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Anesth Analg 2008; 106:1643-1658
© 2008 International Anesthesia Research Society
doi: 10.1213/ane.ob013e3181732c01
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PEDIATRIC ANESTHESIOLOGY

Section Editor:
Peter J. Davis

Strategies and Experimental Models for Evaluating Anesthetics: Effects on the Developing Nervous System

Cheng Wang, MD, PhD, and William Slikker, Jr, PhD

From the National Center for Toxicological Research, U.S. Food & Drug Administration, Jefferson, AR.

Address correspondence and reprint requests to Cheng Wang, MD, PhD, Division of Neurotoxicology, National Center for Toxicological Research/FDA, 3900 NCTR Road, Jefferson, AR 72079-9502. Address e-mail to cheng.wang{at}fda.hhs.gov.

Abstract

Advances in pediatric and obstetric surgery have resulted in an increase in the duration and complexity of procedures requiring anesthesia. It has been reported that anesthetic drugs cause widespread and dose-dependent apoptosis in the developing rat brain. The similarity of the physiology, pharmacology, metabolism, and reproductive systems of the nonhuman primate to that of the human, especially during pregnancy, make the monkey an exceptionally good animal model for assessing potential neurotoxic effects of anesthetics. The window of vulnerability to these neuronal effects of pediatric anesthetics is restricted to the period of rapid synaptogenesis, also known as the brain growth spurt period. To minimize the risks to children resulting from the use of anesthesia, the following questions should be addressed:

  1. What is the relationship between exposure and brain cell loss for drugs commonly used in the practice of pediatric anesthesia (inhaled anesthetics, midazolam, ketamine, and nitrous oxide)?
  2. Are there "class effects," or does each drug need to be considered independently?
  3. Are there important interactions among the drugs used as anesthetics contributing to the risk of brain cell death?
  4. What is the likely period of human vulnerability?

Pharmacogeneomic/system biology approaches have great potential for helping to advance the understanding of brain-related biological processes, including neuronal plasticity and neurotoxicity. Because of the complexity and temporal features of how developmental neurotoxicity is manifested, pharmacogenomic/systems biology approaches may prove to be useful tools for enhancing our understanding of the biological processes induced by anesthetics. Therefore, the main purpose of this review is to describe the application of these approaches and models, as well as protection strategies, especially as regards the issue of anesthetic-induced neuronal cell death during development.

Much of the discussion that follows is based on experiments conducted with ketamine. This is due in part to the use of ketamine in the early studies and the volume of preclinical experimental work performed with this drug, as well as its use in anesthetic studies in developing rodents and nonhuman primates. Although ketamine use in pediatric anesthesia is relatively limited, the findings of the studies are sufficiently strong to merit concern about the N-methyl-d-aspartate antagonist drugs as a class. Our focus on ketamine should not be construed as implying that the risk of neurodegeneration with ketamine is greater, or less, than with other anesthetics. We are simply describing the effects where we have the most preclinical data.

Various anesthetic protocols have been used in pediatric medicine for many decades without systematic assessment concerning drug exposure and possible adverse effects. Most of the currently used general anesthetic drugs have either N-methyl-d-aspartate (NMDA) receptor blocking or {gamma}-aminobutyric 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 earlier developmental events, including synapse formation, neuroplasticity, and survival processes during neural development.

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 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. The functional properties of the NMDA receptor vary throughout the CNS and 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 transmitter in the adult CNS, acts as an excitatory transmitter in the early postnatal stage.11 Functional GABAA receptors are expressed in neurons as early as embryonic stages and investigations by different groups have led to the conclusion that a transient excitatory action of GABA, via GABAA receptors is a general feature of the developing neurons. Activation of GABAA receptors depolarizes neuroblasts and immature neurons in all regions of the CNS studied, including spinal cord,12–14 hypothalamus,15 cerebellum,16 cortex,17 hippocampus,18,19 and olfactory bulb.13 This depolarization is not due to unusual properties of neonatal GABAA channels but rather to an elevated intracellular Cl– concentration, probably from developmental changes in [Cl–]i homeostasis systems.13,20,21 Postsynaptic GABAB receptor-mediated responses, that is, the activation of K+ and inhibition of Ca2+ currents, are absent from 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, but may be due to a lack of coupling between receptors, G proteins, and K+ or Ca2+ channels,22 rather than to 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 ketamine washout, to the excitototoxic effects of glutamate, because these up-regulated NMDA receptors allow for the accumulation of toxic levels of intracellular free calcium under normal physiological conditions. In addition, prolonged supra-physiologic stimulation of immature neurons by GABA agonists enhances the excitatory component in the action of GABA and may contribute to increased excitability during early development.24 This increased excitability, along with NMDA antagonist-induced alteration of NMDA receptors, could lead to 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 an important component of the anesthetic-induced neurotoxicity. Synaptophysin is a synaptic vesicle-associated protein 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 experiments27,28 have been performed to determine the correlation between anesthetics and PSA-NCAM expression, because quantifying the levels of PSA-NCAM after anesthetic exposure would validate the activity states of neuronal synaptic plasticity.

Neuronal susceptibility to neurotoxic insult varies with stage of development. Both in vitro and in vivo approaches have been used to assess the neurotoxicity associated with a wide range of these drugs at a variety of doses and exposure durations. Although comprehensive gene expression/proteomic studies and long-term behavioral assessments remain to be accomplished, in vivo and in vitro models and analysis strategies have been developed to study the biological pathways and behavioral outcomes of anesthetic-induced cell death in the developing nonhuman primate and rodent.

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 Over-activation of NMDA receptors kills neurons (Fig. 1) 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


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  		Figure 1. Enhanced neuronal cell death in 24-h ketamine-infused monkeys. Electron micrographs (EM) show a normal neuron with intact cytoplasm and nuclear membrane from a postnatal day (PND) 5 control monkey (A). EM also shows nuclear condensation (*, in B-1), nuclear fragmentation (>, in B-1) (advanced states of apoptosis), typical mitochondrial swelling and neuronal cell body swelling (©, in B-2) (necrosis) in layers II and III of the frontal cortex from a 24-h ketamine-infused PND 5 monkey. Scale bar = 0.64 µm.

 

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 (by continuously blocking the NMDA receptor in the developing brain). This up-regulation then makes neurons bearing these receptors more vulnerable, after ketamine washout, to the excitotoxic effects of endogenous glutamate. This compensatory hypothesis is supported by the following observations: (1) NR1 subunit mRNA (Fig. 2; in situ hybridization) is up-regulated in ketamine-treated monkey fetuses (gestation day 122) and infants (postnatal day (PND) 5)35, (2) increased expression of NMDA receptor NR1 protein is accompanied by enhanced cell death27, and (3) co-administration of NR1 antisense oligonucleotide (targeted to NR1 NMDA receptor subunit mRNA) is able to block the neuronal cell death induced by ketamine in rat and monkey cortical culture.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 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 reported28 that even low concentrations of ketamine could interfere with dendritic arbor development of immature GABAergic neurons and could potentially interfere with the development of neural networks. Further studies are needed to determine receptor specificity of those effects induced by anesthetics such as ketamine.


