamed, E., Nardi, N., Luria, D., Achiron, A. & Offen, D. (1 994) Dopamine induces apoptosis-like cell death in cultured chick sympathetic neurones. Nmrosci. Letts. 170, 136- 140. Zsnagy, I., Steiber,J. &Jeney, E (1 995) Induction of age pigment accumulation in the brain cells of young male rats through iron-injection into the cerebrospinal fluid. &ontofogy 41, 145-1 56.


Chapter 2

CHANGES IN IONIC FLUXES DURING CEREBRAL ISCHAEMIA Tibor Kristidn and Bo K. Siesjo Laboratory for Experimental Brain Research, Lund University, University Hospital, S-22 I 85 Lund, Sweden

2. I 2.2 2.3 2.4

2.5 2.6

Introduction 2. I.I Ionic fluxes and membrane potential 2. I .2 Pre- and postsynaptic ion fluxes Changes in ionic fluxes Disturbances in ionic fluxes at restricted energy production Bioenergetic failure and ionic fluxes 2.4. I Ionic fluxes accompanying globallforebrain ischaemia 2.4.2 Hypoglycaemia-inducedchanges in ionic fluxes 2.4.3 Changes in intracellular ion concentrations 2.4.4 Normalization of ion gradients during the early recovery period Ion fluxes during focal ischaemia Ionic fluxes in the postinsult period References

27 27 30 31 32 33 34 37 38 39

40 40 42

2. I Introduction

2.1. I

IONIC FLUXES AND MEMBRANE POTENTIAL

Processing and transmission of information by neurones is conveyed by electrical and chemical signals, the former involving temporary changes in the flow of current through cell membranes. This current is carried by positively or negatively charged ions. The membrane consists of a mosaic of lipids and proteins. Since membranes are made up of a double layer of lipids with a hydrophopic inner ‘core’, dissipative movement of charged ions through the lipid layer is extremely unlikely. Thus, ions can cross the membrane only via pores (channels) formed by transmembrane proteins. These channels have two important properties: (i) they recognize and select specific ions, and (ii)they open and close in response to specific signals. The gating of the channels is controlled by voltage (voltage-gated channels), chemical transmitters (transmitterAcademic Press Limited Copyright Q 1997 All @hts ofreproduction in my form reserved

NEUROPROTECTIVE AGENTS AND CEREBRAL ISCHAEMIA, IRN 40 ISBN 0-12-366840-9;0-12-197880-X (pbk)

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T. K R I S T I h AND B.K. SIESJO

gated channels), and pressure or stretch (mechanically gated channels). However, the membranes also contain non-gated channels, which are always open and are not influenced significantly by extrinsic factors. They are important primarily in maintaining the resting membrane potential. The flux of ions through ion channels is passive, requiring no metabolic energy. The direction of this flux, and the equilibrium attained, are determined by the electrochemical driving force across the membrane. This driving force is determined by two factors: the electrical potential difference (membrane potential) and the concentration gradient of the permeate ions across the membrane. Thus, if the membrane potential is identical with the equilibrium potential (EJ for a given ion, a condition when the electrical and chemical forces are equal, there is no net ion flux across the cell membrane, even if the channels conducting this ion are open. Under all conditions, the membrane potential will be close to the E, for the most permeable ion($. Neurones and glial cells in vivo have resting membrane potentials of about -65 mV and -90 mV, respectively (Erecinska and Silver, 1994).This is mainly because the cell membranes contain non-gating channels which are permeable to potassium (K'), and since the equilibrium potential for K' (E,) is about -90 m y The neuronal membrane potential deviates from E, because neurones have a significant number of open channels that are selective to ions other than K'. Thus, in addition to K+, sodium ions (Na') are also involved in generation of the cell membrane potential. In the resting state the membrane permeability for Na+ is low. Since ENais about +50 mV, the resting membrane potential is only slightly shifted from E, towards more positive values. The cell membrane also forms a barrier for other physiological ions such as calcium (Ca2'), hydrogen (H'), and chloride (Cl-). While the intracellular concentration of K+ (K'i) is higher than the extracellular one (K',), the concentration gradients of Na', Ca", and C1- across the cell membrane are in the opposite direction (Erecinska and Silver, 1994). In order to maintain the ion concentration difference between intra- and extracellular fluids, the cell uses metabolically driven ion transporters and exchangers to move K' into the cell and to extrude Na', Ca2' and H+ from intracellular compartments. Thus, dissipation of membrane potential and ion gradients is prevented by the Na'-K+ pump, which extrudes Na' from the cell while absorbing K+. Because the pump moves Na' and K' against their net electrochemical gradients, energy must be provided. This energy comes from the hydrolysis of ATP (see Figure 1). Since Ca2+plays an essential role in regulating numerous cellular functions, its metabolism is subjected to independent regulation (Carafoli, 1987; Blaustein, 1988; Miller, 1991). The concentration gradient for Ca2' is large, with a 10000-fold concentration difference between intra- and extracellular fluids. There are two Ca2+extruding mechanisms in the plasma membrane. The first is a high-affinity, lowcapacity Ca2'-calmodulin ATP-ase, which regulates Ca2' at low concentrations (< mM). When the Ca2' load becomes appreciable, Ca2' ions are removed from the cytosol by a low-&nity, high-capacity 3Na+/ Ca2+exchanger, which is driven by the Na' gradient. To tightly control Ca2'i (the intracellular concentration of Ca"), cells

CHANGES IN IONIC FLUXES DURING CEREBRAL ISCHAEMIA

Active

29

Passive

2K+ ATP ADP+ Pi

U ,

""c,0

K+

0,

8 ""g.:.r+ c1- 4

2H+

c1-

ATP

ADP+ Pi

cl-

ca2' H+

out

In

Out

HCO;

n

In

FIGURE1 Schematic diagram, illustrating active mechanisms for Ca", Na', K+, and H+ extrusion (left panel), and passive conductances and mechanisms for accumulation of Ca", Na+,and H', or loss of K', C1-, and HC0,- (right panel). Reproduced with permission from Kristian et al. 1995b.

also utilize other intracellular mechanisms which sequester calcium, particularly into endoplasmic reticulum (ER) and so-called calciosomes (Berridge, 1993). Cells also possess an array of calcium-binding proteins, which are either fixed or diffusible, which participate in quick Ca2+buffering (Zhouand Neher, 1993).Mitochondria can also take up Ca2+;however, this is of quantitative importance only if Ca2+;reaches values of about 1 mM (Nicholls, 1985). C1- ions are often distributed passively across cell membranes. This means that the equilibrium potential for C1- (E,,) is very close to the resting cell membrane potential. However, if the cell is depolarized, a high C1- permeability will tend to shift the membrane potential back to more negative values. This is why opening of C1- -channels, e.g. by activation of GABA receptors, tends to repolarize membranes, or clamp the membrane potential close to Ecl. H+ ions are distributed actively across the cell membrane. Thus, the intracellular pH @Hi) values are higher than one would expect from a passive distribution. Normally, extracellular pH @He) is around 7.35, and the corresponding pHi value is about 7.0. Energy-dependent extrusion of H + occurs by Na+/H+ exchange, and by Na+-driven CI-/HC03- exchange. H+ probably crosses the membrane via

30

T KRISTIhJ AND B.K. SIESJO

A

I

FIGURE2 Schematic diagram illustrating pre- and postsynaptic ion channels. Presynaptically the voltage-sensitive channels (VSCC) involved in transmitter release are assumed to be the N and P type, whereas the L and T types are assumed to be localized to dendrites and cell bodies. Release of the glutamate (Glu)is shown to activate two types of receptors, selectively sensitive to amino-3-hydroxy-5-methyl-4-isoazole propionic acid (AMPA)and to .%methybaspartate (NMDA), respectively. The AMPA receptor gates a channel that is permeable to monovalent cations (Na', K+,and H+), whereas the NMDA-gated channel is also permeable to Ca2+.Normally this channel is blocked by M$+, but the block is voltage dependent. Thus, AMPA receptor activation and Na' influx lead to depolarization which relieves the Mg" block, allowing Ca2+to enter. Depolarization also allows Ca2+ to enter via VSCC. Inhibition is assumed to be mediated by activation of K+, and C1- conductances. Reproduced with permission from Kriitiin et al., 1995b.

unspecific cation channels, while HC03- is translocated by C1- channels or by C1-/HCO3- exchange. Activation of these channels (and translocases) will lead to passive H + or H C 0 3- fluxes across the cell membrane, causing alterations in pHi and PK*

2. I .2

PRE- AND POSTSYNAPTIC ION FLUXES

During normal neuronal activity, ionic fluxes occur mainly at interneuronal connections, i.e. synapses. The distribution of ion channels at an excitatory synapse is shown schematically in Figure 2. Presynaptic release of glutamate, the major excitatory neurotransmitter, is triggered by presynaptic influx of Ca2+,occurring via the N and P types of voltage-sensitive calcium channels (VSCC). These channels are activated by depolarization of presynaptic membranes due to an increased membrane permeability for Na+ ions. The rise in Ca2+ithen activates exocytotic mechanisms of vesicular neurotransmitter release into the synaptic cleft. Termination of such excitatory cascades occurs by the K+channels opening. The ensuing K+ efflux causing hyper-


31

polarization. An increase in C1- conductance may contribute to shift of the membrane potential back to more negative values. K+ conductances can be activated by the opening of Ca2+-activatedKt channels as a result of Ca2+binding to a regulatory site at the cytoplasmic side of the channel. Repolarization may also occur by activation of ATP-dependent K+ channels. Postsynapticall3 glutamate can bind to both ionotropic and metabotropic receptors. The former encompass those which are selectively activated by a-amino-3hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) and by N-methyl-D-aspartate (NMDA). The AMPA receptor-gated channels are permeable to monovalent cations, and since they preferentially conduct Na+ ions, the opening of these channels will depolarize the postsynaptic membranes. The NMDA receptor-gated channels are normally blocked at resting membrane potential by magnesium ions (hI$+). However, this block is voltage-sensitive and depolarization relieves it, allowing a massive Ca2+influx.Metabotropic receptors are coupled via G proteins to phospholipase C (PLC). Thus, activation of these receptors leads to breakdown of phosphatidyl-inositol bisphosphate (PIP,) to inositol trisphosphate (IP,) and diacylglycerol (DG).These two compounds have further intracellular effects on cellular ionic metabolism, since they participate in signal transmission as second messengers. IPSinduces a release of Ca2+from IP3-sensitiveintracellular calcium stores, thereby rising Ca2+i. At increased Ca2+;levels, DG activates protein kinase C (PKC), an enzyme which phosphorylates membrane proteins, including those forming receptors and ion channels. Phosphorylation usually leads to inhibition of their function, i.e. PKC acts as negative feedback mechanism.

2.2 Changes in ionic fluxes As mentioned above, transmission of signals between neurones is accompanied by transient changes in ionic fluxes at pre- and postsynaptic membranes. However, even if intense, synchronized cellular discharge gives rise to relatively discrete changes in extracellular ion concentrations. For example, epileptic seizures are accompanied by an increase in K+, from a control value of about 3 mM to a value of about 10 mM, and Ca2+, decreases by 0.2-0.4 m~ (Pumain and Heinemann, 1985). Marked disturbances in ion homeostasis at normal energy balance are observed during spreading depression (SD), which can be elicited by local application of K+, by electrical stimulation, or by a stab wound (Bures et a/., 1974; Hansen, 1985). In SD, a propagated disturbance of brain electrical function is accompanied by sudden activation of ion conductances, leading to massive downhill ion fluxes. However, since the energy production is not compromised, the cells are able to quickly reinstitute the ion gradients. Propagation of an SD can be blocked by the NMDA receptor antagonist dizocilpine maleate (MK-80 l), suggesting that activation of postsynaptic NMDA receptor-gated channels is involved in the mechanism of SD generation (Marrannes et al., 1988). Thus, SD is very likely caused by presynatpic release of glutamate, with

T K R I S T m AND B.K. SIESJo

32 A

B

C a':

I

1

Smin

FIGURE3 Typical recording of DC potential shifts and Ca2+,transients from a control animal (A), and from an animal with bilateral carotid artery occlusion (B). The baseline and the rates of Ca2+emux have been marked with stippled lines. Reproduced with permission from Gido el al., 1994b.

subsequent activation of postsynaptic ion conductances. The trigger could be local release of a sufficient amount of Kfto cause presynaptic depolarization End, secondarily, release of glutamate from many nerve endings. However, even if cell ion homeostasis is disturbed during SD, repeated SDs over a 4-5-hour period is not harmful to energy-competent cells (Nedergaard and Hansen, 1988).

2.3 Disturbancesin ionic fluxes at restricted energy production Repumping of ions following the dissipative fluxes that occur during SD requires an extra input of energy. This enhanced energy requirement is reflected in increased blood flow, glucose utilization, and lactate production following SD. None the less, a small perturbation of the phosphorylation potential is observed (Kocher, 1990). Evidently, in energy-competent cells the available ATE and that formed in response to stimulation, is sufficient to restore normal ion gradients (by active transport) and normal membrane permeability (by reuptake of transmitters). Predictably though, if the tissue is energy-compromised, the SD wave should be prolonged because of a reduced cellular capacity to restore ion gradients. As Figure 3 shows, during conditions of restricted energy production there is a delay in the recovery of ion gradients, and normalization takes longer. This delay is probably proportional to the ability of the tissue to increase its rate of energy production (see also Gido et al., 1994a,b). For


33

5 min

FIGURE 4 Typical recording of DC potential shifts and Ca2+,transients before and following bilateral carotid artery occlusion. Arrow indicates the onset of occlusion. Reproduced with permission from Gidd et al., 199413.

example, ifboth common carotid arteries are occluded in rats, the cerebral blood flow (CBF) is reduced to 40-60% of control values and, as Figure 4 illustrates, both the DC potential and calcium transients triggered by SD are markedly prolonged (data from Gido et al., 199413).Furthermore, the reduction in perfusion pressure seems to prevent a compensatory increase in CBF during the SD. This probably restricts the increase in energy production which is necessary for rapid reinstitution of ion gradients. A restriction of energy production should prevent rapid normalization of cell ion homeostasis, thus leading to cell calcium 'overload' and, possibly, to cell death. The relationship between the duration of the calcium transient induced by SD in energy-compromised tissue and cell death was addressed by Gido and collaborators (Gido et al., 1994b). The results failed to show that repeated SDs (even if prolonged for many minutes due to the moderately decreased blood flow) induce brain damage. Possibly, although reduced, CBF was high enough to maintain energy production at levels which would allow cells to protect themselves against Ca2+-inducedtoxicity by energy-dependent intracellular buffering and sequestration of Ca2+,thereby preventing mitochondrial Ca2+overload, but it was not high enough to immediately re-establish the cell ion homeostasis. In other words, the degree of reduction of CBF could be a critical variable.

2.4 Bioenergetic failure and ionic fluxes A dramatic change in ionic fluxes occurs secondary to bioenergetic failure during ischaemia and hypoglycaemia. The former can be divided into two major types: global or forebrain ischaemia of the 'cardiac arrest' type, and focal ischaemia of the 'stroke' type (Siesjo, 1992; Siesjo et al., 1995). In the latter type, one can distinguish between a core of tissue with relatively dense ischaemia and perifocal tissues @enumbra) which are less densely ischaemic.

34

T. KRISTW AND B.K. SIESJO

2.4. I IONICFLUXESACCOMPANYING

GLOBA~FOREBRAINISCHAEMIA

Since ion gradients across nerve and glial cell membranes are upheld at the expense of energy in the form of ATP (see above),energy failure due to ischaemia leads to dissipation of the ionic gradients (Hansen, 1985; Harris and Symon, 1984a; Kristiin et al., 1994). However, as Figure 5 shows, the time course of these fluxes is intriguing, occurring in several phases. In complete ischaemia, when p o p quickly falls to zero, there is initially a slight increase in Na', , Ca2+e,and C1-, (Hansen and Zeuthen, 1981; Kristian et al., 1994). It has been suggested that these changes are caused, at least in part, by shrinkage of the extracellular space (phase I). Changes in K', are somewhat different. The increase in K+, during phase I is more marked than expected on the basis of changes in extracellular space. Usually the initial period of anoxiahchaemia is accompanied by cell hyperpolarization (Hansen et al., 1982; Krnjevic and Leblond, 1987) suggesting activation of K+ conductances and release of K' to extracellular fluids (Hansen et al., 1982; Hansen and Zeuthen, 1981; KristiAn et al., 1994).The triggering factor for this increase in K', has not been established definitively.Rapid failure of the Na+-K+-ATPasecould contribute (Jiang and Haddad, 1991). However, the opening of ATP-sensitive and/or Ca2+activated K+ channels has also been considered (Folbergrovi et al., 1990; Hansen, 1985; Silver and Erecinska, 1990). Since the decrease in ATP during the first minute of ischaemia is very moderate, if detectable (see Folbergrova et al., 1990), the activation of ATP-sensitive K+ conductances seems unlikely. However, since the 'free' ADP concentration increases markedly in this period (Eklholm et al., 1993b), a channel which is sensitive to the ATP/ADP ratio could contribute to the release of K+. In spite of that, the opening of Ca2+-activatedK' channels should be a major factor leading to an increase in K', during phase I. This is supported by the fact that activation of phosphorylase a was observed just 15 seconds after the onset of ischaemia (Folbergrova et al., 1990),probably reflecting a rise in Ca";. An early increase in Ca2+; was also measured in neuronal cells in rat brains subjected to forebrain ischaemia (Silver and Erecinska, 1992). Because of this activation of K+ channels, the increase in K+, is more marked during phase I (about 5-fold, from a baseline level of about 3 mM to about 15 m). Such an increase in K+, must depolarize other membranes, of both glial and neuronal origin. If these encompass presynaptic membranes, the slow rise in K', will lead ultimately to massive transmitter release, thereby triggering ionic fluxes through both voltage-dependent and agonist-operated channels (Hansen, 1985; Siesjij and Bengtsson, 1989). The ensuing sudden cell depolarization is accompanied by massive downhill ionic fluxes, leading to abrupt changes in extra- and intracellular ion concentrations (see Figure 5). In this period (phase II), there is further increase in K',, and uptake of Ca2+,Na', and C1-, with osmotically obliged water (Hansen, 1985; Kristiin et al., 1994; Silver and Erecinska, 1990). The interval between the onset of ischaemia and the ultimate depolarization is called anoxic depolarization (AD) time. Ischaemia also causes a gradual fall in extra- and intracellular pH @Heand pH;, respectively) due to activation of glycolysis, with production of lactate and H +


35

mM 1.2

0.8 0.4

0

mM

140

100

60

mM

140

100

60

I

I

1 mln

FIGURE5 Schematic diagram illustrating changes in extracellular ion concentrations following complete ischaemia. Extracellular potassium (K+,), sodium (Na',), and chloride (We) show an early rise in concentration, and extracellularpH (pH,) steadily falls after the induction of ischaemia (phase I). However, the major ion fluxes occur about 60 sec after interruption of circulation (phase 11). At the time of these rapid ion fluxes, a transient alkaline shift in pH, occurs, suggesting transmembrane fluxes of Hf or HC03-. Slightly modified according to Siesjo et al., 1990.

36

T. KRISTIh AND B.K. SIESJO

(Ljunggren et al., 1974a; Mutch and Hansen, 1984; Smith et al., 1986; Katsura et al., 1992; Silver and Erecinska, 1992). In complete ischaemia, the amount of lactate formed corresponds to the pre-ischaemic tissue stores of glucose and glucogen, the former varying with plasma glucose concentration (Ljunggren et al., 197413). In normoglycaemic animals, the initial pH shift (about 0.4 pH units) is succeeded by an alkaline transient (0.1-0.2 pH unit) which coincides with the AD. It is extremely likely that this shift reflects transient influx of H+ and efllux of HC0,- via activated cation and anion channels (Siesjo, '1988a). The final event is a decline in pH to 6.8-6.7 (Siemkowicz and Hensen, 1981; Siesjo, 1992).In hyperglycaemic animals, ischaemia leads to even greater changes in pH, (Kraig et al., 1986; Smith et al., 1986), where ApH, is related linearly to tissue lactate content (Katsura et al., 1992). However, the higher tissue glucose concentration causes not only marked acidosis but also leads to prolonged AD time (Siemkowicz and Hansen, 1981; Kristian et al., 1994; Ekholm et al., 1995; Erecinska and Silver, 1996). There are at least two reasons why cells depolarize after a longer delay in hyperglycaemic animals. First, high tissue glucose concentrations allow cells to produce enough ATP to fuel membrane pumps for a longer period in the absense of blood flow. Second, in general, low pH inhibits ion fluxes across cell membranes (Moody, 1984), thereby reducing the energy demands for ion extrusion, Thus, hyperglycaemia/acidosis increases the delay before changes in ion concentrations occur and also reduces the rate of ion movements across the cell membranes at the time of AD (Kristian et al., 1994).Changes in Ca2+eare illustrated in Figure 6. In normoglycaemic subjects, depolarization and cellular uptake of calcium are observed after about 60 seconds. This delay is shortened by hypoglycaemia and almost doubled by hyperglycaemia. Furthermore, in hyperglycaemic animals, the rate of fall in Ca2+, is reduced and, in addition, cellular uptake of Ca" occurs in two phases. Since a similar delay and a two-phase reduction in Ca2+,is observed when tissue pH in normoglycaemic animals is reduced by excessive hypercapnia, before ischaemia, or when the animals are pre-treated by the NMDA antagonist dizocilpine maleate (MK-801), it seems clear that the effect of acidosis is exerted largely on the NMDA receptor-gated ion channels (Kristian et al., 1994). These data also suggest that there are at least two pathways for calcium influx during ischaemic depolarization. One obviously represents NMDA receptor-gated channels. The other pathway could be provided by Ca2+/Na+exchange. It has been suggested that since the 2Na+/Ca2+-exchanger is electrogenic, and is therefore affected by the concentration gradients for Na+ and Ca2+and by the cell membrane potential, it is likely that at the time ofAD, the thermodynamic conditions can reverse the exchanger operation and lead to transport of calcium into the cells (Stys et al., 1991; for further discussion see Kristian et al., 1994; Erecinska and Silver, 1996). The results published by Xie et al. (1 995) support this contention. They demonstrated that the ischaemia-induced Ca2+, reduction was slowed down by both MK-801 and NBQX, the latter selectively blocking the AMPA receptor-operated channel. The effect of NBQX suggests inhibition of the reverse operation of 3Na+/Ca2+exchange by maintenance of the Na+ gradient. However, the drugs only marginally affected the

CHANGES IN IONIC FLUXES DURING CEREBRAL ISCHAEMIA Cae2+mM

37

Hyperglycaemia

iq

1 I

0.1

Normoglycaemia

0.1

Hypoglycaernia

0.1 30 sec

-

FIGURE 6 Changes in Ca2+, in the rat cortex in hyper-, normo-, and hypoglycaemic animals following cardiac arrest. The rate of fall and the anoxic depolarization time were clearly influenced by the preischaemicplasma glucose level. Reproducedwith permission from Kristian et al., 199513.

fall in Ca",. Since these experiments were carried out without controlling head temperature, the drugs' effects could have been masked by hypothermia at the time of

AD. 2.4.2 HYPOGLYCAEMIA-INDUCED CHANGES IN IONIC FLUXES In hypoglycaemia, ionic disturbances usually occur when the plasma glucose concentration falls to about 1 m ~Usually, . before cells depolarize, the EEG activity is flattened and the mean arterial blood pressure (MABP) rises above 150 mmHg (Harris el al., 198413; Kristiin et al., 1993). Similarly, as in ischaemia the hypoglycaemia-induced cell depolarization is accompanied by a rise in K+, and a fall in Ca2+,, the latter occurring when K', reaches a value of about 13 mM (Harris el al., 1984b).This first depolarization is usually followed by a slow, transient recovery of ion concentrations, which precedes a persistent increase in K',, and reduction in Ca2', (Harris et al., 1984b; Kristikn et al., 1993). There are some differences in changes of ion concentrations between ischaemia and hypoglycaemic coma. First, no changes in extracellular ion concentrations (notably in K',) have been observed in the period before hypoglycaemic cell

38

T. KRISTrhLN AND B.K. SIESJO

depolarization occurs. Second, in dense ischaemia, K', rises to 60-70 m~ and Ca2+, is reduced to about 0,15 mM (Kristihn et al., 1994),while in hypoglycaemic coma, the K+, level rises to about 40 m ~ and , Ca2+, is reduced below 0.1 mM, reaching an average value of 0.02 m~ (Pelligrino et al., 1982; Harris et al., 1984b; Kriitian et al., 1993). In ischaemia, K+, must rise to a certain threshold value before transmitter release occurs. The hypoglycaemic event probably starts with a very localized cellular depolarization which then spreads throughout the brain, initially affecting only one of the hemispheres. This can also explain why EEG flattening sometimes follows cell depolarization, as recorded by glass microelectrodes (Kristihn et al., 1993). Furthermore, since the changes in extracellular space following AD and hypoglycaemic depolarization are not significantlydifferent (Pelligrino et al., 1981),but the Ca2+,level is lower during hypoglycaemic coma, the cells must take up more Ca2+following hypoglycaemic depolarization. Hypoglycaemic coma does not lead to acidosis, and 2040% ATP content persists (Siesjo, 1988b).This residual energy level may allow cells to better buffer intracellular calcium. The expected events would be lower Ca2+;and reduced Ca",. Thus, if one reduces the ATP level to zero, for example by inducing complete ischaemia during hypoglycaemic coma, Ca2+,increases from 0.02 m~ to Ca2+,levels observed during ischaemia (Kristih et al., 1993).These data suggest that the remaining ATP in brains ofhypoglycaemic subjects is probably used for active uptake of Ca2+into endoplasmic reticulum, thereby preventing mitochondrial calcium overload, and leading to less severe damage when compared to forebrain ischaemia. Furthermore, the lack of acidosis during hypoglycaemic coma probably also favours a better histological outcome. This contention is supported by data showing that superimposed hypercapnia exaggerates hypoglycaemic damage and also causes a slight increase in Ca2+, (Kristian et al., 1995a).

2.4.3 CHANGES IN INTRACELLULAR ION CONCENTRATIONS Changes induced by bioenergetical failure in intracellular ion concentrations are mirror images of the extracellular ones (for review see Erecinska and Silver, 1994). When in vivo data are compared with in vitro experiments, one must consider differences between in vivo and in vitro conditions. These may crucially affect ionic fluxes occurring in the two systems under study. The ion movements across cell membranes in Vim are, at least initially, restricted to intra- and extracellular fluids, the latter occupying only about 20% of total tissue volume. This is because the blood-brain barrier (BBB) tightly controls ion exchange between blood and extracellular fluids and is intact for many hours, even following a transient ischaemic insult (Ohta et al., 1992). Thus, although any eflux or influx of ions markedly influences the extracellular concentrations, the total tissue concentration of ions may be unaltered by a transient ischaemic episode for hours or days (Deshpande et al., 1987; Warner et al., 1987).The situation in vitro is completely different because the extracellular fluid volume is essen-


39

tially unlimited (over 99.9% of total volume). As a result, when cells are exposed to glutamate or NMDA they may take up amounts of calcium (Eimerl and Schramm, 1994). During such experiments neurones may increase their total calcium content several-fold. In uivo, for example, during ischaemia or hypoglycaemic coma, cell calcium uptake is a fraction of that (for discussion see Kristidn et al., 1996). Furthermore, changes in intracellular concentrations of other ions are also more excessive during in uitro experiments (Silver et al., 1996). Thus, while in uitro K+i is decreased about 10-fold and Na'i rises about 5-fold, in uivo K+i is probably reduced by about 20% and Na'i increases 2.5-fold. Therefore, the pathophysiology of cell death in uivo might be different from that in uitro. Possibly, conditions prevailing in uitro may overestimate the excitotoxic component.

2.4.4 NORMALIZATION OF ION GRADIENTS DURING THE EARLY RECOVERY PERIOD If ischaemia is followed by adequate reperfusion following a lag period of 1-2 minutes, the ion gradients are gradually normalized. The recovery at the beginning of reperfusion of K', is very slow. At the time when the cell membranes repolarize, as reflected in a reversal of the shift in DC potential, the reduction of K', is accelerated and the concentration reaches the pre-ischaemic level in about 5 minutes (see Ekholm et al., 1993a).There is subsequently a small overshoot, with K', levels being lower than the normal pre-ischaemic ones. The recovery of Ca", and pH, occurs in two phases. At the time of cell repolarization and accelerated K', reduction, there is a rapid increase in Ca", and pH, up to about 70% of control values, followed by a slow and progressive increase, leading to final normalization of Ca2+, or pH, in 15-20 minutes. The recovery of ion gradients following hypoglycaemic coma is very similar. After glucose injection there is a 2-minute delay before active ion transport across the cell membranes is resumed, and normalization of ion concentrations, except for CaZf, occurs in 5-8 minutes (see Kristidn et al., 1993).The Ca", recovery occurs in a rapid and a slow phase, normal values being reached in about 20 minutes (Kristian et al., 1993). Pre-ischaemic plasma glucose concentration influences not only the changes in ionic fluxes at AD (see above) but also the recovery of ion gradients following reinstitution of a normal perfusion pressure. Both hyperglycaemia and hypercapnia reduce the interval between onset of reperfusion and normalization of Ca2+, (Ekholm et al., 1995).Similarly, Ca2'i also recovers earlier in hyperglycaemic animals (Erecinska and Silver, 1996). Thus, the total depolarization time, or duration of cellular cell calcium 'overload', is shorter in hyperglycaemic/hypercapnic subjects than in normoglycaemic ones. This is due to two factors: the AD time is prolonged and the recovery following termination of the insult is more rapid. This is a potentially important finding since it is believed that the cellular calcium uptake by energy-compromised cells is the major event leading to cell damage, and since hyperglycaemic aggravates damage caused by the ischaemic insult. It should be emphasized that the total Ca2' load to which the cell is exposed is identical in

40

T. K R I S T I h AND B.K. SIESJO

normoglycaemic, hyperglycaemic, and hypercapnic animals. Thus, after 4-5 minutes of ischaemia, Ca2', is reduced to the same level regardless of the pre-ischaemic glucose or intra-ischaemic pH levels. These data clearly indicate that acidosisper se is an exaggerating factor in cell damage induced by energy failure in vivo (for further discussion see Siesjo et al., 1996).

2.5 Ion fluxes during local ischaemia So far, we have described changes in ion fluxes related to the induction of complete/forebrain ischaemia or hypoglycaemia, as well as to the early recovery period following such insults. In focal ischaemia, for example induced by middle cerebral artery occlusion (MCAO), changes in ionic fluxes have been studied mostly in penumbral areas surrounding the dense ischaemic focus. Since the CBF in the ischaemic core is about 20-40°/0 of control, the energy production is insufficient to maintain ion gradients, leading to cell depolarization and downward ion fluxes. However, cell energy potential is higher than in global/forebrain ischaemia since during the first few hours of MCAO, elevated K', or reduced Ca2', are occasionally interrupted by partial, transient normalization of ion homeostasis (Kristiin et al., 1995~). Events in the penumbra zone are characterized by irregularly occurring SD-like waves. Recording of K', revealed that there are two different types of spontaneous depolarizations in cortical tissue surrounding the ischaemic core (Nedergaard and Hansen, 1993).One leads to an abrupt rise in K',, similar to what is seen in SD, while the other involves a two-phase increase in K',, in which a slow progressive increase precedes a rapid one, resembling ischaemic depolarization. While the ischaemia-like depolarizations last for more than 10 minutes, the SD-like transients are short-lasting (4-7 minutes). These data suggest that the prolonged, transient dissipation of ion gradients are probably triggered either by elevated K', levels in the ischaemic focus, with propagation into the penumbra zone, or are evoked by transient decreases in local blood flow. After transient MCAO of 2 hours' duration there is rapid recovery of cell membrane potential and K',, the latter normalizing in 4-6 minutes (Kristiin et al., 1995~). However, Ca", recovery is incomplete, with values typically reaching about 50% of the normal pre-ischaemic level after several hours of recovery.

2.6 Ionic fluxes in the postinsult period Cell death induced by ischaemia or hypoglycaemic coma is conspicuously delayed, occurring hours or days following the initial result. The concept of delayed or secondary post-ischaemic neuronal damage is based on a scenario where the events leading to cell death are divided into three consecutive stages (Siesj6 et al., 1995). The

CHANGES IN IONIC FLUXES DURING CEREBRAL. ISCHAEMIA

41

initial/primary insult is represented by transient ischaemia or hypoglycaemic coma, followed by a free interval characterized by recovery of cell energy state, ion homeostasis, and basic cellular physiological functions. Finally, there is secondary energetic failure accompanied by ultimate cell death. There is little information on a ionic fluxes in the postinsult, free interval period. Since cell death and a perturbed cellular calcium homeostasis is believed to have an intimate relationship, many studies have tried to define changes in cell calcium metabolism. The hypothesis of a sustained perturbation of cell calcium metabolism following a transient insult (Deshpande et al., 1987)is supported by the followingfindings: There is an increased rate of incorporation of 45Ca2+into tissue at a postischaemic time when the total tissue calcium content is obviously not increased (Dienel, 1984; Deshpande et al., 1987; Ohta et al., 1992), suggesting increased calcium cycling across membranes. Following normalization of Ca2+iafter transient ischaemia, there is a secondary rise in Ca2+;in the hippocampal CA 1 neurones at a time when signs of cell death are not yet obvious (Silver and Erecinska, 1992). Thus, these data suggest that a clear perturbation of cell calcium metabolism occurs before cell death. The concept of a postinsult increase in Ca2+;is supported by results showing that, following transient ischaemia there is accumulation of Ca2+by re-energized mitochondria after hours of recirculation (Dux et al., 1987; Zaidan and Sims, 1994). The progressive accumulation of calcium by cells in the post-ischaemic period is probably the result of a perturbed regulation of a calcium transport across membranes and/or dysregulation of intracellular calcium homeostasis due to pathological changes in inositol-phosphate metabolism and intracellular calcium stores. The data published by AndinC et al. (1992) suggest that the cells in CA1 hippocampal sector about 6 hours after transient ischaemia begin taking up more Ca2+in response to a given stimulus than they normally do. The reason could be related to changes in pre- and postsynaptic glutamate metabolism (increased release of glutamate and/or inhibition of its uptake), or a modification of postsynaptic receptors/channels by the preceding ischaemic insult. In support of this contention are data showing that the AMPA receptor-gated channels are modified after a ischaemic insult (Tsubokawa et al., 1995). The conductance of these channels is increased about two-fold. Thus, since the activation of AMPA receptor-gated channels depolarize the cells, their higher conductance probably leads to more profound depolarization and favour an opening of VSCC- and NMDAactivated channels in ischaemic neurones to a level which is relatively rare, or is absent in normal neurones. Therefore, normal physiological activity of neurones in the post-ischaemic brain could also cause a slow, progressive accumulation of calcium. Another possible explanation for the dysfunction of cell calcium homeostasis is a disturbed relationship between calcium leaks and calcium extrusion across intracellular membranes, such as those of the endoplasmic reticulum (ER) or mitochondria. Tsubokawa et al. (1992) used path clamp techniques to study the CA1 cells in

42

T. KRISTIh AND B.K. SIESJO

hippocampal slices following transient ischaemia. Their findings showed that stimulation of excitatory input caused irreversible cell depolarization, and that injection of IPS had similar effects. This fatal depolarization leading to cell death could be prevented by intracellular injection of BAPTA, a fast calcium buffer. In a subsequent article (Tsubokawa et al., 1994), the authors showed that the effect of excitatory stimulation could be elicited by injection of inositol tetrakisphosphate (IP,), and prevented by antibodies against PIP,, or against IPS kinase, the enzyme that converts IPS to IP,. Evidently, these data indicate a coupling between secondary loss of cell calcium homeostasis and disturbances in IP4-regulatedmechanisms of ER refilling by extracellular calcium via cell plasma membrane channels. Taken together, the results suggest that in the postinsult period Ca2+i slowly increases to levels at which re-energized mitochondria start to accumulate calcium. Evidently, this could be due either to a disturbance in the plasma membrane handling of Ca2+or in the corresponding pump/leak relationship at the level of the ER membrane. If these processes are sustained, the mitochondria calcium overloads leads to production of free radicals and to irreversible damage to mitochondrial membranes, energy failure, and cell death (for reviews see Kristian and Siesjo, 1996; Siesjo and Siesjo, 1996).The production of free radicals is also affected by intracellular pH @Hi). As discussed elsewhere (see Siesjo et al., 1996), H+ triggers release of iron from proteins of the transferrin type, which catalyzes a Fenton-type reaction leading to the formation of free radicals. Thus, H+ and Fe2+/Fe3+may have an additional effect on mechanisms causing cell death (see Siesjo et al., 1996). Therefore, apart from calcium ions, H+ ions and iron are important players in the cascade of metabolic events which lead to delayed ischaemic brain damage.

References AndinC, I?, Jacobsen, I. & Hagberg, H. (1992)Enhanced calcium uptake by CAI pyramidal cell dendrites in the postischaemic phase despite subnormal evoked field potentials: excitatory amino acid receptor dependency and relationship to neuronal damage. j ! Cereb. Blood Flow Mehb., 12,773-783. Berridge, MJ. (1993)A tole of two messengers. Nature, 365, 388-391. Blaustein, M. (1988) Calcium transport and buffering in neurones. %ends in Neuroscience, 11, 438443. Bures, J., Buresova, 0. & Krivanek,J. (1974) Th Mechanism and Applications OfLeao's Spreading Depression OfEIectroencephalographicActi@y. Academia, Prague and New York. Carfoli, E. (1987)Intracellular calcium homeostasis, Annu. Reu Biochm., 56, 395-433. Deshpande, J.K., Siesj6, B.K. & Wieloch, T. (1987) Calcium accumulation and neuronal damage in the rat hippocampus following cerebral ischaemia. j ! Cereb. Blood Row Metub., 7 , 89-95. Dienel, G.A. (1984) Regional accumulation of calcium in postischaemic rat brain. j! Neurochem., 43,913-925. Dwc, E., Mies, G., Hossmann, K.-A. & Siklos, L. (1987) Calcium in the mitochondria following brief ischaemia of gerbil brain. Neurosci. Lett., 78, 295-300.


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Eimerl, S. & Schramm, M. (1994) The quantity of calcium that appears to induce neuronal death.3 Nmrochem., 62, 1223-1226. Ekholm, A., Katsura, K., Kristian, T., Folbergrova,J. & Siesjo, B.K. (1993a) Coupling of cellular energy state and ion homeostasis during recovery following brain ischaemia in normoglycemic rats. Brain Res., 604, 185- 191. Ekholm, A., Katsura, K. & Siesjo, B.K. (1 993b) Coupling of energy failure and dissipative K+ flux during ischaemia: role ofpreischaemic plasma glucose concentration.3 Cereb. Blood Flow Metab., 13, 193-200. Ekholm, A., Kristian, T. & Siesjo, B. (1995)Influence of hyperglycemiaand of hypercapnia on cellular calcium during reversible brain ischaemia. Ex$ Brain Res., 104,462-466. Erecinska, M. & Silver, LA. (1994) Ions and energy in mammalian brain. F'rag. Neurobiol., 43, 31-71. Erecinska, M. & Silver, I.A. (1996)Calcium handling by hippocampal neurones under physiological and pathological conditions. In Advances in .Neurology (eds Siesjo, B. & Wieloch, T.), pp. 119-136. Raven Press, New York. Folbergrova,J., Minamisawa, H., Ekholm, A. & Siesjo, B.K. (1 990) Phosphorylase a and labile metabolites during anoxia: Correlation to membrane fluxes of K' and Ca2+.S; Neurocha., 55, 1690-1696. Gido, G., Kristian, T., Katsura, K. & Siesjo, B.K. (1994a)The influence ofrepeated spreading depression-induced calcium transients on neuronal viability in moderately hypoglycemic rats. [email protected] Res., 97, 397403. Gido, G., Kristiln, T. & Siesjo, B. (1994b)Induced spreading depressions in energy-compromised neocortical tissue: calcium transients and histopathological correlates. Neurobwl. Lk., 1,31-41. Hansen, AJ. (1985)Effects ofanoxia on ion distribution in the brain. Physwl. Rev., 65, 101-148. Hansen, A., Hounsgaard, J. & Jansen, H. (1982)Anoxia increases potassium conductance in hippocampel nerve cells. Acta Physz'ol. Scan., 108, 355-365. Hansen, A. & Zeuthen, T. (1981)Extracellular ion concentration during spreading depression and ischaemia in the rat brain cortex. Acta Physil. Scand., 113,437445. Harris, RJ. & Symon, L. (1984a) Extracellular pH, potassium, and calcium activities in progressive ischaemia of rat cortex.3 Cereb, Blood Flow Metab., 4, 178-186. Harris, R., Wieloch, T., Symon, L. & Siesjo, B.K. (198413)Cerebral extracellular calcium activity in severe hypoglycemia: Relation to extracellular potassium activity and energy state. J. Cereb. Blood Flow Metabol., 4, 187-193. Jiang, C. & Haddad, G. (1991) Effect of anoxia in intracellular and extracellular potassium activity in hypoglossal neurons in vitro. j! Neurosci., 66, 103-1 11. Katsura, K., Ekholm, A. & Siesjo, B.K. (1992) Tissue PC02 in brain ischaemia related to lactate content in normo- and hypercapnic rats.3 Cereb. Bloodfiw Metab., 12, 27&280. Kocher, M. (1 990) Metabolic and hemodynamic activation of postischaemic rat brain by cortical spreading depression.J. Cereb. Blood Flow Metab., 10,564-57 1. Kraig, R.P., Pulsinelli, W.A. & Plum, E (1986)Carbonic acid buffer changes during complete brain ischaemia. Am. 5; P h y d , 250, R348pR357. Kriistian, T., Gido, G. & Siesjo, B.K. (1 993) Brain calcium metabolism in hypoglycemiccoma. J. Cereb. Blood Flow Metub., 13,955-961. Kriitian, T., Gido, G. & Siesjo, B. (1995a) The influence of acidosis on hypoglycemic brain damage.3 Cereb. Blood Row Metub., 15, 78-87. Kristiim, T., Katsura, K. & Siesjo, B.K. (199513) Ionic metabolism in cerebral ischaemia. In Pharmazological Control of Calcium and Potassium Homeostasis (eds T. Godfraind, G. Mancia, M.P. Abbracchio, L. Aguilar-Bryan & S. Govoni), pp. 199-208. Kluwer Academic, Milan, Houston. Temporal profile of extracellular ion concentration Kristikn, T., Gido, G. & Siesjo, B.K. (1995~) Nmrosci., 216 (abstract). changes followingtransient middle cerebral artery occlusion in rat. SOC.

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Kristian, T., Katsura, K., Gido, G. & Siesjo, B.K. (1994) The influence of pH on cellular calcium influx during ischaemia. Brain Res., 641, 295-302. Kristian, T., OuYang, Y. & Siesjb, B. (1996) Calcium-related damage in vivo and in vitro: are different mechanisms involved? In Advances in Neurology (eds Siesjo, B. & Wieloch, T.), pp. 107-1 18. Raven Press, New York. Kristian, T. & Siesjij, B. (1996)Calcium-related damage in ischaemia. hi Sci., 59, 357-367. Krnjevic, K. & Leblond,J. (1987)Anoxia reversibly suppresses neuronal calcium currents in rat hippocampal slices. Can.3 Phywl. Pharmacol., 65,2 157-2 161. Ljunggren, B., Norberg, K. & Siesjo, B.K. (1974a)Influence of tissue acidosis upon restitution of brain energy metabolism following total ischaemia. Brain Res., 77, 173-186. Ljunggren, B., Schutz, H. & Siesjo, B.K. (1974b)Changes in energy state and acid-base parameters of the rat brain during complete compression ischaemia. Brain Res., 73,277-289. Marrannes, R., Willems, R., De Prins, E. & Wauquier, A. (1988)Evidence for a role of the Nmethybaspartate (NMDA)receptor in cortical spreading despression in the rat. Brain Res., 457,226-240. Miller, RJ. (1991)The control of neuronal Ca2+homeostasis. Pmg. Neurobiol., 37,255-285. Moody, W. (1984) Effects of intracellular H+ on the electrical properties of excitable cells. Annu. Rev. Neurosci., 7, 154-166. Mutch, W.A. & Hansen, AJ. (1984)Extracellular pH changes during spreading depression and cerebral ischaemia: Mechanisms ofbrain pH regu1ation.J. Cereb.Blood How Melab., 4, 17-27. Nedergaard, M. & Hansen, AJ. (1988) Spreading depression is not associated with neuronal injury in the normal brain. Brain Res., 449, 395-398. Nedergaard, M. & Hansen, AJ. (1993) Characterization of cortical depolarizations evoked in focal cerebral ischaemia.3 Cereb. Blood Flow Metub., 13, 568-574. Nicholls, D.G. (1985) A role for the mitochondrion in the protection of cells against calcium overload? Progr. Brain Res., 63,97-106. Ohta, S., Gido, G. & Siesjb,B.K. (1992)Influence ofischaemia on blood-brain and blood-CSF calcium transport.3 Cmeb. Blood Flow Metub., 12,525-528. Pelligrino, D., Almqvist, L.-0. & Siesjo, B.K. (1 98 1) Effects of insulin-induced hypoglycemia on intracellularpH and impedance in the cerebral cortex of the rat. Brain Res., 221,12!3-147. Pelligrino, D., Yokoyama, H., Ingvar, M. & Siesjo, B.K. (1982)Moderate arterial hypotension reduces cerebral cortical blood flow and enhances cellular release of potassium in severe hypoglycemia. Actu Physhl. Scand., 115,51 1-5 13. Pumain, R. & Heinemann, U. (1985) Stimulus- and amino acid-induced calcium and potas53, 1-16. sium changes in rat neocortex.3 Jvmm~hy~ol., Siemkowicz, E. & Hansen, AJ. (1981) Brain extracellular ion composition and EEG activity following 10 minutes ischaernia in normo- and hyperglycemic rats. Stroke, 12,236-240. Siesjo, B.K. (1988a)Acidosis and ischaemic brain damage. Nmrochem. Pathol., 9, 31-88. Siesjo, B.K. (1988b) Hypoglycemia, brain metabolism, and brain damage. Diab./Met. Rev., 4(2), 113-144. Siesja, B.K. (1992) Pathophysiology and treatment of focal cerebral ischaemia. I. Pathophysiology.j ! Neurosurg., 77, 169-184. Siesjo, B.K. & Bengtsson, E (1 989) Calcium fluxes, calcium antagonists, and calcium-related pathology in brain ischaemia, hypoglycemia, and spreading depression: a unifjring hypothesis.3. Cereb.Blood Flow Metubol., 9, 127-140. Siesjo, B., Katsura, K. & Kristian, T. (1996)Acidosis-related damage. In Advances in Neurology (eds. Siesjd, B. & Wieloch, T)Raven Press, New York. Siesjo, B.K. & Siesjo, F! (1996)Mechanisms of secondary brain damage. Europ. j! Anaestheswl., (in press). Siesjo, B., Zhao, Q, Pahlmark, K., Siesjo, F!, Katsura, K. & Folbergrova,J. (1995) Glutamate, calcium, and free radicals as mediators of ischaemic brain damage. Ann. Thorac. Surg., 59, 1316-20.


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Silver, LA., Deas, J. & Erecinska, M. (1996)Ion homeostasis in brain cells. Differences in intracellular ion responses to energy limitation between cultured neurones and glial cells.j! Cereb. Blood Flow Metub., (in press). Silver, I. & Erecinska, M. (1990)Intracellular and extracellular changes of [Ca2'] in hypoxia and ischaemia in rat brain in viva. j! Gen. Physiol.,95,837-866. Silver, LA. & Erecinska, M. (1992)Ion homeostasis in rat brain in viva: intra- and extracellular Ca2+and H + in the hippocampus during recovery from short-term, transient ischaemia. J. Cereb. Blood Flow Metub., 12,759-772. Smith, M.-L., Hanwehr, R.V & Siesjo, B.K. (1986)Changes in extra- and intracellular pH in the brain during and following ischaemia in hyperglycemic and in moderately hypoglycemic rats.3 Cereb. Blood Flow Metub., 6,574-583. Stys, P.K., Waxman, S.G. & Ransom, B.R. (1991)Na+-Ca'+ exchanger mediates Ca2+influx during anoxia in mammalian central nervous system white matter. Ann. Neural., 30,375-380. Tsubokawa, H., Oguro, K., Robinson, H.P.C., Masuzawa, T. & Kawai, N. (1995)Single glutamate channels in CAI pyramidal neurones after trasient ischaemia. Ndorepmt, 6, 527-53 I . Tsubokawa, H., Oguro, K., Robinson, H.P.C., Masuzawa, T., Kirino, T. & Kawai, N. (1992) Abnormal Ca2+homeostasis before cell death revealed by whole cell recording of ischaemic CAI hippocampal neurones. Neuroscience., 49,807-81 7. Tsubokawa, H., Oguro, K., Robinson, H.P.C., Masuzawa, T., Rhee, T.S.G., Takenawa, T. & Kawai, N. (1 994)Inositol 1,3,4,5-tetrakisphosphateas a mediator of neuronal death in ischaemic hippocampus. Naroscience, 59,29 1-297. Warner, D.S., Smith, M.-L. & Siesjo, B.K. (1 987)Ischaemia in normo- and hyperglycemic rats: effects on brain water and electrolytes. Stroke, 18,464-471. Xie, Y , Zacharias, E., Hoff, P. & Tegtmeier, E (1995)Ion channel involvement in anoxic depolarisation induced by cardiac arrest in rat brain. j! Cereb.Blood Flow Metab., 15,587-594. Zaidan, E. & Sims, N. (1994)The calcium content of mitochondria from brain subregions following short-term forebrain ischaemia and recirculation in the rat. j! Neurochem., 63,

I81 2-18 19. Zhon, Z. & Neher, E. (1993)Mobile and immobile calcium buffers in borine adrenal chromaffin cells.3 Physiol.,469,245-273.


Chapter 3

~

TECHNIQUES FOR EXAMINING NEUROPROTECTIVE DRUGS IN VIVO A. Richard Green and Alan J. Cross* Astra Arcus, Bakewell Road, Loughborough, LEI I SRH, UK *Astra Arcus USA, 755 Jefferson Road, Rochester, New York, NY 14623, USA

3. I 3.2

3.3

3.4

3.5 3.6

General introduction Global models of acute ischaemic stroke 3.2. I Bilateral carotid artery occlusion in the gerbil 3.2.2 Two-vessel occlusion in the rat 3.2.3 Four-vessel occlusion in the rat Focal models of acute ischaemic stroke 3.3. I Introduction 3.3.2 Photochemically induced infarction 3.3.3 Other models of thromboembolism 3.3.4 Middle cerebral artery occlusion models The design of studies using animal models t o discover clinically useful neuroprotective drugs 3.4. I Introduction 3.4.2 Clinically realistic methods of dosing Control of physiologicalvariables 3.4.3 3.4.4 Assessment of damage 3.4.5 The species for investigation Protocols required when using animal models t o discover new therapeutic entities General discussion References

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3. I General introduction Preclinical studies on almost any illness or disease utilize animal models as one of the experimental approaches. There can be two rather distinct, but obviously sometimes related, reasons for this. The first is to model the clinical problem as accurately as possible in order to gain insight into the mechanistic aspects of the problem (for example, the neurochemical changes occurring in the brain following an ischaemic episode) and in this way identlfy potential targets of drug action. The second is to examine putative therapeutic approaches (such as a new drug candidate) in order to predict the Academic Press Limited Copyright 0 1997 All rights ofreproduction in anyform reserved

NEUROPROTECTIVE AGENTS AND CEREBRAL ISCHAEMIA, IRN 40 ISBN 0-12-366840-9; 0-12-197880-X @bk)

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A.R. GREEN AND A.J. CROSS

possible clinical value of that approach. Increasing knowledge of mechanistic changes occurring in a disease state can, in turn, lead to novel therapeutic intervention which can then be examined in the model. Some clinical conditions such as hypertension are amenable to clinically accurate animal models, while others, such as the major psychoses, are more &cult. In the case of psychiatric disorders, a further type of animal model can often be seen to be employed: the screening model. This type of animal model may show few functional similarities to the disease state. Nevertheless, the functional response being examined is altered by drugs known to have clinical efficacy and the model can therefore be used to screen new therapeutic compounds. Some screening models only detect further compounds of the same therapeutic ‘class’ as existing drugs. For example, the antiemetic efficacy of neuroleptic drugs results from a dopamine antagonist action. Therefore, new antischizophrenic drugs being detected by use of this model are almost certainly going to be dopamine antagonists. At present there are no simple (or even complicated!) screening models available which are known to be predictive for neuroprotective activity in the treatment of acute ischaemic stroke. This is for the simple reason that there are currently no drugs available that have been shown to be of clinical benefit. This situation is likely to change in the next three years as there are now several compounds in major clinical trials (see Chapters 8 and 15).Moreover, confidence in clinical trial methodology is strengthened by the positive findings with thrombolytics. Nevertheless, there are a variety of models available which are claimed to model aspects of acute ischaemic stroke in humans. However, there exist a substantial number of variants of these models, reflecting the efforts of neuroscientists to try and model as closely as possible the clinical situation. In return, it has been hoped that we would learn more from these models about the pathogenesis of stroke. This was expected to assist in the discovery of mechanisms responsible for the lesion and help in formulating ideas for therapeutic intervention. However, as pointed out elsewhere (Hunter et al., 1995), there is a significant risk here of producing a circular argument. It is dimcult to obtain patients immediately after the onset of stroke and also extremely dimcult, if not impossible, to study cerebral function in an acutely ill patient. Therefore, much of the knowledge of what is assumed to occur in the brains of patients during an acute ischaemic insult derives from data that has been obtained in experimental animals (e.g. Pulsinelli, 1992). This is not to deny that data obtained in animals, particularly higher species such as primates, is probably of value. However, its relevance must remain unproven. There is therefore an obvious weakness in claiming that efficacy in an animal model is of probable clinical importance because it modifies changes observed in the same or another animal model. This chapter will not discuss and evaluate all the available animal models of stroke; this has been done quite excellently elsewhere by Ginsberg and Busto (1989). It will comment on the strengths and weaknesses of some of the most used models and discuss experimental protocols which are necessary when evaluating potential neuroprotective drugs in order to be able to claim that the compound is worthy of serious evaluation as a candidate drug.

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FIGURE1 Ischaemic damage in gerbil hippocampus following transient forebrain ischaemia. Micrographs shown 20 pm coronal sections of the dorsal hippocampus stained with cresyl violet 4 days post-ischaemia. Note almost complete loss of CAI neurones in ischaemic gerbil compared to control. Data taken from Cross et al. (1 991).

degenerated neurones as a percentage of the entire CAl/CA2 region, thereby correcting for minor differences in sectioning through the hippocampus (Gillet al., 1987; Osborne et al., 1987). It is worth noting that the histopathology in the gerbil after this ischaemic insult is similar to that observed in the human brain following a cardiac arrest (Zola-Morgan et al., 1986; Petito et al., 1987) and that this damage also develops over a 24-hour period (Petito et al., 1987). While it would be naive to suggest that the model can be used for accurate dose-response curves with potential neuroprotective compounds (because of variation in damage seen from experiment to experiment),the model is nevertheless amenable to semi-quantitative evaluation, producing both time- and dose-dependent data (Figure 2).

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49

Stroke is an extremely variable clinical condition which reflects the variability of the underlying disease process. The occlusion can occur at many different sites in the brain and the cause of the occlusion, the severity of the problem, and the degree of reversibilitycan all contribute to the variability in outcome. In addition, other clinical problems, including severe hypertension, can be present in these patients. In contrast, animal models either control or eliminate most of these variables and it is only by taking this approach that the results of the studies can be interpreted. Sometimes a few of the problems are ‘included’ in the models, such as the use of hypertensive rats (e.g Fujishima et al., 1981). However, few workers use aged rats even though stroke is predominantly a disease of later life (Bonita, 1992). Other confounding factors which will need to be taken into account include reperfusion haemorrhagic transformation following thrombolytic treatment. Generally, models are grouped into those producing either global or focal ischaemia. Global ischaemia is produced by transiently occluding blood vessels supplying the brain, thereby resulting in a widespread hypoxic ischaemic episode, usually in the forebrain. Focal ischaemia is generally produced by occlusion (transient or permanent) of specific and selective cerebral vessels, thus producing damage to more defined regions of the brain. It is generally assumed that global models are more relevant to cardiac arrest, while focal models are of greater relevance to acute ischaemic stroke. This may be a reasonable premise but remains unproven until drugs with clinical utility are available to test the hypothesis.

3.2 Global models of acute ischaemic stroke

3.2. I BILATERAL CAROTID ARTERY OCCLUSION IN THE GERBIL This model has been and continues to be widely used. Technically it is a relatively easy model to use since the surgical procedures are straightforward. A transient ischaemic episode is induced in the brain of the Mongolian gerbil (Merionesunguicuhtus)by bilateral occlusion of the common carotid arteries (Crockard et al., 1980). The organization of the cerebral circulation of the gerbil ensures that this two-vessel occlusion results in substantial forebrain ischaemia. The period of occlusion is generally about five minutes: periods greater than 10 minutes generally prove to be fatal. Following shorter periods of occlusion, a characteristic and very specific pattern of neurodegeneration is observed, with the large neurones of the hippocampal CA1/CA2 subfields being particularly affected (Brown et al., 1979).Longer periods of occlusion can result in other brain regions, including the cortex and striatum, also being damaged (e.g. Baldwin et al., 1993a). The degeneration of the hippocampal neurones develops following a period of 24 hours during which no obvious morphological changes are apparent, a process which has been termed ‘delayed neuronal death’ (Kirino, 1982). The value of the model is that the neurodegeneration observed is histologically obvious (Figure 1) and easily quantified, for example, by measuring the length of


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Time post-ischaemia (h) FIGURE2 The effect of chlormethiazole on hippocampal CAI neurone degeneration following transient (5 minute) forebrain ischaemia in the gerbil. Upper: dose-response effect when administered intraperitoneally (i.p.) 1 hour post-ischaemia. Lower: time course of protective effect when given (622 pmol/kg i.p.) at the indicated times post-ischaemia. Data taken from Cross et al. (1991).

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A.R. GREEN AND AJ. CROSS Ischaemidsaline 240

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FIGURE3 Chlormethiazole reduces the hyperlocomotion (upper) and disruption of nestbuilding behaviour (lower) in the gerbil following transient forebrain ischaemia. Data taken from Baldwin et al. (1993a).

Gerbils who have been subjected to an ischaemic insult display behavioural abnormalities such as impaired nest-building and hyperactivity (Baldwinet al., 1993a) and such behaviours can be examined to determine whether neuroprotective compounds have attenuated these abnormal responses (Figure 3).


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3.2.2 TWO-VESSEL OCCLUSION IN THE RAT This model employs bilateral carotid artery occlusion combined with systemic hypotension sufficient to reduce significantly collateral blood flow (e.g. Smith et al., 1984; see also Ginsberg and Busto, 1989). The value of the model again lies in the selective cell death produced in the CA1 neurones of the hippocampus and other vulnerable structures such as the caudoputamen and cortex (Smith et al., 1984). Like the gerbil model, there is one-stage surgery, but unlike the gerbil (which only weighs 50-70 g) the use of rat also allows a variety of physiological measures to be monitored (including cerebral blood flow, p 0 2 , and C 0 2and heart rate). The major problem, which is self-evident, is the necessity to produce hypotension. There are various ways to achieve this, but Ginsberg and Busto (1989) have suggested that even modest variations around the required and generally accepted level of 50 mm Hg may produce variable pathology Other problems can include post-ischaemic seizures (Smith et al., 1984) and the fact that the animals must be anaesthetized, so behavioural evaluation cannot be made.

3.2.3 FOUR-VESSEL OCCLUSION

IN THE RAT

This model allows severe forebrain ischaemia to be produced in awake and freely moving rats and also induces reproducible neuropathology. Therefore, a substantial number of investigations have been made with this model. A limitation is that it is a two-stage operative procedure (Pulsinelli and Brierley, 1979). The first step places a clamp around each carotid artery and an electrocauterization needle is used to coagulate the vertebral arteries (Pulsinelliand Buchan, 1988).The second stage performed 24 hours later requires a brief restraint of the animal while the carotid clips are tightened. These clamps may subsequently be released to permit reperfiusion. Problems with the models include the fact that the first step involves fairly sophisticated surgery (the electrocoagulation step can easily damage the brain if not performed with great skill),and there can be substantial variability not only within one strain but also within the same strain from different suppliers (Pulsinelli and Brierley, 1979). There is also marked mortality after the first stage operation even in laboratories with skilled surgeons (see reviews of Ginsberg and Busto, 1989).

3.3 Focal models of acute irchaemic stroke

3.3. I INTRODUCTION Focal models are broadly grouped into two types, permanent and transient. Permanent focal ischaemia generally results in a dense region of ischaemic damage (the core) and a surrounding penumbral area which is 'at risk', the degenerative damage normally

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spreading out from the core into this region. The damage occurring in this region is due not only to the lessened blood supply but also to chemical factors spreading out from the core area. The aim of protective compounds is therefore to protect the penumbral area, since the core area (havingnear-zero blood supply) is probably fatally damaged very rapidly, and will in any case not receive the neuroprotective agent because of the almost total loss of blood supply. However, it is unlikely that permanent and total cessation of blood flow occurs in a brain region in the majority of clinical strokes because of thrombus disintegration and endogenous thrombolysis (Mohr et al., 1986),and so reversible occlusion models have also been developed. Damage to tissue in these models can therefore result from both the ischaemic episode and also the consequences of the reperfusion. The use of both types of model thereby assists in examining different aspects of the neurodegenerative process.

3.3.2 PHOTOCHEMICALLY INDUCED INFARCTION Many of the focal ischaemia models involve major surgical procedures. The photochemically induced infarction model was developed to try and circumvent this problem. The model is relatively non-invasive and involves intravenous administration of the photosensitive dye Rose Bengal and irradiation of specific areas of the brain with a focused light beam of defined wavelength. No craniectomy is required, merely retraction of the skin over the skull (Watson et al., 1985).A reaction between the light and circulating dye generates free radicals, platelet aggregation and thrombosis (Watson et al., 1985). The size of the initial damage is determined by the diameter of the light beam (Snape et al., 1993).The model is very ‘severe’ in that damage develops rapidly from the core - over 60% of the final area of damage being apparent within 120 minutes of irradiation (Snape et al., 1993; and see Figure 4). There is also the rapid appearance of oedema (Dietrich et al., 1987; Snape et al., 1993; Green and Cross, 1994b)and breakdown of the blood-brain barrier (Snape et al., 1993). However, there is no penumbral region of the type seen with other focal models and despite the superficial similarity of this model to the clinical situation (the non-invasive technique and thrombolytic lesion), this model is now rarely used. This is probably partly because the speed and severity of the lesion results in few agents being neuroprotective, although chlormethiazole (Snape et al., 1993),NBQX (Wood et al., 1993) and the Ca2+channel antagonists (see also Chapter 5),SB201823A (Benham et al., 1993)and flunanarazine (De Ryck et al., 1989)have been reported to be active. Nevertheless, the model has proved useful in examining some of the neurochemical changes that occur in the core region (Baldwin et al., 199313, 1994).

