Developmental Neurotoxicology Research E-Book
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- Herausgeber: John Wiley & Sons
- Kategorie: Fachliteratur
- Sprache: Englisch
This book describes how systems biology, pharmacogenomic and behavioral approaches, as applied to neurodevelopmental toxicology, provide a structure to arrange information in a biological model. Authors review and discuss approaches that can be used as effective tools to dissect mechanisms underlying pharmacological and toxicological phenomena associated with the exposure to drugs or environmental toxicants during development. This book presents cross-cutting research tools and animal models, along with applications to the studies associated with potential anesthetic-induced developmental neurotoxicity; the developmental basis of adolescent or adult onset of disease; risk assessment of methyl mercury and its effects on neurodevelopment; challenges in the field to identify environmental factors of relevance to autism; and the strategy and progress of epilepsy research.
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Veröffentlichungsjahr: 2011
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CONTENTS
COVER
HALF TITLE PAGE
TITLE PAGE
COPYRIGHT PAGE
PREFACE
CONTRIBUTORS
SECTION I: MODELS, APPROACHES, AND CHALLENGES IN NEUROTOXICITY RESEARCH DURING DEVELOPMENT
CHAPTER 1: APPROACHES AND MODELS FOR EVALUATING THE TOXIC EFFECTS OF ANESTHETICS IN THE DEVELOPING NERVOUS SYSTEM
1.1 INTRODUCTION
1.2 NEUROTRANSMISSION, SYNAPTOGENESIS, AND ANESTHETIC-INDUCED NEURONAL CELL DEATH
1.3 In vivo AND in vitro ANIMAL MODELS
1.4 PHARMACOGENOMIC/SYSTEM BIOLOGY APPROACHES (SEE CHAPTER 2 IN THIS BOOK)
1.5 MOLECULAR IMAGING APPROACHES IN THE STUDY OF ANESTHETIC-INDUCED NEURONAL CELL DEATH
1.6 PERINATAL ANESTHETIC ADMINISTRATION AND LONG-TERM BEHAVIORAL DEFICITS (SEE CHAPTER 3 IN THIS BOOK)
1.7 CLINICAL CORRELATION OF PRESENT DATA
1.8 POTENTIAL NEUROPROTECTION
1.9 CONCLUSION
REFERENCES
CHAPTER 2: SYSTEMS BIOLOGY APPROACHES TO NEUROTOXICITY STUDIES DURING DEVELOPMENT
2.1 INTRODUCTION
2.2 ANESTHETIC-INDUCED NEURODEGENERATION VIA N-METHYL-D-ASPARTATE (NMDA) RECEPTORS
2.3 A SYSTEMS BIOLOGY APPROACH TO ANESTHETIC-INDUCED NEUROTOXICITY
2.4 PHARMACOKINETICS AND PHYSIOLOGICAL PARAMETERS ASSOCIATED WITH ANESTHETICS DURING BRAIN DEVELOPMENT
2.5 SUMMARY
REFERENCES
CHAPTER 3: BEHAVIORAL APPROACHES FOR ASSESSING NERVOUS SYSTEM FUNCTION DURING DEVELOPMENT IN ANIMAL MODELS
3.1 INTRODUCTION
3.2 ASSESSMENTS IN RODENTS
3.3 ASSESSING NONHUMAN PRIMATES
3.4 OVERVIEW
REFERENCES
CHAPTER 4: APPLICATIONS OF UNBIASED STEREOLOGY TO NEURODEVELOPMENTAL TOXICOLOGY
4.1 INTRODUCTION
4.2 REFERENCE SPACE VS. REGION OF INTEREST
4.3 ACCURACY VS. PRECISION
4.4 STEREOLOGICAL BIAS VS. UNCERTAINTY
4.5 UNBIASED GEOMETRIC PROBES
4.6 THE DISECTOR PRINCIPLE FOR NUMBER (N)
4.7 FROM NV TO TOTAL N
4.8 THE FRACTIONATOR
4.9 ABSOLUTE PARAMETERS VS. DENSITY
4.10 UNBIASED METHOD = SUM OF DIMENSIONS IN PROBE + PARAMETER ≥ 3
4.11 VARIABILITY ANALYSIS
4.12 DO MORE, LESS WELL
4.13 COMPUTERIZED STEREOLOGY APPROACHES
4.14 PEER REVIEW ISSUES
4.15 SUMMARY
REFERENCES
SECTION II: EFFECTS OF ANESTHETICS AND THEIR POTENTIAL NEUROTOXICITY DURING DEVELOPMENT
CHAPTER 5: NEUROTOXIC EFFECTS OF ANESTHETICS AND POTENTIAL PROTECTIVE AGENTS
5.1 INTRODUCTION
5.2 GENERAL ANESTHETICS
5.3 ANESTHETIC-INDUCED NEUROTOXICITY
5.4 PROTECTIVE AGENTS
5.5 CONCLUSION
REFERENCES
CHAPTER 6: PERINATAL RHESUS MONKEY MODELS AND ANESTHETIC-INDUCED NEURONAL CELL DEATH
6.1 INTRODUCTION
6.2 NEURODEGENERATION OBSERVED IN RODENTS EXPOSED TO ANESTHETICS
6.3 In vitro EVIDENCE OF KETAMINE-INDUCED NEURONAL CELL DEATH IN THE DEVELOPING MONKEY
6.4 In vivo EVIDENCE OF KETAMINE-INDUCED NEURONAL CELL DEATH IN THE DEVELOPING MONKEY
6.5 FUTURE DIRECTIONS AND CHALLENGES
6.6 CONCLUSION
REFERENCES
CHAPTER 7: EFFECTS OF GASEOUS ANESTHETIC COMBINATIONS DURING DEVELOPMENT
7.1 INTRODUCTION
7.2 GABA AGONIST AND NMDA ANTAGONIST CLASSES OF INHALATIONAL (GASEOUS) GENERAL ANESTHETICS
7.3 INHALATIONAL ANESTHETICS CAUSE WIDESPREAD DEVELOPMENTAL NEUROAPOPTOSIS
7.4 ANESTHESIA-INDUCED DEVELOPMENTAL NEUROTOXICITY IS NOT CAUSED BY METABOLIC DISTURBANCES AND/OR HYPOXIA OR HYPERCARBIA
7.5 SEVERITY OF ANESTHESIA-INDUCED DEVELOPMENTAL NEUROTOXICITY DEPENDS ON TIMING RATHER THAN DURATION OF SYNAPTOGENESIS AND/OR ANESTHESIA EXPOSURE
7.6 MECHANISMS OF ANESTHESIA-INDUCED NEUROTOXICITY
7.7 CONCLUSIONS
REFERENCES
CHAPTER 8: PERINATAL ANESTHETIC ADMINISTRATION AND SHORT-LONG-TERM BEHAVIORAL DEFICITS
8.1 INTRODUCTION
8.2 SHORT- AND LONG-TERM BEHAVIORAL DEFICITS IN ANIMALS
8.3 SHORT- AND LONG-TERM BEHAVIORAL DEFICITS IN HUMANS
REFERENCES
SECTION III: THE DEVELOPMENTAL BASIS OF ADOLESCENT OR ADULT DISEASE
CHAPTER 9: DEVELOPMENTAL LEAD EXPOSURE, EPIGENETICS AND LATE ONSET ALZHEIMER’S DISEASE
9.1 INTRODUCTION
9.2 EPIGENETICS AND AD
9.3 EXPOSURE TO LEAD (Pb) AND DEVELOPMENTAL BASIS OF AD
9.4 EPIGENETICS, THE ENVIRONMENT AND LOAD
9.5 DNA METHYLATION AND DNA OXIDATION
9.6 SUMMARY AND CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES
CHAPTER 10: DEVELOPMENTAL TRAJECTORIES OF AUTISM AND ENVIRONMENTAL EXPOSURES—WHAT WE KNOW AND WHERE WE NEED TO GO
10.1 INTRODUCTION
10.2 DIAGNOSIS OF AUTISM
10.3 EARLY DEVELOPMENTAL TRAJECTORIES OF AUTISM
10.4 USE OF HIGH RISK POPULATIONS TO STUDY EARLY SIGNS AND TRAJECTORIES
10.5 DEVELOPMENT OF SCREENING TOOLS FOR INFANTS
10.6 BIOMARKERS OF AUTISM
10.7 AUTISM GENETICS
10.8 EPIGENETICS
10.9 POTENTIAL EXPOSURES OF INTEREST
10.10 TERATOGENS: VALPROIC ACID AND THALIDOMIDE
10.11 PSYCHOSOCIAL STRESSORS
10.12 TERBUTALINE
10.13 PESTICIDES
10.14 MERCURY
10.15 CONFRONTING THE UNIVERSE OF EXPOSURES WITH POTENTIAL RELEVANCE TO AUTISM
10.16 THE PATH FORWARD
10.17 CONCLUSION
REFERENCES
Chapter 11: ACTIONS OF MANGANESE ON PUBERTAL DEVELOPMENT