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  		Figure 2. N-methyl-d-aspartate (NMDA) receptor NR1 subunit mRNA abundance in the frontal cortex of postnatal day (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 IM injection (20 mg/kg) followed by continuous IV infusion at a rate of 20 to 50 mg · kg–1 · h–1 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.

 

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 agonists36–39 and by neuronal ischemia.40–42 Ketamine has a very short half-life in the brain,43,44 and hence some of the inconsistencies could be due to the dose used and the length of time for which neuroprotective concentrations remain.

Prolonged or repetitive pain may occur during critical periods of brain development in hospitalized neonates.45 Rapid brain growth, synaptogenesis, expression of excitatory receptors,46 and developmentally regulated neuronal cell death47 also occur at this time, which may explain why repetitive neonatal pain persistently alters pain processing in rats, mice, and humans.48–52 Very few animal experiments (rodents or nonhuman primates) have studied 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 antiinflammatory effects of anesthetics.

It has been shown in previous studies53 that peak vulnerability to the apoptogenic action of anesthetics is during synaptogenesis, also known as the brain growth spurt. This is a period during which 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. NCAM is believed to be an important regulator of developmental and functional neuroplasticity. In particular, embryonic PSA-NCAM has a vital role in forming connections between neurons.54 Synaptophysin is a synaptic vesicle-associated protein that is involved in synaptogenesis. Interestingly, our data show that PSA-NCAM (Fig. 3A) is partially co-localized with synaptophysin (Fig. 3B) in the neuronal cell membrane in the organotypic slice cultures (control) during development (Fig. 3). Therefore, PSA-NCAM activity seems critical to the process controlling the trafficking and targeting of vesicular proteins to the synapse.


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  		Figure 3. 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 co-localized.

 

The sialylation state of PSA-NCAM is controlled by developmentally regulated Golgi sialyltransferase activity.55 This transferase activity is Ca2+ dependent,56 which may account for its regulation by NMDA receptors.54,57 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.58 In our previous study,27 treatment of frontal cortical cultures from the developing monkey with ketamine caused a substantial decrease in mitochondrial metabolism of [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], along with a concomitant decrease in PSA-NCAM protein expression (Fig. 4). The decrease in PSA-NCAM corresponded to an approximately 40% decrease in PSA-NCAM immunoreactivity, which 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.27,56 The fact that SN-50 (a peptide inhibitor of NF-kB transport) dose-dependently blocked the ketamine-induced cortical neuronal cell death, as well as the loss PSA-NCAM immunoreactivity in culture, argues for the latter mechanism. Future experiments using N-butanoyl-mannosamine to inhibit polysialyl transferase or endoneuramididase N to selectively cleave PSA chains may be able to specifically address this hypothesis.


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  		Figure 4. Effect of ketamine and SN-50 on the decrease in polysialic acid neural cell adhesion molecule (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 3 independent experiments and the data are shown as the means ± sd 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).

 

IN VIVO AND IN VITRO NONHUMAN PRIMATE AND RODENT MODELS

Ketamine-Induced Neuronal Cell Death in the Perinatal Rhesus Monkey (In Vivo)
Although it is clear that anesthetics cause neuronal cell death in the rodent model when given repeatedly during the brain growth spurt period,26,53 it is not yet known whether a similar phenomenon also occurs in primates. To better determine if ketamine-induced neurodegeneration in the developing rat has clinical relevance, ketamine was examined in a nonhuman primate model that more closely mimics the developing pediatric population.27,59 The similarity of the physiology, pharmacology, metabolism, and reproductive systems of the nonhuman primate to that of the human, especially during pregnancy, allow the monkey to be an exceptionally good animal model to detect the potential neurodegenerative effects of ketamine. In our monkey study,35 important maternal and infant physiological variables, including percent oxygen saturation, exhaled carbon dioxide, body temperature, heart rate, arterial blood pressure, glucose, and hematocrit were all monitored and maintained within normal ranges. This is an essential component of any animal model, and is far easier in a primate than a rodent. Because prolonged hypoperfusion can lead to cerebral hypoperfusion and ischemic-related cell death, it is particularly important to ensure normal blood pressure and oxygen saturation.60

Placental transfer of ketamine occurs rapidly. Fetal blood levels have been reported to be comparable to maternal blood levels within 2 min of maternal administration.61 Ketamine is distributed rapidly after IV administration and plasma levels decrease rapidly thereafter.62,63 In our recent publication,35 we reported that the ketamine levels required to maintain anesthesia were 10 to 25 µg/mL, which is 5 to 10 times higher than those observed in humans (2–3 µg/mL). It is important to note that the plasma concentrations of ketamine were highest in the PND 35 monkeys, even though no evidence of neuronal cell death was observed compared with control animals of the same age. In the PND 5 monkeys, where neuronal cell death was evident, plasma levels averaged approximately 10 µg/mL (Fig. 5), which is only three to five times the plasma levels observed in humans.


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  		Figure 5. Plasma concentrations of ketamine and norketamine in pregnant (GD122) or infant (postnatal day(PND)5 or PND35) monkeys. Data points represent mean plasma concentrations (µg/mL) ± sem for ketamine (A) and norketamine (B) from monkeys infused with 20 to 50 mg · kg–1 · h–1 ketamine for 3 or 24 h, followed by a 6-h withdrawal period. Fetal plasma concentrations (GD122) at the time of C-section of pregnant animals are indicated (Fetus). n = 3 animals per time point.

 

The window of vulnerability to the neurotoxic effect of ketamine in the primate is restricted to the period of rapid synaptogenesis, which occurs during the perinatal stages, occurring at least by 75% of gestation and lasting to early postnatal life, ending sometime before PND 35.35 However, in the rat, the window of vulnerability begins 1 day after birth and ends approximately 14 days later.53 This is a period in the rat before the complete development of motor and other relevant systems during intense synaptic remodeling in multiple brain regions. This is also a period of rapid myelin formation during which most afferent pathways are already present in their target areas, although their distribution and synaptic targets are still immature. During these sensitive stages, altered glutamatergic neurotransmission and receptor expression induced by NMDA antagonists (such as ketamine) could affect neuroplasticity and cause neuronal toxicity. Thus, abnormal neuronal development, abnormal synaptic plasticity, and neurodegeneration have been proposed as causal or contributing factors in anesthetic-induced neurotoxicity.27 The period of vulnerability of the immature rat brain to ketamine-induced neuronal cell death coincides with the critical stages of monkey brain development. GD 122 fetuses and PND 5 infants are undergoing synaptogenesis and appear much more vulnerable than PND 35 monkeys that are undergoing much less synaptogenesis. Although the precise correlation between stages of brain development in humans and monkeys and/or species-specific vulnerabilities to anesthesia-induced neuronal damage cannot be specified, clearly, matching such events between human and nonhuman primates is less problematic than matching these phenomena between primates and rodents. For example, according to a recent study, the gestational day 122 monkey fetus is equivalent to the 199 gestational day human fetus as determined by cortical development, and both are in the range of 75% to 80% of normal term.64

It should be mentioned that the pattern of topography, characteristics, and neuronal susceptibility to neurotoxic insult that leads to apoptosis or necrosis are not clear, and may depend on the concentration, the duration of exposure, the receptor subtype activated and the cell type, as well as neuronal stage of development or maturity.32,33,35 Among these factors, the concentration and duration are thought to be the most significant contributors. How do concentration and duration relate to neuronal toxicity?