3.3.3

OTHER MODELSOF CEREBRAL THROMBOEMBOLISM

There have been several approaches used to try and mimic the thromboembolic changes that initiate a stroke. Such approaches have included adaptation of the photochemical method to produce platelet thrombosis in the common carotid artery of rats


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Time post-treatment (h) FIGURE 4 Development of ischaemic damage following photochemical thromboembolism in rat cerebral cortex. Ischaemic damage was assessed by measuring the extent of extravasation of Evans Blue on the surface of the cerebral cortex following ischaemia. Open circles represent saline-treated ischaemic animals and filled circles chlormethiazole-treated animals. Data taken from Snape el al. (1993).

(Futrellet al., 1988).This results in damage in several brain regions, but predominantly in the cerebral cortex (Futrell et al., 1988).Another technique has been the injection of small (35 VM) carbon microspheres into the internal carotid artery of rats (Kogure et al., 1974), thereby producing multifocal infarctions. In essence this approach is the development of a technique that has been known and used for many years, namely the injection of small blood clots into the carotid artery of larger animals (Hillet al., 1955). More recently, this approach has been adapted for use in the rat (Kudo et al., 1982). The problem with all of these models is that one has no control over the distribution of the clots or microspheres. Therefore, there is no uniformity of the size or location of the infarcts. The ‘clot’ models can be used for examining thrombolytic agents but the need for at least semi-quantificationof cerebral infarct lesions makes these models problematic for the examination of neuroprotective agents. Nevertheless, it has been possible to show a protective effect of tissue plasminogen activator (tPA)in this type of model and the advantages of combination therapies have begun to be examined.

3.3.4 MIDDLE CEREBRAL ARTERY OCCLUSION MODELS

The middle cerebral artery (MCA) occlusion models are used extensively and have been suggested to be of particular importance to the drug discovery process because

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of their clinical relevance. This is because the MCA is the most commonly affected vessel in stroke victims (Mohr et al., 1986; Karpiak et al., 1989) and because it is a model that is particularly amenable to techniques that allow reperfusion to occur, as happens in many strokes.In consequence, the MCA occlusion model has been studied extensively and this has resulted in both substantial advantages and disadvantages in the use of this model. There are several obvious advantages. The model is well documented and there is a substantial body of evidence on drugs that do, and do not, protect against neuronal cell death. The model has been applied to several species, including rats, cats and dogs, and variations in the techniques allows some selection as to the cerebral areas affected. Techniques for occluding the arteries include cauterization, clips and threads, intraluminal thread insertion and photochemically induced thrombolytic occlusion (see Ginsberg and Busto, 1989, for review). A major disadvantage of this model is that there are a substantial number of variations in the technique of occluding the artery. This has led to large merences between laboratories in the size and variability of the ischaemic lesion and the efficacy of therapeutic agents. There is also a large strain-dependent effect in rats, also leading to variability The surgical procedures can be extensive and major differences in the degree of damage have been reported by different investigators depending on the site of the occlusion in the MCA. Use ofligatures or clips to allow reperfusion to take place is an important modification because clinically repeated angiography suggests that reperfusion occurs in up to 50% of stroke patients (Saito et al., 1987). The intraluminal thread technique (e.g. Longa et al., 1989; see also Figure 5) is particularly attractive in this regard as the surgical techniques are less severe than with several other methods and the thread can either be withdrawn after a period of time to permit reperfusion (see Sydserff et al., 199513) or left in place when a permanent MCA occlusion model is required (see Sydserff et al., 1995a, 1996). Studies can therefore be performed which allow investigation of the effect of the drugs on the damage induced by both the ischaemic episode and the reperfusion process. Another feature of the MCA occlusion model is the occurrence of brain swelling and oedema (see for example Sydserff et al., 1996), a major clinical problem with stroke. Several studies have investigated the problem of oedema using the MCA occlusion model and it appears to be a consistent feature of this stroke model (e.g. Hayward et al., 1993; Park et al., 1994; Sydserffet al., 1996). There are now many agents that have been reported to be protective when treatment is initiated prior to permanent ischaemia, including NMDA antagonists (Bielenberg and Beck, 1991; Buchan et al., 1992; Park et al., 1994), and the calcium antagonist nimodipine Uacewicz etal., 1990).The damage produced by transient focal ischaemia can also be attenuated by dizocilpine (Park et al., 1988; Gill et al., 1991; Buchan et al., 1992),NBQX (Xue et al., 1994)and isradipine (Kawamura et al., 1991). Chlormethiazole is one of the only compounds to date to be shown to be neuroprotective (Figure 6) when given after the start of repehsion (Sydserff et al., 1995b). Several studies have also shown that neuroprotection demonstrated histologically is


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FIGURE 5 Middle cerebral artery occlusion by use of an intraluminal thread. A nylon suture is introduced into the external carotid artery (ECA) and passed up into the internal carotid artery (ICA).The diameter of the suture is such that it lodges in the anterior cerebral artery (ACA),occluding the middle cerebral artery at its origin. (CCA common carotid artery; PGPA pterygopalatine artery.) Drawing by Simon SydserK

FIGURE 6 The neuroprotectiveeffect of chlormethiazole following transient (1 hour) occlusion of the MCA. Chlormethiazole was administered (1000 pml/kg i.p.) either 1 hour before occlusion (filled bars) or 10 minutes after reperfusion (shaded bars). Both treatments reduced the volume of ischaemic damage in cortex and striature compared to saline controls (open bars). Data taken from Sydserffet al. (199513).

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also accompanied by a decrease in brain swelling and oedema (Hayward et al., 1993; Park et al., 1994; Sydserff et al., 1996). These findings may have clinical significance since oedema and brain swelling may exacerbate the pathological progression of cerebral ischaemia (Katzmann et al., 1977).

3.4 The design of studies using animal models to discover clinically useful neuroprotective drugs 3.4. I INTRODUCTION At present, in the absence of drugs that have been proven to be efficacious clinically, it is possible only to suggest guidelines on the best approaches to use when employing animal models of acute ischaemic stroke, and these have to be personal views. It could be that the best predictive model will finally be showq to be one that appears to have only a limited relationship to the known pathophysiology of stroke. As stated earlier, this is the case in many screening models for the detection ofpsychoactive compounds. What would be generally acceptable in this situation would be a model that gave ‘false negatives’ but not one that gave ‘false positives’. In reality such a simplistic approach does not occur in drug development; a compound would never reach clinical development without much other supportive data, including (usually)a known mechanism of action thought to be relevant to stroke efficacy in one or more in uitro test systems (see for example Chapter 8) and the usual battery of safety evaluation data, all of which could support or stop the development of the compound for clinical use. With regard therefore to the way that compounds should be evaluated in animal models, there are some factors these authors regard as mandatory in the design of experiments evaluating putative protective agents.

3.4.2 CLINICALLY REALISTIC METHODSOF DOSING 3.4.2.1 Time of drug administration At present, any clinical trial on a potential neuroprotective compound will be admitting and treating patients ajer they have had a stroke. It is likely that in a few years’ time when efficacious neuroprotective compounds are available, trials will be undertaken to examine the value of giving such compounds prophylactically to patients who are at high risk of having a stroke. Nevertheless, that is for the future and today any neuroprotective will be given at some time after the ischaemic insult. Potential therapeutic agents must therefore be shown to be efficacious in animal models when given after the ischaemic episode. There are three reasons for this. First, giving the drug before the insult is not a realistic model of the clinical situation. Second, giving the drug before the insult ensures that the drug is in the tissue which


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is compromised and does not have to penetrate into the ischaemic tissue, which is also

unrealistic. Third, giving the drug before the ischaemic episode may result in alterations in cerebral blood flow, plasma glucose concentrations, cerebral oxygen utilization or the temperature of the animals. Every one of these factors has been shown to cause alterations in the degree of ischaemic damage (Pate1 et al., 1991; Needergaard, 1987; Busto et al., 1987). The big problem remains as to what time after the insult is it realistic to administer the drug, that is, what is the size ofthe therapeutic time window? Claims have been made as to the superior value of a particular compound because of its large window of opportunity, as demonstrated by investigations in animal models. The conopeptide SNX- 1 1 1, for example, has been shown to reduce tissue damage up to 24 hours after the insult when using the four-vessel occlusion model (Buchan et al., 1994). This feature, it has been claimed, might confer significant clinical benefit compared to other experimental compounds. This argument may be valid but the problem is that the speed with which damage develops in the brain varies considerably dependent upon the model being used. In general, the damage develops rapidly in focal models, within 60 minutes in the case of the photochemical model for example (Snape et al., 1993). The fact that some NMDA receptor antagonists have to be given within 120 minutes of reperfusion (Hatfield et al., 1992), therefore, does not rule out the possible value of such compounds in humans when given several hours after a stroke where the development of the damage is thought to occur more slowly Thus it has been proposed that treatment to humans be initiated within eight hours of the acute ischaemic insult (Pulsinelli, 1992) and although this proposal appears to be based on data obtained in primate studies (Jones et ul., 198l), positron emission tomography (PET) scans do indicate that this is a reasonable suggestion. Following cardiorespiratory arrest (global ischaemia) in humans it has been demonstrated that damage in the hippocampus develops over many hours (Petito et al., 1987), in a manner that is analogous to global ischaemia in the gerbil (see section 3.2). In conclusion, therefore, while it is felt important that compounds are administered after the ischaemic insult (that is, after the start of reperfusion period in global or reversible focal models, or some time after the start of the ischaemic period in permanent occlusion models), these authors are not convinced that the time window in animals should be taken as reflective of the therapeutic time window in humans. It is likely that questions as to the size of the therapeutic time window available will lessen in the next few years. Early studies with potential therapeutic compounds allowed inclusion of patients for considerable time after the stroke - two days in the case of the nimodipine study (American Nimodipine Study Group, 1992). Trials have now been published in which the inclusion period was less than six hours (Haley et al., 1993; Lenzi et al., 1994),demonstrating that it is possible to get patients into hospital in this time if physicians are educated and alert. Furthermore, when neuroprotective drugs do become available, the admission period will shorten further since all clinicians will be aware of the need for rapid intervention. At present many physicians do not see the need for rapid admission when they know that little is available apart from supportive measures.

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3.4.2.2 Roub ofdrug administration Many stroke patients on admission are not in a condition to swallow drugs, which means that a neuroprotective compound will have to be given by a parenteral route. Furthermore, it is likely, given the fact that neuronal damage develops over a period of time, that sustained plasma (and hence cerebral) levels of a drug will increase the degree ofprotection. This has certainly been the experience of these authors in experimental animals with chlormethiazole (Cross et al., 1995a,b). Experimental drugs should be given, therefore, to animals by sustained intravenous infusion at some stage of the development process. While intravenous infusion can be maintained for relatively short periods of time in anaesthetized animals (Gill et al., 1991),longer periods require different approaches because continuous anaesthesia is impractical. Recent studies have achieved drug infusion for 24 hours in conscious animals, either by lightly restraining the animal (Park et al., 1992) or by catherization of the jugular vein and passage of the tube under the skin to the scalp where it is cemented in place, allowing subsequent attachment of an infusion pump (Cross et al., 1995a). This technique has been found to produce sustained and steady plasma drug concentrations. Use of implanted Alzet minipumps allows a similar approach. 3.4.2.3 Realistic dosiig regimes Doses of drugs given to experimental animals should be realistic. That is, the plasma levels of the drug in experimental animals should be similar to those known or proposed to be well tolerated in humans. A problem with some of the early studies on neuroprotective agents was that compounds were given to anaesthetized animals at very high doses. A case in point were some of the studies with dizocilpine (MK-801) which causes quite severe ataxia and behavioural disruption in rats at doses many times lower than those reported to be neuroprotective (Cross et al., 1995b). It follows that safety constraints were not going to allow clinical studies to be undertaken at doses which were likely to be neuroprotective. A further point is that studies using intravenous infusion techniques should be undertaken only following pharmacokinetic studies which then allow the design of appropriate infusion protocols and which result in defined and steady plasma drug concentrations.

3.4.3

CONTROL OF PHYSIOLOGICAL VARIABLES

It is unlikely that there are any investigators using animal models of stroke who are now unaware of the necessity to control body temperature. There is considerable evidence that hypothermia confers neuroprotection, not only in models of stroke (Busto et al., 1987)but also when neurodegeneration is being induced by neurotoxins such as methamphetamine and ecstasy (Ali et al., 1994; Broening et al., 1995). There has been substantial debate on the ability of NMDA antagonists such as

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dizocilpine to produce neuroprotection in global models. Buchan and Pulsinelli (1 990), for example, claimed that dizocilpine was neuroprotective only because of hypothermia. Others have questioned this conclusion (Gill and Woodruff, 1990)but it is clear that protection by dizocilpine is much more modest in these models when temperature has been controlled than was reported in initial investigation where it was uncontrolled (see Green and Cross, 1994b). What is uncertain is the period during which temperature should be maintained in order to avoid misleading results. One suspects that monitoring over a 24-hour period is reasonable. However, no one appears to be clear as to what temperature constitutes ‘hypothermia’. A brief (20 minutes) decrease of 2-3OC probably has less of a neuroprotective effect than a modest l0C decrease that is sustained for 24 hours or more (Corbett, personal communication). Maintaining temperature therefore remains of paramount importance in experimental studies using laboratory animals since it becomes impossible otherwise to determine the mechanism of action of the investigational drug. Clinically,however, strict body temperature control is unlikely to occur and a modest hypothermic action could be an asset. Certain other physiological functions should also be monitored in any experimental investigation using laboratory animals. Paramount are cardiovascular changes since they could provide an explanation for the neuroprotective effect and be a problem clinically, affecting outcome in both ischaemic and haemorrhagic stroke (Pate1 et al., 1991). As stated earlier, consideration should also be made of cerebral blood flow, oxygen utilization and plasma glucose levels since these can affect the degree of ischaemic damage.

3.4.4 ASSESSMENT OF DAMAGE The primary method of assessment of neuronal damage is that of measurement of histological change (e.g. Osborne et al., 1987). However, histological damage is not always easy to quantify and other techniques are now becoming available which measure the degree of neurological damage. These techniques are primarily biochemical and include the following. Measurement of the degree of gliosis by the use of tissue binding of [3H]-PKl 1195. This is a ligand which is selective for the ‘peripheral’ benzodiazepine sites. These binding sites are not known to be present on neurones but are present on glial cells and macrophages (Starosta-Rubinstein et al., 1987). Ischaemia results in increased [3H]PK 1 1 195 binding in rat (Benavideset al., 1990)and gerbil (Baldwin et al., 1993a) brain, presumably through gliosis and macrophage infiltration (Myers et al., 1991). Measurements of glial fibrillary acidic protein (Miller and O’Callaghan, 1993) and neurone specific enolase (Barone et al., 1993) have also been undertaken in cerebral tissue; both these markers changed after an ischaemic insult (Miller and O’Callaghan, 1993; Barone et al., 1993). Indeed, it has been reported that the increase in neurone-specific enolase correlated with the increase in infarct volume in the rat MCA occlusion model (Hatfield and McKernan, 1992). While these techniques are available to the scientist using animal models, they will

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not be used in general by the neurologist who is assessing whether a treatment has functional benefit in the patient. What is important to the patient is the severity of the neurological deficits. The neurologist will therefore use scales to examine neurological function (see Chapter 15). It is important therefore that measures of behavioural change be developed appropriate for use in the various animal models of stroke. Already there is some evidence which suggests that lesion size and neurological deficit do not correlate (Rogers et al., 1992) and that following an ischaemic episode, drug-treated rodents function more efficiently than controls in behavioural models, even when significant damage is still apparently histologically (Baldwin et al., 1993a). The behaviours assessed will obviously be dependent on the species under investigation. The gerbil, for example, has been examined in tests of locomotion and nest building (see section 3.2.1). Rats can be examined in tests of cognition and attentional skills, locomotion and balancing behaviour, and even dexterity in picking up food with one paw or another. Most of these behaviours have been examined extensively and documented in normal animals over many years. Models using primates are likely to increase because measures of neurological outcome can be made on complex tasks, including manual dexterity,which have exact equivalents in humans.

3.4.5 THESPECIES FOR INVESTIGATION One cannot suggest that there is a ‘preferred’ species for use in modelling stroke. In the case of global ischaemia in the gerbil, the species is self-selecting because of the anatomy ofthe cerebral circulation in these animals. As is apparent in sections 3.2 and 3.3, most models have been developed using rats. This reflects cost considerations and the familiarity ofmost experimenters with this species. It is also a large enough animal to enable physiological parameters to be measured. It has been proposed that the cranial circulation is similar in rat and human pamori et al., 1976),particularly when compared to gerbil, cat and dog, although the rat does not have a good collateral blood supply (Macrae, 1992).The rat brain is also amenable to studies using magnetic reasonance imaging (MRI). This holds promise for the future in that comparisons can be made between histological change, biochemical change, behavioural change and MRI. The rat also has a limited but extensively studied behavioural repertoire. However, as stated earlier (Hunter et al., 1995), the behavioural and neurological equivalence between primates and humans does make it likely that stroke models in primates may be possible in future which are rather close, both physiologically and neurologically, to the pathological state in humans. Pharmacokinetic considerations should also not be forgotten. The differences in the protective effect of dizocilpine in dogs and primates may have been due primarily to pharmacokinetic differences (Shearman, 1989) and a major difference in drug metabolism between the species under investigation and humans will complicate interpretation considerably in terms of the relevance of data obtained to the clinical situation.


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3.5 Protocols required when using animal models to discover new therapeutic entities It is clear from the foregoing sections that no one model fulfils all the requirements for the ‘ideal’model. What can be done is make selections which incorporate desired features. Thus one can select focal or global models, models that produce a permanent ischaemic lesion or allow reperfusion, and those that do or do not allow measurement of physiological parameters or behavioural change. While it is hard to argue strongly against the claims that focal ischaemia using MCA occlusion followed by reperfusion most closely mimics the clinical situation, the fact remains that the predictive value of all models remains unknown. Therefore, it is suggested that any novel compound be examined using at least two models, one of which should be a focal model and one a reperfusion model. Studies should also be undertaken in at least two species. Drugs should be administered post-ischaemia and a reasonable period allowed for recovery so that functional (behavioural) studies can be undertaken. Histology should also be performed after a sustained recovery period where possible to confirm that the drug has produced a true neuroprotective effect, not merely slowed the rate of degenerative process. The effects of the compound on blood pressure, body temperature and other physiological variables should be monitored and, where necessary, controlled. Plasma levels of the drug should be made and the pharmacokinetics of the drug evaluated. Drug administration should at some point be by intravenous infusion and the dose administered reflect the likely clinical dose. Many investigators reading this will doubtless feel that all of this is self-evident. Nevertheless, re-examination of earlier studies (and sadly many current investigations) reveal extravagant claims being made for clinical usefulness of many compounds that have not been tested using these reasonable guidelines. However, there are recent studies which do follow these guidelines rather closely (e.g.Sharkey and Butcher, 1994; Gill et al., 1991 ; Cross et al., 1995a).

3.6 General discussion It seems to these authors that stroke is one therapeutic area where a great deal of reliance appears to be made on animal models in the drug recovery process. This is presumably because it is felt that the pathological process following an ischaemic episode is likely to be similar across species. This may well be true. However, the problem in this thinking lies in the fact that production of an ischaemic episode in an animal is a very controlled and ‘singular’ process and is performed in a healthy animal. In contrast, stroke in humans has a variable aetiology, pathology, clinical presentation and outcome, and occurs in patients who may well have a variety of clinical problems. These facts, coupled with problems in quantlfjring outcome (at present generally changes in neurological assessment),mean that substantial patient numbers

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A.R. GREEN AND A:J. CROSS

(arguably more than 1000)must be included in any major trial to be confident in the results obtained. This situation will probably change when the use of biochemical markers such as changes in neurone specific enolase concentration in plasma and MRI become established. However, such markers will be of value only if they can be shown to correlate with substantial clinical improvement; no one is going to be interested in a drug which improves markers of neuronal damage if it does not also markedly improve the quality of life of the patient. The use of an animal model which is a reliable predictor of clinical outcome would also assist in removing the ethical concerns that exist at present in entering patients into a trial in the absence of a substantial confidence in the outcome. Such concerns will become major after the advent of the first successful therapy, as subsequent trials will not be easy to perform with a placebo group and there will be an expectation that the novel compound be more efficacious than the existing drug. It would be helpful if these expectations could be supported by appropriate animal experimentation. Several authors have questioned the relevance of animal models (Molinari, 1988; Wiebers et al., 1989; Karpiak et al., 1989). Their concerns have centred on both the ability of researchers to accurately model the complex pathology of stroke and the fact that several drugs which have ‘worked’ in animal models have not proved to be of clinical value. Examples include nimodipine (American Nimodipine Study Group, 1992) and thiopental (Brain Resuscitation Clinical Trial Discussion Group, 1986).However, a review of the supporting animal data indicates that rather few of these suggested guidelines were followed. These authors therefore remain positive as to the value of animal models in the development of drugs that will be useful in the treatment of this catastrophic disease.

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Fujishima, M., Ishitsuka, K., Nakatomi, Y, Tamaki, K. & Omae, 1:(1981) Changes in local cerebral blood flow following bilateral carotid occlusion in spontaneously hypertensive and normotensive rats. Stroke, 12, 874-876. Futrell, N., Watson, B.D., Dietrich, W.D., Prado, R., Millikan, C. & Ginsberg, M.D. (1988) A new model of embolic stroke produced by photochemical injury to the carotid artery in the rat. Ann..Meurol., 23, 251-257. Gill, R., Brazell, C., Woodruff, G.N. & Kemp, J.A. (1991) 'The neuroprotective actions of dizocilpine (MK-801) in the rat middle cerebral artery occlusion model of focal ischaemia. Br. 3 Pharmacol., 103, 2030-2036. Gill, R., Foster, A.C. & Woodruff, G.N. (1987) Systemic administration of MK80I protects against ischaemia-induced hippocampal neurodegeneration in the gerbil. J. Neurosci., 7, 3343-3349. Gill, R. & Woodruff, G.N. ( 1 990) The neuroprotective actions of kynurenic acid and MK-80 1 in gerbils are synergistic and not related to hypothermia. Eur.3 Pharmacol., 176, 143-149. Ginsberg, M.D. & Busto, R. (1989) Rodent models of cerebral ischaemia. Stroke, 20, 1627-1642. Ginsberg, A.R. & Cross, A.J. (l994a) Attentuation by chlormethiazole of oedema following focal ischaemia in the cerebral cortex of the rat. .Veurosci. IAttts., 123, 27-30. Green. A.R. & Cross, A.J. (199413) The neuroprotective actions of chlormethiazole. Progr. .Neurobiol., 44, 463-484. Haley, E.C., Brott, T.G., Sheppard, G.L., Barsan, W., Broderick,J.,Marler,J.R. et al. (1 993) Pilot randomised trial of tissue plasminogen act water in acute ischaemic stroke. The TPA bridging study group. Stroke, 24, 1000-1004. Hatfield, R.H., Gill, R. & Brazell, C. (1 992) The dose response relationship and therapeutic window for dizocilpine (MK-80 I ) in a rat focal ischaemia model. Eur.3 Pharmacol., 216, 1-7. Hatfield, R.H. & McKernan, R.M. (1992) CSF neurone specific enolase as a quantitative marker of damage in a rat stroke model. Brain Res., 577, 249--252. Hayward, N.J., McKnight, A.T. & Woodruc G.N. (1993) Neuroprotective effect of the kappa agonist enadoline ((21-977) in rat models of focal cerebral ischaemia. Eur. 3 Neurosci., 5, 96 1-967. Hill, N.D., Millikan, C.H., Wakim, K.C. & Sayre, G.P (1955) Studies in cerebrovascular disease. VII experimental production of cerebral infarction by intracarotid infection of homologous blood clot. Mayo. Clin. Proc., 30, 625433. Hunter, A.J., Green, A.R. & Cross, AJ. (1995) Animal models of acute ischaemic stroke: can they predict clinically successful neuroprotective drugs? Zen& Pharmacol. Sci., 16, 123- 128. Jacewicz, M., Brint, S., Tanabe,.J., Wang, X.J. & Pulsinelli, W. (1990) Nimodipine pretreatment improves cerebral blood flow and reduces brain oedema in conscious rats subjected to focal cerebral ischaemia. 3 Cerebr. Blood Flow Metab., 10, 903-9 13. Jones, T.H., Morawetz, R.B., Crowell, R.M., Marcoux, EW., FitzGibbon, S.J., DeGirolami, VE. & Ojemann, R.C. (1981) Thresholds of focal cerebral ischaemia in awake monkeys.3 Neurosurg., 54, 7 73-782. Karpiak, S.E., Tagliavia, A. & Wakade, C.G. (1989) Animal models for the study of drugs in ischaemic stroke. Annu. Rev. Pharmacol. Exicol., 29, 403-4 14. Katzman, R., Clasen, R., Klatzo, I., Meyer, J.S., Pappios, H.M. & Waltz, A.G. (1977) Brain oedema in stroke. Stroke, 8, 5 12-540. Kawamura, S., Yashui, N., Shirasawa, M. & Fukasawa, H. (1991) Effects of a Ca" entry blocker (nilvadipine) on acute focal cerebral ischaemia in rats. Exb. Brain Res., 83, 434438. Kirino, T. (1982) Delayed neuronal death in the gerbil hippocampus following ischaemia. Brain Res., 239, 57-69. Kogure, K., Busto, R., Scheinberg, I?E. & Reinmuth, O.M. (1974) Energy metabolites and water content in rat brain during the early stage of development of cerebral infarction. Brain, 97, 103- 114.


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Kudo. M., Aoyama, A,, Ichimori, S.E. & Fukunaga, N. (1982) An animal model of cerebral infarction. Homologous blood clot emboli in rats. Stroke, 13, 505-508. Lenzi, G.L., Grigoletto, F., Gent, M., Roberts, R.S., Tech, M., Walker, M. et al. (1994) Early treatment of stroke with nionosialoganglioside G-M-I. Efficacy and safety results of the early stroke trial. ,%’iikf, 25. I552 1558. Lon#a, E.Z., Wciristein, P.R., Carlson, S.E. & Cummins, R. (1989) Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 20, 84 -91. Macrar, I . M . (1!492) New models of focal cerebral ischaemia. Br. 3. Clin. Pharmacol., 34, 302 m. Millcr, D.B. & O’Callaghan, J.P (1993) The interactions of MK-801 with the amphetamine analogues I,-mctharnphetamine (C-METH),3,4 methylenedioxymethamphetamine (DMIIMA) or ~-l-lrnfluramine (D-FEN):neuronal damage and neural protection. Ann. N %rk Acad. Sri, 11SA 679, 32 1-324. Mohr,J.P,Gauticr, J.C., Hier, D. & Stein, R.W. (1986) Middle cerebral artery. In Stroke, Vol. 1: l’athoptysioloLgy, I)ia<po>is andManagemen&(eds Barnett, HJ.M., Stein, B.M., Mohr, J.P & Yatsu, EM.), pp. 377 450. Churchill Livingstone, New Yolk. Moliiiari, G.E (1988)Why model strokes? Stroke, 19, 1 195-1 197. Myers, R., Marijil. L.G., Cullen, R.M., Price, G.W., Frackowiak, R.S. & Cremer, J.E. (1991) Macrophage and astrocyte populations in relation to [3H]-PK 1 1 195 binding in rat cerebral cortex following a local ischaemic lesion. 3. CAreb. Blood Flom Metab., 11, 3 14-322. Needrrgaard, M . ( 1987) Transient focal ischaemia in hyperglycemic rats is associated with increased cerebral infarction. Brain Res., 408, 79-85. Osboriie, K.A., Shigeno, I:, Balarsky, A.M., Ford, I., McCulloch, J., Teasdale, G.M. & Graliam, D.1.( I 987) Quantitative assessment of early brain damage in a rat model of focal cerebral ischarmia. J. .Neural..Neurusug Pyhiat., 50, 402 -4 10. Park, C.K., McCulloch, J., Kang, J.K. & Choi, C.R. (1992) Eficacy of &PPene, a competitivr .N-methyl-!)-aspartateantagonist in focal cerebral ischaemia in the rat. Neurosci. Letts., 147,41 44. Park, C.K., McCulloch, J., Kaiig, J.K. & Choi, C.R. (1994) Pretreatment with a competitive NMDA antagonist 1)-CPPene attenuates focal cerebral infarction and brain swelling in awake rats. ilcla.~eurochzr., 127, 220-226. Park, C.K., Nehls, D.G., Graham, D.I., Teasdale, G.M. & McCulloch, J. (1988)The glutamate antagonist MK-801 reduces focal ischaemic brain damage in the rat. Ann. Neurol., 24, 543 551. Patel, PM., Druniinoiid, J.C. & Cole, 1I.J. (1991) Induced hypertension during restoration of flow after temporary middle cerebral artery occlusion in the rat: effect on neuronal injury arid oedema. Sug .Neural., 36, 195-201. Rtito, C.K., Rlclman, E., Pulsinelli, W.A. & Plum, E ( I 987) Delayed hippocampal damage in humans following cardio-respiratory arrest. Neurology,37, 1281-1 286. Pulsinelli, W. ( 19!)2) Pathophysiology of acute ischaemic stroke. IAncet, 339, 533-536. l’ulsinrlli, W.A. & Brierley.J.B. ( 1 979) A new model of bilateral hemispheric ischaemia in the uiianaesthetised rat. .Stroke, 10, 267-~272. Pulsinelli, W.A. bt Buchan, A.M. (1988) The four vessel occlusion rat model: method for completc occlusion of vertebral arteries and control of collateral circulation. Stroke, 19,9 13-914. Rogers, D.C., Wright, PW., Roberts, J.C., Reavill, C., Rothaul, A.L. & Hunter, AJ. (1992) Photothrombotic lesions in frontal cortex impair the performance of the delayed non-matching to position task by rats. Behav. Brain Res., 49, 231 235. awa, H., Shiokawa, Y., ‘Taniguchi, M. & Tsutsumi, K. (1987) Middle cerebral lusioii: correlation of computed tomography and angiography with clinical outcome. Stroke. 18, 863-868. Sharkey,.J.& Butcher, S.P ( 1 994) lmmunophilins mediate the neuroprotective effects of FK506 in h a 1 ischacinia. .Nature, 371, 336 339.

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Shearman, G.‘I’. (1989)Effect of the NMDA antagonist MK-801 in animal models of focal and global cerebral ischaemia. In C’erebrovascular l h e a e s (eds Giiisberg, M.D. & Dietrich, W.D.), pp. 73-77. Kavrn Press, New York. Smith, M.-L., Bendek, G., Dahlgren, N., Rosen, I., Wieloch, ‘1: & Siesjo, B.K. (1984) Models for studying long-term recovery following forebrain ischaemia in the rat 2. A 2-vessel occlusion model. Acta .Neural. Scand., 69, 385--40I . Snape, M.E, Raldwin, HA.: Cross, A.J. & Green, A.R. ( 1 993) The effects of chlormethiazole and nimodipine on cortical infocrat area after focal cerebral ischaemia in the rat. Neuroscience, 53,837-844. Starosta-Rubinstein, S., Ciliax, B.J., Penney,J.B., McKeerer, l? & Young, A.B. (1987) Imaging of a glioma using peripheral benzodiazepine receptor ligands. Acta .Neuropathol., 8 4 , 8 9 1-895. SydserK, S.G., Cross, AJ. & Green, A.R. (1995a) The rieuroprotective effect of chlormethiazole on ischaemic neuronal damage following permanent middle cerebral artery ischaemia in the rat. .Neurode,gentration, 4, 323-328. Sydserff; S.G., Cross, AJ., West, KJ. & Green, A.R. (l995b) The effect of chlormethiazole on nruronal damage in a model of transient focal ischaemia. Br.J. Pharmacol., 114, 1631- 1635. SydserH; S.G., Green, A.R. & Cross, A:J.(1 996) The effect ofoedema and tissue swelling on the measurement of neuroprotection: a study using chlormethiazole and permanent middle cerebral artery occlusion in rats. Neurodegeneration, 5, 8 I 85. Watson, B.D., Dietrich, W.D., Busto, R., Wachtel, M.S. & Ginsberg, M.D. (1985) Induction of reproductible brain infarction by photochemically initiated thrombosis. Ann. Neurol., 17, 497-504. Wood, N.L., Rothaul, A.L., Meakin, J.E. & Hunter, AJ. (1993) NBQX reduces lesion volume in a rat model of focal cerebral ischaemia. Br. 3 Pharmacol., 108, 265 l? Wiebers, D.O., Adams, H.P & Whisnant, J.P (1 989) Animal models ofstroke: are they relevant to human disease. Stroke, 21, IL3. Xue, I)., Huang, Z.-G., Barnes, K., Lesiuk, HJ., Smith, K.E. & Buchan, A.M. (1994) Delayed trcatmeiit with AMPA, but not NMDA, antagonists reduces neocortical infarction. J. Cerebr. BloodFIouiMetah., 14, 251-261. Yamori, Y.,Horie, R., Honda, H., Sato, M.E. & Fukase, M. (1976) Pathogenetic similarity of strokes in stroke-prone spontaneously hypertensive rats and humans. Stroke, 7, 46-53. Zola-Morgan, S.,Squire, L.R. & Amaral, D.G. (1 986) Human amnesia and the medial temporal region: enduring memory impairment following a bilateral lesion limited to field CAI of the 1iippocampus.J. .Neurosci.,6,2950-2967. ~

Chapter 4

TECHNIQUES FOR ASSESSING NEUROPROTECTIVE DRUGS IN VlTRO Mark f? Goldberg, Uta Strasser and Laura L. Dugan Center for the Study of Nervous System Injury, and Department of Neurology, Washington University School of Medicine, St Louis, Missouri 63 I 10, USA

4. I

4.2

4.3

4.4

4.5

Introduction 4. I.I Goals of in vitm models 4. I .2 Advantages and disadvantages of in vitm models 4. I .3 Methods of assessing neuronal death Simulating ischaemic conditions in vitm 4.2. I Background 4.2.2 Substrate deprivation vs ‘chemical ischaemia’ 4.2.3 Technical problems involved with removal of oxygen and metabolic substrates 4.2.4 An anaerobic workstation for oxygen-glucose deprivation 4.2.5 Modifications t o the anaerobic workstation Operation of anaerobic chamber for oxygen-glucose deprivation 4.2.6 Combined oxygen-glucose deprivation in dissociated cortical neuronal cultures 4.3. I Background 4.3.2 Primary dissociated cortical cell culture model of oxygen-glucose deprivation 4.3.3 Materials 4.3.4 Procedure 4.3.5 Assessment of neuronal death 4.3.6 Notes Combined oxygen-glucose deprivation in organotypic hippocampal cultures 4.4. I Background 4.4.2 Culture methods 4.4.3 Materials Combined oxygen and glucose deprivation (OGD) 4.4.4 4.4.5 Assessment of injury 4.4.6 Notes Comparison of dissociated cell and organotypic slice models of oxygen-glucose deprivation injury Acknowledgements References

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NEUROPROTECTIVE AGENTS AND CEREBRAL ISCHAEMIA, I F W 40 ISBN 0-12-366840-9;0-12-197880-X@bk)

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4. I Introduction 4. I. I GOALS OF IN v i m 0 MODELS

Substantial effort has been devoted to identifymg new agents which can reduce brain damage during stroke. In uitro models of cerebral ischaemia may yield information about pharmacological properties of new drugs at a resolution not possible in uiuo. In uitro models allow determination of drug actions on neuronal biochemistry,physiology, or gene expression. While each of these parameters provide vital information about cellular mechanisms of injury, systems intended to assess potential neuroprotective agents require a different experimental endpoint: the survival or demise of the neurone. Culture systems are especially useful for examining assessing neuroprotection because they allow a sufficient period of observation to distinguish between cells destined to survive or die. This chapter describes two culture models of ischaemic neuronal injury: dissociated cortical neuronal cultures and organotypic hippocampal slice cultures. The models are not unique, and other suitable systems are described elsewhere in this volume. The models are selected because they are currently used in the authors’ laboratories, and because they share several illustrative features. Both models make use of an anaerobic chamber to deprive cells of oxygen and glucose under defined conditions. Cells are maintained in culture before and after neuronal injury. Finally, both models assess potential protective drugs using the specific endpoint of neuronal death or survival. The authors will describe the application of these models in some detail, and consider the advantages and disadvantages of in uitro models in more general terms.

4. I .2 ADVANTAGES AND

DISADVANTAGES OF IN vim0 MODELS

In uitro model systems provide the opportunity to examine mechanisms of hypoxic neuronal injury in a controlled experimental setting. For drug classes which share mechanisms of action, in uitro testing can provide comparative information about potency, efficacy, and selectivity not available with in uiuo methods. In uitro models also permit assessment of cell function during the course of hypoxic injury. Extra- or intracellular electrophysiological recordings have been performed in many preparations during and immediately after hypoxic exposures. Other parameters can also be measured in real time. The open nature of in uitro models also allows the use of ‘opticalphysiology’, taking advantage of the multitude of new fluorescent probes of cellular function, such as the fluorescent calcium indicator, fura-2. Assessment of drug actions in animal models is limited by complex pharmacodynamic interactions involving the experimental compound, the route of delivery, penetration of the blood-brain barrier, diffusion in tissue, and permeability of the target cell membrane. In contrast, in uitro models allow drug delivery to the neuronal extracellular space directly This permits calculation of effective drug concentrations,

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and direct assessment of drug potency. Furthermore, because the extracellular space is under experimental control, it is easy to assess the effects of alterations in extracellular ionic composition, such as extracellular calcium, pH, or osmolarity. In vitro models greatly increase the number of experiments which can be performed using a given number of animals. Since animal trials are time consuming and expensive, it may be efficient to rely on in vitro models for large-scale screening of potential new agents. For example, using dissociated cultures in multi-well culture vessels, it is straightforward to test actions of dozens of drugs each day. A gravid mouse with 12 fetuses prepared for dissociated neocortical cultures can yield approximately 8- 10 multi-well culture plates; each plate may contain 24,48, or 96 individual cultures. If each experimental condition is repeated in quadruplicate, a single culture dissection can provide up to 240 separate data points. Assessment of neuronal viability is also rapid and straightforward. Using semi-automated techniques for measurement of lactate dehydrogenase release (Koh and Choi, 1987), a measure of neuronal cell death, one individual can readily examine hundreds of compounds or combinations of agents each week. The potential limitations of in vitro model systems are widely recognized. The same features of simplified systems which are advantageous experimentally, also represent potentially important differences from the intact brain. The f d three-dimensional architecture and connectivity of the intact brain are not reproduced even in brain slice preparations. Neurones removed from pre- or immediately postnatal rodents may have different phenotypes compared to adult counterparts in the in uivo brain. Finally, while reduction of injury in cultured cells for many classes of experimental agents is associated with reduction in stroke volume in uivo, results of neuroprotective trials using in vitro models do not invariably predict efficacy in vivo. Therefore, it is important to recognize that culture models are effective for drug screening, but that final proof of efficacy in vivo requires careful testing in appropriate animal models. (It should also be noted that, since well-controlled trials of neuroprotective agents in clinical settings are still in their infancy, no animal model has yet been established to predict efficacy in human stroke - see chapter 3.)

4. I .3 METHODS OF ASSESSING NEURONAL DEATH Table 1 reviews tools for assessing neuronal cell death. The reader is referred to a current volume on cell death (Schwartz and Osborne, 1995), which includes detailed protocols for assessing injury in several in vitro systems. Acute neuronal death in the setting of hypoxic-ischaemic brain injury is generally considered to be necrotic, and most current methods for assessing neuronal death in vitro focus on integrity of the cell membrane. Recently, several investigators (Linnick et al., 1993; Gwag et al., 1995)have considered the possibility that hypoxic injury may also demonstrate aspects of apoptosis, a pattern of cell death defined by Kerr et al. (1972)and characterized by cell shrinkage, membrane blebbing, nuclear condensation, and DNA fragmentation. Because current understanding of the cellular events which occur during apoptotic and

TABLE 1 ENDPOINTS OF NEURONAL DEATH Morphology

Function

Pharmacology

Apoptosis

Necrosis

Method

Cell body

Shrinkage

Swelling

Neurophil DNA

Fragmentation Discrete clumping

Varicosities Diffuse shrinkage

Cleavage into 200 bp fragments (ladders)

Generalized cleavage (smear)

Mitochondria Cell membrane

Well-preserved Blebbing with production of small apoptotic bodies

Swelling Large hlebs which may portend imminent cell lysis

Light and electron microscopy (EM) Same Staining of DNA with Hoechst 32558 DNA gel (Southern)for fragmentation;in situ end-labelling for DNA breaks EM, fluorescent mitochondrial dyes Light microscopy and EM

Membrane

Delayed permeability to large molecules

Early permeability

Enzymes

Preserved until late

Lost early

Mitochondria

Preserved

Lost

Protection by macromolecular synthesis inhibitors

No protection by macromolecular inhibitors

Exclusion of vital dyes including trypan blue or propidium iodide Release of LDH De-esterilication of fluorescein diacetate to fluorescent product Potential-sensitive fluorescent dyes for mitochondrial membrane potential

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necrotic cell death is rapidly evolving, the morphological and biochemical criteria which define each entity are still being refined (Table 1). Several caveats should be considered in evaluating neuronal injury as necrotic or apoptotic. First, a single method of assessment does not suffice to define either pattern of cell death. In particular, neither in situ end-labelling of DNA (e.g., TUNEL method), nor blockade of injury by protein synthesis inhibitors, should be used in isolation to classify injury as apoptotic. Second, apoptotic and necrotic patterns ofinjury may not be exclusive, but may reflect a continuum between two extremes. Finally, these patterns describe features of cell death but do not necessarily define cellular mechanisms. It is likely that many distinct mechanisms can result in patterns of neuronal loss which are recognized as necrotic or apoptotic.

4.2 Simulating ischaemic conditions in vitro

4.2. I BACKGROUND An in vitro system cannot reproduce the full constellation of changes which occur

during cerebral ischaemia in viuo. Indeed, the term ‘ischaemia’has little meaning in a model system lacking blood flow. For assessment of neuroprotective agents, many investigators wish to examine the effects of cellular energy deprivation. Additional alterations in the hypoxic-ischaemic extracellular milieu, such as acidic pH or elevated potassium, are readily controlled experimentally

4.2.2 SUBSTRATE DEPRIVATION vs ‘CHEMICAL ISCHAEMIA’ Conditions of severe energy depletion can be reproduced by direct removal of appropriate substrates. Alternatively, pharmacological inhibition of energy production - ‘chemical ischaemia’ - is widely used to model ischaemia in in vitro systems. Iodoacetate, a commonly employed inhibitor of glycolysis, is a nonspecific alkylating agent which modifies cysteines of several enzymes, including those involved in glycolysis such as glyceraldehyde 3-phosphate dehydrogenase (2-deoxyglucose competitively inhibits glycolysis). Cyanide, which inhibits oxidative phosphorylation by binding tightly to cytochrome oxidase (cytochrome b-cl or Complex IV),blocks ATP production by mitochondria. These agents and related compounds are sometimes used in combination. Chemical interruption of cellular metabolism offers several experimental advantages. These techniques can be performed without special equipment, and cells can be examined (e.g., for calcium measurement) directly in room air. Onset of intracellular energy derivation is relatively rapid and does not depend on the rate of cellular removal of substrates from the extracellular space. Although these methods have been applied successfully in many laboratories, these

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authors have preferred when possible to use direct removal of oxygen and metabolic substrates for two reasons. First, this situation may better mimic the actual conditions of ischaemia and reperfusion in the intact brain. Second, there are several potential problems with the use of these metabolic inhibitors to mimic ischaemia. Both drugs are difficultto wash out of cells, so that termination of the metabolic blockade is prolonged and variable. In contrast, reintroduction of oxygen and glucose, which can be accomplished in less than 60 seconds, allows a defined injury exposure, and a return to ‘normal’ conditions in a controlled manner. Third, chemicals used for metabolic blockade may have other cellular consequences. Cyanide not only blocks Complex IV, but inhibits Cu, Zn-superoxide dismutase (SOD1) activity. There is substantial evidence that overexpression of SODl reduces ischaemic injury in many organs (Kinouchi et al., 1991). There is increasing evidence that SOD 1 plays an important role in superoxide radical handling under pathological conditions. Thus, inhibition of SODl by cyanide may enhance oxidative injury artifactually by inhibiting an important anti-oxidant enzyme. Another important consideration in the use of chemical hypoxia is that it is generally performed in normoxia (2 1YO 02). Dubinsky et al. (1 995) reported a substantial reduction in injury ifneurones were exposed to glutamate or NMDA in the absence of oxygen, suggesting that oxygen-requiring pathways contribute in an important manner to glutamate-receptor mediated excitotoxicity. The excitotoxic contribution to neuronal cell death in in zivo and in Vi&o ischaemia may be modified significantly by the presence of atmospheric oxygen (2 1% vs approximately 8% in normal brain, less in hypoxic brain).

4.2.3 TECHNICAL PROBLEMS INVOLVED WITH REMOVAL OF OXYGEN AND METABOLIC SUBSTRATES

These authors describe models of injury produced by combined removal of both oxygen and glucose, in the absence of other potential metabolic substrates. In addition to theoretical considerations, combined oxygen-glucose deprivation (OGD) is advantageous because it offers a high degree of control over the experimental conditions. When cultured neurones are deprived of both oxygen and glucose, they are killed approximately 10-fold more rapidly than in the absence of either oxygen or glucose alone (Goldberg and Choi, 1993). Hypoxic injury to cultured astrocytes is even more strongly dependent on glucose: cortical type I astrocytes can survive pure anoxic insults as long as five days if glucose is provided. Moreover, while the extracellular glucose concentration is critical, it can be dimcult to maintain at non-zero levels in static culture solutions. Under anaerobic conditions, cells can greatly increase utilization of glucose (and production of lactate) to maintain ATP supply Therefore, ifglucose is included in the exposure solution, its concentration depends not only on the starting conditions, but also on cell culture density and metabolic rate. Thorough washing may be required to hlly remove glucose from the exposure medium. Swanson and Choi (1993) found that trace glucose concentrations present after


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repeated medium exchange were sufficient to delay the onset of glucose-deprivation injury. There is no practical way to completely remove oxygen from solutions used in open culture systems. Even if a solution is thoroughly deoxygenated, this situation ends the moment the container is exposed to air (for example, to exchange culture medium). There are two general approaches to exposing cultured cells to anoxic conditions. For experiments performed on the microscope or electrophysiology recording stage, special chambers are used with rapid superfusion of deoxygenated solutions. This model is commonly encountered for hypoxia or ischaemia experiments with acute brain slice preparations. In these experiments, the slice thickness is often such that residual oxygen may be metabolized rapidly by the brain tissue itself. Achieving fully anoxic conditions is more dimcult for dissociated cell cultures. Cummins et al. (1993) observed that even with rapid perfusion of anoxic medium, and with anoxic gas flowing over the stage, oxygen tension was not reduced below 15-30 mm Hg. Anoxia experiments are easier if the cultures can be placed in a sealed chamber. For example, many investigators have used a sealed plastic container with shelves for culture dishes or plates which is flushed with anoxic gas and then placed in a temperature-controlled incubator. This method is straightforward, but the severity of anoxic exposure is influenced by residual oxygen in the culture medium. For example, Goldberg et al. (1986) noted that even with a small volume of cell culture medium, oxygen was detectable in the medium for more than one hour despite rapid superfusion of anaerobic gas. Therefore, the duration of true anoxic conditions depends not only on the time of incubation in the chamber, but also on the rate of diffusion of oxygen out of the medium, and on the rate of consumption of oxygen by cultured cells. This presents a serious potential obstacle, as oxygen utilization is likely to be heavily influenced by culture density and by the experimental compounds under study. For these reasons, these authors prefer a system in which the cultured cells are washed into pre-deoxygenated exposure medium in a closed chamber, as described below. In this way, the duration and intensity of anoxia remain under full experimental control.

4.2.4 AN ANAEROBIC WORKSTATIONFOR OXYGEN-GLUCOSEDEPRIVATION These authors use a glove-box type of anaerobic chamber for oxygen-glucosedeprivation experiments. This apparatus provides an enclosed work space with a controlled oxygen-free atmosphere. Media transfers, microscopic observation, and incubation can all occur under defined anoxic conditions. This approach allows a high volume ofwork flow, using the same techniques which might be performed in a sterile culture hood. Several manufacturers produce anaerobic workstations designed for microbiology laboratories. These authors have modified one from Forma Scientific (Model 1025; Marietta, Ohio, USA) (Figure l), and have four such workstations in operation.

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FIGUREI Anaerobic chamber. Access ifvia two black glove ports mounted in the plastic front. The exchange chamber and control panel are to the right. Visible inside the chamber are the incubator (left) and inverted microscope (centre).

Anoxic conditions are initiated by exchanging chamber air with a special gas mixture consisting of 10% hydrogen, &lo% carbon dioxide (5% is used to match culture incubator conditions), and balance nitrogen. Trace oxygen is removed from the chamber by a palladium catalyst, which combines the H2present in excess with residual 02. The resulting water is removed by passing the chamber atmosphere through a desiccant. A fan circulates chamber atmosphere through catalyst and desiccant containers. The chamber periodically draws anoxic gas mixture to maintain a positive internal pressure and avoid inward leaks. Excessive positive pressure is vented through a mineral oil trap. The anaerobic chamber area is accessed through two sleeve-length vinyl gloves mounted on the clear plastic front. In addition to a stainless steel work surface, the interior of the chamber includes shelves, electrical outlets, and a fluorescent light for general lighting. An incubator inside the chamber allows hypoxia experiments to be performed in a humidified and temperature-controlled setting. There is room to store the usual culture equipment such as pipetters, plasticware, and media. Cultures and supplies are loaded through a smaller exchange chamber attached by a sliding door to the main chamber. The exchange chamber is operated by an automated procedure which sequentially applies vacuum (to approximately -20 psi) followed by introduction of N2 (generally less expensive than the anoxic gas mixture). After three cycles of vacuum, the chamber allows gas equalization between the exchange chamber and the main chamber, and the door can be opened to allow transfer of samples.


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4.2.5 MODIFICATIONS TO THE ANAEROBIC WORKSTATION Several modifications have been made to the chamber to facilitate routine hypoxia experiments in cultured cells. 1. A source of suction is needed to exchange culture medium. A portable electrical pump (Schuco Inc.) with a liquid trap has been installed in the chamber. This approach has the advantage that the system remains closed. Alternatively, laboratory vacuum may be provided by installing a standard laboratory stopcock and needle valve through one metal wall of the chamber. In this case, some attention should be directed to minimizing anaerobic gas flow out of the chamber whenever suction is not required. (Users are advised to consult with the manufacturer before making these modifications.) 2. Solutions are deoxygenated by bubbling with anoxic gas. An anoxic gas inlet may be installed in one wall of the main chamber. It is also possible, although less convenient, to utilize an N2 port provided in the exchange chamber, which is controlled by a switch on the front panel. Since this port must be left open for operation of the exchange chamber, a threaded tubing connector is placed temporarily in the port only after exchange operation is complete. The anoxic gas port is connected through tubing which includes a sterile gas filter and attaches to a Pasteur pipette. 3. An oxygen meter (Microelectrodes, Inc.) is used to monitor oxygen in the chamber atmosphere and experimental solutions. The electrode is mounted on a holder in the chamber, and the control box placed outside the chamber. The electrode cable can be routed through a sealed hole in the chamber. To more readily replace the electrode, or to use it outside the chamber, a custom extension cable can be purchased for this meter. The cable adapter plug is mounted in a sealed hole in a chamber wall or ceiling. 4. Temperature control is critical during hypoxic exposures (see Figure 4).The builtin thermometers in workstation incubators have not proven accurate in the authors’ experience (the temperature control itself is reliable); therefore an additional thermometer should be placed in the same part of the incubator where cultures will be placed. If many cultures will be used at once, it may be desirable to promote uniform temperature distribution within the incubator using a small battery-operated fan. An electric warming plate (e.g. Bench Warmer, BarnsteadThermolyne, Dubuque, Iowa) can be used to maintain cultures near 37% if they must remain outside the incubator during part of the anoxic exposure. 5 . It is sometimes desirable to examine dissociated cultures during anoxic exposures. In one chamber, a small trinocular inverted microscope (TMS, Nikon) has been installed which is equipped for phase-contrast optics (Figure 1). The oculars and eyepiece head are removed, and a closed-circuit video camera is mounted on the video port. A inexpensive surveillance monochrome CCD (charge-coupled device) camera is adequate. The camera is connected to a monochrome monitor outside the chamber, using a BNC-type electrical connector mounted in a chamber wall.

78 4.2.6

M.P. GOLDBERG et al. OPERATION OF ANAEROBIC CHAMBER FOR OXYGEN-GLUCOSE DEPRIVATION

The chamber remains in full-time operation, with a constant supply of the anoxic gas mixture (at 5 psi). This increases anoxic gas mixture use but ensures that the oxygen tension remains low. Catalyst and desiccant wafers are recycled when needed (generally every 3-5 days) by placing them in a 6OoC oven for 2 hours; they should be allowed to cool before returning to the chamber. Both wafers must be replaced every one or two years. If oxygen is allowed into the chamber or the desiccant is no longer active, moisture precipitates within the chamber. This should be avoided because it causes corrosion of metal in the chamber and in other installed equipment. The oxygen electrode membrane should be replaced and recalibrated periodically. All experimental solutions should be prepared, deoxygenated, and warmed in the chamber before the cultures are transferred in. The exchange chamber operates a vacuum; this is not harmful to cultures but centrifuge tubes or flasks must be capped loosely to avoid explosion. For deoxygenation, solutions are placed in a flask or centrifuge tube. A sterile cotton-plugged Pasteur pipette is connected to the anoxic gas inlet tubing. The pipette is placed in the medium and gas pressure slowly increased to provide steady bubbling without displacing the solution from the container. For a 100 ml solution volume (in a 250 ml flask), anoxic gas is bubbled for 10 minutes at 5 psi. Smaller volumes (e.g. 2 ml in a 15 ml centrifuge tube) can be bubbled carefully for 3 minutes at < 1 psi. The chamber vacuum is operated manually to avoid excessive pressure build-up while solutions are bubbling. A large volume of exposure solution is deoxygenated and then used to prepare individual experimental drug solutions in the chamber. Sterile drug stocks are prepared in the highest practical concentrations (ideally 100- 1OOOx) in vehicles lacking glucose. These stock solutions in small volumes are deoxygenated considerably during transfer into the anaerobic chamber. Control solutions (with no added drug) must be prepared using identical procedures, including addition of concentrated vehicle. All solutions are brought to 37OC in the chamber incubator before use.

4.3 Combined oxygen-glucose deprivation in dissociated cortical neuronal cultures 4.3. I BACKGROUNO

Primary dissociated cultures are prepared from embryonic or neonatal rodent brains by enzymatically or mechanically disrupting tissue into a single cell suspension, and plating the cells onto a prepared culture surface or existing cell layer. While synaptic connections are lost initially after dissociation, neurones begin to regrow axons and dendrites within hours of replating, and within 7 days form extensive synaptic connections. Depending on the species, tissue type, and culture methods, dissociated neuro-


79

nal cultures may survive in vitro from days to several weeks. In contrast to many neuronal cell lines, which are often derived from peripheral or central tumours, primary neuronal cultures generally display phenotypes consistent with mature central nervous system brain cells. Primary neuronal cultures express appropriate cell surface receptors and synthesize appropriate neurotransmitters. One strength of the primary cultures model is the ability to selectively culture cells of a defined cell type (e.g., only neurones, astrocytes, oligodendrocytes, or endothelial cells), or from a defined brain region (e.g., neocortex vs hippocampus). Pioneering experiments by Rothman (1984) established that the synaptic connections of primary dissociated neurones determined their vulnerability to hypoxic insults. Mature (but not freshly plated) hippocampal cultures could be killed by overnight exposure in an anoxic chamber, and this could be blocked by agents which interfered with synaptic transmission. Subsequent experiments established that hypoxic neuronal injury in this model was not due to energy depletion alone, but rather to excessive release of the neurotransmitter, glutamate, and toxic overactivation of glutamate receptors (Rothman 1984), specifically of the NMDA receptor subtype (Goldberg et al., 1987). Similar models have been used by many investigators for assessment of neuroprotective agents in vitro.

4.3.2 PRIMARYDISSOCIATEDCORTICAL CELL CULTUREMODEL OF OXYGEN-GLUCOSE DEPRIVATION

Primary cultures prepared from embryonic mouse neocortex have been used by these authors. The cell culture methods are described in detail elsewhere (Rose et al., 1993). Briefly, neocortices from embryonic mice at day 14-1 6 are dissociated using trypsin and mechanical disruption, and plated in serum-containing medium. Cells may be plated on tissue-culture treated polystyrene (Falcon Primaria), or on glass coverslips precoated with poly-D-lysine and laminin. The most consistent results have been found when the neocortical cell suspension is plated on a previously established monolayer of cortical glial cells (mostly type 1 astrocytes). The procedure below assumes that cultures have been plated in 24-well (1 5 rnm) culture vessels. For microscopy applications requiring optical clarity or short working distances, cultures are plated in 35 mm dishes, in which the centre plastic area is replaced by a glass coverslip (MatTek).

4.3.3 MATERIALS

1. BSS2,+02(Earle’s balanced salt solution with 20 mM glucose, oxygenated), containing (mM) NaCl 116.4, KCl 5.4, CaC12 0.8, MgS04 7 H 2 0 0.8, NaH2P04 4 H 2 0 1 .O, NaHC03 26.2, D-glucose 20, phenol red 10 mg/l. Vacuum filter and bubble with 5% C 0 295% air for 10 minutes. Adjust pH to 7.4, ifnecessary with sterile 1~ HCl or NaOH.

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BSSo-Oz (Earle’s balanced salt solution with 0 mM glucose, deoxygenated). Prepare as for BSS,,,+O,, but substitute 20 mM sucrose for D-glucose. Vacuum filter and bubble with 5% CO,, 95% N2for 10 minutes. Adjust pH as above. 3. MEM2,,+0, (minimal essential medium, oxygenated, with 20 mM glucose). Prepare from 100 ml MEM 10X stock (Eagle MEM, with Earle’s salts, without L-glutamine, without bicarbonate, Gibco No. 330-1430), 26.2 ml 1 N NaHC03, 14.5 ml 1 M D-glucose (final 20 mM), balance H 2 0 to make 1000 ml. Bubble for 10 minutes with 5% COP,95% air. Adjust pH as above. 4. 10 mM JV-methyl-D-aspartate.

2.

4.3.4 PROCEDURE 1. Check chamber 0, and incubator temperature. Pipette test drug stock solutions into 5 ml centrifuge tubes. Bring all solutions, equipment, and supplies (but not cultures) into anaerobic chamber. 2. In the workstation, bubble BSSo-Oz with anoxic gas, as described in section 3.2. (Prepare about 60 ml for each 24-well plate.) 3. Prepare experimental solutions (about 1.2 ml for four wells) by adding appropriate volume of BSSo-02 to stock solutions (stocks should be at least 1OOX). Each drug should be prepared at 1.5 times the desired final concentration. The control (no drug) condition should receive an equal volume of vehicle alone. If experimental agents will also be included in the post-anoxic exposure, these solutions should be prepared in MEM+OZ.Place solutions in the chamber incubator to heat to 37OC, for approximately 20 minutes. 4. Transfer cultures into anaerobic chamber. If many different experimental conditions are included, it is practical to expose no more than two 24-well plates at once. 5. Stagger the anoxia start times of each condition (usually four wells per condition) by 1-2 minutes, to ensure that the anoxia duration is always the same. Working one condition at a time, each well is drained to 125 ml and quickly replaced with 750 ml BSS,,-Oz. A repeat pipetter is used to deliver uniform volumes. This is repeated three times. After a fourth drain to 125 ml, the last volume is replaced by 250 ml BSSo-02 containing any experimental drugs (this is added with a standard pipette tip or repeat pipetter). Pipette tips are exchanged, and after the designated delay time (1-2 minutes) the process repeated for the next group of wells. Care is required to avoid damaging cells during washing. 6. Each multi-well plate usually includes a ‘wash control’ condition, consisting of sister cultures not damaged by oxygen-glucose deprivations. These cultures are washed in the identical manner but receive BSS,,+O, rather than BSSo-Oz. Previous experiments have shown that cells can remain in this solution (even in the anaerobic chamber) for well over an hour without measurable injuq. Another set of cultures can be treated the same way, for later determination of complete neuronal injury by addition of concentrated NMDA.


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7. Place cultures in the 37°C anaerobic incubator for the designated injury period (typically 40-90 minutes). If a microscope is installed in the chamber, cultures may be removed from the incubator for brief examination. 8. After the designated interval, terminate OGD in the same order and timing as it was initiated. This is done inside the chamber by washing three times with 750 ml MEM+02, followed by addition of 250 ml M E M + 0 2 (with or without experimental drugs, as needed). Alternatively, exposure may be terminated by simply adding to each culture an equal volume (375 ml) of MEM+02. In the latter procedure, experimental drugs are added at twice the final concentration (but the same concentration if the drugs were also present during the OGD). 9. Remove cultures from the anaerobic chamber and transfer into a normoxic incubator. 10. For determination of complete neuronal death, 300 PM NMDA is added to one set of cultures for 4-24 hours. This results in destruction of all neurones but not glia. 1 1. Assess neuronal death at least one day later, as described below.

