New Animal Models of Human Neurological Diseases

New Animal Models of Human Neurological Diseases

BioValley Monographs Vol. 2

Series Editor

P. Poindron, Strasbourg

Official Publication of the

New Animal Models of Human Neurological Diseases Volume Editors

Philippe Poindron, Strasbourg Pascale Piguet, Basel

28 figures, 5 in color, and 9 tables, 2008

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Singapore · Tokyo · Sydney

Prof. Philippe Poindron

Dr. Pascale Piguet

11, rue Galliéni FR-92100 Boulogne (France)

Pharmazentrum Klingelbergstrasse 50 CH-4056 Basel (Switzerland)

Library of Congress Cataloging-in-Publication Data New animal models of human neurological diseases / volume editors, Philippe Poindron, Pascale Piguet. p. ; cm. - (Biovalley monographs, ISSN 1660–8984 ; v. 2) Includes bibliographical references and index. ISBN 978-3-8055-8405-0 (hard cover : alk. paper) 1. Nervous system-Disease-Animal models. I. Poindron, Phillippe. II. Piguet, Pascale. III. Series. [DNLM: 1. Nervous System Diseases. 2. Disease Models, Animal. WL 140 N943 2008] RC347.N494 2008 616.8-dc22 2007051743

Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and Index Medicus. Disclaimer. The statements, options and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2008 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, Basel ISSN 1660–8984 ISBN 978–3–8055–8405–0

Contents

VII Acknowledgment Poindron, P. (Strasbourg)

1 Theoretical Considerations on Animal Models Poindron, P. (Strasbourg); Callizot, N. (Godmanchester); Piguet, P. (Basel)

11 Mouse Models with Motor Neuron Defects as a Tool for Deciphering Amyotrophic Lateral Sclerosis. Applications, Limits and Future Challenges Echaniz-Laguna, A.; Fricker, B.; Fergani, A.; Loeffler, J.-P.; René, F. (Strasbourg)

39 A Chronic Relapsing Animal Model for Multiple Sclerosis. Myelin Oligodendrocyte Glycoprotein Induced Experimental Autoimmune Encephalomyelitis in Dark Agouti Rats Kinter, J.; Zeis, T.; Schaeren-Wiemers, N. (Basel)

52 Occlusion of the Middle Cerebral Artery in Rats. Refinement of an Animal Model for Human Stroke Cam, E.; Kilic, E. (Zürich); Yulug, B (Bursa); Ritz, M.-F. (Basel)

66 Pyridoxine-Induced Peripheral Neuropathy. Animal Models and Uses Callizot, N. (Godmanchester); Poindron, P. (Strasbourg)

V

81 A Dopamine-Sensitive Inherited Motor Syndrome Linked to the frissonnant Mutation Callizot, N. (Godmanchester); Guenet, J.-L. (Paris); Poindron, P. (Strasbourg)

97 Author Index 98 Subject Index

Contents

VI

Acknowledgment

This second volume of the Biovalley Monographs would never have been published without the generous financial support of Dr. Georg Endress. Dr. Endress is the father of the Biovalley concept. More than 10 years ago, he launched the great adventure which consisted in creating a network of universities, scientists, companies, financial and political institutions to promote the development of biotechnologies in the Upper Rhine Area. As you certainly know, the Biovalley is located in the quadrilateral delimited by Basel, Freiburg im Breisgau, Strasbourg and Mulhouse, and concentrates in a rather narrow space major pharmaceutical firms, prestigious universities and numerous small and middle companies involved in biotechnologies (in the pharmaceutical, environmental as well as in medical or alimentary fields). The idea of Dr. Endress was to give life to a network of cooperating actors decided to share their knowledge and know-how and to create the first international biotechnology cluster in Europe and possibly in the world. Ten years after the launching of this project, Dr. Endress can be proud. The Biovalley is no longer a dream but a living reality. In France, the Région Alsace has got the label of ‘Pôle de Compétitivité à Vocation Internationale’ in ‘Innovations Thérapeutiques’; Basel continues to welcome in its bio-incubators numerous start-ups and spin-offs; Mulhouse has gained an international visibility in ecopharmacology (environmental remediation), fine chemistry and biometry; Freiburg can pride itself on developing innovative tissue engineering techniques or green biotechnologies. Moreover, cooperative scientific programs could be developed between teams from the Louis Pasteur University and Albert Ludwig University, or between Basel, Louis Pasteur and Albert Ludwig Universities.

VII

Regularly, international conferences are held in our golden quadrilateral; the next will take place in Colmar (8th and 9th of November) and will be devoted to ‘The new RNA frontiers’, in the frame of the now classical Colmar Symposia. Certainly we should intensify the trinational cooperation and give up our natural tendency to national chauvinism. But the publication of the Monographs, the papers of which were written by scientists working in the Biovalley, obviously proves that we have progressed toward the settlement of a friendly and efficient cluster. We are indebted for this to Dr. Endress and his somewhat prophetic vision. Thank you again, Dr. Endress. Philippe Poindron, Strasbourg

Acknowledgment

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Poindron P, Piguet P (eds): New Animal Models of Human Neurological Diseases. BioValley Monogr. Basel, Karger, 2008, vol 2, pp 1–10

Theoretical Considerations on Animal Models Philippe Poindrona, Noëlle Callizot b, Pascale Piguet c a Laboratoire de Pathologie des Communications entre Cellules Nerveuses et Musculaires, Faculté de Pharmacie, Université Louis-Pasteur, Strasbourg, France; b Neuropharmacology, Phytopharm plc, Godmanchester, UK; cPharmazentrum, University of Basel, Basel, Switzerland

Abstract Aimed at studying human diseases, animal models emerged in the 1800s and underwent a real boom during the last century. Whether isomorphic or homologous – a categorization based on the extent of similarities between human and animal diseases –, animal models have led to tremendous progress in therapeutics. Following an introduction about history and ethical issues of animal models, this article describes their main features. What are the different kinds of animal models? What characterizes induced (experimental) disease, spontaneous disease, transgenic disease, negative disease and orphan disease models? What are the limits and pitfalls of animal models? What parameters should be taken into consideration for a better predictivity of the models? Various examples taken in the discipline of neuroscience, and even psychiatry, illustrate the difficulties, success and concerns of animal modelling. Copyright © 2008 S. Karger AG, Basel

Animal Models: Definition and Aims

Every researcher acts in a determined framework and according to an experimental scheme supposed to give a satisfying representation of the studied phenomenon. This material is indeed the model. The model has to be as close as possible to the system it represents; it is used under conditions which mimic as best as possible naturally occurring conditions. Importantly, it is not necessary that a model possesses all the properties of a system, since, precisely, modelling aims to create a simplified and useful representation. As mentioned by Segev [1], ‘a model is something simple made by scientists to help them in understanding something complicated. A good model is one that succeeds to reduce

the complexity of the modelled system while preserving its essential features’. Of course, what is most often meant by ‘animal models’ is indeed the modelling of humans. The official definition adopted by the American Research Council Committee on Animal Models for Research and Aging states that ‘in biomedical research, an animal model is a model in which normative biology or behavior can be studied, or in which a spontaneous or induced pathological process can be investigated, and in which the phenomenon in one or more respects resembles the same phenomenon in humans or other species of animal’. Animal models of human diseases offer the advantage – over biochemical (enzymes, receptors) or biological (cell cultures, organites) models – to possess when appropriately chosen a higher degree of isomorphism (see below) with the represented system, namely the human affected by a specific disease. In fact, animals are organisms, just like humans, and a disease concerns an organism in its integrity. If it is a mammal, it is easy to identify, as in the human, homologous subsystems represented by the tissues and organs. When an animal model is validated (even though validation should regularly be reconsidered), it may be used for preclinical studies of new molecules. An example is given by the pmn mouse [see also the article on amyotrophic lateral sclerosis models by Echaniz-Laguna et al., this vol., pp. 11–38] which develops a disease analogous to the Charcot-Marie type 2 disease, namely a rapidly lethal axonopathy [2]. This mouse has been used to study the effects of a new molecule, xaliproden, which delayed significantly the disease progress [3] and is currently assessed for marketing authorization in the USA, Europe as well as Japan for the treatment of sporadic as well as familial amyotrophic lateral sclerosis.

A Few Words of History

Modelling – physiological, pathological or even technological processes – has been a recurrent strategy developed by scientists in order to understand and hopefully planify putative future events. In biology, the use of animal models rose rapidly after 1800 when the scientific thought process recognized that there was little hope of healing human disorders until it was first known how living healthy systems function. The famous physiologist Claude Bernard published in 1865 a book which addressed the physical induction of disease in experimental situations. Other famous advocators of the experimental use of animals, Louis Pasteur in France and Robert Koch in Germany, made their tremendous contribution to the history of medicine. The number of laboratory animal models increased gradually during the last century, to undergo recently a real boom with the emergence of genetic models.

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Animal Models and Ethics: Defining the Necessities

Today, animal models are used virtually in every field of biomedical research: based on ethical matters of testing in humans, this phenomenon, of course, also raises the concern of finding alternatives to the use of animals. Unfortunately, such real substitutes are not yet available to the scientific community in the majority of cases: studies with tissue cultures request a donor organism in order to grow the living cells. Tumorous cell lines play an important role in many studies, but only specific questions can be addressed in a living support endowed with many modified and altered properties. Unforeseen side-effects of inadequately tested new drugs have proven the need for extensive tests before a safe licensing for human applications can be released: thalidomide is a notorious illustration of an insufficient testing in Europe. Studies with inferior organisms such as bacteria provide useful information on cell functioning but cannot of course address the complexity of a network’s activity. Such a network’s functioning can be advantageously studied using computer simulation: nevertheless, a comparison between computer-generated traces and living activity is necessary in order to validate the correctness of the model. How to assess behaviour other than in living animals? Thus, final effective and reliable results request merging data from all the disciplines described above. It would be unfair not to mention that scientists and other people concerned with animal welfare have worked together to include refinement in testing procedures in order to cause less stress to the animals and to reduce the number of animals used in tests, while maintaining reliable data. No doubt that this tendency will develop in parallel with the progress of science, and that animal welfare remains at the centre of every experimenter’s concern.

Identifying Pitfalls

When aimed to understand a biological – normal or not – mechanism, a model is called exploratory. There may also be developed so-called explanatory models: in that case, it is aimed to understand a biological problem, and such models may even be mathematical. Finally, predictive models are used in order to assess the impact of a treatment. The anatomy or morphology of the model structure may be important in all 3 of these model systems. The assumption that Homo sapiens is identical to other animals in his/her bodily functions has however led to a number of errors in the history of medicine. Galen, the Greek physician and philosopher (2nd century AD) studied anatomy almost entirely in apes and pigs, but performed interspecies extrapolation that was not critical enough and resulted in some errors. The prohibition

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by the church of post-mortem dissections of the human body conserved these errors well into the 16th century. Animal models display their limits: a good knowledge of these limits, together with careful choices and extrapolations, is necessary at all steps of a scientific study. The extent of resemblance of the biological structure in the animal with the corresponding structure in humans is called fidelity. Even though a high-fidelity model with close resemblance may seem advantageous, it is important to remember that the discriminating ability may be even more fundamental, e.g. in predictive models. Thus, a model developed to assess the carcinogenicity of a substance should of course be addressed in a species thought to respond in a manner predictive of the human response.

Isomorphism and Homology

Isomorphism An important distinction has led to the classification of animal models into ‘isomorphic’ or ‘homologous’. For an animal model to fulfill the isomorphic criterion, it must show the totality or at least a part of the clinical signs and associated signs (anatomopathological, biochemical, electrophysiological) observed in the humans affected by the disease of relevance. An animal model is considered isomorphic if the animal signs are similar but their cause differs between human and model. An example illustrates how much the use of this criterion is both important and delicate. There is a line of mice presenting a spontaneous mutation for the dystrophin gene. If it was a good model of Duchenne muscular dystrophy (DMD), this line, the mdx mouse [4, 5], should show clinical signs of dystrophy: pseudohypertrophy, motor disorders, myogenic muscular atrophy for example. Moreover, the same anatomopathological and biochemical modifications should be observed in the muscles of this animal as in the ones of children affected by DMD (important fibrosis, fat infiltrations, massive attack of fibres, increase in the serum creatine kinase). This is not the case of the mdx mouse. Indeed, mdx mice present a certain hypertrophy of muscles, but this sign is not accompanied by the fibrosis which is always associated with the degeneration of human muscle [6, 7]. Moreover, the mouse motor disorders are always very inconspicuous, even if they are more pronounced during the weaning period when animals show an increased muscle activity. At this time, a necrosis of muscular fibres may appear, together with an inflammatory disorder and signs of hypercontraction of these fibres. Thus, as for human muscles, the muscles of mdx mice deteriorate when used [8]. But mdx mice escape from the irreversible muscular degeneration which affects human muscles, thanks to a continuous regeneration [9, 10] revealed by the centronucleation

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phenomenon (central position of the nuclei within muscular fibres). In mice aged 3 months and more, 80–90% of muscular fibres display central nuclei indicative of an anterior regeneration. Regeneration is always superior to the muscular degeneration resulting from the absence of expression of dystrophin. Thus, the mdx mouse does not fulfill the criterion of isomorphism whereas it partly fulfills the homology criterion which concerns causes, but also mechanisms (see below). Homology The criterion of homology includes both causes and pathophysiology of the disease. An animal model may be considered homologous if the signs shown by the animal and the cause of the disease are identical to those of humans. A good animal model is the one which develops a disease analogous to the corresponding human disease, for the same reasons and based on the same mechanism. Rabies would be a good example. As exemplified above, the mdx mouse presents a mutation in the dystrophin gene, a mutation which induces in humans DMD or a less severe form, Becker muscular dystrophy; the mdx mouse thus fulfills a part of the homology criterion, and it appears legitimate to attribute to the mutation the disorder the mice develop and which does not affect congenic non-mutated animals. But in the case of this mouse line, the criterion of homology is not strictly fulfilled since apparent causes do not have the same effects at all. In fact, most models are neither homologous nor isomorphic but often termed ‘partial’. Even though these models do not mimic the entire human disease, they may be used to study certain aspects of treatment of the human disease.

Classification of Animal Models

Laboratory animal models can be categorized into 5 groups, namely induced (experimental) disease models, spontaneous disease models, transgenic disease models, negative disease models and orphan disease models. Induced Models Induced models correspond to healthy animals in which the condition under investigation has been experimentally provoked. Cerebral ischaemia induced by a mechanical treatment is an example [see the chapter by Cam et al., this vol., pp. 52–65] of such an experimental disease model, which is indeed the only group that theoretically allows a free choice of species. Care must however be taken in this choice of species, as demonstrated by the lack of effectiveness

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of the chimpanzee models in immunodeficiency syndrome research. Schistosomiasis (mansoni) infection may be studied in experimentally infected mice but not in rats, whose immune system is able to fight the infection effectively [11]. Most induced models are partial or isomorphic because the aetiology of an experimentally induced disease is often different from that of the corresponding disease in humans. The Specific Case of Psychiatric Disorders A very interesting and particular case illustrating the complexity to create appropriate experimental models in the field of neuroscience is the one of psychiatric disorders. Psychiatry has proven to be one of the most difficult disciplines to penetrate for the development of satisfactory in vivo model systems for evaluating novel treatments. During the last century, important advances were made in the study of animal behaviour thanks to the development of ethology (Konrad Lorenz) and comparative psychology (Burrhus Friederich Skinner), which have paved the way to a much better understanding of animal behaviour. If there is no possibility to evaluate depression or schizophrenia in rodents, it is however possible to assess typical behaviours supposed to be characteristic of normal or pathological emotions. In animal investigations of psychiatric disorders, signs suggested to correspond to human symptoms are looked for in animals, and replace the human symptoms (such as anxiety, hallucinations…) which cannot be assessed by definition. In rats, the neonatal ventral hippocampal lesions model is used to simulate some aspects of the symptomatology of schizophrenia: it induces behavioural abnormalities in adulthood [12]. Importantly, varied paradigms exist in schizophrenia (and other psychiatric disorders) research, which give the possibility to compare different protocols. For example, the pharmacological model of phencyclidine injections is known to produce schizophrenia-like symptoms in humans and may as well be applied to animals [13]. Other abnormalities may be looked for, such as deficits in eye tracking or prepulse inhibition, which are thought to mimic some disturbances observed in schizophrenics [12, 14]. Nowadays, about 80 different mutant lines have been reported to display phenotypes interpreted as abnormal ‘depression-related’ or ‘anxiety-related’ behaviour [15]. In some cases, previous knowledge about the role of a gene or gene product was indeed predictive of a given phenotype presenting emotional disturbances. To conclude, experimental models in psychiatry are particularly reminiscent of the overall properties of models in general: in no way may these models pretend to be perfect ones, which would ideally reproduce all aspects of a human disease. However, each model is useful to approach at least one side of the disorder and is characterized by strict limits in its potential use.

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Spontaneous Models Spontaneous animal disease models refer to conditions naturally present in animals and identical to human diseases. Hypertension and type I diabetes are good examples. These models are based on naturally occurring genetic mutants, which exist in high numbers: hundreds of strains of animals with hereditary diseases have been characterized and conserved in specific collections available to researchers. A famous example is Snell’s dwarf mouse which lacks a functional pituitary gland or the pmn and wobbler mouse models which display respectively a defect in tubulin assembly and a degeneration and loss of spinal motor neurons [see the chapter by Echaniz-Laguna et al., this vol., pp. 11–38]. The spontaneous models are often isomorphic, displaying phenotype similarity between the disease in the animal and the corresponding disease in the human, a similarity which often extends to similar reactions to treatment. However, notable exceptions such as the spontaneously mutated mdx mouse (see above) have been described, and such examples should always be kept in mind. Transgenic Models When the object of a study is to define the possible genetic causes of a specific disorder, then comparable genomic segments involved in the aetiology of the disease are a requirement. In transgenic models, the genome of animals has been modified by inserting foreign DNA, replacing or neutralizing (knock-out models) some genes. To be taken into account is however the existence of compensatory mechanisms which may differ between humans and the animal species. Negative Models Negative models are provided by species or strains in which a certain disease does not develop. One famous example investigated in the recent past is the shark, well known for developing cancer with a very low incidence rate. Such a property has led to clinical trials of the anticancer effects of shark cartilage in human patients: although the treatment was ineffective [16], this scientific approach may reveal interesting properties of atypical species. Negative models are mostly related to infectious diseases, which are pathologies often restricted to a limited number of susceptible species: unresponsive species may therefore provide insights into the pathogenic mechanisms and mechanisms of resistance. Orphan Models Orphan model diseases correspond to functional disorders observed in non-human species but not yet described in humans, and which are recognized

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when a similar human disease is later identified. Bovine spongiform encephalopathy is such an example.

Choosing the Appropriate Model

As mentioned by Kinter et al. [this vol., pp. 39–51], ‘…several different models of experimental autoimmune encephalomyelitis were developed, which differ in the immunological reaction, inflammatory processes and the neuropathophysiology in the CNS… Each model shares similarities to multiple sclerosis but also differs in some aspects. Therefore, the proper selection of the most valuable model is essential and strongly depends on the scientific question being addressed’. As mentioned, different types of animal models are endowed with different advantages and drawbacks. Once determined that the use of laboratory animals is necessary, great attention must be paid to the selection of species, breed and strain. Vertebrates, especially mammals, provide essential models for many specific human disease processes. Rats, mice, guinea pigs and hamsters have been favoured because of their small size, ease of handling, short life span and high reproductive rate. Criteria for selection or rejection of particular animal models must take into account varied parameters that might complicate results, such as special physiological features of the animal. Once they are generated, experimental results must be validated with respect to the human. One of the simpler extrapolation processes is illustrated by the determination of safe levels of exposure in people using toxicity data in animals. Of higher complexity is the extrapolation of metabolic patterns and speed: then, body size must be considered. For example, the metabolic rate of small animals is much higher that that of large animals. Given that their organ size expressed as percentage of body weight is similar to humans, this implies an increase in heart frequency in small animals. Thus, scaling is an important factor to consider, and achieving equal concentrations of a substance in the body fluids will not request the same calculation of dosage as achieving a given concentration over a certain time period [17].

The Criterion of Predictivity

Even so, qualitative extrapolation requests many more questions to be answered before relying on comparative data. An important parameter is the criterion of predictivity, which concerns reactivity to the treatments used to fight disease in humans. An animal model is predictive of a human illness if it reacts

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the same way as humans to treatments effective in humans. Thus, L-dihydroxyphenylalanine is the treatment of choice of Parkinson’s disease: an animal model of Parkinson’s disease must improve when treated with this molecule. L-Dihydroxyphenylalanine, in this precise case, is used as a reference compound: every new molecule must be evaluated in comparison with the former. Although the predictive value of animal studies may seem high if they have been conducted thoroughly and have included several animal species, uncritical reliance on the results may be dangerous. Notorious examples remain of drugs developed by large pharmaceutical companies and which produced damage to health in humans. What is noxious or ineffective in non-human species can be innoxious in humans and reciprocally. Thalidomide, which crippled 10,000 children, does not cause birth defects in rats or many other species, but does so in primates. The validity of extrapolation is further complicated by the genetic diversity in humans, which raises the question of possible heterogenous aetiologies of the same disease in humans. A number of different models may be used in complementarity to study a biological phenomenon, and precise questions should be addressed in order to integrate the limits of each model. It is not our aim in this monography to review all animal models of human neurological diseases, but to supply the reader with a few new models, well studied by their authors and susceptible to be used in preclinical research. References 1 2

3

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5 6 7 8 9 10

Segev I: Single neurone models: oversimple, complex and reduced. Trends Neurosci 1992;15: 414–421. Kennel PF, Fonteneau P, Martin E, Schmidt JM, Azzouz M, Borg J, Guenet JL, Schmalbruch H, Warter JM, Poindron P: Electromyographical and motor performance studies in the pmn mouse model of neurodegenerative disease. Neurobiol Dis 1996;3:137–147. Duong F, Fournier J, Keane PE, Guenet JL, Soubrie P, Warter JM, Borg J, Poindron P: The effect of the nonpeptide neurotrophic compound SR 57746A on the progression of the disease state of the pmn mouse. Br J Pharmacol 1998;124:811–817. Heilig R, Lemaire C, Mandel JL, Dandolo L, Amar L, Avner P: Localization of the region homologous to the Duchenne muscular dystrophy locus on the mouse X chromosome. Nature 1987;328: 168–170. Hoffman EP, Brown RH Jr, Kunkel LM: Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987;51:919–928. Hoffman EP, Schwartz L: Dystrophin and disease. Mol Aspects Med 1991;12:175–194. Pastoret C, Sebille A: Further aspects of muscular dystrophy in mdx mice. Neuromuscul Disord 1993;3:471–475. Pastoret C, Sebille A: Fibres of intermediate type 1C and 2C are found continuously in mdx soleus muscle up to 52 weeks. Histochemistry 1993;100:271–276. Pastoret C, Sebille A: Time course study of the isometric contractile properties of mdx mouse striated muscles. J Muscle Res Cell Motil 1993;14:423–431. Anderson JE: Dystrophic changes in mdx muscle regenerating from denervation and devascularization. Muscle Nerve 1991;14:268–279.

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Wang M, Yi XY, Li XP, Zhou DM, Larry M, Zeng XF: Phage displaying peptides mimic schistosoma antigenic epitopes selected by rat natural antibodies and protective immunity induced by their immunization in mice. World J Gastroenterol 2005;11:2960–2966. Le Pen G, Kew J, Alberati D, Borroni E, Heitz MP, Moreau JL: Prepulse inhibition deficits of the startle reflex in neonatal ventral hippocampal-lesioned rats: reversal by glycine and a glycine transporter inhibitor. Biol Psychiatry 2003;54:1162–1170. Le Pen G, Grottick AJ, Higgins GA, Moreau JL: Phencyclidine exacerbates attentional deficits in a neurodevelopmental rat model of schizophrenia. Neuropsychopharmacology 2003;28: 1799–1809. Trillenberg P, Lencer R, Heide W: Eye movements and psychiatric disease. Curr Opin Neurol 2004;17:43–47. Cryan JF, Holmes A: The ascent of mouse: advances in modelling human depression and anxiety. Nat Rev Drug Discov 2005;4:775–790. Loprinzi CL, Levitt R, Barton DL, Sloan JA, Atherton PJ, Smith DJ, Dakhil SR, Moore DF Jr, Krook JE, Rowland KM Jr, Mazurczak MA, Berg AR, Kim GP, North Central Cancer Treatment Group: Evaluation of shark cartilage in patients with advanced cancer: a North Central Cancer Treatment Group trial. Cancer 2005;104:176–182. Calabrese EJ: Principles of Animal Extrapolation. New York, Wiley, 1983.