The dosing paradigms used in animal studies typically do not reflect doses used for pediatric patients. The doses and durations of exposure used in the animal studies typically exceed those used clinically, sometimes substantially. While this is an important observation, it is a key component to preclinical research. Once a toxic dose and effect have been identified, it is possible to evaluate lower doses and/or shorter durations to determine an exposure level associated with no adverse effects, which can than be compared with clinically relevant dosing paradigms to establish the existence, or lack thereof, of a safety margin. This approach is used for drug development. In general, we cannot interpret duration without considering the concentration that was maintained, nor can we consider concentration without consideration of the duration of the exposure.

Another goal is to determine whether there is an anesthetic duration below which no significant ketamine-induced neuronal cell death can be detected. In our study, PND 5 monkeys were evaluated after 3 and 24 h of ketamine anesthesia (IV infusion at a rate of 20 to 50 mg · kg–1 · h–1 to maintain a light surgical plane of anesthesia). The 24-h duration was selected as a relatively long duration, while the 3-h duration was applied as a relatively short duration. No significant neurotoxic effects were observed if the anesthesia duration was 3 h. By contrast, the 24-h anesthetic sessions produced a significant increase in the number of caspase 3-positive neurons, Silver-stained cells, and Fluoro-Jade C-positive cells in layers II and III of the cortex (Fig. 6). These data are consistent with those observed in our time-course studies using a primary cortical culture system established from developing rats and PND 3 monkeys.26,27 In these time-course studies, the cultures were exposed to ketamine for 2, 4, 6, 12, and 24 h. The data indicated that the addition of 10 µM ketamine results in about a 30% loss in cell viability at 6 h, and an approximately 50% to 70% loss after 12 to 24 h of exposure. However, there were no significant differences between control and ketamine-exposed cultures at 2 h. After 4 h of exposure, a slight but nonsignificant increase in neuronal cell death was observed. The data suggest that continuous activation of up-regulated NMDA receptors (compensatory) over 6 to 24 h is critical for ketamine-induced cell death in developing neurons in monkey frontal cortical cultures.27 Ketamine-induced cell death was also exposure-time dependent in vivo.


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  		Figure 6. Quantitative analyses of ketamine-induced neurodegeneration assessed using caspase 3 immunostaining (A), Silver staining (B) and Fluoro-Jade C staining (C). For each condition, three animals were randomly assigned to treatment and control groups (n = 3/group). Data are presented as means ± SD *A probability of P < 0.05 was considered significant (two-way ANOVA). The effect of ketamine treatment at the 3 stage-of-development dates (GD 122. postnatal day (PND) 5, and PND 35) and two durations of exposure (24 and 3 h) was analyzed. The data for the 24 and 3 h exposures were analyzed separately. For the 24-h exposure, a two-way analysis of variance (ANOVA), stage-of-development and treatment, with a repeated measure variable was used to evaluate ketamine effect. The repeated variable is a section variable representing various measurements made in different sections. For each stage of development and treatment combination, the post t-test for the treatment effect was performed. The Tukey test was used to correct for multiple comparisons. The data for the 3-h exposure at PND5 ware analyzed using a one-way ANOVA with a repeated (time or section) variable. The null hypothesis was rejected at the significance level of P < 0.05. All analyses were conducted using Statistical Analysis System (SAS).

 

Application of Rodent and Nonhuman Primate 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 culture26,27,33 and organotypic slice culture34,65) that parallel our in vivo studies28,35 to assess the effects of anesthetic exposure on the developing nonhuman primate and rodent models. Primary frontal cortical culture systems (Fig. 7) and organotypic slice cultures (Fig. 8), established using tissues from monkey and rodent, 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.


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  		Figure 7. Immunofluorescence micrographs of primary monkey frontal cortical cultures. (A) Neuron-specific staining of cultured cells with polysialic acid neural cell adhesion molecule (PSA-NCAM) as revealed by immunofluorescence of anti-mouse IgG conjugated to fluorescein isothiocyanate. (B) Glia-specific staining of cultured cells with GFAP as revealed by immunofluorescence of anti-rabbit IgG conjugated to rhodamine. (C) Hoechst 33285 nuclear staining reveals the total number (nuclei) of the cells in the field. Scale bar = 50 µm.

 

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  		Figure 8. Organotypic cultures prepared from 7-day-old rat pups. The brains are sectioned down the midline and corticostriatal slices containing the anterior commissure are cut at a thickness of 400 µm. The slices are maintained in culture for about 5 to 10 days on a porous and translucent membrane at the interface between the medium and CO2-enriched atmosphere. To characterize this model (A) monoclonal anti-polysialic acid neural cell adhesion molecule (neuronal specific marker), and (B) polyclonal anti- neural cell adhesion molecule antibodies are used for the immunostaining. To demonstrate that neurons in organotypic culture are functional, whole cell patch clamp recording was performed. This slide shows representative sodium current spikes that demonstrate the viability of neurons in an organotypic culture. The sodium current spikes are evoked by applying a depolarizing voltage when the neurons were held at –60 mV.

 

These in vitro preparations are useful for the rapid evaluation of the neurotoxic effects of anesthetic drugs and enable direct study of the brain at various stages of development. Primary (Fig. 7) and organotypic (Fig. 8) 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 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.

PHARMACOGENOMIC/SYSTEM BIOLOGY APPROACHES

Application of Pharmacogenomic/System Biology Approaches at mRNA/DNA Levels (Genomics)
Microarrays
Gene microarray techniques have been used to determine which of several candidate death genes might be associated with anesthetic drug-induced apoptosis. Microarrays facilitate analysis of the relationship between anesthetic drug-induced neurotoxicity and changes in gene expression. Therefore, gene expression profiles for different anesthetic drugs at different time points were compared.