4.3.5 ASSESSMENT OF NEURONAL DEATH Neuronal death is routinely assessed at 24-48 hours after OGD using one or more of the following methods: Microscope observation under phase-contrast or differential interference optics (200G4OOX; Figure 2). Normal neurones appear phase-bright with smooth contours. Dead cells may be absent by phase-contrast, or may have irregular or markedly swollen borders. It is dimcult to distinguish non-viable cells from cells which are damaged but still viable using phase-contrast observation alone. Dye exclusion. Cells are incubated in 0.4% trypan blue (in MEM or BSS) for 5 minutes, then washed in MEM. Cultures are examined under brightfield (not phase) optics. Viable cells exclude the dye and non-viable cells have dark blue nuclei. Alternatively, cultures may be incubated in 5-10 pg/ml of propidium iodide or ethidium bromide for 5 minutes, and then examined using green epifluorescence (rhodamine filters). These dyes need not be washed OK Non-viable cells have condensed, intensely fluorescent orange or yellow nuclei. Measurement of lactate dehydrogenase (LDH) release (Figures 3,4,5): 25-50 ml of medium is removed from each culture and assayed for LDH concentration using a automated assay as described (Koh and Choi, 1987; Klingman et al., 1990), modified for use on a UV-Max plate reader (Molecular Devices). LDH values are normalized by subtracting from all values the concentration of LDH found in drug-free normoxic washed sister control cultures (=O), and then dividing by the amount of LDH found in cultures exposed to 300 p~ NMDA for 4-24 hours (= 100% neuronal death). Maximal neuronal death can also be approximated by exposing cultures to prolonged OGD (>80 minutes) with no added

82


FIGURE2 Early changes in neuronal morphology with oxygen-glucose deprivation. Differential interference contrast (DIC) images of cortical neurones undergoing combined oxygen-glucose deprivation (OGD). To initiate OGD in cortical cell cultures, the medium was replaced with an anaerobic balanced salt solution lacking glucose. Control cultures received medium containing both oxygen and glucose. Control cultures (A) had morphology typical of uninjured cortical neurones, with smooth cellular membranes, and relatively indistinct nuclei. However, after 30 minutes of OGD (B),obvious soma1 swelling, and irregularity of internal cell membranes can be seen. One hour after reintroduction of oxygen and glucose (C), further swelling and the onset of nuclear condensation are observed.

drug. LDH release does not occur during the first 60 minutes of OGD. For longer exposures, LDH shold be sampled before washing to be sure that no LDH is lost from the final assessment of cell death. 4.3.6 NOTES

1.

2.

Washing cultures. It is important to provide a thorough medium exchange to initiate OGD. However, the cultures are vulnerable to wash injury, especially if allowed to dry momentarily Damage is usually apparent by the presence of substantial neuronal death in the centre of wash control cultures. New investigators are advised to practise using coloured solutions in empty wells, to assure the correct volumes and speed of pipetting. Experimental solutions. Solutions have been selected arbitrarily to match the ionic composition of Eagle’s MEM (Earle’s salts) in which cells are maintained for culture. The bicarbonate concentration is balanced for the 5% CO, used in the anoxic and culture incubators. In addition to balanced salts present in BSS, MEM also contains amino acids and vitamins. These are omitted for the sake of simplicity Glutamine is omitted to avoid extracellular hydrolysis to glutamate. Previous experiments have demonstrated that the presence of glutamine and MEM amino acids do influence the duration and pharmacology of oxygen or glucose deprivaton injury (Goldberg et al., 1988; Monyer and Choi, 1990). Special precautions have not been taken to maintain constant osmolarity of all experimental solutions. Although this is theoretically important, even large changes in medium osmolarity were found to have


83

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Duration of oxygen-glucose deprivation (min) FIGURE 3 Time course of injury Cultured neurones are much more vulnerable than glia. In Figure 3, mixed cortical cultures containing either neurones and glia, or glia alone, were deprived transiently of oxygen and glucose for the duration shown (note different time scale). Cell death was assessed one day later by measurement of lactate dehydrogenase (LDH) released into the culture medium. Values are normalized to those found in sister cultures exposed to OGD for 60 minutes (neurones) or 24 hours (glia). Glial cultures consisted of >95% type 1 astrocytes, as assessed by glial fibrillary acidic protein (GFAP)immunoreactivity. ~

little effect on neuronal injury after O G D (Goldberg and Choi, 1993). Experimental variables. The duration of OGD required to produce widespread neuronal injury can vary markedly between culture models, and can vary somewhat from one culture dissectionto the next. In order to obtain a reliable LDH signal, very dense cultures are used, such that most neurones are foundin large aggregates. Sparser culturesreleaseless LDH,and tend to be less vulnerableto OGD. Cultures dissociated from embryonicmouse cortex become vulnerable to OGD only after around 13days in Vitro. Therefore, new model systems should be characterized by developmentaltime course, and all critical comparisons should be performed between sister cultures on the same day Temperature control is critical,as even a few degree reduction can markedly prolong the duration which cells survive OGD (Figure 4). Timing of addition of experimental drugs. This procedure includes wash steps before and after OGD. It is straightforward to include experimental agents only before, during, or after OGD. From a clinical point of view, the most effective agent is one which can be given after the onset of hypoxia-ischaemia. As shown in Figure 5, glutamate receptor blockade is fully protective when administered during the OGD exposure, but the effect vanishes rapidly within a few minutes after the OGD conclusion.

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30 40 50 60 70 80 90 100 Duration of oxygen and glucose deprivation FIGURE 4 Temperature-dependence of neuronal injury following oxygen-glucose deprivation. Reprinted from Bruno et al. (1994), with permission. Figure 4 shows cortical cultures exposed to OGD at either 37% or 30% for the duration indicated. Injury was assessed after incubation at 37OC for one day. Cells survived substantially longer when exposed at 3OOC. Reduction to 34% resulted in an intermediate degree of protection (not shown). These results parallel the situation of animal models in vivo, and demonstrate the importance of accurate temperature control for OGD experiments.

4.4 Combined oxygen-glucose deprivation in organotypic hippocampal cultures 4.4. I BACKGROUND Organotypic cultures of brain slices offer a promising compromise between very complex animal models and more simplified dissociated neuronal cell cultures. The brain area most widely used for organotypic cultures is the hippocampus. Its mainly lamellar organization of cellular connections allows the hippocampus to be cut in slices, retaining almost the same neuronal circuits in every slice. Characterization of organotypic hippocampal cultures during the last few years showed that many tissue-specific characteristics of adult hippocampus are present or develop in this in vitro model. The specific hippocampal organization of neuronal connections is retained in organotypic cultures (Frotscher and GPhwiler, 1988; Caeser and Aertsen, 1991; Stoppini et al., 1991), and the formation of synaptic contacts is comparable to the process in vivo (Buchs et al., 1993; Muller et ab, 1993; Frotscher et al., 1995).Analysis of the expression of calcium-bindingproteins (Bousez-Dumesnil et


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FIGURE 5 Neuroprotectiveeffects of an NMDA antagonist during or after oxygen-glucose deprivation in dissociated cultures. (A) Complete neuroprotection during OGD. Cortical cultures were exposed to OGD for 45 minutes with no added drug (open circle, control), or in the presence of the NMDA antagonist, dextrorphan, at the indicated concentration. (B) Partial protection a& OGD. Cultures were exposed to OGD for 40 minutes, and 100 p~ dextrorphan added at the designated interval after exposure. For this brief exposure, immediate post-treatment provided about 50% reduction of injury but the effect was lost if treatment was delayed by 15 minutes. Values are normalized to those found in sister cultures exposed to OGD with no added drug (= 100). Reprinted from Goldberg and Choi (1993), with permission.

al., 1989) neurotransmitter receptors and other synaptic components (Bahr et al., 1995) in organotypic cultures also showed comparable patterns to the ones in adult hippocampus. Thus, organotypic hippocampal cultures retain many in Vivo features but still offer the advantages of an in Vitro system. Results from in Vivo models have shown that the hippocampus is the most vulnerable brain area to global ischaemia, with selective vulnerability of single neuronal subpopulations. The same selective vulnerability of hippocampus areas could be described in organotypic cultures that

86


were deprived of oxygen and glucose (Strasser and Fischer, 1995a).Pharmacological investigations with glutamate receptor antagonists during oxygen and glucose deprivation also revealed parallels in their protective potential to in u i ~ omodels (Newell et al., 1995; Strasser and Fischer, 1995b; Vornov et al., 1994). Thus organotypic cultures of hippocampal slices offer a valuable tool for in uitro studies of mechanisms involved in ischaemia-induced neurodegeneration.

4.4.2

CULTURE METHODS

When organotypic hippocampal cultures were first established in 1981 (Gahwiler, 1981) the slices were cultured by the so called ‘roller-tube’ method. The slices were put on a glass coverslip, surrounded by a plasma clot and cultured in a rotating tube, so that the tissue was alternating between exposure to air or to medium. In 1991 an alternative method was introduced, in which the tissue is cultured on a cell culture insert (Stoppini et al., 1991).This static culture technique grows the tissue at the interface between culture medium and gas atmosphere. Since the tissue is more easily accessible for pharmacological investigations in this static culture, this method is used for the authors’ own studies of oxygen-glucose-deprivation-induced neuronal death. Cultures are prepared as described previously (Stoppini et al., 1991). Briefly, hippocampi from postnatal rats (P3-P8) are removed rapidly under sterile conditions and cut into 350 mm slices with a tissue chopper. Depending on the quality of the cut slices, 12-24 slice cultures can be prepared from each rat pup. After the slices are washed from the Teflon plate with a few millilitres of cold dissection medium, they are separated with small spatulas and transferred to ice-cold dissection medium. Millicell-CM filters (Millipore) are pre-equilibrated in 24 well plates with 0.3 ml of growth medium per well in a moist 5% C 0 2 atmosphere at 37OC. Each slice is cultured on a single insert for up to three weeks with a total change of medium twice a week. After eight days in vitro, 10 mM MgC12is added to the growth medium, to prevent spontaneously occurring cell death.

4.4.3 MATERIALS 1. Dissection medium @H 7.3)

2. Growth medium @H 7.3):

10Oo/oMEM 25 mM HEPES 4 rnL-glutamine 50% MEM 25 m~ HEPES 4 mM L-glutamine 25% HBSS 25% heat-inactivated horse serum 0.013% NaCH03


87

3. CSS (controlledsalt solution, oxygenated): 120 m~ NaCl 5.4 mM KC1 0.8 mM MgC12X6H 2 0 1.8 mM CaC12X2H 2 0 25 mM Tris 15 mM D-glucose pH 7.2 4. CSSO (controlledsalt solution, deoxygenated): Prepare as for CSS, but substitute 15 m~ sucrose for D-glucose. Bubble the amount of solution needed for each experiment with N2for at least 10minutes in the anaerobic chamber (see Chapter 3.2.5). 5. Propidium iodide stock solution 1 mg/ml (Sigma).

4.4.4 COMBINED OXYGEN AND

GLUCOSEDEPRIVATION

(OGD)

To mimic ischaemic conditions, DIV14 organotypic cultures are exposed to glucoseand oxygen-free buffer (CSS,) in an anaerobic chamber (see sections 3.2.3, and 3.2.4,), as follows. 1. Pipette test drug solutions from lOOX stock solutionsin 5 ml tubes: 1.5 ml of solution should be prepared when three slices are used for each condition. For each 24-well plate, calculate 10 ml of CSSO needed, and pipette the appropriate amount ofpropidium iodide (PI)stock solution into a 50 ml tube, so that the final concentration is 15 mg/ml. The same volume of growth medium containing PI is needed for the recovery period. 2. Bring all solutions, equipment and supplies into anaerobic chamber. 3. In the anaerobic chamber, bubble the CSS, for at least 10 minutes with N2 (10 ml for each 24-well plate), then add the necessary amount to the prepared propidium iodide (PI).Dilute the prepared drug solutions in the 5 ml tubes with CSSo+PI to their final concentration. If experimental agents should be included during the recovery period, these solutions should also be prepared in culture medium containing PI. Place all solutions in the 37OC incubator. 4. Wash slice cultures with sterile CSSo that is not yet bubbled with N2 (0.2 ml on top of the slice and 0.2 ml under the culture insert), to wash out glucose from the medium. (Organotypic cultures are relatively sensitive to this washing step and since the tissue is not covered with medium during the culture period, this washout procedure is not mandatory.) 5. Bring slice cultures into anaerobic chamber, one 24-well plate at a time. 6. Stagger the anoxia start time of each condition by 1 minute, to make sure that the anoxic period is always the same. Lift the culture insert containing the slice with forceps, suck dry the culture well and also drain drops of medium that stick to the insert. With a repeat pipetter, carefully add the solution containing any

88


experimental drug, 0.2 ml in the culture well and 0.2 ml on top of the slice, to assure sufficient diffusion. After 1 minute, repeat for the next experimental condition. 7. Transfer the plate containing the slices to the 37OC incubator for the designated period of time (30-60 minutes). 8. To end OGD conditions, replace CSS,,with growth medium+PI, 0.2 ml in the culture well and 0.2 ml on top of the slice. Termination should be done in the same order and timing as the oxygen and glucose deprivation was initiated. Remove cultures from the anaerobic chamber and keep them in a normoxic incubator until assessment of injury. 9. Wash control conditions are treated outside the anaerobic chamber, by replacing the growth medium with CSS containing PI (0.2 ml on top of the slice and 0.2 ml in the culture well) for the same period as the anoxia.

4.4.5 ASSESSMENT OF INJURY The amount of induced cell death is quantified by measuring the intensity of propidium iodide fluorescence in the cultures, usually at 24 hours after the oxygen and glucose deprivation. This method has recently been described in detail (Strasser and Fischer, 1995a). PI fluorescence pictures of the slice cultures are recorded with a Hamamatsu camera at 4X magnification (Axiovert 405M, Zeiss) and stored for later quantification. For standardization of 100% cell damage, the cultures are fixed in 4% paraformaldehyde for 15 minutes. The fixative is washed away with CSS (two washing steps, each with 0.2 ml on top of the slice and 0.2 ml in the culture well) and then slices are restained with PI in CSS (15 pg/ml) for about 2 hours, to give maximal fluorescence intensity Pictures of the restained cultures are recorded and stored with the same camera setting as the pictures before the fixation. In order to get quantitative measurements, it is essential to keep the camera settings constant during all of the image acquisition. In addition, the orientation and relative position of the slice cultures has to be kept the same during this procedure. Therefore, the fixation and washing steps have to be done carefully, placing the culture insert back into the well in the same orientation that it was before. It might help to mark both the insert and a spot on the edge of the well and to adjust these two marks. Measurement of fluorescence intensity is performed with a Hamamatsu image analysis system (ICMS). In each of the hippocampal areas CAI, CA3 and dentate g y m s , three rectangular measurement windows are placed (Figure 6). For location of these measurement windows, the fluorescence picture of the fixed slice is used, where demarcations of the respective area are clearly visible. The same set of measurement windows is used for measuring the fluorescence intensity in all pictures that have been recorded from one slice. Confocal microscopy can be used to observe the fluorescence intensity of cells in a single plane of the slice culture (Figure 7).


89

FIGURE6 Measurement of propidium iodide fluorescence intensity in organotypic hippocampal cultures, to assess the damage induced by combined oxygen and glucose deprivation. (A) Fluorescence videomicrograph of hippocampal slice culture one day after oxygen-glucose deprivation. Propidium iodide fluorescence is in damaged neurones only. (B) Image of propidium iodide staining in the same slice (same magnification) after fixation and restaining. Boxes illustrate regions of interest for intensity measurements (see section 4.4).

FIGURE7 Confocal images of a rat hippocampal slice culture stained with propidium iodide after oxygen-glucose deprivation injury. Cultures exposed to O G D for 45 minutes were returned to oxygen and glucose-containing medium for 24 hours, and then stained with propidiurn iodide. The C A I region (A) showed extensive cell death in this confocal image (Noran Odyssey confocal microscope; 20X dry objective). The homogeneous nuclear staining of dead neurones (B),consistent with a necrotic injury, can be seen at higher magnification ofthe CAI region (60X, 1.4 NA water-immersion objective).

90


4.4.6 NOTES

This procedure was developed using an anaerobic chamber atmosphere containing 0% carbon dioxide. If a 5% CO2 atmosphere is used (asin the previous section), the exposure media should be buffered with bicarbonate to maintain normal pH. The quality of organotypic slices might vary within one dissection. After 14 days in culture, the hippocampal structure is easily visible at low magnification (40X) and only slices with an intact structure should be included for experiments. In addition to this morphological assessment,cultures can be stained with PI before the experiment, to assure that cell damage in the cultures is not too high before starting the anoxic exposure. Immunohistochemistryfor neurone specific markers (MAPP)in paraffin sections of slice cultures has shown that 24 hours following 60 minutes of combined oxygen and glucose deprivation, all neurones are degeherated, whereas staining for the glial cell marker GFAP is unchanged. Therefore, this prolonged ischaemic period can be used to define 100% neuronal damage. On the other hand, 30 minutes of combined oxygen and glucose deprivation leads only to degeneration of neuronal subpopulations. Deprivation periods between 30 and 60 minutes should therefore be used to produce more or less severe injury.

4.5 Comparison of dissociated cell and organotypic slice models of oxygen-glucose deprivation injury Table 2 compares the features of the two models systems described in this chapter, and Figures 2-7 demonstrate some of the features of oxygen-glucosedeprivation injury in each model. These systems differ in ways additional to the culture method. Dissociated cultures are prepared from neocortices of neonatal mice, and the organotypic cultures are prepared from postnatal rat hippocampi. These differences mostly reflect the historical origins of each model rather than any deliberate preference to study a particular tissue type, species, or developmentalage. Despite these differences, the systems are remarkably similar in their responses to transient oxygen-glucose deprivation. In both cases, OGD durations between 30 and 60 minutes are required to produce widespread neuronal death, this is remarkably similar to the duration of middle cerebral artery occlusion necessaaryto produce focal ischaemic damage in the rat. Neuronal death is not detected immediately after OGD, but develops over the ensuing several hours. In both models, application of glutamate receptor antagonists during OGD reduce subsequent neuronal death. In hippocampal slice cultures, OGD-induced neuronal death is reduced by antagonists of either NMDA or the AMPA/kainate class of glutamate receptors (Strasserand Fischer, 1995b).In contrast, OGD-induced neuronal death in cortical cultures is readily blocked by NMDA antagonists (Goldberg and Choi, 1993),but protection by AMPA/kainate antagonists is observed only when NMDA receptors are also blocked (Kaku et al., 1991).


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TABLE 2 COMPARISON OF ORGANOTYPIC SLICE AND DISSOCIATED CELL CULTURE MODELS Dissociated cell cultures

Organotypic slice cultures

Synaptic connections

Synapses are destroyed during dissociation but reform in culture

Hippocampal intrinsic connections are preserved

Distribution of cell regions

Randomly distributed

Maintain regional distribution of hippocampal cells; assess selective vulnerability

Cell types

Can prepare cultures from specific brain regions, or containing only desired brain cellular elements (neurones, glia, endothelial cells)

Cell types limited to those present in slice model

Three-dimensional architecture

Neurones rest on glial monolayer. Open access to exposure solutions

Slices flattens during culture. Cell layers rest on semipermeable membrane. Astrocyte layer may form at interface

Observations during hypoxia

Phase-contrast morphology

Limited

Assessment of neuronal death

Cell morphology, dye exclusion, LDH release

Cell morphology, dye exclusion

Utility for screening neuroprotective agents

Multiple sister cultures (24-96) can be tested in multi-well plates

More difficult; fewer cultures per experimental animal

Duration of oxygenglucose deprivation needed for neuronal death

-0

3 M O minutes

Glutamate receptor pharmacology

NMDA antagonists protective. AMPA antagonists protective only when NMDA receptors are also blocked

Features of culture system

Assessment of injury

Experimental results minutes

AMPA and NMDA antagonists each neuroprotective

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Each system offers its own experimental advantages and disadvantages. When used in concert with in vivo models of cerebral ischaemic injury, in uitro models may offer substantial promise for understanding mechanisms of hypoxic-ischaemic brain injury, and in developing new therapeutic approaches to stroke.

Acknowledgements

To Dennis W. Choi, Washington University School of Medicine (St Louis, USA) and Gunther Fischer, Hoffmann-La Roche (Basel, Switzerland) for guiding the development of these models in their laboratories. Preparation of this chapter was supported by NIH grants NS 01543 (MPG), AG 00599 (LLD).

References Bousez-Dumesnil, N., Thomasset, M. & Ben-Ari, Y (1989) Calbindin D 28k in hippocampal organotypic cultures. Bruin Res. 486, 165-169. Bruno, VM.G., Goldberg, M.P., Dugan, L.L., Giffard, R.G. & Choi, D.W. (1994) Neuroprotective effect of hypothermia in cortical cultures exposed to oxygen-glucose deprivation or excitatory amino acids.3 Neurochem. 63, 1398-1406. Buchs, R-A., Stoppini, L. & Muller, D. (1993)Structural modifications associated with synaptic development in area CA 1 of rat hippocampal organotypic cultures. Dev.Brain Res. 71,8 1-9 1 . Caeser, M. & Aertsen, A. (1991) Morphological organization of rat hippocampal slice cultures. j! Comp. Neurol. 307,87-106. Cummins, T.R., Agulian, S.K. & Haddad, G.G. (1993) Oxygen tension clamp around single neurones in vitro: a computerized method for studies on O2deprivation. j! Neuroscience Meth. 46, 183-189. Dubinsky,J.M., Kristal, B.S. & Elizondo-Fournier, M. (1995)An obligate role for oxygen in the early stages of glutamate-induced, delayed neuronal death. 3 Neuroscience 15, 707 1-7078. Frotscher, M. & Gahwiler, B.H. (1988) Synaptic organization of intracellularly stained CA3 pyramidal neurones in slice culture of rat hippocampus. Neuroscience 24,541-55 1, Frotscher, M., Zafirov, S. & Heimrich, B. (1995)Development of identified neuronal types and of specific synaptic connections in slice cultures of rat hippocampus, Bog, Neurobwl. 45, vii-xxviii. Gahwiler, B.H. (198 1) Organotypic monolayer cultures of nervous tissue,j! Neuroscience Methods, 4,329-342. Goldberg, M.P. & Choi, D.W. (1 993) Oxygen-glucose deprivation cortical culture: calciumdependent and calcium-independent mechanisms of neuronal injury. 3 Neuroscience 13, 3510-3524. Goldberg, M.P., Monyer, H. & Choi, D.W. (1988) Hypoxic neuronal injury in Vitro depends on extracellular glutamine. Neuroscience htt.94, 52-57. Goldberg, M.P., Weiss, J.W., Pham, PC. & Choi, D.W. (1987) N-methybaspartate receptors mediate hypoxic neuronal injury in cortical culture. j! Pharmacol. Exp. Therap. 243, 784-79 I . Goldberg, WJ., Kadingo, R.M. & Barrett, J.N. (1986) Effects of ischaemia-like conditions on cultured neurones, protection by low Na’, low Ca” solutions.3 Neuroscience 6, 3144-315 1.


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Gwag, B.J., Lobner, D., Koh,J.Y., Wie, M.B. & Choi, D.W. (1995) Blockade ofglutamate receptors unmasks neuronal apoptosis after oxygen-glucose deprivation in vitro. Nmroscience 68, 6 15-6 19. Kaku, D.A., Goldberg, M.P & Choi, D.W. (1991) Antagonism of non-NMDA receptors augments the neuroprotective effect of NMDA receptor blockade in cortical cultures subjected to prolonged deprivation of oxygen and glucose. Brain Res. 554,344-347. Kerr,J.ER., Wylie, A.H. & Currie, A.R. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. BE5: Cancer 26, 239-245. Koh, J.Y & Choi, D.W. (1987) Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efAux assay. j! Neuroscience Meth. 20, 83-90. Lmnick, M.D., Zobrist, R.H. & Hatfield, M.D. (1993) Evidence supporting a role of programmed cell death in focal cerebral ischaemia in rats. Stroke 24, 2002-2009. Monyer, H. & Choi, D.W. (1990) Glucose deprivation neuronal injury in vitro is modified by withdrawal of extracellular glutamine. j! Cereb. Blood Flow Metab. 10, 337-342. Muller, D., Buchs, I?-A. & Stoppini, L. (1993) Time course of synaptic development in hippocampal organotypic cultures, Dev. Bruin Res. 71,93-100. Newell, D.W., Barth, A., Papermaster, V. & Malouf, A.T. (1995) Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures,J Neuroscience. 15, 7702-77 1 I . Rose, K., Goldberg, M.I? & Choi, D.W. (1993) Cytotoxicity in murine cortical cell culture. In In vitro Biologt2al Methods. Methods in roXcology (eds Tyson, C.A. & Frazier,J.M.), pp. 46-60. Academic Press, San Diego. Rothman, S. (1984) Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death.j! Neuroscience4, 1884-1891. Schwartz, L.M. & Osborne, B.A. (eds)(1 995) Cell Death, In Methodr In 0 1 1 Biology, volume 46. Academic Press, San Diego. Stoppini, L., Buchs, I?-A. & Muller, D. (1991) A simple method for organotypic cultures of nervous tissue.3 NmroscienceMethods 37, 173-182. Strasser, U. & Fischer, G. (1995a) Quantitative measurement of neuronal degeneration in organotypic hippocampal cultures after combined oxygen/glucose deprivation, J. Neuroscience Methods 57, 177-186. Strasser, U. & Fischer, G. (l995b) Protection from neuronal damage induced by combined oxygen and glucose deprivation in organotypic hippocampal cultures by glutamate receptor antagonists, Brain Res. 687, 167-1 74. Swanson, R.A. & Choi, D.W. (1993) Glial glycogen stores affect neuronal survival during glucose deprivation in vitro. J. Cereb. Blood Flow Metab. 13, 162-169. Vornov,JJ.,Tasker, R.C. & Coyle,J.T. (1994) Delayed protection by MK-801 and tetrodotoxin in a rat organotypic hippocampal culture model of ischaemia. Stroh 25,457464.


Chapter 5

CALCIUM ANTAGONISTS: THEIR ROLE IN NEUROPROTECTION A. JacquelineHunter SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM I 9 5AW. UK

5. I 5.2 5.3 5.4 5.5

5.6

Introduction The role of calcium in ischaemic stroke Classification of voltage-operated calcium channels In vitm studies with calcium antagonists In vivo studies with calcium antagonists 5.5. I Dihydropyridines 5.5.2 Flunarizine and emopamil 5.5.3 Calcium antagonists with reduced cardiovascular liabilities Clinical studies References

95 95 96 99 100 I00 101 I02 I05 I06

5. I Introduction Earlier studies on the role of calcium in ischaemia and the effects of calcium antagonists were hampered by a lack of tools selective for neuronal versus vascular calcium channel subtypes. More recently, a number of tools have become available which have extended our knowledge of the role of calcium in ischaemia. This chapter will review the exciting new evidence from both in vivo and in z~it70studies. For the purposes of this review the term calcium channel antagonists will be confined to compounds which block Ca2+fluxes through voltage-operatedcalcium channels (VOCCs),although Ca2+ can also enter cells via receptor-operated channels and be released from internal stores.

5.2 The role of calcium in ischaemic stroke Ischaemic stroke is a consequence of the disruption of blood flow following occlusion of a blood vessel in the brain. There is a central core region which receives little or no blood supply and will not survive unless reperfusion can be achieved either Academic Press Limited Copyright 0 1997 All rights of reproduction in any’”yf0rmreserved

NEUROPROTECTIVE AGENTS AND CEREBRAL ISCHAEMIA, IRN 40 ISBN 0- 12-366840-9; 0- 12- 197880-X @bk)

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spontaneously or in response to the administration of a thrombolytic agent. Surrounding this core is a penumbral zone, where blood flow is reduced to 20-25% of normal flow. The infarct spreads from the core region to this surrounding, compromised penumbral zone (Lasson et al., 1990). Hass (1981) was the first person to propose that Ca2+was involved in triggering ischaemia-induced neuronal cell death; a hypothesis that was supported by Siesjo (198 1) and Harris et al. (1 98 1). Subsequently, a number of studies have supported the theory that calcium overload plays a key role in mediating this spreading ischaemic injury (reviewed by Morley et al., 1994). Figure 1 shows, schematically, Ca2+status under both normal and ischaemic conditions in a neurone. The reduction in blood flow and consequent hypoxia causes a decline in ATP levels, leading to the failure of the Na+/K+ pump, which in turn results in membrane depolarization and a breakdown of ion homeostasis. The depolarization leads to opening of VOCCs and Ca2+ influx into the cell. Neuronal Ca2+reaches high mM concentrations and activates a number of calcium-dependent systems, including calcium-dependent kinases (calmodulin-dependent, PKC), phospholipases and proteases (calpains). Sustained activation of these calcium-dependent processes can result in immediate or delayed cell death. In parallel, these increases in Ca2+presynaptically cause the release of glutamate, which acts on excitatory amino acid receptors (NMDA, AMPA and kainate). This results in further influx postsynaptically of both sodium and calcium ions. Stimulation of excitatory amino acid receptors will also lead to the mobilization of Ca2+from intracellular stores via inositol phosphate stimulation (Fransden and Schousboe, 1991). Levels of Ca2+are important for determining subsequent outcome as it has been demonstrated that in focal ischaemia in the cat, changes in Ca2+icorrelate with both histopathology and functional outcome as measured by EEG (Uematsu et al., 1988, 1989).At levels of blood flow below 20%, Ca2+;starts to increase and remains elevated on reperfusion in those animals with poor EEG recovery and the greatest histological damage. It was the level of Ca2+irather than the level of local cortical blood flow that appeared to correlate with recovery of function and histopathology. It was originally thought that cell death caused by ischaemia was due primarily to excitotoxic mechanisms which cause necrosis. Recently it has been realized that apoptotic mechanisms may also contribute to the spread of the infarction in uivo and that calcium may also play a key role in this pathology (Trump and Berezesky, 1995). Therefore calcium appears to play a pivotal role in the two major pathways of ischaemic cell death.

5.3 Classification of voltage-operated calcium channels Calcium channel subtypes were first described on the basis of differences in pharmacology. The L-type channels, which are predominantly found on skeletal, cardiac and smooth muscle, were the most well characterized. At least four other classes of high-

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A Normal 3Na+

A

0

Resting Ca2*,10'M

AOCCs eg NMDA

PI coupled receptors eg rnGluR

dependent pump

A

r \ L

l

v

FIGURE1 The role of calcium and calcium channels in ischaemia. (A) Normal neurone with resting levels of Ca2+iof (B) Ischaemic neurone with elevated calcium levels ( 10+M- 1o-4M).

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TABLE 1 CLASSIFICATION OF HIGH-THRESHOLD

VOLTAGE-GATED CALCIUM CHANNEL SUBTYPES IN RAT BRAIN

Rat a, gene

Pharmacological name

Pharmacology ICSO

rb A

P/Q

rb B

"'

rb C rb D rb E

L L R

100 nM MVIIC 200 nM Aga IVA 2nM GVIA 1 nM MVIIA DHPs DHPs 60 pM Ni

?

P

2 nM Aga IVA

Location Pre- and postsynaptic Pre- and postsynaptic Postsynaptic cell bodies Postsynaptic cell bodies Widespread, pre- and postsynaptic? Purkinje cell bodies

CTX MVIIC Note. MVIIC, w-conotoxin from Conw magus;Aga IVA, w-agatoxin IVA from Agehopsis aperta; GWA, w-conotoxin GVIA from C. geographuc; MVIIA, w-conotoxin from C. magur (SNX-I 1 1); DHP, dihydropyridine.

threshold VOCCs have been identified on neurones on the basis of their biophysical and pharmacological properties: P, N, Q a n d R (Mintz et al., 1992). Selective peptide antagonists have been used extensively to characterize these different subtypes, as shown in Table 1. The diversity of calcium channels was confirmed by molecular cloning studies which have gone some way to resolving the relationship between structural and functional classes. Calcium channels are multimeric complexes composed of a l , a2, and 6 sub-units. A y subunit is also present in skeletal muscle. The pore of the channel, as well as the binding region for the conotoxins, is associated with the a 1 subunit (Varadi et al., 1995).To date, five distinct genes encoding a 1 subunits from the brain have been cloned and expressed, and these are termed A, B, C, D and E (Snutch et al., 1990). Class C and D genes (a l C, a l D ) appear to be responsible for L-type currents. Functional expression of constructs containing the a 1B subunit produces channels with pharmacology similar to that of the native N-type channel. Efforts to match the al-A subunit with one of the known pharmacological classes has proved to be more difficult. Expression of the subunit in oocytes gives a channel with a pharmacology similar to the Qtype found in cerebellar granule cells (Zhange et al., 1993). However, there are also similarities with the P type channel. The a 1A and a 1B subunits share a high degree of sequence homology (70%)but both classes are more distantly related to a l C which produces L-type pharmacology. In the case of the a l B subunit, there is approximately 95% sequence homology between rat and human. Four distinct genes have been found to encode for the p subunit as well as splice variants, whereas the a 2 and y subunits are derived from the same single gene. Detailed reviews on channel subtypes pharmacology and molecular biology are provided by Miljanich and Ramachandran (1995) and Perez-Reyes and Schneider (1994).

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It can be seen from the above that there are a large number of potential different subunit combinations with potentially very different pharmacology in vitro and in vivo. While truly selective antagonists do not yet exist for all the channel subtypes identified, studies with non-selective compounds and the conotoxins have begun to clarifjl the roles of VOCCs in ischaemia.

5.4 In vitro studies with calcium antagonists Most studies have used the dihydropyridines such as nifedipine, nitrendipine and nimodipine. Although many workers have demonstrated the calcium dependence of excitotoxic cell death in vitro (e.g. Pauwels et al., 1990;Abele et al., 1990),studies which have tried to correlate the toxcity with effects on Ca2+levels have produced some confiicting results. Michaels and Rothman (1 990) failed to find a correlation between Ca2+iand the level of toxicity in hippocampal primary cultures. However, Eimerl and Schramm (1994) found that cell death induced by NMDA in cortical cultures was proportional to the increase in Ca2+uptake into the cell. Pizzi et al. (199 1) found that nifedipine (100 n M ) reduced the toxicity caused by 50 VM glutamate in cerebellar granule cells, but only if the compound was present before and during the exposure to glutamate. Isradipine and verapamil produced similar effects in this system. Pauwels et al. (1990) also found that some calcium channel antagonists were neuro-protective against veratridine toxicity in cerebellar granule cells, but the neuroprotective efficacy correlated with the potency of the compounds at Na+ channels rather than at Ca2+channels. In cortical cell culture there appear to be two components to hypoxic injury, each involving NMDA receptor activation and each capable of leading to cell death. Acute swelling is mediated by influx of Na+, C1- and water and is enhanced by the removal of extracellular calcium. A delayed component exists which depends on the presence of extracellular Ca2+and correlates with uptake of Ca45(Dessi et al., 1994; Goldberg and Choi, 1993). Interestingly, this cell injury was increased in the presence of concentrations of EGTA which were nontoxic under normoxic conditions. This delayed cell excitatory amino acid neurotoxicity was attenuated by nifedipine, but concentrations of 10-100 p . of ~ nifedipine were required (Weiss et al., 1990).However, lower concentrations of nimodipine have been reported to reduce potassium-induced calcium influx in cortical cultures (Madden et al., 1990). Thus, the evidence from in i t r o studies does not produce a clear role for DHPs in ameliorating in vitro neurotoxicity. The N channel selective calcium antagonist, o-conotoxin GVIA,has also been tested in vitro (Madden et al., 1990). Cortical cultures were exposed to a hypoxic insult and the effects on Ca2+influx and LDH release compared. GVIA produced a small reduction (20-3O0/0) in Ca2+influx which was not concentration dependent over the range tested (1 nM-1 p ~ )A. greater reduction in LDH release was observed with a maximal reduction of 50% at 1VM. Whether this reflects differences in sensitivity of the two assays or an additional action of GVIA is unclear.

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Recently, calcium antagonists have appeared which are antagonists of neuronal VOCCs and which also possess a reduced propensity to cause haemodynamic effects (Benham et al., 1993; Barone et al., 1994, 1995; Bailey et al., 1995).SB201823A (4-[2(3,4-dichlorophenoxy)ethyl] - 1-pentylpiperidine hydrochloride} has an ICs0of 4.9 p~ against total Ca2+current in sensory neurones and blocks hippocampal Ca2+current. While SB20 1823A shows little selectivity for the subtypes of neuronal Ca2+channels, it is selective for Ca2+channels over Na+ and K+ channels (Benham et al., 1993). In cortical cultures, SB201823A did not block toxicity induced by either glutamate, AMPA or hypoxia (Rogers, 1995). However, in cerebellar granule cells in culture, SB201823A (2.5 pmol/l) totally prevented the initial Ca2+influx and reduced later Ca2+influx by 50% after NMDA/glycine stimulation. Little work has been done to examine the effects of calcium channel antagonists on in Uitro ischaemia (hypoxiaplus hypoglycaemia)models. Recently,AgaIVA has been tested in a hippocampal slice preparation subject to such an ischaemic insult (Small et al., 1995).Both functional electrophysiologicalmeasures and viability assays indicated that 200 nM AgaIVA protected CA1 neurones. This may indicate a role for P/Qtype Ca2+channels in ischaemia-induced damage. However, data obtained to date with other classes of compound suggest that such in vitro ischaemia models also fail to predict the in Uivo neuroprotective profile of these antagonists (e.g. Rogers, 1995).

5.5 In vivo studies with calcium antagonists 5.5. I DIHYDROPYRIDINES

Initial studies on the role of calcium antagonists in ischaemia utilized compounds which had been optimized for their antihypertensive properties such as the dihydropyridines, nicardipine and nimodipine (see Alps et al., 1988, for references). These compounds are poorly brain penetrant and can also cause hypotension at the doses used in some in vivo neuroprotective studies (e.g.Jacewicz et al., 1990). Interestingly, work in Uivo has concentrated almost exclusively on ischaemia-induced cell death rather than on experiments which mirror the in Uitro toxicity experiments, despite some preliminary reports that nimodipine can ameliorate excitoxic damage (Luiten et al., 1995). The use of dihydropyridines (DHPs) as neuroprotective agents was given some support by the finding that increases in [3q-nimodipine were observed after focal ischaemia in rats (Hogan et al., 1990).In this experiment the area with the most severe reduction in blood flow, the striatum, showed an increase in nimodipine binding five minutes after the onset of ischaemia. O n the other hand, the sensory motor cortex, a region with penumbral flow, did not show an increase in [3H]nimodipine uptake until four hours post-occlusion. Similar increases in the binding of L channel antagonists have also been observed after global ischaemia (Magnoni et al., 1988). The literature with regard to the neuroprotective effects of these first generation

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calcium antagonists in stroke models is conflicting. For example, nimodipine has been reported to be neuroprotective in some but not all focal ischaemia studies in the rat (Brown et al., 1995; Hara et al., 1990; Bielenberg et al., 1990). The same is true for global ischaemia models (Alps et al., 1988; Hassman et al., 1988). In part, the reason for the conflicting data may be due to the confounding effects of the systemic hypotension caused by these compounds in some studies. With regard to cerebral blood flow, Smith et al. (1983) found that nimodipine caused hypoperfusion in some areas and hyperperfusion in others. It has also been suggested that the DHPs act preferentially on cerebral blood vessels to restore tissue perfusion at the infarct periphery (Marinov and Wasman, 199 1; Sauter et al., 1989).Additionally, wide variations in the doses and dosing regimes employed (reviewed in Feuerstein et al., 1992) make interpretation of the data difficult. For example, in the gerbil global ischaemia model of bilateral carotid occlusion (BCAO), nimodipine was not neuroprotective when given pre- and post-ischaemia, but nimodipine (1.5 mg/kg) given post-ischaemia reduced neuropathology (Paschen et al., 1988).However nicardipine did produce a significant effect when given pre- and post-ischaemia (Alps et al., 1988). Extensive studies have been conducted with the dihydopyridine, isradipine, in several animal models of ischaemia (Bailey et al., 1995)at a dose which other workers have reported to be neuroprotective, although this dose does produce hypotension in normotensive rats (Barone etal., 1994; Sauter and Rudin, 1991).A dose of 2.5 mg/kg of isradipine, administered i.p. post-ischaemia, failed to produce a reduction in lesion volume in either the rat rose bengal photochemical model or mouse middle cerebral artery occlusion (MCAO) models of focal ischaemia. In the BCAO model of global ischaemia, the same dosing schedule failed to produce a significant reduction in hippocampal CA1 damage or ischaemia-induced hyperactivity. Isradipine also failed to reduce lesion volume in the mouse MCAO when given in a pre- and post-dosing regimen (Bailey et al., 1995). Thus, when administered across several different species and models in a consistent dosing regime, this particular compound did not produce any positive effects, although the neuronal calcium antagonist SB20 1823 did produce a positive effect in the same models. This contrasts with data from the rat MCAO where 2.5 mg/kg of isradipine produced a reduction in lesion size (Sauter and &din, 1990, 1991; Barone et al., 1994).

5.5.2 FLUNARIZINEAND

EMOPAMIL

Emopamil is a calcium channel antagonist and 5HT2 antagonist. It has also been shown in a number of studies to reduce infarct volumes in focal models when given pre- or post-ischaemia (reviewed in Feuerstein et al., 1992). Additionally, Block et al. (1990) reported that administration of emopamil in a global ischaemia model not only reduced hippocampal damage but also improved cognitive performance, that is, functional outcome. Another compound which has been extensively studied in Uiuo is the non-dihydropyridine calcium antagonist, flunarizine. De Ryck and colleagues (1990) examined

AJ. HUNTER

102

20 18 16

[

'4

'i5 12

g

'P

'

10 8 6 4 2 0

Vehicle

SB201823-A

FIGURE2 Mouse MCAO model SB201823 was administeredat 10 mg/kg i.p. 30 minutes post-occlusion and b i d . for 3 days. Mice were sacrificed on day 4 post-ischaemia(from Bailey et al., 1995).

the effects of flunarizine on rose bengal photochemical lesions in rats. These studies demonstrated a significant reduction in lesion size at 4 days, and improved motor performance with flunarizine treatment post-ischaemia @e Ryck et al., 1990).However, as with the DHPs, other workers have not been able to demonstrate a consistent effect of flunarizine in other models of ischaemia, although - again - a range of doses, routes, and times of administration have been employed (Alps et al., 1988; Yoshidomi et al., 1989; Clark et al., 1990).Shimazawa et al. (1995) demonstrated that flunarizine increased cerebral perfusion and inhibited the cortical hypoperfusion resulting from KC1-induced cortical spreading depression (CSD).Flunarizine has also been reported to elevate the threshold for eliciting CSDs in rats (Wauquier et al., 1985).As CSD has been postulated to be involved in the spread of ischaemic damage (Meis et al., 1993), part of any neuroprotective effect observed with flunarizine may be due to indirect effects of the compound on CSD. This lack of consistency across species and models for compoundssuch as nimodipine and flunarizine may be significant in predicting subsequent clinical failure. Both nimodipine and flunarizine have been tested in clinical trials (see below) with no positive findings. Although one does not yet know which model in animals is most predictive of positive effects in man, lack of consistent effects, especially in focal MCAO models, appears to predict lack of efficacy in human stroke patients (see also Chapter 3).

5.5.3 CALCIUM ANTAGONISTSWITH REDUCEDCARDIOVASCULARLIABILITIES The neuronal VOCC antagonist, SB201823A,has been reported to be efficacious in models of both global and focal ischaemia. As shown in Figure 2, SB201283A significantly reduced infarct size in both rat and mouse permanent MCAO models when


-

\ 2.0

g

103

B

*

1.6

c)

3

0

4

i.0

P1 05 z 0.0

VEHICLE

98 201823-A

VEHICLE

SB 201829-A

FIGURE3 The effect of SB201823 10 mg/kg i.v. on neurological grade (A) and hindlimb deficit (B) (from Barone etal., 1995).

given post-ischaemia by either the i.p. (mouse) or i.v. (rat) route (Bailey et al., 1995; Barone et al., 1995).In addition, post-ischaemia dosing of SB20 1283A (10 mg/kg i.p.) produced a 25% reduction in volume in the rose bengal model of photothrombotic lesion (Beham et al., 1993). Importantly, SB20 1823A also produced improvements in neurological grade in the rat MCAO model (Figure 3) and reduced ischaemiainduced hyperactivity in the gerbil BCAO model of global ischaemia (Benham et al., 1993; Barone et al., 1995). Unlike some of the hydropyridines, intravenous dosing of a neuroprotective dose of SB20 1283A (10 mg/kg i.v.) did not produce any significant effects on blood pressure in conscious rats, although a transient reduction in heart rate was seen with this compound (Barone et al., 1995).These studies demonstrate that neuroprotection in a variety of species and models can be observed with a calcium antagonist in the absence of any significant effects on blood pressure. Another neuronal calcium antagonist, SB206284, has also been shown to be neuroprotective in a range of models in the absence of effects on heart rate or blood pressure at neuroprotective doses (Wood et al., 1995).This confirms that a neuronal calcium antagonist can have direct neuroprotective effects in the absence of effects on haemodynamic parameters. While SB201823 and SB206284 described above have displayed impressive efficacy in a range of ischaemia models in vivo, they do not demonstrate any selectivityfor a particular subtype of neuronal calcium channel. In the past five years studies with the peptide N channel selective antagonist, SNX-111 (w-conotoxinMVIIA),have demonstrated efficacy in focal and global models of ischaemia suggestinga key role for ‘N’ type Ca2+channels in mediating ischaemic damage (Takizawa et al., 1995; Valentino et al., 1993).SNX-111 is a slightly more reversible ‘ N channel antagonist than wctx GVIA, which is essentially an irreversible antagonist, although SNX-111 does have a slow off rate in vitro. Although intracisternal wctxGVIA has been reported to be neuroprotective in the gerbil BCAO model (Yamada et al., 1994),icv administration has also been shown to be neurotoxic, probably due to this irreversible channel blockade. Likewise, Madden et al. (1990) failed to find a reduction in spinal cord ischaemia in rabbits with a dose of 0.5 nmol of wctxGVIA administered pre-ischaemia into the subarachnoid space. As can be seen from the above experiments, studies with wctxGVIA have to use direct

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administration into the CNS, whereas SNX-111 has the advantage that it has shown activity when administered either centrally or by the intravenous route. SNX-111 has been demonstrated to be neuroprotective in both focal and global models of ischaemia. Initial studies in global ischaemia models demonstrated that SNX-11 1 decreased the damage observed in the CAI region of the hippocampus and suggested that initiation of therapy could be delayed for up to six hours (Valentino et al., 1993).Buchan et al. (1994)delayed administration of SNX-111 for up to 24 hours post-ischaemia in the rat 4-vessel occlusion (4VO) global ischaemia model. Intravenous administration of 5 mg/kg over 15 minutes produced a significant reduction in hippocampal CA1 damage when first administered at either 6 or 24 hours post ischaemia. This dose of SNX-111 also caused a 20% reduction in blood pressure in a separate group of unoperated rats. Therefore, in most studies with SNX-1 1 1, the compound has been given via a slow infusion (1 hour or longer). Slow infusions of SNX-111 significantly reduced infarct volumes in both nomotensive (Zhao et al., 1994) and spontaneously hypertensive (Buchan et al., 1994) rat MCAO models. In both of these studies, noradrenaline was infused to maintain blood pressure at control levels, although Buchan et al. (1994) also included groups treated with SNX- 11 1 alone. The concurrent administration of noradrenaline did not reduce the neuroprotective effects of SNX-1 11. This neuroprotection was observed in the presence of reduced cerebral blood flow in the ischaemic cortex and so appears to be a direct effect on neurons. A direct action is also supported by the fact that SNX- 1 1 1 is neuroprotective in the global 4VO model when given i.c.v. (Miljanich and Ramachandran, 1995). SNX-11 1 does induce hypothermia, which in itself can be neuroprotective. However, Buchan et al. (1994) maintained the animals by external heating for up to 6 hours postischaemia and so temperature effects are unlikely to have been important in these studies. The hypotension induced by SNX-111 appears to have two components. Sympathetic blockade is important at lower doses, whereas higher doses produce a reduction in blood pressure due to histamine release from mast cells (Miljanich and Ramachandran, 1995). SNX-111 is a peptide which penetrates the brain relatively poorly and therefore, in zivo, relatively high levels of the compound have to be administered compared to potency in vitro (&=9 PM). Development of non-peptide blockers of the ‘N’channel may produce compounds which lack the hypotensive liabilities of SNX-111. This is because they should possess better brain penetration, and hence should be neuroprotective at lower doses than the peptide. The mechanism(s)by which blockade of N channels produces such mixed neuroprotection has not been demonstrated conclusively. SNX-111 is less potent than SNX 230 (w-conotoxin MVIIC) at inhibiting release of glutamate in vitro (Gaur et al., 1994). SNX 230, however, is not neuroprotective despite the fact that it potently blocks neurotransmitter release. Recently, Takizawa et al. (1995) showed that SNX-111 does reduce ischaemia-induced extracellular glutamate release. Whether this is a direct effect on neurotransmitter release or an indirect effect due to reduced neuronal excitability remains to be answered.


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5.6 Clinical studies Currently, the only compound approved for the acute treatment of stroke is nimodipine, which has approval for use in subarachnoid haemorrhage (SAH) but not ischaemic stroke. The leading cause of mortality in SAH patients who survive the initial haemorrhage is cerebral vasospasm. However, it was believed that nimodipine selectively reduced this vasospasm, but controlled trials have not reported a reduction in the incidence of cerebral vasospasm with nimodipine treatment (see reviews by Wong and Clark, 1990; Murphy, 1992). Several large-scale clinical trials have also been carried out with nimodipine treatment in ischaemic stroke, but the results from these trials have been disappointing generally. There is some indication from trials with nimodipine and other DHPs that benefits may be present iftreatment is begun within some hours post-ischaemia (Rosenbaum et al., 1992; American Nimodipine Study Group, 1992). SNX- 11 1 has entered clinical trials and is currently in phase I1 trials for ischaemic stroke. Studies in volunteers confirmed that the hypotensive sideeffects observed preclinically also occur in man after 24-hour infusion (Miljanich and Ramachandran, 1995). Whether these hypotensive effects will limit clinical studies remains to be seen, but the data from the ongoing trials are awaited with interest. Calcium channel antagonist research has entered a new and exciting era. T h e role of different channel subtypes in mediating ischaemic damage is just beginning to be elucidated. Further advances, especially in preclinical in vivo studies, await the development of nonpeptide antagonists selective for each of the different channel subtypes.

References Abele, A.E., Scholz, K.P., Scholz, W.K. & Miller, R.J. (1990) Excitotoxicity induced by enhanced excitatory neurotransmissionin cultured hippocampal pyramidal neurons. Neuron. 2,413419. Alps, BJ., Calder, C., Hass, W.K. & Wilson, A.D. (1988) Comparative protective effects of nicardipine, flunarizine, lidoflazine and nimodipine against ischaemic injury in the hippocampus of the Mongolian gerbil. Bx J. Pharmacol. 93,877-883. American Nimodipine Study Group (1992) Clinical trial of nimodipine in acute ischaemic stroke. Stroke 23, 3-8. Bailey, SJ., Wood, N.I., Samson, N.A., Rothaul, A.L., Roberts,J.C., King, PD., Hamilton, T.C., Harrison, D.C. & Hunter, A.J. (1995) Failure of isradipine to reduce infarct size in mouse, gerbil and rat models of cerebral ischaemia. Stroke 26, 1 1, 2 177-2 183. Barone, F.C., Lysko, P.G., Price, WJ., Feuerstein, G., Al-Baracanji, K.A., Benham, C.D., Harrison, D.C., Harries, M.H., Bailey, SJ. & Hunter, A.J. (1995) SB201823-A antagonizes calcium currents in central neurons and reduces the effects of focal ischaemia in rats and mice. Stroke 26, 1683- 1690. Barone, EC., Price, W.J., Jakobsen, I?, Sheardown, MJ. & Feuerstein, G. (1994) Pharmacological profile of a novel neuronal calcium channel blocker includes reduced cerebral damage and neurological deficits in rat focal ischaemia. Pharmacol. Biochem. Behav. 48, 177-85. Benham, C.D., Brown, TH., Cooper, D.G., Evans, M.L., Harries, M.H., Herdon, H.J., Meakin,J.E., Murkitt, K.L., Patel, S.R., Roberts,J.C., Rothaul, A.L., Smith, S.J., Wood, N.

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& Hunter, AJ. (1993) SB201823-A, a neuronal Ca2+antagonist is neuroprotective in two

models of cerebral ischaemia. Neuropharmmology32, 11, 1249-1257. Bielenberg, G.W., Burniol, M. Rosen, R. & Klaus, W. (1990) Effects of nimodipine on infarct size and cerebral acidosis after middle cerebral artery in the rat. Stroke 21 (Su@ ZV). IV90-IV92. Block, E,Jaspers, R.M.A., Heim, C. & Sontag, K.H. (1990)Semopamil ameliorates ischaemic brain damage in rats: histological and behavioural approaches. Life Sciences 47, 151 1-1518. Brown, C.M., Calder, C., Linton, C., Small, C., Kenny, B.A., Spedding, M. & Patmore, L. (1995) Neuroprotective properties of lifarizine compared with those of other agents in a mouse model of focal cerebral ischaemia. Br.3 Phurmacol. 115, 1425-1 432. Buchan, A.M., Gertler, S.Z., Li, H., Xue, D., Huong, Z.-G., Chaundy, K.E., Barnes, K. & Lesiuk, HJ. (1994)A selective N-type Ca2+channel blocker prevents CA1 injury 24H following severe forebrain ischaemia and reduces infarction following focal ischaemia.3 Cereb. Blood Flow Metab. 14,903-910. Clark, W.M., Madden, K.P. & Zivin,J.A. (1990)The lack of effect of flunarizine on preserving neurological deficit after experimental stroke. Soc. Neurosd. Ab 16,935. De Ryck, M., Van Reempts, J., Borgers, M., Wauquier, A. & Janssen, PAJ. (1989) Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocortical infarcts in rats. Stroke 20,1383-1 390. Dessi, E, Charriaut-Marlangue, C., Ben-Ari, Y. (1994) Glutamate-induced neuronal death in cerebellar culture is mediated by two distinct components: a sodium-chloride component and a calcium components. Bruin Res. 650,49-55. Eimerl, S. & Schramm, V: (1994) The quantity of calcium that appears to induce neuronal death.3 Nmrocha. 62, 1223-1226. Feuerstein, G.S., Hunter, AJ. & Barone, E (1992)Calcium channel blockers and neuroprotection. In Emergirg Strutegies in Nmroprotection (eds Marangos, PJ. & Lal, H.) pp. 129-150. Birkhauser. Fisher, M. (1991) Clinical pharmacology of cerebral ischaemia: old controversies and new approaches. Cerebromc. fi.,1,(suppl. 1) 112-1 19. Frandsen, A. & Schousboe, A. (1991) Dantrolene prevents glutamate cytoxicity and Ca2+ release from intracellular stores in cultured cerebral cortical neurones. 3 Nmrochem. 56, 1075-1078. Gaur, S., Newcomb, R., Rivnay, B., Bell,J.R., Yamashiro, D., Ramachandran, J. & Miljanich, G.P. (1994) Calcium channel antagonist peptides define several components of transmitter release in the hippocampus. Neuropharmmology33, 1211-1 2 19. Goldberg, M.P & Choi, D.W. (1993) Combined oxygen and glucose deprivation in cortical cell culture: calcium-dependent and calcium-independent mechanisms of neuronal injury. 3 Newosc. 13, 3510-3524. Hara, H., Nagasawa, H. & Kogure, K. (1990)Nimodipine attenuates both ischaemia-induced brain oedema and mortality in a rat novel transient middle cerebral artery occlusion model. Acta Nmrochirur. Suppl. 51, 251-253. Harris, RJ., Symon, L., Branston, N.M. & Baynon, M. (1981) Changes in extracellular calcium activity in cerebal ischaemia.3 Cereb. Blood. Row.Metab. 1,203-209. Hass, W.K. (1981) Beyond cerebral blood flow, metabolism and ischaemic thresholds: an examination of the role of calcium in the initiation of cerebral infarction. In Cerebral Vmculor fieme 3 (eds Meyer, J.S., Lechner, H., Reivech, M., Ott, E.O. & Araniber, A.) pp. 3-17. Ecerpta Medicu, Amsterdam. Hogan, M., Gjedde, A. & Hakim, A.M. (1990)Nimodipinebinding in focal cerebral ischaemia. Stroke (Suppl. IV) 21, 78-80. Jacewicz, M., Brint, S., Tanabe,J. & Pulsinelli, W.A. (1990)Continuous nimodipine treatment attenuatescortical infarction in rats subjected to 24 hours offocal cerebral ischaemia.3 Cereb. Blood Row Metab. 10,89-96.


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Lassen, N.A., Fieschi, C. & Lenzi, G.L. (1990) Ischaemic penumbra and neuronal cell death: comments on the therapeutic window in acute stroke with particular reference to thrombolytic therapy. Cerebrovas.Disc. 1991 1 (Suppl. I), 32-35. Luiten, P.G.M., Douma, B.R.K., Van der Zee, E.A. & Nyakas, C. (1995) Neuroprotection against NMDA induced cell death in rat nucleus basalis by Ca2+antagonist nimodipine, influence of ageing and development drug treatment. Nmrodegenerahn 4,307-3 14. Madden, K.P, Clark, W.M., Marcoux, EW., Probert, A.W., Weber, M.L., Rivier,J. & Zivin,J.A. (1990) Treatment with conotoxin, and 'N-type' calcium channel blocker, in neuronal hypoxic-ischaemic injury. Brain Res., 537,256262. Magnoni, M.S., Gotoni, S., Battaini, E & Trabucchi, M. (1988)L-type calcium channels are modified in rat hippocampus by short-term experimental ischaemia. J. Cereb. Blood. Flow Metab., 8 , 9 6 9 9 . Marinov, M. & Wassman, H. (1991).Lack of effect of PN200-110 on neuronal injury and neurological outcome in middle cerebral artery-occluded rats. Stroke 22 (8),1064-1067. Mies, G., Iijma, T. & Hossman, A.K. (1993) Correlation between per infarct DC shifts and ischaemic neuronal damage in rat. NeuroReport 4, 709-7 1 1. Michaels, R.L. & Rothman, S.M. (1990)Glutamate neurotoxicity in vitro: antagonist pharmacology and intracellular calcium concentrations.J. Neuroscience 10, 283-292. Mdjanich, G.P. & Ramachandran, J. (1995) Antagonists of neuronal calcium channels. Annu. Rev. Pharmacol. TToxicoL, 35, 707-734. Mintz, I.M., Venema, VJ., Swiderek,K.M., Lee, TD., Bean, B.P & Adams, M.E. (1992)P-type calcium channels blocked by the spider toxin o-Aga-IVA. Nature 355,827-829. Morley, P, Hogan, M.J. & Hakim, A.M. (1994) Calcium-mediated mechanisms of ischaemic injury and protection. Brain Patholou 4, 37-47. Murphy,J.J. (1 992) The role of calcium antagonists in the treatment of cerebrovascular disease. Drugs BAgang2,1-6. Paschen, W., Hallmayer,J. & Rohn, G. (1 988) Relationship between putrescene content and density of ischaemic cell damage in the brain of Mongolian gerbils: effect of nimodipine and barbiturate. Acute Neuropathol. 76, 388-394. Pauwels, PJ., Van Assouw, H.P, Peeters, L. & Leysen, J.E. (1 990) Neurotoxic action of veratridine in rat brain neuronal cultures: mechanism of neuroprotection by Ca2+antagonists nonselective for slow Ca++channels.j! Pharmacol. Ex$ Ther. 255, 1 117-1 122. Perez-Reyes, E. & Schneider, T. (1994)Calcium channels: structure, function and classification. Drug Develok. Res. 33, 295-3 18. Pizzi, M., Ribola, M., Valerio, A., Memo, M. & Spano, PE (1991)Various Ca2+entry blockers prevent glutamate-induced neurotoxicity.Eur:J. Pharmacol. 209, 169-1 73. Rogers, D.C. (1995)The effects of neuroprotective agents on in vitro and in vivo models of cortical neurotoxicity. PhD Thesis, University of Hertfordshire. Rosenbaum,D., Zabramski,J., Frey,J., Yatsu, E, Marler,J., Spetzler,R. & Grotta,J. (1991)Early treatment of ischaemic stroke with a calcium antagonist. Stroke 22,437-441. Sauter,A., Rudin, M., Wiederhold, K.-H. & Hof, R.P (1 989)Cerebrovascular,biochemicaland cytoprotective effects of isradapine in laboratory animals. A m . 3 Med. 4A,134-146. Sauter, A. & Rudin, M. (199 1)Prevention of stroke and brain damage with calcium antagonists in animals. Am. j'.Hypertens.,4, 121S-127s. Shimazawa, M., Hara, H., Watano, T & Sukamoto, T. (1995)Effects of Ca2+channel blockers on cortical hypoperfusion and expression of c-Fos-like immunoreactivity after cortical spreading depression in rats. Brit. J. Pharmacol. 115, 135S1368. Siesjo, B.K. ( I 98 1) Cell damage in the brain: a speculative synthesis. j! Cereb. Blood Flow Metab. 1, 155-185. Small, D.L., Monette, R., Mealing, G., Buchan, A.M. & Morley, P. (1995) Neuroprotective effects of omega-Aga-IVA against in uitro ischaemia in the rat hippocampal slice. NeuroReport 6, 1617-1620.