Prof. Philippe Poindron 11, rue Galliéni FR-92100 Boulogne (France) Tel. ⫹33 684 01 29 51, Fax ⫹33 684 01 29 51, E-Mail [email protected]

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Poindron P, Piguet P (eds): New Animal Models of Human Neurological Diseases. BioValley Monogr. Basel, Karger, 2008, vol 2, pp 11–38

Mouse Models with Motor Neuron Defects as a Tool for Deciphering Amyotrophic Lateral Sclerosis Applications, Limits and Future Challenges Andoni Echaniz-Laguna a, b, Bastien Fricker a, Anissa Fergani a, Jean-Philippe Loeffler a, Frédérique René a a Laboratoire de Signalisations Moléculaires et Neurodégénérescence, INSERM U-692, Faculté de Médecine, Université Louis-Pasteur, et b Département de Neurologie, Hôpital Civil, Strasbourg, France

Abstract Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder of unknown origin affecting primarily motor neurons. The observation that point mutations in the copper/zinc superoxide dismutase (SOD1) gene are present in some patients with autosomal dominant familial ALS (FALS) has led to the generation of transgenic mice expressing various forms of SOD1 mutants. The transgenic mouse models overexpressing high levels of mutant SOD1 develop motor neuron disease, and the toxicity of mutant SOD1 seems to be unrelated to copper-mediated catalysis but rather to the tendency of a fraction of mutant SOD1 proteins to form misfolded protein species and toxic aggregates. Involvement of cytoskeletal components in ALS pathogenesis is supported by several mouse models of motor neuron disease with neurofilament abnormalities and with genetic defects in microtubule-mediated transport. Implication of vascular factors in ALS is supported by mouse models of motor neuron disease with defects in the vascular endothelial growth factor gene. Mutations in the ALS2 gene are responsible for autosomal recessive forms of FALS, and Als2 knockout mice exhibit motor dysfunction and deficits in endosome trafficking. This review focuses on the applications, limits and prospects of mouse models with motor neuron defects as tools for understanding ALS. Copyright © 2008 S. Karger AG, Basel

Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset human disease of unknown origin in which degeneration of the spinal cord and cortical motoneurons leads to paralysis, respiratory depression and death. The causes for most cases of ALS are unknown, and the clinical course is highly variable, suggesting that multiple factors underlie the disease mechanism.

Approximately 10% of ALS cases are familial (FALS). The discovery in 1993 of point mutations in the gene coding for the Cu/Zn superoxide dismutase 1 (SOD1) in some FALS cases directed most ALS research to elucidating the mechanism of SOD1-mediated disease. Surprisingly, unraveling the mechanisms of toxicity of SOD1 mutants has proved to be extremely difficult. Nevertheless, many neuronal death pathways have been revealed through approaches with transgenic mice expressing SOD1 mutants. A key question in understanding ALS is to determine to what extent cytoskeletal abnormalities such as intermediate filament (IF) accumulations, a hallmark of the disease, actively participate in the neurodegenerative mechanism. Transgenic mouse models have been used to clarify the role of IF proteins in motor neuron death with mixed results, but there is growing evidence that genetic defects in components of the microtubule-based transport might be implicated in the degeneration of motor neurons. Recently, mutations in the ALS2 gene encoding a protein with guanine nucleotide exchange factor (GEF) homology domains have been demonstrated in autosomal recessive forms of FALS. This discovery has led to the generation of transgenic Als2 knockout mice which exhibit motor dysfunction and deficits in endosome trafficking. The latter observation has opened new exciting research avenues in the field of motor neuron degeneration. Surprisingly, genetic experimentally generated defects in the vascular endothelial growth factor (Vegf) gene have been shown to provoke a motor neuron disease mimicking ALS in a transgenic mouse model, suggesting that chronic vascular insufficiency and/or lack of VEGF neuroprotection may be involved in the degeneration of motor neurons in ALS. This review focuses on the mouse studies that contributed to the understanding of the pathogenic pathways of motor neuron disease, and on the limitations of these models. The most studied mouse models with motor neuron dysfunction are shown in table 1.

The Genetics of Familial Amyotrophic Lateral Sclerosis

A genetic basis has been determined for some FALS (table 2). Approximately 20% of FALS are caused by dominantly inherited mutations in the SOD1 gene [1]. The pathological and clinical similarities between familial and sporadic disease have triggered hope that the animal models based on mutant SOD1 might provide insight into physiopathological mechanisms common to both ALS and FALS. However, to date, there is no direct evidence validating this assumption. Using gene mapping methods, two research groups have simultaneously identified a new gene linked to a rare, recessively inherited juvenile- or infantile-onset form of ALS that progresses slowly [2, 3]. The gene, localized on chromosome 2, encodes a 184-kDa protein (named ALS2 or alsin)

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Table 1. Mouse models with motor neuron defects Mouse Protein misfolding Mutant SOD1 Synuclein mutant

Intermediate neurofilament abnormalities Human NF-H or NF-L overexpressor Mutant NF-L NF-L knockout Peripherin overexpressor Microtubule abnormalities p50 dynactin subunit (dynamitin) overexpressor KIF5A knockout Dynein mutations pmn mouse Short tau overexpressor Other models VEGF ␦-HRE Als2 knockout wobbler mouse

Neuropathology

References

Loss of spinal motor neurons IF and SOD1 aggregates, perikaryal inclusions and spheroid-like inclusions in motor neurons

14, 16–18, 44, 45 142

Perikaryal accumulation of NF and axonal atrophy Massive degeneration of spinal motor neurons Developmental loss of 20% motor neurons Loss of spinal motor neurons

76, 77

80 66, 72, 73 82

Loss of motor axons

95

NF accumulations Loss of motor neurons Motor neuron degeneration Loss of motor axons

93 96 100, 101 103

Late-onset loss of motor neurons Late-onset degeneration of Purkinje cells Loss of spinal motor neurons

106 115, 116 110

NF-H, NF-L ⫽ Neurofilament, heavy and light subunits; HRE ⫽ hormone response element.

with 3 putative GEF domains. Small GTP-binding proteins of the Ras superfamily act as molecular switches in signal transduction, affecting cytoskeletal dynamics, intracellular trafficking and other important biological processes. GEFs catalyze the dissociation of the tightly bound GDP from the small G protein in response to upstream signals. Although widely expressed, the ALS2 protein is enriched in nervous tissue, where it is peripherally bound to the cytoplasmic face of endosomal membranes, an association that requires the amino-terminal RCC1-like GEF domain [4]. The G protein(s) controlled by ALS2 GEFs are still unknown, although a report has shown that ALS2 can act

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Table 2. Genetics of FALS Disease

Gene

Chromosomal Inheritance Onset location

References

ALS1 ALS2 ALS3 ALS4 ALS5 ALS6 ALS7 ALS8

SOD1 ALS2 (alsin) – SETX (senataxin) – – – VAPB (vesicle-associated membrane protein/synaptobrevin associated membrane protein B) ANG (angiogenin) DCTN1 (dynactin) – – CHMP2B (charged multivesicular body protein 2B)

21q22.1 2q33 18q21 9q34 15q15.1 16q12 20ptel–p13 20q13.33

dominant recessive dominant dominant recessive dominant dominant dominant

adult adolescence adult adolescence adolescence adult adult adult

1 2, 3 143 7 144 145 146 8

14q11.2 2p13 9q21 9p13.2–21.3 3

4 families 1 family dominant dominant 2 patients

adult adult adult adult adult

6 98 147 148 149

ALS ALS-FTD ALS-FTD ALS-FTD ALS-FTD

FTD ⫽ Frontotemporal dementia.

in vitro as an exchange factor for Rab5a, a small G protein implicated in the control of endosomal trafficking [5]. All of the disease-causing mutants are highly unstable, leading to the conclusion that early-onset motor neuron disease is caused by loss of activity of one or more of the GEF domains of this endosomal GEF [4]. More recently, researchers have identified other genes associated with FALS, including senataxin (SETX), vesicle-associated membrane protein/synaptobrevin-associated membrane protein B (VAPB) and angiogenin (ANG) [6–8]. These findings will undoubtedly lead to the discovery of new molecular pathways implicated in motor neuron death. To achieve this goal, transgenic Setx, Vapb and Ang mouse models will almost certainly be generated in the near future. International efforts to find new genes linked to the remainder of FALS cases are under way, and rapid identification of several new genes is anticipated in the near future. Animal Models of Motor Neuron Disease

Many animal models of motor neuron disease have been described, ranging from aluminum motor neuron toxicity in nonhuman primates to spinal

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muscular atrophy in cows and horses [9–11]. Each of these models may provide important information about the motor neuron and may therefore be relevant to ALS. However, given the close resemblance of the mouse models to human disease, this review focuses exclusively on transgenic mouse models of ALS and their general use in the field of ALS research.

Transgenic Mouse Models Expressing Familial-Amyotrophic-LateralSclerosis-Linked SOD1 Mutants

SOD1 Toxicity Is Not Related to Copper-Mediated Catalysis More than a hundred different mutations have been discovered in the SOD1 gene [1, 12]. SOD1 is an abundant and ubiquitously expressed protein. Because of its normal function in catalyzing the conversion of superoxide anions to hydrogen peroxide, it was first thought that the toxicity of different SOD1 mutants could result from a decreased free-radical-scavenging activity. However, different SOD1 mutants showed a remarkable degree of variation with respect to enzymatic activity. Mice expressing mutants SOD1G93A or SOD1G37R developed motor neuron disease despite elevation in SOD1 activity levels [13]. Surprisingly, the SOD1G86R mutant is devoid of enzymatic activity but transgenic mice develop motor neuron disease [14], suggesting that the toxic gain of function is not related to the enzymatic dismutase activity. Moreover, SOD1 knockout mice did not develop motor neuron disease [15]. The conclusion from these combined results was that the mutations in SOD1 generate a gain of new toxic properties. Unlike transgenic mice overexpressing the wild-type SOD1, the mice bearing the SOD1G93A, SOD1G37R, SOD1G85R or SOD1G86R mutants developed a motor neuron disease with many pathological changes reminiscent of human ALS [14, 16–18]. The major histological hallmarks found in the motor unit are presented in figure 1. Both the nature of the toxicity of SOD1 mutants and the reason for the susceptibility of motor neurons are not fully understood. Initially, studies have focused on aberrant copper-mediated catalysis as potential source of toxicity. One hypothesis was that SOD1 mutations enhanced the ability of the enzyme to use hydrogen peroxide as substrate to generate toxic hydroxyl radicals that can damage cellular targets including DNA, protein and lipid membranes [19]. Another hypothesis was that the misfolding of SOD1 induced by mutations would allow the access of abnormal substrates such as peroxynitrite to the catalytic site leading to the nitration of tyrosine residues [20]. However, neither the peroxidase activity nor peroxynitrite hypotheses were supported by transgenic mouse studies except one [21]. The absence of endogenous SOD1 or the addition of wild-type SOD1 did not affect disease progression in mice expressing

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A

B

C c

D d

E e

F

G

H

Fig. 1. Histological hallmarks of ALS in motor units of SOD1G86R mice. Symptomatic G86R mice display characteristic features of motor unit degeneration. In the ventral horn of the lumbar spinal cord (A–E), the number of motor neurons is decreased, and hyperchromatic pycnotic perikarya of motor neurons (A, arrows) corresponding to dying neurons are seen. Motor neurons exhibit a strong p53 immunoreactivity in the nucleus (B, arrows) as well

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mutant SOD1G85R [22]. Moreover, the gene knockout for the copper chaperone for SOD1 that delivers copper to the SOD1 catalytic site had no effect on disease progression in mutant SOD1 transgenic mice [23, 24]. Finally, transgenic mice overexpressing a mutant form of SOD1 lacking 2 of the 4 histidine residues coordinating the binding of the Cu2⫹ at the catalytic site still developed motor neurodegeneration despite a marked reduction in SOD1 activity [25]. Thus, both studies show that SOD1 mutants cause motor neuron disease through the gain of a new function that appears independent of the enzymatic activity involving the copper catalytic site. Toxicity of SOD1 Mutants Is Related to Misfolded Mutant SOD1 Protein Species and Aggregates To date, the prevailing view is that the toxicity of SOD1 mutants is related to the propensity of mutant SOD1 to form noxious misfolded protein species and aggregates [22, 23, 25–27]. However, the toxicity of these protein aggregates is poorly understood. Deleterious effects could result from the cosequestering of essential cellular components and from overwhelming the capacity of the protein folding chaperones and/or of the ubiquitin proteasome pathway to degrade important cellular regulatory factors [28–30]. In cultured neurons, increasing levels of heat shock protein 70 decreased formation of SOD1 mutant aggregates and toxicity [29]. However, increasing heat shock protein 70 levels by 10-fold did not affect disease pathology in mice expressing SOD1 mutants [31]. Analysis of transgenic mice expressing various SOD1 mutants suggests that the motor neuron death pathways are complex and involve multiple cascades of events including oxidative damage, excitotoxicity, alterations in calcium homeostasis, caspase activation, changes in Bcl-2, BclxL/S, Bax and p53 levels, mitochondrial defects and activation of the Fas transduction pathway [32–35]. Studies on mice expressing mutant SOD1G37R also revealed a deregulation of cyclin-dependent kinase (Cdk) 5 and Cdk4 activities in the spinal cord [36–39]. However, recent studies do not support a role for Cdk5 in the pathogenesis caused by SOD1 mutants [40]. As a matter of fact, the knockout of the

as aggregates of ubiquitin (C, asterisks) and SOD1 (D, arrows) in the cell body. These neuronal modifications are correlated with a strong astrogliosis (E) visualized with an antibody directed against glial fibrillary acidic protein. In the sciatic nerve (F), typical wallerian degeneration of myelinated fibers is found (circles) and on the corresponding muscle, end plates (G), visualized with rhodamine-␣-bungarotoxin, are fragmented, attesting to a denervation process. This process is accompanied by muscular atrophy visible in the gastrocnemius muscle as confirmed by the presence of degenerative fibers (arrows) and fibers with reduced diameter (H). A–F Scale bar ⫽ 25 ␮m. G Scale bar ⫽ 5 ␮m. H Scale bar ⫽ 50 ␮m.

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Table 3. SOD1 mRNA and protein levels in mice expressing various mutant SOD1 transgenes Mouse strain

In vivo stable SOD1 mutants G93A D90A G37R line 29 G37R line 42

Human SOD1 mRNA levels relative to mouse SOD1 n-fold

Human spinal Life span References cord SOD1 days protein levels relative to mouse SOD1 n-fold

40 20 – –

17 20 5 12

124 407 365 154

44 44 17 17

0.90 0.45 low

345 250 210 120

18, 44 44 45 14

In vivo unstable SOD1 mutants G85R 17 G127X 25 L126Z high Mice expressing the murine SOD1G86R mutation (humanG85R equivalent)

p35 gene did not affect disease onset and progression in the SOD1G93A mice [40]. Furthermore, the elimination by gene targeting of the neurofilament medium subunit (NF-M) and heavy subunit (NF-H) tail domains containing the Cdk5 phosphorylation sites conferred protection in SOD1G37R mice, rather than deleterious effects anticipated from a loss of phosphorylation sink for Cdk5 activity [41]. One possible explanation for the benefit of removing the neurofilament tail domains may be an enhancement of anterograde transport as defects in axonal transport have been detected at early stages of disease in mice expressing mutant SOD1 [41–43]. Several transgenic mice have been generated in which ALS-linked SOD1 mutants of different biochemical properties were expressed. Based on mRNA expression levels, the rate of synthesis of mutant SOD1 in the widely used mouse strain SOD1G93A with survival of approximately 130 days corresponds to 40 times the normal synthesis rate of mouse SOD1 [44]. For many other transgenic strains (G85R, D90A and G127X) with later-onset disease, the synthesis rates correspond to approximately 20-fold the synthesis rate of endogenous SOD1 (table 3). Thus, very high levels of mutant SOD1 mRNAs seem to be required for the development of ALS-like phenotypes within the short life span

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of mice. Moreover, the life span of the ALS mice is inversely proportional to gene dosage. For instance, in the SOD1G127X strain, the survival times in hemizygous mice was twice as long as in mice homozygous for the transgene [44]. Yet, the steady-state levels of mutant SOD1 proteins in spinal cords may differ widely between transgenic mouse strains. The level of human SOD1 protein in the young G93A strain is 17-fold higher than the normal mouse SOD1 level, whereas the young G85R and G127X mice exhibit levels that are only 90 and 45% of the mouse SOD1 level [44]. Transgenic mice expressing the mutant SOD1L126Z with mRNA levels comparable to other ALS mouse models also exhibited very low levels of mutant SOD1 proteins [45]. Such widely different steady-state protein levels probably reflect different stabilities and degradation of the various human SOD1 mutants. It is of interest that despite low human SOD1 protein levels in the young G85R, G127X and L126Z mice, their life span remains similar to the G37R or G93A mice that express similar human SOD1 mRNA levels but much higher steady-state protein levels at early stages. Nonetheless, the mutant SOD1 mice (G85R, G127X and L126Z) with low steady-state protein levels showed similar amounts of detergent-insoluble aggregates in the spinal cord at the end stage of disease [18, 44, 45]. To summarize, the motor neuron disease may be caused by long-term exposure to noxious misfolded mutant SOD1 species with propensity to aggregate. However, the exact mechanism of toxicity of the misfolded SOD1 species is still elusive. Nonneuronal Cell Types Are Involved in the Pathogenesis of SOD1-Linked FALS Detailed histological analyses of SOD1G93A mouse neuromuscular junctions, ventral root and spinal cord at various ages revealed that motor neuron pathology most probably begins at the distal axon and progresses in a ‘dying back’ pattern [46]. Thus, the SOD1G93A mice show end plate denervation a long time before ventral root axon loss and motor neuron loss. The failure to detect mutant SOD1L126Z proteins in distal nerve fibers of transgenic mice suggests that direct damage to axons by mutant SOD1 is not a requirement for such an axonal dying back mechanism [45]. This observation might be indicative that, at this anatomical level, other cell types, distinct from the motor neuron, initiate the course of the disease. Studies performed on transgenic mouse models have provided evidence that skeletal muscle may also contribute to neurodegeneration in ALS. Indeed, early upregulation of mitochondrial uncoupling protein 3 and overexpression of genes involved in glucide and lipid metabolism have been demonstrated in skeletal muscle in mice expressing SOD1 mutants [47, 48]. Interestingly, an attenuation of muscular hypermetabolism with a highenergy diet extended the life span of ALS mice, suggesting that skeletal muscle may be able to modulate motor neuron degeneration in SOD1-linked ALS [48].

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Besides the involvement of motor neurons, there is now compelling evidence that nonneuronal cells might contribute to the pathogenic process in mice expressing SOD1 mutants. In transgenic mice or rats expressing mutant SOD1, there is a reduction in levels of the astroglial glutamate transporter excitatory amino acid transporter 2 (EAAT2) that may provoke a glutamate-induced excitotoxicity [18, 49]. Glutamate in excess can cause neuronal death via abnormal activation of glutamate receptors, allowing Ca2⫹ entry into the cell and altering cytosolic free Ca2⫹ homeostasis. Moreover, microglial activation may be involved in the neurodegenerative process. Strong nuclear factor ␬B activity and expression of proinflammatory cytokines and chemokines were detected by in situ hybridization in spinal cords of SOD1G37R mice [50]. The chronic induction of innate immunity by intraperitoneal injection of lipopolysaccharides exacerbated disease in SOD1G37R mice, suggesting that inflammation may contribute to neurodegenerative processes [51]. Conversely, an attenuation of neuroinflammation by minocycline or cyclooxygenase 2 inhibitors extended the longevity of ALS mice [52, 53]. To determine which cell types produce the deleterious effects leading to motor neuron death, transgenic mice expressing SOD1 mutants under astrocyte- or neuron-specific gene promoters have been generated (table 4). Expression of the SOD1G86R mutation under the glial fibrillary acidic protein promoter produced astrocytosis but no motor neuron disease [54]. Recently, transgenic mice expressing a SOD1G37R cDNA under the prion gene promoter were reported to develop motor neuron disease [55]. This demonstrates that expression of mutant SOD1 in the neuromuscular unit is sufficient to cause disease, and that expression of mutant SOD1 in microglia is not required to trigger disease. Surprisingly, neuron-specific expression of SOD1 mutants with NF-L or Thy1 gene promoters in mice did not induce motor neuron disease [56, 57]. However, the possibility remains that the level of transgene expression during aging was below the threshold necessary to provoke disease. This concern has subsequently been addressed by the generation of chimeric mice comprised of mixtures of normal and SOD1-mutant-expressing cells [58]. These chimeric mouse studies with SOD1 mutants have demonstrated that neurodegeneration is delayed or eliminated when motor neurons expressing mutant SOD1 are surrounded by healthy wild-type cells. Moreover, these studies show evidence of damage to wild-type motor neurons by surrounding cells expressing mutant SOD1. More recently, transgenic mice expressing a SODG37R mutation under the Islet-1 transcription factor promoter were developed [59]. The study of these animals showed that expression of the mutation within motor neurons was a primary determinant of disease onset and of an early phase of disease progression [59]. Interestingly, the study of recently generated transgenic mice expressing a SODG37R mutation under the CD11b promoter suggests that

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Table 4. Transgenic mice expressing tissue-specific SOD1 mutants Mouse

Tissue-specific promoter regulating mutant SOD1 expression

Mutant-SOD1expressing tissue

Pathological changes

References

SOD1G86R SOD1G37R

GFAP prion

astrocytes neuromuscular

54 55

SOD1G37R SOD1G93A SOD1G37R

NF-L Thy1 Islet-1 transcription factor CD11b

neurons neurons neurons

astrocytosis motor neuron disease unit no motor neuron disease no motor neuron disease earlier onset of motor neuron disease slowing of late motor neuron disease progression delayed neurodegeneration when motor neurons expressing mutant SOD1 are surrounded by healthy wild-type cells

SOD1G37R

Chimeric mice comprised of mixtures of normal and SOD1mutant-expressing cells

microglia

disease progression is linked to the inflammatory response of microglia and mutant toxicity within these cells [59]. Again, such data emphasize the importance of the cellular environment of motor neurons. It is noteworthy that transgenic mice expressing SOD1 mutants under a muscle-specific gene promoter have not been generated until now. Such a model would be of tremendous value to understand the role of skeletal muscle in the pathophysiology of motor neuron disease.

Transgenic Mouse Models with Intermediate Filament Abnormalities

Neurofilament and peripherin proteins are two types of IFs found in the majority of axonal inclusion bodies, called spheroids, in motor neurons of ALS patients [60, 61]. Multiple factors can potentially cause the accumulation of IF proteins including deregulation of IF protein synthesis, proteolysis, defective axonal transport, abnormal phosphorylation and other protein modifications. Evidence for neurofilament involvement in ALS came from the discovery of codon deletions or insertion in the KSP phosphorylation domain of the NF-H

Mouse Models of ALS

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56 57 59 59

58

gene in a small number of sporadic ALS patients and from the report of mutations in the rod domain of the NF-L gene in cases of Charcot-Marie-Tooth disease type 2 [62–64]. More recently, a peripherin frameshift mutation has also been reported in a case of ALS [65]. Mouse Models with Knockout for Intermediate Filament Genes Transgenic mouse models have been used to address the role of neurofilament and peripherin proteins in neuronal function and disease. None of the mice with knockout for any of the neuronal IF proteins (i.e. neurofilament proteins, peripherin or ␣-internexin) develop gross developmental defects or motor neuron disease [66–71]. However, IF deficiencies are not completely harmless. The reduction in caliber of myelinated axons lacking NF-L was accompanied by 50% reduction in conduction velocity, a feature that would be deleterious for larger animal species [72]. The NF-L-null mice exhibited mild sensorimotor dysfunction, but without overt signs of paresis [73]. Moreover, altered cytochrome oxidase activity in numerous hindbrain regions has been detected in the NF-L-null mice [74]. A significant loss of motor axons has also been observed in these mice and in double NF-M/NF-H knockout mice [66, 69, 71]. In peripherin knockout mice, the number and caliber of myelinated motor and sensory axons in the L5 roots remained unchanged but there was a reduction in the number of L5 unmyelinated sensory axons, demonstrating that peripherin is required for the proper development of a subset of sensory neurons [75]. Mouse Models with Neurofilament Overexpression Overexpression in mice of any of the 3 wild-type neurofilament subunits alone can provoke the accumulation of neurofilaments in neuronal cell bodies [76, 77]. For instance, high-level expression of human NF-H proteins causes large perikaryal neurofilament accumulations [78]. The sequestration of neurofilaments in the cell body resulted in atrophy of motor axons and altered axonal conductances but without motor neuron death even in 2-year-old mice [79]. Intriguingly, overexpressing NF-L in NF-H transgenic mice reduced the perikaryal swellings and rescued the motor neuron dysfunction illustrating again the importance of subunit stoichiometry for proper neurofilament assembly and transport [78]. The proof that neurofilament abnormalities can induce neuronal death came from the expression of an assembly-disrupting NF-L transgene having a leucine-to-proline substitution near the end of the conserved rod domain [80]. Mice expressing this NF-L mutant at only 50% of the endogenous NF-L level exhibited within 4 weeks after birth a massive loss of motor neurons. Increasing the levels of bcl-2 did not protect the large motor neurons from the toxicity of mutant NF-L [81]. Here too, the exact mechanism of toxicity of mutant NF-L is still not understood.