11.1 INTRODUCTION
11.2 BASIC EVENTS LEADING TO FEMALE PUBERTY
11.3 ACTIONS OF MN ON FEMALE PUBERTAL DEVELOPMENT
11.4 ACTIONS OF MN ON MALE PUBERTAL DEVELOPMENT
11.5 GENDER DIFFERENCES TO CHRONIC MN EXPOSURE
11.6 THE HYPOTHALAMIC SITE AND MECHANISM OF MN ACTION
11.7 POTENTIAL RELATIONSHIP BETWEEN ACTIONS OF MN AND PRECOCIOUS PUBERTY
11.8 CONCLUSION
ACKNOWLEDGMENT
REFERENCES
Chapter 12: EXPOSURE OF THE DEVELOPING BRAIN TO POLYCHLORINATED BIPHENYLS INFLUENCES THE SUSCEPTIBILITY OF THE ADULT BRAIN TO STRESS
ACKNOWLEDGMENTS
REFERENCES
CHAPTER 13: A NEURODEVELOPMENTAL ORIGIN FOR PARKINSON’S DISEASE: A LINK TO THE FETAL BASIS FOR ADULT DISEASE HYPOTHESIS
13.1 INTRODUCTION
13.2 DEVELOPMENTAL ANIMAL MODELS OF PD
13.3 DEVELOPMENT AND MAINTENANCE OF THE DOPAMINE SYSTEM
13.4 MODEL EVOLUTION–ROLE OF OTHER RISK FACTORS
13.5 EPIGENETIC CHANGES—A POSSIBLE MECHANISM OF DEVELOPMENTAL NEUROTOXICITY
13.6 SUMMARY
REFERENCES
CHAPTER 14: GENETIC AND ENVIRONMENTAL FACTORS IN ATTENTION-DEFICIT HYPERACTIVITY DISORDER
14.1 INTRODUCTION
14.2 PREVALENCE OF ADHD IN THE UNITED STATES
14.3 CLINICAL CHARACTERISTICS
14.4 NEURODEVELOPMENTAL BASIS OF ADHD
14.5 NEUROANATOMICAL BASIS OF BEHAVIORAL DYSFUNCTION IN ADHD
14.6 NEUROCHEMICAL BASIS OF BEHAVIORAL DYSFUNCTION IN ADHD
14.7 NEUROCHEMICAL ACTION OF DRUGS USED TO TREAT ADHD
14.8 GENETIC FACTORS IN ADHD
14.9 ENVIRONMENTAL RISK FACTORS
14.10 ENVIRONMENTAL TOBACCO SMOKE
14.11 LEAD
14.12 POLYCHLORINATED BIPHENYLS
14.13 ALCOHOL
14.14 PERINATAL HYPOXIA
14.15 HEAD TRAUMA AND LESIONING STUDIES
14.16 GENE–ENVIRONMENT INTERACTIONS
14.17 CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES
SECTION IV: RISK ASSESSMENT OF METHYL MERCURY AND ITS EFFECTS ON NEURODEVELOPMENT
CHAPTER 15: FISH NUTRIENTS AND METHYLMERCURY: A VIEW FROM THE LABORATORY
15.1 INTRODUCTION
15.2 HOW METHYLMERCURY GETS IN FISH
15.3 HUMAN EXPOSURES
15.4 LABORATORY MODELS
15.5 DEVELOPMENTAL EXPOSURES: NONHUMAN PRIMATES
15.6 EXPERIMENTAL STUDIES OF NUTRITION–METHYLMERCURY INTERACTIONS
15.7 CONCLUDING REMARKS
REFERENCES
CHAPTER 16: NEURODEVELOPMENTAL EFFECTS OF MATERNAL NUTRITION STATUS AND EXPOSURE TO METHYL MERCURY FROM EATING FISH DURING PREGNANCY: EVIDENCE FROM THE SEYCHELLES CHILD DEVELOPMENT STUDY
16.1 INTRODUCTION
16.2 METHYL MERCURY IN FISH AS A POTENTIAL RISK TO HUMAN HEALTH
16.3 NUTRIENTS AND CHILD DEVELOPMENT
16.4 THE SEYCHELLES CHILD DEVELOPMENT AND NUTRITION STUDY (SCDNS)
16.5 IMPLICATIONS FOR HEALTH POLICY
16.6 CONCLUSIONS
REFERENCES
CHAPTER 17: METHYLMERCURY NEUROTOXICOLOGY: FROM RARE POISONINGS TO SILENT PANDEMIC
17.1 INTRODUCTION
17.2 HISTORY OF METHYLMERCURY EXPOSURE
17.3 INSIGHT FROM NEUROTOXICITY IN LABORATORY ANIMALS AND WILDLIFE
17.4 CLINICAL APPEARANCE
17.5 MINAMATA DISEASE
17.6 HISTORY REPEATING ITSELF IN NIIGATA
17.7 POISONINGS FROM MERCURY FUNGICIDES
17.8 BUILDING EVIDENCE OF DEVELOPMENTAL NEUROTOXICITY
17.9 EARLY EPIDEMIOLOGICAL STUDIES OF NEURODEVELOPMENT
17.10 MAJOR PROSPECTIVE COHORT STUDIES
17.11 ASSESSMENT OF SAFE INTAKE LIMITS
17.12 RECENT NEUROTOXICITY RISK ASSESSMENTS
17.13 FUTURE PERSPECTIVES
ACKNOWLEDGMENT
REFERENCES
CHAPTER 18: OXIDATIVE STRESS AND METHYLMERCURY-INDUCED NEUROTOXICITY
18.1 INTRODUCTION
18.2 METHYLMERCURY ELECTROPHILICITY
18.3 METHYLMERCURY-INDUCED NEUROTOXICITY AND THE ANTIOXIDANT-GLUTATHIONE SYSTEM
18.4 METHYLMERCURY-INDUCED OXIDATIVE STRESS AND CALCIUM/GLUTAMATE DYSHOMEOSTASIS
18.5 ANTIOXIDANT THERAPY IN METHYLMERCURY POISONING
18.6 CONCLUDING REMARKS
18.7 ACKNOWLEDGMENTS
REFERENCES
CHAPTER 19: LEARNING DEFICITS AND DEPRESSIONLIKE BEHAVIORS ASSOCIATED WITH DEVELOPMENTAL METHYLMERCURY EXPOSURES
19.1 INTRODUCTION
19.2 LEARNING DEFICITS
19.3 DEPRESSIONLIKE BEHAVIOR
19.4 GENDER-RELATED TOXICITY
19.5 MECHANISTIC CONSIDERATIONS
19.6 CONCLUDING REMARKS
ACKNOWLEDGMENTS
REFERENCES
CHAPTER 20: METHYLMERCURY EFFECTS ON NEURAL DEVELOPMENTAL SIGNALING PATHWAYS
20.1 METHYLMERCURY AND THE DEVELOPING NERVOUS SYSTEM
20.2 NEURAL PROGENITOR CELLS AS METHYLMERCURY TARGETS
20.3 DEVELOPMENTAL PATHWAYS AS MeHg TARGETS
20.4 MeHg AND CELL CYCLE PROTEINS
20.5 MeHg AND CYTOKINES
20.6 MeHg AND COMMON TOXICANT PATHWAYS
20.7 CONCLUDING REMARKS
REFERENCES
SECTION V: AUTISM SPECTRUM DISORDERS
CHAPTER 21: NEURODEVELOPMENTAL TOXICOLOGY AND AUTISM SPECTRUM DISORDERS
21.1 INTRODUCTION
21.2 AUTISM
21.3 NEUROANATOMICAL AND NEUROCHEMICAL ALTERATIONS IN AUTISM
21.4 NEUROCHEMICAL ABNORMALITIES
21.5 ENVIRONMENTAL AGENTS AND AUTISM
21.6 ANIMAL MODELS OF AUTISM
21.7 EPIGENETICS OF AUTISM: THE INTERFACE BETWEEN GENETIC AND ENVIRONMENTAL RISK FACTORS
21.8 SUMMARY
ACKNOWLEDGMENTS
REFERENCES
CHAPTER 22: REDOX IMBALANCE AND THE METABOLIC PATHOLOGY OF AUTISM
22.1 INTRODUCTION
22.2 REGULATION OF CELLULAR REDOX STATUS
22.3 REDOX REGULATION IN THE HUMAN BRAIN
22.4 REDOX AND STEM CELL DEVELOPMENT
22.5 METHYLATION AND EPIGENETICS
22.6 REGULATION OF METHIONINE SYNTHASE
22.7 D4 DOPAMINE RECEPTOR-MEDIATED PHOSPHOLIPID METHYLATION
22.8 REDOX AND METHYLATION IN AUTISM
22.9 MITOCHONDRIAL DYSFUNCTION
22.10 EFFECTS OF HEAVY METALS ON REDOX
22.11 CONCLUDING PERSPECTIVE
REFERENCES
CHAPTER 23: NEUROINFLAMMATION AND AUTISM
23.1 INTRODUCTION
23.2 IMMUNOLOGICAL FACTORS IN AUTISM
23.3 NEUROPATHOLOGY OF AUTISM
23.4 MICROGLIA AS IMMUNE CELLS OF THE BRAIN
23.5 DEVELOPMENTAL ASPECTS OF MICROGLIA
23.6 NEUROINFLAMMATION AND ASD
23.7 CONCLUSION
ACKNOWLEDGMENTS
REFERENCES
CHAPTER 24: AUTISM, PERIPHERAL IMMUNITY, AND POLYBROMINATED DIPHENYL ETHERS
24.1 INTRODUCTION
24.2 IMMUNOLOGY BASICS
24.3 IMMUNITY, BEHAVIOR, AND THE NERVOUS SYSTEM
24.4 AUTISM SPECTRUM DISORDERS: NEURAL AND IMMUNE DYSFUNCTION
24.5 POLYBROMINATED DIPHENYL ETHERS (PBDEs)
24.6 CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES
CHAPTER 25: AN EMERGING GENE–ENVIRONMENT INTERACTION MODEL: AUTISM SPECTRUM DISORDER PHENOTYPES RESULTING FROM EXPOSURE TO ENVIRONMENTAL CONTAMINANTS DURING GESTATION