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Smith, M.L., Kagstrom, E., Rosen, I, & Siesjo, B.K. (1983) Effect of the calcium antagonist nimodipine on the delayed hypoperfusion following incomplete ischaemia in the rat. j! Cerebral Blood Flow Metab. 3,543-546. Snutch, T.P., Leonard, J.P., Gilbert, M.M., Lester, H.A. & Davidson, N.P. (1990) Rat brain expresses a heterogeneous family of calcium channels. Proc. Nut1 Acad. SCi. USA 87, 3391. Takizawa, S., Matsushima, K., Fujita, H., Nanri, K., Ogawa, S. & Shinohara, Y (1995)A selective N-type calcium channel antagonist reduces extracellular glutamate release and infarct volume in focal cerebral ischaemiaJ Cereb. Blood Flow and Metabolism 15,611-618. Trump, B.F. & Berezesky, I.K. (1995)Calcium-mediatedcell injury and cell death. The FASEB Journal 9 , 2 19-228. Uematsu, D., Greenberg,J.H., Reivich, M. & Karp, A. (1988) In Uivo measurement of cytosolic-free calcium during cerebral ischaemia and reperfusion. Ann. Neural. 24,420428. Uematsu, D., Greenberg,J.H., Reivich, M. & Hickey, W.E (1989) Direct evidence for calciuminduced ischaemic and reperfusion injury. Ann. Neurol. 26, 280-283. Valentino, K., Newcomb, R., Gadbois, T., Singh, T., Bowersoz, S., Bitner, S., Justice, A., Yamashiro, D., Hoffman, B.B., Ciaranello, R., Miljanich, G. & Ramachandran, J. (1993) A selective N-type calcium channel antagonist protects against neuronal loss after global cerebral ischaemia. Proc. Nut1 Acad. Sci. USA 90,7894-7897. Varadi, G., Mori, Y, Mikala, G. & Schwartz, A. (1995) Molecular determinants of Ca2+ channel function and drug action. 7rad.s Pharmacol. Sci. 16, 4 3 4 9 . Weiss,J.H., Hartley, D.M., Koh,J.,Choi, D.W. (1990)The calcium channel blocker nifedipine attenuates slow excitatory amino acid neurotoxicity. Science 247, 1474-1477. Wong, M.C.W. & Clarke, H.E. Jr (1990) Calcium antagonists: stroke therapy coming of age. Stroke 21, 494-501. Wood, N.I., Benham, C.D., Brown, T.H., Cooper, D.G., Hamilton, T.C., Hunter, AJ., Milkowski, N.A., Patel, S. & Roberts,J.C. (1995)The effects of SB 206284A, a novel calcium channel antagonist, in two species in a focal model of cerebral ischaemia.3 Cerebl: Blood Flow Mefabol. 15, S384. Yamada, K., Teraoka, T., Morita, S., Hasegawa, T. & Naeshima, T. (1994)o-Conotoxin GVIA protects against ischaemia-induced neuronal death in the Mongolian gerbil but not against quinolinic acid-induced neurotoxicity in the rat. Neuropharmacohgy 33, 25 1-254. Yoshidomi, M., Hayashi, T., Abe, K. & Kogure, K. (1989) Effects of a new calcium channel blocker, KB-2796, on protein synthesis of the CAI pyramidal cell and delayed neuronal death following transient forebrain ischemia.3 Neurochem. 53, 1589-1 594. Zhang,J.F., Randall, A.D., Ellinor, PT., Horne, W.A., Sather, W.A., Tanabe, T., Schwartz, T.Z. & Tsien, R.W. (1993) Distinctivepharmacology and kinetics of cloned neuronal Ca2+channels and their possible counterparts in mammalian CNS neurons. Neurofiharrnacology 32, 1075. Zhao, Q, Smith, M.L. & Siesjo, B.K. (1994)The w-conopeptide SNX-I 11, an N-type calcium channel blocker, dramatically ameliorates brain damage due to transient focal ischaemia. Acta. Ptpiol. Scand. 150,459461.

Chapter 6

SODIUM AND POTASSIUM CHANNEL MODULATORS: THEIR ROLE IN NEUROPROTECTION Tihomir F? Obrenovitch Department of Neurochemistry, Institute of Neurology, Queen Square, London, W C I N 3BG, UK

6. I 6.2

Introduction Down-modulation of voltage Na' channels during ischaemia: an inherent adaptive mechanism for neuronal survival 6.3 Na+ channel blockade protects neurones against ischaemia: experimental evidence 6.3. I In vim experiments with Na+-free medium 6.3.2 Protection against ischaemic damage by tetrodotoxin 6.3.3 Protective effect of local anaesthetics 6.3.4 Protective effects of anticonvulsantsacting on Na' channels 6.4 Neuroprotective agents acting on Na' channels 6.4. I Lamotrigine, BW I I 1 4 x 8 7 and BW6 I9C89 6.4.2 Riluzole 6.4.3 Ca2' channel modulators with actions on Na' channels 6.4.4 PD85,639 6.5 Clinical relevance and suitability 6.5. I Clinical relevance 6.5.2 Potential adverse effects 6.6 K' channel openers: introduction 6.7 Effect of ischaemia on K+ channels 6.7. I Initial changes in K' conductance 6.7.2 Changes in K+ conductance associated with early functional loss 6.7.3 Changes in K' conductance and anoxic depolarization 6.7.4 Adenosine, increased Kf conductance and neuroprotection 6.8 Rationale for opening K' channels to protect neurones against ischaemia Potential beneficial actions of K' channel openers 6.8. I 6.8.2 Potential deleterious effects of increased [K'], 6.8.3 Unresolved issues 6.9 K+ channel openers and neuroprotection in ischaemia: experimental evidence 6.10 Concluding remarks Acknowledgements References

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Academic Press Limited Copyright 0 1997 All rights ofreproduction in anyform reserved

NEUROPROTECTIVE AGENTS AND CEREBRAL ISCHAEMIA, IRN 40 ISBN 0-12-366840-9; 0-12-197880-X (pbk)

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6. I Introduction Neuronal damage subsequent to ischaemia results from multifactonal and interrelated processes (for example, excessive glutamate-receptor-mediated excitation, intracellular calcium overload, lipid peroxidation), a feature which justifies the wide range of neuroprotective strategies currently under investigation. This chapter examines two approaches for the protection of neurones in ischaemia, both of which have received relatively little attention so far: (I) down-modulation of voltage-gated Na' channels, and (2) opening of K' channels. The precise meaning of ischaemia is an interruption or reduction of blood flow, but it is more pertinent to consider ischaemia as an imbalance between energy supply and demand when considering neuroprotection. This extended definition signifies that protection may be achieved not only by improving local perfusion in ischaemic regions, but also by reducing the energy consumption of neurones. Decreasing the cerebral metabolic rate with hypothermia or barbiturates has been shown to be cerebroprotective (Spetzler and Hadley, 1989; Gmsberg et al., 1992). Stabilizing membrane potential (that is, opening of K' channels) and decreasing the production of action potential (that is, down-modulation of Na' channels) are two other ways of reducing energy consumption, restricting it to the maintenance of cellular integrity, and thus extending the time to which neurones can be exposed to hypoxia without cellular damage. The fact that functional loss (i.e. loss of consciousness and EEG silence) occurs within seconds of ischaemia onset, whereas energy levels are not depleted for several minutes, strongly suggests that the early anoxia-induced blockade of neuronal function (i.e. EEG silence) may be an adaptive process and not a consequence of energy substrate limitation (Neubauer, 1993). Changes in neuronal membrane ion conductance are clearly involved in this survival strategy, and several lines of evidence suggest that it can be strengthened pharmacologically. Some of the arguments put forward below have been developed in more detail in recent reviews (Obrenovitch and Richards, 1995; Obrenovitch, 1995b; Urenjak and Obrenovitch, 1996).

6.2 Down-modulation of voltage Na' channels during ischaemia: an inherent adaptive mechanism for neuronal survival Voltage-gated Na' channels are responsible for initiation and conduction of the neuronal action potential and, therefore, play a fundamental role in the normal function of the nervous system. In cell bodies and axon initial segments, Na' channels determine the threshold for action potential generation and affect the duration and frequency of repetitive neuronal firing. In synapses, repetitive Na'-influx through voltage-gated Na' channels (i.e. action potentials) trigger neurotransmitter exocytosis (vesicular release). Down-regulationof Na' channels during periods of limited oxygen supply appears

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an effective way of reducing energy expenditure because a large part of the energy consumed by excitable cells is used to maintain Na' and K' gradients across the cellular membrane (Erecinska and Silver, 1989). This mechanism may be an inherent survival mechanism, at least in some neurones (Urenjak and Obrenovitch, 1996).For example, intracellular recordings of neocortical neurones in human brain slices showed that their excitability was markedly decreased within the first 5 minutes of anoxia, and this effect could not be adequately explained by increased K' conductance (see 6.7. l), because it was associated with little or no change in membrane input resistance (R,) and membrane potential (V,). Whole-cell voltage-clamp with isolated human neocortical pyramidal neurones also demonstrated that anoxia and cyanide rapidly decreased a voltage-dependent, tetrodotoxin (TTX)-sensitive Na' current (Cummins et al., 1993). Early down-regulation of Na' channels when oxygen supply is reduced appears especially efficient in freshwater turtles which can survive prolonged anoxia. Under these conditions, the turtle brain EEG and evoked field potentials are reduced, but anoxic depolarization (i.e. sudden increase in the ionic permeability of the plasma membrane) does not occur and ATP levels are preserved (Chih et al., 1989).A number of mechanisms involving large glycolytic capacity, Ca2' channels, K' channels and inhibitory neurotransmitters play a role in turtle brain adaptation to anoxia, but downregulation of voltage-gated Na' channels may be a key contributor. The turtle brain has a much lower density of Na' channels than the rat brain (Xia and Haddad, 1993) and this density is reduced further by anoxia (Pkrez-Pinz6n et al., 1992). Newborn central mammalian neurones are more resistant to anoxia/ischaemia than their adult counterpart (Haddad andJiang, 1993),at least partly because oflower energy requirements (Altman et al., 1993). Low energy expenditure is l i e d to reduced electrical and synaptic activity, presumably because there are fewer neurones, dendritic processes, and synapses in the newborn. However, down-regulation of Na' channels may also play an important role in the tolerance of the newborn CNS to anoxia. Voltage-sensitive Na' currents are much smaller in newborn than in adult cortical neurones (Cummins et al., 1994), and Na' channel density is markedly lower at birth than in the mature brain (Xla and Haddad, 1994).Furthermore, in fetal brain neurones developing in Vitro, a rapid down-regulation of Na' channels occurred whenever Na'-influx was increased by application of Na' channel activators such as veratridine @argent and Couraud, 1990).

6.3 Na' channel blockade protects neumnes against ischaemia: experimental evidence

6.3.I

IN VlTRO EXPERIMENTS WITH

NA'-FREE MEDIUM

The contribution of Na' channels to the mechanisms leading to anoxic white matter injury have been well characterized by Waxman and coworkers in the optic nerve.

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This model offers the advantages that it consists predominantly of axonal fibres and glia, without neuronal cell bodies, and the compound action potential (CAP)provides a reliable estimate of the number of functional nerve fibres. Application of Na+-free medium before 60 minutes' anoxia, markedly improved the recovery of the rat optic nerve CAP in vitro, whereas increasing the transmembrane Na" gradient at various times before or during anoxia worsened the injury (Stys et al., 1991, 1992b). In this preparation, the main protective action of reducing Na' influx during anoxia may have been avoiding the reversal of the Na"-Ca2+ exchanger, thus preventing intracellular Ca2+ loading (Stys et al., 1992b). Replacement of Na' in the incubation medium with the impermeant cation &methyl-D-glucamine also prevented anoxiainduced membrane injury to dissociated rat CA 1 hippocampal neurones (Friedman and Haddad, 1994). The route for sustained entry of Na' into white matter cells during anoxia is a relevant and intriguing question because the classical voltage-gated Na+ channels which initiate the rapid upstroke of axonal action potential (that is, membrane depolarization) also contribute to its termination by fast and complete inactivation, implying that these channels should rapidly close during anoxic depolarization. One possibility, suggested by Stys et al. (1991), is that a non-inactivating Na' conductance, persisting at depolarized membrane potentials, was involved. Measurements of the rat optic nerve CAP at rest, or depolarized by K" (15-40 mM), supported this hypothesis. A TTXsensitive Na" conductance which was present at rest, persisted in nerves depolarized sufficiently to abolish classical transient Na" currents (Stys et al., 1993). It is important to mention that non-inactivating Na' conductance, which rapidly activates like the classical Na" channels, but inactivates either very slowly or incompletely even with prolonged depolarization, have been identified in central neurones (French et al., 1990;Lynch et al., 1995).Although such non-inactivating Na+ current (also called sustained or persistent Na" current) may only represent 1-3% of the peak amplitude of Na' current, they may play a critical role in situations where membrane depolarization is sustained (Taylor, 1993). It is interesting to note that R56865, a benzothiazolamine acting on slow Na" currents (Kiskin et al., 1993), significantly attenuated the reduction of extracellular Na' concentration occurring after anoxic depolarization (?he, Y et al., 1995).

6.3.2 PROTECTIONAGAINST ISCHAEMICDAMAGE BY TETRODOTOXIN Selective blockade of voltage-gated Na+-channels by tetrodotoxin (TTX) slowed down extracellular acidosis produced by complete ischaemia in the isolated perfused rat brain and markedly delayed anoxic depolarization (Prenen et al., 1988; Xie et al., 1994).As a direct consequence, TTX also delayed the dramatic ionic changes associated with anoxic depolarization, that is, Na"-entry, Ca2+-entryand K+-efltlux(xle et al., 1994).In rat hippocampal slices exposed to anoxia, T T X reduced the fall in ATP concentration and improved the recovery of evoked population spike from dentate granule neurones and CA1 pyramidal neurones (Boening et al., 1989). These effects


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support the notion that Na+-channel blockade reduces energy demand, even in ischaemic conditions severe enough to abolish electrical and synaptic activity. It is important, however, to note that TTX does not prevent anoxic depolarization, because this implies that voltage-gated Na+-channels do not play an essential role in the sudden increase in the ionic permeability of the cellular membrane which provides anoxic depolarization. In the rat optic nerve, T T X substantially improved post-anoxic functional recovery, at concentrations that had little effect on the amplitude of the control CAP (Stys et al., 1992b). Conversely, veratridine-induced Na+ influx potentiated anoxic injury (Stys et al., 1992b).Direct application of TTX to the rat hippocampus also reduced, dose-dependently, neuronal death subsequent to transient global ischaemia in rats and gerbils (Yamasaki et a/., 1991;Lysko et al., 1993), and improved functional recovery (Prenen et al., 1988). Finally, this toxin protected hippocampal cultured neurones against hypoglycaemia- and potassium cyanide-induced injury, even when applied after the insult (Tasker et al., 1992; Vornov et al., 1994). All these experimental findings clearly support the notion that blockade of voltage-gated Na+-gated channels is potentially neuroprotective.

6.3.3

PROTECTIVE EFFECT OF LOCAL ANAESTHETICS

Local anaesthetics bind to a specific site inside the pore of Na+ channels, promote their inactivation and this action results in a block of Na+ currents with complex voltage- and frequency-dependent properties (Catterall, 1987; Buttenvorth and Strichartz, 1990). These drugs also alter the conductance of K+ channels, although to a lesser extent (Stolc, 1988). Despite some conflicting in uivo results, studies with local anaesthetics generally support the concept that down-modulation of Na+ channels is potentially neuroprotective. Stys and co-workers (1992a) demonstrated that tertiary amine local anaesthetics (lidocaine, procaine) and their quaternary analogues QX-3 14 and QX-222 provide significant protection from anoxic injury in the rat optic nerve. In rat hippocampal slices, the recovery rate of synaptic function following 15 minutes of hypoxia was improved significantly by prior incubation with local anaesthetics (Lucas et al., 1989), and lidocaine prevented CA1 pyramidal cell damage produced by 12 minutes of anoxia/aglycaemia (Weber and Taylor, 1994). It is important to record that, as with TTX, functional recovery was improved markedly with anaesthetics concentrations that caused little suppression of the normal CAP in the optic nerve, and synaptic function in hippocampal slices. The effect of lidocaine treatment against ischaemia has been extensively tested in vivo, but the variety of animal models and dose regimes used preclude a reliable synthesis of the data (Urenjak and Obrenovitch, 1996). Nevertheless, three relevant features emerge: (i)As with other drugs, lidocaine treatment may be effective only with focal or incomplete global ischaemia; (ii)systemic administration of lidocaine must be sustained; and (iii) high doses may not be effective because of cardiovascular toxicity.

114

6.3.4

TI?OBRENOVITCH PROTECTIVE EFFECTS OF ANTICONVULSANTS ACTING ON

NA' CHANNELS

A number of anticonvulsants interact primarily with Na' channels at therapeutic concentrations. The most prominent compounds of this type are phenytoin, carbamazepine and lamotrigine (for lamotrigine see section 6.4.1). Their primary action is use-dependent inhibition of Na' conductance by stabilization of the channel inactivation state, leading to selective block of burst firing (Catterall, 1987). In the rat optic nerve, both phenytoin and carbamazepine protected against anoxic injury at concentrationsbelow those inhibiting the CAP, and well below the therapeutic range used to control epilepsy (Fern et al., 1993). Pretreatment with phenytoin (20 mM) also protected rat hippocampal slices against 10 minutes of hypoxia, as assessed by improved recovery of synapticallyevoked population spikes(Kennyand Sheridan, 1992). In the same preparation exposed to hypoxia/glucose-free medium, phenytoin (5-1 00 mM) concentration-dependently delayed negative shifts of the direct current (DC)potential (that is, anoxic depolarization) improved recovery of synaptic potentials and protected against histological damage (Weber and Taylor, 1994). It is important to emphasize that, as with TTX (in some models) and lidocaine, effective neuroprotection by phenytoin was achievable without blocking synaptic potentials or presynaptic fibre volleys. In contrast, 10 and 100 mM ofphenytoin alone failed to protect murine-cultured cortical neurones from injury induced by oxygen-glucosedeprivation; phenytoin became effective only when combined with glutamate receptor blockade (Lynch et al., 1995). Several studies indicate that phenytoin minimizes residual energy demand during ischaemia, delays anoxic depolarization, and thereby increases tolerance (Artru and Michenfelder, 1980; Watson and Lanthorn, 1995). Phenytoin pretreatment (200 mg/kg) significantly protected hippocampal CAI neurones in gerbils subjected to 5 minutes of forebrain ischaemia produced by bilateral carotid artery occlusion under various experimental conditions (Taft et al., 1989),but Deshpande and Wieloch (1986) failed to demonstrate protection by phenytoin in a rat model of global ischaemia. A much lower dose (15 mg/kg) of this drug attenuated both necrosis and neurological deficits in the rabbit brain subjected to transient global ischaemia (Cullen etal., 1979). Phenytoin and carbamazepine were also effective in models of focal cerebral ischaemia (for review, see Urenjak and Obrenovitch, 1996). For example, when administered 30 minutes and 24.5 hours after insult, phenytoin (2 X 100 mg/kg) and carbamazepine (2 X 50 mg/kg) reduced the infarct size produced by MCA occlusion in rats by 40 and 24%, respectively (Rataud et al., 1994).

6.4 Neuroprotectiveagents acting on Na' channels 6.4. I

hMOTRIGINE, BwI003C87 AND

BW6 I9C89

In addition to their anticonvulsant action, lamotrigine and its derivatives BW1003C87 and BW6 19C89 protect the brain against ischaemic and traumatic injury. In the gerbil model of global ischaemia, high doses of lamotrigine (30-50 mg/kg; i.e. approximately


115

given before and shortly after carotid occlusion, pro6 X the anticonvulsant ED50) tected against behavioural deficits and reduced hippocampal damage (Wiard et al., 1995).With permanent MCA occlusion in rats, lamotrigine (20 mg/kg) administered intravenously over 10 minutes immediately after ischaemia onset reduced the volume of total infarct by 3 1YO and cortical infarct volume by 52% (Smith and Meldrum, 1995). The protective actions of BW1003C87 and BW619C89 have been studied extensively in models of global ischaemia (Gilland et al., 1994; Lekieffre and Meldrum, 1993; Meldrum et al., 1992)and focal ischaemia (Graham et al., 1993, 1994b; Leach et al., 1993). Both analogues appear to be more potent neuroprotectors than lamotrigine. For example, at 20 mg/kg (i.v.; 5 minutes after MCA occlusion) BW619C89 reduced total infarct volume by 57'10, and protected even the basal ganglia, a region reputedly refractory to protection in this model (Leach et al., 1993).In the optic nerve preparation, BW6 19C89 (1-1 00 mM) dose-dependently prevented the axonopathy induced by oxygen and glucose deprivation, without impairing axonal conduction (Garthwaite et al., 1995).BW619C89 is currently in phase 11 clinical trails for the treatment of stroke and traumatic brain injury. Loading doses of up to 2.0 mg/kg, followed by 1.O mg/kg every 8 hours as repeated bolus or continuous infusion over 72 hours, have been well tolerated after acute stroke (Muir and Lees, 1995). Lamotrigine and its derivatives are often referred to as (presynaptic) glutamate release inhibitors because they inhibit veratridine-induced glutamate release (Meldrum et al., 1992; Leach et al., 1993; Gilland et al., 1994; Okiyama etal., 1995), but this hypothetical mechanism is inconsistent with a number of important experimental findings on the pathophysiology of cerebral ischaemia (Obrenovitch and Richards, 1995), and strong evidence suggests that their primary, direct action at therapeutic doses is usedependent inhibition of Na+ channels (Xle, X. et d., 1995). In binding experiments carried out with rat brain synaptosomes, lamotrigine concentration-dependently inhibited the binding of batrachotoxinin (BTX-B), a specific ligand for Na+ channel neurotoxin binding site 2 (Cheung et al., 1992), and BW1003C87 was also mentioned as sharing this property (M. Leach, personal communication to Graham et al., 1994a). Lamotrigine and BW6 19C89 inhibited veratridine-evoked neurotransmitter release, but not that produced by Kf (Leach et al., 1986, 1991). Using mouse neuroblastoma cells, Lang et al. (1993) found that 100 mM lamotrigine, as well as phenytoin and carbamazepine, produced a use-dependent inhibition of Na' channels, shifting the voltagedependency of steady-state inactivation towards more negative potentials by 7-1 5 mV, and slowing the rate of recovery from inactivation. Whole-cell voltage clamp recordings of recombinant rat brain Na' channels expressed in Chinese hamster ovary (CHO)cells have suggested recently that BW6 19C89 has similar actions, and may be more potent than lamotrigine p i e , X. et al., 1995; Xie and Garthwaite, 1995).

6.4.2 RILUZOLE

Repeated doses of riluzole (4-8 mg/kg) significantly reduced degeneration of hippocampal CAI pyramidal cells, prevented memory loss (Malgouris et al., 1989), and

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T.P. OBRENOVITCH

improved the EEG (Pratt et al., 1992)in gerbils subjected to transient bilateral carotid artery occlusion. In the rat MCA occlusion model, riluzole reduced the volume of infarcted cortex (Pratt et al., 1992; Wahl et al., 1993; Rataud et al., 1994)with an efficacy similar to that of lamotrigine and carbamazepine (Rataud et al., 1994).However, it failed to improve neurological and memory deficits in this model (Wahl et al., 1993) and, as other drugs, did not reduce the striatal lesion (Pratt et al., 1992). The actions of riluzole against ischaemia-induced brain damage (Malgouris et al., 1989) and amyotrophic lateral sclerosis (ALS) (Bensimon et al., 1994) are often attributed to an antiglutamate action because of unusual effects on glutamatergic transmission (for review, see Urenjak and Obrenovitch, 1996). Riluzole also inhibited glycinergic inhibitory postsynaptic currents in hypoglossal motoneurones (Umemiya and Berger, 1995). However, this drug clearly acts on Na' channels at therapeutic concentrations. Riluzole displaced BTX-B in binding studies, and inhibited both acetylcholine release (Hays et al., 1991b) and intracellular Ca2' loading evoked by veratridine of 0.3 1 mM; Hubert et al., 1994).In rat cortical slices, riluzole suppressed ['*C]-guanidine uptake (an index of Na' flux) with an ICs0 of 4.1 mM (versus 23 mM for phenytoin) (Hays et al., 1994).Voltage-clamp studies confirmed the interaction of riluzole with voltage-gated Na' channels. For example, in isolated myelinated nerve fibres of the frog, riluzole was a highly specific blocker of inactivated Na' channels, 300 times more effectively on these channels than on K' or resting Na' channels (Benoit and Escande, 1991). Furthermore, in cultured neurones, riluzole (1-30 mM) produced at 5-30 mV negative shift of the Na' current steady-state inactivation curve, with modest effects on Na' channel activation and recovery from inactivation, and inhibition of Na' currents was frequency-dependent only at activation frequencies exceeding 30 Hz (Randle et al., 1994). These actions were confirmed with rat brain IIA Na' channels expressed in oocytes (Hebert et al., 1994). RP66055 (3-{ 2- [4-(4-fluorophenyl)-1-piperazinyl]ethyl}-2-imino-6-trifluoromethoxybenzothiazoline),a riluzole derivative, is also a potent neuroprotective agent in rodent models of ischaemia (Stutzmann et al., 1993; Rataud et al., 1994). So far, this compound is described as an Na' channel blocker because binding assays only revealed an affinity for voltage-gated Na' channels.

6.4.3

CA"

CHANNEL MODULATORS WITH ACTIONS ON

NA' CHANNELS

A number of drugs, classified as Ca2' channel blockers with anti-ischaemia properties, also interact strongly with Na' channels. Typical examples of such compounds are diphenylpiperazine analogues (flunarizine, lifarizine, and KB-2 796) and benzothiazole derivatives (R56865, lubeluzole and sabeluzole) (for review, see Urenjak and Obrenovitch, 1996). This section focuses on lifarizine and lubeluzole because they are in clinical trials (Muir and Lees, 1995). Lifarizine protected the gerbil striatum against ischaemia-induced dopamine depletion (Brown et al., 1993), and several brain regions against damage produced by transient forebrain or global cerebral ischaemia in rats (Alps et al., 1990, 1995;


117

McBean et al., 1995a,b).With permanent MCA occlusion in cats, 2 mg/kg lifarizine administered intravenously after ischaemia onset, followed by infusion at 0.7 mg/kg per hour over 12 hours, reduced the size ofthe infarct by 70%. The highest dose tested in this study (50 mg/kg intravenously, with 17.5 mg/kg per hour maintenance dose) was able to reduce the infarct size by as much as 88% (Kucharczyk et al., 1991). Monitoring of the developing lesions using magnetic resonance imaging (MRI) and spectroscopy also suggested that energy levels were preserved, and tissue acidosis and oedema reduced in animals treated with lifarizine (Kucharczyk et al., 1991). Lifarizine was introduced initially as a Ca'+-modulator (Alps et al., 1990), but ligand binding and functional assays have shown marked interaction with voltagegated Nat channels. Lifarizine displaced BTX-B from rat cortical membranes with an IC,,) of 55 nM (MacKinnon et al., 1995), and protected cultured cortical neurones against veratridine neurotoxicity with an IC5, of 0.4 mM (May et al., 1995). Patchclamp studies in mouse and human neuroblastoma cells have demonstrated that lifarizine is a voltage-dependent inhibitor of Na' currents, subsequent to interaction with the inactivated state ofthe channel (Brown et al., 1994; McGivern etal., 1995).It is interesting to note that, in comparison to the anticonvulsants phenytoin and lamotrigine, the lifarizine block of Na' channels showed limited use- and frequencydependence, which suggests that this property may be essential for anti-epileptic activity but not for neuroprotection. Lubeluzole (R87926; the S-isomer of a 3,4-difluoro-benzothiazole)is a close structural analogue of R56865. When administered as a single intravenous bolus, 5 minutes after induction of photochemical infarcts in the rat sensorimotor cortex, it protected neurological function with an ED50 of 0.16 mg/kg @e Ryck et al., 1994). Protection remained effective when treatment was delayed for up to 1 hour after infarct induction. With a different regimen (intravenous bolus of 0.3 1 mg/kg starting 5 minutes post infarct, followed by a 1 hour infusion of 0.3 1 or 0.63 mglkg), the infarct volume was reduced by around 22-24% (De Ryck et al., 1994). In the same model, lubeluzole prevented the slow rise in extracellular glutamate (Scheller et al., 1995)and delayed functional alterations (Buchkremer-Ratzmann and Witte, 1995) in the peninfarct region. Prolonged lubeluzole pretreatment also protected cultured hippocampal neurones against glutamate toxicity with an ICSOof 32 nM (Lesage et al., 1995). An interesting property of this drug is the much lower cerebroprotective potency of its (-)-R-isomer (R091154) as this may help to clan@ the mechanism of action (see below). A phase I1 trial has investigated the effect of 5-day treatment with lubeluzole (10 or 20 mg per day, versus placebo) on neurological and functional recovery, and mortality in 232 ischaemic stroke patients. The low dose of lubeluzole showed a trend for more favourable outcome than placebo for all the efficacy parameters considered (Diener et al., 1995). Phase I11 trials are planned in order to confirm this therapeutic effect. The primary molecular target(s) of this compound remain to be clarified (Lesageet al., 1995; Osikowska-Everset al., 1995).Lubeluzole displaced BTX-B (0.1-1 mM), reduced veratridine-induced Na' influx into rat synaptosomes (IC50 1.04 mM), and inhibited voltage-gated Na' currents in voltage clamped neurones (K& 2.4 mM). Lubeluzole also had moderate agonist affinity for the 5HTIAreceptor, sigma site 1 and

118

T.F? OBRENOVITCH

2, and the histamine H I receptor. Finally, lubeluzole inhibited glutamate-induced cGMP elevation, but not the corresponding rise in intracellular Ca2', suggesting an action on glutamate-activated nitric oxide production. Among all these actions, only the last was stereospecific, that is, as for neuroprotection, lubeluzole was more effective than its (-)-R-isomer.However, the fact that both lubeluzole and its R-isomer interacted with Na' currents measured in whole-cell voltage-clamped neurones (Osikowska et al., 1995), but that lubeluzole was a much more potent neuroprotector against photochemical infarcts, does not rule out the hypothesis that the antiischaemic action of lubeluzole results from an action on Na' channels. The differential neuroprotective potency of the two isomers remains to be confirmed in stroke models which do not rely on endothelial damage, and lubeluzole may be a more potent inhibitor of sustained or persisting Na' currents than its R-isomer (see section 6.3.1). 6.4.4 PO85639 PD85,639 belongs to a novel series of phenylacetamides structurally related to both local anaesthetics and phenytoin (Thomsen et al., 1993; Roufos et al., 1994).Th'is compound may have some neuroprotective potential, as it inhibited hypoxia-induced LHD release from cultured rat brain neurones with an of 89 mM (Roufos et al., 1994). PD85,639 interacts strongly with voltage-gated Na' channels: (i) it displaces BTX-B binding to rat neocortical membranes with a Ki of 0.26 mM (Roufos et al., 1994)and binds specifically to the local anaesthetic receptor (Thompsen et al., 1993); (ii) it inhibits veratridine-stimulated influx of [I4C]guanidine and Na' (Hays et al., 1991a; Roufos et al., 1994);and (iii)it protects rat brain neuronal cell cultures against veratridine toxicity = 5 mM; Roufos et al., 1994).Voltage-clamp recordings from CHO cells expressing brain type IIA Na' channel and dissociated rat brain neurones have confirmed that PD85,639 strongly attenuated Na' currents when applied either in the external bath or in the internal pipette solution, with properties close to those of local anaesthetics (Ragsdale et al., 1993).

6.5 Clinical relevance and suitability 6.5. I CLINICAL RELEVANCE The elements outlined in sections 6.2 and 6.3 support the rationale of down-regulating voltage-gated Na' sodium channels as a neuroprotective intervention during or preceding ischaemia (Figure 1).It also appears possible to interact selectively with specific Na' channels, or Na+ channel states, to provide protection without complete blockade of neuronal function. As such, this strategy is relevant to patients at high risk of cerebral ischaemia (for example, those undergoing cardiopulmonary bypass,


119

Potential actions

Reduction of residual energy demand Reduction of slowly inactivating (i.e. after cessation of functional or persistent Na' currents activity) +

Delayed anoxic depolarization

\

-

Reduction of intracellular Na' loadinc

/

Resultant beneficial effects

I Preservation of

I I

Ca2+homeostasis Acid-base regulation Neurotransmitter (alutamate)- uptake . Cell volume regutaiion

FIGURE1 Nat channel down-modulation during ischaemia: potential actions and resulting beneficial effects on vital transmembrane processes (for details, see Urenjak & Obrenovitch, 1996). Potential benefits

Reduced energy demand and preservation of ionic gradients Focal ischaemia: Enhanced tolerance to recurrent spreading depression Global, transient ischaemia:

Increased tolerance to persistent enhancement of synaptic efficiency Effects on altered voltage-gated Na+channels

FIGURE2 Potential benefits of postischaemic modulation of Na' channels (for details, see Urenjak & Obrenovitch, 1996).

carotid endarterectomy or aneurysm surgery),and represents a potentially major clinical application (Fisher et al., 1994). In addition, a number of experimental findings strongly suggest that downmodulation of Na+ channels remains beneficial even when it is delayed, that is, after occlusion of a major cerebral artery (stroke) or following transient global ischaemia (Urenjak and Obrenovitch, 1996). Therefore, this therapeutic strategy may remain beneficial in stroke patients who are admitted to hospital several hours after the onset of the symptoms (Hantson et al., 1994).In these situations, the basis for protection may still be linked, at least partly, to reduced energy demand and preservation of ionic gradients (Figure 2). In delayed interventions after stroke, down-modulation of Na' channels may reduce to some extent the occurrence of recurrent spreading depression (SD;that is,

120

T.P. OBRENOVITCH

transient suppression of electrical activity with membrane depolarization propagating across grey matter regions), together with enhancing the tolerance of the tissue to this deleterious phenomenon (Urenjak and Obrenovitch, 1996). Indeed, experimental studies of focal ischaemia have established that recurrent SD propagates from the ischaemic core to adjacent regions, contributing to the development of tissue damage (for review, see Hossmann, 1994; Obrenovitch, 1995a). With delayed neuronal death subsequent to transient global ischaemia, modulation of Na+ channels may alleviate the effects of intracellular Ca2+and Na+ overload, which could result from a variety of potentially deleterious abnormalities. Current hypotheses include delayed excessive release of excitatory amino acids (Szatkowski and Attwell, 1994; see, however, Obrenovitch and Richards, 1995), synchronous and long-lasting enhancement of the efficiency of excitatory synapses (Crtpel et al., 1993; Gozlan et al., 1994; Obrenovitch and Richards, 1995) and persistent alteration of voltage-gated Na' channels (Urenjak and Obrenovitch, 1996).

6.5.2

POTENTIAL ADVERSE EFFECTS

One potential problem of drugs interacting with voltage-gated Na' channels may be cardiovascular effects. Several Na+ channel-blocking compounds have significant cardiac effects (for example, lidocaine; Artru et al., 199l), particularly prolongation of the QTc interval of the ECG (for example, liifarizine, lubeluzole)with the risk of initiating arrhythmia. This peripheral action may limit dosing or restrict clinical use (Muir and Lees, 1995). The worsening of outcome previously observed with nimodipine in clinical trials could have been linked to hypotension, and similar trends were noted with the highest dose of lifarizine tested in clinical trials. The loss of neuroprotective efficacy with lifarizine in a photochemical rat model of focal cerebral ischaemia also appeared to be associated with the hypotensive action of the compound at high doses (1 mg/kg i.v.) (McBean et al., 199513).

6.6 K+ channel openers: introduction A heterogeneous array of K' channels has been identified in the CNS, which remain classified according to their electrophysiologicalproperties because selective drugs are still lacking. Some K+ channels are voltage-gated (for example, delayed rectifiers); others are voltage-dependent and activated by increases in cytoplasmic Ca2+(Ca2+activated K+ channels) or Na' (Na+-activatedK' channels); and others are activated when cellular ATP decreases (ATP-sensitive K+ channels) (HaUiwell, 1990). ATPsenstive K+ channels have recently attracted considerable interest because they link bioenergetic metabolism to membrane excitability They are particularly abundant in endocrine cells, smooth muscle and skeletal muscle cells, as well as in neurones (Lazdunski, 1994).ATP-sensitive K' channels are inhibited by antidiabetic sulphon-


121

ylureas (e.g. glibenclamide, tolbutamide) and activated by ATP-sensitive K+ channel openers such as cromakalim, nicorandil and pinacidil (Edwards and Weston, 1993). The equilibrium membrane potential for K+ is approximately -90 mV under physiological conditions, because the resting cell membrane is selectively permeable to K+. For this reason, activation of K+ channels opposes depolarization, or causes repolarization or hyperpolarization. In this sense, K+ channel activation results in an outward current which tends to reduce membrane excitability, and therefore downregulates neuronal activity (Wann, 1993).

6.7 Effects of ischaemia on K+ channels The first functional change to occur immediately after the onset of ischaemia in the mammalian brain is a temporary increase in EEG activity, which lasts for only a few seconds (Astrup et al., 1980; Hanssen and Nedergaard, 1988) and may correspond to the early, brief and small depolarization commonly observed with in uitro preparations exposed to anoxia (Hansen et al., 1982; Fujiwara et al., 1987; Leblond and Kmjevic, 1989).This short period of activation is followed by functional loss, in which the EEG becomes silent and evoked potentials are abolished. If ischaemia is severe and sustained, anoxic depolarization (a sudden increase in the ionic permeability of the plasma membrane) occurs within a few minutes. The contribution of K+ channel opening to changes in function and extracellular K+ during these various phases is discussed below.

6.7. I

INITIAL CHANGES IN K+ CONDUCTANCE

In chloral hydrate anaesthetized rats, Zetterstrom et al. (1995) have shown that repeated periods of anoxia for 30 seconds, which transiently reduced arterial PO2by 80%, produced small, reversible increases of extracellular K+ concentration ([1K+],) in the dorsal hippocampus. These changes in [K'Ie were markedly inhibited by pretreatment with 1 mg/kg of 4-aminopyridine (4-AP), but were unaffected by the inhibitor ofATP-sensitive K+channels, gliquidone. In addition, it is unlikely that ATP levels were reduced significantly by such brief anoxic challenges (Lowry et al., 1964; Obrenovitch et al., 1990).As 4-Al? inhibits voltage-dependent K+channels, these findings suggest that the very early K+ efflwr may be triggered by the brief, initial increase in neuronal activity outlined above.

6.7.2 CHANGES IN K+ CONDUCTANCE ASSOCIATED WITH EARLY FUNCTIONAL

LOSS

In zntro electrophysiologicalstudies have shown repeatedly that the early functional loss of brain function is associated with hyperpolarization of neurones subsequent to

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T.P. OBRENOVITCH

increased K+ conductance (Hansen et al., 1982; Fujiwara et al., 1987; Leblond and Krnjevic, 1989).This event, superimposed onto progressive impairment of Na+-K+ ATPase, may underlie the slow and gradual increase in E'lewhich precedes anoxic depolarization (Sick et al., 1982; Hansen, 1985). The experimental findings outlined below suggest that this increase in K+ conductance may originate from the opening ofCa2+-dependentK+ channels subsequent to a rise in intracellular free Ca", and/or ATP-sensitive K+ channels. In sensory neurones isolated from mouse dorsal root ganglia, an increased K+ conductance developed within seconds of metabolic block, hyperpolarizing neurones and shunting action potentials (Duchen, 1990). This effect was reduced by incubation of cells in Ca2+-freesolutions or by increasing the Ca2+buffering capacity of the pipettefilling solutions used for patch-clamp. Ionomycine, a Ca2+- H + ionophore, which should raise the intracellular concentration of free Ca2+ ([Ca"]i) without directly affecting energy availability, produced a very similar increase in K+ conductance. In contrast, inclusion of ATP or an ATP-regenerating system in the pipette-filling solution used for patch clamp did not alter the increase in K+ conductance subsequent to metabolic inhibition. These results suggested that, in these neurones, the early increase in K+ conductance subsequent to energy metabolism deficiency was Ca2+ dependent (that is, presumably due to opening of Ca2+-activatedK + channels), and did not involve ATP-sensitive Kf channels (Duchen, 1990). A number of other observations support the hypothesis that, in some preparations, ATP-sensitive K+ channels may not contribute to the increase in Kf conductance associated with early functional loss: (i) two well-established K+ antagonists of ATPsensitive K+ channels (tolbutamide and glibenclamide) failed to reduce the effects of brief periods of anoxia on CAI neurones in rat hippocampal slices (Leblond and Krnjevic, 1989);(ii) the initial increase in pC'Ie did not correlate with changes in brain ATP concentrations produced by cardiac arrest in rats (Katsura et al., 1992);and (iii) the level ofATP does not decrease immediately subsequent to ischaemia onset, but is maintained at the expense of phosphocreatine (Obrenovitch et al., 1988). In an isolated rat brain model, 30% of tissue ATP (4.3 mM) was still present when depolarization occurred (Obrenovitch et al., 1990), that is, a level considerably above the 1 mM level where isolated ATP-sensitive channels are activated (Ashcroft, 1988). With regard to arguments (ii) and (iii) outlined above, it is important to note that ATP is not distributed evenly inside the cell and, therefore, the average ATP (tissue) level may not reflect marked local changes at critical intracellular sites. In addition, some ATP-sensitive Kf channels can be activated directly by a variety of hormones and neurotransmitters, via direct G-protein interactions, or via CAMP and protein kinase A (Kirsch et al., 1990; Honor6 and Lazdunski, 1993) (see section 6.7.4). In contrast to the above findings, Mourre et al. (1989) showed that the rat hippocampal CA3 region is rich in binding sites for glibenclamide, and that this drug blocked the early hyperpolarization produced in these neurones by hypoxia. In the substantia nigra, the brain region richest in binding sites for ATP-sensitive K', treatment with cyanide to mimic ischaemia caused the activation of a K+ current in a subpopulation of neurones, and this response was abolished by the sulphonylurea


123

tolbutamide (Murphy and Greenfield, 1991). These studies and some of the data outlined below (section 6.7.3, below) suggest that, at least in some neurones, ATPsensitive K+ channels may open early in ischaemia.

6.7.3

CHANGES IN

K+ CONDUCTANCE AND ANOXIC DEPOLARIZATION

In CA3 neurones of rat hippocampal slices, activators of ATP-sensitive K+ channels (diazoxide, somatostatin and galanine) reduced depolarization produced by brief anoxic episodes (Ben-Ari and Lazdunski, 1989; Ben-Ari et al. 1990), whereas their blockade with glibenclamide facilitated this event (Ben-Ari, 1989, 1990).With regard to p'],, glibenclamide blockade of ATP-sensitive K+ channels halved the increase in [K'Ie produced by anoxia in brain stem slices from adult rats (Jiang et al., 1992). These findings were confirmed recently in the cortex of rats subjected to complete ischaemia by cardiac arrest. Glibenclamide significantly reduced the [K+],threshold at which anoxic depolarization occurred (that is, it facilitated this event), as well as the [K'], increase measured 5 minutes after anoxic depolarization (Xie etal., 1995).Taken together, these data support the hypothesis that the opening ofATP-sensitive K + channels in anoxdischaemic conditions contributes to reduce neuronal excitability and dampens anoxic depolarization. However, cromakalin (an opener of ATP-sensitive K+ channels; Schmid-Antomarchi et al., 1990) altered neither anoxic depolarization nor [K'], increase following cardiac arrest, suggesting that thresholds (time and [KfIe) K+ channels may be fully open when ischaemia is complete (Xie et al., 1995).

6.7.4 ADENOSINE, INCREASED K+ CONDUCTANCE AND

NEUROPROTECTION

Recent evidence strongly suggests that adenosine controls, or at least influences, the opening of K+ channels in ischaemia. Pretreatment with the adenosine A1 receptor antagonists theophylline (100 mM) or 8-cylopentyl- 1,3-dipropylxanthine (DPCPX, 0.1 mM) significantly reduced the early rise in [K'], in the CAI pyramidal cell layer of rat hippocampal slices produced by anoxia alone (Croning et al., 1994) or superimposed on to glucose deprivation (Croning et al., 1995).Unexpectedly, in contrast to what was obtained when blocking K+ channels, adenosine receptor antagonists delayed the onset of anoxic depolarization (Croning et al., 1995). Preconditioning with either sublethal ischaemia (Kiriino et al., 1991; Heurtaux et al., 1995) or SD (Kawahara et al., 1995), both ofwhich induce the expression of early genes (c-fos,c-jun, and so on) and heat shock protein genes (Herdegen et al., 1993), is known to render the brain more resistant to a subsequent ischaemic insult. Heurtaux et al. (1 995) recently proposed that the mechanism behind this increased tolerance to ischaemia involved the following cascade of events: liberation of adenosine, stimulation of adenosine A, receptors, and, via these receptors, the opening of ATP-sensitive K+ channels. Their hypothesis is supported by the following findings: (i) extracellular levels of adenosine increase early after ischaemia onset, presumably because of the

124

T.F! OBRENOVITCH

breakdown of intracellular ATP, and/or its direct release as a neuromodulator (Obrenovitch and Richards, 1995); (2) pretreatment with either the selective highaffinity A, antagonist DPCPX (1 mg/kg) or glibenclamide blocked the beneficial effects of ischaemic preconditioning (Heurtaux et al., 1995); and (iii)the Al adenosine receptor agonist A@-cyclopentyladenosine (CPA) and the Kt channel opener levoromakalin (i.e. the single enantiomer of the racemate cromakalin) inhibited expression of the heat shock protein HSP70 mRNA in the CA1 region, and this inhibitory effect was blocked by glibenclamide in both cases (Heurtaux et al., 1995).

6.8 Rationale for opening K+ channels to protect neumnes against ischaemia 6.8. I

POTENTIAL BENEFICIAL ACTIONS OF

K+ CHANNEL OPENERS (FIGURE3)

The function of the early opening of K+ channels (see section 6.7, above) may be to reduce neuronal activity by decreasing membrane excitability and stimulus-coupled transmitter release from presynaptic terminals, and thus to complement the downregulation of voltage-gated Na' channels (section 6.2) in reducing the energy consumption of neurones and promoting their survival (Mourre et al., 1990; Miller, 1990; Xie, Y. et al., 1995).An alternative benefit may be linked to reduced ischaemiainduced excitotoxicity, either by inhibition of synaptic glutamate release (Heurtaux et aL, 1993; see, however, Obrenovitch, 1995b) or postsynaptic hyperpolarization (e.g. strengthening of the NMDA-receptor M$+ block). Whatever the mechanism(s), the potential beneficial effect of the activation ofATP-sensitive K+ channels was demonstrated by the finding that neurones from rat brain stem slices failed to recover depolarization after 5-6 minutes' anoxia when glibenclamide was present in the lntracellular ATP (-1

Extracellular adenosine (+)

Activation of ATP-sensitive K+ channels

c

Hyperpolarization

.)

Reduced excitability Inhibition of neurotransmitter release (glutamate, GABA) Postsynaptic hyperpolarization -. Reduced excitotoxicity Vasodilation of cerebral vessels

FIGURE3 Effects of ischaemia on ATP-sensitive K+ channels, and the resulting consequences on neuronal excitability, neurotransmitter release, and cerebral vessels.


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incubation medium, but repolarized and recovered when not exposed to this agent (Jiang et al., 1992). ATP-sensitive K+ channel openers may also be beneficial through vascular effects (Standen et al., 1989; Edwards and Weston, 1990). Several antihypertensive agents used in clinical practice, including pinacidil, minoxidil sulfate and diazoxide, operate by opening plasmalemnal K+ channels in vascular smooth muscle. They are direct arterial vasodilators and their hypotensive effects result primarily from a fall in total peripheral resistance, but they also act on cerebral vessels. In vitro, nicorandil, pinacidil and lemakalim vasodilated cerebral arteries from dogs (Zhang et al., 1992). In viuo, topical application of nicorandil and cromakalim concentration-dependently dilated both pial arterioles and veinules, and this effect was reduced by glibenclamide (Ishiyama et al., 1994).

6.8.2

POTENTIAL DELETERIOUS EFFECTS OF INCREASED [K+IE

Paradoxically, promoting neuronal efflux of intracellular K+ may not be exclusively beneficial. As proposed by Katsura et al. (1982), high [K'Ie may stimulate the metabolism of glial cells and thus reduce overall energy availability (Salem et al., 1975; Gardner-Medwin, 1981). Exacerbating high extracellular K+ could also be detrimental in view of its potential negative effect on glutamate uptake (Szatkowski and Attwell, 1994). As ATP-sensitive K+ channels have been associated with the control of presynaptic neurotransmitter release, with activation of these channels leading to inhibition of release, K+ channel openers may be protective by repressing excitatory glutamatergic systems; equally, they could be detrimental by decreasing the inhibitory capacity of GABAergic pathways (see chapter 10).Indeed, in substantia nigra slices, ATP-sensitive K+ channels have been found to play a central role in the control of GABA release (Amoroso et al., 1990), and ATP-sensitive K+ channel openers concentrationdependently blocked the release of this inhibitory neurotransmitter (SchmidtAntomarchi et al., 1990).

6.8.3 UNRESOLVED ISSUES A number of important issues need to be resolved before K+ channel openers can enter clinical trials for the prevention or treatment of cerebral ischaemia. Brain tissue penetration is one potential limitation; protective effects have only been obtained in Vzuo with K+ channel openers applied directly to the brain (Xie, Y. et al., 1995; Heurtaux et al., 1993, 1995). Neuronal selectivity is another requirement to avoid the unwanted peripheral effects of ATP-sensitive K+ channels, especially on pancreatic p-cells as this would lead to reduced insulin secretion, increased blood glucose (Petersen and Findlay, 1987), and potentially damaging exacerbated acidosis of the ischaemic brain (Siesjij et al., 1993). As K+ channel openers shorten the action

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potential duration, another potential adverse effect of this class of drugs is myocardial arrhythmia. Clinical trials with ATP-sensitive K+ channel openers tested as antihypertensives have not reported pro-arrhythmic effects (e.g. levcromakalim; Cavero and Premmereur, 1994), but neuroprotection may require a much higher dosage. Finally, it remains to be verified whether K+ channel openers are effective when administered after the onset of ischaemia. Testing whether K+ channel openers inhibit recurrent SD in focal ischaemia would be relevant because blocking K+ conductance with 4AP or tetraethylammonium (TEA) favoured both spontaneous and hypoxia-induced SD in hippocampal slices (Aitken et al., 1991; Psarropoulou and Avoli, 1993).

6.9 K+ channel openers and neuroprotection in ischaemia: experimental evidence A number of studies support the hypothesis that K+ channel openers may strengthen the inherent protective action of increased K + conductance in ischaemia. Pretreatment with nicorandil(30-100 mg/kg), a hybrid vasodilator with a dual mechanism of action as a K+ channel opener and a nitrate, improved the recovery of reflex potentials after spinal cord ischaemia in cats (Suzuki et al., 1995).This beneficial action of nicorandil, which was shared by pinacidil (100 mg/kg) (another K+ channel opener), was abolished by co-administration of 3 mg/kg glibenclamide. The fact that none of the K+ channel openers improved the spinal cord blood flow during ischaemia and reperfusion strongly suggests a direct neuroprotective effect of drug-induced opening of ATP-sensitive K+ channels (Suzuki et al., 1995). Nicorandil (10 and 20 mg/kg per minute infused intravenouslyfor 60 minutes before ischaemia)also showed a direct protective effect against the dysfunction of the central vagal baroreflex system following transient global cerebral ischaemia in dogs (Kurihara et al., 1993). The ATP-sensitive K+ channel openers cromakalin, nicorandd and pinacidd, administered intracerebroventricularly both before ischaemia and during the reperfusion period, totally blocked the expression of c-jios, c-jun, heat shock protein, and amyloid f3-proteinprecusor induced in the rat hippocampus by 20 minutes of forebrain ischaemia. These drugs also markedly protected against delayed neuronal death (Heurtaux et al., 1993). The mechanism of neuroprotection presumably involved opening of ATP-sensitive K+ channels, since glipizide, a specific blocker of that type of channel, abolished the beneficial effects of K+ channel openers (Heurtaux et al., 1993). Levoromakalin (10 nmol administered intracerebroventricularly 30 minutes before the induction of cerebral ischaemia and once each day during recirculation) protected hippocampal CAI neurones against delayed neuronal death produced by 6 minutes of forebrain ischaemia in rats (Heurtaux et al., 1995). It is relevant to recall here the finding of Leblond and Krnjevic (1 989) that inhibition of ATP-sensitive K+ channels did not reduce the effects of brief periods of anoxia on CAI neurones. This in uitro data suggests that the neuroprotective effects reported


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by Heurtaux and co-workers may not be linked to an action of the K+ channel openers early in ischaemia, but rather after occurrence of anoxic depolarization or during recirculation. Finally, the anti-ischaemic potential of K+ channel openers has been substantiated in experimental models of myocardial ischaemia. For example, aprikalim (RP5289 1) significantly reduced ischaemic damage to the heart (Grover et al., 1990; Auchampach et al., 199l),presumably by inducing an earlier termination of the action potential, thus shortening the action potential duration and reducing the action potential frequency.

6. I 0 Concluding remarks Down-regulation of voltage-gated Na+ channels is an inherent mechanism to reduce the energy expenditure of neurones and favour their survival during periods of anoxia or energy metabolism deficiency. The fact that a number of neuroprotective drugs, which are structurally unrelated, share the property of down-modulating Na+ channels, indicates that selective modulation of these channels is a valid strategy for the protection of the brain against ischaemic damage. In addition, a number of findings suggest that neuroprotection can be achieved without conspicuous adverse effects on the normal function of the brain and heart. The K+ channels constitute another exciting target for neuroprotection, but a number of issues must be resolved before K+ channel openers can enter clinical trials for the treatment or prevention of stroke. Finally, a note of caution. Voltage-gated Na+, Ca", and K+ channels are members of a related-gene family (for example, 55% sequence homology remains between the principal subunits of Na+ and Ca2+channels), and they share strong functional analogies such as voltage-dependent activation and inactivation. This may explain why a number of drugs are only partially selective to a given cation channel. In this chapter, emphasis is placed on the primary interaction of drugs with either Na+ or K+ channels. However, the contribution of secondary actions on Ca2+channels (e.g. lifarizine; Spedding et al., 1995; Sheridan, 1995) or of other pharmacological properties (for example, actions of flunarizine on adenosine, of riluzole on the NMDA-receptor, and of lubeluzole on the nitroc oxide pathway) to neuroprotection cannot be ruled out.

Acknowledgements

The work of the author's team on cerebral ischaemia and neuroprotection was supported by The Medical Council, The Wellcome Trust, The Brain Research Trust, The Mihara Trust (Tokyo),Pfizer Central Research (UK),and Johnson and Johnson (UK).The author thanks Dr D.A. Richards (Department of Pharmacology, School of Pharmacy, London) for his editorial comments.

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Leach, MJ., Swan,J.H., Eisenthal, D., Dopson, M. & Nobbs, M. (1993)BW619C89, a glutamate release inhibitor, protects against focal cerebral ischaemic damage. Stroke 24, 1063-1067. Leblond,J. & Krnjevic, K. (1989)Hypoxic changes in hippocampal neurones. 3; Nmroflhyd. 62, 1-14. Lekieffre, D. & Meldrum, B.S. (1993)The pyrimidine-derivative,BW1003C87, protects CAI and striatal neurones following transient severe forebrain ischaemia in rats. A microdialysis and histological study. Neuroscience 56, 93-99. Lesage, AS., De Loore, K.L., Osikowska-Evers, B., Peeters, L. & Leysen, J.E. (1995) Lubeluzole,a novel neuroprotectant, inhibits the glutamate-activatedNOS pathway3 Cereb. Blood Flow Metab. 15 (Suppl. l), S432. Lowry, O.H., Passonneau,J.K, Hasselberger, EK. & Schultz, D.W. (1964) Effect of ischaemia on known substrates and cofactors of the glycolytic pathway in brain. 3; Biol. C h . 239, 18-30. Lucas, L.F., West, C.A., Rigor, B.M. & Schurr, A. (1989)Protection against cerebral hypoxia by local anesthetics: a study using brain slices.3; Nmroxci. Meth. 28, 47-50. Lynch,JJ.111, Yu, S.P., Canzoniero, L.M.T., Sensi, S.L. & Choi, D.W. (1995)Sodium channel blockers reduce oxygen-glucose deprivation-induced cortical neuronal injury when combined with glutamate receptor antagonists.3; Pharmacol. Exfl. Ther. 273,554-560. Lysko, P.G., Yue, T.L., Gu,J.L., Webb, C.L. & Feuerstein, G. (1993)Neuroprotectiveeffects of tetrodotoxin in cultured cerebellar neurones and in gerbil global brain ischaemia. SOG. Neurosci. Abstr. 19, 286. Mackinnon, A.C., Wyatt, K.M., McGivern,J.G., Sheridan, R.D. &Brown, C.M. (1995) [3H]lifarizine, a high A n i t y probe for inactivated sodium channels. Br. 3; Pharmacol. 115, 1103-1 109. Malgouris, C., Bardot, E, Daniel, M., Pellis, E, Rataud, J., Uzan, A., Blanchard, J.-C. & Laduron, P.M. (1989) Riluzole, a novel anti-glutamate, prevents memory loss and hippocampal neuronal damage in ischaemic gerbils. 3; Neurosci. 9, 3720-3727. May, G.R., Rowand, W.S., McCormack,J.G. & Sheridan, R.D. (1995)Neuroprotective profile of lifarizine (RS-87476) in rat cerebrocortical neurones in culture. Br. 3; Pharmacol. 114, 1365-1370. McBean, D.E., Winters, K, Wilson, A.D., Oswald, C.B., Alps, BJ. & Armstrong,J.M. (1995a) Neuroprotective efficacy of lifarizine (RS-87476)in a simplifiedrat survivalmodel of 2 vessel occlusion. Br.3; Pharmacol. 116, 3093-3098. McBean, D.E., Winters, X, Wilson, A.D., Oswald, C.B. & Armstrong,J.M. (1995b)The ion channel modulator lifarizine (RS-87476)reduces the size of the infarct produced in the rat by the rose Bengal photochemical focal cerebral ischaemia. Br. 3; Phurmacol. 114 (Suppl.), 332P. McGivern,J.G., Patmore, L. & Sheridan, R.D. (1995) Actions of the novel neuroprotective agent, lifarizine (RS-87476),on voltage-dependent sodium currents in the neuroblastoma line, N1E-115. Br.3; Pharmacol. 114, 1738-1744. Meldrum, B.S., Swan,J.H., Leach, MJ., Millan, M.H., Gwinn, R., Kadota, K., Graham, S.H., Chen, J. & Simon, R.P. (1992) Reduction of glutamate release and protection against ischaemic brain damage by BW 1003C87.Brain Res. 593, 1-6. Miller, RJ. (1990) Glucose-regulated potassium channels are sweet news for neurobiologists. TrmdsNmrosci. 13, 197-199. Mourre, C., Ben-Ari, Y, Bernardi, H., Fosset, M. & Lazdunski, M. (1989)Antidiabetic sulphonylurea: location of binding sites in the brain and effects on the hyperpolarization induced by anoxia in hippocampal slices. Brain Res. 486, 15S164. Mourre, C., Smith, M.L., Siesjo, B.K. & Lazundski, M. (1990) Brain ischaemia alters the density of binding sites for glibenclamide, a specific blocker of ATP-sensitive K+ channels. Brain Res. 526, 147-152.


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Muir, K.W. & Lees, K.R. (1995) Clinical experience with excitatory amino acid antagonists drugs. Stroke 26, 503-5 13. Murphy, K.PSJ. & Greenfield, S.A. (1991) ATP-sensitive potassium channels counteract anoxia in neurones of the substantia nigra. Exp. Brain Res. 84, 355-358. Neubauer,J.A. (1993)Anoxia? Don't get excited!J. Clin. Invest. 91, 377. Obrenovitch, T.P. (1 995a) The ischaernic penumbra: Twenty years on. Cerebrovasc.Brain Metab. Rev. 7 , 297-323. Obrenovitch, T.P. (1 995b) Origins of glutamate release in ischaemia. Acta Eurochirg (Wien), 66 (Suppl.),50-55. Obrenovitch, T.P. & Richards, D.A. (1 995) Extracellular neurotransmitter changes in cerebral ischaemia. Cerebrovasc. Brain Metab. Rev. 7 , 1-54. Obrenovitch, T.P., Garofalo, O., Harris, R.J., Bordi, L., Ohno, M., Momma, E, Bachelard, H.S. & Symon, L. (1988) Brain tissue concentration of ATP, phosphocreatine, lactate and tissue pH in relation to reduced cerebral blood flow following experimental acute middle cerebral artery occ1usion.J. Cereb. Blood Flow Metab. 8, 866-874. Obrenovitch, T.P., Scheller, D., Matsumoto, T., Tegtmeier, E, Holler, M. & Symon, L. (1990) Rapid redistribution of hydrogen ions is associated with depolarization and repolarization subsequent to cerebral ischaemia-reperfusion. J. Nmrophyxiol. 64, 1 125-1 133. Okiyama, K., Smith, D.H., Gennarelli, T.A., Simon, R.P., Leach, M. & McIntosh, T.K. (1995) The sodium channel blocker and glutamate release inhibitor BW1003C87 and magnesium attenuate regional cerebral oedema following experimental brain injury in the rat. J. Neurochm. 64, 802-809. Osikowska-Evers, B.A., Wilhelm, D., Nebel, U., Hennemann, I?, Scheufler, E. & Tegtmeier, F. ( I 995) The effect of the novel neuroprotective compound lubeluzole on sodium current and veratridine induced sodium load in rat brain neurones and synaptosomes.3 Cereb. Blood Row Metab. 15 (Suppl. I), S380. Ptrez-Pinzh, M.A., Rosenthal, M., Sick, T.J., Lutz, EL., Pablo,J. & Mash, D. (1992) Down regulation of sodium channels during anoxia: a putative survivalstrategy of turtle brain. Am. J. Physiol. 262 (4 Pt 2), R7 12-R7 15. Petersen, O.H. & Findlay, I. (1987) Electrophysiology of the pancreas. Phyiol. Rev. 67, 1054-1 116. Pratt,J., Rataud,J., Bardot, E,Row, M., Blanchard,J.-C., Laduron, PM. & Stutzmann,J.-M. (1992) Neuroprotective actions of riluzole in rodent models of global and focal ischaemia. Neurosci. Lett. 140, 225-230. Prenen, G.H.M., Go, G.K., Postema, E, Zuiderveen, F. & Korf,J. (1988)Cerebral ation shifts in hypoxic-ischaemic brain damage are prevented by the sodium channel blocker tetrodotoxin. Exp. .Neural. 99, 118-132. Psarropoulou, C. & Avoli, M. (1 993) 4-aminopyridine-inducedspreading depression episodes in immature hippocampus: developmental and pharmacological characteristics. Neuroscience 55, 57-68. Ragsdale, D.S., Nurnann, R., Catteral, W.A. & Scheuer, T. (1993)Inhibition of Na' channels by the novel blocker PD85,639. Mol. Pharmacol. 43, 949-954. Randle, J.C.R., Leguern, S., Boudeau, PH. & Bohme, G.A. (1994) Riluzole inhibition of sodium channel currents and synaptic transmission is increased at high frequencies of activation. Can.3 Physiol. Pharmacol. 72 (Suppl. l), 425. Rataud, J., Debarnot, E, Mary, V, Pratt, J. & Stutzmann, J.-M. (1994)Comparative study of voltage-sensitive sodium channel blockers in focal ischaemia and electric convulsions in rodents. Nmrosci. Lett. 172, 19-23. Roufos, I., Hays, SJ., Dooley, D.J., Schwarz, R.D., Campbell, G.W. & Probert, A.WJr. (1994) Synthesisand pharmacological evaluation of phenylacetamidesas sodium-channelblockers. J. Med. Chm. 37, 268-274. Salem, R.D., Hammerschlag, R., Bracho, H. & Orkand, R.K. (1975) Influence of

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potassium ions on accumulation and metabolism of ['4C]glucoseby glial cells. Bruin Res. 86, 499-503. Scheller,D., Kolb,J., Szathmary, S., Zacharias, E., De Ryck, M., Van Reempts,J., Clincke, G. & Tegtmeier, E (1995)Extracellular changes of glutamate in the periinfarct zone. Effect of lubeluzole.3; Cueb. Blood Flow Metub. 15 (Suppl.l), S379. Schmid-Antomarchi,H., Amoroso, S., Fosset, M. & Lazdunski, M. (1990)K+ channel openers activate brain sulfonylurea-sensitive Kf channels and block neurosecretion. Roc. Natl Acad. Sci USA 87,3489-3492. Sheridan, R.D. (1995) Selectivity of the neuroprotective agent lifarizine. %ends Pharmucol. Sn'. 16, 292. Sick, TJ., Rosenthal, M., Lamanna,J.C. & Lutz, PL. (1982) Brain potassium ion homeostasis during anoxia and metabolic inhibition in the turtles and rats. Am. 3. Physiol. 243, R28 1-R288. Siesjo, B.K., Katsura, K., Mellergird, P, Ekholm, A., Lundgren, J. & Smith, M.-L. (1993) Brain Res. 96, 23-48. Acidosis-relatedbrain damage. hg, Smith, S.E. & Meldrum, B.S. (1995) Cerebroprotective effect of lamotrigine after focal ischaemia in rats. Stroke 26, 117-122. Spetzler, R.F. & Hadley, M.N. (1989) Protection against cerebral ischaemia: the role of barbiturates. Cerebrovasc. Brain Metub, Rev. 1,2 12-229. Spedding,M., Kenny, M. & Chatelain, P (1995)New drug binding sites in Ca2+channels. Emds Phurmucol. Sci. 16, 139-142. Standen, N.B., Quayle,J.M., Davies, N.W., Huang, Y. & Nelson, M.T. (1989)Hyperpolarizing vasodilators activate ATP-sensitive Kf channels in arterial smooth muscle. Science 245, 177-180. Stolc, S. (1988) Comparison of effects of selected local anesthetics on sodium and potassium channels in mammalian neurones. Gen. Physiol. Biophys. 7, 177-189. Stutzmann, J.M., Mignani, S., Debarnot, E, Rataud, J., Piot, O., Pauchet, C., Jimonet, P, Reibaud, M., Malgouris, C., Uzan, A., Pratt, J., Blanchard, J.-C. & Barreau, M. (1993) Pharmacologicalprofile and neuroprotective activities of RP 66055, a riluzole derivative, in rodents. Sod. Narosci.Abstr. 19, 1647. Stys, PK., Waxman, S.G. & Ransom, B.R. (1991)Na+-Ca2+exchanger mediates Ca2+influx during anoxia in mammalian central nervous system white matter. Ann. Neurol. 30,375-380. Stys, PK., Ransom, B.R. & Waxman, S.G. (1992a)Tertiary and quaternary local anesthetics protect CNS white matter from anoxic injury at concentrations that do not block excitability.3; Nmrophysiol. 67, 236-240. Stys, PK., Waxman, S.G. & Ransom, B.R. (199213) Ionic mechanism of anoxic injury in mammalian CNS white matter: Role of Naf channels and Na+-Ca2+exchanger.J. Neurosci. 12,4301139. Stys, PK., Sontheimer, H., Ransom, B.R. & Waxman, S.G. (1993) Noninactivating, tetrodotoxin-sensitiveNaf conductance in rat optic newe axons. Roc. Nut1 Acad. Sci. USA 90, 697643980. Suzuki, T., Sekikawa,T., Nemoto, T., Moriya, H. & Nakaya, H. (1995)Effects ofnicorandil on the recovery of reflex potentials after spinal cord ischaemia in cats. Br. 3; Phurmacol. 116, 1815-1820. Szatkowski, M. 81 Attwell, D. (1994) Triggering and execution of neuronal death in brain ischaemia: two phases of glutamate release by different mechanisms. Eends Neurosci. 17, 359-365. Taft, W.C., Clifton, G.L., Blair, R.E. & Delorenzo, RJ. (1989) Phenytoin protects against ischaemia-produced neuronal cell death. Bruin Res. 483, 143-1 48. Tasker, R.C., Coyle, J.T. & Vornov, JJ. (1992) The regional vulnerability to hypoglycemiainduced neurotoxicity in organotypic hippocampal culture: protection by early tetrodotoxin or delayed MK-801. 3; Neurosci. 12,4298-4308.