Tissue (In Vivo and In Vitro).
Frozen tissue was sectioned using a cryostat before laser capture microdissection (LCM). Briefly, the tissue was embedded in a mold containing Optical Cutting Temperature compound. This tissue-containing cryomold was allowed to equilibrate on dry ice and was then frozen onto the cutting stage of the cryostat. Approximately 7 to 10 µm sections were cut and immediately adhered to chilled microscope slides which were kept on dry ice until ‘same-day’ processing for LCM or stored at –80°C for future processing. Keeping the tissue frozen before LCM ensures that high quality mRNA is obtained from the cells collected. Cells from the same brain region were collected from each subject to be used as part of the systems biology study. Total RNA was isolated from these cells and an RNA amplification kit was used to generate labeled cRNA for microarray experiments. Briefly, double-stranded (ds) cDNA was synthesized from the isolated RNA before in vitro transcription to generate antisense RNA, which was then used in a second round of amplification to generate additional ds cDNA. This ds cDNA was amplified along with cyanine 3-CTP or cyanine 5-CTP dye to generate labeled antisense cRNA.

Oligonucleotide Microarrays (In Vivo and In Vitro).
The labeled RNA, along with labeled reference RNA, was co-hybridized onto a rat oligo microarray containing over 22,000 sequences. The microarray slides were scanned and the data analyzed using ArrayTrack in-house software (Figs. 9 and 10).


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  		Figure 9. Schematic of microarray procedures used in the systems biology approach. To determine which of several candidate death genes are associated with anesthetic drug-induced apoptosis, microarray techniques are applied to the anesthetic drug models. Laser capture microdissection is used to collect cells from a specific brain region and total RNA is isolated from these cells. The first round of RNA amplification is performed to generate double stranded cDNA. The cDNA is then amplified along with cyanine 3-CTP or cyanine 5-CTP dye in order to generate labeled antisense cRNA. The labeled antisense cRNA is hybridized onto a rat oligo microarray containing over 22,000 sequences which is then scanned and the data are analyzed using ArrayTrack software (NCTR).

 


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  		Figure 10. Volcano plot illustrating the significant gene expression changes in the thalamus 2 h after ketamine treatment. Using a P value criterion of P < 0.05 and a fold-change threshold of 1.5 produced 624 significant gene expression changes. All 22,000 genes contained on the microarray are featured on the volcano plot. Features in red indicate "fully significant" genes with P < 0.05 and fold-change >1.5, features in pink indicate "significant P value only" genes with P < 0.05 and fold-change <1.5, features in yellow indicate "significant fold-change only" genes with P > 0.05 and fold-change >1.5, and features in black indicate "non-significant" genes with P > 0.05 and fold-change <1.5.

 
An In Vivo Example.
Repeated administration of ketamine produces neuronal apoptosis in neonatal rats.53 To determine whether a single dose of ketamine also produces apoptosis, 7-day-old rats were injected subcutaneously with either water or 40 mg/kg ketamine. Brain tissue was harvested 1, 2, and 4 h after the injection to identify significant gene expression changes in the thalamus that might be related to acute ketamine exposure. LCM was used to collect approximately 500 cells from the thalamus and the microarray procedures used were identical to those described above. Microarray analyses identified 18 significant (>1.5-fold change at P < 0.05) gene expression changes in the brain 1 h after treatment, 624 significant gene expression changes 2 h after treatment, and 52 significant gene expression changes 4 h after treatment. Of these, several genes were specifically associated with apoptosis (CYCS, ELL, PDCD8, PRKCB1, and RIPK1) and others associated with oxidative stress were found to be up-regulated in the brain after a single injection of ketamine.35

Application of Pharmacogenomic/System Biology Approaches at Protein Levels
Proteomics
To identify specific proteins that are correlated with NMDA antagonist/GABA agonist-induced apoptosis, 2-dimensional polyacrylamide gel electrophoresis or a Phosphoprotein Isotope-coded Solid-phase Tag method can be used to separate proteins from tissue samples. Excision or digestion followed by affinity isolation can also be used to separate specific proteins. Quantification and identification can be performed by matrix-assisted laser desorption/ionization-mass spectrometry.35

Western Blot Analysis (In Vitro and In Vivo)
Antisense oligonucleotides were used to determine the effect of co-administration of NMDA receptor antisense oligonucleotides on anesthetic drug-induced neurotoxicity and to determine whether the administration of antisense oligonucleotides targeted to the NR1 NMDA receptor subunit blocked steady-state protein levels. To determine whether decreased PSA-NCAM expression is associated with ketamine-induced loss of cortical neurons or to local NMDA receptor blockade (as determined by polysialyl transferase activity), Western blot analyses of PSA-NCAM/NR1 protein/Actin ratios and the protective effects of the antisense oligonucleotides were examined.26 Co-administration of NR1 antisense oligonucleotide was able to almost completely block the neuronal cell death induced by ketamine (Fig. 11). Our data indicate that ketamine significantly up-regulated NMDA receptor NR1 subunit protein. Co-administration of antisense oligonucleotide specifically prevented NR1 up-regulation and blocked the reduction of PSA-NCAM expression induced by ketamine.


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  		Figure 11. Western blot analysis of the effect of ketamine and NR1 antisense oligonucleotide on the regulation of N-methyl-d-aspartate receptor NR1 subunit protein and polysialic acid neural cell adhesion molecule (PSA-NCAM) expression (A). β-actin was used as a loading control and was not affected by ketamine. The data from three independent experiments were quantified using densitometry and expressed as the ratio of PSA-NCAM to actin and NR1 to actin (B). Statistical comparisons consisted of a one-way ANOVA with the Holm-Sidak test. A probability of *P < 0.05 was considered significant.

 

PERINATAL ANESTHETIC ADMINISTRATION AND LONG-TERM BEHAVIORAL DEFICITS

The loss and maintenance of synapses during development are believed to be controlled by an energy-dependent mechanism involving the NMDA receptor, similar to those involved in long-term potentiation.66,67 The administration of NMDA receptor antagonists, such as ketamine, phencyclidine (PCP), and MK-801, to rats during a critical time of perinatal development results in neuronal cell death in several major brain areas.10,53 In 1999, Ikonomidou et al.53 demonstrated severe widespread apoptotic degeneration throughout the rapidly developing brain of the 7-day-old rat pup after ketamine administration. It has been reported that exposure to a drug combination (midazolam, nitrous oxide, and isoflurane) causes widespread neurodegeneration in the developing rat brain and persistent learning deficits.68 Our previous studies have also demonstrated that repeated administration of PCP (a non-competitive NMDA antagonist) results in a sensitized locomotor response in rats subjected to later PCP challenge.69 This sensitization is associated with apoptotic cell death and an increase in NMDA receptor NR1 subunit mRNA and immunoreactivity of the NMDA receptor in rat forebrain.70,71 The neurodegeneration and associated deficits in prepulse inhibition were prevented by treatment with a superoxide dismutase mimetic, M40403,72 suggesting an important role of superoxide anions in NMDA antagonist-induced apoptosis and behavioral alterations.