25.1 INTRODUCTION
25.2 BACKGROUND
25.3 DEVELOPMENT OF A SUSCEPTIBILITY–EXPOSURE PARADIGM TO ACCESS PRENATAL POLYCYCLIC AROMATIC HYDROCARBON EXPOSURE-INDUCED PERTURBATIONS IN NEOCORTICAL DEVELOPMENT
25.4 IDENTIFICATION OF A GENETIC VARIANT OF MET THAT DIFFERENTIALLY BINDS SP1 TRANSCRIPTION FACTOR COMPLEXES IS SHOWN TO BE ASSOCIATED WITH ASD
25.5 TEMPORAL PATTERNS OF MET EXPRESSION DURING FOREBRAIN DEVELOPMENT OVERLAP THE PERIOD OF B(A)P-INDUCED MODULATION OF SP1–DNA BINDING
25.6 SUMMARY AND IMPLICATIONS FOR FUTURE STUDIES
REFERENCES
SECTION VI: STRATEGIES AND PROGRESS IN EPILEPSY RESEARCH
CHAPTER 26: NEONATAL SEIZURES
26.1 EPIDEMIOLOGY AND DIFFERENTIAL DIAGNOSIS
26.2 DIAGNOSIS OF NEONATAL SEIZURES
26.3 CURRENT THERAPEUTIC STRATEGIES FOR NEONATAL SEIZURES
26.4 FUTURE TREATMENT DIRECTIONS
26.5 CONCLUSIONS
REFERENCES
CHAPTER 27: EXPERIMENTAL MODELS OF EPILEPTOGENESIS
27.1 INTRODUCTION
27.2 MODELING ACQUIRED EPILEPSY IN IMMATURE RODENTS
27.3 STATUS EPILEPTICUS
27.4 PROLONGED HYPERTHERMIC SEIZURES
27.5 HYPOXIC–ISCHEMIC BRAIN DAMAGE (MODELS OF STROKE)
27.6 TRAUMATIC BRAIN INJURY
27.7 METHODOLOGICAL CONSIDERATIONS
27.8 BRAIN DEVELOPMENT IN RODENTS
27.9 FUTURE DEVELOPMENT
REFERENCES
CHAPTER 28: EFFECT OF SEIZURES ON THE DEVELOPING BRAIN: LESSONS FROM THE LABORATORY
28.1 INTRODUCTION
28.2 SEIZURE-INDUCED INJURY AND EPILEPTOGENESIS: AGE EQUIVALENCE BETWEEN SPECIES
28.3 KAINIC ACID–INDUCED STATUS EPILEPTICUS
28.4 CORTICOTROPIN-RELEASING HORMONE-INDUCED SEIZURES
28.5 PERFORANT PATH STIMULATION–INDUCED STATUS EPILEPTICUS
28.6 PILOCARPINE AND LITHIUM–PILOCARPINE-INDUCED STATUS EPILEPTICUS
28.7 INFLAMMATION AMPLIFIES SEIZURE-INDUCED INJURY
28.8 COGNITIVE AND BEHAVIORAL EFFECTS OF SEIZURES IN THE DEVELOPING BRAIN
28.9 CONCLUSION
REFERENCES
PLATES
INDEX
DEVELOPMENTAL NEUROTOXICOLOGY RESEARCH
Copyright © 2011 by John Wiley & Sons, Inc. All rights reserved.
Published by John Wiley & Sons, Inc., Hoboken, New Jersey.Published simultaneously in Canada.
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Library of Congress Cataloging-in-Publication Data:
Wang, Cheng, 1954–Developmental neurotoxicology research : principles, models, techniques, strategies, and mechanisms / edited by Cheng Wang, William Slikker Jr.p.cm.Includes bibliographical references and index.ISBN 978-0-470-42672-2 (cloth)1. Neurotoxicology. 2. Developmental toxicology. I. Slikker, William. II. Title.[DNLM: 1. Nervous System–drug effects. 2. Nervous System–growth &development. 3. Neurotoxicity Syndromes–etiology. WL 102 W2457d 2011]RC347.5.W36 2011616.8′0471–dc222010021928
PREFACE
In preclinical and clinical studies, early life stress has been shown to cause neuroanatomical and biological alterations and homeostasis unbalance. These alterations lead to disruptions in regulatory systems and a heightened risk for pathology. Our goal in writing this book is to highlight ways in which preclinical research approaches can help inform clinical interventions and vice versa.
Because of the complexity and temporal features of the manifestation of function and structure in the developing brain, the developing nervous system may be more susceptible to neurotoxic insults. The study of neurodevelopmental toxicology has great potential for helping to advance the understanding of brain-related biological processes, including neuronal plasticity, neurodegeneration/regeneration, toxicity, and effectiveness of many products. In this book, we delineate systems biology and pharmacogenomic and behavioral approaches as applied to neurodevelopmental toxicology to provide a structure for arranging information in a biological model. Approaches that can be used as effective tools to dissect mechanisms underlying pharmacological and toxicological phenomena associated with the exposure to drugs or environmental toxicants during development are reviewed and discussed. This book presents cross-cutting research tools and animal models, along with applications to studies associated with potential anesthetic-induced developmental neurotoxicity, the developmental basis of adolescent or adult onset of disease, risk assessment of methyl mercury and its effects on neurodevelopment, challenges in the field to identify environmental factors of relevance to autism, and the strategy and progress of epilepsy research.
In a sense, the book is also revolutionary; it attempts to incorporate new, postgenomic techniques while addressing all levels of developmental neurotoxicology research from genetic and systems biology/pharmacogenomic characterization, as well as presenting biochemical applications to behavioral effects in animal test paradigms. These paradigms ranged from in vitro to in vivo examples, from rodent to nonhuman primate models, and finally to clinical applications. Also, because we believe that students should appreciate that science is an evolutionary process in which the great discoveries of today are built upon the brilliant and groundbreaking work of our scientific forebears, many topics are introduced with a historical account of early research in that area.
The book is organized into six main parts. Section I describes models, approaches, and challenges in neurotoxicity research during development. Section II covers potential anesthetic-induced developmental neurotoxicity. Section III discusses the developmental basis of adolescent or adult onset of disease. Section IV delineates the effects of methylmercury on neurodevelopment and the development of a safety assessment paradigm. Section V discusses challenges in the field to identify environmental factors of relevance for autism. Finally, Section VI describes the strategy and progress of epilepsy research.