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Taylor, C.P. (1993)Na+ currents that fail to inactivate. TrmdsNeurosci. 16,455460. Thomsen, W., Hays, SJ., Hicks,J.L., Schwartz, R.D. & Catterall, W.A. (1993)S specific binding of the novel Na' channel blocker PD85,639 to the a subunit of the rat brain Na' channel. Mol. Pharmacol. 43,955-964. Umemiya, M. & Berger, AJ. (1995)Inhibition by riluzole of glycinergic postsynaptic currents in rat hypoglossal motoneurones. Br.3 Pharmacol. 116, 3227-3230. Urenjak, J. & Obrenovitch, T.P. (1996) Pharmacological modulation of voltage-gated Na+ channels: A rational and effective strategy against ischaemic brain damage. Pharmacol. Rev. 48, 2 1-67. Vornov,J.J.,Tasker, R.C. & Coyle,J.T. (1994)Delayed protection by MK-801 and tetrodotoxin in a rat organotypic hippocampal culture model of ischaemia. Stroke 25,457465. Wahl, E, Allix, M., Plotkine, M. & Boulu, R.G. (1993) Effect of riluzole on focal cerebral ischaemia in rats. Eur. 3 Pharmacol. 230,209-2 14. Wann, K.T. (1993) Neuronal sodium and potassium channels: structure and function. Br. j! Anaesth. 71,2-14. Watson, G.B. & Lanthorn, T.H. (1995)Phenytoin delays ischaemic depolarization, but cannot block its long-term consequences, in the rat hippocampal slice. Nmropharmacology 34, 553-558. Weber, M.L. & Taylor, C.P. (1994) Damage from oxygen and glucose deprivation in hippocampal slices is prevented by tetrodotoxin, lidocaine and phenytoin without blockade of action potentials. Brain Res. 664, 167-177. Wiard, R.P., Dickerson, M.C., Beek, O., Norton, R. & Cooper, B.R. (1995)Neuroprotective properties of the novel antiepileptic lamotrigine in a gerbil model of global cerebral ischaemia. Stroke 26, 466-472. Xia, Y. & Haddad, G.G. (1 993) Neuroanatomical distribution and binding properties of saxitoxin sites in the rat and turtle CNS.3 Comp. Neurol., 330, 363-380. Xia, Y. & Haddad, G.G. (1994)Postnatal development of voltage-sensitive Na' channels in rat brain.3 Comp. Neurol. 345, 279-287. Xie, X., Lancaster, B., Peakman, T & Garthwaite,J. (1995)Interaction ofthe antiepileptic drug lamotrigine with recombinant rat brain type IIA Na+ channels and with native Na' chanArch. 430,437446. nels in rat hippocampal neurones. Xie, X.M. & Garthwaite,J. (1995)State-dependent block of recombinant rat brain type IIA Na channels by the neuroprotectant 619C89. Brain Res. Assoc. Abstr. 12, 74. Xie, Y , Dengler, K., Zacharias, E., Wiffert, B. & Tegtmeier, E (1994) Effects of the sodium channel blocker tetrodotoxin (TTX) on cellular ion homeostasis in rat brain subjected to complete ischaemia. Brain Res. 652, 2 16-224. Xie, Y , Zacharias, E., Hoff, P. & Tegtmeier, E (1995) Ion channel involvement in anoxic depolarization induced by cardiac arrest in rat brain.3 Cereb. Blood Flow Metub. 15,587-594. Yamasaki, Y , Kogure, K., Hara, H., Ban, H. & Akaike, N. (1 99 1) The possible involvement of tetrodotoxin-sensitiveion channels in ischaemic neuronal damage in the rat hippocampus. Neurosci. h t t . 121, 251-254. Zetterstrom, T.S.C., Vaughan-Jones, R.D. & Grahame-Smith, D.G. (1995) A short period of hypoxia produces a rapid and transient rise in [K'Ie in rat hippocampus in Vivowhich is inhibited by certain K+-channel blocking agents. Neuroscience 6 7 , 8 15-82 1. Zhang, H., Stockbridge, N., Weir, B., Vollrath, B. & Cook, D. (1992)Vasodilatation of canine cerebral arteries by nicorandil, pinacidil and lemakalim. Gen. Pharmacol. 23, 197-201.


Chapter

7

NMDA ANTAGONISTS: THEIR ROLE IN NEUROPROTECTION Daniel L. Small and Alastair M. Buchan* Cellular Neurobiology Group, Institute for Biological Sciences, National Research Council of Canada, Building M-54, I200 Montreal Road, Ottawa, Canada K I A OR6 *Clinical Neurosciences, University of Calgary, Foothills Hospital, 1403-29 Street NW, Calgary, Alberta 2TN 2T9, Canada

7. I 7.2 7.3 7.4 7.5 7.6

7.7

7.8 7.9

Introduction Excitotoxicity hypothesis NMDA receptor molecular biology NMDA receptor biophysics NMDA receptor pharmacology NMDA antagonist neuroprotection in in vitro models of ischaemia 7.6. I Introduction 7.6.2 Neuronal cultures 7.6.3 Brain slices 7.6.4 Organotypic cultures NMDA antagonist neuroprotection in in vivo models of ischaemia 7.7.I Introduction 7.7.2 Global o r severe forebrain ischaemia models 7.7.3 Focal models of cerebral ischaemia How to determine what goes to trial Conclusion Acknowledgements References

i37 138 140 142 145 150 150 150 151 152 153 153 153 155 157 158 159 159

7. I Introduction At 1 1.13 am on 5 December, 1995 patient recruitment in all phase I11 clinical trials for cis-4-(phosphono-methyl)-2-piperidinecarboxylic acid (CGS 19755; Selfotel), were terminated by Ciba-Geigy. This decision was made upon receiving a recommendation from an independent data and safety monitoring board reporting that the ‘benefit-to-risk’ was not significant to warrant continuation of the trials at this time. In the early 1990s a wave of clinic trials testing the efficacy of NMDA antagonists in the treatment of cerebral ischaemia was launched. The verdict returned on the success of CGS 19755 in clinical trials is one of the first. Is Academic Press Limited Copyright 0 1997 All rights ofreproduction in ay&m reserved


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it the beginning of the end for NMDA antagonists in the treatment of cerebral ischaemia? It is the opinion of these authors that clinical trials for NMDA antagonists in stroke may have been launched prematurely with ‘less than perfect’ compounds, and the failure of these early trials may adversely affect the development of better NMDA antagonists and the chances of their use in future clinical trials. CGS 19755 had impressive preclinical data demonstrating dramatic cytoprotection in gerbil models of severe transient forebrain ischaemia (Boast, 1988; Boast et al., 1988). Furthermore, it was efficacious when administered post-ischaemically (Boast et al., 1988) and did not produce psychotomimetic effects in monkeys at doses which were neuroprotective (France et al., 1989). Given the potent neuroprotection with mild hypothermia (Buchan and Bulsinelli, 1990),it is of importance to note, however, that data on body temperature in these studies (either during or followingthe ischaemia)is sparse at best. In retrospect, the failure of CGS 19755 in clinical trials might have been predicted had more careful attention been paid to monitoring and maintaining control over physiological variables such as temperature in the preclinical animal studies. This review will attempt to critically assess the utility of NMDA antagonists in the treatment of cerebral ischaemia. Concepts of a modified excitotoxicity will be presented with a description of the NMDA receptor physiology and pharmacology as it pertains to excitotoxicity Published data for in uitro and in uivo models of ischaemia using NMDA antagonists will be reviewed and proposals for new directions will be offered.

7.2 Excitotoxicity hypothesis Although the concept of excitotoxicity, namely, ‘a paradoxical property, shared by glutamate and specific excitatory amino acid (EAA) analogues, of causing acute neuronal degeneration by excessive stimulation of postsynaptic E M ionotropic receptors’, was advanced more than two decades ago (Olney et al., 197 l), Lucas and Newhouse (1957) had reported the neurotoxic effects of exogenous glutamate in infant mice 14 years earlier. Later it was shown that the effects of exogenous glutamate could be mimicked with NMDA (Coyle, 1983). Following the observation that exogenously applied glutamate (Olney et al., 197 1) or NMDA (Coyle, 1983)were neurotoxic, Beneviste and colleagues (1984), demonstrated that, during ischaemia, extracellular glutamate levels in the brain rise. As will be discussed in more depth later, drugs which block the NMDA subtype of glutamate receptors can inhibit NMDA-induced neurotoxicity (Foster et al., 1987). Moreover, consistent with the idea that glutamate activation of postsynaptic EAA receptors underlies excitotoxicity transection of afferent glutamatergic fibres travelling to vulnerable neurones is neuroprotective (Johansen et al., 1986;Jorgensen et al., 1987; Ondera et al., 1986; Wieloch et al., 1985). The molecular mechanisms underlying excitotoxicity are not hlly understood, but

NMDA ANTAGONISTS: THEIR ROLE IN NEUROPROTECTION

139

FIGUREI Schematic cartoon of proposed excitotoxicity cascade illustrating potential targets of therapeutic intervention within the cascade. See text for details. (Reproduced with permission from Small & Buchan, 1996.)

it is thought that the initial events are the activation of EAA receptors, an influx of Na' and, more importantly, Ca2+,which depolarizes the neurone, further potentiating the activation and Ca2+ permeation of EAA receptors. As Ca2+ accumulates within the neurone under these unregulated conditions (Figure l), a cascade of events is triggered which ultimately results in the death of the cell. The activation of metabotropic glutamate receptors (Figure 1) results in increases in protein kinase C (PKC) and decreases in protein b a s e A (PKA), through activation of G-proteins. These second messengers are important players in the excitotoxic cascade as their targets include EAA receptors and voltage-gated ion channels. The late events include activation of immediate early genes (IEGs),production of nitric oxide (NO),acidosis and activation of lipases and endonucleases. Although extracellular glutamate levels rise during ischaemia (Beneviste et al., 1984), Obrenovitch and Richards (1995) have shown that levels begin to return to normal within 20 minutes of a cardiac arrest model of ischaemia. The most vulnerable neurones, however, do not succumb for days (Pulsinelliet al., 1982).It would seem that the profound neuroprotection with EAA antagonists and agents which inhibit glutamate release given 12 hours (Liand Buchan, 1993) and 24 hours (Buchan et al.,

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D.L.SMALL AND A.M. BUCHAN

1994) after the ischaemic insult are incongruent with Beneviste and colleagues' observations that extracellular glutamate levels are returning to normal only minutes after an ischaemic insult (Beneviste et al., 1984). Recent evidence suggests that EAA receptors themselves may be modified following ischaemia (Pellegrini-Giampietroet al., 1992, 1994; Perez-Velazquez and Zhang, 1994; Pollard et al., 1993a; Tsubokawa et al., 1994, 1995; Zhang et al., 1995), in a manner which results in a greater Ca2+influx and depolarization. These changes are thought to be at the level of transcription (Pellegrini-Giampietroet al., 1992, 1994; Perez-Velazquez and Zhang, 1994; and Pollard et al., 1993a),rather than behavioural changes in the receptors due to modulatory agents like phosphorylating enzymes or protons. That is not to say that neurotoxic EAA behaviour is not further enhanced by modulatory agents, just that the receptors are different.

7.3 NMDA receptor molecular biology NMDA receptors are heteromeric pentamers which form ligand-gated ion channels (BCht et al., 1995). The subunits are products of two gene families; the NR1 gene which undergoes alternative splicing to yield 8 difTerent products, and the four NR2 genes A, B, C, and D. Similar in size to other glutamate receptor subunits but about twice the size of other ligand-gated ion channels, the predicted molecular weights of the gene products are between 103-1 63 kDa with an open reading frame comprising between 920 and 1456 amino acids. There is approximately 40-50% homology within the NR2 gene family, but as little as approximately 20% homology among the NRl and NR2 gene families. This is comparable to the homology of NMDA receptor subunits with other glutamate receptor subunits (Mishina et al., 1993). The NR1 splice variants and their proposed nomenclature (Zukin and Bennett, 1995),based on the presence or absence of three alternatively spliced exons, are presented in Table 1. The eight functional splice variants are denoted with subscripts that indicate the presence (1) or absence (0)of the three alternatively spliced exons from the 5" to 3" end (Durand et al., 1993).Thus, the first NRl subunit to be cloned (and the most prominent receptor isoform in rat forebrain) is denoted NRloll; the NRloll subunit lacks N1 but has both C1 and C2. The NRloolsubunit has the N1 insert but lacks C1 and C2. An X is used to indicate that the presence of an exon is indeterminant. ThusNR1 IxxdenotesNR1variantsthat have theNl insertwithorwithout C 1 or C2. NRlooxdenotes NR1 receptors that lack N1 and C 1, with or without C2. The N-terminal (exon 5) is a 2 1 amino acid chain with 6 positively charged residues arranged at either end and 3 negative residues in the middle. Exons 2 1 (C 1) and 22 (C2) make up the C-terminal with 37 and 38 amino acids, respectively. On the Cterminal there are 4 or 5 residues which are phosphorylated by protein h a s e C (PKC). Until recently, the membrane topology of the NMDA receptor was assumed to be similar to other ligand-gated ion channels having 4 transmembrane domains and an extracellular N- and C-terminal, but it is now depicted to have 3 trans-


141

TABLE 1 NR1 RECEPTOR SPLICE VARIANTS AND NOMENCIATURE Splice

Insert

variant NI NRlwo NRlool NRlolo NRloll NRlloo + NRIlol + NRI 110 NRIII,

+ +

Other names (with references)

(4 (4

(4

C1

C2

(a)

-

-

R1E NRlc R1C SS 51-2 NMDA-R1C R1D R1A NRla SL 51 NMDA-R1A RIG NRlb RlF LS (Not yet found in a cDNA library) RIB LL NMDA-R 1B

+ + -

+ +

+ -

+ -

+ +

-

(b)

(f)

NMDARl4a NMDARl-2a NMDAR1-3a NMDARl-la NMDAR14b NMDARI-2b NMDAR 1-3b NMDAR 1-1b

Nok. (a) Sugihara et al., 1992; (b) Durand et al., 1992; (c)Anantharam et al., 1992; (d) Yamazaki et al., 1992; (e) Nakanishi et al., 1992; (f) Hollman et al., 1993. (Reproduced from Zukin & Bennett, (1995),with permission.)

membrane domains (TM 1,3 and 4), such that the N-terminal is extracellular and the C-terminal is intracellular (Figure 2) (Bennett and Dingledine, 1995; Hollmann et al., 1994; Stern-Bach et al., 1994). There is a fourth hydrophobic domain, TM2, but it does not span the membrane. Instead, TM2 doubles back through the membrane so that it both enters and exits the membrane on the intracellular side. TM2 is analogous to H5 or the pore region of the voltage-gated potassium channels (Hartmann et al., 1991) in that it, too, forms a channel pore (Figure 2). A critical residue within the pore which determines permeability is an asparagine (N).There are two domains S1 and S2 (Figure 2), located on the N-terminal as it enters the membrane and TM3 as it leaves the membrane, which are thought to form the glutamate- and glycine-binding domains due to their homology with QBPl and QBP2 (bacterial glutamate-binding protein) (O’Hara et al., 1993; Kuryatov et al., 1994). NR 1 is expressed ubiquitously throughout the mammalian brain and expression of NRl alone in oocytes is sufficient for expression of NMDA-activated channel behaviour similar to native channels (Moriyoshi et al., 1991). Every NMDA receptor contains at least one NRl subunit with one or more NR2 subunits. Although the subunit composition in Uiuo which accounts for the functional diversity of NMDA receptors is not well understood, there exists evidence that NRl can couple with NRPA, B, C, or both A and B, or A and C together (Sheng et al., 1994; Didier et al., 1995; Wafford et al., 1993). The expression of NR1 splice variants is not uniform throughout the brain. NRlou and NRlXolappear first in embryonic brain, followed by NR1 which are gradually expressed at embryonic day (E)19, NRl xoo at postnatal day (P)7, and NRlXlonot until P12 (Zukin and Bennett, 1995). The NRl1, expression peaks in the hippocampus and cerebellum at P 14 and in thalamus at P3 1. The expression of NRlou is uniform throughout the hippocampus in CA1-4 and dentate gyrus, while the expression of NR1 is most abundant in CA3 and weakest in CAI (Laurie et al., 1995). The expression of NR2 subunits is not uniform throughout the

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FIGURE2 Schematic illustration of proposed transmembrane topology of an NMDA receptor subunit depicting the N (N 1) and C (C1, C2) terminal cassettes of NR1 I II as well as the asparagine residue (black oval),within the pore (TM2), and the functional domains S 1 and S2 associated with ligand binding. Asterisked arrowhead indicates the position of an essential residue for glycine binding; the crosses on C 1 are proposed phosphorylation sites; the positive and negative areas on the N terminal cassette indicate the approximate position of positively and negatively charged residues.

brain. NR2A is found in most regions of the brain including the cortex, hippocampus and cerebellum. NRPB is found in the cortex and hippocampus, but not the cerebellum, while NR2C is found only in the cerebellum, and NRPD is found only in the olfactory bulb (Figure 3; Buller et al., 1994).During development, NR2B is expressed predominantly in the forebrain region until P12 when NRPA and C begin to appear (Monyer et al., 1994). NRPC is transiently expressed in the hippocampus during development (Pollard et al., 1993b) and has been reported to reappear following a transient hypoxic-hypoglycaemic insult (Zhang et al., 1995).The significance of a reemergence of developmental NMDA subunits following ischaemia will become apparent later following a description of their biophysics and pharmacology

7.4 NMDA receptor biophysics The ionic channel associated with the NMDA receptor has a conductance and subconductance of 50 and 38 pS for both NR1-NRPA and NR1-NRPB combinations, and 36 and 11 pS for NR1-NRSC combinations (Seeberg et al., 1995).The channel is cationic, allowing the passage of Na', K' and Ca2+.The NMDA receptor has extracellular and cytoplasmicmouths that can accommodate large cations up to 7.3 A


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FIGURE 3 In situ hybridization of oligonucleotide probes specific for (A)NRPA; (B) NRPB; (C) NRZC, and (D) NRZD subunits are shown in horizontal sections of rat brain. (Modified with permission from Buller et al., 1994.) Key: MS - medial striatum; VP - ventral posterior nucleus of thalamus; GL glomerular layer; M T - midline thalamic nuclei; A - anteroventral nucleus; MG - medial geniculate; PG periaquaductal grey. ~

~

in mean diameter, but it has a narrow pore with a mean diameter of 5.5 A, which functions in part to restrict the passage of the larger cations (Villarroel et al., 1995). Although the relative permeability of NMDA receptors for Ca2+has been determined under a variety of experimental conditions, only a couple of physiologically relevant

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measures have been attempted, yielding a percentage of inward current carried by Ca2+of 12.4% (Rogers and Dani, 1995), and 6.8% (Schneggenburger et al., 1993). The NMDA receptor is at least 5 times more permeant to Ca2+ than any other ionotropic glutamate receptor (Schneggenburger et al., 1993). Replacing asparagine (N) with glutamine (Q) or arginine (R) in the TM2 of the NR1 subunit results in a decrease in the Ca2+permeability of the NMDA receptor (Burnashev et al., 1992b), analogous to molecular manipulations of the Q / R site of AMPA receptors (Burnashev et al., 1992a). The change in the N residue of TM2 of NR1 (Figure 2) does not affect the Mg2+binding, but the same substitution of TM2 in NR2 subunits decreases the Mg2+ block and does nothing to the Ca2+ permeability (Burnashev et al., 1992b). Physiological concentrations of extracellular Mg2+block the NMDA receptor in a voltage-dependent manner. The physical plugging of the cation permeation path is relieved at depolarizations beyond -30 mV; Intracellular Mg2+can also block the NMDA receptor but only under non-physiological conditions such as high Mg2+ concentrations (>1mM) or extremely depolarized potentials. The sensitivity to Mg2+ block depends on both the activity of intracellular kinases (PKC decreases external M g ' block; Chen and Huang, 1992),and the NMDA receptor subunit combination (NR 1-NR2C exhibits a weaker M$+ block than other NR 1-NR2 subunit combinations (Kutsuwada et al., 1992; Meguro et al., 1992; Monyer et al., 1992). The weaker MgZfblock exhibited by NR2C is consistent with the observations of a weaker Mg2+ block in the immature hippocampus (Bowe and Nadler, 1990; Morrisett et al., 1990), and the expression patterns of NR2C in the hippocampus during development (Pollard et al., 1993b). Zn" also blocks NMDA receptors, but at a different site@)than Mg2+.At micromolar concentrations, the block is voltage-independent (Westbrook and Mayer, 1987), while a flickery block is produced at higher micromolar concentrations. At submicromolar concentrations, Zn2+actually potentiates NMDA responses (Hollmann et al., 1993). The effects of Zn2+on NMDA receptors are subunit specific. Zn2' potentiation of the NRl subunit is twice that of the NRloll subunit because of the positive residues on the N1 cassette of NRlloo (Figure 2; Zukin and Bennett, 1995). Furthermore, the Zn2+potentiation is lost when NR1 subunits are combined with any of the NR2 subunits (Hollmann et al., 1993). Polyamines can also either block (Mayer and Westbrook, 1987) or potentiate (McGurk et al., 1990) NMDA responses, depending on the concentration of polyamines and the NMDA subunit involved. There are thought to be as many as three distinct sites of polyamine interaction to account for the different effects on NMDA responses (Hollmann et al., 1993). The flickery block occurs at higher concentrations and is the result of polyamines getting physically stuck in the permeation pathway of the channel. However, at low micromolar concentrations, polyamines potentiate NMDA responses. These qualitatively different effects with different concentrations are relevant, given the 14-fold increases in polyamine concentration following ischaemia (Paschen et al., 1987). Like Zn2+,the polyamine potentiation of NMDA responses on homomeric NMDA receptors is greater with the NRl subunit (Zukin


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and Bennett, 1995). However, unlike the Zn2+potentiation, rather than being lost when NR1 is combined with NR2 subunits, the potentiation by the NRl ,oo subunits is enhanced. The polyamine potentiation is exhibited with NR2B and not NR2A or NR2C subunit combinations (Williams et al., 1994). Polyamines also modulate the proton sensitivity of the NMDA responses (Trayneliset al., 1995). Polyamines potentiate NMDA responses by relief of the tonic block by protons at physiological pH. This effect is less pronounced with NRl subunits than NRloxx subunits due to the fact that the positive charges of the N1 cassette, like those of polyamines, serve to relieve the tonic proton inhibition of NMDA responses. The acidosis which occurs following ischaemia would result in a greater block of NMDA receptors especially if the NMDA receptors were those which lacked the N terminal splice variant. Glycine serves as a co-agonist on NMDA receptors by interacting with a distinct extracellular site@)within the S1 and S2 region (O’Hara et al., 1993; Figure 2), with an affinity for glycine in the nanomolar range (Johnson and Ascher, 1990).Given that physiological glycine concentrations are in the micromolar range (Curtis and Johnston, 1974), the NMDA receptor usually exists in the ‘glycine-primed’state. The on-rate of glycine is rapid, whereas the relaxation rate is 147- 1000 ms, depending on the NMDA receptor subunit combination (Ascher andJohnson, 1994).Glycine a h ity varies with subunit composition as well; the NR2A subunit confers a lower a h ity for glycine than NRPB, C, or D (Kutsuwada et al., 1992; Stern et ab, 1992). Deactivation and desensitization of NMDA receptors occur much more slowly than for AMPA receptors, and the affinity of NMDA receptors for glutamate is much higher than it is for AMPA receptors (Sather et al., 1992), so glutamate activates NMDA receptors more efficiently than AMPA receptors. There is also variability in the affinity for glutamate among the NMDA receptors, depending on their subunit composition. The affinity for the NRl ollsplice variant subunit is 5-fold higher than for the NRl IMI subunits. NMDA receptor deactivation is the major determinant of the slow NMDA excitatory postsynaptic potential (EPSC) decay. NMDARl subunits coexpressed with NMDARP subunits A, B, C, and D give current decays with time constants of 90,400, 370, and 4800 ms, respectively (Seeberg et al., 1995).

7.5 NMDA receptor pharmacology The many modulatory sites on the NMDA receptor serve as pharmacological targets, thus resulting in a number of classes of antagonists based on their site of action (Table 2). Competitive antagonists act at the NMDA/glutamate binding site, while ‘noncompetitive’ antagonists bind to sites within the ion channel pore, resulting in a physical plugging of the permeation pathway. Other targets are the glycine and polyamine sites on the receptor. Competitive antagonists can be displaced by glutamate, necessitating very high concentrations of antagonist to overcome the elevated levels of glutamate during

TABLE 2 NMDA ANTAGONISTS Class

Compound and company

Neuroprotection?

Competitive

CGS 19 755/Selfotel (Ciba) h4DL 100453 (Merrell Dow) ATA (Eli Lily) dCPP-ene (SDZ EAA 494; Sandoz) CGP 40 116 (Ciba-Geigy) NPC 12626 (Nova Pharmaceuticals Corp.) NPC 17 742 (Nova Pharmaceuticals Corp.) AP5 AP7

dish(-)'/slice( -)293/focalG7/g10balE10

Non-competitive, high a f i i t y

MK80 1 /Dizocilpine (Merck) Phencyclidine CNS 1 1OS/Aptiganel HCL (Cambridge Neurosciences) FRI 15 427 (Fujisawa Pharmaceutical Co.) MDL 27 266 (Merrell Uow)

Intermediate affinity

Dextrorphan

Low affinity

Ketamine Kyurenate Dextromethorphan (Hoffmann LaRoche) Memantine (Merz) Amantadine Magnesium Remacemide (Astra)

Glycine site

C-W 554/Felbamate (Carter-Wallace) ACEA 1021 (ACEA Pharmaceuticals)

Partial agonist

HA-966 L-687 4 14 (Hoffmann La Roche) ACPC/SYM 2030 (Symphony)

Polyamine site

SL 82.07 1S/Eliprodil (Synthelabo/Lorex) Ifenprodil (Synthelabo/Lorex) CP 10 1 606 (F'lizer)

fOCal&W focal893 &h7.91 /focalg'

Note. ACEA 1021, halogenated quinoxaline-2,3-dione;ACPC, 1-aminocyclopropanecarboxylicacid; AP5,2-amino-5-phosphonopentanoicacid; AP7,2amino-7-phosphonoheptanoicacid; ATA, aurintricarboxylicacid; CGP 40 1 16, D-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid; CGS 19 755, cis-4@hosphonomethyl)-2-piperidinecarboxylic acid; d-CPPene, d-3-(carboxypiperazin-4-yl)-propenyl1-phosphonic acid; dextrorphan, d-3-hy&oxy;N-methyl-morphinan; FR 1 15427, l-rnethyl- l-phenyl- I ,2,3,4-tetrahydroisoquinolinehydrochloride; E4966, 3-amino- l-hydroxyWrrolid-2-one;L 687 414, 3R-(+)-&-4-methyl-HA966; MDL 27 266, 5-(4-chlorophenyl)-4-ethyl-2,4dihydm-2-~ethyl-3H-1,2,4-triazol-3-one; MDL I00 453, [R]4oxo-5cyclohepten-5,1O-imine; NPC 17 742, 2R,4R,5S-[2-amino-4,5-(1,2-cyclohexyl)-7phosphononowaline; MK-80 1, 5-methyl- 10, 11-dihydro-5H-dibenzo[a,~ phophonoheptanoic acid]; NPC 12 626, 2-amin0-4,5-(1,2-cyclohexyl)-7-phophonoheptanoicacid. Re&mces: (1) Aizenman & Hartnett, 1992; (2)Schurr et al., 1995; (3) Schurr et al., 1993; (4) Simon & Shiraiihi, 1990; (5)Takizawa et al., 1991; (6)Sauer et al., 1993; (7) Sauer etal., 1994; (8)Boast, 1988; (9) Grotta etal., 1990; (10) Boast, 1988; (1 1) Weller el al., 1993; (12) Zeevalk etal., 1993; (13)Zeevalketd., 1995; (14) Samples & Dublinsky, 1993; (15)Roberts-Lewis ef al., 1993; (16)Schramm et af., 1990; (1 7) Bulloch el al., 199Oa; (18) Bulloch el al., 1990b; (19)Bulloch etaf., 19%; (20)Herrling, 1994; (21)Sauer et al., 1995; (22)Ferkany et d.,1989; (23)Nishikawa et d., 1994; (24)Choi et nl., 1988; (25) Goldberg et al., 1987a; (26) Manev et al., 1989; (27)Aitken et al., 1988; (28)Donevan & Rogawski, 1993; (29)Tarnawa et al., 1989; (30)Rothman & Olney, 1987; (31) Clark & Rothman, 1987; (32)Roman et al., 1989; (33) Swan et al., 1988; (34)Block & Pulsinelli, 1987; (35)Jensen & Auer, 1989; (36)Swan et al., 1988; (37) Simon et nl., 1984; (38) Favaron el al., 1988; (39) Kochhar et al., 1988; (40) Ozyurt et al., 1988; (41) Park et al., 1988a; (42)Park et al., 1988b; (43) Bielenberg & Beck, 1991; (44)Dezsi et al., 1994, (45)Persson et al., 1989; (46)Wieloch et al., 1988; (47) Fleischer et d.,1989; (48)Buchan & Pulsinelli, 1990; (49) Lanier et af., 1990; (50) Gill et af., 1987; (5 1) Gill et af., 1988; (52) Sauer et al., 1988; (53) Minematsu et al., 1993; (54) Keana et al., 1989; (55)Park et al., 1993; (56) Gamzu & McBurney, 1994; (57) Katsuta el nl., 1995; (58)Nakanishi et nl., 1994; (59)Warner et al., 1995; (60) Goldberg et al., 1987a; (61)Kent et d., 1989; (62) Steinberg et al., 198913; (63) Steinberg et al., 1989a; (64)Swan & Meldrum, 1993; (66)Steinberg et al., 1988a; (67)Jensen & Auer, 1988; (68)Marcoux et al., 1988; (69) Germano et nl., 1987; (70)Roussel et al., 1990; 1990; (65)Aronowski et d., (71) Germano et al., 1987; (72) Steinberg et al., 1988b; (73) Chen et al., 1992; (74)Erdo & Schafer, 1991; (75) Osborne & Quack, 1992; (76) Seifel Nasr et al., 1990; (77)Lustig et al., 1992; (78) Cox et al., 1989; (79) Bennan et al., 1994; (80)Ordy etal., 1992; (81)Wallis el al., 1992; (82)Wasterlain et al., 1992; (83)Warner et d., 1991; (84) Boje etal., 1993; (85)Boje etal., 1992; (86)Gill et al., 1995; (87) von Lubitz et af., 1992; (88)Gotti et al., 1990; (89) Carter et al., 1988; (90) Gotti etal., 1988; (91)Pagnozzi et al., 1995; (92)Menniti etal., 1995; (93) Hasegawa etal., 1994; (94)Muir & Lees, 1995.

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ischaemia. With the high antagonist concentrations, normal, non-ischaemic NMDA receptor activity would be blocked, resulting in adverse side-effects, as are seen with competitive antagonists such as CGS 19755, NPC 12626 and dCPP-ene, to name a few (Muir and Lees, 1995). An ideal therapeutic candidate must exhibit a good therapeutic ratio (concentration of drug which produces desired effect/concentration of drug which produces undesired ‘side-effects’) or ‘benefit-to risk’. As mentioned previously, although CGS 19755 is neuroprotective in some models of ischaemia, clinical trials have been suspended because the ‘benefit-to-risk’ was not significant. Many, ifnot all, of the non-competitive NMDA antagonists are use-dependent by virtue of the fact that they bind within the ion channel pore and, therefore, can only reach their binding site when the channel is activated and the channel is open. The theoretical advantage of a use-dependent compound is that as glutamate levels rise, the antagonist works better, so that only excess excitatory activity is blocked. With these compounds the normal excitatory activity, such as that necessary for learning and memory, is not disturbed (Kemp and Leeson, 1993).This advantage has yet to be realized in any clinical trials for the treatment of stroke. However, this could be due to the fact that the trials for cerebral ischaemia to date have focused on high-affinity compounds. It has been postulated (Jones and Rogawski, 1992; Rogawski, 1993)that low-affinity NMDA receptor antagonists reach equilibrium more quickly relative to high-affinity antagonists, and that the time required by high-affinity compounds to reach a steady-state block allows enough Ca2+influx that damage still occurs. Given that the onset of block is concentration-dependent, this early Ca2+ influx can be blocked by increasing the concentration of the high-affinity antagonist, but the result is a complete and long-lasting block of all NMDA receptor activity including normal activity. Therefore, for high-affinity agents to be neuroprotective, concentrations must be high enough so that negative side-effects are likely. This is the case with compounds such as MK-80 1, PCP, and CNS 1 102. They are said to have a low therapeutic ratio. The side-effects associated with these types of agents are psychotomimetic and cardiovascular in nature, including paranoid ideation, hallucinations, peripheral vasoconstriction and catatonia (Muir and Lees, 1995). Conversely, the low-affinity agents are better tolerated and are not associated with similar side-effects as they have a better therapeutic ratio (Muir and Lees, 1995). Consequently agents like memantine, ADCI, remacemide and the active desglycinyl metabolite of remacemide are receiving more attention (Jones and Rogawski, 1992; Parsons et al., 1995; Subramaniam et al., 1996).Although some of these agents work well for epilepsy (Meldrum, 1988), their efficacy in stroke requires further investigation. Hopefully, the failure of the high-affinity NMDA antagonists in clinical trials will not adversely affect the likelihood of considering the low-affinity compounds in clinical trials, should they prove effective in animal models of ischaemia. Glycine site and polyamine site antagonists are fewer in number and have received less attention than the other two groups of NMDA antagonists. However, they have been gaining in popularity as safer alternatives for stroke therapy because they have few adverse affects (Muir and Lees, 1995). Further studies of the neuroprotective effi-


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cacy of these agents are now required. Some agents like HA 966 and ACPC are partial agonists at the glycine site (Table 2). Glycine levels in the brain are more than sufficient to saturate the glycine site of the NMDA receptor (Ascher and Johnson, 1994). A partial agonist with a very high affinity would be predicted to work well by providing protection from excess activity, yet permitting normal NMDA receptor activity. Although these agents have been shown to provide neuroprotection in focal models of cerebral ischaemia (Muir and Lees, 1995; Warner et al., 1995; Gill et al., 1995), failure of the glycine antagonists to ameliorate damage in a model of global cerebral ischaemia (Warner et al., 1995), suggests that further studies are required. A compound must prove neuroprotective in several models by different laboratories before even being considered for clinical trials, given the tremendous expense of trials and the potential backlash by policy makers and administration if a compound fails in clinical trials. Of the few polyamine site antagonists, most are reported to be subunit-specific. Eliprodil (Williams, 1993), ifenprodil (Williams, 1993) and CP 101 606 (White et al., 1995), are all NRPB-specific antagonists. These compounds are devoid of psychotomimetic effects and brain vacuolization associated with other NMDA antagonists (Scatton et al., 1995; Pagnozzi et al., 1995). These compounds have all proven to be neuroprotective in a number of models (Scatton et al., 1995; Toulmond et al., 1993; Menniti et al., 1995). Of the polyamine site antagonists, eliprodil has made it the furthest, to phase I11 clinical trials (Muir and Lees, 1995), but is overshadowed by concern over the lack of specificityfor NMDA receptors. Although it is specific for the NR2B receptor subunit, it also blocks Ca2+channels as well as Na+ channels (Scatton et al., 1995). This lack of specificity could underlie the cardiovascular side-effects in clinical trials, namely the dose-dependent prolongation of the corrected QT interval. So far, C P 101 606 has not demonstrated any activity at Ca2+channels or Na+ channels, but the development of this agent is still in its infancy. Although the NR2B subunit is highly expressed in the normal hippocampus (Buller et al., 1994),the recent report of the expression of the NR2C subunit mRNA following an in Uitro hypoxic/hypoglycaemic insult (Perez-Velazquezand Zhang, 1994) warrants testing of agents specific for NRPC subunits. There are no known NRPC-specific agents, but there are agents such as PCP which slightly prefer NR2C subunits over NRSA, and there are NR2B subunits and agents like remacemide and memantine which slightly prefer NRPA and NR2B subunits over NRPC subunits, thus enabling some pharmacological differentiation of the NR2 subunits. A full characterization of the post-ischaemic EAA receptor subunits would provide insight into which compounds may show neuroprotective efficacy. It may also explain why delayed treatment of global ischaemia with NMDA antagonists is not efficacious, while treatment with AMPA antagonists and glutamate release inhibitors is. Pharmacological investigation of the NMDA receptor subunit composition is hampered, however, by the dearth of agents which can be used as tools. The best methods of investigating post-ischaemic NMDA receptor subunit composition remain electrophysiological characterization of the receptor behaviour and molecular biological characterization of the mRNA for the various subunits.

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7.6 NMDA antagonist neuroprotectionin in vitm models of ischaemia 7.6. I INTRODUCTION

As much time has been spent attempting to demonstrate mechanistic similarities between in uitro and in viva models of ischaemia as has been spent testing compounds for neuroprotection in the in uitro models. Pursuit of an in vitro replica of in viva animal models of ischaemia tends to dominate much of the work on in uitro ‘ischaemia’. This has been the impetus for the marriage of the hippocampal slice model and the culture dish model, known as the organotypic slice or organotypic culture model. These models have advantages and disadvantages over the animal models but it is important to maintain perspective when using in vitro models. Moreover, it has been far too easy for some investigators to lose perspective and criticize whole animal findings based on an extrapolation of observations made in culture dishes.

7.6.2 NEURONAL CULTURES

The observation that brief periods of exposure to low concentrations of glutamate resulted in delayed injury of primary neuronal cultures (Rothman et al., 1987),similar to that observed in selectively vulnerable brain regions after transient forebrain ischaemia (Kirino, 1982), initiated a plethora of in vitro excitotoxicity studies using primary neuronal cultures. The models vary considerably, as follows: the timing of the insults varies because some investigators apply EAAs for a brief period (5-30 minutes) (Durkin et al., 1996; May and Robison, 1993),and some for extended periods (12-24 hours) (Rothman et al., 1987; Koh et al., 1990; Zorumski et al., 1990);(2) the severity of the insults varies in that the concentration of NMDA applied range from 1-50 VM (Koh and Choi, 1988); (3) different brain regions are used to obtain the primary cultures, namely, cerebellar (Eimeri and Schramm, 1991a), hippocampal (May and Robison, 1993), and cortical (Koh et al., 1990) regions; (4)the endpoints used to measure viability vary, including morphology (Koh et al., 1991; Schramm et al., 1990; Goldberg et al., 1987a; Choi et al., 1988), LDH release (May and Robison, 1993; Goldberg et al., 1987b; Choi et al., 1988),and live/dead stains like propidium iodide (Durkin et al., 1996; Felipo et al., 1993; Favaron et al., 1988). In many of these models, cell death can be inhibited by competitive or noncompetitive NMDA antagonists (Peterson et al., 1989; Eimeri and Schramm, 1991a and 1991b, Kaku et al., 1993), but not (or only marginally) inhibited by AMPA or kainate antagonists (Hartley et al., 1993; Eimeri and Schramm, 1991a; Kaku et al., 1993). The inhibition of cell death by inhibiting the expression of NMDA receptors with antisense oligonucleotides (Wahlestedt et al., 1993) lends further support for the role of NMDA receptors in the death of cultured neuronal cells. Moreover, transfection of NMDA receptors in a non-neuronal cell l i e leads to cell death following NMDA exposure (Anegawa et al., 1995).


151

It is important to keep in mind, however, that the lethality of glutamate can be altered dramatically by changing the experimental conditions. One important factor is the age of the primary cultures. Younger cultures (i.e. 8 days as compared to 14 days in u i b ) are more resistant to glutamate exposure (Choi etal., 1987).O n the other hand, neuronal explants suffer a substantial degree of spontaneous degeneration when kept in vitro for more than one week (Peterson et al., 1989; Favit et al., 1992). Changes have to be made to the medium to maintain the viability of the cultures, such as increasing the concentration of glucose (Choi et al., 1987) or KCl (Eimeri and Schramm, 1991a), and in many cases serum is added to the culture medium (Eimeri and Schramm, 1991a and 1991b; Dux et al., 1992; Erdo et al., 1990).Although the serum is withdrawn during and following the glutamate exposure for technical reasons, it has been shown that returning the serum to the medium substantially reduces neuronal vulnerability, presumably due to the protective trophic factors present in the serum (Dux et al., 1992). The merences between excitotoxicity modelled using neuronal cultures and that which occurs in uiuo could be due in part to the fact that the population of EAA receptors present in the neuronal cultures is different from that in the vulnerable brain regions ofthe adult. It has been shown that the developmental changes in EAA receptor expression is arrested in primary neuronal cultures (Paschen et al., 1995).There is also evidence suggesting that, following an ischaemic insult in Vivo, there is an altered gene expression programme (Somogyi et al., 1995; Pelligrini-Giampietro et al., 1992; Pollard et al., 1993a), similar to that observed following myocardial infarction (GidhJain et al., 1995; Izumo et al., 1987), which may include EAA receptors (PelligriniGiampietro et al., 1992; Pollard et al., 1993a).The utility of neuronal cultures in studies of excitotoxic mechanisms should not be understated. Some striking similarities have been uncovered including hypothermic neuroprotection (Bruno et al., 1994) and neuroprotection with delayed application of AMPA antagonists but not NMDA antagonists (Prehn et al., 1995).Nevertheless, care must be taken when extrapolations are made to in uivo ischaemia.

7.6.3 BRAINSLICES

In uitro systems in general, and brain slice preparations in particular, offer many advantages over in uivo techniques. First there is total control of the extracellular environment by the investigator due in part to the removal of the blood-brain barrier, permitting the investigator to apply drugs directly to the region of interest. Second, the investigator can visualize the brain region under study. Third, brain slices are ammenable to functional, biochemical and morphological analysis. Fourth, anaesthetics are not required. Another reason is that the natural architecture is somewhat preserved so that functional synapses can be assayed. Finally, slices from one animal can be separated into several treatment groups to be compared to one another. There are some disadvantages, however; severed afferent and efferent nerves mean the neuronal environment is artificial; there is a lack of behavioural output; and there is a lack of blood-borne components.

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Hippocampal brain slices are used in an attempt to examine a system which is more representative of in vivo ischaemia than that of neuronal cultures. By reducing glucose concentrations in the presence of mild hypoxia, conditions resembling those of ischaemia can be achieved (Schiffand Somjen, 1987),including a selective vulnerability of the CA1 neurones (Aitken and Schiff, 1986). Similar to the culture models, many of these experiments are designed with the expectancy of not only identlfylng basic mechanisms of tissue damage but also using the slice as a screen for testing possible protective therapies. Many studies have demonstrated that NMDA antagonists, especially those which are non-competitive like MK-801 (Table 2), are neuroprotective in slice models (Armstrong, 1991; Bickler and Hansen, 1994; Clark and Rothman, 1987; Rader and Lanthorn, 1989;Wallis et al., 1992).Others have reported a lack of protection with NMDA antagonists, especially those which are competitive like AP5 and CGS 19755 (Aitken et al., 1988; Schurr et al., 1995; Donevan and Rogawski, 1993; Tarnawa et al., 1989). Some speculate that the neuroprotection demonstrated with the non-competitive NMDA receptor antagonist MK-80 1 is due, in part, to its efficacy as an L-type calcium channel blocker (Schurr et al., 1993). Therefore, caution should be exercised when extrapolating to in vivo models. Positive results in an in vitro screen for a protective agent should always require further testing in in vivo models. Conversely, given that so many agents have been shown to be neuroprotective in in vitro models, more in culture models than in slice models, perhaps agents should first be screened in those in vivo models of cerebral ischaemia which are least likely to yield a successful candidate, thus limiting the number of neuroprotective candidates that need to be tested in in vitro models. The in vitro models increase the stringency of the screening process and could provide significant information on the mechanism(s) of action of an agent under consideration. Stroke in humans is an incredibly heterogeneous disease which would be best represented by the combined effects of a drug in a number of different in vitro models.

7.6.4

ORGANOTYPlC CULTURES

Although brain slice models offer distinct advantages over primary culture models, the metabolic state of brain slices is compromised, thus limiting their survival in vitro to several hours. This seriously hinders attempts to study the phenomenon of delayed neuronal death common to in vivo models ofischaemia. Organotypic culture models (Straser and Fischer, 1995; Vornov et al., 1991, 1994; Buchs et al., 1993; Caeser and Aertsen, 1991; Gahwiler, 1981, 1988; Newell et al., 1990; Stoppini etal., 1991; Torp etal., 1992; Vornov and Coyle, 1991) were developed to overcome this barrier. These in vitro models have reproduced elements of the time course (Vornov et al., 1994),regional vulnerability (Vornov et al., 1991; Newell et al., 1990)and pharmacological sensitivities, including NMDA receptor-dependent excitotoxicity (Vornov and Coyle, 199l), of in vitro ischaemic hippocampal injury. In fact, NMDA antagonists like MK-801 can provide significant protection against an ischaemic insult even when applied to the organotypic hippocampal culture after a delay of 30 minutes (Vornov et al., 1994).Although these


153

cultures are made with slices from neonatal rats, the tissue appears to mature to resemble the adult hippocampus over the course of weeks in culture (Vornov et al., 1994), unlike primary neuronal cultures whose maturation is stunted (Paschen et al., 1995).To what degree is the regional vulnerability to ischaemia due to the preservation of these intrinsic neuronal properties and to what degree is it due to the pattern of synaptic connections maintained in the explant? Further studies of this model should reveal the answer and provide insight into the development of neuroprotective strategies.

7.7 NMDA antagonist neuroprotection in in vivo models of ischaemia 7.7. I INTRODUCTION Much of the current understanding of the clinical condition of stroke has come from an ability to carefully model cerebral ischaemia in animals. Given that human ischaemic stroke is a cerebrovascular disease, the presence of an intact vasculature, something obviously lacking in in uitro brain slice and cell culture models, should be an integral component of experimental models aimed at the investigation of ischaemic damage caused by vascular occlusion. The reversibility of experimental ischaemia and selective vulnerability, in terms of anatomical space and temporal profile, has been well characterized by the early pioneering work of several investigators (e.g. Hossman and Kleihues, 1973; Siesjo, 1981; Speilmeyer, 1925; Pulsinelli and Brierley, 1979). Experimental models initially concentrated on global ischaemia for mechanistic studies, but their popularity has waned as a push to develop models which more closely approximate the clinical condition has resulted in a myriad of both transient and permanent rodent focal models (McAuley, 1995) for use in therapeutic testing. Unfortunately, in spite of the fact that focal models more closely represent the pathophysiology of stroke most frequently observed in the clinic, these models fail to accurately predict the efficacy of therapeutic strategies. A model which is shown to be predictive will be desirable by those interested in producing therapeutic agents, even if it has limited similarity to the known pathophysiology of stroke (see Chapter 3). Currently, global ischaemia models are among the most stringent in testing for potentially neuroprotective agents, yet for a compound to be accepted for costly and timeconsuming clinical trials, positive results should be obtained from a number of different models. The heterogeneous nature ofstroke as a disease means that it is likely to be best represented by a number of different in vitro and in vivo models.

7.7.2 GLOBAL OR SNERE FOREBRAIN ISCHAEMIA MODELS Global ischaemia, by definition, requires reperfusion. Following what is ordinarily brief (5- 15 minutes), severe forebrain ischaemia, there is selective injury to cells in the striatum (medium and small cells), pyramidal neurones in hippocampal regions CA1 and

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CA4, and neurones in layers 3, 5 and 6 of the cortex (Pulsinelli et al., 1982). These neurones die after variable periods of time and often after a delay. Although striatal neurones die within 6 hours following the insult, there are very slow mechanisms which cause continued degeneration such that cells in the hippocampal CA1 region can remain viable for up to 7 days. The most popular models ofglobal ischaemia are the 4-vessel occlusionin rat and 2-vessel occlusion in rat and gerbil, but the methods of inducing severe forebrain ischaemia range from cerebrospinal fluid compression and cardiac arrest, to neck tourniquet and decapitation (Ginsberg and Busto, 1989). The advantages of the 2-vessel occlusion model over other models are: (i) it is a one-stage surgical procedure; (ii)ventilation can be controlled, thus ensuring normoxia and normocarbia; (iii) cerebral recirculationcan be instituted easily; (iv)it is suitable for chronic survival studies; (v) there is a lower experimental failure rate; (vi) it is a well-established model, having undergone rigorous histopathological, neurochemical and neurobehavioural studies; (vii) it gives predictable and reproducible neuronal damage; and (viii)the severity of injury correlates with ischaemic duration. The disadvantages associated with this model are as follows: (i)anaesthetics and drugs are used, complicating the interpretation of the outcome; (ii) there is some interanimal inconsistency in cerebral blood flow and pathological outcome; (iii)post-ischaemic seizures may occur following longer periods of ischaemia; and (iv) the degree of ischaemia cannot be assessed immediately by observing behavioural changes. The 4-vessel occlusion model is performed on awake animals so that monitoring behavioural changes permits immediate assessment of the degree of ischaemia induced. This model shares many of the advantages of the 2-vessel occlusion model, namely: (i)ease of instituting cerebral recirculation; (ii) suitability for chronic survival studies; (iii) it is a well-established model, having undergone rigorous histopathological, neurochemical and neurobehavioural studies; (iv) predictable and reproducible neuronal damage results; and (v) the severity of injury correlates with ischaemic duration. However, considerable technical skill and finesse are required to surgically prepare the animals for the induction of ischaemia. In some laboratories approximately 50% of the rats will survive the firststage procedure, have forebrain ischaemia of a sufficiently high grade, and avoid the possible complications of acute death from brain stem ischaemia and post-ischaemic seizures. There is an apparent discrepancy in the efficacy of NMDA antagonists in models of severe forebrain ischaemia, even among studies using a common model and species (Buchan, 1990).This is almost certainly due to the huge number of variables remaining, many of which are within the investigator’s control. These include choice of the severity and duration of the ischaemic insult, the antagonist to be tested, the dose and route of administration, the timing of administration in relation to ischaemia and reperfusion, and the assessment of outcome, be it behavioural, biochemical or pathological following one or more periods of recovery. O n examination of studies investigating the neuroprotective efficacy of MK-801 in various models of global ischaemia, it was found that positive results were obtained by laboratories in which severity of insult was less, than in laboratories in which results were negative (Pulsinelli and Buchan, 1990). Physiological variables are also a big source of the apparent vari-


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ability in the efficacy of NMDA antagonists. Blood and brain glucose can be altered by drugs and/or diet. Control of body temperature is essential because mild hypothermia, either as a result of anaesthesia or NMDA antagonists like MK-80 1, result in significant neuroprotection against cerebral ischaemia (Buchan and Pulsinelli, 1990). Moreover, during cerebral ischaemia, brain temperature may fall more than core or rectal temperature. There are a few reports of neuroprotective agents such as FRll5427, a high-affinity non-competitive NMDA antagonist, which do not result in hypothermia (Katsuta et al., 1995), but there is still the issue of adverse side-effects; FRll5427, like MK-801, produces vacuolization. Thereapeutic ratio aside, when all of the variables associated with the global models are considered, the efficiency of NMDA antagonists tested in global models of cerebral ischaemia is less than that obtained with either AMPA receptor antagonists or N-type Ca2+channel antagonists (Buchan et al., 1994; Xue et al., 1994). The mechanisms of neuronal injury in global models may include altered expression of EAA receptors, and agents specific for these receptors may prove more efficacious. There is an initial up-regulation of NR1, NR2A and NR2B receptor subunit expression in the most regions of the hippocampus following ischaemia (Heurteux et al., 1994), followed by a significant drop in expression preceding the death of the vulnerable regions (Sugimoto et al., 1994). Following myocardial infarction there is a re-emergence of fetal or developmental gene programmes (Gidh-Jian et al., 1995). Although few studies have yet addressed whether a similar phenomenon occurs following cerebral ischaemia, the results are encouraging: (i) nestin, a developmental form of a cytoskeletal protein, and embryonic forms of GAD are expressed following an EAA-induced hippocampal injury (Somogyiet al., 1995);(ii) there is an increase in the expression of a developmental isoform of a microtubule-associated protein (MAP~c),following a model of global ischaemia in rats (Saito et al., 1995);(iii) following global ischaemia in rats, GluRB receptor subunits, which are not expressed until 2 weeks after birth, decrease relative to the subunits which are present in abundance throughout development (Pelligrini-Giampietro et al., 1992); (iv) following an in Vitro model of global ischaemia there is expression in the hippocampus of NR2C, an NMDA receptor subunit found only in the hippocampus during development (PerezVelazquez and Zhang, 1994; Zhang et al., 1995); and (v) the adult GABAA receptor subunits are decreased (Inglefield et al., 1995; Li et al., 1993). NMDA antagonists have been demonstrated to be neuroprotective using focal models of cerebral ischaemia. The pathophysiologicalfeatures of global ischaemia are distinct from focal ischaemia, allowing for a variety of alternative explanations for the differences in efficacy of NMDA antagonists in focal and global models of ischaemia.

7.7.3 FOCAL MODELS OF CEREBRAL ISCHAEMIA Rat models of middle cerebral artery occlusion are by far the most prevalent models of focal ischaemia, but there exist a vast number of variations on this model (McAuley, 1995; Ginsberg and Busto, 1989). Models of focal ischaemia employ either

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permanent or reversible occlusions. With permanent occlusion, a densely ischaemic core of infarcted tissue results, in which the cells are irreversibly damaged and no pharmacological intervention is possible. Surrounding the core is a penumbral region (1-2 mm perimeter) in which the cells are altered ischaemically in such a way that they are not irreversibly damaged but are at risk of succumbing to the insult within a period of time if some therapeutic intervention is not attempted. Blood flow to the core is zero, or near zero, while the blood flow to the penumbra is compromised to varying degrees, depending on the particular model (McAuley, 1995).The occlusions can be induced by a variety of techniques which include cauterization, clips, threads (ligation or intraluminal insertion), endothelin- 1 administration, injection of emboli or microspheres, or photochemically induced thrombosis (McAuley, 1995). The techniques which require little surgical manipulation, like those involving thromboembolytics, suffer from a heterogeneity in the size and/or location of the infarct. Although this characteristic is a better representation of stroke in humans, the need for control over variables is absolute in any experimental model. The variables which have been identified as leading to inter- and intra-model variability (McAuley, 1995; Duverger and MacKenzie, 1988) include: rat strain, weight, age and supplier; anaesthetic used; physiological variables such as arterial pressure, body temperature, brain temperature, blood gases and blood glucose hematocrit; and type, location, extent, and duration of occlusion, as well as histological artifact due to fixation procedures. Also to avoid inter- and intra-model variability, care should be taken in the analysis of the data obtained. Precise language and strict guidelines must be used in reporting histological damage. In focal ischaemia models, NMDA antagonists have typically reduced the area of ischaemic damage by about 50% but in almost all cases this protection is maximal in the cortex and minimal in the striatum. The reason for a much greater efficacy of NMDA receptor antagonists in the cortex over the striatum may be due in part to the fact that there are fewer NMDA receptors in the striatum than in the cortex. Alternatively, the poor efficacy in the striatum may be related to the severity of ischaemic in this area relative to the cortex. However, in order to get protection with most NMDA antagonists, they must be given either before, or in a narrow temporal window (1-3 hours) within the onset of occlusion. This is in clear opposition to neuroprotection with N-type channel Ca2+ antagonists in models of global ischaemia, where agents can be given as late as 24 hours after the insult (Buchan et al., 1994). Generally, the neuroprotective efficacy of NMDA antagonists is less in models of global ischaemia than in models of focal ischaemia. In addition to differences in blood flow, many other pathophysiological features of global ischaemia are distinct from focal ischaemia, allowing for a variety of alternative explanations for the differences in the neuroprotective efficacy of NMDA antagonists. The large number of mechanisms underlying the pathophysiology of focal ischaemia (Siesjij, 1992a,b) increases the likelihood that NMDA receptors may be involved and hence that NMDA receptor antagonists would prove neuroprotective. NMDA receptor antagonists from all classes have demonstrated neuroprotection (Table 2). Some NMDA antagonists, like dextromethorphan (Lo and Steinberg,


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199l), CGS- 19755(Takizawa et al., 199l), and MK-80 1 (Buchan et al., 1992),enhance regional cerebral blood flow and thus lessen the severity of the insult. There are, however, some reports of neuroprotection with these compounds without there being an effect on regional cerebral blood flow (Hossman, 1994; Park et al., 1988b).It is difficult to separate the blood flow effects from the inhibition of excitotoxicity by antagonizing NMDA receptor activity on the neurones at risk. It is of paramount importance to demonstrate neuroprotection based on the comparison of identical insults. If the severity of the insult varies between control and treatment groups within experiments designed to predict the efficacy of a neuroprotective compound, the accuracy of the predictions made would be far less than anticipated.

7.8 How to determine what goes to trial Above all else, a candidate for the therapeutic intervention of stroke in humans must be safe and devoid of serious side-effects, regardless of how neuroprotective the compound proves to be in in vivo animal models. A compound must have a good therapeutic ratio, unlike many of the high afiity, non-competitive NMDA receptor antagonists, like MK-80 1. The psychotomimetic effects and vacuolization associated with these compounds have become benchmarks to the extent that the importance of the cardiovascular side-effects associated with compounds like eliprodil are easily overlooked in preclinical trials. Not all non-competitive compounds will necessarily exhibit the negative side-effects associated with the high-afiity compounds. Much attention is being given to some of the low-affinity non-competitive compounds which are being looked at for the treatment ofepilepsy (Rogawski, 1993; Kemp and Leeson, 1993). Another issue to be considered in the development of a candidate for the therapeutic intervention ofstroke is the timing of treatment. How soon does treatment need to be initiated following the onset of ischaemia? This issue is particularly germane to NMDA receptor antagonists since they are protective in animals only when administered within 3 hours of the onset of ischaemia. The patient may have to recognize the symptoms associated with stroke (not a trivial matter for nalve victims of a transient ischemic attack (TIA)), get to a clinic or hospital, be seen by a physician, and have treatment initiated all within this narrow temporal window. In the USA, the median delay from onset to first contact with a physician has been reported to be 4 hours, and median time to contact with a neurologist 10.5 hours (Feldman et al., 1993).This may be unsatisfactory, however (see also the discussion in section 3.4.2). Alternatively, for a few cases such as prophylactic treatment for ischaemia (Fisher et al., 1994),indicated for patients with a history of TIAs or at risk of cerebrovascular disease, compounds which can be given before the insult would be useful. The duration of treatment then becomes more of a concern. Even the safest candidates to date are not likely to be well tolerated for extended periods of time. The duration of treatment is also pertinent for treatment following

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ischaemia. How long do the neurones at risk have to be treated before they are no longer at risk? Presently, there are few laboratories actively addressing this question. The outcome measure in most animal models is not more than 7 days for global and 3 days for focal models. As mentioned earlier, there is some experimental evidence suggesting that there is a change in the EAA receptor subtypes following cerebral ischaemia. Are they capable of reverting back to pre-ischaemic subtypes? If yes, how long do they take to revert? This would impact the duration of treatment. Again, tolerability of the compound becomes more of an issue if long-term treatment is hdicated. Could treatment initiate a reversion to the pre-ischaemic subtypes? Treatment with thyroid hormone signals the post-infarcted myocardium to re-express the adult isoform of myosin heavy chain and terminates the expression of the fetal isoform (Izumo et al., 1987). Another important issue in determining what goes to trial is which outcome measure(s) is chosen for the experimental models of cerebral ischaemia. Most outcome measures of studies of ischaemia in animal models are either quantified in terms of neuronal damage in the hippocampus in global models or cortical infarct volumes in focal models. The variables that affect these outcome measures like temperature¶blood pressure, blood gases, and severity of the insult, are controlled. These outcome measures have been chosen so that they may be measured precisely and comparisons made between studies, and so that they are easily affected. Such is the case for proximal MCAO focal models in which the striatum is always infarcted and irreversibly damaged. The relationship of the outcome measure of this model (the reduction in volume of cortical infarct) has little relevance to hnctional outcome. Damage to the striatum would leave the subject paralysed. Ideally, the outcome measures would be multiple, and include morphological, biochemical, functional and behavioural assays. Within reason, the outcome measures should be made at several time points, extending the window of observation following reperfusion, as the recovery of a patient is ideally over years and not just within one week.