Previous in vivo studies have also demonstrated that perinatal PCP administration results in profound behavioral abnormalities in the adolescent rat that may be related to enhanced apoptotic cell death of neurons in the frontal cortex.73 These cortical deficits may have a significant impact on the function of subcortical structures such as the nucleus accumbens, which serves as an important regulatory center by integrating the functions of the basal ganglia and the limbic system. The nucleus accumbens receives glutamatergic afferents from several brain regions, in particular, the frontal cortex.74–77 Medium spiny neurons account for the majority of the neostriatal cell population and are a major synaptic target of dopaminergic input to the striatum.78–81 Thus, alteration of the cortical input to these neurons during development may play a significant role in mediating the behavioral effects of perinatal PCP/ketamine treatment later in life (Fig. 12).


Figure 1210
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  		Figure 12. The effect of perinatal phencyclidine (PCP) treatment on the acquisition of a delayed spatial alternation task. At the outset of training there was no difference in percent accuracy between the saline- and PCP-treated rats. Between postnatal day (PND) 38 and PND 49, saline-treated rats were significantly more accurate than PCP-treated rats. Nevertheless, by PND 70, both groups had acquired the task with approximately equal accuracies.

 

CLINICAL CORRELATION OF PRESENT DATA

Sedatives and general anesthetics have been used for years in pediatric patients without overt clinical evidence of CNS sequelae. Although the doses and durations of ketamine exposure that resulted in neurodegeneration were substantially larger than those used in the clinical setting, those associated with isoflurane were in the same range as the human.68 There are insufficient human data to either support or refute the clinical applicability of rodents and nonhuman primate findings. However, moderate adverse effects related to CNS function in pediatric populations may be difficult, if not impossible, to detect.

Drugs that block the NMDA receptor subtype of glutamate and/or positively modulate or gate the GABAA receptor have been associated with apoptotic neuronal cell death in developing rodents.35,65,68,82 To induce or maintain a surgical plane of anesthesia, it is a common practice in pediatric or obstetrical medicine to use a combination of drugs 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 postsynaptic GABA receptor, are commonly used together. Thus, another important goal of our anesthetic studies was to determine if a combination of NMDA antagonists and GABA agonists would prevent or enhance each other’s effects (including potential 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 to 8 h of exposure. There were no significant effects from 2 h of exposure (Fig. 13) in PND 7 rat brain. These findings are consistent with previous studies performed using the same animal model (68). Our data28 indicate that a low dose of isoflurane (0.55%) showed no significant enhancement of apoptotic cell death in the brain. In contrast, relatively high doses of isoflurane (0.75, 1.0, and 1.5%)68 may produce neurodegeneration in multiple brain regions.


Figure 1310
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  		Figure 13. Representative immunocytochemical pictures and quantitative analysis of caspase-3. A combination of anesthetics (75% nitrous oxide + 0.55% isoflurane) resulted in an exposure time-dependent neuronal neurotoxicity in the frontal cortex. No significant enhancement of neurotoxicity was detected from animals treated with this anesthetic regimen for 2 or 4 h. However, a significant increase in the number of caspase-3 positive neuronal cells was noted when the exposure-time reaches 6 or 8 h. Scale bar, 500 µm. For each condition group, at least three animals were randomly assigned. Data are presented as means ± sem. A probability of *P < 0.05 was considered significant (one-way ANOVA).

 

Immature GABA receptors are excitatory early in development and convert to an inhibitory role in mature neurons.83 It can be postulated that prolonged supra-physiologic stimulation of immature neurons by GABA agonists enhances the excitatory component in the action of GABA and may contribute to increased excitability during early development.24 This increased excitability, along with NMDA antagonist-induced alteration of NMDA receptors, could lead to neuronal cell death. Our data28 indicate that a significant effect was observed in the frontal cortex when this low dose of isoflurane (0.55%) was combined with nitrous oxide (75%). Therefore, apoptosis is found in many neuronal populations after a higher dose of isoflurane (0.75%, 1.0%, and 1.5%) combined with nitrous oxide (75%), but is limited to the cortex after a low dose of isoflurane (0.55%) combined with nitrous oxide (75%).

Additive toxicity between a nontoxic concentration of an NMDA antagonist and a GABAmimetic drug is also seen with combinations such as ketamine and midazolam. Additional experimental models (in vitro and in vivo studies of nonhuman primate and rodent models) will be necessary to confirm this conclusion.

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.84 Another important property of this agent is the neutralization of toxic acylCoA production in the mitochondria,85 which correlates with various pathological processes, including organic aciduria86 and numerous diseases of the CNS such as neurodegenerative diseases,87–89 ornithine transcarbamylase deficiency,90,91 and other mitochondrial diseases.85 l-carnitine administration may offer a straightforward approach to mitigating neurotoxic effects. Such studies are underway.

Another group of molecules regulating mitochondrial function and stability is BCL-2. 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.92 Among the potential downstream regulators, the effect of inhaled anesthetics on Bax and BCL-XL has been measured. 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.93 The anesthetic combination [nitrous oxide (75%) with isoflurane (0.55%)] resulted in a significant up-regulation of Bax protein compared with control (Fig. 14), and this effect was blocked by the co-administration of l-carnitine (300 or 500 mg/kg). These data suggested that increased superoxide anion activates an apoptotic pathway involving regulation of many genes, including Bax and BCL-XL.


Figure 1410
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  		Figure 14. 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 ± sem 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.94 It has been 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 the apoptotic cascade activation.95 Yon et al.96 reported that co-administration of an anesthesia cocktail (midazolam, isoflurane, and nitrous oxide) with melatonin reduces the severity of anesthesia-induced damage in the developing rat brain. This study has demonstrated that melatonin provides significant protection against anesthesia-induced neuroapoptotic damage in the developing brain of the immature rats. Although the exact mechanism by which melatonin protects against apoptotic cell death is not known, this study has show that the neuroprotective effect is mediated, at least in part, via mitochondria-dependent apoptotic cascade. It appears that key elements involve the stabilization of the inner mitochondrial membrane, which in turn control cytochrome c release and apoptotic cascade activation. Several mechanisms involved in inner membrane stabilization by melatonin have been suggested. One involves a decrease in mitochondrial protein and DNA damage and the improvement of adenosine triphosphate synthesis by scavenging oxygen97 and nitrogen-based reactants generated in mitochondria, which in turn control the loss of the intra-mitochondrial glutathione.98 Melatonin may also stabilize the inner membrane by increasing the efficiency of the electron transport chain and by controlling the reduction potential.99 In addition, direct action of melatonin on the control of currents through the mitochondrial transition pores100 has been demonstrated. Jevtovic-Todorovic and her colleagues95 have shown that melatonin stabilizes the inner mitochondrial membrane by increasing the protein levels of Bcl-XL. It is most likely that multiple, rather than a single, mechanisms contribute to melatonin-induced restoration of mitochondrial function.