Achieving the goals set forth for this book was most challenging. We thank the contributing authors who have conveyed the excitement of the new findings they have described and for identifying the remaining knowledge gaps concerning neurobehavioral toxicology. We would appreciate any comments you wish to offer about Developmental Neurotoxicology Research: Principles, Models, Techniques, Strategies, and Mechanisms.
CHENG WANG, M.D., PH.D.WILLIAM SLIKKER, JR., PH.D.
CONTRIBUTORS
MICHAEL ASCHNER Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA
PAUL ASHWOOD Division of Rheumatology, Allergy and Clinical Immunology, Department of Medical Microbiology and Immunology, Center for Children’s Environmental Health, University of California Davis, Davis, CA, USA
ANGELA BAKER Department of Environmental and Occupational Medicine, Robert Wood Johnson Medical School and Division of Toxicology Environmental and Occupational Health Sciences Institute, Piscataway, NJ, USA
RIYAZ BASHA Cancer Research Institute, M. D. Anderson Cancer Center Orlando, Orlando, FL, USA
ROBERT F. BERMAN Department of Neurological Surgery, Center for Children’s Environmental Health and the UC Davis M.I.N.D. Institute, School of Medicine, University of California, Davis, CA, USA
MAXINE P. BONHAM Department of Nutrition and Dietetics, Faculty of Medicine, Nursing and Health Sciences, Monash University, Melbourne, Australia
DANIEL CAMPBELL Department of Psychiatry and the Behavioral Sciences, Zilkha Neurogenetic Institute Center for Genomic Psychiatry, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
SANDRA CECCATELLI Department of Neuroscience, Karolinska Institutet, 171 77 Stockholm, Sweden
ANNA L. CHOI Department of Environmental Health, Harvard School of Public Health, Landmark Center 3E, 401 Park Drive, Boston, MA, USA
THOMAS W. CLARKSON Department of Environmental Medicine, School of Medicine and Dentistry, University of Rochester, Rochester, NY, USA
PHILIP W. DAVIDSON Department of Pediatrics, School of Medicine and Dentistry, University of Rochester, Rochester, NY, USA
W. LES DEES Department of Integrative Biosciences, College of Veterinary Medicine, Texas A&M, College Station, TX, USA
RICHARD C. DETH Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, USA
EMEIR M. DUFFY Northern Ireland Centre for Food & Health, University of Ulster, Coleraine, BT 52 1SA, Northern Ireland
MARCELO FARINA Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil
PAULA GOINES Division of Rheumatology, Allergy and Clinical Immunology, Department of Medical Microbiology and Immunology, Center for Children’s Environmental Health, University of California Davis, Davis, CA, USA
PHILIPPE GRANDJEAN Department of Environmental Medicine, University of Southern Denmark, J.B.Winslowsvej 17, 5000 Odense, Denmark, Department of Environmental Health, Harvard School of Public Health, Landmark Center 3E, 401 Park Drive, Boston, MA, USA
ALYCIA HALLADAY Director of Research for Environmental Sciences, Autism Speaks, 2 Park Avenue, New York, NY, USA
G. JEAN HARRY Neurotoxicology Group, Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA
JERROLD J. HEINDEL National Institute of Environmental Health Sciences, NIH/DHHS, Research Triangle Park, NC, USA
JILL K. HINEY Department of Integrative Biosciences, College of Veterinary Medicine, Texas A&M, College Station, TX, USA
NATHANIEL HODGSON Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, USA
DARRYL B. HOOD Department of Neuroscience and Pharmacology, Institute for Environmental-Health Disparities and Medicine, Meharry Medical College, Nashville, TN, USA
MICHAEL R. HUNSAKER Department of Neurological Surgery and Neuroscience Program, School of Medicine, University of California, Davis, CA, USA
SHAUN HUSSAIN Division of Pediatric Neurology, Room 22-474 MDCC; and Division of Pediatric Neurology; 22-474 MDCC, David Geffen School of Medicine and Mattel Children’s Hospital, Los Angeles, CA, USA
VESNA JEVTOVIC-TODOROVIC Department of Anesthesiology, University of Virginia Health System, Charlottesville, VA, USA
KYUNG HO KIM Department of Molecular Biosciences, UC Davis School of Veterinary Medicine, Davis, CA, USA
CLAIRE M. KOENIG Department of Neurological Surgery and Center for Children’s Environmental Health, University of California, Davis, CA, USA
HANA KUBOVÀ Institute of Physiology, Department of Developmental Epileptology, Academy of Sciences of the Czech Republic, Vídeská 1083, Prague 4, Czech Republic
JANINE M. LASALLE Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, CA, USA
CINDY P. LAWLER Cellular, Organ and Systems Pathobiology Branch Division of Extramural Research and Training National Institute of Environmental Health Sciences Keystone Building, Room 3022, 530 Davis Drive, Research Triangle Park, NC, USA
PAMELA J. LEIN Department of Molecular Biosciences, UC Davis School of Veterinary Medicine, Davis, CA, USA
PAT LEVITT Department of Cell and Neurobiology, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles CA, USA
FANG LIU National Center for Toxicological Research, U.S. Food & Drug Administration, Jefferson, AR, USA
PETER R. MOUTON University of South Florida School of Medicine, Tampa, FL, USA
KATSUYUKI MURATA Department of Environmental Health Sciences, Akita University School of Medicine, Akita, Japan
CHRISTINA MURATORE Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, USA
GARY J. MYERS Department of Neurology, School of Medicine and Dentistry, University of Rochester, Rochester, NY, USA
M. CHRISTOPHER NEWLAND Department of Psychology, Auburn University, Auburn, AL, USA
NATALIA ONISHCHENKO Department of Neuroscience, Karolinska Institutet, 171 77 Stockholm, Sweden
TUCKER A. PATTERSON National Center for Toxicological Research, U.S. Food & Drug Administration, Jefferson, AR, USA
MERLE G. PAULE National Center for Toxicological Research, U.S. Food & Drug Administration, Jefferson, AR, USA
ISAAC N. PESSAH Department of Molecular Biosciences and Center for Children’s Environmental Health, School of Veterinary Medicine, University of California, Davis, CA, USA
MATTHEW D. RAND Department of Anatomy and Neurobiology, College of Medicine, University of Vermont, Burlington, VA, USA
JASON R. RICHARDSON Department of Environmental and Occupational Medicine, Robert Wood Johnson Medical School and Division of Toxicology Environmental and Occupational Health Sciences Institute, Piscataway, NJ, USA
JOÃAO BATISTA TEIXEIRA ROCHA Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
RAMAN SANKAR Division of Pediatric Neurology, Room 22-474 MDCC; and Division of Pediatric Neurology; 22-474 MDCC, David Geffen School of Medicine and Mattel Children’s Hospital, Los Angeles, CA, USA
BRADLEY J. SCHNACKENBERG National Center for Toxicological Research, U.S. Food & Drug Administration, Jefferson, AR, USA
CONRAD F. SHAMLAYE Ministry of Health, Government of Seychelles and Department of Environmental Medicine, School of Medicine and Dentistry, University of Rochester, Rochester, NY, USA
RENÉE A. SHELLHAAS Division of Pediatric Neurology, Department of Pediatrics & Communicable Diseases, University of Michigan Medical School, Ann Arbor, MI, USA
WILLIAM SLIKKER JR. National Center for Toxicological Research, U.S. Food & Drug Administration, Jefferson, AR, USA
VINOD K. SRIVASTAVA Department of Integrative Biosciences, College of Veterinary Medicine, Texas A&M, College Station, TX, USA
J.J. STRAIN Northern Ireland Centre for Food & Health, University of Ulster, Coleraine, BT 52 1SA, Northern Ireland
MICHELE M. TAYLOR Department of Environmental and Occupational Medicine, Robert Wood Johnson Medical School and Division of Toxicology Environmental and Occupational Health Sciences Institute, Piscataway, NJ, USA
M. THIRUCHELVAM Environmental and Occupational Health Sciences Institute, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ, USA
SALLY THURSTON Department of Biostatistics and Computational Biology, School of Medicine and Dentistry, University of Rochester, NY, USA
D. URBACH-ROSS Environmental and Occupational Health Sciences Institute, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ, USA
JULIE M.W. WALLACE Northern Ireland Centre for Food & Health, University of Ulster, Coleraine, BT 52 1SA, Northern Ireland
MOSTAFA WALY Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, USA
CHENG WANG National Center for Toxicological Research, U.S. Food & Drug Administration, Jefferson, AR, USA
JUDY VAN DE WATER Division of Rheumatology, Allergy and Clinical Immunology, Department of Medical Microbiology and Immunology, Center for Children’s Environmental Health, University of California Davis, Davis, CA, USA
GENE WATSON Eastman Department of Dentistry and Center for Oral Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY, USA
PAL WEIHE Department of Environmental Medicine, University of Southern Denmark, J.B.Winslowsvej 17, 5000 Odense, Denmark, Department of Occupational and Public Health Faroese Hospital System, Sigmundargòta 5, PO Box 14, Tórshavn, Faroe Islands
NASSER H. ZAWIA Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI, USA
XUAN ZHANG National Center for Toxicological Research, U.S. Food & Drug Administration, Jefferson, AR, USA
XIAOJU ZOU National Center for Toxicological Research, U.S. Food & Drug Administration, Jefferson, AR, USA
SECTION I
MODELS, APPROACHES, AND CHALLENGES IN NEUROTOXICITY RESEARCH DURING DEVELOPMENT
TUCKER A. PATTERSON PH.D.