7.9 Conclusion A treatment for stroke using NMDA receptor antagonists seemed promising at the beginning of the decade, when several clinical trials were launched. The less than desired success of some of these trials has dampened enthusiasm for NMDA receptor antagonists. The substantial number of patients and expense necessary to detect a therapeutic candidate’s beneficial effect in the clinic promises to dissuade further efforts with NMDA receptor antagonists unless reliable and predictive animal data are obtained with compounds which exhibit better therapeutic ratios. The interest in lowaffinity non-competitive antagonists, given their good therapeutic ratios, may yield successful candidates. Investigation into the expression profile of the post-ischaemic NMDA receptor subtypes may provide insights into new targets as yet unexplored. Currently, interest is with NRPB-specific antagonists like eliprodil and CP 101 606,

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given the high expression levels of NR2B in the hippocampus under normal conditions. Perhaps an NR2C-specific compound would be a better choice given the observation of an increase in expression following ischaemic-like conditions in vitro (Perez-Velazquez and Zhang, 1994).Regardless of the class of compounds considered for the next round of clinical trials, several predictive animal models, even if the resemblance to the pathophysiology of stroke is minor, must be used to demonstrate both efficacy and safety. Furthermore, the efficacy and safety of a compound must be considered over extended reperfusion periods in the eventuality that treatment is required for prolonged periods. Studies of this nature, performed over the next few years, should bolster confidence about the probability of a successful outcome with NMDA receptor antagonists.

Acknowledgements This work was supported in part by the Heart and Stroke Foundation of Ontario, grant No. ST27 17, and the National Research Council of Canada. We wish to thank Paul Morley for his critical reading of the manuscript and helpful comments.

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Chapter 8

DEVELOPMENT OF THE NMDA ION-CHANNEL BLOCKER, APTIGANEL HYDROCHLORIDE, AS A NEUROPROTECTIVE AGENT FOR ACUTE CNS INJURY Robert N. McBurney Cambridge Neuroscience, Inc., One Kendall Square, Cambridge, MA 02139, USA 8. I

8.2 8.3 8.4 8.5

8.6 8.7

Introduction 8. I.I The medical, societal and economic need is substantial 8. I.2 Regeneration of the CNS: a long-term goal 8. I.3 Protecting the CNS against damage: a near-term goal 8. I.4 Neuroprotective therapies pursued in the clinic Difficulty of developing drugs for acute CNS injury 8.2. I It can’t be done! 8.2.2 It can be done! Development of NMDA antagonists for acute CNS injury 8.3. I Discovery of NMDA antagonists: excitement of the 1980s Development of NMDA antagonists: realities of the 1990s 8.3.2 Aptiganel hydrochloride: from laboratory to clinic 8.4. I Basic concepts Preclinical studies of aptiganel hydrochloride 8.4.2 Clinical experience 8.5. I Normal subjects 8.5.2 TBI patients 8.5.3 Stroke patients 8.5.4 Clinical experiences: human pharmacokinetics Comments on the progress of aptiganel hydrochloride The future Ac knowledgements References

i73 173 I74 I 74 175 i76 176 176 I77 177 178 I 79 I79 I80 182 182 182 186 192 192 193 194 I94

8. I Introduction

8. I. I THEMEDICAL, SOCIETAL AND

ECONOMIC NEEDIS SUBSTANTIAL

Stroke and severe traumatic injuries of the head and spine are among the most devastating of medical incidents. Together, each year, they kill or severely disable many Academic Press Limited Copyright 0 1997 All rights ofreproduction in anyfarm reserved


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hundreds of thousands of people in the USA and millions worldwide. Stroke is the third most common cause of death in the USA, and severe head injury is the most common cause of disability in young males. The annual economic cost of these medical disasters for the USA has been estimated at up to $80 billion. Beyond simple financial estimates, it is dimcult to quantifir the personal, family and societal effects of these medical conditions, which can leave survivors with profound dysfunction of the nervous system. As a result of loss of mobility, cognitive deficits, inability to communicate effectively or lack of control of basic body functions, senior citizens can face the prospect of long-term dependency, including institutional care; healthy young people can have most of their hopes and dreams for the future destroyed. In common with many major medical conditions, in one way or another these nervous system-damaging injuries affect a vast number of people beyond those who are directly injured.

8. I .2 REGENERATION OF THE CNS A LONG-TERM GOAL Unlike the nervous systems of some more primitive organisms, the mammalian central nervous system (CNS) does not have the capacity to regenerate following damage. Although neuroscientists are searching for the clues that will make it possible, at some time in the future, to generate new cells and appropriate interconnections, it is unlikely that the regeneration of complex functions of the nervous system like speech, motor programmes and cognitive prowess will be effectively regenerated in the same time frame. At present, scientists can only just see the faintest glimmer of the ‘starburstofknowledge’that they will need to achieve the ultimate goal for a victim of CNS damage: a complete return to normal function.

8. I .3 PROTECTING THE CNS AGAINST DAMAGE: A NEAR-TERM GOAL The immediate hope for a breakthrough in the medical approach to acute CNS damage is to prevent irreversible damage, or, at the very least, to limit its extent by some form of pharmacological intervention. Over the past decade or so there have been tremendous advances in understanding pathophysiological mechanisms which lead to nerve cell destruction in stroke and CNS trauma. Perhaps the most surprising revelation has been that all cells at risk do not die in a matter of minutes to an hour, even though they may lose their ability to function; the total brain or spinal cord damage resulting from a stroke or trauma can take hours or even days to reach its maximum extent. The CNS tissue damage progresses over this time from the regions which are most metabolically compromised by lack of adequate blood flow, to those regions that are less compromised. The cascade of biochemical processes leading to cell destruction usually begins with a deficiency in the supply of essential metabolic substrates (oxygen and glucose) and involves excessive activity of excitatory neurotransmitters (glutamate and aspartate), cellular

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calcium ion overload, the abnormal activation of calcium-activated proteases and lipases, and the generation of toxic free radicals. Crucial in determining the fate of any individual nerve cell in a metabolically compromised region of the CNS is the relationship between the extent of its calcium ion overload and its ability to generate adenosine trisphosphate (ATP) as the energy source needed to extrude calcium ions from a cell or to ‘hide’ calcium ions within a cell and thereby limit cytosolic calcium ion levels to those below which cell-destructive processes are abnormally activated. In regions of the CNS where even suboptimal amounts of ATP can be synthesized from a reduced supply of metabolic substrates, cells can avoid their ultimate fate for extended periods of time. Animal studies have definitively shown that a variety of interventions, commenced after the initial insult to the brain or spinal cord, can limit the extent of CNS damage.

8. I .4 NEUROPROTECTIVE THERAPIES PURSUED IN THE CLINIC A number of ‘front running’ therapeutic approaches to limiting acute CNS tissue damage have arisen from research into the pathophysiological mechanisms of stroke and CNS trauma. The first approach is to reduce the rate of all metabolic processes in the CNS by reducing the temperature of the tissue. In small animals just a few degrees Celsius reduction in tissue temperature has been shown to be effective in limiting tissue damage (Ginsberg et al., 1992)and, while technically dimcult to undertake in man, this approach is now the subject of a full-scale clinical trial for head injury victims. The second general approach applies to a subset of stroke victims. For the forms of stroke in which cerebral blood vessels are blocked by blood clots, it is possible to restore tissue blood flow and the ATP-generating capacity of cells by ‘dissolving’ the clots through local stimulation of ‘clot-busting’ enzymes. Clinical trials have shown some success, although they are not without dangers. The third general approach is to limit the extent of calcium ion overload by shutting down the main pathways through which these ions enter nerve cells in metabolically compromised CNS tissue. Since the predominant pathway for calcium ion entry into nerve cells in ischaemic CNS tissue is via the excitatory-amino-acid-activatedK methyl+-aspartate (NMDA) receptor-channel complex, a considerable effort has focused on the discovery and development of small organic molecules which can inhibit the ion fluxes induced by activation of this receptor-channel complex. One example of such an effort, the discovery and development to date of aptiganel hydrochloride (CNS 1102, CERESTAT@, a registered trademark of Cambridge Neuroscience, Inc.), a molecule which blocks the open ion-channel of this receptorchannel complex, is the main topic of this chapter. By the time this chapter is available for general reading, the interim findings outlined below will have been presented elsewhere in a number of forms and will almost certainly be superseded. Furthermore, within a year from the date of publication, the interim and final results ofpivotal clinical trials with this particular compound in stroke and traumatic brain injury FBI) should be known. Therefore, to make the article

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worth writing (and, hopefully, reading), the author has included some general observations on the development of therapies for the acute phase of CNS injury and some of the rationale behind the discovery and development of apitganel hydrochloride.

8.2 Difficulty of developing drugs for acute CNS injury 8.2. I ITCAN’T

BE DONE!

About 10 years ago a distinguished neurosurgeon was heard to say something to the effect that if one desired to guarantee the failure of a drug in clinical trials then one should develop the drug as a treatment for stroke! But for two examples that come to mind, his point of view remains unassailed. The two examples, nimodipine for the prevention of recurrent cerebral vasospasms in subarachnoid hemorrhage and recombinant human tissue plasminogen activator (rt-PA)given within three hours of the onset of an occlusive stroke, are applicable to such a small number of stroke victims that they do not qualif\j as having sufficient impact to overthrow the neurosurgeon’s assertion. This is particularly true since, at the time of writing, only the nimodipine example has resulted in a therapeutic approach that is approved by health authorities for widespread clinical practice. The history of the clinical development of drugs, and other interventions, which are designed to limit the extent of CNS tissue damage in stroke, TBI and spinal cord injury is indeed a chronicle of failed projects and unmet expectations. Two major factors have probably contrived to bring about this disappointing situation: (1) the complexity of the clinical situation, and (2) the lack of animal models of acute CNS injury which can predict the clinical effectiveness of the therapeutic intervention. On the clinical side, every individual acute CNS injury has a different set of characteristics, including concomitant medical conditions, and it is almost impossible to select, particularly in the time period available, a homogeneous population of patients for a clinical trial. More importantly, until recently, only patients whose CNSdamaging incidents were associated with acute trauma were given the status of medical emergencies and managed aggressively. Since, from the metabolic argument above, time to therapeutic intervention is clearly an important factor, a therapy was doomed to be ineffective if it was given to a patient at a time after the last ‘deathdefying’ nerve cell had lost the battle to get control of its calcium overload.

8.2.2 ITCAN

BE DONE!

The positive results from the NIH-sponsored clinical trial of rt-PA (National Institute of Neurological Disorders and Stroke WINDS] rt-PA Stroke Study Group, 1995) provide the first indication that the clinical research community is now on the right track towards the successful development of treatments for the acute phase of CNS


177

injury. Restricting participation in the clinical trial to patients who could be treated within three hours from the onset of the stroke was probably an important determinant of the positive outcome of the trial. Other trials in which a variety of thrombolytic agents (streptokinase,urokinase and rt-PA) have been used at times longer than three hours have failed to demonstrate a beneficial efficacy to safety profile for this general therapeutic approach (see del Zoppo, 1995). Although it is unlikely that the results of this trial will have a substantial impact on the management of most stroke patients, the positive outcome for patients treated within three hours of their strokes will certainly give impetus to those groups who are trying to raise community awareness of the early symptoms of a stroke. The benefits of increased awareness will be twofold (1) an increase in the number of patients who can benefit from such early intervention when it is approved for use in normal medical practice; and (2) an increase in the population of patients who will be eligible for clinical trials which allow entry into a trial at later than three hours after a stroke. Some additional observations on the clinical investigations and animal studies to date with thrombolytics are as follows. The therapeutic approach of restoring blood flow to metabolically compromised tissue seemed obvious. Moreover, many animal studies clearly demonstrated the beneficial effects of CNS tissue reperfusion following a period of occlusion of the blood supply to the tissue. Nevertheless, it has been a formidable task to transform an obvious idea with good support from animal studies to a statistically significant beneficial effect on a clinically meaningful outcome measure in a cohort of stroke patients. The entire process, from concept through to the production of rt-PA by the techniques of genetic engineering, to the successful clinical trial, has taken about 40 years and has consumed the attention of a large number of research workers and the equivalent of hundreds of millions of dollars of both private and public finances.

8.3 Development of NMDA antagonists for acute CNS injury

8.3. I DISCOVERY OF NMDA ANTAGONISTS: EXCITEMENTOF THE 1980s

In the mid-to late 1980s, one of the most exciting advances in neuroscience was the characterization of the NMDA receptor-channel complex and the recognition of its role in normal physiological processes and pathophysiological mechanisms (Dingledine, 1986; Cotman and Iversen, 1987; Barnes, 1988). The efforts of academic research groups yielded new information about the sites on the receptor-channel complex through which the complex could be activated or its activity modulated (Wong and Kemp, 1991).A number of pharmacological tools for studying the NMDA receptor-channel complex became available through work in synthetic chemistry. In addition, some compounds that were already known, like phencyclidine, dextrorphan and MK-801, were surprisingly found to have powerful inhibitory effects on the activity of the complex (Kemp et al., 1987).

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The availability of the pharmacological tools, particularly highly selective, blood-brain-barrier-permeable molecules like MK-80 1, resulted in an explosion of in uivo studies on the roles of the NMDA receptor-channel complex. Perhaps the most remarkable studies were those which demonstrated that NMDA antagonists could dramatically limit the extent of brain damage in animals who had been subjected to experimental strokes (see McCulloch et al., 1992).Moreover, the treatment was effective, even when administered after the experimental stroke had been induced. This chapter will not comment further on the animal studies in which NMDA antagonists have been studied. Chapter 7 provides an excellent overview of the characteristics of the NMDA receptor-channel complex and of the dramatic research developments that led to the concept of post-ictus neuroprotection for victims of acute CNS injury.

8.3.2 DEVELOPMENT OF NMDA ANTAGONISTS: REALITIES OFTHE 1990s In the late 1980s the pharmaceutical industry was galvanized by the dramatic effects ofNMDA antagonists in animal models of acute CNS injury. Recognizing the medical need for and commercial potential of treatments for stroke and severe injuries of the head or spine, many pharmaceutical companies initiated projects to discover compounds suitable for advancement to clinical trials. A large number of drug candidates rapidly emerged from these efforts. Notable among these compounds were: the NMDA ion-channel blockers MK-801 (dizocilpine maleate, Merck and Co.), dextrorphan (Roche),remacemide (Fisons [now Astral) and CNS 1102 (aptiganel hydrochloride, Cambridge Neuroscience); competitive antagonists of the EAA binding site CGS 19 755 (selfotel, Ciba-Geigy [now Novartis]) and CPP-ene (Sandoz [now Novartis]); and the polyamine-site antagonist eliprodil (Synthelabo). Each of these compounds had demonstrated its ability to limit the extent of brain damage in animal models of stroke and, in some cases, in animal models of CNS trauma. The flagship compound in the demonstration of the neuroprotective effects of NMDA antagonists was MK-80 1, a highly specific and very potent ligand for a binding site within the ionchannel of the NMDA receptor-channel complex. From a host of animal studies, it was obvious that MK-801 was a powerful CNS-acting molecule with excellent blood-brain-barrier-crossing properties and the ability to preserve CNS tissue at risk of destruction as a result of brain ischaemia. At the time of writing, the number of these compounds still advancing towards definitive proof of efficacy in the clinic is greatly reduced. The development of MK801 was the first to stall and the development of others such as dextrorphan, CPPene, CGS 19 755 and eliprodil either has been terminated or has not progressed as hoped. By the time this chapter is published, there should be more information on the progress of all the members of this first group of NMDA antagonists to challenge the gauntlet of development for stroke and TBI. The exact reasons for each compound’s success or failure will probably never be understood completely, but each development campaign will surely provide a great deal of valuable information for future


179

efforts to develop therapies to limit the extent of tissue destruction in acute CNS injury. Preliminary information on the early stages of the development of some of the compounds mentioned above can be obtained by reading Grotta et al. (1 995), Albers et al. (1995), Lyden et al. (1 996), Muir and Lees (1 995), and Chapter 15. Aptiganel hydrochloride continues to progress towards pivotal clinical trials in stroke and TBI under a collaboration between Cambridge Neuroscience and Boehringer Ingelheim. There follows an overview of the progress of that compound from concept to the clinical trials.

8.4 Aptiganel hydrochloride:from laboratory to clinic 8.4. I BASICCONCEPTS At the commencement of the drug discovery project that eventually resulted in the selection of aptiganel hydrochloride as a drug development candidate, the desired characteristics of the molecule were established by the project team at Cambridge Neuroscience. Of primary importance was that the molecule should block the open ion channel of the NMDA receptor-channel complex. This choice of mechanism of action was based on the following considerations. For a non-competitive antagonist of responses mediated via the NMDA receptorchannel complex, the percentage inhibition of calcium (and other) ion fluxes would be unaffected by the extent of activation of the complex by the excessive amounts of excitatory amino acids (EAAs). Molecules with this mechanism of action were thought to be more robust in their ability to inhibit responses of the receptor-channel complex than molecules which act through competitive inhibition of either the EAA receptor site or the glycine co-agonist site on the complex. Since the percentage inhibition produced by a competitive antagonist is reduced as the agonist concentration increases, for any given concentration of a competitive inhibitor, the percentage inhibition could be reduced to zero in areas of high EAA or glycine concentrations. 2. Molecules which are cation-channel blockers commonly have two general chemical characteristics, a cationic region centred on a protonatable nitrogen and a hydrophobic region. A focus on molecules of this type was considered much more likely to generate compounds with the ability to cross the blood-brain barrier than a focus on molecules designed to interact with the sites that were naturally occupied by highly polar amino acids, especially the acidic amino acids such as glutamate. Since time to therapeutic intervention was a critical factor in the proposed clinical setting, rapid blood-brain barrier penetration of the therapeutic agent was considered essential. Concentrating on ion-channel blockers, because of their general chemical characteristics, was thought to be the best way of finding a compound with the speed of action appropriate for the envisaged clinical use. 1.

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FIGURE1 Chemical structure of aptiganel hydrochloride.

Other important properties of the drug candidate were considered to be: 1. An affinity for the ion-channel site that was high enough to afford the molecule selectivity for the NMDA ion-channel but not so high as to make the biological effect of the molecule dimcult to reverse. 2. A relatively short plasma half-life which, combined with reversibility of interaction at the ion-channel site and good blood-brain barrier permeability, would ensure that drug action could be readily terminated. 3. Good water solubility for an intravenous-use formulation. Reversibility of drug action was thought to be particularly important because of the emergency medicine context for the use of the therapeutic. Furthermore, since side-effects (especiallyCNS side-effects)were likely to occur, a readily reversible drug would allow for a more complete exploration of patient-dosing paradigms in the search for optimal efficacy-to-safety profile than could be achieved with a poorly reversible therapeutic approach.

8.4.2 PRECLINICAL STUDIESOF APTIGANEL

HYDROCHLORIDE

The drug candidate selected from this discovery effort was aptiganel hydrochloride (N-(1-naphthyl)-Nf-(3-ethylphenyl)-N’-methyl-guanidine hydrochloride, CNS 1102, CERESTATQ), a potent and selective ligand for the ion-channel site of the activated NMDA receptor-channel complex (Reddy et al., 1994). The structure of aptiganel hydrochloride is presented in Figure 1. In in vitro studies, aptiganel hydrochloride protected cultured brain neurones against exposure to toxic concentrations of glutamate (ED5,=0.38 PM) at concentrations consistent with its a h i t y (Ki) of 28 nM for the NMDA receptor ion-channel site (Kirk et al., 1994).In a simple animal model of focal and global brain ischaemia, aptiganel hydrochloride was also able to protect neonatal rats against brain damage induced by ligation of the common carotid artery followed by hypoxia (Wang et aL, 1995).An intraperitoneal dose of 6 mg/kg provided >95% protection in this model. In the rat middle cerebral artery occlusion (MCAO) model of human ischaemic stroke, intravenous administration of aptiganel hydrochloride substantially reduced (by 40-70%) the volume ofbrain damage and the concomitant neurological dysfunction. The compound was active in both permanent (Minematsu et al., 1993a; Park et


181

al., 1993) and reversible (Minematsu et al., 199313) rat MCAO models in which a variety of different intravenous dosing regimens were used. Protection was observed when aptiganel hydrochloride was administered up to one hour post-occlusion (Meadows et al., 1994). In rats, the lowest maintained plasma concentration of aptiganel hydrochloride associated with neuroprotection was -10 ng/ml, which was achieved and maintained by a bolus dose of 0.25 mg/kg followed by a three-hour continuous infusion of 0.17 mg/kg/hr. This plasma concentration of aptiganel hydrochloride was associated with a mild degree of ataxia and/or sedation. Increased sedation occurred with higher doses of the compound and higher plasma levels, but the degree of neuroprotection was maintained. Results from general toxicology studies performed to date, including 14- and 90day intravenous studies in rats and cynomolgus monkeys, indicate that aptiganel hydrochloride can be administered safely for the projected clinical usage. In specialized toxicology studies, the propensity of aptiganel hydrochloride to induce neuronal vacuolization (Olney et al., 1989)in the cingulate gyrus and retrosplenial cortex of the female rat brain was examined. For subcutaneous administration, the threshold dose for inducing vacuoles is approximately 1 mg/kg. Vacuolization was reversible within 12 hours, even following doses as high as 10 mg/kg S.C.No vacuoles were observed after 0.25 or 2.5 mg/kg, administered intravenously. The pharmacokinetics of aptiganel have been examined across species. Following intravenous administration, aptiganel distributes rapidly (9 Yo of Total

5

5 2

0 0

3 1

31%

44%

4

2

7 3 4 0

2 1%

48%

0 3 1

Severe Vegetative/dead 1

5

5 0 1

1 1 0

28%

28%

These included personality changes such as mood swings, depression, and anxious agitation. Two patients had continuing seizure activity that was directly attributed to the injury. None of these or any changes in clinical laboratory measures were associated directly with aptiganel hydrochloride. A second study (Study 005) has been completed recently. The objectives of the study were to assess the safety of extended (1 2-72 hours) infusions of aptiganel hydrochloride administered within 8 hours of a severe traumatic head injury (GCS 4-8). Assessments of safety were based on vital signs, ICE cerebral perfusion pressure (CPP) and clinical laboratories. The drug dose was the highest examined in the previous study - 100 pg/kg followed by an infusion of 40 pg/kg/hour (approximately 1 mg/kg/24 hours). Six patients were treated in each of the four ascending duration groups of 12, 24, 48 and 72 hours. Nine patients received placebo in this phase. In addition, 14 patients were treated in a second, open-label phase of the study using

DEVELOPMENT OF Ah' NMDA ION-CHANNEL BLOCKER

185

a fixed dose calculated to deliver and maintain a plasma drug level of at least 30 ng/ml. The first patient enrolled in the study was discontinued due to the occurrence of seizures observed within the first hour of infusion. An error by the pharmacist resulted in the patient receiving 10 times the prescribed amount of drug. The patient received 1 mg/kg/l5 minutes followed by 400 pg/kg/hour for approximately two hours. There were no clinically significant changes in vital signs, including blood pressure. The next day, a repeat C T showed an increase in cerebral oedema. The standard therapy was continued with no recurrence of seizures. Within a week, the patient was greatly improved with a GCS 14 from an initial GCS 6. He had occasional episodes of confusion but was fully oriented and alert with a discharge GOS of 2 (moderately disabled) at one week. There did not appear to be any short-term sequelae. At day 90, the patient's GOS was 2 and his disability rating scale (DRS)score was 4. Plasma drug concentrations for this patient were monitored over the 24 hours after administration of the drug was halted. At the end of the infusion, the level was 148.9 ng/ml. After 24 hours, the level had declined to 16.8 ng/ml. The clearance was estimated to be 16.88 ml/min/kg, which is within the range observed for normal volunteers and elderly stroke patients. Over the entire study, there were no adverse drug effects observed on any of the physiological parameters measured (intracranial pressure, mean arterial pressure, cerebral perfusion pressure). With the exception of the first patient described above, discontinuation of the drug infusion occurred only when a patient was determined to have died. The target plasma level of 30 ng/ml was achieved for all infusion durations. The plasma kinetics of the drug were unchanged by the length of the infusion. In addition, a non-weight adjusted dose yielded a consistent plasma level independent of body weight. The clearance was approximately 22 ml/min/kg, which does not differ from that observed in elderly stroke patients or normal volunteers. The open-label portion of the study enrolled a number of patients with a poor prognosis, including patients with no activity on EEGs due to extended periods of increased ICP (> 30 mm Hg). Seven of the 14 patients died or were removed from life support within the first week after the injury. Over the entire study, the death rate was within the expected range (30%) for this severity of injury. The number of placebo patients (9) was too small to permit a comparative analysis of outcome measures. Five of these patients were admitted with a GCS of 7-8, and were under 35 years of age. These variables are indicators of a better outcome. In contrast, the drug-treated groups included an older population. As of this date, outcome data are available for 34 patients. Even though increasing age is considered a risk factor, the older patients (>36 years) who had received aptiganel had outcomes at three months that were better than expected from historical data. According to the Traumatic Coma Data Bank (TCDB), statistics for six-month outcome (the good and moderate disability outcome) is expected only for approximately 30% of patients in the 36-55 age range (Choi et al., 1994). In contrast, 55% of the patients in this age range who received aptiganel in Study 005 had a good or moderate disability

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outcome. Moreover, based on the TCDB, one could expect further improvement from three to six months.

8.5.3 STROKE PATIENTS Three clinical studies have been conducted with aptiganel hydrochloride in stroke patients. The first was a double-blind, dose-escalating evaluation (Block, for CNS1102-003 Study Group, 1995).This trial was intended to assess safety and not efficacy, so a longer time period after onset of the stroke was allowed in this study than was envisaged for an efficacy study. Patients presenting within 18 hours of an ischaemic stroke in the carotid or vertebrobasilar artery territories were screened for entry. They were required to have a minimal neurological deficit score of at least 4 on the NIH Stroke Scale (NIHSS),with the exception of those with isolated hemianopia or aphasia. A CT or magnetic resonance image (MRI)was required for confirmation of clinical diagnosis. Patients suitable for the trial were randomized (central randomization) to either an active or placebo (saline) treatment administered first as a bolus dose over 15 minutes, followed immediately by a second dose infused over 4 or 6 hours. Patients were monitored continuously for changes in vital signs and emerging clinical signs and symptoms. Standard clinical laboratory testing was performed along with neurological assessments at 12 hours, weekly during hospitalization, at discharge, and again at 30-60 days post-treatment. The Barthel Index was assessed at discharge and at follow-up. Blood samples were taken periodically to determine plasma drug concentrations. Patients were treated with either drug or placebo administered first as a loading dose over 15 minutes, followed immediately by a continuous 4-hour infusion. The dose was administered in an escalating fashion after review of each dose level for effects on vital signs, side-effects and general observations by the attending physicians as to tolerability The dose escalation was halted during administration of the drug at 30 pg/kg/ 15 minutes followed by an infusion of 20 pg/kg/hr for 6 hours (total dose 150 pg/kg) because of the preponderance of severely compromised patients enrolled in that group. No patient’s treatment was discontinued and all patients effectively completed the protocol with the exception of three patients lost to follow-up. The number of patients by group are summarized in Table 4. There was an equal distribution of sexes, with most patients being over 70 years old (within-group mean ages ranged from 69-76 years). The population was typical in that concurrent diseases such as hypertension, obesity, coronary artery disease, and diabetes mellitus were present. The within-group mean times from stroke to the beginning of treatment ranged from 9.1-12.0 hours. The stroke aetiology was classified on diagnostic and clinical assessments prior to discharge from hospital (Table 5). Most patients were hypertensive on entry and had been determined to be stable during the baseline evaluation. Isolated instances of increasing blood pressure were treated with labetalol hydrochloride or other anti-

187


TABLE 4 EXPOSURE AND MOR'TALIIT

IN STROKE PATIENTS EXPOSED T O ASCENDING INTRAVENOUS DOSES OF APTIGANEL HYDROCHLORIDE OR PLACEBO IN STUDY 003

Aptiganel HCI [total dose (pg/kg/ I5 mins pg/kg/hr X 4 hrs)]

+

Drug

30 (10+5) 32 (20+3) 30 (30+0) 50 (30+5) 70 (30+ 10) 90 (30+ 15) 110 (30+20) 150 (30+20/6 hrs)

8 5 14 9 13 13 5

3 3 3 4 2

3

Total

74

20

8

Number entrolled

Number of deaths

Placebo

7

*rug

Placebo

1 1 1 1 1

1

2 2 1

1

TABLE 5 PERCENTAGE OY PATIENTS WITH A GIVEN STROKE AKTIOLOGY BY TOAST CLASSIFICATION WITHIN EACH DOSE GROUP OF STUDY 003

Total dose (pg/kg) Stroke aetiology

50 70 90 110 150 Placebo 30-32 (n=20) (n=20) (n=14) (n=9) (n=12) (n=12) (n=4)

Large artery atherosclerosis Cardioembolism Small artery occlusion Other determined Undetermined

25% 30 20 0 25

25% 15 30 0 30

43% 21 7 7 21

33% 33 0 0 33

33% 17 8

8 33

17% 42 17 0 25

25% 50 0 0 25

hypertensive medications during the test article administration. Within-group averages for mean arterial pressures (MAP)are presented in Figure 3. Elevations in heart rate were observed, but there was no clear dose-response relationship. In the highest dose-group, increases in heart rate of -10 beadminute were recorded during the fifth and sixth hours of the infusion. A total of 94 patients were enrolled, with 74 receiving active drug at differing doses, and 20 receiving saline as the placebo. The death rate up to two months' post-stroke was typical for this population; 1/20 (5%)for placebo and 8/74 (1 1Yo) for patients who received active drug. The primary causes of death included cardiac and respiratory arrest, pneumonia and recurrent cerebrovascular events. The deaths occurred from 2-38 days following treatment, with all but two occurring at least one week posttreatment. All deaths but one were ascribed to the underlying diseases or ischaemic stroke. The exception involved a 68-year-old male treated with 30 pg/kg/ 15 minutes

188

5

R.N. McBURNEY

I

I I N = 18,20, 14,9, 13, 12 and 5, respectively I I, I I I I 70 -

I

80

I I

I

I

I

1

I

followed by 20 pg/kg/hour of aptiganel hydrochloride for 6 hours. The patient had presented with a right MCA infarct and normal blood pressure. The patient’s blood pressure was elevated prior to treatment (190/ 100)and increased during the infusion. This latter increase was treated successfullywith labetalol hydrochloride. The patient was treated for nausea, vomiting and agitation following the end of the infusion. A second C T scan the next day showed a large left hemisphere parenchymal haemorrhage contralateral to the right infarct, the cause of which could not be determined definitively because no autopsy was performed. A safety committee of three non-investigator physicians felt that it was unlikely that the haemorrhage was directly related to the administration of aptiganel hydrochloride. However, the rise in blood pressure could have precipitated the process and the drug may have contributed to the hypertensive episode. All non-fatal serious adverse events through the entire follow-up period (60 days) were recorded. None was attributed to drug administration. These included the occurrence of pneumonia, extension of stroke, and other cardiovascular events such as a myocardial infarction or sporadic episodes of dysrhythmias. These were expected events in this population, with the incidence of 7/20 (35%)placebo and 12/74 (16%) drug patients reporting. Expected side-effects (based on the signs and symptoms reported by normal volunteers) were recorded during the infusion and up to 12 hours thereafter. Table 6 presents these signs and symptoms as a per cent of all placebo or drug patients taken together. During the course of the study, all adverse events were recorded. Table 7 lists the most common of all other adverse events that occurred with an incidence of 3 5% of all patients that received active drug at any dose.

DEVELOPMENT O F AN NMDA ION-CHANNEL BLOCKER

189

TABLE 6 PERCENTAGE OF EXPECTED SIDE-EFFECTS REPORTED UP TO 12 HOURS POST-INFUSION IN STUDY 1 102403

Side-effect

Placebo (n=20)

Blurred/double vision Catatonia/non-responsive Cold extremities Disorientation/detached feeling Dry mouth/thirst Flushing Lightheaded/dizzy Nausea Nystagmus Paraesthesia Sedation/decreased responses Speech/dysarthric or sluggish Sweating Tired/weak/malaise Vomiting

0Yo 0 0 5 0 0 0 0 0 5 5 0 0 0 0

Drug (n= 74) 4 YO 4

1 10 5 3 7

11 5 5 12 7 3

10 8

TABLE 7 PERCENTAGE OF ADVERSE EFFECTS REPORTED BY 3 5% OF APTIGANEL-TREATED PATIENTS FOR THE ENTIRE STUDY003 PERIOD

Side-effect None Hypertension Tachycardia Agitation Abnormal ECG (i.e. PVCs, sagging S-T) Chest pain Headache Increased blood glucose Pneumonia

Placebo ( ~ 2 0 )

Drug

10% 5 5 5 15

20% 15 10 11 10

(n=74)

0 20 0 0

The overall incidence of any one event is low and associated with an elderly population with concurrent diseases (such as diabetes, hypertension) and medications. The mean change from baseline to follow-up (approximately 6 weeks) on the NIH Stroke Scale (NIHSS) by dose group is presented in Table 8. Between-group comparisons were not attempted since the trial had not been designed to assess efficacy. Not only was entry permitted up to 18 hours poststroke, but because of the escalating-dose and non-parallel entry design, there

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TABLE 8 MEANNIH STROKE SCALE SCORES*

IN STROKE PATIENTS EXPOSED TO VARIOUS DOSES OF AITIGANEL OR PLACEBO IN STUDY 003

Total dose (CLg/kg)

Placebo 30-32 50 70 90 110 150

Baseline Number Mean

FOIIOW-UP Number Mean

20 20 13 8 12 12 5

17 19 12 8 10 11 2

9.8 9.5 12.1 13.1 10.8 14.2 11.8

5.8 3.7 8.0 6.1 8.3 11.0 1.5

No&.*Score range: 0-42 (lowered score indicates improvement)

were differences in severity of the neurological impairment between dose groups at entry into the study (as reflected in the NIHSS scores). The Barthel Index was used as an assessment of the quality of daily living. As might have been expected, the mean Barthel values at follow-up were correlated with the mean neurological impairment at baseline, and ranged from 50-83, with the placebo having a value of 74. In summary, the drug has been well tolerated in patients experiencing an acute ischaemic event. The incidence of central nervous system complaints was low and did not approach the severity observed in the normal male volunteers. Isolated cardiovascular effects, such as increasing blood pressure and heart rate, were manageable with treatment. Two additional studies in stroke patients have completed enrolment, but neither database is finalized at the time of writing. The first (Study 008), a parallel, doubleblind comparison of three doses of aptiganel (30-110 pg/kg) versus placebo will examine the dose-response relationship by utilizing the NIH stroke scale at 7 days and 1 month after treatment. In addition, the Barthel Index and a modified Rankin will be examined at 3 months; 132 patients with an ischaemic stroke within 6 hours of the event entered into the trial. Final blinded observations are expected to occur in early 1996. In order to determine an appropriate tolerable non-weight adjusted dose for phase I11 trials, an additional dose-escalating study in patients within 24 hours of an ischaemic stroke was undertaken (Study 0 10).The primary measure for safety evaluations was an assessment of any dose-dependent significant rises in blood pressure or any other untoward cardiovascular event (e.g. hypotension) (Fayad et al., 1996). The first phase of the study included only a single dose administered over 5 minutes in increments of 1.5 mg to groups of at least 4 patients each (3 drug, 1 placebo). This phase administered doses of 3-7.5 mg. It was determined that a dose of 7.5 mg would probably be unacceptable to most physicians and patients, due to a consistent significant increase in systolic blood pressure (in some cases greater than 30 mm Hg). In


0.0 1

0

1

I

4

8

I

I

12 16 Time (hours)

1

20

191

1 24

FIGURE4 Mean ( 21 s.e.m.) plasma aptiganel levels over time for two different 12-hour intravenous dosing regimens of aptiganel hydrochloride (Study 0 10).

addition, the frequency and severity of CNS side-effects were deemed not to be adequately tolerable for large-scale studies. The second phase of the study examined several bolus doses in conjunction with an infusion selected to maintain a given plasma level for up to 12 hours. After the first 6 hours, the duration of the infusion was extended at the discretion of the investigator and research staff, with input in some cases from the patient’s family members. The highest dose studied was 6 mg initially, followed by 1 mg/hour infusion. At this dose level, most of the patients exhibited some degree of sedation, including a non-responsiveness to commands. Of the 8 patients receiving drug, only 3 underwent the complete 12 hours’ treatment. Subsequently, all but 2 of 12 patients tolerated 12 hours of a dosing regimen comprised of a 4.5 mg bolus, followed by 0.75 mg/hour. One of the 2 patients was treated for 6 hours, at which point he had to be transferred to another hospital. The second patient’s treatment was stopped after 5 hours due to hypotension secondary to the use of morphine sulphate for pain. This dose regimen achieved and maintained a drug plasma level of over 10 n g / d (Figure 4). There were three deaths in the 30-day follow-up period: 1 of 10 in the placebotreated patients (loolo)and 2 of 36 in aptiganel-treated patients (5%). Although not designed to assess efficacy, neurological function was evaluated by the NIHSS and the Scandinavian Stroke Scale (SSS) at baseline and at day 7 (or discharge, if earlier). Summary descriptive statistics are shown in Table 9 and indicate that, on average, patients receiving aptiganel exhibited greater neurological improvement than patients receiving placebo. Based on considerations similar to those discussed for Study 003, no statistical comparisons were made.

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TABLE 9 NEUROL~GICAL STATUS AS INDICATED BY MEAN NIH STROKE SCALE SCORES AND '10 IMPROVED ON THE MOTOR COMPONENT OF THE SCANDINAVIAN STROKE SCALE FOR PLACEBO AND NTIGANEL-TREATED GROUPS IN STUDY 010 Group

n

Baseline

Placebo All bolus 4.5mg+0.75/hr 6.0 mg+ 1.O/hr

10 15 12 8

9.8 10.0 12.5 8.5

Mean NIHSS scores Change -7 days 8.6 5.9 7.8 4.6

1.2 4.0 4.7 3.8

SSS Oh improved* motor score 50 80 75 75

Nok. *Gait improved by 2 3 points, or aU 3 motor components improved by 1 grade (2 points).

8.5.4

CLINICAL EXPERIENCES: HUMAN PHARMACOKINETICS

The plasma aptiganel concentration half-life in normal male volunteers was found to be longer than the half-lives of the compound in either rats or cynomolgus monkeys: approximately 4 hours, with a mean clearance of 18 ml/min/kg (Muir et al., 1994). Pharmacokinetic parameters (C,,, AUC) were found to be linear with dose in man in a similar manner to that which was found in rats, cynomolgus monkeys and baboons. The pharmacokinetics of the drug in man were not affected by the age, sex or disease state of the patients. In stroke patients (most over 70 years old), the phamacokinetics of aptiganel were found to be similar to those in normal volunteers (Block, for CNS 1102-003 Study Group, 1995). In patients with severe traumatic brain injury, drug clearance was sometimes observed to be more rapid than in normal volunteers or stroke patients (Gamzu, for CNS 1 102-002 Study Group, 1994).

8.6 Comments on the progress of aptiganel hydrochloride To date, more than 300 patients and volunteers have been exposed to aptiganel hydrochloride at doses which have produced plasma levels of the drug in the range of plasma levels that were associated with neuroprotection in animal models of CNS injury. In TBI patients it has been possible, rapidly and safely, to establish plasma levels of the drug which are well within the range of plasma levels of the compound that have been associated with neuroprotection in animals and to maintain those levels for periods up to 72 hours. In stroke patients, the cardiovascular side-effects of the compound have limited the dosing to a regimen that can rapidly produce plasma levels of the drug which are above the threshold plasma level necessary to produce neuroprotection in animals. The favourable physicochemical properties of the compound, combined with its rapid blood-brain barrier penetration and excellent reversibility of biological effects have definitely facilitated a reasonably complete exploration of safe and tolerable dosing regimens in the two patient populations. It is unlikely that such


193

an exploration would have been possible with a compound which did not have such favourable characteristics. Eventually, these characteristics might have more influence in determining the success of a particular therapeutic in stroke and TBI patients than the particular detailed mechanism of action of an agent which is designed to shut down the operation of the NMDA receptor-channel complex. Aptiganel hydrochloride is now well positioned to advance to a definitive test of the hypothesis that an NMDA antagonist can limit the extent of brain damage following a stroke or severe traumatic injury to the head or spine.

8.7 The future During 1996 aptiganel hydrochloride will enter pivotal clinical trials in both TBI and stroke patients. A statistically significant and clinically meaningful beneficial effect on the outcome for the drug-treated patients in either of these trials will provide support for the general concept of neuroprotection and for the specific therapeutic approach which focuses on NMDA ion-channel blockers. Moreover, the relationship will be strengthened between the ability of a therapeutic agent to generate positive efficacy in certain animal models and its ability to produce beneficial effects in a clinical setting. Most importantly, if the therapeutic is approved by the relevant authorities for use in normal clinical practice, the prognosis will be improved for the better for patients who suffer these forms of acute CNS injury. Success in the clinic with a neuroprotective agent will almost certainly lead to further clinical studies in which the combination of a neuroprotective drug and a thrombolytic agent will be investigated. These investigations will test the prediction of animal studies that the combination of neuroprotection and reperfusion can prevent more damage to CNS tissue than either therapeutic approach alone (Minematsu et al., 1993b). An additional and important prediction of these animal studies is that early treatment with a neuroprotective agent can extend the time period after the onset of a cerebrovascular occlusion during which reperfusion can be used safely and with efficacy The confirmation of this finding with stroke patients would greatly expand the applicability of thrombolysis to this patient population. Right now, the positive clinical benefit demonstrated in stroke patients with rt-PA is a great encouragement for ongoing efforts to demonstrate that other therapeutic approaches can also prevent or limit the extent of CNS tissue damage in stroke and severe traumatic injuries to the head and spine. Further positive results from these ongoing clinical investigations will undoubtedly convert to ‘true believers’ those who are currently sceptical of any practical therapeutic intervention being able to effect a profound change in prognosis for a patient who suffers acute CNS injury. With a sure foothold on the firm ground of clinical success, the scientific and medical communities wiU be seeking ‘new and improved’ therapies. The success of any individual therapeutic agent, particularly in this complex medical area where so many intrepid voyagers have seen their ‘molecular ships of

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hope’ wrecked on unforeseen reefs, should be attributed not just to the design of the successful agent, nor to the crew which manned the project, nor to the navigators who charted the project’s course. Because so much information on physiological and pathophysiological mechanism, molecular design, in uitro and in Vivo pharmacology, medical practice and clinical trial design will have been provided by d the successful and unsuccessful attempts, the attribution of success should go to all who have sought to improve the prognosis for victims of stroke and severe traumatic injuries to the head and spine.

Acknowledgements

I should like to thank all those who have contributed to the discovery and development of aptiganel hydrochloride, especially to Drs Bill Holt, E k a n Gamzu, Andy Knapp and John Wecker and to Ms Laima Mathews who, at various times, have led the preclinical or the clinical development. Additional thanks go to Ms Jane Wagner and Mr Mario Pita for their assistance in the preparation of the manuscript.

References Albers, G.W., Atkinson, R.P., Kelley, R.E. & Rosenbaum, D.M. on behalf of the Dextrorphan Study Group (1995) Safety, tolerability, and pharmacokinetics of the N-methybaspartate antagonist dextrorphan in patients with acute stroke. Stroke 26,254-258. Barnes, D.M. (1988)NMDA receptors trigger excitement. Science 239, 254-256. Block, G.A. for the 1 102403 Study Group (1995) Final results from a dose-escalatingsafety and tolerance study of the non-competitive NMDA antagonist CNS 1 102 in patients with acute cerebral ischaemia. Stroke 26, 185. Choi, S.C., Barnes, M.S., Bullock, R., Germanson, T.A., Marmarou, A. & Young, H. (1994) Temporal profile of outcomes in severe head injury. j! Neurosurg. 81, 169-1 73. Cotman, C.W. & Iversen, L.L. (1987) Excitatory amino acids in the brain - focus on NMDA receptors. Zen& Nirosci. 10, 263-265. del Zoppo, GJ. (1995) Acute stroke. On the threshold of a therapy? New Engl. j! Med. 333, 1632-1633. Dingledine, R. (1986)NMDA receptors: what do they do? ZeendsNncrosci.9 , 4 7 4 9 . Fayad, PB., Edwards, K., Hormes, J. & Lees, K.R. (1996) The safety and tolerability of non weight-adjusted doses of aptiganel HC 1 ( C E R E S T A ~ )in acute ischaemic stroke patients. A. H. A. February 1996. Gamzu, E.R. for the CNS 1 102402 Study Group (1 994) CERESTAT~~(CNS 1 102) an NMDA antagonist in severe traumatic brain injury. A. Jv: A. October 1994. Ginsberg,M. (1995a)Neuroprotection in brain ischaemia: an update Part I.Nirosci. 1,95-103. Ginsberg, M. (1995b) Neuroprotection in brain ischaemia: an update Part 11. Neurosci. 1, 164-175. Ginsberg,M.D., Sternau, L.L.,Globus, M.Y-T., Dietrich, W.D. & Busto, R. (1992)Therapeutic modulation ofbrain temperature: relevance to ischaemic brain injury. Cerebmvasc.Brain Metab. Rev. 4, 189-225.


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Grotta, J., Clark, W., C o d , B., Pettigrew, L.C., Mackay, B., Goldstein, L.B., Meissner, I., Murphy, D. & LaRue, L. (1995) Safety and tolerability of the glutamate antagonist CGS 19 755 (selfotel)in patients with acute ischaemic stroke. Stroke 26, 602-605. Kemp, J.A., Foster, A.C. & Wong, E.H.E (1987) Non-competitive antagonists of excitatory amino acid receptors. Trendr Neurosci. 10, 294-298. Kirk, CJ., Reddy, N.L., Fischer,J.B., Wolcott, T.C., Knapp, A.G. & McBurney, R.N. (1994)In Vitro neuroprotection by substituted guanidines with varying affinities for the N-methy1-Daspartate receptor ionophore and for sigma sites:j! Pharmacol. Ex!. Thher. 271, 1080-85. McBurney,R.N. ( 1994)Therapeutic potential of NMDA antagonists in neurodegenerativediseases. Neurobwl. Aging. 15, 271-273. McCulloch,J., Bullock, R. & Teasdale, G.M. (1992)Excitatory Amino AcidAntagonish: Opkortunities for the Treatment oflschaemic Brain Damage in Man (ed. Meldrum, B.), pp. 287-326. Blackwell Scientific, Oxford. Meadows, M.-E., Fisher, M. & Minematsu, K. ( 1 994) Delayed treatment with a noncompetitive 4, 26-31. NMDA antagonist, CNS-I 102, reduces infarct size in rats. Cerebrovmc, a. Minematsu, K., Fisher, M., Li, L., Davis, M.A., Knapp, A.G., Cotter, R.E., McBurney, R.N. & Sotak, C.H. (1993a) Effects of a novel NMDA antagonist on experimental stroke rapidly and quantitatively assessed by diffusion-weightedMRI. Neurol. 43, 397403. Minematsu, K., Fisher, M., Li, L. & Sotak, C. (199313)Diffusion and perfusion MRI studies to evaluate a non-competitive NMDA antagonist and reperfusion in experimental stroke in rats. Stroke 24, 2074-2081. Muir, K.W., Grosset, D.G., Gamzu, E. &Lees, K.R. (1994)Pharmacological effects of the noncompetitive NMDA antagonist CNS 1 102 in normal volunteers. Br. 3 Clin. Pharmacol. 38, 33-38. Muir, K.W. & Lees, K.R. (1995) Clinical experience with excitatory amino acid antagonist drugs. Stroke 26, 503-5 13. NINDS rt-PA Stroke Study Group (1995) Tissue plasminogen activator for acute ischaemic stroke. New Eng1.J Med. 333, 1581-1587. Olney,J.W., Labruyere,J. & Price, M.T. (1989)Pathological changes induced in cerebrocortical neurones by phencyclidine and related drugs. Science 244, 1360-1 362. Park, C.K.,McBurney, R.N., Holt, W.E, Cotter, R.E.,McCulloch,J., Kang,J.K. &Choi, C.C. (1993) The dose-dependency of the antischaemic efficacy and of the side effects of a novel NMDA antagonist, CNS 1102.3 Cereb. Blood Flow Metab. 13,641. Reddy, N.L., Hu, L.-Y., Cotter, R.E., Fischer, J.B., Wong, W.J., McBurney, R.N., Weber, E., Holmes, D.L., Wong, S.T., Prasad, R. & Keana, J.EW. (1994)Synthesis and structure-activity studies of N,N-diarylguanidine derivatives. N-( I-Naphythyl)-N’-(3-ethylphenyl)-Nfmethylguanidine: A new, selective non-competitive NMDA receptor antagonist. j! Med. C h a . 37,260-267. Wang, S., Zhou, D., Fischer,J.B., Knapp, A.G. & Holt, W.E (1995)CNS 1102 protects against brain damage due to hypoxic ischaemia (HI) in the neonatal rat. Soc. Neurosci. Abstr. 21,995. Wong, E.H.R. & Kemp,J.A. (1 99 I ) Sites for antagonism on the N-methybaspartate receptor channel complex. Ann. Rev. Pharmacol. liwcicol. 31, 40 1425.


Chapter 9

PHARMACOLOGY OF AMPA ANTAGONISTS AND THEIR ROLE IN NEUROPROTECTION Rarnrny Gill* and David Lodge+ *Hoffmann La Roche, Pharma Division, PRPN, BAU 6814 10, Grenzacher Strasse, 4002 Basel, Switzerland; +LillyResearch Centre Limited, Erl Wood Manor, Windlesham, Surrey, GU2 6PH, UK

9. I 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9

Discovery of glutamate receptor subtypes AMPA agonists Elucidation of more potent and selective AMPA antagonists Molecular biology of AMPA receptors Antagonist pharmacology of recombinant AMPA receptors AMPA antagonists and cerebral ischaemia Role of AMPNkainate antagonists in focal ischaemia models Mechanism of protection following focal ischaemia Role of AMPNkainate antagonists in transient forebrain ischaemia models 9.9. I Gerbil ischaemia model 9.9.2 Rat transient forebrain ischaemia models 9.9.3 Mechanism of protection in global ischaemia models 9.10 Side-effect profile of AMPNkainate antagonists and relevance t o clinical testing References

I97 I98 200 202 203 203 206 213 214 214 214 216 220 222

9. I Discovery of glutamate receptor subtypes It is now generally recognized that the amino acid, L-glutamate, is the major transmitter of fast synaptic excitation in the mammalian brain and spinal cord. Starting from the work of Hayashi (1952)and Curtis et al. (1959), L-glutamate has been shown to excite neurones throughout the central nervous system. Studies with numerous analogues of glutamate demonstrated the need for one amino and two acidic groups in the molecule for functional excitation of all central neurones (Curtis and Watkins, 1963). Further studies with L-glutamate and L-aspartate in the thalamus and spinal cord demonstrated the existence of subsets of neurones with differential pharmacology between these two transmitter candidates (McLennan et al., 1968;Duggan, 1974). This was the first hint of receptor subtypes. As a result of the early medicinal chemistry, substitution of a methyl on the amino group of D-aspartate yielded N-methyl-DAcademic Press Limited Copyright 0 1997 All rights ofreproduction in anyform reserved

NEUROPROTECTIVE AGENTS AND CEREBRAL ISCHAEMIA, IRN 40 ISBN 0-12-366840-9;0-12-197880-X(pbk)

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aspartate as a potent excitant (Curtis and Watkins, 1963). During the early 1970s studies using natural products provided quisqualic, kainic and ibotenic acids, respectively, as highly potent neuronal excitants in the mammalian CNS (Shinozaki and Konishi, 1970; Shinozaki and Shibuya, 1974;Johnston et al., 1974; Biscoe et al., 1976). Potency ratios of such compounds varied between cells, further suggesting receptor subtypes. Final acceptance of subtypes, however, depended on the emergence of some early antagonists, such as D-a-amino-adipate (DAA),y-glutamyl-amino-methyl-sulphonate (GAMS) and glutamate diethyl ester (GDEE; Haldemann and McLennan, 1972; Biscoe et al., 1977; Lodge et al., 1978; Davies and Watkims, 1979, 1985; McLennan and Lodge, 1979; Evans et al., 1978).These three agents proved to be weak and somewhat non-selective antagonists of NMDA, kainate and quisqualate, respectively. Excitation by aspartate and NMDA were also blocked selectively by increasing levels of extracellular Mg2+(Evans et al., 1977; Davies and Watkins, 1977; Ault et al., 1980) and by %amino- 1-hydroxy-2-pyrrolidone (HA-966; Haldeman et al., 1972; Davies and Watkins, 1973; Biscoe et al., 1977; Evans et al., 1978). Such antagonists, though weak, also blocked synaptic excitations in the central nervous system (CNS), confirming the role of glutamate as a neurotransmitter. Although the potency and selectivity of many of these early agonists and antagonists left much to be desired, the elucidation of three classes of NMDA antagonist (DAA,HA-966 and Mg') supported the concept of the NMDA receptor as a separate pharmacological entity The evidence for a subdivision of the non-NMDA receptors was increased by the emergence of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA, Figure l), as a potent GDEE-sensitive excitatory amino acid agonist clearly distinguishable from kainate (Krogsgaard-Larsen et al., 1980).

9.2 AMPA agonists

AMPA was demonstrated to be the most selective agonist for this subtype of excitatory amino acid receptors (Krogsgaard-Larsen et al., 1980). Structural modifications of AMPA have given rise to various agonists, partial agonists and some antagonists (see review, Watkins et al., 1990). The most potent agonist of these was the bicyclic

FIGURE1 Structures of the agonist a-amino-3-hydroxy-5-rnethyl-4-isoxazole propionate (AMPA) and various AMPA antagonists: 2-amino-3[3-carboxymethoxy)-5-methyl-isoxazol-4yllpropionate (AMOA);6,7-dinitroquinoxaline-2,3-dione (DNQX), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX); 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline(NBQX); 64 1 -imidazolyl)-7-nitroquinoxaline-2,3( 1 H,4H)-dione (Yh49OK); 3SR,4aRS,SRS,8aRS)-S[2-(1 H-tetrazol-5-yl)-ethyl]-1,2,3,4,4a,5,6,7,8a-decahydroisoquinoline-3-carboxylic acid, (LY215490); 1,2,3,6,7,8,-hexahydro-3(hydroxy~ino)-~~,7-trimethy1-2-oxobenzo 2,1-b:3,4c')dipyrrole-5-sulfonamide(NS 257); 1 -(aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3benzodiazepine ( G Y K I 52466) and methohexital sodium.

PHARMACOLOGY OF AMPA ANTAGONISTS

AMPA

AMOA

DNQX

CNQX

NBQX

YM90K H3C\

Me2N02S

NS257

N-oH

*

LY215490

CHI

C3H5

CEHQ

0

Methohexital Sodium

6,7-dichloro-2( 1H)-oxoquinoline-3phosphonic acid

'q 0

-N

Q NH2

GYKI 52466

199

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R. GILL AND D. LODGE

homologue of AMPA (R~-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[ 1,2)d]isoxazol-4-yl)propanoicacid (4-AHCP) which was shown to be a potent agonist in the cortical wedge preparation and the cat spinal cord in vivo, with greater potency than AMPA in this latter preparation (Hansen and Krogsgaard-Larsen, 1990). The other class of agonists have been developed by Watkins et al. (1990) and are based on the naturally occurring compound, willardiine. Willardiine is a potent agonist of AMPA receptors (Evans et al., 1980; Patneau et al., 1992) but has little action at the pure kainate receptors of the dorsal root C-fibres (Agrawal and Evans, 1986). There are other naturally occurring AMPA agonists such as f3-N-oxalyl-amino+alanine (BOAA), which occurs naturally in Luttyrus sativus, a chickling pea. BOA4 is thought to be responsible for the neurodegenerative disease lathyrism (Spencer et al., 1986). Binding studies indicate that it has greater affinity for AMPA rather than kainate receptors (Bridges et al., 1988). Another similar toxin, f3-N-methyl-amino alanine (BMAA), which is a component of cycad seeds, has been proposed to be responsible for amyotrophic lateral sclerosis and Parkinsonian dementia on the island of Guam (Spencer et al., 1987; Weiss et al., 1989).

9.3 Elucidation of more potent and selective AMPA antagonists The 6-cyano-7-nitro- and 6,7-dinitro- quinoxalinediones, CNQX and DNQX (Honor et al., 1988; Figure 1) were the first of a new generation of AMPA antagonists. Both in receptor binding and electrophysiological studies, they showed an approximately 5fold selectivity for AMPA over kainate (Honor6 et al., 1988; Fletcher et al., 1988). However, in patch clamp studies these compounds were competitive antagonists, showing similar effects against responses to AMPA and kainate, but were less effective at NMDA receptors (Lester et al., 1989).The NMDA antagonist action of these cornpounds has now been attributed to an interaction at the glycine site (Birch et al., 1988). Further substitution and modification of the quinoxalinedionesresulted in the compound 2,3-dihyclroxy-6-nitro-7-sulphamoyl-benz(F)-quinoxaline (NBQX; Figure l), which had 30-fold selectivity toward AMPA and was devoid of activity at the NMDA receptor (Sheardown et al., 1990; Lodge et al., 199 1). This was the first AMPA/kainate antagonist with selectivity and systemic activity, and an obvious candidate to examine the role of non-NMDA glutamate receptors in ischaemia. The discovery of this compound resulted in many in vivo studies, although its physico-chemical characteristics and its phamacokinetics were not in favour of its development as a drug. Precipitation in the kidney led to its withdrawal from clinical studies (Xue et al., 1994). Further manipulation of the quinoxalinediones has led to the synthesis of yet another competitive AMPA antagonist, 6-(1-imidazolyl)-7-nitroquinoxaline2,3(1H,4H)-dione (YMSOK, Figure 1). Binding studies demonstrated that it was highly selective against AMPA binding and although it did display some shift of glycine binding, it was 30 times less potent at this site (Ohmori et al., 1994; ShimizuSasamata et al., 1996).


20 1

Another novel competitive AMPA antagonist is the racemic decahydroisoquinoline, LY215490, of which LY293558 is the active isomer (Ornstein et al., 1993, 1995; Figure 1). This compound displaces AMPA rather than kainate in binding studies, selectively blocks AMPA-evoked depolarizations on cortical slices, and crosses the blood-brain barrier to antagonise AMPA receptor agonists (Ornstein et al., 1993; Schoepp et al., 1996). As with NBQX, selectivity between AMPA and kainate is lost with LY293558 on single spinal neurones in vlvo, presumably because kainate excites these neurones via AMPA receptors. Similar types of data have been reported for two analogues of AMPA called AMOA and AMNH, which are substituted with a 3-carboxymethoxy and a 2methylisoxazole, respectively, on the isoxazole ring of AMPA (Krogsgaard-Larsen et al., 199 1). In binding studies, AMOA displaces AMPA selectively, whereas AMNH additionally displaces some kainate binding. On cortical slices, however, AMOA and AMNH were weakly selective for AMPA- and kainate-induced depolarizations, respectively (Krogsgaard-Larsen et al., 199 1). Another novel, selective and competitive AMPA receptor antagonist is 1,2,3,6,7,8,hexahydro-3(hydroxyimino)-NJ,7-trimethyl-2-oxobenzo 2,l -b:3,4-c’)dipyrrole-5sulfonamide (NS 257; Wgtjen et al., 1994; Nielsen et al., 1995; Figure 1). This compound has been demonstrated to be selective in vltro and in vivo for blocking AMPA induced toxicity. Other quinoxalinediones have also been identified using these basic structures (see Bigge et al., 1995). All of these competitive AMPA antagonists appear to have similar potency and selectivity to NBQX. Desos et al. (1996) reported on the structure activity of a new series of quinolones as competitive AMPA antagonists, the lead compound being water soluble and equipotent in vivo to NBQX (Figure 1). In addition to these competitive AMPA receptor antagonists, a class of noncompetitive antagonists based on the 2,3-benzodiazepine structure of GYKI 52466 (Tarnawa et al., 1990; Ouardouz and Durand, 1991; Lodge et al., 1992; Figure 1) has emerged. These antagonists, which do not show voltage- or use-dependency, are easily bioavailable to the CNS from both intravenous and oral routes. O n AMPA receptors of rat hippocampal neurones, GYKI 52466 had an IC,, of 4-7 PM (Donevan and Rogawski, 1993; Zorumski et al., 1993),whereas on kainate receptors of rat dorsal root ganglion (DRG) neurones the ICsOwas greater than 200 PM (Wilding and Huettner, 1995).GYKI 53655 (LY300168),the 3-methyl-carbamoyl derivative of GYKI 52466, is approximately 10 times more potent than the parent compound on Ah4PA receptors but not on kainate receptors (Wilding and Huettner, 1995; Paternain et al., 1995). The barbiturates which act on GABA receptors to potentiate their inhibitory action have also been shown to antagonise responses to AMPA and kainate. Simmonds and Horne (1988) studied a range of barbiturates in the rat cortical wedge preparation and reported the following potency profile as antagonists of AMPA and kainate receptors: quinalbarbitone > pentobarbitone > phenobarbitone > butobarbitone. The barbiturate methohexitone has recently been demonstrated to be the most potent AMPA/kainate antagonist among this class of compounds (Zeman and Lodge, 1992)with some selectivity for kainate as opposed to AMPA receptors. A number of low molecular weight, polyamine-containing, arthropod toxins

202


(Jackson and Usherwood, 1988) have also been reported to be potent AMPA/kainate antagonists in studies in vivo (Jones et al., 1990). They appear to be acting at the ionchannel site and demonstrate a use-dependent block, as seen with ion-channel blockers of the NMDA receptor complex. However, other studies suggest that some of these toxins are not selective for AMPA/kainate receptors but are, in fact, more potent NMDA receptor antagonists (Priestleyet al., 1989; for review, see Lodge andJohnson, 1990). Argiotoxin (from the spider venom) has now been reported to be a voltagedependent open channel blocker which demonstrates selectivityfor the GluR1,3 and 4 subunits of AMPA receptors (Herlitze et al., 1993). Thus, this compound may become an important tool for synthesis of further subtype-selective AMPA receptor antagonists.