CONCLUSION

Exposures of developing mammals to anesthetics, including those that block NMDA-type glutamate receptors and those that activate GABA receptors, affect the endogenous neuronal transmission systems and enhance neuronal cell death in a dose and developmental stage-dependent manner. This review emphasizes the incorporation of in vivo models with more circumscribed in vitro preparations in the developing rodent and nonhuman primate. These combined models provide the opportunity for the rapid evaluation of anesthetics over a wide range of doses and exposure durations. This combination of models enables 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 loss in simplified nonhuman primate or rodent systems.

In comparing the rodent and the nonhuman primate data, it is clear that the same principles apply: drug exposure (measured as dose, concentration, duration of exposure, or area under the concentration versus time curve) and neuronal developmental stage. The duration of anesthetic exposure to induce cell death, as measured by minimal exposure requirements, is similar (4–6 h) for nonhuman primate and rodent brain cells in culture. The duration of anesthetic exposure is also similar between rodents in vivo (4–6 h) and nonhuman primates (more than 3 h, although less well defined). The susceptible stage or period of development has not been completely described but begins somewhere before the last quarter of pregnancy and continues to shortly after birth in the nonhuman primate. This developmental stage is similar to the period of rapid synaptogenesis in the rodent nervous system that occurs between 1 and 14 days postgestational age.

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 nonhuman primate and 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 elucidate pathways to predict anesthetic-induced brain cell death, and help discover treatments to ameliorate the consequences (if any) of anesthetic toxicity in pediatric patients.

Footnotes

Accepted for publication March 10, 2008.