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Mouse Models with Peripherin Abnormalities Overexpression of wild-type peripherin in mice caused the selective loss of motor neurons during aging [82, 83]. The onset of neuronal death was precipitated by the absence of NF-L, as revealed by cross-breeding of peripherin transgenic mice with NF-L knockout mice. This context is reminiscent of the findings in ALS in which a reduction of NF-L mRNA levels in affected motor neurons is seen [84]. In addition, it induced formation of perikaryal and axonal IF inclusions resembling spheroids in motor neurons of human ALS. Thus, the toxicity of peripherin overexpression in mice may be related in part to the axonal localization of IF aggregates. This is supported by the rescue of peripherin-mediated disease in mice by the overexpression of NF-H transgene [85]. A possible explanation is that perikaryal sequestration of an excess peripherin may reduce the formation of deleterious axonal IF accumulations. Other mechanisms may also contribute to the toxicity of peripherin overexpression. For example, in vitro studies have shown that dorsal root ganglion neurons from peripherin transgenic embryos die when grown in a proinflammatory CNS culture environment rich in activated microglia, suggesting that peripherin aggregates might predispose neurons to deleterious effects of a proinflammatory environment [86]. To investigate the role of peripherin in disease caused by SOD1 mutations, SOD1G37R mice that lack peripherin or that overexpress peripherin have been generated [87]. The excess or absence of peripherin did not affect the onset and progression of motor neuron disease in mutant SOD1 mice. Thus, peripherin is obviously not a key contributor to motor neuron degeneration associated with toxicity of mutant SOD1. Nevertheless, because mutations in SOD1 are responsible for only a subset of all ALS cases, it remains possible that peripherin might contribute to motor neuron loss in ALS of other etiologies. However, recent support for the peripherin involvement in disease came from the findings of toxic peripherin splice variants and from the discovery of a frameshift mutation in the peripherin gene of a human ALS case [65, 88]. Studies with transgenic mice bearing this peripherin frameshift mutant might spark new insights into the molecular mechanisms of motor neuron disease.

Transgenic Mouse Models with Microtubule-Based Transport Abnormalities

A feature that distinguishes motor neurons from other cells is their extreme asymmetry (axon length up to 1 m) and large volume (up to 5,000 times that of a typical cell). This size is the cause of an enormous metabolic load on the more normally sized cell body that synthesizes the components for this large cell.

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Most proteins are synthesized in cell bodies and transported to nerve terminals through axonal transport. Various molecular motors, which are multisubunit ATPase members of the kinesin family and dynein, move cargos along microtubules in the anterograde and retrograde directions, respectively. Impairment of axonal transport has recently emerged as a common factor in several neurodegenerative disorders. Mutations that disrupt either kinesin or the dynein complex cause impairment of axonal transport, blockade of membranous cargoes and axonal degeneration. In SOD1 mutants, several arguments support the hypothesis of impairment in axonal transport. G37R and G85R mice show an early decline of slow axonal transport [43]. In G93A, Zhang et al. [42] reported a modulation of fast axonal transport, and Warita et al. [89] showed a decrease in kinesin accumulation in the proximal end of ligated sciatic nerve, suggesting an early impairment of fast axonal transport in the anterograde direction. In G86R mice, there is an upregulation of fast axonal transport [90]. All these data suggest an early impairment of axonal transport in ALS mouse models. Mouse Models with Kinesin Defects Mice heterozygous for disruption of the kinesin KIF1B gene have provided the proof that defects in axonal transport can provoke neurodegeneration [91]. As a matter of fact, these mice showed a defect in transporting synaptic vesicle precursors, and they suffer from progressive muscle weakness similar to human neuropathies. This discovery subsequently led to the identification of a loss-offunction mutation in the motor domain of the KIF1B gene in patients with Charcot-Marie-Tooth disease type 2A [91]. Moreover, missense mutations in the KIF5A gene are responsible for a hereditary form of spastic paraplegia, and disruption of the KIF5A gene in mice was reported to cause neurofilament transport impairment [92, 93]. Mouse Models with Dynein Abnormalities Dynein is a molecular motor involved in retrograde axonal transport of organelles along microtubules. Dynein activity requires association with dynactin, a multiprotein complex that activates the motor function of dynein and participates in cargo attachment [94]. The overexpression of the p50 subunit of dynactin, dynamitin, disrupts the dynein/dynactin complex and thereby inhibits motor activity. Transgenic mice overexpressing dynamitin developed a lateonset and progressive motor neuron disease resembling ALS with neurofilamentous swellings in motor axons [95]. Other mouse mutants, called legs at odd angles (Loa) and cramping 1 (Cra1), that arose by mutagenesis with N-ethyl-Nnitrosourea, were found to carry missense mutations in the dynein heavy chain 1 gene [96]. The Loa and Cra1 mice bearing heterozygous dynein mutations develop progressive motor neuron disease due to impairment in retrograde

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transport. The notion that impairment of retrograde axonal transport may play a causative role in pathogenesis is further supported by the discovery of missense mutations in the dynactin/p150glued causing lower motor neuron disease in humans [97, 98]. Surprisingly, recent studies performed on double-transgenic SOD1G93A-Cra1/⫹ mice demonstrated that mutations in dynein led to a decrease in motor neuron death in SOD1G93A mice [99]. Again, these results underline the importance of axonal transport to understand the selective degeneration of motor neurons. A Spontaneous Mouse Model with a Defect in Tubulin Assembly: The pmn Mouse This autosomal recessive motor neuron disease was discovered by spontaneous mutation in mice. Animals homozygous for the pmn mutation develop a progressive caudocranial degeneration of their motor axons from the age of 2 weeks and die 4–6 weeks after birth. Evidence for the importance of the axonal transport machinery in motor neuron disease came from the identification of a gene mutation responsible for the pmn in the mouse. Two groups identified the pmn mutation as a substitution at the last residue of the tubulin-specific chaperone E protein [100, 101], which is essential for proper tubulin assembly and for the maintenance of microtubules in motor axons. Thus, it can be extrapolated that altered function of tubulin cofactors might be implicated in human motor neuron diseases. Mouse Models with Tau Defects Tau is a microtubule-associated protein involved in stabilization of microtubules. There are 6 tau isoforms that are derived from a single gene via alternative splicing of the primary gene transcript. Abnormalities of tau in human disease are known as tauopathies that include Alzheimer’s disease, frontotemporal dementia with parkinsonism linked to chromosome 17, progressive supranuclear palsy and ALS/parkinsonism-dementia complex of Guam [102]. Transgenic mice overexpressing the shortest tau isoform developed axonal degeneration of spinal neurons and motor weakness [103]. These tau transgenic mice are characterized by the presence of filamentous aggregates of hyperphosphorylated tau, not only in cortical and brainstem neurons, but also in spinal neurons [104]. The inclusions contain 10- to 20-nm tau-positive straight filaments. Gliosis has been detected in the spinal cord with degeneration of axons in ventral roots. Neurofilaments are also associated with these aggregates, demonstrating that abnormalities in tau protein can directly affect neurofilaments. Cross-breeding experiments with tau transgenic mice and NF-L knockout mice demonstrated an alleviation of tau-mediated disease by reducing the neurofilament content [105].

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Transgenic Mice with Targeted Disruption of the Hypoxia Response Element in the Vascular Endothelial Growth Factor Gene

Vascular endothelial growth factor (VEGF) is a cytokine essential for angiogenesis. Targeted disruption in mice (Vegf d/d) of the hypoxia response element sequence in the Vegf gene unexpectedly resulted in severe motor deficits at 5–7 months of age with pathological changes resembling ALS [106]. However, the mechanism of disease is still unclear. It has been suggested that chronic vascular insufficiency and possibly a lack of VEGF neuroprotection might result in motor neuron degeneration. The SOD1G93A mice crossed with Vegf d/d mice die earlier because of severer motor neuron degeneration [107]. Furthermore, some studies found an association between some human haplotypes in the VEGF upstream promoter sequence with risk of ALS, and there is evidence for a VEGF deregulation in response to hypoxia in patients with ALS [101–103, 107–109]. Thus, altered responses to hypoxia may be a contributor to motor neuron survival.

A Spontaneous Mouse Model of Motor Neuron Disease: The wobbler Mouse

The wobbler mice originated from a spontaneous mutation that is transmitted by an autosomal recessive gene wr mapping to chromosome 11. This animal is a model of motor neuron disease which has been extensively investigated [110]. However, the exact genetic defect has not yet been identified. The pathological signs of wobbler mice can be recognized early in the postnatal period. The disease is associated with the degeneration and loss of spinal motor neurons. Pathology of cortical motor neurons has also been reported [110]. The wobbler phenotype is characterized by perikaryal vacuolar degeneration and swelling of motor neurons, astrogliosis and microglial activation. There is evidence of dysfunctional mitochondrial respiration in the wobbler with decreased activity of complex IV in a manner similar to what has been reported in the spinal cord of patients with sporadic ALS [111]. Ubiquitin and hyperphosphorylated NF-H immunoreactivities have also been detected in cortical neurons of affected animals [112]. Moreover, increased expression of neurofilament NF-M protein has been observed in affected motor neurons in the wobbler mice [112].

Transgenic Mouse Models with Knockout for Als2

Mutations were observed in coding exons of a new gene mapping to chromosome 2q33, ALS2 coding for alsin, from patients with an autosomal

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recessive form of juvenile ALS, primary lateral sclerosis and infantile-onset ascending hereditary spastic paralysis [2, 3, 113, 114]. The ALS2 gene is ubiquitously expressed. It encodes a protein with GEF homology domains that are known to activate small guanosine triphosphatase (GTPase) belonging to the Ras superfamily. The RCC1-like, DH/PH and VPS9 domains are GEF for small GTPase Ran (Ras-related nuclear), Rho (Ras-homologous member) and Rab5 (Ras-related in brain 5), respectively. Als2 knockout mice have been described by 2 groups [115, 116]. These studies have demonstrated that the absence of Als2 does not produce a severe phenotype in mice. However, the studies by Cai et al. [115] showed that the Als2-null mice develop age-dependent deficits in motor coordination, and primary cultures of motor neurons lacking Als2 were more susceptible to oxidative stress in vitro. Whereas Cai et al. [115] detected no neuropathological changes in their Als2-null mice, Hadano et al. [116] showed that Als2-null mice develop an age-dependent and slow progressive loss of cerebellar Purkinje cells, a reduction in ventral motor axons during aging, astrogliosis and evidence of deficits in endosome trafficking. As a consequence, it is anticipated that the alsin knockout mice might be useful to investigate some aspects of Als2 functions in endosomal trafficking and the mechanisms of long-term degeneration of large neuronal groups.

Transgenic Mouse Models with Motor Neuron Defects as a Tool to Test Therapeutic Strategies for Amyotrophic Lateral Sclerosis

To date, there is no effective pharmacological treatment for ALS. Transgenic animal models that exhibit many of the pathological changes in human ALS provide useful tools for drug testing. Many of the pharmacological approaches tested so far have produced only modest beneficial effects. Vitamin E, gabapentin and salicylate had no effect on survival of SOD1G93A mice [117–119]. Riluzole, a glutamate antagonist and the only drug currently approved for ALS treatment, extended the life span of SOD1G93A mice by 10–15 days without affecting disease onset. More neuroprotection was provided in SOD1G93A mice by the intracerebroventricular administration of zVAD-fmk, a broad caspase inhibitor [120]. Celecoxib treatment significantly delayed the onset of weakness and weight loss and prolonged survival by 25% [53]. Celecoxib is an inhibitor of cyclooxygenase 2, an enzyme that plays a role in inflammatory processes and in the production of prostaglandins that can stimulate the release of glutamate from astrocytes. Minocycline, a second-generation tetracycline with anti-inflammatory properties, has been shown to increase survival in at least 3 independent

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laboratories, using 2 different mutant SOD1 mouse lines, and using various modes of drug delivery [52, 121, 122]. Minocycline may confer neuroprotection through multiple pathways as it can reduce microglial activation, caspase 1, caspase 3, inducible nitric oxide synthase, p38 microtubule-associated protein kinase activation and mitochondrial cytochrome c release. Moreover, the addition of riluzole and nimodipine further enhanced the effect of minocycline on survival [123]. Minocycline is a clinically well-tolerated drug. There is an ongoing trial in humans to test the effectiveness of minocycline in ALS. Another antibiotic, the ␤-lactam antibiotic ceftriaxone, can confer some protection in the mouse model of ALS presumably through elevation of expression of the glutamate transporter EAAT2 [124]. An elevation in EAAT2 would attenuate glutamate neurotoxicity. A clinical trial is also in progress to test the efficacy of ceftriaxone in human ALS. Another compound that can catalytically decompose oxidants as peroxynitrite, the manganese porphyrin AEOL 10150, was found to extend the survival of SOD1G93A mice by approximately 20 days when administered intraperitoneally or subcutaneously at sign onset [125]. Apoptotic pathways might represent targets for disease intervention in ALS. Thus, many neurotrophic factors such as insulin-like growth factor 1, glial cell-line-derived neurotrophic factor, ciliary neurotrophic factor and VEGF might confer protection to motor neurons. The failure of neurotrophic factors in human trials so far may be due in part to the limited delivery of the proteins to the target neurons. Recently, the intracerebroventricular delivery of recombinant VEGF was found to delay disease onset and to extend survival in a SOD1G93A rat model [126]. Another approach for treatment of motor neuron disease that may be considered in the future would involve the delivery of viral vectors to mediate expression of growth factors such glial cell-line-derived neurotrophic factor, insulin-like growth factor 1 and VEGF [127–129]. Another strategy for treatment of familial ALS cases would be to use viral vectors encoding RNA interference molecules to target SOD1 mRNA for degradation [130–132]. Indeed, SOD1 silencing with lentiviruses almost doubled the longevity of mice expressing the SOD1G93A mutant [130].

Limits of Transgenic Mouse Models of Amyotrophic Lateral Sclerosis

Obviously, one should keep in mind that mice are not humans and sometimes therapeutic approaches that confer benefits with mouse models might fail in a human trial. A good example is creatine, a compound believed to improve mitochondrial function. Creatine administered in drinking water was found to extend the longevity of mice expressing mutant SOD1 [133, 134]. However, a

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clinical trial of creatine was found to be ineffective in human ALS [135]. As a consequence, the results in mouse models should be interpreted with caution. Many factors must be considered including pharmacokinetics, as well as routes and timing of drug administration. There is also the question of validity of the mutant SOD1 mice as models of gene defects that account for only 2% of ALS cases. More largely, one should wonder whether results of drug testing in ALS mouse models are predictive of human outcomes. Is the widely used SOD1G93A mouse strain a valid preclinical model for drug testing? The synthesis rate of the human SOD1 mutant in this mouse corresponds to 40 times the synthesis of endogenous mouse SOD1. It has been proposed that extreme levels of mutant SOD1 proteins in such mouse models can produce artifacts such as vacuoles, which may not be relevant to human ALS pathogenesis [44]. In keeping with this concept, it has recently been demonstrated that high human SOD1 mutation expression rates in the mouse can cause artificial loading of mitochondria, suggesting that the vacuolar pathology in transgenic mouse models is probably an artifact caused by the extreme human SOD1 loading of mitochondria [136]. Perhaps, it would be more appropriate to test therapeutic approaches and potential drugs in experimental models with late onset of disease that mimic better the human situation, i.e. with lower gene copy number encoding the mutant SOD1 proteins. For example, overexpression of a human NF-H transgene extended the life span by several months in mice overexpressing SOD1G37R by 5-fold but the same human NF-H transgene had little effect in mice overexpressing SODG37R by 12fold [36]. Another key question is whether mouse models with motor neuron defects truly reflect human ALS. Indeed, human ALS is a disease characterized by a selective motor neuron death not only in spinal cord and brainstem (lower motor neurons, LMN), but also in motor neocortex (upper motor neurons, UMN). Clinical and/or electrophysiological signs suggesting both LMN dysfunction (muscle atrophy and weakness, fasciculations, cramps) and UMN dysfunction (spasticity, brisk tendon reflexes, Babinski and Hoffmann signs) are strictly required to diagnose ALS in man [137]. In contrast, although LMN dysfunction is usually obvious in mouse models with motor neuron defects, UMN dysfunction is difficult, if not impossible, to evaluate ‘clinically’ in such models. Indeed, the rodent corticospinal tract and motor neocortex are anatomically, morphologically and electrophysiologically different from their human counterparts [138, 139]. It seems therefore likely that UMN defects in the mouse and man probably have different functional consequences in both species. However, it has been demonstrated that cerebral neurons of transgenic SOD1 mice display subtle abnormalities that may be a distant reflection of the UMN dysfunction observed in human ALS [140, 141].

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Future Challenges

ALS is a complex disease with probably multiple causes, making the discovery of effective therapies challenging. As a result, a combination of different therapies acting in synergy will probably be needed for effective ALS treatment. In a near future, new strategies might include a search for agents that can prevent the abnormal aggregation of mutant SOD1 or IF proteins. Gene therapy approaches involving the use of recombinant viruses offer a promising strategy for the delivery of genes to enhance motor neuron survival or to silence specific deleterious genes such as mutant SOD1. The next few years should provide some perspective on the potential of neural stem cells to replace or to repair damaged neurons. Eventually, a better understanding of ALS pathogenesis will require the discovery of new genes associated with the disease.

Acknowledgements A.E.L. was supported by grants from the Association pour la Recherche sur la Sclérose Latérale Amyotrophique (France) and from the Académie Nationale de Médecine (France, Bourse Collery). B.F. was supported by an MENRT grant. A.F. was supported by grants from the Association pour la Conservation des Embryons (France) and by the Region Alsace. J.P.L.’s laboratory is supported by the Association Française contre les Myopathies (France) and the Association pour la Recherche sur la Sclérose Latérale Amyotrophique (France), Biovalley and AREMANE.

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137 Brooks BR: El Escorial World Federation of Neurology criteria for the diagnosis of amyotrophic lateral sclerosis. Subcommittee on Motor Neuron Diseases/Amyotrophic Lateral Sclerosis of the World Federation of Neurology Research Group on Neuromuscular Diseases and the El Escorial ‘Clinical limits of amyotrophic lateral sclerosis’ workshop contributors. J Neurol Sci 1994; 124(suppl):96–107. 138 De Felipe J, Alonso-Nanclares L, Arellano JI: Microstructure of the neocortex: comparative aspects. J Neurocytol 2002;31:299–316. 139 Terashima T: Anatomy, development and lesion-induced plasticity of rodent corticospinal tract. Neurosci Res 1995;22:139–161. 140 Tovar YRLB, Tapia R: Cerebral neurons of transgenic ALS mice are vulnerable to glutamate release stimulation but not to increased extracellular glutamate due to transport blockade. Exp Neurol 2006;199:281–290. 141 Vijayvergiya C, Beal MF, Buck J, Manfredi G: Mutant superoxide dismutase 1 forms aggregates in the brain mitochondrial matrix of amyotrophic lateral sclerosis mice. J Neurosci 2005;25: 2463–2470. 142 Giasson BI, Duda JE, Quinn SM, Zhang B, Trojanowski JQ, Lee VM: Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein. Neuron 2002;34:521–533. 143 Hand CK, Khoris J, Salachas F, Gros-Louis F, Lopes AA, Mayeux-Portas V, Brewer CG, Brown RH Jr, Meininger V, Camu W, Rouleau GA: A novel locus for familial amyotrophic lateral sclerosis, on chromosome 18q. Am J Hum Genet 2002;70:251–256. 144 Hentati A, Ouahchi K, Pericak-Vance MA, Nijhawan D, Ahmad A, Yang Y, Rimmler J, Hung W, Schlotter B, Ahmed A, Ben Hamida M, Hentati F, Siddique T: Linkage of a commoner form of recessive amyotrophic lateral sclerosis to chromosome 15q15–q22 markers. Neurogenetics 1998; 2:55–60. 145 Abalkhail H, Mitchell J, Habgood J, Orrell R, de Belleroche J: A new familial amyotrophic lateral sclerosis locus on chromosome 16q12.1–16q12.2. Am J Hum Genet 2003;73:383–389. 146 Sapp PC, Hosler BA, McKenna-Yasek D, Chin W, Gann A, Genise H, Gorenstein J, Huang M, Sailer W, Scheffler M, Valesky M, Haines JL, Pericak-Vance M, Siddique T, Horvitz HR, Brown RH Jr: Identification of two novel loci for dominantly inherited familial amyotrophic lateral sclerosis. Am J Hum Genet 2003;73:397–403. 147 Hosler BA, Siddique T, Sapp PC, Sailor W, Huang MC, Hossain A, Daube JR, Nance M, Fan C, Kaplan J, Hung WY, McKenna-Yasek D, Haines JL, Pericak-Vance MA, Horvitz HR, Brown RH Jr: Linkage of familial amyotrophic lateral sclerosis with frontotemporal dementia to chromosome 9q21–q22. JAMA 2000;284:1664–1669. 148 Vance C, Al-Chalabi A, Ruddy D, Smith BN, Hu X, Sreedharan J, Siddique T, Schelhaas HJ, Kusters B, Troost D, Baas F, de Jong V, Shaw CE: Familial amyotrophic lateral sclerosis with frontotemporal dementia is linked to a locus on chromosome 9p13.2–21.3. Brain 2006;129:868–876. 149 Parkinson N, Ince PG, Smith MO, Highley R, Skibinski G, Andersen PM, Morrison KE, Pall HS, Hardiman O, Collinge J, Shaw PJ, Fisher EM: ALS phenotypes with mutations in CHMP2B (charged multivesicular body protein 2B). Neurology 2006 DOI: 10.1212/01.wnl.0000236199. 1182185.

Jean-Philippe Loeffler Laboratoire de Signalisations Moléculaires et Neurodégénérescence INSERM U-692, Université Louis-Pasteur Faculté de Médecine 11, rue Humann FR–67085 Strasbourg Cedex (France) Tel. ⫹33 3 90 24 30 81, Fax ⫹33 3 90 24 30 65, E-Mail [email protected]

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Poindron P, Piguet P (eds): New Animal Models of Human Neurological Diseases. BioValley Monogr. Basel, Karger, 2008, vol 2, pp 39–51

A Chronic Relapsing Animal Model for Multiple Sclerosis Myelin Oligodendrocyte Glycoprotein Induced Experimental Autoimmune Encephalomyelitis in Dark Agouti Rats Jochen Kinter, Thomas Zeis, Nicole Schaeren-Wiemers Neurobiology, Department of Research, University Hospital Basel, Pharmacenter, Basel, Switzerland

Abstract Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). One of the mostly used animal models for MS is experimental autoimmune encephalomyelitis (EAE). Until now several different EAE models have been developed, differing in the immunological reaction, inflammatory processes and the neuropathophysiology in the CNS. Here, we present a model induced in Dark Agouti rats by immunization with the N-terminal fragment of myelin oligodendrocyte glycoprotein. This specific model shows several similarities to MS, such as a relapsing-remitting disease course, demyelination and axonal degeneration. By immunohistochemical characterization, lesions could be detected mostly in the spinal cord, but also in the optic nerve, brainstem, cerebellum and in different areas of the forebrain. The mimicking of particular features of MS and the occurrence of special disease entities like optic neuritis, Devic’s disease and the acute MS form of Marburg’s type makes this EAE type an excellent model for investigating certain aspects of the pathophysiology seen in MS. Copyright © 2008 S. Karger AG, Basel

Multiple sclerosis (MS) is the most common neurological disorder of young adults in the western countries. The hallmarks of the disease are demyelinating lesions in the central nervous system (CNS). The current prevailing hypothesis is that MS is an autoimmune disorder directed against CNS antigens leading to inflammation and demyelination [for reviews, see 1–4]. The primary cause and the pathogenesis of MS are still unknown. A common animal model used to study possible pathological mechanisms of MS is experimental autoimmune encephalomyelitis (EAE). Since the initial experiments by Rivers et al.