9.4 Molecular biology of AMPA receptors The use of molecular biological techniques has revolutionized scientists' understanding of glutamate receptors over the last six or seven years. The first glutamate receptor was cloned and expressed by Hollmann, Heinemann and colleagues in 1989. This was named GluRl and was subsequently identified as an AMPA receptor. Since then, three further AMPA receptors, GluR2-4, have been identified (for reviews see Seeburg, 1993; Wisden and Seeburg, 1993; Hollman and Heinemann, 1994; Nakanishi and Masu, 1994; Dani and Mayer, 1995; Bettler and Mulle, 1995). Each subunit is subject to splice variants and potential for RNA editing. Four transmembrane (TM)spanning domains were located in each subunit, although TM2 is not strictly trammembrane since it loops into and out of the membrane from the intracellular face (Woand Oswald, 1994; Dani and Mayer, 1995; Bettler and Mulle, 1995). This TM2 loop is thought to be the pore-lining sequence in each of the subunits of the presumed pentameric receptor. From a neurotoxicological viewpoint, a very important difference between AMPA receptor subunits is their permeability to Ca2+.GluR2 subunits, unlike the other subunits, display a low permeability to Ca2+.This Ca2+permeability is now known to be controlled by RNA editing of a single amino acid in the TM2 loop, which results in an arginine (R)in GluR2 instead of the glutamine (Q in the other AMPA receptor subunits at this site. The same site appears to determine the ability ofpolyamine toxins from spiders and wasps to block glutamate channels. Argiotoxin, lo-' M, and philanthotoxin, M, block GluR1, 3 and 4, but have considerably less effect on GluR2-containing AMPA receptors (Herlitze et al., 1993; Blaschke et al., 1993; Brackley et al., 1993). Mutagenesis studies have confirmed the crucial importance of this Q / R site in controlling both Ca2+permeability and toxin sensitivity of the glutamate ion channels. A further feature of AMPA receptors relevant to neurotoxicity is their varied rate of desensitization. From studies on native receptors using patch clamp recording, it


203

was known that AMPA and glutamate produced rapidly desensitizing responses (JSiskin et al., 1986; Mayer and Vyklicky, 1989)and this has been confirmed in recombinant AMPA receptors. Characterization of recombinant AMPA receptors GluRl-4 confirmed this to be the case (Seeburg, 1993; Hollmann and Heinemann, 1994).This rapid desensitization of both native and recombinant AMPA receptors can be slowed by certain benzothiazides such as diazoxide and more potently by cyclothiazide (Yamada and Rothman, 1992; Wong and Mayer; 1993; Partin et al., 1993, 1994; Moudy et al., 1994). Interestingly, these compounds do not reduce desensitization at kainate receptors.

9.5 Antagonist pharmacology of recombinant AMPA receptors With respect to competitive antagonists, CNQX has a potency in the 50-400 nM range on G l u R 1 4 subunits (Stein et al., 1992).LY293558 blocks the GluR1-4-mediated currents with ICs0values in the low micromolar range (Bleakman et al., 1995). Quinoxalinediones, CNQX and NBQX also block recombinant kainate receptors GluR5 and 6, and LY293558 blocks GluR5 but not GluR6. The 2,3-benzodiazepines (Tarnawa et al., 1990) are potentially selective AMPA receptor antagonists. Thus, the ICsosof GYKI 52466 and 53655 were about 5 and 0.5 p ~respectively, , on recombinant AMPA receptors with very little effect on recombinant kainate receptors at 100 p~ (EJ. Fletcher and D. Bleakman; unpublished observations). Similarly, on GluRl4 dominated rat hippocampal neurones, GYKI 52466 reduced AMPA-induced currents with an IC,,, of 4-7 p~ (Donevan and Rogawski, 1993; Zorumski et al., 1993; Parsons et al., 1994), whereas on GluR5-expressing DRG neurones, the IC50 was greater than 200 p~ (Wilding and Huettner, 1995). Although at this early stage, there is good correspondence between results in cell lines expressing recombinant glutamate receptors and those in native neurones where expression of glutamate receptors has been identified by molecular biological techniques. The wide variety of tools from competitive antagonists and allosteric modulators through to channel blockers is enabling a detailed analysis of the roles of defined glutamate receptors in CNS function.

9.6 AMPA antagonists and cerebral ischaemia

1 I

1

1

!

,

Starting with the initial studies on glutamate receptors (Hayashi, 1952; Curtis and Watkins, 1960, 1963), there has been the realization that the excitatory role of glutamate could have important therapeutic implications, particularly in epilepsy More relevant to the neuroprotective potential of glutamate antagonists, however, was the observation that glutamate receptors mediate neurotoxicity. Hence, their potential

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role in acute and chronic neurodegeneration was also being realized (Olney, 1969; Rothman and Olney, 1987; Choi, 1988). Glutamate receptors have been implicated in many neurodegenerative diseases, including stroke, Alzheimer's and Huntington's disease, amyotrophic lateral sclerosis, and so on (for reviews see Rothman and O h q , 1987; Choi, 1988; Zeman et al., 1994).However, the most compelling evidence for the involvement of glutamate-mediated excitotoxicity comes from cerebral ischaemia. Glutamate appears to play a critical role in ischaemia induced neuronal degeneration (for reviews see Rothman and Olney, 1987; Choi, 1988; Benveniste, 1991; McCulloch et al., 1991). Increased extracellular levels of glutamate have been demonstrated using in uivo microdialysis in models of global and focal ischaemia (for review see Benveniste, 1989; Benveniste et al., 1984; Hagberg et al., 1985; Butcher et al., 1990; Lo el a/., 1993; Matsumoto et al., 1996; Figure 2). Glutamate acts on NMDA, AMPA/kainate and metabotropic receptors to produce an increase in cytosolic free Ca2+.The cytosolic Ca2+interacts with diacylglycerol to activate protein kinase C (PKC),which acts via a number of mechanisms (primarily by altering membrane ionchannels), to increase neuronal excitability and further increase cytosolic Ca2'. Elevated cytosolic Ca2+also activates several enzymes (calpains, endonucleases and phospholipases) capable of either directly or indirectly (through free radical formation) destroying cellular structure. The increased levels of intracellular Ca" may also activate the nitric oxide pathway and thus, through free radical formation, produce cell damage The cytosolic Ca2+levels are normally maintained via the Na+-Ca2+ exchanger. The activation of AMPA receptors may also be involved in reversal of the Na+-Ca2+ exchanger. Therefore, all of these mechanisms contribute to cell death. Furthermore, the glutamate released from synaptic terminals or leaking nonspecifically from ruptured neurones may contribute to additional injury propagation. The high levels of glutamate in the synaptic cleft triggers a cascade of excitotoxic events, resulting in an uncontrolled influx ofCa2+into the post-synaptic neurone. The high concentrations of intracellular Ca2+ are cytotoxic, perhaps by activation of calcium-sensitive proteases (calpains) or by stimulation of nitric oxide which in turn leads to formation of free radicals. These various triggers ultimately result in cell death (Siesjo, 1981; Siesjo and Bengtsson, 1989; Kemp, 1994; Figure 2). In the clinical setting, cerebral ischaemia can result from a cardiac arrest, stroke, head trauma or even following cardiac bypass surgery. Animal models of cerebral ischaemia have been developed to give insight into the pathological, neurochemical and pharmacological changes occurring following ischaemia. For some time, NMDA receptor antagonists were the main focus of therapeutic intervention in pathophysiological processes because of the high Ca2+permeability of the NMDA receptor-linked ion channel and the involvement of this cation in processes leading to cell death (Siesjo, 1981; Choi, 1988; Figure 2). However, as stated above, AMPA (and kainate) receptors have appreciable Ca2+permeability and in any case resultant depolarization would allow calcium entry via voltage-dependent calcium channels. The NMDA receptor antagonists were reported to be neuroprotective in the gerbil model of transient forebrain ischaemia (Gill et al., 1987; Boast

FIGURE 2 Possible mechanisms by which intracellular calcium may be increased in neurones during ischaemia, and the putative intracellular targets which may mediate cell damage and eventually cell death. (Reprinted with permission from Cerebrovasnrlnrand Bruin Metaboltmr Reviews 6 (3), Gill, R. ‘The pharmacology ofAMPA/kainate antagonists and their role in cerebral ischaemia, pp. 225-256, copyright 1994, Lippincott-Raven Publishers, 227 E. Washington Square, Philadelphia, PA, USA.)

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et al., 1988; Warner et al., 1991a), and in models of focal ischaemia (for review see McCulloch et al., 1991). However, in models of severe forebrain ischaemia in rats, these compounds were found to produce equivocal results, and more attention was focused on Ah4PA antagonists (Simon et al., 1984; Church et al., 1988; Gill et al., 1989; Buchan et al., 1991a; for review see Buchan, 1990). The animal models of severe forebrain ischaemia in the rat, such as in the rat 2vessel occlusion model with hypotension (2-VO; Smith et al., 1984) or 4-vessel occlusion model (4-VO; Pulsinelli et al., 1982) and the gerbil model of bilateral carotid occlusion (Kirino, 1982), result in hippocampal degeneration of CAI neurones. This neuronal degeneration occurs over a period of > 24 hours and is described as ‘delayed neuronal degeneration’ (for reviews see Buchan, 1990; Meldrum, 1990; Benveniste, 1991). Studies using these models demonstrated that NMDA antagonists were neuroprotective in models where a mild ischaemic lesion was present, such as in the gerbil model, but were less effective in models of severe forebrain ischaemia. However, in animal models of focal ischaemia in which the damage appears to develop more rapidly, around 24 hours of permanent or temporary middle cerebral artery (MCA) occlusion (Kaplan et al., 1991; Gill et al., 1995),NMDA antagonists were highly neuroprotective, providing up to 70% protection (for review see McCulloch et al., 1991, and Chapter 7, p. 155).Permanent MCA occlusion for 24 hours results in a lesion consisting of a well-defined core area, in which blood flow has been reduced to < 10% of normal, and a so-called ‘penumbral’ region in which the blood flow is around 20% of normal (Astrup et al., 1981; Tamura et al., 198la,b). The NMDA antagonists are able to reduce cortical infarction in the penumbral areas but generally not in the core areas (Park et al., 1988a,b; Bullock et al., 1990; Gill et aL, 1991). In this model it was demonstrated that the most efficaciouscompound MK-80 1 may be neuroprotective by reducing cortical spreading depression-like activity in the penumbral areas (Gill et al., 1992a).

9.7 Role of AMPNkainate antagonists in focal ischaemia models NBQX was the first AMPA antagonist to be tested in a focal ischaemia model in the rat (Gill and Lodge, 1991; Gill et al., 199213). NBQX was neuroprotective when administered as two bolus doses of 30 mg/kg intravenously, 30 and 60 minutes postMCA occlusion (see Figure 3). This resulted in 24% and 27% protection against hemispheric and cortical ischaemic damage, respectively. However, NBQX has a plasma Ti,*of only 30 minutes so an infusion dosing regime was used. NBQX was administered as an intravenous bolus of 30 mg/kg followed by 10 mg/kg/h for 4 hours. This dosing regimen resulted in a mean plasma level over the 4 hours of 17 pg/ml, and 29% and 35% protection against the volume of hemispheric and cortical damage (Figure 3). NBQX was also reported to be neuroprotective in a rat model of temporary MCA occlusion for 2 hours plus permanent occlusion of the ipsilateral common carotid artery in spontaneously hypertensive rats (Buchan et al., 1991b; Xue et al., 1994;


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FIGURE3 Volume of ischaemic damage in the cerebral hemisphere, cortex and caudate nucleus for the different dosing regimes with NBQX- and vehicle-treated animals (open bars). [A] Data for the dose-response curve for NBQX. Doses of 3, 10 or 30 mg/kg i.v. were given immediately after MCA occlusion and again 1 hour post-occlusion. The dose of 2 X 30 mg/kg resulted in significant (*P[115. Olney, J.W. (1969) Glutamate-induced retinal degeneration in neonatal mice. Electron microscopy of the acutely evolving lesion. 5; Jveuropathol. Exp, Neurol. 28, 455474. Olney, J.W., Labruyere, J. & Price, M.T. (1989) Pathological changes induced in cerebrocortical neurones by phencyclidine and related drugs. Science 244, 1360-1 362. Opitz, T. & Reymann, K.G. (1991)Blockade of metabotropic glutamate receptors protects rat CAI neurones from hypoxic injury. Neuroreport 2,455457.


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Chapter 10

GABA AND NEUROPROTECTION Patrick D.Lyden UCSD Stroke Center, Department of Neurosciences, University of California, 200 W. Arbor Drive 8466, San Diego, C A 92 103-8466 and Veteran's Administration Medical Center, Department of Neurology, 3350 La JollaVillage Drive, San Diego, CA 92 I 6 I , USA

10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.9 10.10

233 233 235 236 236 238 240 250 252 25 3 254

Introduction Excitotoxicity The GABA strategy The excitotoxic index Anatomy of the GABA receptor Response of GABA to ischaemia GABA, agonists are neuroprotective Pharmacology of GABA mirnetics Combinatorial strategies Future directions References

I 0. I Introduction Gamma aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in mammalian brain. GABA neurotransmission results in increased chloride flux across the postsynaptic membrane, and hyperpolarization. In many ways these actions counterbalance the physiological and toxic effects of glutamate. Recently, agonists of the GABAAreceptor have been shown to provide neuroprotection during ischaemia. The potency of these agents equals or exceeds that of glutamate antagonists, yet with fewer side-effects. In this chapter the rationale for using GAl3A agonists as neuroprotectants will be described. Data showing efficacy of these agents will be presented and a proposal for future studies of drug combinations will be presented.

10.2 Excitotoxicity The excitotoxic hypothesis is well described elsewhere in this volume. Relevant to a discussion of GABA, however, are a few aspects of the excitotoxin story. Several lines of evidence indicate that excitatory neurotransmitters such as glutamate and NEUROPROTECTIVE AGENTS AND CEREBRAL ISCHAEMIA, IRN 40 ISBN 0- 12-366840-9;0- 12-197880-X @bk)

Academic Press Limited Copyright 0 1997 All @htr ofreproa'uchn in anyform reserved

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aspartate play a central role in mediating cell death after a variety of cerebral insults, including ischaemia, trauma, seizure and hypoglycaemia (Greenamyre, 1986; Cotman and Iversen, 1987; Auer and Siesjo, 1988).In cell culture, excitotoxic amino acids cause death of neurones but not glia, and synaptic activity appears to be essential in this process (MacDonald et al., 1987; Choi et al., 1987).At least three glutamate receptor subtypes are identified based on ligand binding studies: Xmethyl-D-aspartate (NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-kainate, and metabotropic (Nakanishi, 1992). Of these subtypes, the NMDA receptor appears to be critical in mediating the effects of ischaemia including rapid and delayed neuronal death (Hartley et al., 1993).Although the AMPA/kainate receptor plays a critical role in normal synaptic transmission, it may not be involved in ischaemic excitotoxicity since specific receptor subtype antagonists are not particularly neuroprotective. The metabotropic receptor, which is coupled to second messenger G-proteins, may participate in the mobilization of calcium from intracytoplasmic stores such as endoplasmic reticulum. The importance of this receptor in mediating excitotoxicity is unknown at present. Depolarization of the postsynaptic cell occurs in response to application of glutamate, and appears to be a necessary step in the sequence of events leading to cell death (Rothman, 1983; Rothman et al., 1987). During excitation of the postsynaptic membrane, there is an influx of sodium, chloride, calcium and water into the cell (Choi, 1985, 1987; Hartley et al., 1993). Inflow of ions and water leads to oedema and, if severe or prolonged, such oedema may lead to cell lysis and death (Choi, 1987). Glutamate does not cause oedema or lysis of mature neurones in culture if sodium and/or chloride are not present in the culture medium (Choi, 1987).Inflow of calcium may lead to delayed neuronal death (over 24-72 hours) through unknown mechanisms after comparatively brief exposure to ischaemia or excitotoxins (Choi, 1985; Goldberg and Choi, 1993). This effect persists in culture if sodium is not present in the medium but is blocked by M$+or by removing calcium from the medium (Choi, 1987;Rothman et al., 1987).Some calcium channels are voltage gated, and glutamate causes depolarization to a membrane voltage that opens some of these channels, so the membrane potential may be a critical determinant of calcium influx (Choi, 1985; Rothman et al., 1987). However, some calcium appears to enter the cell through the NMDA receptor itself, and this ligand-gated influx appears to continue even in voltage-clamped cells (Rothman et al., 1987; MacDermott et al., 1986). Nevertheless, it seems reasonable to suspect that prevention of glutamate-stimulated depolarization ought to prevent some of the early cellular oedema due to sodium, chloride and water movement, and some of the calcium flow that leads to delayed toxicity, In support of this expectation, it was observed that hyperpolarization reduced or blocked calcium inflow into neurones (Riveros and Orrego, 1986) and reduced the probability of discharge (Hirayama et al., 1990). Several glutamate antagonists (MK-80 1, CGS- 19755, and dextrophan) appear to protect the brain during experimental focal ischaemia (Ozyurt et al., 1988; Yum and Faden, 1990; Park et al., 1988; Kochhar et al., 1988; Boast et a/., 1988; George et al., 1988;Prince and Feeser, 1988; Steinberg et al., 1988),and some agents are proceeding

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to clinical trial. These drugs resemble dissociative anaesthetics such as ketamine and phencyclidine, and humans experience significant side-effects during treatment with NMDA antagonists. In prior studies ofMK-80 1 in rats and rabbits, considerable sedation at effective doses has been observed (Kochhar et al., 1988). In humans, relatively low doses of dextrorphan resulted in unacceptable levels of confusion, agitation and hallucination (Alberts et al., 1995). Grotta studied several doses of CGS-19755 in humans and found the same side-effectprofile in the higher dose groups (Grotta et al., 1995). Lower doses were tolerated, but the efficacy of the low dose remains to be proven. Indeed, animal studies suggest that efficacy and side-effects are closely linked and that a tolerable yet effective dose may be dimcult to find. Therefore, it may be desirable to pursue an alternative strategy for brain protection during cerebral ischaemia that blocks the effect of excitotoxins without causing side-effects. GABA agonists were chosen for this purpose.

10.3 The GABA strategy

In mammalian brain, GABA is considered the principal inhibitory neurotransmitter (Roberts, 1974). Inhibitory neurotransmitters lower the resting membrane potential of the neurone and reduce the probability that glutamate stimulation leads to action potential (Roberts, 1974; Bachelard, 1981; Hirayama et al., 1990).GABA mediates its effects through two receptor subtypes, A and B (Figure 1). The GABAA receptor is a ligand-gated chloride channel found throughout the brain that mediates a fast inhibitory response (Allenet al., 199 1). The GABABreceptor is linked to a second messenger G-protein and is found on neurones and glia within and outside the central nervous system. GABABstimulation appears to reduce presynaptic release of several neurotransmitters and to mediate the late inhibitory postsynaptic potential via a potassium channel (Karlsson and Olpe, 1989). When GABA or a suitable analogue occupies the postsynaptic GABAA receptor, chloride flux increases markedly, the resting membrane potential may not increase and voltage-gated calcium channels remain closed (Riveros and Orrego, 1986; Scharfman and Sarvey, 1985; Hirayama et al., 1990). GABA-stimulated changes in membrane potential would block only voltage-gated calcium influx, however. Ligand-gated calcium flux via glutamate receptors would probably remain unaltered, although this point remains to be studied. The effect of membrane potential on the release of intracytoplasmic calcium stores is also unknown. In addition to chloride permeability changes, GABAA agonists reduce the cerebral metabolic rate for glucose at doses that do not cause sedation or impair respiration or cardiac function (Kelly and McCulloch, 1982, 1983). GABAA receptors are found on cerebral blood vessels and mediate dilation of cerebral, but not extracranial, vessels. This effect is blocked by competitive antagonists of the GABA receptor (Edvinsson and Krause, 1979). Thus, it was predicted that GABAA agonists would prove to be neuroprotective for at least three reasons. First, increased chloride conductance could block voltage

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FIGURE1 Schematic of the GABAergic synapse. The details of the anatomy and biochemistry are detailed in the text. Drugs that may influence GABA release, re-uptake or post-synaptic binding are illustrated. (Reproduced with permission of Raven Press, LTD from Suzdak andJansen, 1995).

mediated postsynaptic effects of glutamate by preventing induced action potentials and holding voltage-gated ligand channels closed. Second, in the face of ischaemia, the reduced metabolic respiratory rate would preserve substrate and reduce the build-up of toxic byproducts of anaerobic glycolysis. Third, vasodilation would improve blood flow into the ischaemic penumbra via unobstructed collateral channels. It could not be predicted whether any or all of these mechanisms might serve this purpose, but the availability of several agonists led to an attempt of simple pharmacologicalobservations of the effect of GMAAagonists on outcome after focal ischaemia.

10.4 The excitotoxic index The ultimate destiny of an ischaemic neurone may be viewed as the outcome of the interplay between excitatory and inhibitory neurotransmitters. Although this notion is quite simplified, it allows the formulation of testable hypotheses and perhaps will facilitate the rational design of stroke therapies. In order to describe the balance of opposing transmitter effects, the excitotoxic index was proposed (Mordecai et al., 1991). The index is computed from the sum of the extracellular concentrations


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Voltage Gated Ca2+channel

FIGURE2 During ischemia excessive depolarization of the membrane potential by glutamate enables calcium to enter the cell by removing a voltage dependent block of the NMDAreceptor and by activating voltage gated calcium channels. Hyperpolarization of the membrane potential by GABA could block calcium entry by both of these routes and hence prevent calcium from reaching neurotoxic levels. Figure prepared by Tim Ashwood.

of dopamine and glutamate, divided by the concentration of GABA. The absolute value of the result is not so important as the concept, illustrated graphically in Figure 2. One very attractive corollary to this idea is that it justifies an attempt to combine two treatments, such as a GABAA agonist and an NMDA antagonist. Hopefully, the GABAA agonist would block voltage-mediated effects. The NMDA antagonist would add to this effect by, hopefully, blocking ligand-gated channel effects. Together, the two drugs might confer superior neuroprotection, using complementary mechanisms, than either agent used alone at higher doses. It was thought possible to afford the subject adequate neuroprotection with fewer side-effects. In fact, it was hoped that the combination strategy would block one of the known side-effects of NMDA antagonists, namely the so-called toxic neuronal vacuolation. These cytoplasmic vacuoles are sometimes noted in neurones of the cingulate and retrosplenial cortex, but their functional significance is unknown (Olney, 197 1).

10.5 Anatomy of the GABA receptor The GABA receptor complex is a pentamere, made up of three main subunits named a , fi and y, and two minor subunits, 6 and p (see Figure 1). Each subunit may occur in one of several isoforms, for example, al-6, fil-fi3, yl-y3, pl and p2. The subunits included in the ionopore determine the binding characteristics of the receptor complex, which vary throughout the brain, but exact stoichiometries remain to be elucidated (Persohn et al., 1992). The genetic sequences of most of the main GABA

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receptor subunits are known, but the typical distribution of subunits remain to be mapped in humans (Wisden and Seeburg, 1992). The distribution of the al, 82 and y2 subunit mFWAs (messenger RNA) closely parallel the distribution of tritiated muscimol, a specific ligand for the GABAA receptor, and this combination of subunits appears to be the most abundant in rat brain (Persohn et al., 1992). The functional consequences of different subunit combinations in the ionophore are not fully known. The y2 subunit is necessary for the binding of benzodiazepines to the GABAA receptor, although the a subunit confers ligand specificity. GABA and other GABAA agonists, such as muscimol, bind to a distinct site on the ionophore. There are also binding sites for modulatory substances. Notably, the benzodiazepines bind to a site that is separate from the muscimol site. The distribution of GABA receptors is ubiquitous and heterogeneous. Thus, relatively higher densities are found within the cerebellum and striatum, lesser densities in the septum, and intermediate densities in hippocampus and cortex (Zilles, 1992; Zukin et al., 1974).Within the cortex the distribution of receptors parallels the laminar structure of the cortex. GABA is found mainly in non-pyramidal aspiny interneurones, which comprise about 20% of all cortical neurones. A comparison of the densities of glutamate to GABAA receptors yields some interesting observations (Zilles, 1992). The ratio of the two densities is highest in the hippocampus, favouring glutamate by 4.3 times. Glutamate to GABAA receptor density ratios of about 2.0 are found in cortex, amygdala and caudate/putamen. It is interesting to note that of these regions the hippocampus is known to be most particularly susceptible to ischaemic damage (Collins et al., 1989). This observation echoes the excitotoxic index idea noted above, and suggests further that selective vulnerability in the central nervous system may reflect the differential distribution of the two receptor classes.

10.6 Response of GABA to ischaemia Permanent ischaemia is followed by significant increases in the extracellular concentrations of glutamate, dopamine and GABA (Globus et aL, 1988; Hagberg et al., 1985).The peak levels are 2-4 times the basal levels. GABA increases as does glutamate, but returns to normal more quickly. This experiment has been accomplished once in humans (Kanthan et al., 1995). During temporal lobe resections, dialysate probes were inserted and samples collected while the temporal lobe was rendered partially ischaemic, at the outset of the resection. Glutamate levels increased 100-fold over baseline. GABA and other extracellular amino acids also increased, although not as significantly The mechanism of ischaemia-induced amino acid release is not known but several potential avenues exist (Phillis et al., 1994b). Amino acids can be stored in vesicles for release in response to action potential, or may reside in the cytosol of the presynaptic neurone. Some amino acid neurotransmitters, notably glutamate and GABA, are also present in glia following uptake from the synaptic cleft or as


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byproducts of neurotransmitter metabolism. The release of vesicle-stored neurotransmitter depends on calcium and energy, and occurs in response to depolarization. Ischaemia leads to depolarization that could cause substantial release of stored neurotransmitter. O n the other hand, the release of non-vesicular cytosolic substances from neurones or glia does not require calcium or energy. Sodium-dependent reuptake systems in neurones and glia may reverse in the face of ischaemia and depolarization, thus discharging from the cell this material that is not stored in vesicles. Finally neurotransmitter can also be released as the cell membrane disintegrates under the action of calcium-activated phospholipases and oxygen-free radicals. Such release does not proceed via any specific transporter protein, but merely occurs as the cell membrane disintegrates. Limited evidence suggests that during ischaemia, neurotransmitter release proceeds by all three mechanisms, but that calcium-dependent mechanisms account for only a small fraction of the total amino acid release (Phillis etal., 1994a; Torp et al., 1993). Ischaemic synaptic release of glutamate and aspartate clearly proceed by different mechanisms from GABA. Tetrodotoxin (which blocks the sodium channels necessary to propagate depolarizing action potentials) reduces ischaemia-evoked GABA release but has no effect on glutamate efRux (Phillis et al., 1994b). Inhibitors of the glutamate transporter inhibit the rise of glutamate but not GABA, while inhibitors of the GABA transporter reduced GABA ef€lux without altering glutamate release. Thus, reversal of the specific amino acid transporters during ischaemia clearly plays some role in mediating neurotransmitter release (Phillis et al., 1994b).However, despite such differences, the rise in extracellular concentrations of all amino acids occurs over the same time period after ischaemia. Therefore, it is likely that much of the neurotransmitter release after ischaemia occurs because of membrane damage and leakage of vesiclestored glutamate and GABA down the intra-to-extracellular concentration gradient. This suggests that amino acid efAux via damaged membranes plays a significant role, since it affects the extracellular concentrations of glutamate and GABA equally and further that inhibitors of presynaptic release mechanisms are unlikely to prove efficacious (Phillis et al., 1994b). It appears that reperfusion may result in a somewhat different picture. After 10 minutes of ischaemia followed by reperfusion, both glutamate and GABA return to basal concentrations over about 30 minutes (Ravindran et al., 1994). After 20 or 60 minutes of focal ischaemia, followed by 40 minutes of reperfusion, glutamate levels remain increased. GABA concentrations increase until reperfusion begins and then rapidly return to basal levels, again within about 30 minutes (Phillis et al., 1994a).It is not clear why glutamate concentrations remain elevated after the longer durations of ischaemia. Glial cells take up a large portion of the released extracellular glutamate. Glutamate is converted to glutamine in an energy-dependent reaction by glutamine synthetase. Recent studies of intracellular concentrations of amino acids revealed that ischaemia retards or blocks this reaction (Torp et al., 1993) which could further increase the extracellular glutamate concentration. Indeed, in this study it appeared that failure of glial uptake mechanisms was a critical step mediating the increase in extracellular glutamate.

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10.7 GABAAagonists are neuroprotective In cultures of cortical cells, the excitotoxin NMDA causes neuronal death in a concentration-dependent manner. Cells that labelled positively with tritiated-GABA (i.e. presumed GABAergic) appeared to resist the insult (Tecoma and Choi, 1989).These results suggest that either GABAergic cells resist ischaemia for some unknown reason, or that the NMDA receptor is critical in mediating cell death, while the GABA receptor is not. GABAAagonists appear to block kainate-stimulated cell death in culture, although GABA itself appeared to accelerate cell death (Erdo et al., 1991). Transient occlusion of both cartoid and both vertebral arteries (4-vesselocclusion, 4-VO) results in a reproducible hippocampus lesion in rats (Pulsinelliet al., 1982).The lesion consists of neurone loss in the hippocampal CAI and CA3 layers, a process that is mediated by glutamate. In addition, there is loss of NMDA receptors in the hippocampus and dorsal striatum (Francis and Pulsinelli, 1982). In this model, GABAergic neurones appear to resist ischaemia, in that there is selective loss of glutamatergic neurones and relative sparing of GABAergic neurones. These neurones function during and after ischaemia and appear to protect target neurones from excitotoxic cell death Uohansen et al., 1991). GABAergic neurones in the hippocampus show resistance to ischaemia in other models as well (Nitsch et al., 1991). However, global ischaemia causes loss of GAFJAergic cortical interneurones in infant primates, which may lead to the development of primary epilepsy (Sloper et al., 1980). These apparently contradictory results give rise to the speculation that there may be two subpopulations of GABAergic neurones (Shuaib et al., 1993). One group, including hippocampal and some striatal neurones, show greater resistance to ischaemia, while another group, including other striatal and cortical neurones, show relatively lesser resistance. Thus, it remains to be shown whether GABAergic neurones are resistant to ischaemia and, if they are, whether this is true throughout the cerebrum or only in some locations (Obenaus et al., 1993). Support for GABAergic neuroprotection comes from other sources as well. Elevated GABA after ischaemia may explain the observation that adrenalectomy prior to ischaemia reduces the extent of hippocampal cell loss (Ravindran et al., 1994). In this experiment, GABA levels rose to high levels, compared to non-adrenalectomized controls, while glutamate levels were no different. The mechanism mediating the effect is unknown, but the operated animals did not secrete any hydrocortisol. Steroids could alter the vulnerability of hippocampal cells by modulating GABA release, as was observed in this experiment, or by other speculative mechanisms. The greater preservation of GABAergic neurones during ischaemia in some brain regions suggests that GABA uptake inhibitors could be neuroprotective. Uptake inhibitors increase the synaptic concentration of a neurotransmitter by blocking reuptake without affecting release. Uptake inhibitors would be neuroprotective because release of the neurotransmitter is assured, i.e. some GABA-releasing neurones still function during and after ischaemia. Unfortunately, since the active agent is GABA itself, receptor binding would occur at GABA, as well as at presynaptic GABAB sites. Presynaptic GABAB receptors may be linked to release of other neurotransmitters,


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including glutamate. In the 4-VO model, GABA uptake inhibitors are clearly protective when given before and after ischaemia (Johansen and Diemer, 1991). As mentioned, dopamine is considered excitotoxic and its effects are counterbalanced by inhibitory neurotransmitters. If the source of inhibitory neurotransmitters is eliminated, a state of relative disinhibition can be studied. For example, cells in the substantia nigra pars reticulata receive GABA from cells in the caudate nucleus. Lesioning the caudate nucleus leaves the nigral cells subject to the action of unopposed dopamine (excitotoxicity)and results in delayed cell death 20 days later (Saji and Reis, 1987). The neuronal degeneration is completely blocked by infusions of muscimol, implying that (i) the neuronal death is due to an imbalance of inhibitory and excitatory inputs, and (ii) the imbalance can be corrected pharmacologically with exogenous GABA analogues. Although elegant, this experiment does not address the scenario of an imbalance resulting from excess excitotoxins, rather than insufficient inhibitory inputs. Sternau et al. (1989) have published a provocative series of experiments involving GABA agonists. They chose a model of forebrain ischaemia in which both carotid arteries of the gerbil are occluded (2-vessel occlusion model, 2-VO) resulting in loss of CA1 hippocampal neurones. A variety of agents were tested for neuroprotection against CA 1 neurone loss. Many GABAergic agents were effective if given before the onset of ischaemia, including diazepam, pentobarbital, valproic acid, baclofen and muscimol. Pentobarbital, baclofen and diazepam were given after the onset of ischaemia and were not neuroprotective. Also using the 2-VO model, Shuaib and colleagues (1 993) have studied GABA agonists extensively. Muscimol infused into the ventricle for 7 days protected the cortex, hippocampus, substantia nigra, striatum and thalamus from three episodes of 2minute ischaemia. In this study, infarctions were not seen; rather, silver impregnation methods were used to label degenerating neurones. In muscimol-treated animals, no degenerating neurones were seen in the cortex or hippocampus, areas where neurones receive both GABAergic and glutamatergic inputs. However, in the striatum and thalamus, where cells also receive dopaminergic input, some degenerating neurones were seen, albeit far fewer than in control subjects. One criticism of the 2-VO model is that gerbils tend to suffer seizures during ischaemia. Since GABA-mimetic agents are all anticonvulsants, it is possible that the protective effect seen in these models is due to a mixture of anticonvulsant and antiischaemia effects. To sort out this question, Madden (1994) used a model of spinal cord ischaemia in which the distal portion of the cord was rendered ischaemic by reversibly occluding the infrarenal aorta (Zivin and DeGirolami, 1980). Muscimol given 5 minutes after ischaemia significantly protected the animals, while bicuculline (a GABA, antagonist) significantly reduced the tolerance to ischaemia. Bicucullinetreated animals did not suffer generalized seizures, and neither drug affected any vital signs. This work shows that the protective effect of muscimol is very likely directly related to ischaemia, not an anticonvulsant mechanism. Prior to presenting one’s own findings with GABA agonists, it is necessary to clarify the design of the exprimental stroke models. Initially, the lack of a reasonable animal model was a serious obstacle. Animal systems designed to investigate pathophysio-

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logical mechanisms do not generally serve well for pharmacological screening. In most systems the behavioural and morphometric response to ischaemia is variable, necessitating relatively large numbers of study subjects (Hsu, 1993).This variability is partly due to variations in blood supply, such as an incomplete circle of Willis or differing degrees of collateral blood flow through pial anastomoses. To cope with this inherent variability, and to develop an efficient pharmacological screening tool, Zivin and colleagues devised a quantal bioassay procedure (Waud, 1972; Zivin et al., 1987). The quantal bioassay is an extension of the method of Probits, in which the responses of groups of subjects are related to the amount of ischaemia given to the subjects. Following small amounts of ischaemia, all subjects are normal; after large amounts of ischaemia, all subjects are abnormal, and after intermediate amounts, a fraction of the animals are abnormal. The ischaemic insult administered to the subjects is quanitified either by counting the number of minutes of reversible vascular occlusion, or the number of microemboli trapped in the brains after embolization (Zivin et al., 1987; Zivin and DeGirolami, 1980). Some time after the onset of ischaemia, usually 48 hours, each subject's behavioural response is evaluated with a quantal rating scale: normal, abnormal, dead. Although the quantal scale is not designed to detect the subtle deficits that clinicians are used to eliciting, it has three advantages over a more detailed scoring system: (i)inter-observer agreement is extremely high, thus reducing experimental error and reducing the number of animals used; (ii) the system is biased towards finding very effective treatments as slightly effective compounds that may not prove to be effective in humans will be screened out; and (iii) the scale lends itself to probit analysis which requires well-defined ordinal rankings (Waud, 1972; Finney, 1952).The function that is fit to the data is the logistic equation. After iteratively fitting the equation to the data, one can derive the amount of ischaemia (either duration or quantity of microemboli)that renders 50% of the subjects abnormal. This quantity is called the ED50, which is analogous to the toxicological LD50. The quantal bioassay method has proven to be extremely useful as a pharmacological screening tool, and was used in the first demonstration of tPA efficacy for stroke treatment (Zivin et al., 1985). To study the anti-ischaemia effects of GABAAagonists, studies of muscimol were begun using the microsphere embolism model (Lyden and Hedges, 1992; Lyden and Lonzo, 1994). Muscimol was protective during ischaemia in both rats and rabbits (Figure 3). To groups of rats, the following was administered muscimol 1.5 mg/kg (n= 16);MK-80 1, 1.O mg/kg (n = 28); or saline (n = 30), intravenously 5 minutes after embolization (Figure 3A). The ED, values (mean t SE) for saline-treated rats were 3.53 2 0.96 X lo2,for MK-801 7.18 2 2.07 X lo2,and for muscimol 11.19 f 2.28 X 10' microspheres. The ED,, for muscimol was significantly greater than that for saline(t= 3.05,P60%) of infarct volume measured 24 hours after occlusion (Relton and Rothwell, 1992). Subsequently,it has been demonstrated that this protection was equivalent to that seen with peripheral administration of the NMDA receptor antagonist MK-80 1 (Loddick and Rothwell, 1996). However, unlike MK-80 1, neuroprotection from rIL- 1ra treatment is evident throughout the cortex and striaturn (Loddick and Rothwell, 1996, and see Figure 1). Furthermore, rIL- 1ra does not affect physiological parameters (blood pressure, heart rate or body temperature) in ischaemic animals, indicating the neuroprotective action of rIL- 1ra is due to an inhibition of the neurochemical processes that lead to neuronal death (Loddick and Rothwell, 1996). It has been confirmed that the protection offered by rIL-Ira is permanent, and is almost identical when assessed 24 hours or 7 days after MCAo (Loddick and Rothwell, 1996). Recombinant IL-lra offers almost equal protection when administered before or at the time of ischaemia. However when administration is delayed until 30 minutes after MCAo, cortical protection is still evident, but there is no protection of the striatal tissue (Loddick and Rothwell, 1996). These data are consistent with studies showing that progression of infarction in the striatum occurs before that in the cortex (Garcia et al., 1995a; Shigeno et al., 1985), and suggest that IL- 1 is an important mediator in the progression of infarction. Recombinant IL- lra also results in significant inhibition of neuronal damage caused by MCAo when administered peripherally (Garcia et al., 1995b; Relton et al., 1996).This protein has a molecular weight of about 17 kDa, and therefore is unlikely to cross readily the blood-brain barrier. However, a transport system has been described whereby circulating IL-lra enters the brain (Gutierrez et al., 1994). Furthermore, rIL- 1ra could act on the systemic side of the vascular endothelium or penetrate the brain after damage caused by ischaemia, although the latter is unlikely

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to occur until several hours after the ischaemic episode. Importantly, Garcia et al. (1 995b) have found that systemic injection of rIL-lra markedly inhibits neuronal death induced by occlusion of the MCA via the monofilament suture technique. This procedure does not require craniotomy, or damage to the blood-brain barrier through surgery, indicating that rIL-lra can enter the brain rapidly after cerebral ischaemia even when the brain has not been penetrated by surgery. The studies of Garcia et al. (1995b), and Relton and coworkers (1996) demonstrated that repeated systemic administration (intravenous bolus, with subsequent subcutaneous injections of rIL-lra (100 mg/kg 0, 4,8, 12 and 18 hours after MCAo) dramatically reduces neuronal damage and brain oedema caused by MCAo, without affecting arterial blood pressure or heart rate. Recombinant IL- 1ra (10 Fg, icv) produced similar protection to that seen after administration of the NMDA receptor antagonist, MK-801 (4 mg/kg, intraperitoneally) when it was injected 30 minutes before MCAo (Loddick and Rothwell, 1996). However, when both compounds are administered systemically at the time of MCAo, rIL-lra still offers some neuroprotection, while MK-801 is without longer effect (Relton et al., 1996). Although most studies on the effects of rIL- 1ra on ischaemic brain damage have utilized histological stains to measure the infarct volume, recent studies performing neuronal counting have confirmed that rIL- 1ra reduces significantly the number of dead neurones seen after MCAo in the rat (Garcia et al., 1995b) and the mouse (Rothwell, Davies and Rothwell, unpublished data). Furthermore, Garcia et al. (1 995b) reported that the reduction in infarct volume caused by rIL- 1ra is accompanied by improved neurological scores. Systemic administration of rIL-lra, at the time of induction of ischaemia, markedly inhibited behavioural dysfunction in ischaemic rats. This difference was observed in postural reflex testing, increased paw use and weight bearing, and exploratory rearing behaviour when compared to untreated animals. (Garcia et al., 1995b). The novel approach of gene transfer has been utilized to demonstrate that experimentally induced sustained over-expression of brain IL- 1ra dramatically reduces brain damage caused by MCAo (Betz et al., 1995).This was achieved by injection into the brain of rats of a non-replicating adenovirus into which the IL-1ra gene had been inserted. Animals injected with the virus encoding IL-lra had a significantly reduced infarct volume (70%) when compared to untreated animals. However, injection of normal adenovirus (with no insert) also resulted in a slight, though non-significant, reduction in infarct volume (Betz et al., 1995). Recombinant IL-lra presumably acts by blocking the effects of IL-1 at the type 1 receptor, and therefore does not distinguish the relative importance of IL-la or IL1p. However, recent data reported that intracerebroventricular (icv)injection of a specific anti-IL-lp antibody caused a significant reduction of brain oedema and infarct volume after transient focal ischaemia in rats, suggesting that IL- 1p mediates neuronal damage after ischaemia (Yamasaki et al., 199513).These data do not negate the role of IL-la in neuronal death, but taken together with the authors’ data describing induction of IL- 1f3, but not IL- 1a , mRNA after MCAo, suggest that the beta form of IL-I is the primary mediator of neuronal death.

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12.5 Other interleukins in stroke The above findings strongly implicate endogenous IL-1 in the pathogenesis of ischaemic brain damage, and suggest that IL- Ira is a potent inhibitor of such damage. These authors have further proposed that IL- 1ra is an endogenous neuroprotective agent which probably acts to limit neurodegeneration by inhibiting effects of IL- 1. ILIra is expressed in response to ischaemic injury, about 30 minutes after expression of IL- 1fi (Loddick, Licinio, Wong and Rothwell, unpublished data) or traumatic brain injury, predominantly in neurones which surviue the insult (Toulmond and Rothwell, 1995b).Inhibition of the action of endogenous IL- 1ra, by icv injection of anti-rat ILIra antiserum, has no effect on normal brain tissue but greatly increases (by over 70%) damage caused by MCAo or fluid percussion injury in the rat (Loddick, Toulmond and Rothwell, unpublished data). Studies relating to the role of other cytokines in cerebral ischaemia are largely circumstantial, as few studies have investigated the effects of blocking endogenous brain cytokines. Preliminary data indicate that central administration of physiological relevant doses of recombinant IL-6 inhibit ischaemic brain damage (Loddick, Turnball and Rothwell, unpublished data). In contrast, transgenic mice over-expressing IL-6 in astrocytes develop severe neurological disease (Campbell et al., 1993). Indirect evidence implicates IL8 in ischaemic brain damage as IL-8/CINC expression is rapidly induced after cerebral ischaemia (Yamasaki et al., 1995a) injection (icv) of an antibody which blocks endogenous IL-8 activity markedly attenuates damage caused by transient forebrain ischaemia in the rat (Kogure, data presented at The Pharmacology of Cerebral Ischaemia, Margurg, 1994).This protection may be due to inhibition of IL-&induced neutrophil invasion, since neutrophil invasion contributes to neuronal death due to reversible ischaemia (Chen et al., 1992; Lindsberg et al., 1991; Shiga et al., 1991; Takeshima et al., 1991).

12.6 Effects of rlL=I ra on other forms of neurodegeneration Many forms of neurodegeneration, although not classified as stroke, may result indirectly from, or share common underlying mechanisms with, cerebral ischaemia. Therefore, therapies developed for the treatment of stroke are likely to be beneficial in a number of other conditions. Recombinant IL- 1ra (rIL-1ra) has now been tested in several forms of experimental neurodegeneration or brain inflammation. Fetal hypoxia is a common clinical condition, which frequently results in cerebral damage due to ischaemia. Martin et al. (1 995) have demonstrated that systemic injection of rIL-lra protects against neontal hypoxic brain damage in the rat. Brain trauma which results in secondary ischaemic brain damage leads to rapid expression of cytokines. Lateral fluid percussion injury in the rat causes cortical damage and secondary neurodegeneration over a period of 2-3 days after the impact (Toulmond et al., 1993). Injection of rIL-lra (10 pg icv) reduces the extent of damage

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caused by this form of injury by approximately 50% when assessed 3 days after injury (Toulmond and Rothwell, 1995a). As with cerebral ischaemic damage, the effect of rIL-lra is sustained, as the extent ofprotection was almost identical when assessed 7 days after injury. Recombinant IL- 1ra is effective even when first administered 4 hours after injury, which is consistent with the hypothesis that rIL- 1ra is preventing delayed, secondary, ischaemic damage (Toulmond and Rothwell, 1995a). Cytokine overexpression has been reported in patients with multiple sclerosis and in experimental models of the disease such as experimental allergic encephalomyelitis (M E ) (see Hopkins and Rothwell, 1995). Administration of rIL-lra significantly inhibits the clinical symptoms of EAE in the rat (Martin and Near, 1995b)implying a role for IL-1 in this condition and probably in multiple sclerosis. Common mechanisms of neuronal death may be responsible for diverse forms of neurodegenerative disease. In particular, excitotoxic processes resulting from excessive activation of excitatory amino acid receptors (Lipton and Roenberg, 1994; Meldrum and Garthwaite, 1990; Meldmm, 1993) and the consequent excessive cellular influx of Ca” (Orrenius et a/., 1989) is of fundamental importance in many neurodegenerative conditions. For this reason, great effort has been expended in the development of inhibitors of glutamate release or action, with particular focus on inhibitors of modulators of NMDA and AMPA receptors (Albers et al., 1989, 1992). The role of IL- 1 in excitotoxic damage has been investigated by studying neuronal damage induced in uivo by local infusions of selective agonists at NMDA or AMPA receptors into the rat brain. Cis-2,4, methanoglutamate (MGlu), is a selective NMDA receptor agonist, which causes extensive localized neuronal death that is blocked by MK-801 but not the AMPA antagonist CNQX (Allan et al., 1995; Relton and Rothwell, 1992).Co-infusion of rIL- 1ra (5 pg) with MGlu reduces the volume of excitotoxic damage by approximately 50% (Relton and Rothwell, 1992). Similarly, SAMPA-induced striatal damage (which is blocked by CNQX, but not MK-80 1) is also reduced by co-administration of rIL- 1ra (Allen et al., 1995). The finding that rIL- Ira blocks damage caused by both NMDA and AMPA receptor activation suggests that rIL-lra may be beneficial in several forms of ischaemic damage and may provide some clues about its mechanisms of action. Further studies on excitotoxic damage with IL- 1 suggest that this cytokine acts at specific sites in the brain, probably the striatum, to cause distant and extensive damage in cortical areas (Lawrence and Rothwell, unpublished data).

12.7 Mechanisms of action of IL- I and rlL- I ra In view of the diversity and complexity of cytokine actions, and the relatively recent nature of studies on the brain, it is perhaps not surprising that the mechanism of action of the interleukins in ischaemic brain damage remains elusive. Studies on the rat have failed to detect any effect of rIL-lra in body temperature or cardiovascular function in normal or ischaemic animals (see above). This, together with the fact that rIL-lra

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protects against excitotoxic brain damage caused by NMDA or AMPA receptor activation, indicates that rIL- 1ra acts at some point in the cellular processes leading to neuronal death beyond the point of excitatory amino acid release, rather than on general physiological parameters. Infusion of IL-1 into the brain of normal animals does not cause overt neuronal death, at least not at concentrations of the cytokine found in neurodegenerative conditions (Lawrenceand Rothwell, unpublished data). However, IL- 1 does markedly exacerbate ischaemic (Loddick and Rothwell, 1996; Minami et al., 1992b; Yamasaki et al., 1995b; and see Figure 1) or traumatic brain damage (Toulmond and Rothwell, unpublished data). This indicates that either it influences only compromised neurones, or that it interacts with other molecules or cells released after tissue damage. In contrast, co-infusion of IL-1 with an NMDA or an AMPA agonist into the striatum does not exacerbate local damage. However, infusion of IL- 1 and S-AMPA into the rat striatum leads to extensive exacerbation of damage to the cortex (Lawrence and Rothwell, unpublished data). Thus, IL-1 may interact with AMPA receptors in the striatum to stimulate pathways to the cortex which lead to neuronal death, probably via release of glutamate (Lawrence and Rothwell, unpublished data). Since rIL- 1ra protects against a variety ofbrain insults, it is likely to influence some fundamental mechanism(s) involved in several forms of neurodegeneration. An obvious example of such a process is release of excitatory amino acids, such as glutamate. No effect of IL-1 or rIL-lra on release of radiolabelled glutamate from brain synaptosomes or brain slices has been detected (Allan et al., 1995). This observation does not exclude the possibility that rIL- 1ra influences release of glutamate or other exatatory amino acid (EAA) in vivo, but indicates that modulation of EAA release is not likely to be its primary site of action. IL- 1 has a number of actions on cells of the CNS which may participate in neurodegeneration (see Table 2). These actions of IL-1 include damage to the blood-brain barrier, effects on the endothelium, and release of nitric oxide and arachidonic acid by neurones and glia, all of which could cause neuronal death either directly or through their products such as free radicals, eicosanoids or platelet activating factor (PAF) (see Rothwell and Relton, 1993). Several actions of I L 1 in the brain, for example, on fever, appetite and particularly adrenal activation, are dependent on the release of the neuropeptide corticotrophin-releasingfactor (CRF) (see Rothwell and Hopkins, 1995).Several studies now indicate that CRF, originally identified as a mediator of stress responses, itself participates in neurodegeneration. CRF gene expression is induced rapidly after MCAo (Wong et al., 1995),and icv injection of a CRF receptor antagonist inhibits global and focal ischaemia or excitotoxic brain damage (Lyons etal., 1991; Strijbosetal., 1994).AdirectrelationshipbetweenIL-1 andCRFinneurodegeneration has not been demonstrated, but remains an attractive hypothesis. Much of an understanding of the mechanisms of underlying ischaemic neuronal death, and particularly of the excitotoxic processes, is derived from studies on primary cultured neurones (see Choi, 1994). However, it seems that this approach may not be valid for studies on I L 1 or IL- 1ra. In marked contrast to effects in vivo, rIL- 1ra does not inhibit neuronal death caused by glutamate or agonists of NMDA, AMPA or

INTERLEUKINS AND CEREBRAL ISCHAEMIA

29 1

TABLE 2 ACTIONS OF IL- 1 WHICH MAY CONTRIBUTE TO ISCHAEMIC BRAIN DAMAGE

Beneficial

Detrimental

NGF synthesis

NO synthesis

Reduced Ca entry

Arachidonic acid and prostanoid synthesis

Inhibits LTP Enhanced GABA activity

CRF expression

Glial activation

ICAM- 1 expression Neutrophil invasion Microglial activation Free radical release

Key: NGF - nerve growth factor; LTP - long term potentiation; NO - nitric oxide; CRF corticotrophm releasing factor; ICAM-1 - intercellular adherin molecule-1 . ~

kainate receptors in rat primary cultured striatal or cortical neurones (Strijbos and Rothwell, 1995). Furthermore, application of IL- 1 for 24 hours at picomolar or low nanomolar concentrations protects against rather than enhances these forms of excitotoxic damage. Similar protective properties have been reported for other cytokines, including transforming growth factor p and IL-6 (e.g. Prehn et al., 1993; Toulmond et al., 1992). Exposure of cortical neurones to IL-lp for 72 hours at higher concentrations (over 50 nM) does lead to neuronal death, which is not blocked by EAA receptor antagonists or by a nitric oxide (NO) synthase inhibitor, but were blocked by rIL-lra (Strijbos and Rothwell, 1995).It is possible that these concentrations, although considerably higher than the a f i t y of known IL- 1 receptors (less than 1 nM), are required because immature neurones in culture are insensitive to neurotoxic effects of IL- 1, or because IL- 1 is degraded rapidly in these cultures. However, the absence of any effect of rIL-lra in this system indicates that it is fundamentally different to processes which occur in uivo. The marked discrepancy between these in uivo and in vitro studies is frustrating, but may provide some insight into the mechanisms of action of IL-lra. Primary cultured neurones differ in many ways to neurones in their normal environment. In particular, the presence of non-neuronal cells such as glia or endothelial cells is normally low in such cultures, and it was also observed that rIL- 1ra is ineffective in pure neuronal cell lines (unpublished data). There is now considerable evidence that cytokines such as IL- 1 could influence neuronal function and survival indirect& through effects on glia. For example, IL- 1, TNFa and IFNy can all stimulate microglia to produce neurotoxic factors (see Banati et al., 1993; Giulian, 1993; Meda et al., 1995; Piani et al., 1991, 1994). Recent interest has focused on the manner in which neurones die, since it has been

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proposed that in the adult nervous system, neuronal death can occur via apoptosis as well as necrosis (Bowen, 1993; Linnick et al., 1993; Gordon, 1995). Apoptosis is an active or ‘programmed’ form of cell death and may therefore be liable to intervention. It is not known if IL- 1 or IL- 1ra affect apoptosis after ischaemic brain damage, though preliminary data suggest that apoptotic neurones are relatively rare (65 years, risk factors such as heart disease, hypertension and diabetes mellitus, for hypertension at entry over 160/95 mm Hg, mild versus moderate to severe initial deficit, and start of treatment between 13-24 hours, no significant difference between nimodipine and placebo was seen. The authors suggested that this finding should be

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confirmed in a controlled trial focused on the dose of 120 mg and a time period of 12 hours after onset of symptoms for initiation of the treatment. The early treatment approach is now being evaluated in a Dutch study, the Very Early Nimodipine Use in Stroke (VENUS) Study (Limburg, 1996). 15.2.I . I Intravenous nimodipine

Nimodipine, given as an intravenous infusion in acute ischaemic stroke, has been evaluated in four trials (Heiss et al., 1990; Bridgers et al., 1991; Norris et al., 1994; Wahlgren et al., 1994). The first study by Heiss et al. of 27 patients assessed the effect of intravenous nimodipine or placebo on cerebral metabolism by positron emission tomography Patients treated with nimodipine had significantly better recovery of the cerebral glucose metabolism in regions surrounding the core of dense ischaemia. A significantly better functional improvement, as evaluated with the Barthel scale, was seen in the nimodipine-treated group compared to controls. Nimodipine was given in a dose of 2 mg/h for 5 days followed by an oral dose of 120 mg daily until day 2 1, or matching placebo. The INWEST trial (Wahlgreen et al., 1994) and the trial by Bridgers et al. (1991) used the same treatment plan as in the first study by Heiss et al. (1990), although they added an initial parallel group receiving a dose of 1 mg/h of nimodipine. Both these trials were terminated prematurely because of safety concerns after inclusion of 295 and 204 patients, respectively. Patients treated with an initial intravenous dose of 2 mg/h had a statisticallysignificantworse neurological and functional outcome compared to the placebo groups. The results of the 1 mg/h treatment were less clear. In the study by Bridgers et al., an improvement trend was noted in a posthoc subgroup of patients with an initial moderate deficit in which treatment was started within 12 hours after the stroke. In the INWEST study, however, the trend was very similar to outcome in the 2 mg/h treated group, although less pronounced, and the difference was not statisticallysignificant. An intravenous dose of 2 mg/h for 10 days, followed by a daily oral dose of 180 mg for 6 months in 164 patients showed no improvement compared to placebo treatment in the study by Norris et al. (1 994).

15.2.1.2 Nimodipine and dely oftreahnent initiation The meta-analysis of nine oral nimodipine studies revealed a significant reduction of the number of patients with an unfavourable outcome if treatment was started within 12 hours after onset of symptoms. The study included all patients with a daily dose of 120 mg of nimodipine, but a recomputation of patients on daily doses of 60 mg and 240 mg, did not essentially change the result. Being a significant result in one of 13 subgroups, the risk of a chance effect is substantial. This risk was obvious to the authors, who concluded that the result should be verified in a controlled trial focused on the dose of 120 mg and a time period of 12 hours. Such a trial is now ongoing (Limburg, 1996). Some further support for the validity of the observation was given in a later study by Kaste et al. (1994), who found that if medication was started withii

A REVIEW OF CLINICAL STUDIES

34 1

12 hours, mobility improved better during treatment with nimodipine. If the observation of a positive effect of nimodipine for patients treated within 12 hours is correct, and treatment later after onset is of no value or even harmful, the overall negative effect of nimodipine in acute stroke can be understood from the perspective that only 17% of the patients in the nine studies were actually treated in the early time interval. 15.2.1.3 Nimodipine-indued hypotmsion Intravenous nimodipine treatment caused a signhcant dose-dependent reduction of systolic and diastolic blood pressure for the patients in the INWEST study. An explorative analysis indicated a correlation between diastolic and mean arterial blood pressure in the nimodipine treated groups and unfavourable neurological outcome. The authors suggested that the unfavourable outcome may have been caused by a reduction of the cerebral perfusion pressure during intravenous nimodipine treatment, particularly in the 2 mg/h group. Little data on the effect of nimodipine on blood pressure were given in other intravenous nimodipine trials. The INWEST study found no difference in blood pressure between the groups during the oral treatment phase starting 5 days after randomization. An oral nimodipine study by Kaste et al. (1994), however, found a significant lowering effect on the blood pressure. The authors of the INWEST study noted that for patients who did not fall in blood pressure, nimodipine treated had a better outcome than those on placebo. However, these patients were very few and the results did not permit conclusions.

15.2.1.4 Overall evaluation ofnimodipine in acute stroke The overall conclusion from the meta-analysis mentioned earlier (Mohr et al., 1994), two other systematic overviews (Gelmers and Hennerici, 1990; Di Mascio et al., 1994) and a meta-analysis of mortality data for all published nimodipine studies (Wahlgren, 1995) is that nimodipine treatment does not improve neurological or functional outcome or mortality compared to placebo treatment. There are two major concerns regarding the validity of the negative results of the nimodipine studies. The first is that a minority of the patients have been included within a reasonable time interval of 12 hours after onset of the neurological symptoms. The positive subgroup findings for this group suggest that a nimodipine effect may exist. The second is that a hypotensive effect of nimodipine may have outweighed any neuroprotective effect, in particular in the intravenous studies. In general, the reports of an overall negative outcome of the nimodipine studies seem to have been generally accepted, although one study is still ongoing. One reason for the limited interest for calcium antagonists in cerebral ischaemia may be the awareness that the L-type of voltage-sensitive calcium channel is only one of several alternative pathways resulting in an increased intracellular calcium concentration. In the perspective of an accumulating interest for combination therapy strategies, calcium antagonists (and in particular the most extensively evaluated agent, nimodipine) may again be of interest for further trials.

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15.2.2 PY 108468 PY 108-068 is another dihydropyridine calcium antagonist, which in animal models of ischaemic stroke had favourable effects on oxygen delivery, neurological recovery and mortality (Wiernsperger et al., 1984). In small doses, the drug increases cerebral blood flow in humans with acute stroke, but higher doses may worsen blood flow in the peri-infarcted area (Vorstrup et al., 1986). The drug has previously been under consideration for treatment of hypertension and effort-induced angina. In a pilot study of PY 1OM68 in acute ischaemic cerebral infarction by Oczkowski et al. (1989), no difference was found in neurological outcome and mortality between active treatment and placebo controls. The drug was given as an oral daily dose of 150 mg within 48 hours after onset of symptoms. A trend to improvement in functional recovery was seen in the active treatment group, but the authors estimated that a larger trial, involving at least 254 patients, would be necessary to prove an effect. The drug was found to be safe. Transient hypotension was seen in 2 patients in the PY 108-068 group but in none of the patients in the control group.

15.2.3 ISRADIPINE Isradipine, also a dihydropyridine calcium antagonist, reduced infarct size in experimental models of acute ischaemic stroke (Sauter et al., 1990).A randomized controlled trial of isradipine has been undertaken, the ASCLEPIOS study (Azcona and Lataste, 1990).The study has been terminated prematurely as a result of safety considerations, and details on the results are not yet revealed (Lataste et al., 1992).

15.2.4 FLUNARIZINE Flunarizine is used for treatment of vestibular disorders, prevention of migraine and is said to provide symptomatic relief from different vascular conditions. The therapeutic effect is thought at least partly to be due to inhibition of calcium entry through VSCC (Holmes et al., 1984; Van Nueten and Vanhoutte, 1984).Two randomized controlled trials have been reported on the effect of flunarizine in acute ischaemic stroke, a pilot study by Limburg and Hijdra (1 990) involving 26 patients and a larger study with 433 included patients by Prange et al. (1991). Both of these studies were negative. There are no data on the effect on blood pressure of this treatment.