National Center for Toxicological Research, U.S. Food & Drug Administration, Jefferson, AR, USA
This section addresses models and approaches that are used in neurotoxicity research during development. Some of the current challenges that are encountered when performing developmental neurotoxicology studies are also discussed. The section begins with Chapter 1 describing approaches and models used to evaluate potential anesthetic-induced neurotoxicity on the developing nervous system. In Chapter 2, this area of anesthetic-induced neurotoxicity during development is further examined using a systems biology approach. This is followed by Chapter 3, which describes various behavioral approaches that are used to assess nervous system function during development. Chapter 4 uses examples of environmental toxicant-induced neurotoxicology to examine the practices of design-based stereology.
Chapter 1 (Slikker et al.) highlights ways in which preclinical research can help inform clinical interventions and vice versa and presents cross-cutting research tools and animal models, along with applications to studies associated with potential anesthetic-induced developmental neurotoxicity. Although comprehensive gene expression/ proteomic studies and long-term behavioral assessments remain incomplete, in vivo and in vitro models and analytical strategies have been developed to help identify the biological pathways and behavioral outcomes of anesthetic-induced cell death in the developing nonhuman primate and rodent.
The application of a systems biology approach has great potential for helping advance the understanding of brain-related biological processes, including neuronal plasticity and neurotoxicity. This approach may also allow for monitoring efficacy of treatment regimens. In addition, by using in vivo and in vitro rodent models, this approach may enhance our understanding of complex biological processes such as neuronal cell death (apoptosis and/or necrosis) induced by anesthetics in the developing brain. Understanding these complex biological processes will clarify pathways that will hopefully allow us to predict anesthetic-induced brain cell death and help discover treatments to ameliorate the consequences (if any) of anesthetic toxicity in pediatric patients.
In Chapter 2 (Patterson et al.), the value of a systems biology approach to enhance the understanding of complex biological processes such as neurodegeneration in the developing brain after potential neurotoxic insults is discussed. The goal of systems biology is to predict the functional outcomes of component-to-component relationships using computational models that allow for the directional and quantitative description of the complete organism in response to environmental perturbations. A systems biology approach can also be used to clarify the mechanisms involved in the toxicological phenomena associated with exposure to toxicants. Chapter 2 addresses the development of predictive models that integrate responses from different organizational levels.
The degree to which the nervous system is resistant to neurotoxic insults is highly dependent upon the stage of development. Due to the complexity and temporal features of developmental neurotoxicity, this area of toxicology would greatly benefit from a systems biology approach. In Chapter 2, the systems biology approach is applied to representative general anesthetics, such as ketamine, in order to delineate how specific receptor subunit and intracellular signaling events are involved in potential anesthetic-induced neurotoxicity. Biochemical and molecular mechanisms are explored along with gene expression profiles that underlie potential anesthetic-induced neurotoxicity during sensitive developmental stages.
Chapter 3 (Paule) discusses several behavioral approaches (both nonoperant and operant) for assessing nervous system function during development in both rodent and nonhuman primate animal models. Multiple behavioral paradigms that are used for preweaning versus postweaning assessments in rodents are presented, as well as operant procedures. In addition, using an operant test battery to assess behavior in a nonhuman primate model is discussed.
The ability to assess nervous system function, especially during development or after developmental exposures or insults, is incredibly valuable not only because it provides opportunities to learn about the biological substrates that subserve critical brain function, but also because it provides researchers with invaluable metrics of nervous system integrity. These metrics can then serve as biomarkers of health and act as sensitive indicators of the effects of chemicals that affect the nervous system. The results produced from these assessments demonstrate that animal models can serve as valuable surrogates for the study of human brain function and dysfunction. By examining the effects of developmental exposure to potentially neurotoxic agents on nervous system function in rodents and nonhuman primates, the ability to predict the adverse effects of these agents on related brain functions in humans is greatly enhanced.
Changes in the morphological structure of the developing brain support normal neurological function. Environmental toxins that perturb normal brain development may cause acute and chronic disturbances in neurological function and lead to greater susceptibility to later neurological damage. Unbiased or design-based stereology provides the state-of-the-art methodology to generate accurate, precise, and efficient quantification of temporal and spatial changes in brain structure, including neuronal plasticity, neurodegeneration, regeneration, atrophy/hypertrophy, and other manifestations of neurotoxicity. Chapter 4 (Mouton) reviews the principles and practices of design-based stereology, with an emphasis on assessment of changes caused by environmental toxicants. Specific examples are included that outline design-based approaches to evaluate the effects of neurotoxins on total numbers of neurons and synapses at the light and electron microscopy levels.
These four chapters use very different approaches to address a single issue: the study of developmental neurotoxicity. The authors also describe many of the challenges researchers face when delving into the brain during various stages of development and attempting to study the plethora of potential mechanisms involved in neurotoxicity during development.
CHAPTER 1
APPROACHES AND MODELS FOR EVALUATING THE TOXIC EFFECTS OF ANESTHETICS IN THE DEVELOPING NERVOUS SYSTEM
WILLIAM SLIKKER, JR., XUAN ZHANG, FANG LIU, MERLE G. PAULE, and CHENG WANG
National Center for Toxicological Research, U.S. Food & Drug Administration, Jefferson, AR, USA
1.1 INTRODUCTION
Early-life stress has been shown to cause neuroanatomical and biological alterations and to disturb homeostasis in preclinical and clinical studies. These alterations, in turn, lead to disruptions in regulatory systems and to a heightened risk for pathology. This review highlights ways in which preclinical research can help inform clinical interventions and vice versa and will present crosscutting research tools and animal models along with applications to studies associated with potential anesthetic-induced developmental neurotoxicity.
Various anesthetic protocols have been used in pediatric medicine for many decades without systematic assessments of possible adverse effects. It is known that most of the currently used general anesthetics have either N-methyl-D-aspartate (NMDA) receptor blocking or gamma amino butyric acid (GABA) receptor–enhancing properties. These receptors mediate their actions by the activation of ionotropic (ligand-gated ion channels) and metabotropic (G protein-coupled) receptors and act to influence early neuronal developmental events including synapse formation, neuroplasticity, and survival.
The amino acid L-glutamate is generally recognized as the major excitatory neurotransmitter of the mammalian central nervous system (CNS) and glutamate receptors play a major role in fast excitatory synaptic transmission. NMDA-type glutamate receptors are widely distributed throughout the CNS and operate ligand-activated ion channels that are primarily composed of three families of NMDA receptor subunits: NR1 with eight known splice variants, NR2 (A–D) [1-3], and NR3A and B [4, 5]. The NR1 subunit is essential for receptor/channel function. Functional properties of the NMDA receptor vary throughout the CNS; the binding affinities of various ligands for recombinant NMDA receptors depend on subunit composition [6]. NMDA receptors are involved in a variety of physiological and pathological processes, including memory and learning [7], neuronal development [8], epileptiform seizures, synaptic plasticity, and acute neuropathologies associated with stroke and traumatic injury [9]. During the brain growth spurt, blockade of the NMDA receptor for a period of hours triggers widespread apoptotic neurodegeneration in the rodent brain [10].