REFERENCES

  1. Kutsuwada T, Kashiwabuchi N, Mori H, Sakimura D, Kushiya E, Araki K, Meguro H, Masaki H, Kumanishi G, Arakawa M, Mishina M. Molecular diversity of the NMDA receptor channel. Nature 1992;358:36–41[Medline]
  2. Monyer H, Sprengel R, Schoepfer R, Herb A, Higuchi M, Lomeli H, Burnashev N, Sakmann B, Seeburg PH. Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science 1992;256:1217–21[Abstract/Free Full Text]
  3. Moriyoshi K, Masu M, Ishii T, Shigemoto R, Mizuno N, Nakanishi S. Molecular cloning and characterization of the rat NMDA receptor. Nature 1991;354:31–7[Medline]
  4. Nishi M, Hinds H, Lu HP, Kawata M, Hayashi Y. Motoneuron-specific expression of NR3B, a novel NMDA-type glutamate receptor subunit that works in a dominant-negative manner. J Neuroscience 2001;21:RC185[Abstract/Free Full Text]
  5. Wong HK, Liu XB, Matos MF, Chan SF, Perex-Otano I, Boysen M, Cui J, Nakanishi N, Trimmer JS, Jones EG, Lipton SA, Sucher NJ. Temporal and regional expression of NMDA receptor subunit NR3A in the mammalian brain. J Comp Neurol 2002;450:303–17[Web of Science][Medline]
  6. Laurie DJ, Seeburg PH. Regional and developmental heterogeneity in splicing of the rat brain NMDAR1 mRNA. J Neurosci 1994;14:3180–94[Abstract]
  7. Collingridge GL, Kehl SJ, McLennan H. Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus. J Physiol 1983;334:33–46[Abstract/Free Full Text]
  8. D’Souza SW, McConnell SE, Slater P, Barson AJ. Glycine site of the excitatory amino acid N-methyl-D-aspartate receptor in neonate and adult brain. Arch Dis Child 1993;69:212–15[Abstract/Free Full Text]
  9. Beal MF. Mechanisms of excitotoxicity in neurologic diseases. FASEB J 1992;6:3338–44[Abstract]
  10. Scallet A, Schmued LC, Slikker W, Grunberg N, Faustino PJ, Davis H, Lester D, Pine PS, Sistare F, Hanig JP. Developmental neurotoxicity of ketamine: morphometric confirmation, exposure parameters, and multiple fluorescent labeling of apoptotic neurons. Toxicol Sci 2004;81:364–70[Abstract/Free Full Text]
  11. Ben-Ari Y, Khazipov R, Leinekugel X, Caillard O, Gaiarsa JL. GABAA, NMDA and AMPA receptors: developmentally regulated ‘ménage à trois’. TINS 1997;20:523–9[Web of Science][Medline]
  12. Wu WL, Ziskind-Conhaim L, Sweet MA. Early development of glycine- and GABA-mediated synapses in rat spinal cord. J Neuroscience 1992;12:3935–45[Abstract]
  13. Serafini R, Valeyev AY, Barke JL, Poulter MO. Depolarizing GABA-activated Cl- channels in embryonic rat spinal and olfactory bulb cells. J Physiol 1995;15, 488(Pt 2):371–86
  14. Rohrbough J, Spitzer NC. Regulation of intracellular Cl– levels by Na(+)-dependent Cl– cotransport distinguishes depolarizing from hyperpolarizing GABAA receptor-mediated responses in spinal neurons. J Neuroscience 1996;16:82–91[Abstract/Free Full Text]
  15. Obrietan K, van den Pol AN. GABA neurotransmission in the hypothalamus: developmental reversal from Ca2+ elevating to depressing. J Neuroscience 1995;15:5065–77[Abstract]
  16. Connor JA, Tseng H-Y, Hockberger PE. Depolarization- and transmitter-induced changes in intracellular Ca2+ of rat cerebellar granule cells in explant cultures. J Neurosci 1987;7:1384–400[Abstract]
  17. Luhmann HJ, Prince DA. Postnatal maturation of the GABAergic system in rat neocortex. J Neurophysiol 1991;65:247–63[Abstract/Free Full Text]
  18. Ben-Ari Y, Cherubini E, Corradetti R, Gaiarsa JL. Giant synaptic potentials in immature rat CA3 hippocampal neurones. J Physiol 1989;416:303–25[Abstract/Free Full Text]
  19. Mueller AL, Taube JS, Schwartzkroin PA. Development of hyperpolarizing inhibitory postsynaptic potentials and hyperpolarizing response to gamma-aminobutyric acid in rabbit hippocampus studied in vitro. J Neuroscience 1984;4:860–7[Abstract]
  20. Zhang L, Spigelman I, Carlen PL. Development of GABA-mediated, chloride-dependent inhibition in CA1 pyramidal neurones of immature rat hippocampal slices. J Physiol 1991;444:25–49[Abstract/Free Full Text]
  21. Staley K, Smith R, Schaack J, Wilcox C, Jentsch TJ. Alteration of GABAA receptor function following gene transfer of the CLC-2 chloride channel. Neuron 1996;17:543–51[Web of Science][Medline]
  22. Fukuda A, Mody I, Prince DA. Differential ontogenesis of presynaptic and postsynaptic GABAB inhibition in rat somatosensory cortex. J Neurophysiol 1993;70:448–52[Abstract/Free Full Text]
  23. Gaiarsa JL, Tseeb V, Ben-Ari Y. Postnatal development of pre- and postsynaptic GABAB-mediated inhibitions in the CA3 hippocampal region of the rat. J Neurophysiol 1995;73:246–55[Abstract/Free Full Text]
  24. Khazipov R, Khalilov I, Tyzio R, Morozova E, Ben-Ari Y, Holmes GL. Developmental changes in GABAergic actions and seizure susceptibility in the rat hippocampus. Eur J Neurosci 2004;19:590–600[Web of Science][Medline]
  25. Muller D, Wang C, Skibo G, Toni N, Cremer H, Calaora V, Rougon G, Kiss, JZ. PSA-NCAM is required for activity-induced synaptic plasticity. Neuron 1996;17:413–22[Web of Science][Medline]
  26. Wang C, Sadovova N, Fu X, Scallet A, Hanig J, Slikker W. The role of NMDA receptors in ketamine-induced apoptosis in rat forebrain culture. Neuroscience 2005;132:967–77[Web of Science][Medline]
  27. Wang C, Sadovova N, Hotchkiss C, Fu X, Scallet AC, Patterson TA, Hanig J, Paule MG, Slikker W. Blockade of N-methyl-D-aspartate receptors by ketamine produces loss of postnatal day 3 monkey frontal cortical neurons in culture. Toxicol Sci 2006;91:192–201[Abstract/Free Full Text]
  28. Zou X, Sadovova N, Patterson TA, Divine RL, Hotchkiss CE, Ali SF, Hanig JP, Paule MG, Slikker W, Wang C. The effects of L-carnitine on the combination of inhalation anesthetic-induced developmental neuronal apoptosis in the rat frontal cortex. Neuroscience 2008;151:1053–65[Web of Science][Medline]
  29. Komuro H, Rakic P. Modulation of neuronal migration by NMDA receptors. Science 1993;260:95–7[Abstract/Free Full Text]
  30. Williams K, Dichter MA, Molinoff PB. Up-regulation of N-methyl-D-aspartate receptors on cultured cortical neurons after exposure to antagonists. Mol Pharmacol 1992;42:147–51[Abstract]
  31. Rothman DL, Behar KL, Hetherington HP, den Hollander JA, Bendall MR, Petroff OA, Shulman RG. 1H-Observe/13C-decouple spectroscopic measurements of lactate and glutamate in the rat brain in vivo. Proc Natl Acad Sci USA 1985;82:1633–7[Abstract/Free Full Text]
  32. Ankarkona MM, Dybukt JM, Bonfoko E, Zhivotovsky B, Orrenius S, Lipton SA, Nicotera P. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 1995;15:961–73[Web of Science][Medline]
  33. Wang C, Kaufmann JA, Sanchez-Ross MG, Johnson KM. Mechanisms of N-methyl-D-aspartate-induced apoptosis in phencyclidine-treated cultured forebrain neurons. J Pharmacol Exp Ther 2000;294:287–95[Abstract/Free Full Text]
  34. Wang C, Anastasio N, Popov V, LeDay A, Johnson KM. Blockade of N-Methyl-D-aspartate receptors by phencyclidine causes the loss of corticostriatal neurons. Neuroscience 2004;125:473–83[Web of Science][Medline]
  35. Slikker W, Paule MG, Wright LKM, Patterson TA, Wang C. Systems biology approaches for toxicology. J Appl Toxicol 2007;27:201–17[Web of Science][Medline]
  36. Lees GJ. Effects of ketamine on the in vivo toxicity of quinolinate and N-methyl-D-aspartate in the rat hippocampus. Neurosci Lett 1987;78:180–6[Web of Science][Medline]
  37. Beal MF, Kowall NW, Swartz KJ, Ferrante RJ, Martin JB. Systemic approaches to modifying quinolinic acid striatal lesions in rats. J Neurosci 1988;8:3901–8[Abstract]
  38. Keihoff G, Wolf G, Stastny F, Schmidt W. Quinolinate neurotoxicity and glutamatergic structures. Neuroscience 1990;43:235–42
  39. Massieu L, Thedinga KH, Mcvey M, Fagg GE. A comparative analysis of the neuroprotective properties of competitive and uncompetitive N-methyl-D-aspartate receptor in vivo: implications for the process of excitotoxic degeneration and its therapy. Neuroscience 1993;55:883–92[Web of Science][Medline]