[5], several different models of EAE have been developed, which differ in the immunological reaction, inflammatory processes and the neuropathophysiology in the CNS [for reviews, see 6–8]. Each model shares similarities to MS but also differs in some aspects. Therefore, the proper selection of the most valuable model is essential and strongly depends on the scientific question being addressed. The various models differ in the choice of species, strains, antigens and immunization protocols are used. There exist models for nonhuman primates like marmosets and rhesus monkeys, as well as for rodents like guinea pigs, rats and mice. The latter has become the mostly used animal with the advantage of the availability of genetically modified mice and the good knowledge about the mouse genome. In rats, as well as in other animals, the susceptibility and the type of EAE observed are strongly strain dependent. Furthermore, there exists a large variety of antigens to induce EAE: whole spinal cord preparation, purified myelin proteins, recombinant myelin proteins or synthetic peptides. The known CNS antigens that can induce an autoimmune response and EAE are myelin basic protein, myelin oligodendrocyte glycoprotein (MOG), myelin-associated glycoprotein, proteolipid protein (PLP) and S-100 protein. Generally, there are two different ways to induce EAE: by active immunization with neuroantigens or by passive transfer of neuroantigen-specific T cells, which have been stimulated in vitro with the antigen for 3–4 days. Even the immunization protocols necessary for induction of EAE vary. For example, Dark Agouti (DA) rats develop severe paralytic EAE after immunization with myelin basic protein in incomplete Freund’s adjuvant, which lacks mycobacteria, whereas mice may require multiple injections of antigen as well as additional pertussis toxin injections for EAE induction [9, 10]. As MOG-induced EAE in particular rat strains shares the major features of MS such as a relapsing-remitting disease course and demyelination [11, 12], we describe here a protocol for MOG-induced EAE in DA rats. The inbreeding of these rats was initiated by Odell at the Oak Ridge National Laboratory and completed at the Wistar Institute in 1965. This strain is susceptible to the induction of autoimmune thyroiditis [13] as well as collagen-induced arthritis following immunization with type II collagens. Protocols were established for MOG-induced EAE in DA rats using either recombinant rat [14] or mouse MOG [15] as antigen. In the chronic relapsing EAE model which is presented here, animals are actively immunized with a recombinant N-terminal fragment of mouse MOG, which leads to the formation of inflammatory demyelinating lesions depending both on T cells and anti-MOG antibodies [16]. The pathology in the described MOG-induced EAE model reflects the spectrum seen in MS in many ways. Not only a relapsing-remitting disease course like in classical MS can be observed, but special disease entities like

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optic neuritis, Devic’s disease and the acute MS form of Marburg’s type are also observed in MOG-immunized DA rats [15]. Inflammatory demyelinating lesions and axonal degeneration are both typical characteristics seen in MS, as well as in the described EAE model. For these reasons, the MOG-induced chronic relapsing EAE model in the DA rat represents a suitable model for studying the pathophysiological mechanisms in MS.

Materials and Methods All protocols for animal experimentations must first be reviewed and approved by an Institutional Animal Care and Use Committee or must conform to governmental regulations regarding the care and use of laboratory animals. Purification and Expression of Recombinant MOG Expression of Recombinant Mouse MOG For the expression of recombinant mouse MOG, the bacterial expression vector pRSET A (Invitrogen Corp.) was used containing the amino acids 1–116 of the mature mouse protein fused to several histidine residues (HIS-tag) [17, 18]. An overnight culture was used for inoculation of a large expression culture in SOB (2% Bacto tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM MgSO4, 10 mM MgCl, ampicillin, kanamycin). The OD600 was measured until it reaches 0.5 and expression was induced by the addition of isopropyl-␤-D-thiogalactopyranoside at 1 mM final concentration. After 4 h the bacteria were harvested by centrifugation (15 min, 4,000 g). The pellet was then frozen and stored until purification was performed. Purification of HIS-Tagged MOG For immobilized metal ion affinity chromatography, the Talon purification system (Clontech) was used. The bacterial pellet was resuspended in lysis buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris-HCl, pH 8) and sonicated to disrupt the bacteria. After a further centrifugation (20 min, 10,000 g), the pellet was again resuspended in lysis buffer and the centrifugation step was repeated. Both supernatants were pooled and subjected on the immobilized metal ion affinity chromatography column for purification at room temperature. After loading, the column was washed with 2 vol lysis buffer and 2 vol washing buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris-HCl, pH 6.3). The purified recombinant protein was collected by eluting the column with elution buffer (8 M urea, 100 mM NaH2PO4, 10 mM TrisHCl, pH 4.5). To obtain soluble (not refolded) recombinant MOG, the purified protein was dialysed 4 times (dilution factor 1:200 each) against 20 mM sodium acetate buffer (pH 3.6) at 4⬚C. Finally, the purified and soluble protein was concentrated (Centricon, 10,000 MWCO) until the protein concentration was at least 2 mg/ml. The protein was aliquoted and stored at ⫺80⬚C. Aliquots once thawed were not frozen again. Induction of EAE in DA Rats Animals Female 10- to 12-week-old DA/OlaHsd rats were purchased from Harlan (the Netherlands). The animals were housed in light- and temperature-regulated rooms under

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Table 1. Clinical score Score

Clinical signs

0 1 2 2.5 3 3.5 4 5

no clinical signs tail weakness monoparesis or monoplegia mild paraparesis paraparesis or paraplegia paraplegia with spasticity hemiplegia, quadriparesis quadriplegia, moribund state

specific pathogen-free conditions with free access to water and food. Note that the housing conditions can also influence the clinical course of EAE. For example, particular transgenic mice can develop spontaneous EAE when housed under nonsterile conditions, whereas those maintained in a specific pathogen-free facility did not [19]. Antigen Preparation and Immunization A 1:1 emulsion of 2 mg/ml recombinant MOG solution and incomplete Freund’s adjuvant was prepared. The incomplete Freund’s adjuvant/antigen mixture was drawn up into a glass syringe with an 18-gauge needle. The needle was removed, and a syringe was attached to a double-ended locking hub connector (Luer-Lok, Becton Dickinson) or plastic 3-way stopcock. At the other end, an empty glass syringe was attached, and the mixture was forced back and forth from one syringe to the other repeatedly. When the mixture was homogeneous and white, the connecter was disconnected, a 22-gauge needle was attached, and air bubbles were removed. (When extruding a small drop on the surface of water, a good oil-in-water emulsion should hold together as a droplet and not disperse.) The emulsion was prepared just before the immunization and kept on ice. The DA rats were first anesthetized with isofluorane, then the base of the tail was shaved and disinfected. Immunization was performed with application of 100 ␮l of the emulsion subcutaneously at the base of the tail. The tail of each animal was marked for identification. Rats were weighed daily, and the clinical score was monitored as described below. Monitoring the Clinical Score One week after immunization, the rats were monitored every day for neurological deficits, which start about 14 days after immunization. Each rat was graded daily and assigned a score from 0 to 5 as shown in table 1. The rats were sacrificed after 60 days, and brains, as well as spinal cords, were removed for further analysis. When they had a clinical score of more than 3 for 2 days in a row, they had to be killed for ethical reasons. The clinical stages of the disease are defined as follows: the acute phase is the period of the first clinical signs, in which rats show ascending paralysis following active disease induction. The phase of clinical improvement that follows a clinical episode was described as remission. A remission was defined as a reduction of the clinical score by a minimum of 1

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grade for at least 2 days after the peak of the acute phase or a disease relapse has been reached. A relapse is the phase of increasing neurological deficits seen after remission. This is normally defined as an increase of at least 1 grade in the clinical score maintained for a minimum of 2 days after remission has occurred. The animals were grouped in 4 different categories depending on the disease course obtained by the clinical score recorded during the experiment. The animals without any obvious neurological deficits represented one group. A second group was composed of animals with an acute or progressive disease course showing no remissions and not establishing a stable chronic phase. Animals showing a stable chronic state after an initial acute phase represented a third group, and a fourth group consisted of rats with a relapsing-remitting disease course, which was defined by at least 1 relapse of 1 score for a minimum of 2 days. Immunohistochemistry The animals were anesthetized by inhalation of 2–3 vol% isoflurane (Abbott, Switzerland) and sacrificed by decapitation. The brain and the spinal cord were removed and either fixed for 24 h in 4% paraformaldehyde (in phosphate-buffered saline, pH 7.5), cryoprotected and embedded, or directly embedded in O.C.T. compound (Tissue-Tek, Sakura Finetek, NL) and freshly frozen using dry ice. Cryostat sections (12 ␮m) were either mounted on gelatin-coated slides or processed as free-floating sections for further analysis. Tissue sections were fixed in 4% paraformaldehyde for 30 min or in acetone for 1 min depending on the antibody used. For staining of PLP or neurofilament, the tissue sections were incubated in 70% ethanol overnight at room temperature. After blocking the sections in blocking solution (phosphate-buffered saline, pH 7.5, 2% fish gelatine, 2% normal goat serum, 0.2% Triton X-100) for 1 h, sections were incubated with the appropriate primary antibodies overnight at 4⬚C in blocking solution [20]. When peroxidase was used, endogenous peroxidase was quenched by incubation of slides for 20 min in methanol plus 0.3% H2O2. Incubation of secondary antibodies was performed either with fluorescence-labeled or biotinylated antibodies in blocking solution. When biotinylated secondary antibodies were used, further incubations were performed with premixed avidin and biotinylated peroxidase complex (Vectastain ABC kit; Vector Laboratories) according to the manufacturer’s instructions. The immunohistochemical signal was revealed by a color reaction with 3-amino-9-ethylcarbazole. Counterstaining was either performed with hematoxylin for 1 min followed by rinsing the slide in running tap water, or with 4⬘,6-diamidino-2-phenylindole when using fluorescence-labeled antibodies. Antibodies The following primary antibodies were used: CD68 (ED1; Serotec; 1:500); PLP (MCA839G; Serotec; 1:500); CD11b (MCA275R; Serotec; 1:500); glial fibrillary acidic protein (GFAP, G-A-5; Sigma; 1:2,000). As a secondary antibody, a biotinylated goat antimouse IgG (115–065–166; Jackson Immunoresearch; 1:500) was used.

Results and Discussion

Clinical Course of Disease The first clinical signs, typically the loss of tail tonicity, were observed about 2 weeks after the induction of EAE. In the MOG-induced EAE model in

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Table 2. Spectrum of disease course Disease course

Number

Percent of total

No clinical signs Acute Chronic Relapsing-remitting

18 15 8 14

33 27 15 25

EAE type distribution: clinical scores of the animals were analyzed over time. Animals which had to be sacrificed after less than 7 days after disease onset were counted as acute EAE. An improvement of at least one score point for more than one day was counted as remission. Animals having a clinical course with a steady worsening and no improvement over at least 20 days were counted as chronic-progressive EAE. In total, we analyzed 70 animals from which 15 were control and 55 were EAE animals.

DA rats, the animals show a broad spectrum of different disease courses. By varying the amount or the solubility of the antigen, the spectrum of disease courses can be influenced. Immunization using precipitated recombinant MOG leads to an increase in animals that develop an optic neuritis but fail to develop clinical signs [15]. Increasing the amount of antigen leads to a larger fraction of animals showing a relapsing-remitting disease course [21]. In our study, we used 100 ␮g MOG resulting in 4 groups of animals defined in respect of the clinical disease course. Typical EAE disease courses are shown in table 2 and figure 1. One group of up to 30% was composed of animals showing no obvious neurological deficits (fig. 1a). A second group of rats displayed an acute progressive form without a remission or a stable chronic phase (fig. 1b). These animals have to be sacrificed early after the disease onset because of the lack of disease amelioration or stabilization. After the initial acute phase, a third group of animals stabilize in a chronic phase and show neither any detectable disease progression nor amelioration of the disease (fig. 1c). Although it cannot be excluded that there is still a slow disease progression that is not detectable by the grading system, these rats resemble a group of animals with chronic EAE. The last group is defined by rats developing a relapsing-remitting course (fig. 1d) mimicking the typical disease course seen in MS patients during the early disease phase. Although the classification into 4 groups is a simplification, it demonstrates the spectrum of disease courses seen in MOG-induced EAE in DA rats, which to some extent corresponds to the different disease

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5 No obvious clinical deficits

Clinical score

4

Acute disease course

3 2 1 0 0

10

a

20

30

40

50

60

0

10

b

Days after injection

20

30

40

50

60

Days after injection

5 Chronic disease course

Clinical score

4

Relapsing-remitting disease course

3 2 1 0 0

c

10

20

30

40

Days after injection

50

60

0

d

10

20

30

40

50

Days after injection

Fig. 1. Different disease courses in MOG-induced EAE in DA rats. Four typical disease courses can be observed in an EAE experiment induced by immunization with 100 ␮g recombinant MOG. Whereas some animals do not show any clinical signs (a), others develop an acute disease onset without remission (b). In some cases, animals stabilize after an initial acute phase and show a chronic disease course without further relapses or remissions (c). The fourth group includes animals which have a relapsing-remitting disease course characterized by at least one relapse (d).

courses observed in MS patients. An experiment with a more homogenous disease outcome can be obtained by varying the dose and solubility of the recombinant MOG used for immunization. Lesion Characterization Lesion identification and characterization within the CNS were done by immunohistological analysis with several antibodies to identify inflammation (CD68), demyelination (PLP), astrogliosis (GFAP) or neuronal degeneration (neurofilament). The pathology of MOG-induced EAE was characterized by large inflammatory demyelinating lesions within the CNS. The highest incidence of lesions was found in the spinal cord (fig. 2), optic nerve and tract, brainstem, and cerebellum. In some animals, lesions can also be detected in regions of the forebrain (fig. 3). The anatomical localization of inflammatory

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60

CD68

Control

a

df

wm

d

PLP

GFAP

b

c

e

f

h

i

gm

vf

g EAE

dr

vr

j

k

l

Fig. 2. Examples of inflammatory demyelinating lesions in the spinal cord. Lesion characterization was performed by immunohistological analysis to identify inflammation (CD68; a, d, g, j), demyelination (PLP; b, e, h, k) and astrogliosis (GFAP; c, f, i, l). In contrast to control animals (a–c), inflammatory demyelinating lesions were present in animals developing EAE (d–l). Inflammation in the white matter (d and g, arrows) was leading to focal demyelinating lesions (e and h, arrows) and astrogliosis (f and i). Interestingly, demyelination did not always correlate with infiltration (d and e, arrowheads). Note that astrocytes are already activated in areas where demyelination was not yet evident (f, arrowheads). In some animals, a widespread infiltration as well as an extensive demyelination could be observed (j and k). As a consequence, a strong activation of astrocytes could be detected throughout the whole width of the spinal cord (l). df ⫽ Dorsal funiculus; dr ⫽ dorsal root; gm ⫽ gray matter; vf ⫽ ventral funiculus; vr ⫽ ventral root; wm ⫽ white matter.

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a

b

c

d

e

f

Fig. 3. Demyelinating lesions in the brain. The MOG-induced EAE model in DA rats also induces demyelinating lesions in the brain. Lesions are observed often in the brainstem and the cerebellum, but also in periventricular regions of the forebrain, e.g. the septum (a). The lesions are characterized by a loss of myelin proteins like PLP (b and c) and the presence of reactive astrocytes (d). In the lesion, numerous CD11b-positive cells (f) can be seen, which are composed of activated microglia and macrophages showing also phagocytotic activity identified by the marker CD68 (e).

infiltrates influences the clinical score, which is based mainly on motor deficits caused by spinal cord lesions. Additionally, ataxia can be observed primarily due to lesions in the brainstem or cerebellum. Moreover, the anatomical distribution of lesions within the CNS is influenced by the antigen specificity of T cells [22]. For example, adoptive transfer of myelin-basic-protein-specific T cells in Lewis rats results in widespread inflammation of the spinal cord and only minor involvement of the forebrain, whereas MOG-specific T cells induce lesions also within the forebrain and the optical nerve.

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Cellular Composition of Lesions In most models of EAE, the cellular composition of lesions consists of infiltrating T cells, macrophages and activated microglia. To identify infiltrating phagocytotic cells, like activated macrophages and microglia, an antibody against CD68 was used (fig. 2d, g, j, 3e). A high density of activated macrophages/microglia could be detected in demyelinated areas within the spinal cord (fig. 2d, g, arrows) and the brain (fig. 3e). Even areas adjacent to the demyelinated lesions were often populated by CD68-positive cells, even though they were apparently normal (fig. 2d, arrowheads). These might be signs of ongoing white-matter destruction. Activation of macrophages/microglia could be confirmed by using CD11b as an additional marker (fig. 3f). Strong immunoreactivity of antibodies against GFAP demonstrated pronounced astrogliosis in and around the lesions (fig 2f, i, l, 3d). In actively demyelinating lesions, signs of antibody-mediated myelin destruction were observed, which was evidenced by the presence of complement components and immunoglobulin G [23]. In some cases, widespread demyelination, inflammation and astrogliosis were observed throughout the whole width of the spinal cord (fig. 2j, k, l). Demyelination The extent of demyelination within the spinal cord (fig. 2e, h, k) and brain (fig. 3a–c) was visualized by using an antibody recognizing the major myelin protein PLP. MOG-induced EAE induces not only an encephalitogenic T-cell response, but also an autoantibody response which initiates demyelination and enhances disease severity in animals actively immunized with MOG. Passive transfer of activated MOG-specific T cells only leads to inflammation, and additional administration of antibodies is necessary to obtain demyelination [16]. The strong demyelinating activity seen in animals actively immunized with MOG is unique among the available EAE models. This demyelination is mediated through a combination of complement- and antibody-dependent mechanisms [14, 16]. Besides the strong demyelinating activity, signs of remyelination can also be observed. This is evident after 10 days from onset of disease and characterized by thin myelinated internodes [21]. Axonal Pathology The rediscovery of axonal damage as an important component of MS has led to new insights into the pathology of MS lesions [24, 25]. Axonal injury and loss is thought to be responsible for the progressive exacerbation of the disease, and the need for neuroprotective therapies has become apparent [for a review, see 26]. Experimental studies showed parallel findings in EAE [27, 28]. Furthermore, specific molecular abnormalities such as redistribution of ion channels on chronically demyelinated axons were identified, which may play an

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important role in the axonal pathology of MS [27, 29, 30]. The degree of axonal loss correlates with clinical severity in progressive as well as relapsing-remitting forms of MOG-induced EAE [21]. In contrast, demyelination and inflammation do not show any significant correlation with the clinical severity scores in animals having a relapsing-remitting disease course [21]. Conclusions

There are several EAE protocols available, and the choice of which model should be applied strongly depends on the scientific question to be addressed. Compared to MS, where the primary cause of the disease remains still unknown, the EAE models are autoimmune mediated diseases. The MOGinduced EAE in DA rats closely mimics some of the main clinical features of MS, which makes it an attractive model to study the pathophysiology of the disease. A spectrum of disease courses can be observed in MOG-induced EAE in DA rats ranging from animals showing no clinical sign to severe fatal forms. An advantage is the possibility to influence the disease outcome in favor of a specific disease course, such as relapsing-remitting EAE. The presence of demyelinating lesions and remyelinating activity makes the model a viable tool for investigations of the mechanisms involved in remyelination. In MS patients, axonal loss has been observed which correlates with disease progression. Axonal degeneration and loss are also present in the CNS of DA rats immunized with recombinant MOG, and this axonal pathology in inflammatory demyelinating lesions closely reflects that observed in MS. In summary, the MOG-induced chronic relapsing EAE in DA rats may serve as a good model for investigating certain aspects of the pathobiology seen in MS. Acknowledgments We thank Dr. Danielle Pham-Dinh (INSERM U546, Paris, France) for providing the mouse MOG cDNA construct, Dr. Robert Weissert (Hertie Institute for Clinical Brain Research, Tübingen, Germany; present address: Serono, Geneva, Switzerland) for helpful discussion and Prof. Richard Reynolds (Imperial College, London, UK) for providing recombinant protein and helping to set up the EAE model. This work was supported by the National Societies for Multiple Sclerosis from Switzerland, France, the UK and the USA.

References 1

Wekerle H, Lassmann H: The immunology of inflammatory demyelinating disease; in Compston A, Confavreux C, Lassmann H, McDonald J, Miller D, Noseworthy J, Smith K, Wekerle H (eds):

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12

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15

16

17

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McAlpine’s Multiple Sclerosis. 4th Edition. New York, Churchill Livingstone Elsevier, 2005, pp 491–540. Hemmer B, Archelos JJ, Hartung HP: New concepts in the immunopathogenesis of multiple sclerosis. Nat Rev Neurosci 2002;3:291–301. Noseworthy JH, Lucchinetti C, Rodriguez M, Weinshenker BG: Multiple sclerosis. N Engl J Med 2000;343:938–952. Lassmann H, Bruck W, Lucchinetti C: Heterogeneity of multiple sclerosis pathogenesis: implications for diagnosis and therapy. Trends Mol Med 2001;7:115–121. Rivers TM, Sprunt DH, Berry GP: Observations on attempts to produce acute disseminated encephalomyelitis in monkeys. J Exp Med 1933;58:39–53. Wekerle H: Experimental autoimmune encephalomyelitis as a model of immune-mediated CNS disease. Curr Opin Neurobiol 1993;3:779–784. Steinman L, Zamvil SS: Virtues and pitfalls of EAE for the development of therapies for multiple sclerosis. Trends Immunol 2005;26:565–571. Gold R, Linington C, Lassmann H: Understanding pathogenesis and therapy of multiple sclerosis via animal models: 70 years of merits and culprits in experimental autoimmune encephalomyelitis research. Brain 2006;129:1953–1971. Lenz DC, Wolf NA, Swanborg RH: Strain variation in autoimmunity: attempted tolerization of DA rats results in the induction of experimental autoimmune encephalomyelitis. J Immunol 1999; 163:1763–1768. Martin R, McFarland HF, McFarlin DE: Immunological aspects of demyelinating diseases. Annu Rev Immunol 1992;10:153–187. Adelmann M, Wood J, Benzel I, Fiori P, Lassmann H, Matthieu JM, Gardinier MV, Dornmair K, Linington C: The N-terminal domain of the myelin oligodendrocyte glycoprotein (MOG) induces acute demyelinating experimental autoimmune encephalomyelitis in the Lewis rat. J Neuroimmunol 1995;63:17–27. Johns TG, Kerlero de Rosbo N, Menon KK, Abo S, Gonzales MF, Bernard CC: Myelin oligodendrocyte glycoprotein induces a demyelinating encephalomyelitis resembling multiple sclerosis. J Immunol 1995;154:5536–5541. Rose NR: Differing responses of inbred rat strains in experimental autoimmune thyrioditis. Cell Immunol 1975;18:360–364. Weissert R, Wallstrom E, Storch MK, Stefferl A, Lorentzen J, Lassmann H, Linington C, Olsson T: MHC haplotype-dependent regulation of MOG-induced EAE in rats. J Clin Invest 1998;102: 1265–1273. Storch MK, Stefferl A, Brehm U, Weissert R, Wallstrom E, Kerschensteiner M, Olsson T, Linington C, Lassmann H: Autoimmunity to myelin oligodendrocyte glycoprotein in rats mimics the spectrum of multiple sclerosis pathology. Brain Pathol 1998;8:681–694. Linington C, Bradl M, Lassmann H, Brunner C, Vass K: Augmentation of demyelination in rat acute allergic encephalomyelitis by circulating mouse monoclonal antibodies directed against a myelin/oligodendrocyte glycoprotein. Am J Pathol 1988;130:443–454. Pham-Dinh D, Mattei MG, Nussbaum JL, Roussel G, Pontarotti P, Roeckel N, Mather IH, Artzt K, Lindahl KF, Dautigny A: Myelin/oligodendrocyte glycoprotein is a member of a subset of the immunoglobulin superfamily encoded within the major histocompatibility complex. Proc Natl Acad Sci USA 1993;90:7990–7994. Reynolds R, Dawson M, Papadopoulos D, Polito A, Di Bello IC, Pham-Dinh D, Levine J: The response of NG2-expressing oligodendrocyte progenitors to demyelination in MOG-EAE and MS. J Neurocytol 2002;31:523–536. Goverman J, Woods A, Larson L, Weiner LP, Hood L, Zaller DM: Transgenic mice that express a myelin basic protein-specific T cell receptor develop spontaneous autoimmunity. Cell 1993;72: 551–560. Schaeren-Wiemers N, Bonnet A, Erb M, Erne B, Bartsch U, Kern F, Mantei N, Sherman D, Suter U: The raft-associated protein MAL is required for maintenance of proper axon-glia interactions in the central nervous system. J Cell Biol 2004;166:731–742.