15.3 Monogangliosides Randomized controlled trials of monoganglioside (GM1) therapy in stroke were first initiated by Bassi et al. (1 984) and Battistin et al. (1985) after reports that GM 1


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promotes functional recovery of injured dopaminergic and cholinergic activities (Wojcik et al., 1982; Toffano et al., 1983) and protects neurones against retrograde degeneration Uonsson et al., 1984; Karpiak and Mahadik, 1984).Gangliosides are a heterogeneous group of sialic-acid-containing glycosphingolipids in the outer leaflet of the plasma membrane lipid bilayer. The concentration of these compounds is high in the brain (Ledeen, 1983; Svennerholm, 1984; Dal Toso et al., 1988). Gangliosides may influence transmembrane signalling by interacting with cell surface glycoproteins, membrane-embedded proteins and calcium ions (Tettamanti et al., 1985).Exogenous gangliosides, in particular GM 1, have been found to enter plasma membranes of neurones and mimic endogenously occurring gangliosides (Carolei et al., 1991).GM1 may antagonize pathological effects of excitatory amino acids in ischaemia without affecting physiological activation of excitatory amino acid receptors, a property called abuse-dependent antagonism (Coolingridge and Bliss, 1987; Manev et al., 1990). Increasing experimental data have focused on the capacity of the mature brain to reorganize and functionally recover following a focal brain damage (Bjorklund and Stenevi, 1979; Seifert, 1981). Consequently, early trials on GM 1 aimed to restore function after ischaemic injury and treatment was initiated in the subacute phase, 10-15 days after onset of the neurological symptoms. The trials by Bassi et al. (1984) and Battistin et al. (1985) included 78 patients treated with GM 1, extracted and purified from bovine brain, 40 mg daily as intramuscular injections or placebo for 6 weeks. The trial treatment had been preceded by corticosteroids for 10 days for the purpose of reducing oedema. A modification of the Mathew score (Mathew et al., 1972)by Frithz and Werner (1975)was used for evaluation of the results by Bassi et al. (1984) and a semi-quantitative global score including results of a clinical evaluation, electroencephalography, flash-evoked potentials and computer tomography of the brain by Battistin et al. (1985). These studies were important because they reported a statistically significant positive outcome for efficacy parameters and absence of adverse effects by the study treatment. The results initiated several new randomized controlled trials, mostly with a higher dose - 100 mg daily intramuscularly or intravenously - and an earlier start of treatment, between 5-72 hours after onset of symptoms. In total, about 1500 patients have been included in 14 randomized controlled trials between 1984 and 1994. One of the largest trials was reported by Argentino et al. (1989),in which 502 patients with a first ever hemispheric cerebral infarction were randomized at 31 clinical centres within 12 hours after onset of symptoms to either an intravenous dose of 100 mg GM 1 daily or placebo for 15 days. Half of the patients in each group were also given haemodilusion. The effect of the treatment was evaluated after 15, 2 1 and 120 days concerning mortality rate, neurological disability measured by a modified Rankin scale (Rankin, 1957) and change of score in the Canadian Neurological Scale (CNS; C o d et al., 1986). An intention-to-treat analysis showed no differences in the outcome parameters between the groups. An efficacy analysis in which patients were excluded if they proved to have had an earlier stroke or if they refused to continue in the study showed a statistically significant higher degree of

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neurological improvement in the GMl-treated group during the first 10 days. However, this effect was no longer present after 120 days. The initial positive results motivated the authors to recommend a larger multicenter trial of GM1 in acute stroke. A few smaller published trials (Jamieson et al., 1989; Giraldi et al., 1990; Monaco et al., 1991, Angeleri et al., 1992, Wender et al., 1993) and two unpublished trials by Abraham, UK, and Rheuter, Germany (Carolei et al., 1991), revealed a variety of non-significant and partly positive results for the GM1 treatment. Recently, two large trials, the Sygen Acute Stroke Study (SASS; Alter 1994) and the Early Stroke Trial (EST, Lenzi et al., 1994) reported negative results for main outcome parameters. The SASS trial involved 13 clinical centres in North America, recruting 287 acute stroke patients within 48 hours after onset of symptoms. Computer tomography should be compatible with an anterior or middle cerebral artery ischaemic stroke. Patients received 100 mg GM1 intramuscularly (i.m.) or placebo for 28 days and evaluated at regular intervals up to 84 days after randomization. Primary endpoint measures were the Toronto Stroke Scale (Norris, 1982), Barthel Index (Mahoney and Barthel, 1965) and mortality There were neither significant differences between active treatment and placebo for these primary parameters, nor for secondary endpoints including other neurological and neuropsychological impairment scales. Another trial was recommended because a statistically significant better outcome in the motor component of the Toronto Stroke Scale was found in the GMl group at day 28 when the treatment was stopped (B0.02) and a borderline significance at the final follow-up at day 84 (-0.057). The EST involved 16 clinical centres, mainly in Europe. The trial recruited 805 patients with acute stroke within 5 hours after onset of a unilateral motor deficit, clinically attributable to an ischaemic lesion of one cerebral hemisphere. A C T was required before randomization to rule out other causes to the symptoms than focal cerebral ischaemia. Patients received a first dose of 200 mg GM1 intravenously (i..) or placebo, and a second dose of 100 mg GM1 i.v. or placebo 12 hours later. Daily intravenous injections of 100 mg followed until day 10, and intramuscular injections from day 11 to day 2 1. Follow-up evaluations were made at 15 days, at day 2 1 and 2 and 4 months. Primary endpoint measures were mortality and change in the Canadian Neurological Score (CNS) between baseline and the final 4 months followup evaluation. Survival was similar in the two treatment groups. Improvement in neurological status, as measured by the change in CNS score, was greater in the group receiving GM 1, with a statistical borderline result ( e 0 . 0 6 ) . Aposthoc analysis showed a statistically significant result (-0.0 16) for patients included within 4 hours after onset, and the authors concluded that the efficacy of this treatment was greater when given soon after onset of stroke. Although theposthoc analyses may be encouraging, the protocol-defined primary parameters failed to show convincing evidence for the efficacy of GM1 treatment. Treatment with GM1 in the doses and administrative routes used in these trials were reported to be safe. No differences were seen between treatment arms for adverse events.


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15.4 Naloxone The observation that the opiate antagonist naloxone improved recovery after experimental spinal cord injury (Faden etal., 1981) and induced cerebral ischaemia in gerbils (Hosobuchi et al., 1982) supported the hypothesis that activation of opiate receptors may cause neurological damage. The results stimulated efforts to evaluate the effect of the drug in stroke patients (Fallis et al., 1984; Perraro et al., 1984; Czlonkowska and Cyrta, 1988; Czlonkowska et al., 1992). Fallis et al. evaluated the effect of naloxone in a double-blind trial conducted with 15 stroke patients whose deficits ranged from 8-60 hours in duration. Two injections were given of naloxone to achieve a total dose of 0.4 mg in 3 patients and 4.0 mg in 12 patients, or matching placebo. Four naloxone patients improved slightly, while 5 patients improved in the placebo group. There were no significant elevations of plasma beta-endorphin among the stroke patients. Chzlonkowska and Cyrta (1988) and Chzlonkowska et al. (1992) observed neurological improvement for naloxone patients, but the results of the different trials remain inconclusive.

15.5 Piracetam Piracetam increases cyclic adenosine monophosphate in the brain and stimulates adenylate kinase (Herrschaft, 1988). In a positron emission tomography (PET) study, Heiss et al. (1983) found increased glucose metabolism in the ischaemic part of the brain in patients with acute stroke. Three pilot studies (Kartin et al., 1979; Herrschaft, 1988; Platt et al., 1992) have stimulated a larger randomized multicenter trial including 927 patients with an acute stroke within 12 hours after onset of symptoms. For the whole group of patients, no significant difference was seen between active and placebo-treated patients using the Barthel and Orgogozo scales. In a preplanned subgroup analysis, those included within 6 hours had a better outcome on the Orgogozo scale, but this was not statisticallysignificant (P=0.07). O n the Barthel scale, there was a significant difference at 4 and 12 weeks favouring placebo (De Deyn, 1995).

15.6 GABA agonistslclomethiazole Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain (Fagg and Foster, 1983).GABAAreceptors gating a chloride channel seem to be present in all neurones. Activation of the receptor/ion channel complex results in hyperpolarization of the cell membrane, thus inhibiting action potentials elicited by depolarization (Curtis et al., 1968; Kelly d al., 1969). Three subunits of the receptor, a, fi and y, all bind GABA, although the highest affinity is with the a-subunit (Figure 2). Barbiturates bind to the a-and f3-subunits, while benzodiazepines bind only to the

N.G. WAHLGREN

346

Benzodiazepine

FIGURE2 The GABA receptor.

y-subunit (Barnard et ab, 1987). The binding a f i i t y of one of the ligands seems to increase if any of the others bind to the receptor. GABA’s main physiological role is to balance the action of the major excitatory transmitter, glutamate. GABA is released immediately from presynaptic terminals following the onset of cerebral ischaemia, like other neuroactive amino acids, including glutamate (Green et al., 1992).The synthesis of GABA is decreased for up to four hours, probably through a feedback mechanism. It has been suggested that the GABAergic inhibitory mechanism is impaired during ischaemia and that stimulation of the GABAA receptor might balance the excitotoxic cascade (Green et al., 1992; see also Chapter 10). Elevation of GABA levels by the use of the GABA transaminase inhibitor y-VinylGABA, which inhibits elimination of GABA, in bilateral cerebral ischaemia in Mongolian gerbils resulted in increased levels of energy metabolites compared to controls, indicating reduced energy utilization (Abel and McCandless, 1992). In a gerbil model of repetitive forebrain ischaemia, y-vinyl-GABA gave significant protection to the hippocampus CA1 region and to substantia nigra reticulata in treated animals compared to controls (Shuaib et al., 1992).Muscimol, a GABA agonist, also protected the cerebral cortex, striatum, hippocampus, thalamus and substantia nigra reticulata from the effects of repetitive ischaemia in gerbils (Shuiab et al., 1993).Chlomethiazole has been used for many years in clinical practice as an anticonvulsant and sedative. It seems to increase the effect ofGABA by an interaction with the GABA, receptor-gated chloride channel. Chlomethiazole does not seem to function as a direct agonist at the receptor. Neither has chlomethiazole any effect on GABA synthesis or release (Cross et al.,1989). Chlomethiazole in a dose of 100 mg/kg intraperitoneally (i.pJ has been shown to protect against hippocampal neurodegeneration in a gerbil model of transient forebrain ischaemia when given up to 3 hours after a 5-minute episode of bilateral carotid artery occlusion (Cross et al., 1991). In a rat model of focal cerebral ischaemia induced by means of a photochemically induced thrombosis of cerebral arteries, 200 mg CMZ i.p. reduced the area ofthe infarct when given 5 minutes postischaemia (Snape etal., 1993).

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Glycine

I

I

PCP

FIGURE 3 The NMDA receptor.

15.7.I.I Dizolcipine Dizolcipine (MK-80 1) demonstrated a marked reduction of infarct size in the focal ischaemic stroke models with rats, cats and rabbits, with treatment administered before and after induction of ischaemia (Kocchar et al., 1988; Ozyurt et al., 1988; Park et al., 1988, 1989; Buchan et al., 1989). Results from global stroke models were conflicting, in particular when treatment was started after onset of ischaemia (Swan and Meldrum, 1989; Gill et al., 1988, 1989; Fleischer et al., 1988).A neuroprotective effect in some of the pre-treatment global models in rats may have been caused by hypothermia rather than by dizolcipine, as prevention of hypothermia abolished this effect and hypothermia alone reproduced it (Buchan, 1990). The clinical development of dizolcipine was discontinued as a consequence of safety concerns: a vacuolization effect of relatively low doses ofthe drug (Olney et al., 1989),EEG changes ( h u n g and Desborough, 1988),behavioural side-effects such as cataplexy, locomotor disturbance and reduced spatial learning at higher doses (Koek et al., 1988). Other effects of dizolcipine which caused uncertainty about the future development were transient hypotension (Park et al., 1989) and dose-dependent depression of the level of consciousness (Buchan, 1990). 15.7.1.2 Dextromethorphan and dextrorphan

Dextrorphan and the related compound dextromethorphan, a cough suppressant, is a non-competitive NMDA antagonist which interacts with the phencyclidine binding site using a different mechanism from that of dizolcipine (Wong et al., 1986). Dextromethorphan and dextrorphan reduced infarct volume when given before (George et al., 1988; Steinberg et al., 1988a) or after (Steinberg et al., 1988b, 1989) occlusion of the temporary anterior cerebral artery in a rabbit model. In a pilot study of 10 patients, oral dextromethorphan in a dose of 240 mg daily for three weeks did


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Hyperactive behaviour induced by transient bilateral carotid occlusion in gerbils during 7 days following the insult was attenuated when given CMZ 200 mg/kg or 100 mg/kg i.p. 60 minutes after ischaemia. The degree of hyperactivity was linearly related to the amount of neurodegeneration 7 days after the ischaemia (Baldwin et al., 1993). A phase I1 dose-finding safety study of patients with acute ischaemic stroke has confirmed that a dose of 75 mg/kg during 24 hours is safe and practical to use. The results of a phase I11 trial, including about 1350 patients within 12 hours after an acute stroke, is planned to be released in early 1997. The study has passed its final safety interim analysis, based on 900 patients, and the conclusion of the independent monitoring committee was to continue recruitment in accordance with the study protocol.

15.7 NMDA antagonists The role of NMDA antagonists has been questioned lately as a result of the decision to terminate trials with CGS 19755 (Selfotel), a competitive NMDA antagonist, because of an insufficient efficacy/safety ratio. An acute stroke study of another competitive NMDA-antagonist, eliprodil, was also recently stopped as an interim analysis revealed that there was no difference between active treatment and placebo. No safety problems were reported. At least one competitive NMDA antagonist (CerestatB) is currently being evaluated in a controlled trial (see Chapter 8). The key role of excitatory amino acids (EAA) for neurotoxicity induced by cerebral ischaemia became increasingly evident during the 1980s (Choi, 1990; Fagg et al., 1986). Glutamate is the primary of these compounds, although aspartate and other amino acids, such as cysteate, homocysteate, homocysteine sulphinate and cysteine sulphate, also may take some part in excitatory neurotransmission. The N-methybaspartate (NMDA) receptor is the best known of the EA.4 receptors (Figure 3). Stimulation by glutamate or, specifically for this receptor, by NMDA, opens the related ion channel for passage of calcium (Ca"), sodium (Na') or potassium (K+) ions. Other EAA receptors, 'the non-NMDA receptors', are the AMPA (quisqualate), the kainate, the L-AP4 and the metabotropic receptors (Morley et al., 1994). In cerebral ischaemia, excess release of glutamate from nerve terminals results from failure of Na+/K+-pumps and depolarization of voltage-gated presynaptic sodium and N-type voltage-sensitive calcium channels. Postsynaptically, glutamate activates NMDA and non-NMDA receptors finally resulting in a dramatic increase in intracellular calcium, calcium-mediated cell death and spreading neuronal excitation.

15.7. I NON-COMPETITIVE NMDA ANTAGONISTS Non-competitive antagonists like phencyclidine (PCP), ketamine, dizolcipine (MK801), dextromethorphan and dextrorphan (Wong et al., 1986; Sills and Loo, 1989) bind to the phencyclidine recognition site in the NMDA-gated ion channel.


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not cause any severe side-effects (Albers et al., 1991). Intravenous dextrorphan induced hallucinations, agitation, sedation and gastrointestinal symptoms. Hypotension occurred at the highest doses (Albers et al., 1995).The clinical development of dextrorphan has been terminated. 15.7.1.3 Cerestut

Cerestat (CNS 1 102; Reddy et al., 1994), another non-competitive NMDA antagonist, reduced infarct volume by 66% when given 15 minutes after permanent occlusion of the middle cerebral artery in rats (Minematsu et al., 1993). Diffusion weighted magnetic resonance imaging exposed even greater ischaemic lesion reduction during a three-hour treatment period. Delayed treatment initiation with the same dose also reduced the infarct volume by 50% (Meadows et al., 1994). A dose-finding safety trial with Cerestat in over 60 acute stroke patients approached neuroprotective dose levels (Fisher, 1994). Transient catatonia and mild agitation occurred in a few patients. No pronounced effects on blood pressure or heart rate were noted. A pivotal safety and efficacy trial of Cerestat in acute ischaemic stroke patents is in progress (Turrini, 1996; see Chapter 8). 15.7.1.4 Mapesium

Mg2+causes a voltage-dependent block of the ion channel of the NMDA-receptor (Nowak et al., 1984), and acts as a non-competitive NMDA antagonist at higher concentrations (Harrison and Simmonds, 1985). Focal ischaemia rat models demonstrated reduced infarction volume with magnesium therapy (McDonald et al., 1990; Izumi et al., 1991). In a rat 4-vessel occlusion model, hippocampal CAI necrosis decreased significantlywhen MgC12was given 24 hours after initiation of ischaemia (Tsuda et al., 1991). A safety pilot study of 13 patients found that the drug was well tolerated (Strand et al., 1993).A larger proportion of the Mg*+-treatedpatients improved neurologically, and the need for institutional care 6 months after stroke was reduced. The authors recommended further evaluation in large scale trials.

I 5.7.2

COMPETITIVE NMDA ANTAGONISTS

Competitive NMDA antagonists in general penetrate the blood-brain barrier poorly, a problem which may be less important in the ischaemic area with barrier damage. Compounds in this group, such as APH, CPP, CGS- 19755 and MDL 100,453 block the NMDA receptor site (Warkins and Olverman, 1987).CGS-19755, CPP (Boast et al., 1988) and MDL 100,453 (Hasegawa et al., 1994) reduce infarct size when the treatment is started after onset of ischaemia.

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15.7.2.1 SeEftel (CGS-19755) Selfotel (CGS-19755) is the most extensively tested drug in this group. It is thought to act directly on the glutamate-NMDA binding site. Phase I11 clinical trials have recently been terminated because of a statisticallynon-significant increased mortality in the active treatment groups and a low likelihood that the trial will be able to prove efficacy for stroke patients (Ciba-Geigy, personal information). The final phase I11 trials were preceded by two phase I1 safety studies. A safety and dose-finding study (Grotta, 1994) found side-effectsin all 6 of patients treated within 12 hours of a hemispheric ischaemic stroke with 2 mg/kg as one or two intravenous bolus doses, in 4 of 6 with 1.75 mg/kg and 3 of 6 with 1.5 mg/kg. Side-effects noted were agitation, hallucinations, confusion, paranoia and delirium appearing up to 22 hours (mean 1-3 hours) after treatment and lasting 2-60 hours (mean 24 hours). Three patients died of the total of 24 treated with Selfotel as compared to 1 of 8 in a placebo-treated control group. Despite the side-effects reported, doses up to 1.5 mg/kg were concluded to be safe and tolerable in acute ischaemic stroke patients. The degree of improvement in the National Institute of Health (NIH) stroke scale was 7 1YO in all active treated patients versus 36% in the placebo group of the survivors. A Barthel index of 270 was reached in 95% of the actively treated group versus 29% of the controls. It was not stated in the report how much of the clinical improvement that was related to the doses over 1.5 mg/kg. Another multicenter randomized placebo-controlled trial (Coull, 1994) of 109 patients treated within 6 hours after onset of symptoms with 1.5 mg/kg as a single intravenous dose of Selfotel showed neurological adverse events (agitation, confusion and hallucinations) in 57% (13% severe) of the actively treated as compared to 18% in the placebo group (P=O.OOOl); 4% versus 0% had respiratory adverse events.

15.7.2.2 Eliprodil The reality of an NMDA receptor regulatory site for polyamines has been suggested following observations that the endogenous polyamines, spermine and spermidine, increase the binding of open-channel blockers such as Dizolcine and increase NMDAelicited currents in cultured neurones (Williams et al., 1991). Eliprodil, a polyamine site antagonist, has been found to reduce infarct size in stroke models (Gotti et al., 1990; Poignet et al., 1992). An intraperitoneal injection in mice of 10 mg/kg of eliprodil5 minutes, 6 hours and 18 hours after occlusion of the middle cerebral artery and then twice daily until sacrifice reduced the infarct volume by 60-70% compared to controls (Williamset al., 1991). A safety pilot study involving 114 patients revealed mild side-effects such as reversible QQ prolongation and dizziness at high doses (Fisher, 1995). A large phase I11 trial was terminated recently since a sequential efficacy analysis of the first 483 patients failed to demonstrate a statisticallysignificant difference between the eliprodil group and the placebo control group (Synthtlabo Recherche, 1996).


35 1

15.8 Inhibition of glutamate release 15.8. I LUBELUZOLE Lubeluzole is a benzothiazole compound which prevents the increase in extracellular glutamate concentrations (Scheller et al., 1995),normalizes neuronal excitabilityin the peri-infarct region (Buchkremer-Ratzmann and Witte, 1995) and inhibits glutamateinduced nitric oxide-related neurotoxicity (Lesage et al., 1994). Lubeluzole reduced infarct volume in photochemically induced thrombotic cerebral infarcts in rats and improved the neurological outcome @e Ryck, 1994). A phase I1 trial of a potentially neuroprotective substance, lubeluzole, has been terminated recently (Diener et al., 1996)and phase I11 trials are now ongoing (Lubeluzole Study Group, 1996). In the phase I1 trial, patients with an acute stroke within 6 hours were randomized to receive intravenous treatment with placebo or with one or two dosages of lubeluzole, 7.5 mg over one hour as a loading dose following by a continuous infusion of 10 mg/day for 5 days, or a loading dose of 15 mg followed by a daily dose of 20 mglday for 5 days. Neurological outcome assessed by the National Institute of Health (NIH) scale and the European Stroke Scale (ESS), functional outcome according to the Barthel scale and mortality at day 28 were selected as study endpoints. Initially, the trial aimed at the inclusion of 270 patients, but was terminated prematurely following a recommendation from the safety committee. The committee had noted an imbalance in mortality between the treatment groups. Mortality rates in the three treatment groups (placebo, low-dose and high-dose) were 18%, 6% and 35%, respectively. It was concluded that the higher mortality in the high-dose group could at least in part be explained by an imbalance at randomization resulting in a higher number of patients with severe ischaemic stroke at baseline. The low dose was found to be safe and to reduce mortality (P=O.O19). The first results of the phase I11 trials are expected in the second part of 1996.

15.8.2 BW619C89 BW6 19C89 is a derivative of BW 1003C87, a substance related to the anticonvulsant, lamotrigine. BW619C89 reduces the release of neurotransmitters from slices of rat cerebral cortex. The mechanism is not clarified completely, but there are some indications of an action at presynaptic voltage-sensitive sodium channels. BW6 19C89 reduces infarct size in global and focal stroke models in rats (Leach et a/., 1993; Smith et al., 1993; Graham etal., 1994).Its predecessor, BW1003C87, also protects the brain in similar models (Lustiget al., 1992; Meldrum et al., 1992),but has been found unsuitable for development because of antif'olate properties. A dose-finding phase IIa trial of BW619C89 in acute ischaemic stroke has been terminated and a phase 111 randomized controlled trial is planned to start during 1996 after some delay

352 15.8.3

N.G. WAHLGREN PHENYTOIN AND FOS-PHENYTOIN

The anticonvulsant drug phenytoin and its derivative fos-phenytoin are sodium channel antagonists with cytoprotective properties (Fisher, 1995).A clinical trial of fosphenytoin in cerebral ischaemia is to be launched.

15.9 Free radical scavengers Oxygen-free radicals apparently damage brain cells during reperfusion into the ischaemic area following spontaneous or induced recanalization, when antioxidative defence mechanisms are insufficient (Siesjo et al., 1989; Chan, 1994). Formation of superoxide, hydrogen peroxide and hydroxyl radicals may result in peroxidation injury of lipid membranes, protein oxidation and damage of DNA. Potential agents include superoxide dismutase (SOD), catalase, vitamin E, glutathione, lazaroids, iron chelators and phenyl-t-butyl-nitrone (PBN; see Chapter 13). SOD, a scavenger of superoxide radicals, has been shown in a rat ischaemic stroke model to reduce infarct volume (Kinouchi et al., 1991), but this approach has not yet been evaluated at a clinical level.

15.9. I TIRILAZAD Tirilazad, a lipid peroxidation inhibitor, has been found to reduce infarct size in several stroke models (Xue et al., 1992). A safety study of tirilazad in acute ischaemic stroke including 1 11 patients randomized to one of three dose levels (0.6, 2.0 and 6.0 mg/kg/day) or placebo, revealed that the treatment was well tolerated (STIPASinvestigators, 1994).The authors concluded that a larger trial would be needed to demonstrate eficacy. Recently, tirilazad was found to reduce mortality and increase the number of patients with a good recovery in patients with subarachnoidal haemorrhage (Kassell et al., 1996). A safety and efficacy trial of tirilazad in patients with acute stroke was recently terminated after inclusion 414 f d y eligible patients within 6 hours after onset on neurological symptoms (Peters et al., 1996). Patients were given a dose of 6 mg/kg/day of tirilazad intravenously or placebo. A favourable outcome was seen in 5 1.O% of the controls and 48.1°/o in the active treated patients, as estimated using the Glasgow Outcome scale. A favourable outcome with the Barthel scale was seen in 55.0% of controls and in 50.9% of patients treated with tirilazad. These differences were not statistically significant. It was concluded that 6 mg/kg/day of tirilazad did not improve functional outcome in patients with acute cerebral ischaemia. A study with a similar protocol including 556 fully eligible patients provided corresponding results (RANTTAS investigators, 1996). Phase I11 trials using a higher dose of tirilazad are in progress.


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15.9.2 PHENYL-T-BUML-NITRONES (PBN)

Free radicals react with nitrones to form stable nitroxides which can be identified with ESR spectroscopy. The spin-trapping agent phenyl-t-butyl-nitrones (PBN) reduced ischaemia-induced forebrain oedema and hippocampal CA 1 neuronal loss in gerbils and rats, supporting the concept that free radicals contribute to brain injury following ischaemia (Yue et al., 1992; Cao and Phillis, 1994; Sen and Phillis, 1993).

IS.I0 Inhibition of leucocyte adhesion Inflammatory processes may contribute to ischaemic cell damage after occlusion of a cerebral artery (Barone et al., 1992; Hallebeck et al., 1986). Specific cell-surface integrins, the CD 18 receptor complex, take part in the regulation of leucocyte migration into the ischaemic tissue (Price el al., 1987; Youker et al., 1992; Zimmerman and McIntyre, 1988). The expression of ICAM- 1 (intercellular adhesive molecule), a cell surface glycoprotein, on vascular endothelium facilities leucocyte adhesion (Dustin et al., 1986; Smith et ul., 1988; Staunton et al., 1989).Adhering neutrophils may release free radicals, proteases, and toxic oxidative metabolites that initiate a cascade of damage (Hernandez et al., 1987).Mechanical obstruction of the microcirculation may cause a ‘no-reflow’ - phenomenon (Ames et al., 1968). Treatment with anti-ICAM antibody reduces neurological deficits after embolic stroke in the rabbit and in a rat model (Bowes et al., 1993; Zhang et al., 1994). In a recent study, ischaemic cell damage was found to be promoted by postischaemic inflammatory response after 2 hours of transient middle cerebral artery occlusion and reduced by administration of an anti-ICAM antibody during reperfusion (Zhang et al., 1995).A clinical trial of anti-ICAM antibody is in progress.

15. I I General discussion

It can be seen from the review above that by now a range of possible therapeutic approaches have been, or are now being, investigated for the treatment of acute ischaemic stroke. Many of these are based on a knowledge of the pathophysiology of ischaemic stroke as studied in experimental animals. It is reasonable to assume that the next two or three years will see the first successes in the treatment of stroke by neuroprotective agents. However, which approach will be successful is unknown. Furthermore, the first success will not halt the trials of drugs based on other mechanistic approaches. One only has to look at the way that hypertension can be treated by p-adrenoceptor antagonists, ACE inhibitors and calcium antagonists to realize that research into and clinical trials on new drugs will continue for many years to come.

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A Adenosine, 259-280 and ATP-sensitive potassium channels, 123-124 formation, 260 levels, 260-262 drugs affecting, 267-268 mechanisms of action, 269-272 excitatory amino acid regulation, 270-27 1 free radical formation, 27 1-272 in preconditioning, 272 schematic summary, 270 neuroprotective effects in ischaemia, 259-260 receptor agonist effects, 268-269 long-term, 272-274 receptor antagonist effects, 269 long-term, 272-274 receptors characteristics, 264-265 distribution, 262, 263, 266 hypoxia/ischaemia adaptation, 266-267 Adrenergic system, and aging, and nitrones, 306 AgaIVA, in vih studies, 100 Aging, and nitrone free radical traps, 306-307 4-AHCR 200 Alpha-amino-3-hydroxy-5-methyl-4isoxazole propionate see AMPA Alzheimer’s disease, DNA strand breaks, 6 Amanitu muscaria, 25 1 see also Muscimol Amino(hydroxydihydrocycloheptaisoxazoly1) propanoic acid, 200 I-Aminopyridine, and potassium conductance, 121 AMNH, 201 AMOA, 201 AMPA, 198 structure, 199 see also NMDA antagonists 4MPA agonists, 198, 200

IL-I inhibition, 289 AMPA receptor-gated ion channels, 30, 3 1 AMPA receptors and excitotoxicity, 234 molecular biology, 202-203 recombinant, antagonist pharmacolog, 203 AMPAIkainate antagonists, 198, 200-202 in focal ischaemia models, 206-212 NBQX, 206-208 neuroprotective effects, 209 neuroprotection mechanisms, 2 13-2 14, 2 16-220 side-effect profile, 220-22 1 structures, 199 therapeutic potential, 22 1 in transient forebrain ischaemia models, 214-220 gerbil model, 2 14 neuroprotection mechanism, 2 16-220 rat models, 2 14-2 16 Amyotrophic lateral sclerosis (ALS), 15 Anaerobic workstations, 75-78 Anaesthetics, local, as neuroprotectors, 1 13 Animal models of stroke, 47-68 for acute ischaemic stroke focal models, 53-58, 155-157 global models, 49-53, 153-155 AMPA/kainate antagonist neuroprotection focal models, 206-2 12 gerbil model, 2 14 mechanism, 216-220 rat model, 2 14-2 16 model selection, 63 NMDA antagonist study, 153-157 protocol guidelines, 63 reasons for use, 47--18,64 relevance, 48,64 screening models, 48 study design, 58-62 and variability of stroke, 49,63 see also In vitro models Anoxic depolarization time, 34 Anticonvulsants, sodium channel interactions, 1 14

365

366

INDEX

AP- 1 transcription factor, redox modulation, 31 1 ApopTag method, 4 , 6 Apoptosis, neuronal, 2-16,71-73 agents causing, 3-4 drugs for reducing, 15- 16 genedproteins in, 10, 12 identification, 4-6 and IL-l/IL-ha, 292 in ischaemia-hypoxia, 7-1 0 and trophic factors, 14-15 mitochondria in, 12-1 4 vs necrosis, 4 and nitrone free radical traps, 305-306 NO in, 326 trophic factor insufficiency in, 2-3 see alro Necrosis, neuronal Aprikalim, and cardiac ischaemic damage, 127 Aptiganel hydrochloride, 175, 178, 179 clinical experience, 182-1 92,349 normal subjects, 182 pharmacokinetics, 192 side effects, 182 stroke patients, 186-1 92 trauma patients, 182-186 preclinical studies, 180- 181 progress summary, 192-1 93 structure, 180 L-Arginine analogues, as NOS inhibitors, 323-324 and iNOS activity, 32 1 see also LNAME/L-NA/GNMMA Argiotoxin, 202 ASCLEPIOS study, 342 Astroqtes, hypoxic injury, and ischaemia simulation, 74 A n , and ischaemia, 122 ATP-sensitive potassium channels, 120-121 in ischaemia, 122-123 opening beneficial effects, 124-125 deleterious effects, 125

B Barbiturates, as AMPA/kainate antagonists, 20 1 Batrachotoxinin, binding inhibition, 1 15 bax genes, in apoptosis, 10, 12 bcl genes, in apoptosis, 10, 12, 14

BCL-2 in neuronal apoptosis, 13, 14 in neurones, and ischaemia, 10 Bicuculline,24 1 Blood flow, cerebral, and NO, 325 Blood pressure, and nimodipine, 34 1 Blood-brain barrier, and ion fluxes in ischaemia, 38 BMAA, 200 BOAA, 200 Body temperature control, in animal model drug studies, 6W1 and IL-1 fl, 285-286 see also Hypothermia Brain injury see Traumatic brain injury Brain slices for NMDA antagonist studies, 151-152 see also Organotypic hippocampal cultures BW1003C87/BW619C89, as neuroprotectors, 114-1 15 BW619C89, clinical development, 35 1 C

c-JUN, in apoptosis, 6, 7, 12 Caffeine, 269 long-term effects, 272,273 Calcium intracellular control, 28-29 intracellular increase, 204,205 and tissue damage, 175 ion fluxes, 34, 35 in ischaemic stroke, 96,97 and neuronal necrosis, 1-2 post-ischaemic metabolism, 4 1 4 2 Calcium antagonists,99-108 clinical studies, 105, 339-342 in uiho studies, 99-1 00 in Uivo studies, 10&105 dihydropyridines, 1OCL101 emopamil, 101 flunarizine, 101-102 SB201283A, 102-103 S N X l l l , 103-104 Calcium channels, 30 classification,96,98-99 in ischaemic stroke, 97 modulators, as neuroprotectors, 1 16-1 18, 127 Carbamazepine, as neuroprotector, 114

INDEX Ced-9, in apoptosis, 13 Cell death, post-ischaemic, 40-42 see also Apoptosis, neuronal, Necrosis, neuronal Central nervous system injury, drug development, 176-1 77 protection, as therapeutic goal, 174-175 regeneration as goal, 174 Cerebral blood flow, and NO, 325 Cerestat see Aptiganel hydrochloride CGS 19755, 148, 178,234-235 clinical studies, 350 failure, 137, 138 preclinical data, 138 Chloride ions, membrane fluxes, 28, 29 Chlormethiazole clinical studies, 346-347 neuroprotection gerbil model, 5 I , 52 middle cerebral artery occlusion model, 56-58 Cholinergic system, aging changes, and nitrones, 306 Ciliary neurotrophic factor (CNTF), therapeutic use, 15 Clinical studies, 176-177, 337-363 aptiganel hydrochloride normal subjects, 182 pharmacokinetics, 192 side effects, 182 stroke patients, 186-192 trauma patients, 182-1 86 calcium antagonists, 339-342 chlormethiazole,346-347 free radical scavengers, 352-353 future prospects, 353 glutamate inhibitors, 35 1-352 leucocyte adhesion inhibitors, 353 monogangliosides, 342-344 naloxone, 345 NMDA antagonists, 347-350 competitive, 34!+50 non-competitive, 347-349 piracetarn, 345 tabulated summary, 338 CNQX, 200 pharmacology, 203 structure, 199 CNS 1 102 see Aptiganel hydrochloride o-Conotoxins, GVIA, in vitro studies, 99 MVI see SNX-111; SNX-230 Cortical spreading depression see Spreading depression (SD)

367

Corticotrophin-releasingfactor (CRF), in neurodegeneration, 290 CP 101 606, 149 Cromakalim, 125, 126 Cyanide, in ischaernia simulation, 73, 74 Cyclic-GMP in brain, and NO, 324 and NO mediation, 322-323 fl-Cyclohexyladenosine (CHA), 268,269 Cytokines, and nitrones, 307-308 see also Interleukins

D (-)-Deprenil, apoptosis reduction, 15-1 6 Dextromethorphan, clinical study, 348-349 Dextrorphan, 178,234-235 clinical study, 348-349 Diacylglycerol(DG), in cellular ionic metabolism, 3 1 Diazoxide, 125 Dihydropyridines clinical studies, 339-342 in vitro studies, 99 in viuo studies, 10&101 see also partinrlnr chemical 2,3-Dihydroxy-6-nitro-7-sulphamoylbenz(F)-quinoxalinesee NBQX Dilazep, and reperfusion brain damage, 267 5,5-Dimethyl-1-pyrroline N-oxide see DMPO Dizolcilpine see MK-80 1 DMPO pharmacological actions, 302 structure, 301 DNA NO toxicity, 325-326 strand breaks, in apoptosis, 6 DNQX, 200 structure, 199 Dopamine age-associated release, and nitrones, 306 excitotoxicity, neurotransmitter inhibition, 24 1 Dye exclusion, for neuronal death assessment, 81

E Eliprodil, 149, 178 clinical development, 350

368

INDEX

Emopamil, in vivo studies, 101 Endotoxaemia, murine models, nitrone effects, 307-309 Excitatory amino acids adenosine regulation, 270-27 1 receptors, 138-140 see also AMPA, Glutamate; NMDA Excitotoxic index, 236 Excitotoxicity, 233-235 damage, IL-1 in, 289 hypothesis, 138-1 40

F Fetal hypoxia, I L 1ra neuroprotection in, 288 Flunarizine clinical trials, 342 in vivo studies, 101-102 Focal ischaemia ion fluxes in, 40 neuroprotection mechanisms, 2 1 3-2 14 Fos-phenytoin,clinical trial, 352 Free radicals, adenosine actions, 27 1-272 sce also Nitrone spin traps G

G-protein-coupled amplification cascade, 311-312 GABA, 233-258 excitotoxicindex, 236, 237 excitoxicity and, 233-235 inhibitory effect, receptor subtypes in, 235 and ischaemia, 238-239 metabolism, 250 mimetics, 250-252 in combination therapies, 252-254 future therapeutic development, 253-254 GABA receptors, 235, 345-346 anatomy, 237-238 GABAAreceptor agonists, 235-236, 240-150 muscimol in viuo studies, 242-250 Gamma-aminobutyric acid see GABA Genes induction, and nitrones, 309-3 10 in neuronal apoptosis, 10, 12 transfer studies, IG1ra neuroprotection, 287

Glia, amino acid neurotransmitters, 238 Glibenclamide,and Kt channels in anoxia, 122, 123 Glucose, and ischaemia simulation, 74-75 Glutamate antagonists in combination therapies, 252-254 neuroprotection, 234-235 cell depolarization, 234 IL- 1/IL1ra effects, 290 and ischaemia, 238-239 NMDA responses, 145 receptors and cerebral ischaemia, 203-204 subtypes, 197-198,234 release inhibitors, clinical studies, 35 1-352 see also AMPA; NMDA antagonists Glycine antagonists, 148-1 49 NMDA receptor agonist, 145 Glycolysis, activation, in ischaemia, 34, 36 GM 1, clinical studies, 342-344 GMP see Cyclic GMP Guanylate cyclase, activation, and haem/NO interaction, 322 GVIA, in nitro studies, 99 GYKI 52466,201 neuroprotective effect, 2 12 mechanism, 2 16,220 pharmacology, 203 structure, 199 GYKI 53655,201 pharmacology, 203

H Haem groups, NO interactions, 322-323 Haemoglobin, in NO removal, 321-322 Head injury, consequences, societal/personal, 173-1 74 see also Traumatic brain damage Hippocampal cultures see Organotypic hippocampal cultures Hydrogen ions, membrane fluxes, 28,29 Hyperglycaemia ion gradient normalization, 39-40 ischaemia, and cell depolarization, 36, 37 Hyperthermia, and I L l fJ285-286 Hypoglycaemia ion gradient normalization, 39 ionic fluxes in, 37-38

INDEX Hypotension, nimodipine-induced, 34 1 Hypothermia neuroprotection, and drug studies, 60-6 1 tissue protection, 175 Hypoxia adenosine levels in, 26 1 fetal, IL- I ra neuroprotection in, 288 neuronal apoptosis in, 7-10 and trophic factors, 14-15

I ICAM- 1, in neurotoxicity,353 ICE genes in apoptosis, 10, 12 see also Interleukin-p converting enzyme (ICE) IL- 1,282 in brain, and ischaemia, 282-284 in ischaemic brain damage, 284-287, 288, 290 mechanisms of action, 289-292 modulation, pharmacological, 292 see also Interleukins IL- 1 ra mechanisms of action, 289-292 in neuroprotection, 288-289 cerebral ischaemia, 286287,288 in stroke therapy, 293 IL-6, in ischaemic brain damage, 288 IL-8, in ischaemic brain damage, 288 In vitro models, 69-93 advantages, 70-7 1 goals, 70 ischaemia simulation, 73-8 anaerobic workstation, 75-78 ‘chemical’vs substrate deprivation, 73, 74 comparison of culture models, 90-92 organotypic hippocampal cultures, 84-90 in primary neuronal dissociated cultures, 78-84 technical problems, 73-5 limitations, 7 1 neuronal death assessment, 7 1-73 oxygen-glucose deprivation comparison of models, 9&92 dissociated neuronal cultures, 78-84 organotypic hippocampal cultures, 84-90 see also Animal models of stroke see also under NMDA antagonists

369

In Uivo models see Animal models of stroke Injury see Head injury; Traumatic brain injury Inositol trisphosphate, in cellular ionic metabolism, 3 1 Interleukin-p converting enzyme (ICE), 292 see also ICE genes Interleukins, 28 1-298 in brain, and ischaemia, 282-284 in ischaemic brain damage, 284-288 modulation, pharmacological, 292-293 therapeutic considerations, 293 see also IL- 1; IL- 1ra; IL-6; IL-8 INWEST clinical trial, 340, 34 1 Iodoacetate, in ischaemia simulation, 73 5-Iodotubericidin, and ischaemic damage, 267 Ion channels, flux across, 28 see also by particular element Ionic fluxes, 27-45 bioenergetic failure and, 3 3 4 0 in global/forebrain ischaemia, 33, 34-37 in hypoglycaemia, 37-38 intracellular ion concentrations, 38-39 ion gradient normalization, 3 9 4 0 in focal ischaemia, 40 and membrane potential, 27-30 postinsult period, 40-42 pre-/postsynaptic, 30-3 1 and restricted energy production, 32-33 in spreading depression, 3 1-32 Isradipine clinical studies, 342 in uitro studies, 99 in viuo studies, 10 1

K Kainate see AMPA/kainate

L L-NAMEIL-NA/L-NMMA, as NOS inhibitors, 323-324 Lactate dehydrogenase, in neuronal death assessment, 8 1-82 Lamotrigine, as neuroprotector, 114-1 15 Lemakalim, 125 Leucocyte adhesion inhibitors, therapeutic potential, 353

370

INDEX

Leukocytes, neuronal damage, and adenosine, 27 1 Levcromakalin, as neuroprotector, 126 Lidocaine, as neuroprotector, 113 Lifarizine, as neuroprotector, 1 16-1 17 Lipwortin, 292 Lipopolysaccharide,and cytokine cascade, 307-308 Local anaesthetics, as neuroprotectors, 1 13 Lubeluzole, as neuroprotector, 117-1 18 clinical studies, 35 1 LY215490,201 neuroprotective effect, 2 10-2 12 structure, 199 LY293558,201 neuroprotective effect, 2 12 pharmacology, 203

M Magnesium clinical development, 349 NMDA receptor blockade, 144 MCPG, neuroprotectiveeffect, 219 Metabotropic receptor, 234 Methohexital sodium, structure, 199 Methohexitone, neuroprotectiveeffect, 2 12 Methylxanthines, 269 long-term effects, 272,273 Microglia, as IL-lP source, 283-284 Microsphere thromboembolismmodels, 55 Minoxidil sulfate, 125 Mitochondria, in neuronal apoptosis, 12-14 MK-801,178,234-235 animal model studies, 60-6 1, 348 clinical studies, 348 neuroprotective effect, 152 in combination, 208,210,252-254 Morris water maze test, 244 in uivo studies, vs muscimol, 244-246, 250 pharmacokinetics,62 Models see Animal models of stroke; In uitro models Monogangliosides,clinical studies, 342-344 Monomethyl-L-argininesee GNAME/L NA/L-NMMA Morris water maze, 242,244 Multiple sclerosis, fetal, 289 Muscimol, 25 1 neuroprotectiveeffect, 241, 346 in uivo studies, 242-250

N N-acetyl cysteine (NAC), apoptosis reduction, 15, 16 N-methyl-D-aspartatesee NMDA Naloxone, clinical studies, 345 NBQX, 200 neuroprotective effect, 206-208,2 11,2 12 in gerbil model, 214 mechanism, 213,216,220 in rat transient forebrain model, 214-216 pharmacology, 203 side-effect profile, 22 1 structure, 199 Necrosis, neuronal, 7 1-73 vs apoptosis after ischaemia, 7,8-9, 11 and toxin concentration, 4 stroke and, 1-2 see also Apoptosis, neuronal Nerve growth factor (NGF), in neuronal apoptosis, 3 Neurodegenerativediseases, therapeutic trophic factors, 15 Neurological deficit, after stroke, assessment, 62 Neurones cultures comparison vs brain slices, 90-92 dissociated cortical, 78-84 for NMDA antagonist studies, 150-1 5 1 death assessment, 71-73 in hippocampal cultures, 88-89 in primary cultures, 81-82 NO cytotoxicity, 325-327 NO modulation, 324-325 see ako Apoptosis, neuronal, Necrosis, neuronal Neutrophil leukocytes, neuronal damage, 27 1 NFKB,redox modulation, 3 10-3 11 Nicardipine, in uivo studies, 100 Nicorandil, 125, 126 Nimodipine, 176 clinical studies, 105, 339-341 in uivo studies, 100-10 1 Nipecotic acid, 25 1 Nitric oxide, 3 19-336 actions cytotoxic, 319-320 molecular mechanisms, 322-323 physiological, 319, 320 and cerebral ischaemia, 327-330

INDEX assays, 322 functions in brain, 324-325 metabolism, 321-322 in neurotoxicity, 325-327 and NMDA receptor, 327 and NOS inhibition, 323-324 synthesis, 320-32 1 Nitric oxide synthase inducible and nitrones, 308-309 regulation, 32 1 inhibitors, 323-324 in NO synthesis, 320-32 1 Nitro-L-argininesee L-NAME/LNA/LNMMA Nitrobenzylthioinosine,and ischaemic damage, 267 Nitrone spin traps, 298-3 17 chemisny, 300,301 in endotoxaemia models, 307-309 cytokine cascades, 307-308 and inducible NOS, 308-309 mechanisms of action, 309-3 12 neuroprotection, 312 in aging, 306-307 gene induction suppression, 305-306 pharmacological actions, 302-303 uses, 301, 303 see also PBN NMDA antagonists, 198 adverse effects, 220-22 1 classes, 145- 149 clinical studies, 347-350 failure, 137- 138 discovery, 177-1 78 neuroprotection, 204,206 mechanism, 2 13 in Eriho models, 150-153 in Vivo models, 153-157 therapeutic candidate development, 157-159, 175, 178-179 future prospects, 193- 194 properties of candidates, 179-80 see d o Aptiganel hydrochloride see also AMPA NMDA receptor agonists, IL- 1 inhibition, 289 NMDA receptor-gated ion channels, 30, 31 NMDA receptors biophysics, 142-145 molecular biology, 140- 142 NO and, 327 pharmacology, 145-1 49

37 1

NR genes, 140-142 NS 257,201 structure, 199 0

Organotypic hippocampal cultures, 84-90 vs dissociated cell system, 90-92 for NMDA antagonist studies, 152-153 oxygen-glucose deprivation studies, 87-90 see also Brain slices

P Parkinson’s disease, DNA strand breaks, 6 PBN, 300 free radical scavenging, 301 and inducible NOS, 308-309 neuroprotection, 303,353 in aging, 306-307 postischaemic damage, 304-305 postischaemic gene induction supression, 305-306 pharmacological actions, 302-303 structure, 301 PC 12 cells, apoptosis, 3 PD85,639, as neuroprotector, 1 18 Peroxynitrite, 326-327 pH, in global/forebrain ischaemia, 34-36 Phenyl-tert-butyl nitrone see PBN fl-R-Phenylosopropyl adenosine (R-PIA), 268,269 Phenytoin, 114 clinical trial, 352 Pinacidil, 125, 126 Piracetam, clinical studies, 345 rt-Plasminogen activator, 176 clinical trials, 177 Polyamines NMDA responses, 144-1 45 NMDA site antagonists, 149 Potassium channels activation, in global/forebrain ischaemia, 34 classification, 120 and ischaemia, 121-1 24 openers, 120-12 1 beneficial effects, 124-1 25 clinical trials, 126 deleterious effects, 125 in neuroprotection, 126-127

372

INDEX

Potassium channels - continued potential limitations, 125-126 Potassium fluxes, 30-31 in focal ischaemia, 40 in global/forebrain ischaemia, 34, 35 in hypolycaemia, 37-38 Preconditioning, 123-1 24 adenosine in, 272 Procaine, as neuroprotector, 1 13 Programmed cell death, neuronal, 2-3 see also Apoptosis, neuronal; Necrosis, neuronal Propentofylline and adenosine excitatory amino acids, 27 1 and adenosine transport, 267-268 Propidium iodide fluorescence, 88,89 Psychiatric disorders, drug screening model, 48 PY 108-068, clinical studies, 342

Q Quantal bioassay, for ischaemia, 242 Quinoxalinediones,200, 20 1 see also NBQX, NS 257 QX-222/QX-3 14, 1 13

R R56865, 112 R-PIA, 268,269 Remacemide, 178 Riluzole, as neuroprotector, 1 15-1 16 RP66055, as neuroprotector, 1 16

Simulation of ischaemia, see under In vitro models SNX- 1 11 clinical studies, 105 in viuo studies, 103-104 results, 59 SNX-230, 104 SOD 1, cyanide inhibition, and ischaemia simulation, 74 sod 1 gene, in apoptosis, 12, 14 Sodium channels, voltage-gated, in neuroprotection, 110-120, 127 agents, 114-1 18 adverse effects, 120 lamotrigine, 1 14-1 15 lifarizine, 1 16rl17 lubeluzole, 1 17- 1 18 PD85,639, 118 riluzole, 115- 1 16 blockade, 111-1 14 anticonvulsants, 1 14 local anaesthetics, 1 13 Na+-free medium experiments, 111-1 13 tetrodotoxin, 1 12-1 13 clinical relevance, 11a- 120 down-modulation, 110-1 1 1 Sodium nitroprusside, neuroprotection, 323 Sodium-potassium pump, 28,29 Spreading depression (SD) flunarizine inhibition, 102 ion fluxes in, 31-32 NMDA/non-NMDA receptor antagonist and, 213 and restricted energy production, 32-33 Superoxide dismutase therapeutic potential, 352 see also SOD 1; sod 1 gene

S SASS clinical trial, 344 SB201823A in vitro studies, 100 in Uiuo studies, 102-103 Screening of drugs animal models, 48 psychiatric disorders, 48 in uitro advantages, 7 1 Selfotel see CGS 19755 Senescence,cellular, and nitrone spin traps, 307 see also Apoptosis, neuronal; Necrosis, neuronal

T 3,3,5,5-TetramethylpyrmlineN-oxide, structure, 301 Tetrodotoxin and GABA release in ischaemia, 239 sodium channel blockade, neuroprotection, 1 12-1 13 Theophylline and ischaemia damage, 272,273 neuroprotection, 269 Thiol groups, NO interactions, 323 Thromboembolism, cerebral, models, 54-55

INDEX Thrombolytic agents, clinical trials, 177 Tiagahine, 251-252 Tirilazad, clinical trials, 352 TM domains, of NMDA receptor, 14G14 1, 142 TMPO, structure, 30 1 Transcription factors, phosphorylation regulated, redox modulation, 310-311 Transmembrane (TM)domains, AMPA receptors, 202 Traumatic brain injury aptiganel hydrochloride studies, 182-1 86 IL- Ira neuroprotection in, 288-289 see aDo Head injury TUNEL method, 4 , 6

Verapamil, in uitro studies, 99 Veratridine, toxicity, in uitro studies, 99 Vigabatrin, 25 1 y-Vinyl-GABA, 346

W Wdardine, 200

Y YMSOK, 200 neuroprotective effect, 208, 2 1 1 in gerbil ischaemia model, 2 14 structure, 199

V Z Vasodilation,NO in, 325 VENUS clinical trial, 340

Zinc, NMDA receptor blockade, 144

373


CONTENTS OF RECENT VOLUMES

Volume 30

Biochemistry of Nicotinic Acetylcholine Receptors in the Vertebrate Brain 3akob Schmidt The Neurobioligy of XAcetylaspartylglutamate M y D. Blakcb andjkseph Z Coyb Neuropeptide-Processing, -Converting, and -Inactivating Enzymes in Human Cerebrospinal Fluid Lars Ttenius and Fred Nybtg Targeting Drugs and Toxins to the Brain: Magic Bullets Lance L Simpson

and Angiotensin Receptors: Quantitative Autoradiographic Studies 3 m M . Saavedra, E t o Castrh, 30rge S. Gutkind, and AdilJ. Nmarali Schizophrenia, Affective Psychoses and Other Disorders Treated with Neuroleptic Drugs: The Enigma ofTardive Dyskinesia,Its Neurobiological Determinants, and the Conflict of Paradigms John L. Waddington Nerve Blood Flow and Oxygen Delivery in Normal, Diabetic, and Ischemic Neuropathy Phillip A. Low, Tmence D. L q t l u n d , and Philip G. McManis INDEX

Neuron-Glia Interrelations Antonia V e r M Cerebral Activity and Behavior: Control by Central Cholingeric and Serotonergic Systems c . ff. Vandenvolj INDEX

Volume 32

On the Contribution of Mathematical Models to the Understanding of Neurotransmitter Release H. Parnas, I. Parnas, and L. A. Segel Single-Channel Studies of Glutamate Receptors M. S. I? Sansom and €?N R. Ushenuood

Volume 3 I

Animal Models of Parkinsonism Using Selective Neurotoxins: Clinical and Basic Implications MUhaelJ. .&pond and Edward M . Strich Regulation of Choline Acetyltransferase Paul M . Saluaima andJames E. Vaughn Neurobiology of Zinc and Zinc-Containing Neurons ChristopherJ. Frednickson Dopamine Receptor Subtypes and Arousal Ennw Ongini and Ymcerqo G. Long0 Regulation of Brain Atrial Natriuretic Peptide

Coinjection of Xenofius Oocytes with cDNA Produced and Native mRNAs: A Molecular Biological Approach to the Tissue-Specific Processingof Human Cholinesterases Shlorno Seidman and Hermonn Soreq Potential Neumtrophic Factors in the Mammalian Central Nervous System: Functional Significance in the Developing and Aging Brain Dalia M . Araujo, Jean-Guy Chabot, and R h i Qinbn Myasthenia Gravis: Prototype of the Antireceptor Autoimmune Diseases Simone Schonbeck, Susanne Chrestel, and Reinhardt Hoh&ld

375

376


Presynaptic Effects of Toxins Alan L. Harvey Mechanisms of Chemosensory Transduction in Taste Cells Myles H. A h b a s Quinoxalinedionesas Excitatory Amino Acid Antagonists in the Vertebrate Central Nervous System Stephen N Davies and Graham L. Collingmjge Acquired Immune Deficiency Syndrome and the Developing Nervous System Douglas E. Brmnman, Swan K McCune, and Illana Gozes

Activity-Dependent Development of the Vertebrate Nervous System R. Dough Fieldc and Phillip G. Nelson A Role for Glial Cells in Activity-Dependent Central Nervous Plasticity? Review and Hypothesis Chitian M. Miiller Acetylcholine at Motor Nerves: Storage, Release, and Presynaptic Modulation by Autoreceptors and Adrenoceptors I g n ~Wesssler INDEX

Volume 35

INDEX

Volume 33

Biochemical Correlates of Long-Term Potentiation in Hippocampal Synapses Satoru Otani and %he&l Ben-Ari

Olfaction S. G. Shirlqr

Molecular Aspects of Photoreceptor Adaptation in Vertebrate Retina Satoru Kawamura

Neuropharmacologicand BehavioralActions of Clonidine: Interactions with Central Neurotransmitters 9 3. J. Buccajmo Development of the Leech Nervous System Gun& S.Sten&,William B. Kitan, 37., Stephen A. iiirrence, Kathleen A. French, and DavidA. Wkblat GABA, Receptors Control the Excitability of Neuronal Populations Armin Stelzer Cellular and Molecular Physiology ofAlcohol Actions in the Nervous System Forrest l? W&ht INDEX

The Neurobiology and Genetics of Infantile Autism Linda j! Latspeich and Roland D. Ciaranello Humoral Regulation of Sleep Levente Ka@} Fermc Obd,3r.and James M. Krueger Striatal Dopamine in Reward and Attention: A System for Understanding the Symptomatology of Acute Schizophrenia and Mania Robert Miller Acetylcholine Transport, Storage, and Release Stanley M. Parsons, Chni Prior and Ian G. Marshall Molecular Neurobiology of Dopaminergic Receeptors D a d R. Siblej Frederick3 Monsma}Jr.,and rOng Shm

Volume 34 INDEX

Neurotransmitters as Neurotrophic Factors: A New Set of Functions Joan P Schartz

Volume 36

Heterogeneity and Regulation of Nicotinic AcetylcholineReceptors RonaMj! h k a r and Merouane Benchmy

CA2+,N-Methyl-D-aspartate Receptors, and AIDS-Related Neuronal Injury Stuart A. Lipton


377

Processing of Alzheimer A/3-Amyloid Precursor Protein: Cell Biology, Regulation, and Role in Alzheimer Disease Sam Gandy and Paul Gremgard

Population Activity in the Control of Movement Apostolos I? Georgopoulos

The Role of the Amygdala in Emotional Learning Michael Dauis

Temporal Mechanisms in Perception Ernst Po$pel

Section 111: Functional Segregation and InteMolecular Neurobiology of the GABAA gration in the Brain Receptor Reentry and the Problem of Cortical InteSusan M . J. Dunn, Alan N Bateson, and gration Ian L. Ma& Giulw TTononi The Pharmacology and Function of Central Coherence as an Organizing Principle of GABAB Receptors Cortical Functions David D.Mott and Darrell R Lewis woyskgm

Excitotoxicity and Neurological Disorders: Involvement of Membrane Phospholipids Akhkq A. Farooqui and Uoyd A. Horroch Injury-Related Behavior and Neuronal Plasticity: An Evolutionary Perspective on Sensitization, Hyperalgesia, and Analgesia .G&ar Z Walters INDEX

Section IV: Memory and Models Selection versus Instruction: Use of Computer Models to Compare Brain Theories George N Reeke, 9. Memory and Forgetting: Long-term and Gradual Changes in Memory Storage Lamy R. Squire Implicit Knowledge: New Perspectives on Unconscious Processes Daniel L. Schacter Section V: Psychophysics, Psychoanalysis, and Neuropsychology

Volume 37

Section I: Selectionist Ideas and Neurobiology Selectionist and Instructionalist Ideas in Neuroscience Olaf Sporns Population Thinking and Neuronal Selection: Metaphors or Concepts? Ernst M q r Selection and the Origin of Information Manzed Eigen Section 11: Development and Neuronal Populations Morphoregulatory Molecules and Selectional Dynamics during Development Kathryn L. Crossin Exploration and Selection in the Early Acquisition of Skill Esther Thelen and Daniela Corbetta

Phantom Limbs, Neglect Syndromes, Repressed Memories, and Freudian Psychology R S. Rmnachandran Neural Darwinism and a Conceptual Crisis in Psychoanalysis Arnold H. Model1 A New Version of the Mind Olium Sack INDEX

Volume 38

Regulation of GABAAReceptor Function and Gene Expression in the Central Nervous System A. L e ~ lM0770W i Genetics and the Organization of the Basal Ganglia Robert Hitzmnn, Y i n g Qpn, Stephen X i s , Katherine Dains, and Barbara Hitzmnn

378

CONTENTS O F RECENT VOLUMES

Structure and Pharmacology of Vertebrate GABA, Receptor Subtypes Paul3 Whiting, Ruth M. McKernan, and KeithA. Waword Neurotransmitter Transporters: Molecular Biology, Function, and Regulation Beth Borowsky and Beth j? Ho@n Presynaptic Excitability Myer B. Jackson Monoamine Neurotransmitters in Invertebrates and Vertebrates: An Examination of the Diverse Enzymatic Pathways Utilized to Synthesize and Inactivate Biogenic Amines B. D. Sl&y abd A. RJuorio Neurotransmitter Systems in Schizophrenia Gauin I? Rqwlds Physiology of Bergmann Glial Cells Thomas Miilh and Helmut Kettmmann INDEX

Volume 39

Modulation of Amino Acid-gated Ion Channels by Protein Phosphorylation StephenJ.Moss and Bevor G.Smart

Use Dependent Regulation of GABAA Receptors Eugm M. Barnes, J x SynapticTransmissionand Modulation in the Neostriatum D a d M. Lovinger and Elizabeth 7yh The Cytoskeleton and Neurotransmitter Receptors Valericj? WhatrCy and R. Adron Havk Endogenous Opioid Regulation of Hippocampal Function C h r h Chavkin Molecular Neurobiology of the Cannabinoid Receptor Mary E. Abood and Bilh R. Martin Genetic Models in the Study of Anesthetic Drug Action Victoriaj? Simpson and Thomas E. johnson Neurochemical Bases of Locomotion and Ethanol's Locomotor Stimulant Effects 'limara3 Phillips Ethanol Effects on Ion Channels Fulton Crews, Leslie Morrow, Hugh Cnhell and George Breese INDEX

Neuroprotective Agents and Cerebral Ischaemia

Cerebral Palsy

Gating in Cerebral Networks

The Cerebral Circulation

Agents and Lives

Objects, Agents, and Features

Agents and patients

Oxidizing and reducing agents

Agents and Their Actions

Agents And Adepts

Mobile Agents and Security

Agents And Adepts

Gating in Cerebral Networks

La Onda Cerebral

Handbook on Cerebral Artery Dissection

Cerebral Monitoring in the OR and ICU

Cerebral Monitoring in the OR and ICU

Rapid Detection of Infectious Agents (Infectious Agents and Pathogenesis)

Foreign Agents)

Antifungal Agents

Mobile Agents

Autonomous Agents

Trading agents

Foreign Agents

Antiviral Agents, Vaccines and Immunotherapies

Agents of Light and Darkness

Artificial Intelligence agents and Environments

Agents of Light and Darkness

Agents of Light and Darkness

Agents of Light and Darkness

Agents of Light and Darkness

Neuroprotective Agents and Cerebral Ischaemia

Cerebral Palsy

Gating in Cerebral Networks

The Cerebral Circulation

Agents and Lives

Objects, Agents, and Features

Agents and patients

Oxidizing and reducing agents

Agents and Their Actions

Agents And Adepts

Mobile Agents and Security

Agents And Adepts

Gating in Cerebral Networks

La Onda Cerebral

Handbook on Cerebral Artery Dissection

Cerebral Monitoring in the OR and ICU

Cerebral Monitoring in the OR and ICU

Rapid Detection of Infectious Agents (Infectious Agents and Pathogenesis)

Foreign Agents)

Antifungal Agents

Mobile Agents

Autonomous Agents

Trading agents

Foreign Agents

Antiviral Agents, Vaccines and Immunotherapies

Agents of Light and Darkness

Artificial Intelligence agents and Environments

Agents of Light and Darkness

Agents of Light and Darkness

Agents of Light and Darkness

Agents of Light and Darkness

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