Developmental Neurotoxicology Research: Principles, Models, Techniques, Strategies, and Mechanisms - Hardcover

Hardcover

ISBN 10: 0470426721 ISBN 13: 9780470426722

Publisher: Wiley, 2011

Synopsis

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.

About this title

About the Author

Cheng Wang is Senior Neurobiologist in the Division of Neurotoxicology at the National Center for Toxicological Research, U.S. Food and DrugAdministration. He is the author of over fifty-five peer-reviewed publications and book chapters. Dr. Wang was awarded the Outstanding Performance Award at the Society of Toxicology 44th Annual Meeting and the 2007 FDA Scientific Achievement Award for Excellence in Laboratory Science.

William Slikker, Jr., is the Director of the National Center for Toxicological Research, U.S. Food and Drug Administration. He has over 260 peer-reviewed scientific publications. Dr. Slikker has served as coeditor of several books and is an Associate Editor of the journals NeuroToxicology and Toxicological Sciences.

From the Back Cover

How preclinical research approaches can inform clinical interventions and vice versa

Because of the complexity and temporal features of 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. Developmental Neurotoxicology Research delineates how systems biology, pharmacogenomic, and behavioral approaches, as applied to neurodevelopmental toxicology, provide a structure to arrange information in a biological model.

The 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

• The strategy and progress of epilepsy research

Incorporating new, post-genomic techniques, this book provides researchers with effective tools for dissecting the mechanisms underlying pharmacological and toxicological phenomena associated with the exposure to drugs or environmental toxicants during development.

From the Inside Flap

How preclinical research approaches can inform clinical interventions—and vice versa

Because of the complexity and temporal features of 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. Developmental Neurotoxicology Research delineates how systems biology, pharmacogenomic, and behavioral approaches, as applied to neurodevelopmental toxicology, provide a structure to arrange information in a biological model.

The 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

• The strategy and progress of epilepsy research

Incorporating new, post-genomic techniques, this book provides researchers with effective tools for dissecting the mechanisms underlying pharmacological and toxicological phenomena associated with the exposure to drugs or environmental toxicants during development.

Excerpt. © Reprinted by permission. All rights reserved.

Developmental Neurotoxicology Research

Principles, Models, Techniques, Strategies, and MechanismsBy Cheng Wang William Slikker, Jr

John Wiley & Sons

Copyright © 2011 John Wiley & Sons, Ltd
All right reserved.
ISBN: 978-0-470-42672-2

Chapter One

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-Daspartate (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), and NR3A and B. 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. NMDA receptors are involved in a variety of physiological and pathological processes, including memory and learning, neuronal development, epileptiform seizures, synaptic plasticity, and acute neuropathologies associated with stroke and traumatic injury. During the brain growth spurt, blockade of the NMDA receptor for a period of hours triggers widespread apoptotic neurodegeneration in the rodent brain.

GABA, the principal inhibitory neurotransmitter in the adult CNS, acts as an excitatory transmitter in the early postnatal stages. Functional GABA_A 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 GABA_A receptors represents a general feature of developing neurons. Activation of GABA_A receptors depolarizes neuroblasts and immature neurons in all regions of the CNS examined to date, including spinal cord, hypothalamus, cerebellum, cortex, hippocampus, and olfactory bulb. This depolarization is not due to unusual properties of neonatal GABA_A channels but to an elevated intracellular Cl^- concentration, probably from developmental changes in [Cl^-]_i homeostatic systems. Postsynaptic GABA_B 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. The reasons for this delayed maturation of postsynaptic GABA_B 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, rather than to the late development of receptors.

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. 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. PSA-NCAM is also a neuronspecific marker known to be an NMDA-regulated molecule important in synaptogenesis during development. Some experiments 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 longterm 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. 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. 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. More recently, it has been shown that NMDA receptor activation can also lead to apoptotic cell death.

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]; (2) there is increased expression of NMDA receptor NR1 protein accompanied by enhanced cell death; 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. 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 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). Additionally, a study by Turner et al. 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.

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 and by neuronal ischemia. Ketamine has a very short half-life in the brain 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. Rapid brain growth, synaptogenesis, expression of excitatory receptors, and developmentally regulated neuronal cell death also occur at this time, which may explain why repetitive neonatal pain persistently alters subsequent pain processing in rats, mice, and humans. 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 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. 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.

The sialylation state of PSA-NCAM is controlled by developmentally regulated Golgi sialyltransferase activity. 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. In our previous study, 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. 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.

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 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.

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 and organotypic slice cultures that parallel our in vivo studies 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.

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.

(Continues...)

Excerpted from Developmental Neurotoxicology Researchby Cheng Wang William Slikker, Jr Copyright © 2011 by John Wiley & Sons, Ltd. Excerpted by permission of John Wiley & Sons. All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
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Edition details

• Publisher: Wiley
• Publication date: 2011
• Language: English
• ISBN 10: 0470426721
• ISBN 13: 9780470426722
• Binding: Hardcover
• Edition number: 1
• Number of pages: 672
• Editor: Wang Cheng, Slikker Jr William