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Papadopoulos D, Pham-Dinh D, Reynolds R: Axon loss is responsible for chronic neurological deficit following inflammatory demyelination in the rat. Exp Neurol 2006;197: 373–385. Berger T, Weerth S, Kojima K, Linington C, Wekerle H, Lassmann H: Experimental autoimmune encephalomyelitis: the antigen specificity of T lymphocytes determines the topography of lesions in the central and peripheral nervous system. Lab Invest 1997;76:355–364. Linington C, Morgan BP, Scolding NJ, Wilkins P, Piddlesden S, Compston DA: The role of complement in the pathogenesis of experimental allergic encephalomyelitis. Brain 1989;112:895–911. Ferguson B, Matyszak MK, Esiri MM, Perry VH: Axonal damage in acute multiple sclerosis lesions. Brain 1997;120:393–399. Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mork S, Bo L: Axonal transection in the lesions of multiple sclerosis. N Engl J Med 1998;338:278–285. Bjartmar C, Trapp BD: Axonal and neuronal degeneration in multiple sclerosis: mechanisms and functional consequences. Curr Opin Neurol 2001;14:271–278. Kornek B, Storch MK, Weissert R, Wallstroem E, Stefferl A, Olsson T, Linington C, Schmidbauer M, Lassmann H: Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions. Am J Pathol 2000; 157:267–276. Wujek JR, Bjartmar C, Richer E, Ransohoff RM, Yu M, Tuohy VK, Trapp BD: Axon loss in the spinal cord determines permanent neurological disability in an animal model of multiple sclerosis. J Neuropathol Exp Neurol 2002;61:23–32. Craner MJ, Hains BC, Lo AC, Black JA, Waxman SG: Co-localization of sodium channel Nav1.6 and the sodium-calcium exchanger at sites of axonal injury in the spinal cord in EAE. Brain 2004;127:294–303. Craner MJ, Newcombe J, Black JA, Hartle C, Cuzner ML, Waxman SG: Molecular changes in neurons in multiple sclerosis: altered axonal expression of Nav1.2 and Nav1.6 sodium channels and Na⫹/Ca2⫹ exchanger. Proc Natl Acad Sci USA 2004;101:8168–8173.

Dr. Nicole Schaeren-Wiemers Neurobiologie, Pharmazentrum 7007 Departement Forschung Klingelbergstrasse 50 CH–4056 Basel (Switzerland) Tel. ⫹41 61 267 15 41, Fax ⫹41 61 267 16 28, E-Mail [email protected]

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Poindron P, Piguet P (eds): New Animal Models of Human Neurological Diseases. BioValley Monogr. Basel, Karger, 2008, vol 2, pp 52–65

Occlusion of the Middle Cerebral Artery in Rats Refinement of an Animal Model for Human Stroke Ertugrul Cama, Ertugrul Kilicb, Burak Yulugc, Marie-Françoise Ritz d a

Department of Anatomy, Institute of Veterinary Science, University of Zürich, and Department of Neurology, University Hospital Zürich, Zürich, Switzerland; c Department of Neurology, University of Uludag, Bursa, Turkey; dNeurosurgery Laboratory, Department of Research, University Hospital Basel, Basel, Switzerland b

Abstract To mimic cerebral ischemia leading to stroke in man, many animal models have been made which occlude brain arteries, transiently or permanently. These models are aimed at studying the morphological changes and the pathophysiology of stroke, and at screening and testing new neuroprotective or thrombolytic drugs. When compared with the model making use of a craniotomy to permanently occlude the cerebral arteries, the model of the middle cerebral artery occlusion using the intraluminal filament in rats considerably decreased the additional surgical trauma due to the experimental procedure. Despite the advantages of this widespread method, a refinement of the model was needed to reduce the mortality rate, ameliorate the reproducibility of the ischemic lesion and to better imitate the pathophysiological situation of stroke in man. We report here the refinement of the permanent cerebral ischemia models in rats leading to an exclusive and selective occlusion of the middle cerebral artery, a full restoration of the blood flow in the ipsilateral common carotid artery and a less severe and invasive surgical intervention. Moreover, some of these improvements may also be used to improve the transient cerebral ischemia model. Copyright © 2008 S. Karger AG, Basel

Stroke is one of the most life-threatening neurological diseases, the third most common cause of death after heart disease and cancer, and the leading cause of disability and hospitalization [1, 2]. Approximately 80–85% of all strokes are caused by ischemia. Cardiogenic emboli lodge in the middle cerebral artery (MCA) or its branches in 80% of cases [3]. If an embolus is large enough to occlude the prox-

imal stem of the MCA (3–4 mm), a major stroke results. Injury from ischemic stroke is the result of a complex serie of cellular metabolic events that occur rapidly after the interruption of nutrient blood flow to a region of the brain. The duration, severity and location of focal cerebral ischemia determine the extent of brain damage and thus the severity of stroke. Approximately 80% of strokes are ischemic [4]. About 50% of strokes are caused by cerebral thrombosis (formation of a blood clot within cerebral arteries damaged by atherosclerosis), which falls into 2 subcategories: large-vessel thrombosis and small-vessel thrombosis. Large-vessel thrombosis (e.g. carotid, MCA or basilar arteries) accounts for approximately 30% of strokes, while approximately 20% involve small, deeply penetrating arteries (e.g. lenticulostriate, basilar penetrating, medullary) that cause a type of thrombotic stroke known as lacunar stroke. Understanding the mechanisms of injury and neuroprotection in these diseases is critical if we are ever to discover new target sites to treat ischemia. There are many animal models available to investigate injury mechanisms and neuroprotective strategies [for a review, see 5]. The major models of stroke can be divided into 3 subgroups: global ischemic, focal ischemic and hemorrhagic. Global models involve blocking (occluding) the major blood vessels that supply the forebrain and result in ischemia over a large proportion of the brain. These models are used much less now – as compared with a few years ago – because they are now generally considered to model the cerebral consequences of cardiac arrest rather than stroke [6]. Focal models occlude a specific vessel, usually the MCA, because the majority of human ischemic strokes results from an occlusion in the region of the MCA. MCA occlusion (MCAO) models are either permanent or transient (removal of the blockade to allow reperfusion). Together, these two MCAO models allow examination of the damage that results from both ischemia and reperfusion. A relatively noninvasive method to produce either permanent or transient MCAO in rodents is the use of an intraluminal filament. This technique has been very popular since its inception in the late 1980s [7, 8] for studying mechanisms of both cellular injury and neuroprotection. Although it was suggested that animal models cannot produce a reliable prediction of clinical efficacy [9], they have continued to play a key role in the evaluation of putative neuroprotectants because they provide information about efficacy in vivo. However, the recent clinical failures have, again, resulted in questioning the validity of animal models. Therefore it has recently been proposed that animal models should be modified to reflect more accurately the complexity of human stroke [10]. The permanent MCAO models in rats reported in this article have been improved for this purpose, and the comparison with the models by Koizumi et al. [7] and Longa et al. [8] is documented here.

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S

T

C

a

b 5mm

Fig. 1. Guide and silicone cap used to occlude the MCA origin. S ⫽ Stopper (silicone drop) at a distance of 20 mm from the cap; T ⫽ abraded tip of the guide; C ⫽ silicone cap (a) that is positioned for usage (b).

Material and Methods Preparation of an Intraluminal Occluder (Silicone Cap) An intraluminal occluder for permanent MCAO was created. For this purpose, a surgical nylon monofilament (Dermalon®; diameter 0.13 mm) was coated at the top with a tiny silicone drop (Xantopren®, Kulzer, Hanau, Germany) forming a cap and hardened by polymerization (Activator NF Optosil®, Kulzer). The silicone cap was then carefully removed from the monofilament and cut to a definite length of 1.5 mm. A polyamide guide (Ethilon®; diameter 0.23 mm) was used and prepared by abrading the tip with sandpaper (No. 1000) to a diameter of approximately 0.13 mm. Thus, the silicone cap could be guided and released in place after pushing it at the origin of the MCA. The length of the guide was 6 cm. The guide, together with the cap, is shown in figure 1. Anesthesia Anesthesia was induced in a box with 4% isoflurane in a gas mixture of 30% oxygen and 70% air for about 10 min. During surgery the anesthetic state was maintained with 2% isoflurane in the same oxygen and air mixture by a nose mask. General Management of the Animals All animal experiments were performed according to the Swiss Federal Animal Protection Act. Thirty-four adult male Wistar rats (285–370 g) were kept under the regulation of the Swiss Federal Animal Protection Ordinance. The animals had free access to standard chow for rats and water both before and after surgical intervention. During surgery, the body temperature was continuously monitored by a rectal probe and maintained at 37.0 ⫾ 0.5⬚C with an electronically regulated heating blanket (Harvard Apparatus, UK). Anesthetized rats were put in dorsal recumbency. The fur around the surgical areas was cut. Additionally the frontal area between ears and eyes was shaved to perform laser Doppler flowmetry (LDF) measurements. Furthermore the inner aspect of the femoral region and finally the ventral area of the neck for the midline incision were shaved. Afterwards the 3 surgical operating areas were cleaned with 70% alcohol and disinfected. The left femoral artery was catheter-

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ized for continuous monitoring of the mean arterial blood pressure, heart rate and to gain blood samples for blood gas analysis (ABL510, Radiometer). At the end of the surgical procedure, the rats were transferred singly to cages. After regaining consciousness completely, the animals were transferred to their home cages, with free access to food and water. Surgical Procedures Laser Doppler Flowmetry For the cerebral blood flow measurement, a frontal midline incision was made beginning between the eyes up to the level of the ears about 3–4 cm long. The skin was retracted with the microdissecting retractors. The underlying tissue was separated, and the cranial skull was visualized. A burr hole was made with a drill at 1 mm posterior and 5 mm lateral to the bregma under the control of an operating microscope. The dura mater was left intact. A polythene tube (inside diameter 1.67 mm and outer diameter 2.47 mm, 800/110/600, Sims Portex Ltd., UK) was placed on the drilled hole and fixed with Histoacryl®. The laser Doppler probe was inserted into this tube on the surface of the dura mater and fixed in order to perform successive measurements before, during and after occlusion. The ‘Refined’ Permanent Middle Cerebral Artery Occlusion Model Using the Silicone Cap Nine male adult Wistar rats with a body weight between 330 and 370 g were used. A sagittal skin incision was made (approximately 2–3 cm long) on the ventral midline of the neck; the glandula mandibularis and muscles (m. sternohyoideus, m. omohyoideus, m. sternothyroideus) were separated carefully. The common carotid artery (CCA) was isolated from the truncus vagosymphaticus, and the carotid bifurcation was exposed. Then the internal carotid artery (ICA) was isolated and carefully separated from the adjacent nervus vagus. The pterygopalatine artery (PA) was separated from the nervus glossopharyngeus and exposed but not intercepted. The CCA was temporarily clamped by using a microvascular clip 5 mm caudal to the carotid bifurcation. Another microvascular clip was placed onto the carotid bifurcation. A 4-0 silk (Silkam®, Braun, Melsungen, Germany) ligature was prepared loosely proximal of the rostral clip. A small incision opened the CCA at a distance of about 5 mm downstream the caudal clip. The silicone cap, which was plugged at the tip of a 3-0 polyamide filament (Ethilon), was introduced into the lumen and gently advanced up to the microvascular clip. A prepared ligature was pulled smoothly around the inserted filament to prevent bleeding out of the vascular incision after removal of the microvascular clip. The silicone occluder was guided to the ICA under control of microscopic visualization to avoid slipping into the external carotid artery (ECA) or PA. The silicone cap was gently advanced from the CCA into the ICA for a length of approximately 18 mm beyond the bifurcation of the CCA. Thus the origin of the MCA could be occluded safely (fig. 1). Mild resistance indicated that the silicone cap was properly lodged in the right coronary artery and so the blood flow to the MCA was blocked. This was controlled by LDF before and after occlusion of the MCA. A 60% decrease in the LDF indicated the typical low cerebral blood flow. After these procedures, the guide was pulled back and removed totally while a microvascular clip was attached. In order to restore the carotid perfusion, the incision on the CCA was closed with simple interrupted sutures using 10-0 nylon (Ethilon). The microvascular clips were then removed to provide physiological blood flow. Finally the skin wound was closed with simple interrupted sutures. Blood samples were taken at 2 time points: 10 min before occlusion and 10 min after occlusion of the MCA. LDF

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measurements were evaluated immediately before and immediately after occlusion and then every 10 min for a period of 30 min. The ‘Standard’ Models for Permanent Middle Cerebral Artery Occlusion Modified Koizumi Method of Cerebral Ischemia The left MCA was occluded in 8 rats (290–325 g) by using a modified method firstly described by Koizumi et al. [7] in 1986. Briefly, the CCA and the carotid bifurcation were exposed through a sagittal midline incision of the neck, and the CCA was carefully separated from surrounding nerves and fascia up to its bifurcation at the base of the skull. The ICA was isolated and carefully separated from the adjacent vagus nerve, and the PA was ligated close to its origin with a 5-0 (Silkam, Braun) suture material. A microvascular clip was placed 5 mm caudally of the carotid bifurcation, and another microvascular clip was placed on the carotid bifurcation. A 4-0 silk suture material (Silkam, Braun) was tied loosely near the rostral clip. Another purse string suture was tightly made by using 10-0 monofilament (Ethilon) near the proximal clip. A small incision was made between the two clips. The filament was introduced through the incision and advanced up to the caudal clip. The loosely tied silk suture was used for sealing the approach to prevent bleeding. After removal of the caudal clip, the filament was advanced slowly and carefully through the ICA up to the rostral cerebral artery. Thus, an introduction into the PA could be avoided. Complete MCAO was controlled by the typical decrease in the LDF values. A prepared purse string suture was tied carefully to prevent hemorrhage. Thus, a permanent occlusion was performed for 24 h with the filament in situ. LDF values and blood samples were taken before and after MCAO. Modified Longa Method Eight male Wistar rats (285–340 g) were used in this control group. The method was described by Longa et al. [8] in 1989. The authors developed the following procedure: the CCA, ECA and ICA were exposed and isolated together with the carotid bifurcation using an operating microscope. The occipital artery, a branch of the ECA, was then isolated and ligated cranially to the origin of the occipital artery. The PA, an extracranial branch of the ICA, was isolated and ligated caudally and near its origin. The ligation was performed using a 5-0 silk suture material. A first microvascular clip was placed on the CCA 5 mm caudally to the carotid bifurcation and a second one on the ICA caudally to the origin of the PA. Another 40 silk suture material was slackly tied on the ECA after the carotid bifurcation. Caudally to this ligature, a small incision was made in the ECA. The filament was introduced at this site and advanced into the ICA near the clip. A slackly tied ligature around the vessel and the filament in it prevented bleeding after removal of the clip. The filament was then advanced 18 mm rostral to the carotid bifurcation and further to occlude the MCA. Afterwards, the ECA was ligated before the clip on the CCA was removed. Finally the skin incision was closed with single interrupted sutures (silk 4-0). LDF values and blood samples were taken before and after MCAO. Histology Twenty-four hours following the surgical intervention of MCAO, the animals were deeply anesthetized with 4% isoflurane in a mixture of oxygen and air. Thereafter, the animals were decapitated and the brains quickly removed. The brains were inspected for appropriate placement of the intraluminal occluder (filament and silicone cap method) and then placed in embedding cassettes (Vasopath, 90200.02, Tespa, Giessen, Germany) and

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immersed/fixed in neutralized formalin (4% v/v) for 5 days at room temperature. After fixation, the silicone caps or the filaments were removed, and the brains were washed under running water for at least 1 h and automatically processed for embedding in paraffin (Histomaster, Haska AG, Berne, Switzerland). Sections of 10 ␮m were cut through the whole brain in steps of 100 ␮m. A mean of 13 sections per brain was obtained for staining. The sections were mounted on slides and dried. The slides were then put in xylol to remove paraffin, rehydrated and stained with toluidine blue for 20 s. After dehydration, the slides were covered automatically with a plastic film. The volume measurements of the ischemic lesions were performed using the C.A.S.T.grid program (Computer-Assisted Stereological Toolbox; Olympus, Denmark). The volumes were determined using consecutive sections. In every section, the ischemic area was delineated. Then a point grid was laid over the image of the tissue, the distances from point to point being 1,500 nm in the x- and y-directions, respectively. The number of points falling into the area was counted in every section. The sum of points obtained in the areas of all inspected sections was used to calculate the volume of the damaged brain tissue according to the following formula: volume (mm3) ⫽ P (total) ⫻ X (mm) ⫻ Y (mm) ⫻ t (mm) where P is the sum of points counted in all sections, X and Y are the distances between points in x- and y-directions (1.5 mm each), t is the distance between the sections (1 mm). This procedure was applied for 3 different parts of the brain (striatum, cortex and total hemisphere). Statistical Analysis All values were given as means ⫾ SEM. Differences between groups were compared by using one-way ANOVA followed by Fisher’s probable least-squares difference test. p ⬍ 0.05 was considered to indicate statistical significance.

Results

Comparison between the Different Methods for Permanent Focal Cerebral Ischemia in the Rat The three different methods are schematically illustrated in figure 2. Whereas in the cap method, a silicone cap occludes exclusively the origin of the MCAO, leaving the blood flow in the ipsilateral CCA intact (fig. 2a), the filaments used to occlude the MCA in the two other methods also block blood flow from the collaterals and the CCA (fig. 2b, c). The localization of the silicone cap in the ICA is shown in figure 3, and the closure of the incision made in the CCA in the silicone cap model is illustrated in figure 4. Laser Doppler Flowmetry LDF measurements, 10 min before and 10 min after MCAO, are presented in figure 5. After filament or silicone cap insertion, the mean local blood flow

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Fig. 2. Diagrams of the different methods of MCAO. C ⫽ The silicone cap; B ⫽ The filament;. 1 ⫽ a. carotis communis (CCA); 2 ⫽ a. carotis externa (ECA); 3 ⫽ a. occipitalis; 4 ⫽ a. carotis interna (ICA); 5 ⫽ a. pterygopalatina; 6 ⫽ a. communicans caudalis; 7 ⫽ a. cerebri media (MCA); 8 ⫽ a. cerebri rostralis; 10 ⫽ a. basilaris; 11 ⫽ a. cerebri caudalis. a The ‘new’ silicone cap method. b The Koizumi (CCA route) method. c The Longa (ECA route) method.

declined to approximately 40% of the preischemic levels in a similar manner in all 3 groups. Physiological Parameters Blood gas values are summarized in table 1, mean arterial blood pressure and heart rate are shown in figure 6 for the 3 permanent cerebral ischemia methods. Blood gas analysis showed no significant difference between the groups. Mean arterial blood pressure remained fairly constant throughout the experiment, and there were no significant differences before and during ischemia in each group. There were also no significant differences in heart rate among the groups. Induction of Ischemia and Related Mortality A total of 25 rats were used to compare the 3 methods tried for MCAO. All rats were decapitated after 24 h of occlusion, and the infarct area was measured. Eight rats were used for the modified Longa model, 8 rats for the modified Koizumi model, 9 rats for the silicone cap method. During surgical procedures,

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----

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--- ICA

2 mm Fig. 3. Ventral aspect of a rat brain showing the silicone cap (black arrow, surrounded by the dashed line) placed in the left ICA, thereby occluding the origin of the MCA. The MCA and the middle cerebral vein (MCV) of the right hemisphere are indicated by dashed lines.

A B CCA

Fig. 4. Closure of the CCA by interrupted sutures (10-0 nylon; indicated by the arrow) before the bifurcation into the ECA (A) and the ICA (B). This suture allows the reestablishment of a normal carotid blood flow after placement of the silicone cap.

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Fig. 5. Variation of the blood flow in the 3 models before and during 20 min of MCAO measured with LDF. Values are means ⫾ SEM. Table 1. Blood gas parameters before and after MCAO in the 3 methods of permanent occlusion Group Koizumi method (n ⫽ 6) Before MCAO After MCAO Longa method (n ⫽ 5) Before MCAO After MCAO Silicone cap method (n ⫽ 9) Before MCAO After MCAO

O2, mm Hg

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pH

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166.54 ⫾ 14.87 163.46 ⫾ 17.59

48.72 ⫾ 3.52 49.02 ⫾ 3.79

7.363 ⫾ 0.022 7.351 ⫾ 0.023

162.40 ⫾ 7.02 158.90 ⫾ 6.46

43.4 ⫾ 2.47 44.1 ⫾ 2.71

7.381 ⫾ 0.015 7.395 ⫾ 0.018

body temperature was normal in all rats (37.0 ⫾ 0.5⬚C). With the modified Longa (ECA route) method performed in 8 rats, successful measurements were achieved in only 5 rats, 3 of them died prematurely (mortality rate: 37.5%). Death was caused by subarachnoid hemorrhage in 2 rats, and 1 developed a cerebral infarct. The modified Koizumi (CCA route) method was also proofed in 8 rats. Successful estimation and calculation of the infarct areas were achieved in only 6. One rat died because of subarachnoid hemorrhage and the other one as the result of cerebral infarction (mortality rate: 25%). The silicone cap method was performed in 9 rats. In this group no death was observed, and all animals showed a selective MCAO (mortality rate: 0%).

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Fig. 6. Comparison of the mean arterial blood pressure (MABP) and heart rate (HR) in the 3 models of MCAO before and during occlusion. Values are means ⫾ SEM.

Size of the Infarcted Areas 24 h after Middle Cerebral Artery Occlusion Independently of the method used, the successfully occluded animals demonstrated an ischemic damage encompassing the entire striatum and cortex. Figure 7 gives an overview of the size of the infarcted area 24 h after permanent MCAO obtained with the 3 methods. In all rats subjected to MCAO, the infarcted region extended to the whole striatum and most of the ipsilateral cortex excluding the cingulate cortex, which is supplied with blood from the rostral cerebral artery. All intraluminal vascular occlusion methods resulted in a significant brain infarction in both cortex and striatum at the ipsilateral side. The infarct volumes were quantified from the histological sections and summarized in figure 8. The infarct areas in the cortex and striatum were significantly smaller with the Longa and the silicone cap methods than with the Koizumi method. However, the total infarct size in the animals treated with the silicone cap was significantly smaller than the total volume of the infarcts obtained with the two filament models (Koizumi method: 261.38 ⫾ 12.42 mm3, Longa method: 191.25 ⫾ 21 mm3, silicone cap method: 128.0 ⫾ 15.3 mm3). These results suggest that, with the

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a

b

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5 mm

Fig. 7. Size of the cerebral infarct induced by 24 h of MCAO in the 3 different models. Sections were stained with toluidine blue. Ischemic infarcts, depicted by the dotted lines, are weakly stained and are covering different areas in the Koizumi (a), Longa (b) or the silicone cap models (c).

Koizumi and Longa methods, the filament may reduce collateral flow in addition to the flow going through the MCA and therefore produce extensive ischemic damage. The silicone cap method may however occlude the MCA more specifically, allowing normal collateral flow, and therefore induce a limited infarction.

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Fig. 8. Volume of the ischemic lesions after 24 h of MCAO with the Koizumi, the Longa and the silicone cap methods in the total ipsilateral hemisphere, the cortex and the striatum. ap ⬍ 0.05 compared with the Koizumi method; bp ⬍ 0.05 compared with the Longa method.

Discussion

Various compounds (e.g. nimodipine and barbiturates) have been shown to be ineffective clinically despite positive experimental data found with several animal methods [11–13]. This raised the question of the value of these animal methods of MCAO. In animal studies, all variables are controlled that might affect the outcome such as temperature, blood pressure, blood gases and pH. The animals used are always young and healthy, whereas human patients may have other existing pathologies such as age, diabetes, hypertension and arrhythmia. These other pathologies may alter the way stroke or global ischemia presents in humans, but we have not studied these additional comorbid pathologies in the animal models. Some animal studies ignored mortality by discarding the animals that died prematurely [14]. In most of the studies reported, the postischemic observation period is too short and usually limited from 6 h to a few days [15], while in clinical studies the outcome at 3 months is most often the primary endpoint [16]. Another potential reason for the lack of translation of therapies to humans after ischemia is that the patient clinical trials may not be designed precisely on the basis of preclinical data. However, most of our current knowledge of brain ischemia is largely based on the data acquired from animal studies [17]: (1) a major part of our understanding of the normal brain energy metabolism, oxygen and substrate utilization or the effects of anesthesia, sleep, brain activity and pathological states (e.g. epilepsy, hypoglycemia, hypoxia and ischemia) has been derived from rodent studies; (2) sophisticated methods for the quantitative mapping of local cerebral blood flow, glucose utilization and protein synthesis were completely developed and largely validated in the rat; (3) the major disease mechanisms

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now regarded as primarily responsible for engendering hypoxic-ischemic brain injury-excitotoxicity, intracellular calcium ion accumulation and free radical generation were all acquired mainly in rodent investigations, or in slices or cell cultures derived from their brains; (4) a full range of promising neuroprotectant strategies has emerged largely from studies in rodents. Despite their limitations, animal experiments are the most reliable method for studying stroke pathophysiology and testing new drugs for efficacy and safety. Regarding animal welfare, it is mandatory to take the 3R (replacement, reduction, refinement) guidelines into consideration. The proposed method should result in a reduction of pain and in the number of the animals used and should be well reproducible. In our case, 2 of the 3 Rs are fulfilled. Additionally refinement should be provided by using advanced surgery techniques in order to imitate the clinical situation of stroke. During the recent decades, a lot of experimental stroke methods have emerged, each of them with unfavorable technical features. The most used stroke method worldwide is the intraluminal filament technique. This method is certainly well reproducible, but it generates as large cerebral infarcts as cerebral hemorrhage, because the coated filament completely obstructs the MCA trunk and the major vessels of the collateral blood flow. Thus, this method induces a high mortality rate that may explain the limits of the long-term observation studies. In the study described here, a new method of reproducible permanent focal cerebral ischemia has been established and was compared with both of the mostly used permanent focal cerebral ischemia methods. The new method of permanent focal cerebral ischemia is standardized by the intraluminal placement of a silicone cap. This silicone is introduced through a small incision into the CCA and advanced 18 mm distal to the carotid bifurcation to occlude the MCA. The most important benefits of this method consist in a selective and exclusive occlusion of the MCA, and in the full restoration of the cerebral blood flow from the ipsilateral CCA. This is achieved by suturing the incision of the CCA with single interrupted sutures (nylon 10-0). The models of transient MCAO using intraluminal filament may also be refined by the restoration of the carotidal perfusion after removing the filament by this technique.