GABA, the principal inhibitory neurotransmitter in the adult CNS, acts as an excitatory transmitter in the early postnatal stages [11]. Functional GABAA receptors are expressed in neurons early in development (embryonic stages), and investigations by several research teams have led to the conclusion that a transient excitatory action of GABA via GABAA receptors represents a general feature of developing neurons. Activation of GABAA receptors depolarizes neuroblasts and immature neurons in all regions of the CNS examined to date, including spinal cord [12-14], hypothalamus [15], cerebellum [16], cortex [17], hippocampus [18, 1], and olfactory bulb [13]. This depolarization is not due to unusual properties of neonatal GABAA channels but to an elevated intracellular Cl− concentration, probably from developmental changes in [Cl−]i homeostatic systems [13, 20, 21]. Postsynaptic GABAB receptor-mediated responses, that is, the activation of K+ and inhibition of Ca2+ currents, are absent from the embryonic and neonatal rat hippocampus and neocortical neurons until the end of the first postnatal week of life [22, 23]. The reasons for this delayed maturation of postsynaptic GABAB receptor-mediated inhibition are not yet well understood. It may be due to a lack of coupling between receptors, G proteins, and K+ or Ca2+ channels [22], rather than to the late development of receptors [23].
It has been hypothesized that exposure of the developing brain to NMDA antagonists induces neuronal cell death, most likely through compensatory mechanisms. An important working hypothesis is that exposure of developing brains to individual anesthetics (such as ketamine), with continuous blockade of NMDA receptors, causes a compensatory up-regulation of these receptors. This up-regulation makes neurons bearing these receptors more vulnerable, after removal (washout) of the offending compound, to the excitotoxic effects of glutamate because these up-regulated NMDA receptors allow for the influx of toxic levels of intracellular free calcium under normal physiological conditions. In addition, prolonged supraphysiologic stimulation of immature neurons by GABA agonists enhances overall neuronal excitation and may contribute to increased excitability during early development [24]. This increased excitability, along with NMDA antagonist-induced alteration of NMDA receptors, could contribute to abnormal neuronal cell death.
Modifications of synaptic efficacy are believed to play an important role in information processing and storage by neuronal networks. It has been suggested that synaptic abnormalities are important components of anesthetic-induced neurotoxicity. Synaptophysin is a synaptic vesicle-associated protein that is involved in synaptogenesis. The sialic acid polymer on neural cell adhesion molecules (PSA-NCAM) is an important regulator of cell surface interactions [25]. PSA-NCAM is also a neuron-specific marker known to be an NMDA-regulated molecule important in synaptogenesis during development [26]. Some experiments [27, 28] have been performed to determine the correlation between anesthetics and PSA-NCAM expression because quantifying the levels of PSA-NCAM following anesthetic exposure serves to validate the activity states of neuronal synaptic plasticity.
Neuronal susceptibility to neurotoxic insult varies with the stage of development. Both in vitro and in vivo approaches have been used to assess the neurotoxicity associated with a wide range of anesthetic drugs at a variety of doses and exposure durations. Although comprehensive gene expression/proteomic studies and long-term behavioral assessments remain to be completed, in vivo and in vitro models and analytical strategies have been developed to help identify the biological pathways and behavioral outcomes of anesthetic-induced cell death in the developing nonhuman primate and rodent.
1.2 NEUROTRANSMISSION, SYNAPTOGENESIS, AND ANESTHETIC-INDUCED NEURONAL CELL DEATH
Glutamate promotes certain aspects of neuronal development including migration, differentiation, and plasticity [29]. The NMDA-type glutamate receptor NR1 subunit is widely distributed throughout the brain and is the fundamental subunit necessary for NMDA channel function. NMDA receptor density has been shown to increase in cultured cortical neurons after exposure to the NMDA receptor antagonists D-AP5, CGS-19755, and MK-801 but not after exposure to the AMPA/kainate receptor antagonist CNQX [30]. Overactivation of NMDA receptors is known to kill neurons via a necrotic mechanism characterized by excessive sodium and calcium entry accompanied by chloride and water entry that leads to cell swelling and death [31]. More recently, it has been shown that NMDA receptor activation can also lead to apoptotic cell death [32-34].
Of particular interest are the possible mechanisms by which NMDA antagonists such as ketamine enhance neuronal cell death as a result of ketamine-induced compensatory up-regulation of NMDA receptors. This is postulated to occur because of continuous blockade of the NMDA receptor in the developing brain. This up-regulation then makes neurons bearing these receptors more vulnerable to the excitotoxic effects of endogenous glutamate after ketamine washout. This compensatory hypothesis is supported by the following observations: (1) NR1 subunit mRNA (Fig. 1.1; in situ hybridization) is up-regulated in ketamine-treated monkey fetuses (gestation day 122) and infants [postnatal day (PND) 5] [35]; (2) there is increased expression of NMDA receptor NR1 protein accompanied by enhanced cell death [27]; and (3) coadministration of NR1 antisense oligonucleotide (targeted to NR1 NMDA receptor subunit mRNA) is able to block neuronal cell death induced by ketamine in rat and monkey cortical cultures [26, 27]. Given the key role of the NR1 subunit, it is not surprising that up-regulated NR1 expression along with alterations in other NMDA receptor subunits (such as those in the NR2 family) and the composition of receptor subunits play an important role in determining the pharmacological properties of the receptor. In addition, it has been reported [28] that even low concentrations of ketamine can interfere with dendritic arbor development in immature GABAergic neurons and could potentially interfere with the development of neural networks. In a prepulse inhibition (PPI) behavior assay, administering another NMDA receptor antagonist, MK-801, to neonatal rats (PND 6, 8, and 10) increases prepulse-induced delays in startle response times in adult rats (PND 56) [36]. Additionally, a study by Turner et al. [37] demonstrated that GAD 67 (a GABAergic marker) expression is highly regulated in a variety of brain regions during the postnatal period and that the molecular environment in the PND 7 brain is significantly different from that found on PND 21. Further studies are needed to determine the role of the GABA system in neuronal apoptosis induced by anesthetics such as ketamine.
FIGURE 1.1 NMDA receptor NR1 subunit mRNA abundance in the frontal cortex of PND 5 monkeys. The autoradiograph grain density (labeling) for NR1 subunit mRNA is up-regulated in 24-h ketamine-infused monkeys (B) compared with controls (A). For treated monkeys, ketamine was given as an initial intramuscular injection (20 mg/kg), followed by continuous intravenous infusion at a rate of 20–50 mg/kg/h to maintain a light surgical plane of anesthesia (as evidenced by lack of voluntary movement, decreased muscle tone, and minimal reaction to physical stimulation with maintenance of an intact palpebral reflex) for 24 h. Quantitative analysis (relative labeling density) of the effects of ketamine infusion on the in situ hybridization signal of NMDAR1 subunit mRNA expression in layer II of the frontal cortex of PND 5 monkeys is also shown (C). A comparison between 24-h ketamine infusion and control indicates a significant increase (*P < 0.05) for NR1 mRNA in situ hybridization signals in ketamine-treated monkeys; however, no significant effect was observed between the 3-h ketamine-treated and control monkeys. Scale bar = 60 μm. See color insert.
On the other hand, studies in vivo on the protective effects of NMDA antagonists, such as ketamine, have given inconsistent results. Both no (or minimal) and substantial protective effects have been found against the lesions produced in vivo by NMDA agonists [38-41] and by neuronal ischemia [42-44]. Ketamine has a very short half-life in the brain [45, 46] and, hence, some of the inconsistencies could be due to the dose used and the length of time for which neuroprotective concentrations were maintained.
Prolonged or repetitive pain may occur during critical periods of brain development in hospitalized neonates [47]. Rapid brain growth, synaptogenesis, expression of excitatory receptors [48], and developmentally regulated neuronal cell death [49] also occur at this time, which may explain why repetitive neonatal pain persistently alters subsequent pain processing in rats, mice, and humans [50-54]. To date, very few animal experiments (rodents or nonhuman primates) have studied the effects of surgical or other noxious stimuli during exposure to anesthetics. It is important, therefore, to study the mechanisms by which repetitive pain alters development in the neonatal brain through factors altering cell survival, neuronal activity, and plasticity and the relationship between pain and the analgesic and anti-inflammatory effects of anesthetics.