Final Conclusion

In conclusion, the minimally invasive nature of the described method reduces considerably the severity of the surgical intervention. The ischemic part of the brain corresponded very well with the field of the arterial supply of the MCA. The reliability of the method makes it most suitable for the studies in focal cerebral ischemia. Moreover, improvement of the mortality rate shown with this method makes it the method of choice for approximating the pathol-

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ogy and symptoms of human stroke. Regarding the 3R guidelines, it can be stated that the ‘classical’ methods of permanent occlusion should be replaced by this ‘cap method’ in the future.

References 1 2 3 4 5 6 7

8 9 10 11 12 13 14

15

16 17

Bonita R: Epidemiology of stroke. Lancet 1992;339:342–344. Kaste M, Fogelholm R, Rissanen A: Economic burden of stroke and the evaluation of new therapies. Public Health 1998;112:103–112. Kistler JP: The risk of embolic stroke: another piece of the puzzle. N Engl J Med 1994;331: 1517–1519. Mohr JP, Caplan LR, Melski JW, Goldstein RJ, Duncan GW, Kistler JP, Pessin MS, Bleich HL: The Harvard Cooperative Stroke Registry: a prospective registry. Neurology 1978;28:754–762. Traystman RJ: Animal models of focal and global cerebral ischemia. ILAR J 2003;44:85–95. Green AR, Cross AJ: Techniques for examining neuroprotective drugs in vivo. Int Rev Neurobiol 1997;40:47–68. Koizumi J, Yoshida Y, Nakazawa T, Ooneda G: Experimental studies of ischaemic brain edema. I. A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischaemic area. Jpn J Stroke 1986;8:1–8. Longa EZ, Weinstein PR, Carlson S, Cummins R: Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 1989;20:84–91. Ginsberg MD: Adventures in the pathophysiology of brain ischemia: penumbra, gene expression, neuroprotection: the 2002 Thomas Willis Lecture. Stroke 2003;34:214–223. Legos JJ, Tuma RF, Barone FC: Pharmacological interventions for stroke: failures and future. Expert Opin Invest Drugs 2002;11:603–614. Zivin JA, Grotta JC: Animal stroke models: they are relevant to human disease. Stroke 1990;21: 981–983. Wiebers DO, Adams HP Jr, Whisnant JP: Animal models of stroke: are they relevant to human disease? Stroke 1990;21:1–3. Millikan C: Animal stroke models. Stroke 1992;23:795–797. Zhang B, Tanaka J, Yang L, Sakanaka M, Hata R, Maeda N, Mitsuda N: Protective effect of vitamin E against focal brain ischemia and neuronal death through induction of target genes of hypoxia-inducible factor-1. Neuroscience 2004;126:433–440. Hermann DM, Kilic E, Hata R, Hossmann KA, Mies G: Relationship between metabolic dysfunctions, gene responses and delayed cell death after mild focal cerebral ischemia in mice. Neuroscience 2001;104:947–955. Bohannon RW, Lee N: Association of physical functioning with same-hospital readmission after stroke. Am J Phys Med Rehabil 2004;83:434–438. Ginsberg MD: The validity of rodent brain-ischemia models is self-evident. Arch Neurol 1996;53: 1065–1067, discussion 1070.

Dr. Marie-Françoise Ritz University Hospital Basel Department of Research Neurosurgery Laboratory Klingelbergstrasse 50 CH–4056 Basel (Switzerland) Tel. ⫹41 61 267 15 35, Fax ⫹41 61 267 16 28, E-Mail [email protected]

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Poindron P, Piguet P (eds): New Animal Models of Human Neurological Diseases. BioValley Monogr. Basel, Karger, 2008, vol 2, pp 66–80

Pyridoxine-Induced Peripheral Neuropathy Animal Models and Uses Noëlle Callizot b, Philippe Poindron a a

Laboratoire de Pathologie des Communications entre Cellules Nerveuses et Musculaires, Faculté de Pharmacie, Université Louis-Pasteur, Strasbourg, France; b Neuropharmacology, Phytopharm plc, Godmanchester, UK

Abstract The neurotoxicity induced by an excess of vitamin B6 in animals has been known for many years but the first human clinical cases have only recently been reported. All subjects showed paraesthesia and numbness as well as ataxia. The clinical examination showed a large sensory deficit with Achilles’ reflex loss. The electromyographic examination showed a large sensory wave amplitude decrease but no change in the motor conduction. Different rat models of pyridoxine-induced neuropathy exist. Here, we present results with a modified and improved intoxication schedule of an existing rat model. We describe in detail the evolution of the disease and show for the first time that 4-methylcatechol, an inducer of nerve growth factor (NGF) synthesis, improves the clinical status of the intoxicated animals and restores the morphological integrity of the large fibres. We conclude that (a) the pyridoxine-induced sensory neuropathy provides the pharmacologists with a valuable model for studying and evaluating new neurotrophic factors endowed with NGF-like properties and (b) this model can be included in the palette of experimental sensory neuropathies used in preclinical research for evaluating new putative neuroprotective drugs, the mechanisms of action of which are not known. Copyright © 2008 S. Karger AG, Basel

In humans, peripheral neuropathies comprise a heterogeneous group of disorders in terms of aetiology, clinical manifestation and prognosis. The diversity of the clinical symptoms is dependent on the types of peripheral nerve fibres involved in the pathology [1]. Impairment in sensory function may involve mixed modalities, producing multiple symptoms (as in diabetic neuropathy) in which damage may occur to all sensory neuron types. In contrast, a specific large-fibre neuropathy (with severe loss of proprioceptive function) is encountered clinically after vitamin B6 (pyridoxine) intoxication [2–27] or,

more commonly, as a consequence of treatment with the chemotherapeutic drugs – such as taxol or cisplatin – in humans suffering from cancer, and are the most important limitation to the use of these drugs. The neurotoxicity induced by an excess of vitamin B6 in animals has been known for many years but the first human clinical cases were only reported in 1983 [27]. The authors described 7 adults (5 female and 2 male subjects) with ataxia and severe sensory-nervous-system dysfunction after daily high-level pyridoxine consumption (2–6 g/day for 2–40 months, respectively). In 1985, Parry and Bredesen [23] described 16 patients displaying a neuropathy associated with a lower dose of pyridoxine abuse (0.2–5 g/day). All subjects showed paraesthesia and numbness as well as ataxia. The clinical examination showed a large sensory deficit with Achilles’ reflex loss, associated with Romberg’s signs (loss of proprioceptive control in which increased unsteadiness occurs when standing with the eyes closed compared with standing with the eyes open). The electromyographic examination showed a large sensory wave amplitude decrease but no change in the motor conduction. In 1940 the first animal model of pyridoxine-induced neuropathy was described in rats [28]. Since these studies, many others were done in the mouse [29], rat [13, 29, 30], guinea pig [29] and dog [15–19]. A general comment could be extracted from these studies: the sensitivity for pyridoxine changed from one species to the other. The mouse [29] is highly resistant to massive pyridoxine intoxication [1,800 mg/kg/day for 1 week, 1,200 mg/kg/day for 6 weeks (Swiss mice), 3,200 mg/kg/day for 2 days or 2,400 mg/kg/day for 2 weeks (C3H mice)]. Whatever the intoxication protocol (dose as well as time), mice did not show any neuropathological abnormality. The guinea pig [29] develops ataxia, walking deficit and muscular tone deficit after 1,800 mg/kg/day for 5–6 days of pyridoxine. Similar anatomopathology deficits with the rat are observed. The beagle dog shows some axonal lesions of dorsal root ganglion (DRG) neurons [15]. In the rat [15], high doses of pyridoxine (1,200 or 600 mg/kg/day) from 6 to 10 days cause a neuronopathy with necrosis of DRG sensory neurons, associated with centrifugal axonal atrophy and breakdown of peripheral and central sensory axons. Large-diameter neurons with long processes and large cytoplasmic volumes are mainly affected. By contrast, smaller doses (150–300 mg/ kg/day) up to 12 weeks display minor effects on the DRG neuron cell body but produce a neuropathy with axonal atrophy and degeneration [19]. Helgren et al. [13] showed in the rat intoxicated with pyridoxine (400 mg/kg, twice a day), large-calibre argyrophilic axonal profiles in the dorsal columns. According to the authors, these profiles would be ascending collaterals from I␣-afferent fibres degenerated as a consequence of pyridoxine administration. Intoxicated rats with very high doses (1,200 mg/kg/day) show a

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decrease in the muscular tone and hesitating walk. After 5–6 days of intoxication, a severe member ataxia appears and important loss of body weight is observed (between 8 and 35%). Rats intoxicated with low doses of pyridoxine (300 mg/kg/day) do not display any signs or body weight decrease [29]. In preliminary experiments, we have shown that rats intoxicated for 4 days with pyridoxine already displayed a significant deficit in the walking test, which evaluates motor performances depending on the integrity of proprioceptive sensitivity (data not shown). In addition, after 5–7 days of intoxication, animals presented a complete loss of proprioceptive sensitivity, particularly affecting the hindpaws. Electromyographic measurements performed after a 7day intoxication revealed that only sensory, not motor functions were affected. In more than 80% of pyridoxine-treated animals, the H wave disappeared and the remaining animals showed a substantial reduction in H wave amplitude. Finally sensory nerve conduction velocity (SNCV) was lower in pyridoxinetreated animals than in control animals [3]. We present here results showing that modifying the intoxication schedule of the rats improves the results obtained by Helgren et al. [13] by reducing the mortality rate of pyridoxine-treated animals, which in our hands was rather elevated when the protocol of these authors was used. We describe in detail the evolution of the disease and prove that 4-methylcatechol, an inducer of nerve growth factor (NGF) synthesis, improves the clinical status of the intoxicated animals and restores the morphological integrity of the large fibres.

Materials and Methods Animals Female Sprague-Dawley rats (200–250 g; Janvier, Le Genest-Saint-Isle, France) were used throughout this study. They were group based (2 animals per cage) and maintained in a room with controlled temperature (21–22⬚C) and a reverse light–dark cycle (12/12 h) with food and water available ad libitum. All experiments were carried out in accordance with institutional guidelines. Pharmacological Treatment Pyridoxine (Sigma, Saint-Quentin-Fallavier, France) was diluted in a 0.9% (weight/vol) sterile aqueous solution of sodium chloride and administered intraperitoneally twice a day, morning and afternoon, for 7 days. Pyridoxine intoxication was initiated during the second week of the study (day 8). The basal values of parameters the evolution of which should be followed during the disease was determined during the first week of the study, subsequently referred to as week of basal value determination. Pyridoxine solution was prepared immediately before each injection. 4-Methylcatechol (Sigma) was diluted in a 0.9% (weight/vol) sterile aqueous solution of sodium chloride and administered intraperitoneally. 4Methylcatechol solution was prepared every day and administered starting from day 8 of the

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experiment – that is on the same day as the initiation of pyridoxine intoxication – for 3 weeks. Three randomly established groups (n ⫽ 11) [22] of animals were used for the study: (a) animals of the control groups received vehicle (iso-osmotic sterile aqueous solution of sodium chloride); (b) untreated, intoxicated animals were intraperitoneally administered 350 mg/kg body weight pyridoxine, in a volume of 400 ml/100 g body weight, twice a day; (c) treated, intoxicated animals were intraperitoneally administered as above 350 mg/kg body weight pyridoxine and 10 mg/kg 4-methylcatechol once a day (in separate injections performed in the morning, in a volume of 100 ml/100 g body weight). It was shown that, whichever the parameter considered, no difference could be observed between the animals of the three experimental groups during the week of basal value determination. The groups were therefore considered as homogeneous. Motor Coordination Measurements Sensitivity Test: Hot Plate Test The animals were placed in a glass cylinder of 17 cm height and 21 cm diameter on a hot plate maintained at 52⬚C. The animal behaviour was observed (licking of one foot or jump to escape the heat). The latency before the first reaction – whichever it was – was recorded. Electrophysiological Recordings All recordings were made with a standard electromyographic apparatus (Neuromatic 2000M, Dantec, Les Ulis, France) in accordance with the guidelines of the American Association of Electrodiagnostic Medicine [1]. Rats were anaesthetized by intraperitoneal injection of 60 mg/g body weight of ketamine hydrochloride (Imalgene, Merial, Lyon, France). A monopolar needle electrode (Dantec, code 13L70; diameter, 0.3 mm) was inserted into the back of the animal to ground the system. Throughout the procedure, the animals were kept under a heating lamp to maintain the muscles at a physiological temperature. The temperature was verified on the surface of the tail with a contact thermometer and was maintained at 31⬚C. Amplitude and distal latency of M and H waves were recorded in the plantar hindpaw muscle after stimulation of the sciatic nerve. A reference electrode and an active needle electrode were placed in the right hindpaw [18]. The sciatic nerve was stimulated with a single 0.2-ms square pulse, at a suboptimal intensity of 12.8 mA. The amplitude and the latency of H and M waves were expressed in millivolts and milliseconds, respectively. The SNCV of the caudal nerve was also recorded. The tail skin electrodes were placed as it follows: a reference needle electrode was inserted at the base of the tail and an electrode needle placed 30 mm away from the reference needle toward the extremity of the tail. A ground needle electrode was inserted between the anode and reference needles. The caudal nerve was stimulated with a series of twenty 0.2-ms square pulses at an intensity of 12.8 mA. The mean of the amplitude of the evoked responses was calculated and retained as the characteristic value. Morphological Analyses Morphological analyses were performed on day 28, which is 2 weeks after the end of the intoxication period. As already mentioned, 3 animals per group were used for these analyses. They were anaesthetized by intraperitoneal injection of 60 mg/kg body weight Imalgene 500. The L4 and L5 DRG from a given animal were fixed overnight with a 4% solution of glutaraldehyde (Sigma) in iso-osmotic phosphate buffer (pH 7.4). DRG were then treated for 2 h in a 2% solution of osmium tetroxide (Sigma) in phosphate buffer; they were dehydrated in

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Intoxication 4-Methylcatechol treatment

Fig. 1. Effect of 4-methylcatechol on the body weight of the pyridoxine-intoxicated versus control animals. The values represent the mean ⫾ SEM.

serial alcohol solutions and embedded in Epon (Epikon 812, Carl Roth KG, Karlsruhe, Germany). Embedded tissues were then placed at 170⬚C for 3 days to promote polymerization of Epon. Transverse sections of 6 mm were made with a microtome, stained with a 1% solution of toluidine blue (Sigma) in phosphate buffer, for 2 min, and dehydrated and mounted in Eukitt (O. Kindler GmbH & Co, Freiburg im Breisgau, Germany). Sections were observed using an optical microscope, and morphometric analyses were performed with the aid of the Visiolab 2000 software (Biocom, Paris, France). Five sections per animal, two fields per section, were analysed. Statistical Analyses A global analysis of the data was performed using one-factor or repeated-measure analysis of variance (ANOVA); one- and two-way ANOVA tests were used. Dunnett’s test was used whenever the results of the ANOVA test were significant. Moreover, a non-parametric test (Kruskal-Wallis test) was also used to analyse some data. The level of significance was set at p ⬍ 0.05.

Results

Effect of Pyridoxine Intoxication on the Growth of Animals As shown in figure 1, pyridoxine intoxication significantly affected the growth of the animals – whether they were treated or not with 4-methylcatechol – the body weight of which decreased by 15% during the first 2 days of pyridoxine administration. Beyond this period, the weight of intoxicated animals

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25 Control/vehicle Pyridoxine/vehicle Pyridoxine/4-methylcatechol

Time (s)

20

15

** *

10

5

0

4

11

15

18

22

Days

Intoxication 4-Methylcatechol treatment

Fig. 2. Effect of 4-methylcatechol on the precision walking test of the pyridoxineintoxicated versus control animals. The values represent the mean ⫾ SEM. *p ⬍ 0.05, **p ⬍ 0.01 (Dunnett’s test).

increased again, in parallel to that of control animals. The mean weight of 4methylcatechol-treated, intoxicated animals remained consistently greater than that of untreated, intoxicated animals, but the difference was not significant (p ⫽ 0.31, repeated-measure ANOVA). Hot Plate Test Figure 2 shows that, in the hot plate test, on day 11, intoxicated animals, whether they were treated or not, reacted later to heat than the control animals, as shown by the delay in appearance of the first licking or jump, but the differences between the three groups were not significant. On day 15, the first reaction time of the treated, intoxicated animals returned to normal values, whereas that of the untreated, intoxicated animals significantly (p ⬍ 0.05, Dunnett’s test) differed from that of the control animals. On day 18, this difference was no longer detectable. Electromyographic Recordings Amplitude and Latency of Motor M Wave As shown in figure 3a and b, intoxication, combined or not with 4methylcatechol treatment, did not modify either amplitude or latency of the

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15

5

Control/vehicle Pyridoxine/vehicle Pyridoxine/4-methylcatechol

4 Latency (ms)

Amplitude (mV)

20

10 5 0

2

16 Intoxication

23

30

0

37 Days

b

4-Methylcatechol treatment

***

3

2

16 Intoxication

23

30

37 Days

4-Methylcatechol treatment 12.5

** ***

10.0 Latency (ms)

Amplitude (mV)

4

c

2 1

a

* 2 1 0

3

7.5 5.0 2.5 0

2

16 Intoxication

23

30

4-Methylcatechol treatment

37 Days

d

2

16 Intoxication

23

30

37 Days

4-Methylcatechol treatment

Fig. 3. Effect of 4-methylcatechol on the M wave amplitude (a) and latency (b), and on the H wave amplitude (c) and latency (d) of the pyridoxine-intoxicated versus control animals, in comparison with control animals. The values represent the mean ⫾ SEM. *p ⬍ 0.05, **p ⬍ 0.005, ***p ⬍ 0.001.

motor M wave, as compared with control animals, whichever the time of measurement. Amplitude and Latency of H Wave Figure 3c and d shows that pyridoxine intoxication induced on day 16 a very significant (p ⬍ 0.001, ANOVA test) decrease in amplitude of the H wave as compared with that of control animals, whether the animals were treated or not with 4-methylcatechol (fig. 3c). On day 23, there always existed a very significant difference in H wave amplitude, between untreated, intoxicated and control animals (p ⬍ 0.001, Dunnett’s test), and between treated, intoxicated and control animals (p ⬍ 0.005, Dunnett’s test). On day 30, only the untreated, intoxicated animals displayed significant differences (p ⬍ 0.005, Dunnett’s test) with control animals. On day 37, untreated, intoxicated animals had recovered a normal H wave amplitude. It is worth noting that the

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Table 1. Percentage of animals displaying an H wave before and after pyridoxine intoxication Experimental group

Day 2

Day 16

Day 23

Day 30

Day 37

Control Pyridoxine-intoxicated 4-Methylcatechol, pyridoxine-intoxicated

100 100 100

100 12.5 87.5

100 25 87.5

100 75 100

100 100 100

Day 16 is the first day after the end of pyridoxine intoxication.

distal latency of the H wave was never affected by intoxication, whichever the time of the measurement. Analysing in more detail the results of these experiments revealed (table 1) that the number of animals developing an H wave was significantly (p ⬍ 0.001, Kruskall-Wallis test) higher in the treated, intoxicated than in the untreated, intoxicated group. In fact, on day 16, only 12.5% of the untreated, intoxicated animals displayed such a wave, whereas 97.5% of the treated, intoxicated animals developed it. The same observation was made on day 23: an H wave was detected in 25% of the untreated, intoxicated animals and 87.5% of the treated, intoxicated animals. On day 30, these proportions were 75 and 100%, and on day 37, all animals presented the H wave. Sensory Nerve Conduction Velocity As shown in figure 4, on day 16, the untreated, intoxicated animals showed a significant (p ⬍ 0.005, Dunnett’s test) difference in SNCV as compared with the control animals, whereas no noticeable change in this parameter was observed in 4-methylcatechol-treated, intoxicated animals. On day 23, analogous results were observed with a lesser degree of significance (p ⬍ 0.01, Dunnett’s test). Curiously, on day 30, the treated, intoxicated animals presented a decrease in SNCV as compared with control animals (p ⬍ 0.01, Dunnett’s test), which could indicate that the effect of 4-methylcatechol was transient. However, in this group, SNCV was significantly higher than that of untreated, intoxicated animals. On day 30, all treated, and untreated, intoxicated animals had recovered a normal SNCV. Morphological Analyses Morphological analyses of the fibres of DRG neurons showed that the untreated, intoxicated animals presented a very significant (p ⬍ 0.001,

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70 60

Control/vehicle Pyridoxine/vehicle Pyridoxine/4-methylcatechol ** *

Velocity (m/s)

50

**

* ***

*

40 30 20 10 0

2

16

23

30

37

Days

Intoxication 4-Methylcatechol treatment

Fig. 4. Effect of 4-methylcatechol on the SNCV in electromyographic testing for the pyridoxine-intoxicated versus control animals. The values represent the mean ⫾ SEM. *p ⬍ 0.05, **p ⬍ 0.01, ***p ⬍ 0.001 (Dunnett’s test).

Dunnett’s test) decrease in their number (fig. 5) as compared with that of control or treated, intoxicated animals. Their size was also affected. There was no difference in these parameters between treated, intoxicated and control animals. In addition, sensory neuron cell bodies were shown not to be affected by the pyridoxine intoxication (data not shown).

Discussion

Role of Pyridoxine-Induced Neuropathy in the Toxic Neuropathies The pyridoxine-induced neuropathy can be classified in sensory toxic neuropathies. Sensory toxic neuropathy could be induced with (a) industrial pollutants (i.e. acrylamide, dimethylaminopropionitrile, ethylene oxide, hexane and derivatives or trichloroethylene), (b) metals (organic or inorganic mercury, thallium, gold, arsenic, lead), (c) drugs (antimitotic agents such as cisplatin, vincristine or taxol; antituberculosis agents such as isoniazid or streptomycin; antiparasite and antibacterial agents such as metronidazole, nitrofurantoin, chloroquine or clioquinol; antiviral agents such as zidovudine, dideoxyinosine, dideoxycytidine or suramide; neuropsychiatry drugs such as lithium, phenytoin

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250

Fibres per field (n)

200

150

Control/vehicle Pyridoxine/vehicle Pyridoxine/4-methylcatechol

*

*

100

50

0

Fig. 5. Number of total fibres in the sciatic nerve per analysed fields in the pyridoxineintoxicated versus control animals. The values represent the mean ⫾ SEM. *p ⬍ 0.001 (Dunnett’s test).

or amitriptyline; rheumatological drugs such as gold salts or colchicine;.. dermatological drugs such as thalidomide; cardiovascular drugs such as amiodarone, and other drugs and compounds such as almitrine, pyridoxine, cimetidine or carbimazole). Concerning pyridoxine-induced neuropathy models, the interesting question is to know if this neuropathy is a peculiar case with a limited interest as a model of sensory toxic neuropathy or if it shares similar characteristics with other pure sensory toxic neuropathies. Then this model of neuropathy could be used additionally for studying neuroprotective drugs for the treatment of toxic (such as cisplatin- or taxol-induced) or metabolic (diabetes-associated) sensory neuropathies. First of all we need to recall that to be a good model of human toxic induced neuropathy, our animal model should fulfil three criteria: (a) isomorphism of the symptoms, (b) homology of the causes and (c) reactivity to drugs known to be active, if any, against the human disease it is supposed to mimic. This third criterion is also called predictivity. We showed, using a new schedule of pyridoxine administration, that it was possible to induce a non-lethal neuropathy which is of rapid onset, practically devoid of side effects – except slight growth retardation – reversible and having the features of an exclusively sensory deficit. Obviously, the clinical and electrophysiological signs presented by pyridoxine-intoxicated animals were identical to those presented by humans overdosed with pyridoxine. The sensorimotor impairment induced in rats by this vitamin was consistent with a proprioceptive deficit due to sensory neuropathy involving large fibres. Moreover, in contrast

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Table 2. Symptomatology of sural nerve from patients suffering from different toxic sensory neuropathies Inductors

Antimitotic agents Vincristine (vinblastine)

Cisplatin Taxol Antituberculosis agent Isoniazid Antibactericide and antiparasite agent Metronidazole Antiviral agents Dideoxycytidine, dideoxyinosine Drugs Thalidomide Colchicine

Type of neuropathy

Neuropathy characteristics

Reference No.

Sensory and axonal sensorimotor peripheral neuropathy Sensory peripheral neuropathy Sensory peripheral neuropathy

Reflex loss, paraesthesia, autonomic nerve damage, sometimes cranial nerve damage Paraesthesia, ataxia, no loss of thermal pain, reflex loss Sensory symptoms with quick apparition

8

Peripheral sensory neuropathy

Interfering with the pyridoxine metabolism, paraesthesia

20

Peripheral sensory neuropathy

Painful paraesthesias

10

Peripheral sensorimotor neuropathy (axonal degeneration?)

Painful paraesthesias, motor deficit, decrease in Achilles’ reflex

4

Sensory neuropathy (of longest and biggest fibres) Neuronomyopathy, axonal sensory neuropathy

Pain and cramps of members

24

Muscular deficit, fibrillation and polyphasic action potential

7

5–8 21

to Helgren et al. [13], small fibres were also involved as shown by the decreased SNCV and the altered thermosensitivity detected in the hot plate test. The same signs are observed in humans suffering from pyridoxine-induced neuropathy. We can conclude therefore that the criterion of isomorphism was fulfilled. There are many pure toxic sensory neuropathies displaying similar symptoms with those of pyridoxine-induced neuropathy and mainly those induced by drugs used in human therapeutics (such as: vincristine, cisplatin, taxol, isoniazid, metronidazole, colchicine, thalidomide, almitrine). Table 2 shows sural nerve symptomatology from patients suffering from different toxic sensory neuropathies. It could be noted that these neuropathies

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mainly involved large sensory myelinated fibres (i.e. cisplatin, almitrine, colchicine, vincristine, thalidomide and pyridoxine) and a regeneration process with an increase in small unmyelinated fibre number (cisplatin, vincristine, thalidomide). In most of the cases, no neuropathy is noted (except with taxol and thalidomide). We can see slight lesions of demyelinization in metronidazole, vincristine and taxol neuropathies and wallerian degeneration in metronidazole and isoniazid neuropathies. This succinct analysis shows that there are some similarities in the cellular pathology between neuropathies induced by cisplatin, vincristine, colchicine, thalidomide and pyridoxine. Concerning the homology criteria, morphological analyses of fibres emerging from L4 and L5 DRG indicated that pyridoxine induced a severe loss of large and small sensory fibres without affecting sensory neuron cell bodies suggesting that cellular pathogenesis of the neuropathy is analogous to the human disease. Although the molecular pathogenesis of pyridoxine-induced sensory neuropathy is not yet elucidated, we can speculate that both human and rat pyridoxine-induced neuropathies resulted from the same molecular lesions. We can consider therefore that the criterion of homology was almost entirely fulfilled. It could be noted that the agents inducing toxic sensory neuropathies are really different and in most cases their molecular mechanisms are unknown. By contrast the cellular pathology is monotone, in most cases there is axonopathy by a ‘dying-back process’. The predictivity to reference treatment is the trickiest criterion to evaluate. The reason is the lack of treatment for all these toxic neuropathies. We proved in our study that it was possible to shorten the duration of the disease by treating the intoxicated animals with a compound known to induce NGF, namely 4-methylcatechol [17]. To the best of our knowledge, this drug has never been used to treat human toxic and metabolic neuropathies or neurodegenerative peripheral disorders or pyridoxine-induced neuropathies. However, it has been shown that chronic treatment of pyridoxine-intoxicated animals with NT3, a neurotrophic factor involved in the survival of sensory neurons of cranial ganglia [6], induced a reversal of the neurological symptoms. We observe here that 4-methylcatechol improved the clinical status of intoxicated animals and promoted nerve regeneration or prevented nerve degeneration. Interestingly, it was observed that the SNCV of treated, intoxicated animals did not decrease as long as 4-methylcatechol was administered, but was slightly affected 1 week after cessation of treatment. This could mean that the beneficial effect of this drug is transient. However, since the pyridoxine-induced sensory neuropathy was reversible, it was not possible to draw definitive conclusions from our results. 4-Methylcatechol is known to induce NGF synthesis in the peripheral organs innervated by sympathetic nerves. NGF is

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physiologically transported into the neuron cell body by means of receptormediated axonal retrograde transport [9, 17, 26]. In the literature many authors have proved the neuroprotective effect of 4-methylcatechol in different models of induced neuropathies. Hanaoka et al. [11, 12] have shown that intraperitoneal administration of 4-methylcatechol prevents the deterioration of SNCV and the decrease in NGF content in sciatic nerves of rats suffering from experimental diabetic neuropathy. In addition, a 2-week treatment with 4-methylcatechol reduced the symptoms of acrylamide-induced neuropathy in Sprague-Dawley rats [14–25]. This treatment also resulted in the promotion of peripheral nerve regeneration in sciatic-nerve-lesioned Wistar rats [16, 17]. We have shown in a rat model of vincristine- as well as cisplatin-induced neuropathy that a 3-week treatment was able to decrease the symptoms of neuropathy (internal data). It is interesting to note that these toxic, metabolic or traumatic neuropathies can be treated using the same drug, although their pathogeneses were extremely different. In the light of these results it seems that pyridoxine-induced neuropathy is an excellent model of sensory neuropathy sensitive to NGF.

Overall Conclusion

To summarize, (a) the pyridoxine-induced sensory neuropathy provides the pharmacologists with a valuable model for studying and evaluating new neurotrophic factors endowed with NGF-like properties; (b) this model can be included in the palette of experimental sensory neuropathies used in preclinical research for evaluating new putative neuroprotective drugs, the mechanisms of action of which are not known.

References 1 2 3 4 5

6

American Association of Electrodiagnostic Medicine: Guidelines in electrodiagnostic medicine. Muscle Nerve 1992;15:229–253. Albin R, Albers JW, Greenberg HS, Townsend JB, Lynn RB, Burke JM, Alessi AG: Acute sensory neuropathy-neuronopathy from pyridoxine overdose. Neurology 1987;37:1729–1732. Callizot N, Warter JM, Poindron P: Pyridoxine-induced neuropathy in rats: a sensory neuropathy that responds to 4-methylcatechol. Neurobiol Dis 2001;8:626–635. Carey P: Peripheral neuropathy: zalcitabine reassessed. Int J STD AIDS 2000;11:417–423. Chaudhry V, Rowinsky EK, Sartorius SE, Donehower RC, Cornblath DR: Peripheral neuropathy from taxol and cisplatin combination chemotherapy: clinical and electrophysiological studies. Ann Neurol 1994;35:304–411. Davies AM, Lumsden AG, Rohrer R: Neural crest-derived proprioceptive neurons express nerve growth factor receptors but are not supported by nerve growth factor in culture. Neuroscience 1987;20:37–46.

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7 8 9

10 11

12

13

14

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17

18

19

20 21 22 23 24 25

26 27 28 29

De Deyn PP, Ceuterick C, Saxena V, Crols R, Chappel R, Martin JJ: Chronic colchicine-induced myopathy and neuropathy. Acta Neurol Belg 1995;95:29–32. Foreman A: Peripheral neuropathy in cancer patients: clinical types, etiology, and presentation. Part 2. Oncology (Williston Park) 1990;4:85–89. Furukawa Y, Fukazawa N, Miyama Y, Hayashi K, Furukawa S: Stimulatory effect of 4-alkylcatechols and their diacetylated derivatives on the synthesis of nerve growth factor. Biochem Pharmacol 1990;40:2337–2342. Gupta BS, Baldwa S, Verma S, Gupta JB, Singhal A: Metronidazole induced neuropathy. Neurol India 2000;48:192–193. Hanaoka Y, Ohi T, Furukawa S, Furukawa Y, Hayashi K, Matsukura S: Effect of 4-methylcatechol on sciatic nerve growth factor level and motor nerve conduction velocity in experimental diabetic neuropathic process in rats. Exp Neurol 1992;115:292–296. Hanaoka Y, Ohi T, Furukawa S, Furukawa Y, Hayashi K, Matsukura S: The therapeutic effects of 4-methylcatechol, a stimulator of endogenous nerve growth factor synthesis, on experimental diabetic neuropathy in rats. J Neurol Sci 1994;122:28–32. Helgren ME, Cliffer KD, Torrento K, Cavnor C, Curtis R, Distefano PS, Wiegand SJ, Lindsay RM: Neurotrophin-3 administration attenuates deficits of pyridoxine-induced large-fiber sensory neuropathy. J Neurosci 1997;17:372–382. Hopkins AP, Gilliat RW: Motor and sensory nerve conduction velocity in the baboon: normal values and changes during acrylamide neuropathy. J Neurol Neurosurg Psychiatry 1971;34: 415–426. Hoover DM, Carlton WW: The subacute neurotoxicity of excess pyridoxine HCl and clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) in beagle dogs. II. Pathology. Vet Pathol 198;18: 757–768. Kaechi K, Furukawa Y, Ikegami R, Nakamura N, Omae F, Hashimoto Y, Hayashi K, Furukawa S: Pharmacological induction of physiologically active nerve growth factor in rat peripheral nervous system. J Pharmacol Exp Ther 1993;264:321–326. Kaechi K, Ikegami R, Nakamura N, Nakajima M, Furukawa Y, Furukawa S: 4-Methylcatechol, an inducer of nerve growth factor synthesis, enhances peripheral nerve regeneration across nerve gaps. J Pharmacol Exp Ther 1995;272:1300–1304. Kennel PF, Fonteneau P, Martin E, Schmidt JM, Azzouz M, Borg J, Guenet JL, Schmalbruch H, Warter JM, Poindron P: Electromyographical and motor performance study in the pmn model of neurodegenerative disease. Neurobiol Dis 1996;3:137–147. Krinke G, Naylor DC, Skorpil V: Pyridoxine megavitaminosis: an analysis of the early changes induced with massive doses of vitamin B6 in rat primary sensory neurons. J Neuropathol Exp Neurol 1985;44:117–129. Mahashur AA: Isoniazid induced peripheral neuropathy. J Assoc Physicians India 1992;40: 651–652. Michaud LB, Valero V, Hortobagyi G: Risks and benefits of taxanes in breast and ovarian cancer. Drug Saf 2000;23:401–428. Moses LE, Oakford RV: Tables of Random Permutations. Stanford, Stanford University Press, 1963. Parry GJ, Bredesen DE: Sensory neuropathy with low dose pyridoxine. Neurology 1985;35:1466–1468. Rao DG, Kane NM, Oware A: Thalidomide neuropathy: role of F-wave monitoring. Muscle Nerve 2000;23:1301–1302. Saita K, Ohi T, Hanaoka Y, Furukawa S, Furukawa Y, Hayashi K, Matsukura S: A catechol derivative (4-methylcatechol) accelerates the recovery from experimental acrylamide-induced neuropathy. J Pharmacol Exp Ther 1996;276:231–237. Saporito MS, Wilcox HM, Hartpence KC, Lewis ME, Vaught JL, Carswell S: Pharmacological induction of nerve growth factor mRNA in adult rat brain. Exp Neurol 1993;123:295–302. Schaumburg H, Kaplan J, Windebank A, Vick N, Rasmus S, Pleasure D, Brown MJ: Sensory neuropathy from pyridoxine abuse: a new megavitamin syndrome. N Engl J Med 1983;309:445–448. Unna K, Antopol W: Toxicity of vitamin B6. Proc Soc Exp Biol Med 1940;43:116. Xu Y, Sladky JT, Brown MJ: Dose-dependent expression of neuropathy after experimental pyridoxine intoxication. Neurology 1989;39:1077–1083.

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Windebank AJ, Low PA, Blexrud MD, Schmelzer JD, Schaumburg HH: Pyridoxine neuropathy in rats: specific degeneration of sensory axons. Neurology 1985;35:1617–1622.

Prof. Philippe Poindron 11, rue Galliéni FR-92100 Boulogne (France) Tel. ⫹33 684 01 29 51, Fax ⫹33 684 01 29 51, E-Mail [email protected]

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Poindron P, Piguet P (eds): New Animal Models of Human Neurological Diseases. BioValley Monogr. Basel, Karger, 2008, vol 2, pp 81–96

A Dopamine-Sensitive Inherited Motor Syndrome Linked to the frissonnant Mutation Noëlle Callizot a, Jean-Louis Guenet b, Philippe Poindron a a Laboratoire de Pathologie des Communications entre Cellules Nerveuses et Musculaires, Faculté de Pharmacie, Université Louis-Pasteur, Strasbourg, et b Unité de Génétique des Mammifères, Institut Pasteur, Paris, France

Abstract The frissonnant (fri) mutation is an autosomal recessive mutation which spontaneously appeared in 1977 in the stock of C3H mice of the Pasteur Institute in Paris. fri/fri mutation causes locomotor instability and rapid tremor. The tremor ceases when mutated animals sleep or are anaesthetized. In the light of these symptoms, it seems that fri/fri shares similarities with human Parkinson disease (PD). Here we give a precise and full description of the fri phenotype with an accurate behavioural, electrophysiological and histological analysis. The results show that fri/fri mice present an important motor deficit with visible tremor and stereotypies. We observed that fri/fri mice display some memory deficits as in human PD. We also show thatL-3,4-dihydroxyphenylananine and apomorphine (dopaminergic agonists), ropinirole (selective D2 agonist) and selegiline (a monoamine oxidase B inhibitor) improve the clinical status of these animals. Finally, we found that fri/fri mice did not display any anatomopathological evidence of nigrostriatal lesion nor a decrease in tyrosine hydroxylase production. We conclude that fri/fri mice present a dopamine-sensitive inherited motor syndrome associated with a parkinsonian syndrome, but are not a mouse model of PD disease. They could be used as a tool for studying and screening dopaminergic compounds with central activity. Copyright © 2008 S. Karger AG, Basel

Introduction

The mutation frissonnant (fri) is an autosomal recessive condition that appeared spontaneously in 1977 in the stock of C3H mice of the Pasteur Institute in Paris. fri/fri mutation causes locomotor instability and rapid tremor.

The tremor ceases when mutated animals sleep or are anaesthetized. In the light of these symptoms, it seems that fri/fri shares similarities with human Parkinson disease (PD). PD is characterized by akinesia, tremor, rigidity and postural instability [1]. Other symptoms such as mild cognitive defects, autonomic dysfunction and affective disorders may also be observed [2]. PD usually affects people in their middle or later years, with an incidence rate of 5–20/100,000 [3]. Ninety percent of PD cases are sporadic and 10% inherited [4]. The behavioural deficits are primarily due to progressive degeneration of nigrostriatal dopaminergic neurons which results in motor syndrome. The 3 main symptoms appear when more than 55–60% of dopamine have disappeared from the nigrostriatal structures because of degeneration of dopaminergic neurons [5]. The first aim of the present study was to run a complete behavioural and histological phenotyping of fri/fri mice, the second aim was to verify whether the fri/fri mice could be validated as an animal model of inherited PD, by addressing 3 questions: (a) do the fri/fri mice present the behavioural deficits observed in PD, (b) is the clinical status of fri/fri mice improved by drugs known as efficient antiparkinsonian compounds, i.e. L-3,4-dihydroxyphenylananine (L-dopa) and apomorphine, and finally (c) do the fri/fri mice display degeneration of nigrostriatal dopaminergic neurons as in PD? In this study, we made a precise and full description of the fri phenotype with an accurate behavioural, electrophysiological and histological analysis. The results show that fri/fri mice present an important motor deficit with visible tremor (allowing to phenotypically recognize fri/fri mice at birth) and stereotypies. We observed that fri/fri mice display some memory deficits as in human PD. We also show that L-dopa and apomorphine (dopaminergic agonists), ropinirole (selective D2 agonist) and selegiline (a monoamine oxidase B inhibitor) improve the clinical status of these animals. Finally, we found that fri/fri mice did not display any anatomopathological evidence of nigrostriatal lesion nor a decrease in tyrosine hydroxylase (TH) production.

Materials and Methods Animals The fri/fri mice were from the Unité de Génétique des Mammifères of the Pasteur Institute in Paris. Although fri/fri mice were fertile, heterozygous ⫹/fri mice (subsequently referred to as heterozygous mice) were used for the reproduction. Homozygous fri/fri mice were identified at birth by the characteristic tremor. For control, ⫹/⫹ mice from the same genetic background were used. The ⫹/fri animals were identified by 2 backcrosses with 2 different ⫹/fri animals and detection of the presence of fri/fri in the progeny. Animals were housed in plastic cages at a constant temperature of 22⬚C (⫾0.5⬚C) with a 12-hour light:12-

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hour dark inverted cycle. They had free access to food and water. All animals used in this study were handled in compliance with approved institutional care and the usual protocols, according to the guidelines of the French Ministry of Agriculture and Fisheries. For the experiments, both male and female mice were used; they were 5–34 months old. Behavioural Testing Motor Coordination Measurements Rotarod Test. The rotarod test consisted in automatically measuring how long the mouse could maintain itself on a rotating axle (rotarod apparatus from Bioseb, Paris, France; diameter of the axle: 3 cm; speed of rotation: 12 rpm) without falling, the test being stopped after an arbitrary limit of 300 s. If the animal fell down before 300 s, the time was recorded and an additional trial was performed (3 maximum), and the mean time of these 3 trials was calculated and retained as characteristic value. String Test. The string test consisted in hanging the animal to a thread (diameter: 1 mm) – stretched horizontally 40 cm above a table – by its forepaws and measuring the time required for the animal to grasp the thread with its hindpaws. Three trials (maximal duration: 60 s each) were performed, and the mean time of these 3 trials was calculated and retained as characteristic value. If the animal fell down, it was scored with a maximum of 60 s. Muscular Power: Maximal Strength The maximal muscle strength was measured with an isometric transducer (Bioseb) attached to a piece of grid. When the animal held the wire with its 4 legs, it was slowly moved backwards until it released the grid; the transducer measured the maximal strength. Results are given in newtons. Two trials were performed, and the mean value was calculated and retained as characteristic value. Assessment of Sensory Dysfunction: Tail Flick Test The tail of the mouse was placed under a shutter-controlled lamp as a heat source. The latency before the mouse flicked its tail from the heat was recorded. A sensory alteration increases the latency of flick. Two trials were performed, and the mean value was calculated and retained as characteristic value. Footprint Analysis The animals were placed in a 9.5-cm-wide, 58-cm-long corridor the floor of which was covered with white absorbing paper. The animals were first trained to explore the corridor; then their hindpaws were dipped in black ink and they were placed into the corridor for the trial. Under these conditions, they left permanent footprints when they walked. During the trial the animals had to walk straightforward without stopping. If they did not, a new trial was performed, a maximum of 3 consecutive trials being accepted. The following parameters were recorded: (a) width of foot placement; (b) intrastep distance; (c) step length. Locomotor Activity in Open Field A video track was placed over a plexiglass open field (OF; length: 52 ⫻ 52 cm; height: 40 cm) and recorded the activity of the animal during 10 or 30 min. The floor of the OF was divided into 9 equal squares. The locomotor activity was expressed in terms of mean number

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of squares crossed per minute. The total number of rearings was also recorded at the same time. The initiation of the movement was recorded by measuring the time spent by the animal to walk over a distance of 2 squares after a stop of 10 s. Five measurements were performed, and the mean value was calculated and retained as characteristic value. Electrophysiological Measurements All recordings were made with a standard electromyographic apparatus (Neuromatic 2000M, Dantec, Les Ulis, France) in accordance with the guidelines of the American Association of Electrodiagnostic Medicine (1992). Mice were anaesthetized by intraperitoneal injection of 60 ␮g/g of ketamine hydrochloride (Ketalar®; Parke Davis, Courbevoie, France). A monopolar needle electrode (Dantec, code 13L70, diameter: 0.3 mm) was inserted into the back of the animal to ground the system. Throughout the procedure, the animals were kept under a heating lamp to maintain a physiological muscle temperature. The temperature was verified on the surface of the tail with a contact thermometer. Compound Muscle Action Potential Compound muscle action potentials (CMAP) were recorded in the gastrocnemius muscle. Amplitudes (in millivolts) from the right muscle-evoked responses were measured. For the recording of the gastrocnemius CMAP, supramaximal square pulses (1 Hz), of 0.2 ms duration, were delivered through a needle electrode (Dantec, code 13L81) to the sciatic nerve at the sciatic notch level. An anode needle electrode was inserted at the base of the tail. The active recording unipolar needle electrode (Dantec, code 13RS1) was inserted in the medial part of the gastrocnemius. The reference recording electrode was inserted over the Achilles tendon. The myo-electric signal was bandpass filtered (2–5 Hz) to eliminate artefacts. An initial negative deflection and biphasic waveform indicated recording at the motor point. The CMAP amplitude was measured from peak to peak. Sensory Nerve Conduction Velocity The sensory nerve conduction velocity (SNCV) was determined by the rate at which the action potential propagated along the caudal nerve during orthodromic conduction. This rate is variable and depends on current flow along the axon, the depolarization threshold of the membrane and temperature. To elicit the sensory nerve action potential, the tail was strapped to a polystyrene board. The unipolar needle electrodes (Dantec, code 13R24) were inserted as follows: (a) the stimulating cathode was placed exactly two thirds of the length of the tail, distally to its hairline; (b) the stimulating anode was inserted 3 mm distally to the stimulating cathode; (c) the recording cathode and anode were inserted 5 and 2 mm distally to the hairline of the tail, respectively, and (d) a ground electrode was inserted half way between the stimulating and recording cathodes. Twenty sensory evoked responses were averaged for each recording. The SNCV of the caudal nerve was calculated from the latency of the stimulus artefact to the onset of the negative peak of the action potential elicited, and the distance between the stimulating and the recording cathodes. Treatment of fri Mice with L-Dopa, Apomorphine, Selegiline and Ropinirole L-Dopa (Sigma, L’Isle d’Abeau-Chesnes, France), apomorphine (Sigma), selegiline [(R)-(–)-N,a-dimethyl-N-(2-propynyl)phenetylamine hydrochloride; Sigma], an inhibitor of B-type monoamine oxidase, and ropinirole, a selective D2 receptor agonist (Requip®;

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SmithKline-Beecham, Nanterre, France), were dissolved in 9‰ aqueous solution of sodium chloride. L-Dopa (5, 10, 20, 40 and 60 mg/kg), selegiline (10 mg/kg) and ropinirole (20 mg/kg) were injected intraperitoneally 9, 45 or 30 min, respectively, before the behavioural testing, and apomorphine (0.1, 0.2 and 2 mg/kg) was injected subcutaneously 30 min before the behavioural testing. Locomotor activity, rearing and initiation of movement were recorded before and after the drug administration. The locomotor performances were measured and expressed in percent relatively to the performances measured before treatment and set at 100%. Object Recognition Test The object recognition test [6] was performed in the plexiglass OF placed in a dark room. The objects to be discriminated were a marble and a dice. Animals were first habituated to the OF for 15 min. The next day, they were submitted to a 10-min acquisition trial (first trial) during which they were individually placed in the OF in the presence of an object A (marble or dice). Activity, that is the number of squares crossed, and the time spent to explore the object were recorded. A 10-min retention trial (second trial) was then carried out 3 or 24 h later. For this trial, the object A and the object B were placed in the OF, and locomotor activity and exploratory times (TA and TB) were recorded. The recognition index (RI) is defined as [TB/(TA ⫹ TB)] ⫻ 100. There is no recognition when RI does not differ from 50. Histopathology Brains of 2 controls and 2 fri/fri mice were collected and fixed in a 4% aqueous solution of paraformaldehyde and then immersed in an 18% aqueous solution of sucrose. Olfactory bulbs, diencephalon and mesencephalon were preserved in all specimens studied. Tissues were frozen in liquid nitrogen, mounted in OCT embedding medium and cut into 40␮m-thick sections using a Reichert-Jung cryostat (Vienna, Austria). The sections were collected and placed in phosphate-buffered saline. Sections were then processed for TH immunostaining [7]. They were treated overnight at 4⬚C with a rabbit polyclonal anti-TH antiserum (Chemicon International Inc., Temecula, Calif., USA) diluted to 1/500 in a 0.1 mol/l phosphate-buffered saline, pH 7.4, containing 0.4% Triton X100 (Sigma) and 10% fetal calf serum (Gibco, Life Technologies, CergyPontoise, France). After 3 washes in buffer of 5 min each, the primary antibody was detected by the avidin-biotin method using the Vector Laboratories staining kit (Burlingame, Calif., USA). Briefly, preparations were treated for 1 h with a diluted horse biotinylated anti-rabbit polyclonal antiserum and then washed 3 times for 5 min with buffer. The diluted avidinbiotin peroxidase complex (Sigma) was then applied for 1 h at room temperature, and the preparations were washed 3 times for 5 min with buffer. Finally, the sections were treated with a solution of diaminobenzidine hydrochloride and hydrogen peroxide to localize the antigen/peroxidase-labelled antibody complex. A number of sections was also Nissl stained.