Previous studies have shown in [55] that peak vulnerability to the apoptogenic action of anesthetic agents is during a period of rapid synaptogenesis, also known as the brain growth spurt. The brain grows at an accelerated rate because newly differentiated neurons throughout the brain are rapidly expanding their dendritic arbors to provide the required surface area to accommodate new synaptic connections during this period. It is believed that the neural cell adhesion molecule is an important regulator of developmental and functional neuroplasticity. In particular, embryonic PSA-NCAM plays a vital role in forming connections between neurons [56]. Synaptophysin is a synaptic vesicle-associated protein that is also involved in synaptogenesis. Interestingly, our data show that PSA-NCAM (Fig. 1.2A) is partially colocalized with synaptophysin (Fig. 1.2B) in neuronal cell membranes in organotypic slice cultures (control) during development (Fig. 1.2). PSA-NCAM appears to be associated with the processes controlling the trafficking and targeting of vesicular proteins to the synapse.
FIGURE 1.2 Double immunostaining micrographs showing polysialic acid neural cell adhesion molecule (PSA-NCAM; A) and synaptophysin (B) neuronal surface staining in an organotypic culture. Note that PSA-NCAM and synaptophysin are partially colocalized.
The sialylation state of PSA-NCAM is controlled by developmentally regulated Golgi sialyltransferase activity [57]. This transferase activity is Ca2+ dependent [58] and this may account for its regulation by NMDA receptors [56, 59]. The regulation of PSA-NCAM expression by NMDAergic activity plays a critical role in neuroplasticity during development, particularly in NCAM-mediated cell–cell interactions and synapse formation [60]. In our previous study [27], treatment of frontal cortical cultures from the developing monkey with ketamine caused a substantial decrease in mitochondrial metabolism of MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], along with a concomitant decrease in PSA-NCAM protein expression (Fig. 1.3). The decrease in PSA-NCAM corresponded to an approximately 40% decrease in PSA-NCAM immunoreactivity. This decrease could be the direct result of local NMDA receptor blockade (subsequent reduction in Ca2+-regulated polysialyl transferase activity) or the indirect result of neuronal loss [27, 58]. The fact that SN-50 (a peptide inhibitor of NF-kB transport) dose dependently blocked ketamine-induced cortical neuronal cell death, as well as the loss of PSA-NCAM immunoreactivity in culture, argues for the latter mechanism. Future experiments using N-butanoyl-mannosamine to inhibit polysialyl transferase or endoneuraminidase N to cleave PSA chains selectively may be able to address this hypothesis specifically.
FIGURE 1.3 Effect of ketamine and SN-50 on the decrease in PSA-NCAM expression in monkey frontal cortical cultures. PSA-NCAM immunoreactivity was intense in the control culture (A) and diminished in the ketamine-treated culture (B). Scale bar = 50 μm. Densitometry measurements were used to calculate a ratio of PSA-NCAM to actin in each lane for each of three independent experiments and the data are shown as the means ± S.D. of those ratios (C). SN-50 (2.5 μM) effectively prevented the reduction of PSA-NCAM induced by ketamine. No protective effect was observed from the inactive control peptide for SN-50 (2.5 μM). See color insert.
1.3 In vivo AND in vitro ANIMAL MODELS
1.3.1 Ketamine-Induced Neuronal Cell Death in the Perinatal Rat (in vivo)
Our recent developmental neurodegenerative study in rat pups demonstrated apoptotic cell death of neurons in several brain regions following postnatal exposure to ketamine [61] On PND 7. Rat pups were subcutaneously injected with different doses of ketamine (5, 10, or 20 mg/kg) using single or multiple injections at 2-h intervals; neurotoxic effects were examined 6 h after the last injection. In rats that were administered six injections of 20 mg/kg ketamine, a significant increase in the number of caspase-3- and Fluoro-Jade C–positive neuronal cells was observed in the frontal cortex and other brain regions. Typical apoptotic characteristics of typical nuclear condensation and fragmentation were seen in electron microscopic findings. Additionally, in situ hybridization showed a remarkable increase in mRNA signals for the NMDA NR1 subunit in the frontal cortex. Ketamine administration resulted in a dose-related and exposure time–dependent increase in neuronal cell death during development. Ketamine-induced cell death is apoptotic in nature and closely associated with enhanced NMDA receptor subunit mRNA expression. This result is consistent with other findings that anesthetics cause neuronal cell death in the rodent model when given repeatedly during the brain growth-spurt period [26, 55].
1.3.2 Application of Rodent in vitro Models in the Evaluation of Anesthetics during Development
Both in vitro and in vivo approaches have been used to assess the neurotoxicity associated with a wide range of drugs at a variety of doses and exposure durations. We have used in vitro systems [primary cultures [26, 27, 33] and organotypic slice cultures [34, 62] that parallel our in vivo studies [32, 61] to assess the effects of anesthetic exposure in rodent models. Organotypic slice cultures (Fig. 1.4), established using brain tissue from rodents, provide parallel in vitro models that assist in evaluating the neurotoxicity of various anesthetics at a variety of doses using a minimal number of animals in a short period of time.
FIGURE 1.4 Organotypic cultures prepared from 7-day-old rat pups. The brains were sectioned down the midline and corticostriatal slices containing the anterior commissure were cut at a thickness of 400 μm. The slices were maintained in culture for 5–10 days on a porous and translucent membrane at the interface between the medium and the CO2-enriched atmosphere. To characterize this model, (A) monoclonal antipolysialic acid neural cell adhesion molecule (neuronal specific marker) and (B) polyclonal anti-NCAM antibodies were used for immunostaining. Whole-cell patch clamp recordings were performed to demonstrate that neurons in organotypic culture were functional. This slide shows representative sodium current spikes that demonstrate the viability of neurons in an organotypic culture. The sodium current spikes were evoked by applying a depolarizing voltage when the neurons were held at –60 mV [92]. See color insert.
These in vitro preparations are useful for rapidly evaluating the neurotoxic effects of anesthetic drugs and enable direct study of the brain at various stages of development. Primary (Fig. 1.5) and organotypic (Fig. 1.4) cultures maintain important anatomical relationships and synaptic connectivities, allow for direct assessment of cell death, and are reliable models for screening and evaluating the neurotoxicity of different anesthetic drugs. In addition, these preparations allow for the direct application of antisense oligodeoxynucleotides (ODN) that target specific receptor genes, as well as direct enzymatic and therapeutic drug treatment. This approach allows for the collection of a large amount of data from a minimal number of subjects and allows for the investigation of cellular mechanisms associated with anesthetic-induced cell damage in simplified primate or rodent systems.
FIGURE 1.5 Immunofluorescence micrographs of primary monkey frontal cortical cultures. (A) Neuron-specific staining of cultured cells with PSA-NCAM as revealed by immunofluorescence of antimouse IgG conjugated to fluorescein isothiocyanate. (B) Glia-specific staining of cultured cells with GFAP as revealed by immunofluorescence of antirabbit IgG conjugated to rhodamine. (C) Hoechst 33285 nuclear staining reveals the total number (nuclei) of the cells in the field. Scale bar = 50 μm. See color insert.
1.4 PHARMACOGENOMIC/SYSTEM BIOLOGY APPROACHES (SEE CHAPTER 2 IN THIS BOOK)
1.5 MOLECULAR IMAGING APPROACHES IN THE STUDY OF ANESTHETIC-INDUCED NEURONAL CELL DEATH
Molecular imaging is an emerging approach that unites molecular biology and in vivo imaging. With minimal intervention, it can be used to observe aspects of cellular function and enables the follow-up of the molecular processes in living organisms. Probes, or biomarkers, interact chemically with their molecular targets, and, in turn, alter the image contrast according to molecular changes occurring within the area of interest. Therefore, molecular imaging can help to visualize particular targets and/or pathways.
Positron emission tomography (PET), one of the modalities applied in molecular imaging, allows noninvasive, in vivo measurements of multiple molecular processes in various organs to be obtained. The development of microPET imaging applications has provided us with the ability to collect sensitive and quantitative three-dimensional molecular information from the living brains of small animals such as rats and mice [63-68]. Because it is important to obtain sufficient data from living animals to allow for repeated assessment of the neurotoxic effects associated with early exposure to ketamine, microPET was used to image neuronal apoptosis in the living rat brain using the tracer [18F]-labeled annexin V [69]. On PND 7, rat pups in the experimental group were exposed to six injections of ketamine (20 mg/kg at 2-h intervals) and control rat pups received six injections of saline. On PND 35, 37 MBq (1 mCi) of [18F]-annexin V was injected into the tail vein of treated and control rats, and static microPET images were obtained over 2 h following the injection. The uptake of [18F]-annexin V was significantly increased in the regions of interest (ROI) in the brains of ketamine-treated rats compared with saline-treated controls. Additionally, there was a prolonged duration of annexin V tracer washout in the ketamine-treated animals. These results demonstrate that microPET imaging is capable of distinguishing differences in retention of [18F]-annexin V in a selected brain region and suggests that this compound may provide a minimally invasive biomarker of neuronal apoptosis in rats [69].