The mean number of nigrostriatal neurons per microscopic field was determined using the Visiolab 2000 software (Biocom, Paris, France). Statistical Analysis All data were analysed by repeated analysis of variance, followed, when appropriate, by a Dunnett’s test. In some cases, a paired or unpaired t test was also used. A value of p ⬍ 0.05 was considered as significant.

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Fig. 1. Performances in the rotarod test (a) and in the string test (b) for the ⫹/⫹ (n ⫽ 8), ⫹/fri (n ⫽ 8) and fri/fri (n ⫽ 8) groups. The values represent the mean ⫾ SEM. Dunnett’s test, *p ⬍ 0.005, **p ⬍ 0.001.

Results

Behaviour Sensorimotor Tests fri mice were not able to stay on a rotarod for more than 10 s, whereas control and heterozygous ⫹/fri (data not shown) mice could stay for at least 300 s, the upper time limit fixed for the test (fig. 1a). fr/fri mice were unable to catch the string with their hindpaws, in contrast with control and heterozygous mice which could catch it very rapidly (fig. 1b). The gait analysis showed that the width of foot placement of fri/fri mice was greater than for control or heterozygous mice (fig. 2b). The step length and the intrastep distance were also shorter (fig. 2a, c). By contrast, there was no difference in the time of first reaction to the tail flick test between the different groups (fig. 3). It could be noted that all tests were performed on animals of different ages (5, 6, 8, 10, 12, 28 and 34 months). No significant evolution in the value of parameters studied (data not shown) could be seen in the 3 groups of animals (fri/fri; ⫹/fri; ⫹/⫹). Locomotor Activity In the OF, significant differences were detected between fri/fri and control mice (⫹/fri and ⫹/⫹; fig. 4a). In addition, the number of rearings was less in fri/fri mice compared to control animals (fig. 4b).

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Fig. 2. Footprint analysis: the intrastep distance (a), width between steps (b) and the step length (c) for the ⫹/⫹ (n ⫽ 8) and fri/fri (n ⫽ 8) groups. The values represent the mean ⫾ SEM. Dunnett’s test, *p ⬍ 0.05, **p ⬍ 0.005.

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Fig. 3. Performances in the thermosensitivity test (tail flick test) for the ⫹/⫹ (n ⫽ 8) and fri/fri (n ⫽ 8) groups. The values represent the mean ⫾ SEM. Unpaired t test.

Electromyography No difference was detected in electromyographic parameters, caudal nerve (SNCV) or CMAP (gastrocnemius) amplitude or latency, between homozygous, heterozygous and control mice (fig. 5a–c). Memory Test A significant difference in RI could be detected between the homozygous (RI: 45.93 ⫾ 7.65) and control mice (RI: 72.65 ⫾ 2.77). The homozygous mice were unable to recognize object A that they were supposed to know. This deficit was not due to the locomotor deficit since fri/fri mice explored the 2 objects as long as the control group (fig. 6) and indicated that these mice probably suffered from a short-term memory deficit.

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Fig. 4. Locomotor activity (a) in the OF and total number of rearings (b) for the ⫹/⫹ (n ⫽ 8), ⫹/fri (n ⫽ 8) and fri/fri (n ⫽ 8) groups. The values represent the mean ⫾ SEM. Dunnett’s test, *p ⬍ 0.01.

Sensitivity to L-Dopa, Apomorphine, Selegiline and Ropinirole Ninety minutes after administration of L-dopa (all doses), fri mice improved their motor performances (fig. 7a). However, the only significant differences (one-sample t test) were observed at doses of 10 mg/kg (p ⬍ 0.01), 20 mg/kg (p ⬍ 0.05) and 40 mg/kg (p ⬍ 0.05). Within this range of doses, the improvement depended on the dose administered. Unlike the fri/fri mice, the control mice did not improve their locomotor activity after being treated with Ldopa. Moreover, the number of rearings was significantly increased in the case of fri/fri mice at all doses ranging between 10 and 60 mg/kg (p ⬍ 0.05; paired t test; fig. 7b). Analogous results were observed for the moving speed and movement execution which were significantly improved after L-dopa administration at all doses ranging between 5 and 60 mg/kg (paired t test; fig. 7c). No difference could be seen in the case of control mice. Finally, frequency of tremor was obviously less elevated after L-dopa administration at doses ranging between 10 and 40 mg/kg.

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Fig. 5. SNCV (a), amplitude (b) and latency (c) of the CMAP in the electromyogram for the ⫹/⫹ (n ⫽ 7) and fri/fri (n ⫽ 7) groups. The values represent the mean ⫾ SEM. Unpaired t test.

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Fig. 6. RI in the object recognition test in the OF for the ⫹/⫹ (n ⫽ 8) and fri/fri (n ⫽ 8) groups. The values represent the mean ⫾ SEM. Unpaired t test, *p ⬍ 0.05. Percentages versus a theoretical mean of 50%.

Thirty minutes after administration of apomorphine (0.1 and 0.5 mg/kg), the fri/fri mice displayed increased locomotion (p ⬍ 0.01, paired t test) and rearing (fig. 8a, b). The only significant difference in the number of rearings was seen at 0.5 mg/kg (p ⬍ 0.05, paired t test). The motor performances of control mice were not improved by the treatment and even worsened, probably because of habituation of the animals in the OF test. The apparent heterogeneity in the results obtained in the different experimental groups for a given condition was due to intragroup variability; it was why each animal was its own control. Selegiline-treated (10 mg/kg) fri/fri mice showed improved motor performances 45 min after administration of the drug (fig. 9; p ⬍ 0.05, unpaired

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Fig. 7. Locomotor activity (a) expressed in percentage, movement speed (b) and total number of rearings (c) before and 90 min after the injection of 5, 10, 20, 40 and 60 mg/kg of L-dopa for the ⫹/⫹ (n ⫽ 8) and fri/fri (n ⫽ 7) groups. The values represent the mean ⫾ SEM. Paired t test, *p ⬍ 0.05, **p ⬍ 0.01.

t test). Here again, the control mice displayed a decreased locomotion after the treatment, probably for the same reasons as mentioned above for the apomorphine-treated mice. Ropinirole (20 mg/kg) significantly (p ⬍ 0.01, paired t test) increased the motor performances of fri mice within 20 min (fig. 10). Histological and Immunocytochemical Studies Examination of the totality of the substantia nigra and striatum did not reveal obvious differences between fri/fri and control animals (data not shown). Similarly, the immunocytochemical study of TH expression [7] did not show

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Fig. 8. Locomotor activity (a) and total number of rearings (b) before and 30 min after the injection of 0.1 and 0.5 mg/kg of apomorphine for the ⫹/⫹ (n ⫽ 8) and fri/fri (n ⫽ 7) groups. The values represent the mean ⫾ SEM. Paired t test, *p ⬍ 0.05, **p ⬍ 0.01, ***p ⬍ 0.005.

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Fig. 9. Locomotor activity before and 45 min after the injection of 10 mg/kg of selegiline for the ⫹/⫹ (n ⫽ 7) and fri/fri (n ⫽ 7) groups. The values represent the mean ⫾ SEM. Paired t test, *p ⬍ 0.05.

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Fig. 10. Locomotor activity before and 45 min after the injection of 20 mg/kg of ropinirole for the ⫹/⫹ (n ⫽ 7) and fri/fri (n ⫽ 7) groups. The values represent the mean ⫾ SEM. Paired t test, *p ⬍ 0.01.

any differences between fri/fri and control mice neither in the distribution of TH in these areas of the brain (fig. 11) nor in the number of cell bodies (fri/fri: 152.00 ⫾ 32.5; ⫹/⫹: 103.0 ⫾ 12.5 cell bodies/field, plane 57 [8], n ⫽ 3 sections). Diencephalon, olfactive bulb and mesencephalon sections were also examined. No difference in the number of neurons or morphological alteration could be detected between ⫹/⫹ and fri/fri mice.

Discussion

Animal Models of PD The rodent animal models most widely used as PD models are (a) 6hydroxydopamine-induced unilateral lesion of rats and (b) N-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP)-treated mice. The 6-hydroxydopamine-induced unilateral lesion of rat striatum is most widely used to study effects of putative antiparkinsonian drugs [9]. This model is based on the fact that in such lesioned animals, injection of dopaminergic compounds makes the lesioned animal rotate to the less stimulated side of the striatum. The MPTP model consists in systemically injecting mice with high doses of this toxin which induces a severe degeneration of catecholaminergic neurons [10]. Following this treatment, depletion of dopamine in the striatum has been observed [10–12]. However, the behavioural deficits induced by the treatment have been shown to be short-lived [11, 13–15] probably because of

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a

b Fig. 11. TH activity in the substantia nigra in the immunocytochemical study of the fri/fri (a) and ⫹/⫹ (b) animals. Note that there is no difference in the staining intensity and number of immunoreactive cells (plane 57 [8]). ⫻50.

regeneration of lesioned dopaminergic neuron terminals [10] and heterogeneity. fri/fri Mice as PD Model? The present results show that the fri/fri mice share many features with currently accepted animal models of PD. They display important movement poverty and very slow movements (as shown by the rotarod and string tests). Moreover, these animals present a significant decrease in their locomotor activity in the OF test: the number of rearings is smaller for fri/fri than for control and heterozygous mice. Execution of movement is slower and weaker in the case of fri/fri than in that of control and heterozygous mice. The footprint study reveals that fri/fri mice have a step shorter than that of the control mice and cannot easily synchronize their movements which they execute with difficulty. When the movement was triggered off, it was performed quickly and with large amplitude, similar to the ‘roue dentée’ (cogwheel) symptom in PD patients. fri/fri mice do not display any impairment of sensitivity as shown by the tail flick test. They do not have impairment of thermoregulation since their body temperature is the same as that of control and heterozygous mice (data not shown). Electromyography did not show any peripheral disorder therefore we could conclude that there is neither neurodegenerescence nor dysfunction of sensory or motor nerve fibres. fri/fri mice present impairment in cognitive functions. This feature is shared by MPTP-treated mice [16] and PD patients who may have some alterations of cognitive functions – developing during the progress of the disease [17] – memory deficits [18] and depression [18].

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We observed fri/fri mice stock for 1 year. We did not see any change in clinical signs during this time indicating no evolution of the disease. The animals presented symptoms of the fri disease from birth to death. In summary, fri/fri mice share some major signs of a parkinsonian syndrome, we can conclude that the criterion of isomorphism of the model is almost totally fulfilled. For the predictivity criterion, we showed that fri/fri disease is dopamine sensitive. We studied first the reactivity of fri/fri mice to L-dopa, a dopamine precursor. An acute treatment of fri/fri mice with this compound improved locomotor activity (more rearings, faster movements) whereas it had no effect on control mice; this improvement depended on the dose used. The optimal dose of L-dopa was 20 mg/kg (above that, no further improvement was recorded). Beneficial effects were transient and stopped approximately 4 h after drug administration. Analogous results were obtained by treating MPTP-intoxicated mice with L-dopa [10, 19, 20]. Similarly, apomorphine, selegiline and ropinirole improved the clinical status of fri/fri mice. Selegiline is not an antiparkinsonian drug per se but is often used in association with L-dopa, since it inhibits monoamine oxidase B (brain form) and then increases the life-span of dopamine. Unfortunately, because of the severe symptoms affecting fri/fri mice, we could not study the effect of neuroleptic drugs, such as sulpiride, and observe the clinical worsening expected in case of parkinsonian syndrome. However, taken together, the present results indicate that the fri/fri disease is sensitive to drugs active on the dopaminergic pathways; the criterion of predictivity is at least partly fulfilled. The last but probably the most important criterion, the homology, is not fulfilled. Indeed, we were unable to find any anatomopathological lesions of the nigrostriatal systems or to detect any decrease in TH activity (enzyme involved in the dopamine synthesis) in the fri/fri mouse brain. There could be many reasons to explain why fri/fri mice react positively to L-dopa and selegiline. One could be a deficit in dopamine concentration in the synaptic cleft of central dopaminergic neurons. There is no decrease in TH immunoreactivity but other hypotheses could be formulated: D receptors could have an altered affinity for dopamine; impairment in dopamine release in synapses or hyperactivity of monoamine oxidase B involving a high degradation of dopamine could be the cause. To the best of our knowledge there do not exist numerous animal models mimicking inherited human parkinsonian disease. One of the most promising was described by Craig et al. [21]. Briefly, these authors showed that rats harbouring a mutation in the gene coding for protein kinase C␥ had altered behaviour, movement disorder and age-progressive dysfunction and death of neurons

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in the substantia nigra pars compacta. Since no pharmacological study of this strain was performed, it was not possible to determine whether these animals were sensitive to drugs used in the treatment of human PD and related diseases. Conversely, we do not know if fri/fri mice harbour a mutation in a gene coding for protein kinase C␥. To summarize, fri/fri mice present a dopamine-sensitive inherited motor syndrome associated with a parkinsonian syndrome, but are not a mouse model of PD disease. However, behavioural, histological and pharmacological features of both mouse and rat [21] models render them complementary for studying the molecular pathogenesis of parkinsonian syndrome and related diseases. They could be used as a tool for studying and screening dopaminergic compounds with central activity whereas AS/AGU rats could be used for exploring the molecular mechanisms of dopaminergic neuron death.

Overall Conclusions of Animal Models

In the light of this study, an overall conclusion could be drawn on animal models of human disease. First of all, we need to remember that to be confirmed as a valuable model of human disease an animal model should imperatively fulfill 3 criteria [22]: (a) isomorphism (it should present common symptoms with the human disease it is supposed to mimic); (b) predictivity (it should be sensitive to drugs known to improve the clinical status of humans suffering from this disease); (c) homology (it should share some common causes with the human disease). Secondly, facing any animal presenting a peculiar clinical status, a global and precise analysis of the phenotype has to be done. This analysis must be rigorous and should collect the clinical, biochemical, anatomopathological and pharmacological information. Third, it is necessary to be critical towards any animal models used. The ‘ideal’ model does not exist; the closer to the human pathology a model is, the better and more relevant the results will be. The limits of the validity must be evaluated for any model used; to extrapolate too much is dangerous for the relevancy of results. By contrast, underestimating results would be dangerous. A good model of a human disease will show a correct balance between the relevance of the model and the use by the scientist. We could conclude with a model definition given by Segev [23] in 1992: ‘A model is something simple made by scientists to help them in understanding something complicated. A good model is one that succeeds to reduce the complexity of the modelled system while preserving its essential features.’

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2 3 4 5 6 7

8 9 10 11

12 13 14 15 16 17 18 19 20 21 22 23

Hughes AJ, Daniel SE, Kilford L, Lees AJ: Accuracy of clinical diagnosis of idiopathic Parkinson’s disease: a clinico-pathological study of 100 cases. J Neurol Neurosurg Psychiatry 1992;55:181–184. Parkinson J: An Essay on the Shaking Palsy. London, Whittingham & Newland, 1817. Rajput AH, Offord K, Beard CM, Kurland LT: Epidemiological survey of dementia in parkinsonism and control population. Adv Neurol 1984;40:229–340. Duvoisin R: Genetics of Parkinson’s disease. Adv Neurol 1986;45:307–312. Fearnley JM, Lees AJ: Ageing and Parkinson’s disease: substantia nigra regional selectivity. Brain 1991;114:2283–2301. Ennaceur A, Delacour J: A new one-trial test for neurobiological studies of memory in rats: behavioral data. Behav Brain Res 1988;31:47–59. Hokfelt T, Smith CB, Norell G, Peters A, Crane A, Goldstein M, Brownstein M, Sokoloff L: Attempts to combine 2-deoxyglucose autoradiography and tyrosine hydroxylase immunohistochemistry. Neuroscience 1984;13:495–512. Franklin KBJ, Paxinos G: The mouse brain in stereotaxic coordinates. Academic Press, 1997, New York and London. Ungerstedt U: Postsynaptic supersensitivity after 6-hydroxy-dopamine induced degeneration of the nigro-striatal dopamine system. Acta Physiol Scand Suppl 1971;367:69–93. Sundström E, Fredriksson A, Archer T: Chronic neurochemical and behavioral changes in MPTPlesioned C57BL/6 mice: a model for Parkinson’s disease. Brain Res 1990;528:181–188. Hallman H, Olson L, Jonsson G: Neurotoxicity of the meperidine analogue N-methyl-4-phenyl1,2,3,6-tetrahydropyridine on brain catecholamine neurons in the mouse. Eur J Pharmacol 1984; 97:133–136. Heikkila RE, Hess A, Duvoisin RC: Dopaminergic neurotoxicity of N-methyl-4-phenyl-1,2,3,6tetrahydropyridine in mice. Science 1984;224:1451–1453. Guze BH, Barrio JC: The etiology of depression in Parkinson’s disease patients. Psychosomatics 1991;32:390–395. Rozas G, Lopez-Martin E, Guerra MJ, Labandeira-Garcia JL: The overall rod performance test in the MPTP-treated-mouse model of parkinsonism. J Neurosci Methods 1998;83:165–175. Willis GL, Donnan GA: Histochemical, biochemical and behavioral consequences of MPTP treatment in C-57 black mice. Brain Res 1987;402:269–274. Dluzen DE, Kreutzberg JD: N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) disrupts social memory/recognition processes in the male mouse. Brain Res 1993;609:98–102. Lieberman A: Emerging perspectives in Parkinson’s disease. Neurology 1992;42:5–7. Sagar HJ, Sullivan EV, Gabrieli JD, Corkin S, Growdon JH: Temporal ordering and short-term memory deficits in Parkinson’s disease. Brain 1988;111:525–535. Fredriksson A, Plaznik A, Sundtrom E, Jonsson G, Archer T: MPTP-induced hypoactivity in mice: reversal by L-Dopa. Pharmacol Toxicol 1990;67:295–301. Fredriksson A, Archer T: MPTP-induced behavioral and biochemical deficits: a parametric analysis. J Neural Transm 1994;7:123–132. Craig NJ, et al: A candidate gene for human neurodegenerative disorders: a rat PKC gamma mutation causes a parkinsonian syndrome. Nat Neurosci 2001;4:1061–1062. Meunier J: Psychopharmacology. Paris, Masson,1995, pp 1–5. Segev I: Single neurons models: oversimple, complex and reduced. Trends Neurosci 1992;15: 414–421.

Prof. Philippe Poindron 11, rue Galliéni FR-92100 Boulogne (France) Tel. ⫹33 684 01 29 51, Fax ⫹33 684 01 29 51, E-Mail [email protected]

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Author Index

Callizot, N. 1, 66, 81 Cam, E. 52

Kilic, E. 52 Kinter, J. 39

Schaeren-Wiemers, N. 39

Echaniz-Laguna, A. 11

Loeffler, J.-P. 11

Yulug, B. 52

Fergani, A. 11 Fricker, B. 11

Piguet, P. 1 Poindron, P. VII, 1, 66, 81

Zeis, T. 39

Guenet, J.-L. 81

René, F. 11 Ritz, M.-F. 52

97

Subject Index

ALS2 function and mutation in amyotrophic lateral sclerosis 12–14 knockout mouse model of amyotrophic lateral sclerosis 26, 27 Amyotrophic lateral sclerosis (ALS) ALS2 mutation 12–14 animal models ALS2 knockout mouse 26, 27 drug testing in transgenic mouse models 27, 28 intermediate filament transgenic mice knockout mice 22 neurofilament overexpression 22 overview 21, 22 peripherin models 23 limitations of transgenic mouse models 28, 29 microtubule-based transport transgenic mice dynein defects 24, 25 kinesin defects 24 overview 23, 24 pmm mutants 25 Tau defects 25 overview 14, 15 prospects 30 SOD1 transgenic mice cell types in pathogenesis 19–21 copper-mediated catalysis role 15, 17 histology 16 protein misfolding and aggregation 17–19

vascular endothelial growth factor hypoxia response element disruption in transgenic mice 26 wobbler mouse 26 clinical features 11, 12 SOD1 mutation 12 Classification, animal models 5–8 Clinical relevance, animal models 3, 4 Compound muscle action potential (CMAP), frissonnant mutation 84, 87 Dynein, mouse model of amyotrophic lateral sclerosis 24, 25 Ethics, animal models 3 Experimental autoimmune encephalitis (EAE) antigens for induction 40 myelin oligodendrocyte glycoprotein rat model advantages 49 axonal pathology 48, 49 clinical scoring and course 42–45 demyelination 48 immunohistochemistry 43 induction 41, 42 lesions cellular composition 48 characterization 45–47 pathology 40, 41 protein expression and purification 41 overview 39, 40

98

frissonnant mutation behavioral testing muscle strength 83 rotarod test 83, 86 string test 83, 86 compound muscle action potential 84, 87 drug testing 84, 85, 88–90 footprint analysis 83 histopathology 85, 90, 92 locomotor activity 83, 84, 86 object recognition test 85, 87 origins 81 Parkinson’s disease similarities 82, 93–95 sensory nerve conduction velocity 84, 87 statistical analysis 85 tail flick test 83 Growth, pyridoxine intoxication effects 70, 71 Homology, animal models 5, 95 Induced models overview 5, 6 psychiatric disorders 6 Intermediate filament, transgenic mouse models of amyotrophic lateral sclerosis knockout mice 22 neurofilament overexpression 22 overview 21, 22 peripherin models 23 Isomorphism, animal models 4, 5, 95 Kinesin, transgenic mouse models of amyotrophic lateral sclerosis 24 Laser Doppler flowmetry, middle cerebral artery occlusion in rat 55, 57, 58 4-Methylcatechol, pyridoxine-induced peripheral neuropathy testing 77, 78 Middle cerebral artery occlusion (MCAO), rat advantages 63–65 anesthesia 54 animal welfare 64 comparison of techniques 57, 58

Subject Index

histology 56, 57 infarct area 1 day after occlusion 61, 62 intraluminal occluder preparation 54 laser Doppler flowmetry 55, 57, 58 modified Koizumi method 56, 58, 60 modified Longa method 56, 58, 60 mortality 60 overview 53 physiological parameters 58 silicone cap refined model 55, 56, 64 statistical analysis 57 stroke etiology 52, 53 surgery 54, 55 Minocycline, testing in transgenic mouse models of amyotrophic lateral sclerosis 27, 28 Model aims 1, 2 definition 1, 95 history of animal models 2 MPTP, Parkinson’s disease models 92 Multiple sclerosis (MS) animal models, see Experimental autoimmune encephalitis clinical features 39 Myelin oligodendrocyte glycoprotein (MOG), experimental autoimmune encephalitis in rat advantages 49 axonal pathology 48, 49 clinical scoring and course 42–45 demyelination 48 immunohistochemistry 43 induction 41, 42 lesions cellular composition 48 characterization 45–47 pathology 40, 41 protein expression and purification 41 Negative models, overview 7 Orphan models, overview 7, 8 Parkinson’s disease (PD) frissonnant mutation similarities 82, 93–95 MPTP models 92

99

Peripheral neuropathy clinical heterogeneity 66 pyridoxine induction historical perspective and features 67, 68 intoxication schedule modification in rats animals 68 clinical relevance 75–77 drug testing 77, 78 electromyography 69, 71–73 growth effects 70, 71 hot plate test 71 induction 68, 69 morphological analysis 69, 70, 73, 74 motor coordination measurements 69 sensory nerve conduction velocity 69, 73 statistical analysis 70 sensory toxic neuropathy inducers 74–76 Peripherin, transgenic mouse models of amyotrophic lateral sclerosis 23 pmm mutants, transgenic mouse models of amyotrophic lateral sclerosis 25 Pyridoxine-induced peripheral neuropathy historical perspective and features 67, 68 intoxication schedule modification in rats animals 68 clinical relevance 75–77 drug testing 77, 78 electromyography 69, 71–73 growth effects 70, 71 hot plate test 71 induction 68, 69 morphological analysis 69, 70, 73, 74 motor coordination measurements 69

Subject Index

sensory nerve conduction velocity 69, 73 statistical analysis 70 Riluzole, testing in transgenic mouse models of amyotrophic lateral sclerosis 27 Selection, animal models 8 Sensory nerve conduction velocity (SNCV) frissonnant mutation 84, 87 pyridoxine-induced peripheral neuropathy 69, 73 SOD1 mutation in amyotrophic lateral sclerosis 12 transgenic mouse models of amyotrophic lateral sclerosis cell types in pathogenesis 19–21 copper-mediated catalysis role 15, 17 histology 16 protein misfolding and aggregation 17–19 Spontaneous models, overview 7 Stroke, see Middle cerebral artery occlusion, rat Tau, transgenic mouse models of amyotrophic lateral sclerosis 25 Transgenic models, overview 7 Vascular endothelial growth factor (VEGF) hypoxia response element disruption in transgenic mouse model of amyotrophic lateral sclerosis 26 testing in transgenic mouse models of amyotrophic lateral sclerosis 28 wobbler mouse, amyotrophic lateral sclerosis model 26

100