1.6 PERINATAL ANESTHETIC ADMINISTRATION AND LONG-TERM BEHAVIORAL DEFICITS (SEE CHAPTER 3 IN THIS BOOK)
1.7 CLINICAL CORRELATION OF PRESENT DATA
Sedatives and general anesthetics have been used for decades in pediatric patients without overt clinical evidence of CNS sequelae. Although the doses and durations of ketamine exposure that have resulted in neurodegeneration in our animal models were substantially greater than those used in the clinical setting, doses and durations associated with isoflurane were in the same range as those used in humans [70]. So far, there are insufficient human data to either support or refute the clinical applicability of rodent and nonhuman primate findings. However, moderate adverse effects related to CNS function in pediatric populations may be difficult, if not impossible, to detect.
As stated previously, agents that block the NMDA subtype of glutamate receptor and/or positively modulate or gate the GABAA receptor have been associated with apoptotic neuronal cell death in developing rodents [35, 62, 70, 71]. To induce or maintain a surgical plane of anesthesia, it is common practice in pediatric or obstetrical medicine to use a combination of agents from these two classes.
NMDA antagonists and GABA agonists are often used in combination during general anesthesia. For example, the anesthetic gas nitrous oxide, an NMDA receptor antagonist, and isoflurane, a volatile anesthetic that acts on multiple receptors including the postsynaptic GABA receptor, are commonly used in combination. Thus, another important goal of our studies was to determine if a combination of NMDA antagonists and GABA agonists would prevent or enhance each other’s effects (including neuronal cell death). In PND 7 rat pups, an enhancement in brain damage was noted when nitrous oxide (75%) was combined with isoflurane (0.55%). Maximal neuronal cell death was observed after 6–8 h of exposure. There were no significant effects after only 2 h of exposure in the PND 7 rat brain. These findings are consistent with previous studies performed using the same animal model [70]. Our data [28] indicate that a low dose of isoflurane (0.55%) alone caused no significant enhancement of apoptotic cell death in the brain. In contrast, higher doses of isoflurane (0.75, 1.0, and 1.5%) [70] produced neurodegeneration in multiple brain regions.
Immature GABA receptors are excitatory during development but convert to being inhibitory in mature neurons [72]. We postulated that prolonged supraphysiologic stimulation of immature neurons by GABA agonists generally enhances CNS excitation during early development [24]. This increased excitability, along with NMDA antagonist-induced action at NMDA receptors, could lead to neuronal cell death. Our data [28] indicate that a significant effect was observed in the frontal cortex only when the low dose of isoflurane (0.55%) was combined with nitrous oxide (75%). Apoptosis was found in many neuronal populations, however, following exposures to higher doses of isoflurane (0.75, 1.0, and 1.5%) combined with nitrous oxide (75%).
Additive toxicity between a nontoxic concentration of an NMDA antagonist and a GABAmimetic agent has also been observed with other combinations such as ketamine and midazolam [73]. Additional experimental models (in vitro and in vivo nonhuman primate and rodent models) will be necessary to confirm and extend these observations.
1.8 POTENTIAL NEUROPROTECTION
L-carnitine plays an integral role in attenuating neurological brain injury associated with mitochondria-related degenerative disorders. L-carnitine is an L-lysine derivative and its main role lies in the transport of long-chain fatty acids into mitochondria to enter the β-oxidation cycle [74]. Another important property of this agent is the neutralization of toxic acylCoA production in mitochondria [75], which correlates with various pathological processes including organic aciduria [76] and numerous diseases of the CNS including neurodegenerative diseases [77-79], ornithine transcarbamylase deficiency [80, 81], and other mitochondrial diseases [75]. L-carnitine administration may offer a straightforward approach to mitigating neurotoxic effects, and such studies are underway.
Another group of molecules regulating mitochondrial function and stability is the BCL-2 family of proteins. Although the precise mechanism by which BCL-2 family members act remains unclear, it has been established that they play a key role in the mitochondrial apoptotic pathway [82]. The effect of inhaled anesthetics on Bax and BCL-XL was measured among potential regulators. Bax is a proapoptotic protein, a pore-forming cytoplasmic protein that translocates to the outer mitochondrial membrane, influencing its permeability and inducing cytochrome-c release from the intermembrane space of the mitochondria into the cytosol, subsequently leading to cell death [83]. An anesthetic combination [nitrous oxide (75%) with isoflurane (0.55%)] resulted in a significant up-regulation of Bax protein compared with a control (Fig. 1.6), and this effect was blocked by the coadministration of L-carnitine (300 or 500 mg/kg).
FIGURE 1.6 Western blot analysis of the effect of combined anesthetics (75% nitrous oxide + 0.55% isoflurane) and L-carnitine on the regulation of BCL-XL and Bax protein expression (A). Densitometry measurements were used to calculate a ratio of BCL-XL to Bax (by stripping the membranes) in three independent experiments, and the data are shown as the means ± S.E.M. of the ratio (B). L-carnitine effectively prevented reduction of BCL-XL/Bax ratio induced by anesthetics.
Melatonin is produced at night by the pineal gland and promotes sleep. Melatonin functions as a direct free oxygen radical scavenger and indirect antioxidant, reducing the toxicity of a large number of drugs [84]. It was recently reported that melatonin suppresses apoptosis in cultured pineal cells by up-regulating Bcl-XL, which in turn inhibits cytochrome-c release and caspase-3 activation, thus blocking activation of an apoptotic cascade cascade [85]. Recently, Jevtovic-Todorovic’s group has reported that coadministration of an anesthetic cocktail (midazolam, isoflurane, and nitrous oxide) with melatonin reduces the severity of anesthesia-induced damage in the developing rat brain [86]. This study demonstrated that melatonin provides significant protection against anesthesia-induced neuroapoptotic damage in the developing brain of immature rats. Although we do not know the exact mechanism by which melatonin protects against apoptotic cell death, the neuroprotective effect is mediated, at least in part, via a mitochondria-dependent apoptotic cascade. It appears that key elements involve the stabilization of the inner mitochondrial membrane, which sequentially controls cytochrome-c release and apoptotic cascade activation. Several mechanisms involved in inner mitochondrial membrane stabilization by melatonin have been suggested. One mechanism involves a decrease in mitochondrial protein and DNA damage and the improvement of ATP synthesis by the scavenging of oxygen [87] and nitrogen-based reactants that are generated in mitochondria, which, in turn, control the concentration of intramitochondrial glutathione [88]. Melatonin may also stabilize the inner membrane by increasing the efficiency of the electron transport chain and by controlling the reduction potential [89]. In addition, the direct action of melatonin in controlling currents through the mitochondrial transition pores [90] has been observed. Jevtovic-Todorovic and colleagues have shown that melatonin stabilizes the inner mitochondrial membrane by increasing the protein levels of Bcl-XL. It is most likely that multiple mechanisms contribute to melatonin-induced restoration of mitochondrial function.
1.9 CONCLUSION
Exposures of developing mammals to anesthetics, including those that block NMDA-type glutamate receptors and those that activate GABA receptors, affect endogenous neuronal transmission systems and enhance neuronal cell death in a dose- and developmental stage-dependent manner. This chapter emphasized the parallel use of in vivo models with more circumscribed in vitro preparations in the developing rodent. These combined models provide the opportunity for the rapid evaluation of anesthetic agents over a wide range of doses, exposure durations, and drug combinations and enables the collection of a large amount of data from a minimal number of subjects. The in vivo models provide the functional anchors to the in vitro investigation of cellular mechanisms associated with anesthetic-induced cell loss [91].
The application of pharmacogenomic and systems biology approaches has great potential for helping advance the understanding of brain-related biological processes, including neuronal plasticity and neurotoxicity. These approaches may also allow for monitoring efficacy of treatment regimens. In addition, by using in vivo and in vitro rodent models, these approaches may enhance our understanding of complex biological processes such as neuronal cell death (apoptosis and/or necrosis) induced by anesthetics in the developing brain. Understanding these complex biological processes will clarify pathways that will hopefully allow us to predict anesthetic-induced brain cell death and help discover treatments to ameliorate the consequences (if any) of anesthetic toxicity in pediatric patients.
Disclaimer: This document has been reviewed in accordance with United States Food and Drug Administration (FDA) policy and approved for publication. Approval does not signify that the contents necessarily reflect the position or opinions of the FDA. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the FDA.
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