  40. Meldrum BS Evans MC, Swan JH, Simon RP. Protection against hypoxic/ischaemic brain damage with excitatory amino acid antagonists. Med Biol 1987;65:153–7[Web of Science][Medline]
  41. Church J, Zeman S, Lodge D. The neuroprotective action of ketamine and MK-801 after transient cerebral ischemia in rats. Epilepsia 1988;31:382–90
  42. Ridenour TR, Wanner DS, Todd MM, Baker MT. Effects of ketamine on outcome from temporary middle cerebral artery occlusion in the spontaneously hypertensive rat. Brain Res 1991;565:116–22[Web of Science][Medline]
  43. White PF, Marietta MP, Pudwill CR, Way WL, Trevor AJ. Effects of halothane anesthesia on the biodisposition of ketamine in rats. J Pharmac Exp Ther 1976;196:545–55[Abstract/Free Full Text]
  44. De Sarro GB, De Sarro A. Anticonvulsant properties of non-competitive antagonists of the N-methyl-D-aspartate receptor in genetically epilepsy-prone rats: comparison with CPPene. Neuropharmacology 1993;32:51–8[Web of Science][Medline]
  45. Simons SHP, ven Dijk M, Anand KJS, Roofthooft D, ven Lingen RA, Tibboel D. Do we still hurt newborn babies? A prospective study of procedural pain and analgesia in neonates. Arch Pediatr Adolesc Med 2003;157:1058–64[Abstract/Free Full Text]
  46. Chahal H, D’Souza SW, Barson AJ, Slater P. Modulation by magnesium of N-methyl-D-aspartate receptors in developing human brain. Arch Dis Childhood Fetal Neonatal Ed 1998;78:F116–F120
  47. Rainowicz T, de Courten-Myers GM, Petetot JM, Xi G, de los Reyes E. Human cortex development: estimates of neuronal numbers indicate major loss late during gestation. J Neuropathol Exp Neurol 1996;55:320–8[Web of Science][Medline]
  48. Anand KJS, Coskum V, Thrivikraman KV, Nemeroff CB, Plotsky PM. Long-term behavioral effects of repetitive pain in neonatal rat pups. Physiol Behav 1999;66:627–37[Medline]
  49. Sternberg WF, Ridgway CG. Effects of gestational stress and neonatal handling on pain, analgesia, and stress behavior of adult mice. Physiol Behav 2003;78:375–83[Medline]
  50. Anand KJS, Runeson B, Jacobson B. Gastric suction at birth associated with long-term risk for functional intestinal disorders in later life. J Pediatr 2004;144:449–54[Web of Science][Medline]
  51. Peters JW, Schouw R, Anand KJS, ven Dijk M, Duivenvoorden HJ, Tibboel D. Does neonatal surgery lead to increased pain sensitivity in later childhood? Pain 2005;114:444–54[Web of Science][Medline]
  52. Grunau RE, Weinberg J, Whitfield MF. Neonatal procedural pain and preterm infant cortisol response to novelty at 8 months. Pediatrics 2004;114:e77–e84[Abstract/Free Full Text]
  53. Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vockler J, Dikranian K, Tenkova TI, Stefovska V, Turski L, Olney JW. Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 1999;283:70–4[Abstract/Free Full Text]
  54. Butler AK, Uryu K, Rougon G, Chesslet MF. N-methyl-D-aspartate receptor blockade affects polysialylated neuronal cell adhesion molecule expression and synaptic density during striatal development. Neuroscience 1999;89:1169–81[Web of Science][Medline]
  55. Breen K, Regan CM. Developmental control of N-CAM sialylation state by Golgi sialyltransferase isoforms. Development 1988;104:147–54[Abstract]
  56. Bruses JL, Rutishauser U. Regulation of neural cell adhesion molecule polysialylation: evidence for nontranscriptional control and sensitivity to an intracellular pool of calcium. J Cell Biol 1998;140:1177–86[Abstract/Free Full Text]
  57. Wang C, Pralong WF, Schulz MF, Rougon G, Aubry JM, Pagliusi S, Robert A, Kiss JZ. Functional N-methyl-D-aspartate receptors in O-2A glial precursor cells: a critical role in regulating polysialic acid-neural cell adhesion molecule expression and cell migration. J Cell Biol 1996;135:1565–81[Abstract/Free Full Text]
  58. Szele FG, Dowling JJ, Gonzales C, Theveniau M, Rougon G, Chesslet MF. Pattern of expression of highly polysialylated neural cell adhesion molecule in the developing and adult rat striatum. Neuroscience 1994;60:133–44[Web of Science][Medline]
  59. Haberny KA, Paule MG, Scallet AC, Sistare FD, Lester DS, Hanig JP, Slikker W Jr. Ontogeny of the N-methyl-D-aspartate (NMDA) receptor system and susceptibility of neurotoxicity. Toxicol Sci 2002;68:9–17[Abstract/Free Full Text]
  60. Slikker W, Zou X, Hotchkiss CE, Divine RL, Sadovova N, Twaddle NC, Doerge DR, Scallet AC, Patterson TA, Hanig J, Paule MG, Wang C. Ketamine-induced neurodegeneration in the perinatal rhesus monkey. Toxicol Sci 2007;98:145–58[Abstract/Free Full Text]
  61. Ellingson A, Haram K, Sagen N, Solheim E. Transplacental passage of ketamine after intravenous administration. Acta Anaesthesiol Scand 1977;21:41–4[Web of Science][Medline]
  62. Grant IS, Nimmo WS, Clements JA. Lack of effect of ketamine analgesia on gastric emptying in man. Br J Anaesth 1981;53:1321–3[Abstract/Free Full Text]
  63. Clements JA, Nimmo WS. Pharmacokinetics and analgesic effect of ketamine in man. Br J Anaesth 1981;53:27–30[Abstract/Free Full Text]
  64. Clancy B, Finlay BL, Darlington RB, Anand KJS. Extrapolating brain development from experimental species to humans. Neurotoxicology 2007;5:931–7
  65. Wang C, Fridley J, Johnson KM. The role of NMDA receptor upreglation in phencyclidine-induced cortical apoptosis in organotypic culture. Biochem Pharmac 2005;69:1373–83[Web of Science][Medline]
  66. Constantine-Paton M. Effects of NMDA receptor antagonists on the developing brain. Psychopharmac Bull 1994;30:561–5
  67. Sceeetz AJ, Constantine-Paton M. Modulation of NMDA receptor function: implications for vertebrate neural development. Fedn Proc Fedn AM Socs Exp Biol 1994;8:745–52
  68. Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat rain and persistent learning deficits. J Neurosci 2003;23:876–82[Abstract/Free Full Text]
  69. Johnson KM, Phillips M, Wang C, Kevetter GA. Chronic phencyclidine induces behavioral sensitization and apoptotic cell death in the olfactory and piriform cortex. J Neurosci Res 1998;52:709–22[Web of Science][Medline]
  70. Hanania T, Hillman GR, Johnson KM. Augmentation of locomotor activity by chronic phencyclidine is associated with an increase in striatal NMDA receptor function and an upregulation of the NR1 receptor subunit. Synapse 1999;31:229–39[Web of Science][Medline]
  71. Wang C, Showalter VM, Jillman GR, Johnson KM. Chronic phencyclidine increases NMDA receptor NR1 subunit mRNA in rat forebrain. J Neurosci Res 1999;55:7762–9
  72. Wang C, McInnis J, West JB, Bao J, Anastasio N, Guidry JA, Ye Y, Salvemini D, Johnson KM. Blockade of phencyclidine-induced cortical apoptosis and deficits in prepulse inhibition by M40403, a superoxide dismutase mimetic. J Pharmacol Exp Ther 2003;304:266–71[Abstract/Free Full Text]
  73. Wang C, McInnis J, Ross-Sanchez M, Shinnick-Gallagher P, Wiley JL, Johnson KM. Long-term behavioral and neurodegenerative effects of perinatal phencyclidine administration: implications for schizophrenia. Neuroscience 2001;107:535–50[Web of Science][Medline]
  74. Groenewegen HJ, Berendse HW, Meredith GE, Haber SN, Voorn P, Wotters JG, Lohman AHM. Functional anatomy of the ventral limbic system-innervated striatum. In: Willner P, Scheel-Kruger J, eds. The mesolimbic dopamine system: from motivation to action. London: John Wiley & Sons; 1991:19–59
  75. Albin R, Makowiec RL, Holligsworth ZR, Dure IVLS, Penny JB, Young AB. Excitatory amino acid binding sites in the basal ganglia of the rat: quantitative autoradiographic study. Neuroscience 1992;46:135–48[Web of Science][Medline]
  76. Zahm DS, Brog JS. On the significance of subterritories in the ‘accumbens’ part of the rat ventral striatum. Neuroscience 1992;50:751–67[Web of Science][Medline]
  77. O’Donnell P, Grace AA. Synaptic interactions among excitatory afferents to nucleus accumbens neurons: hippocampal gating of prefrontal cortical input. J Neuroscience 1995;15:3622–39[Abstract]
  78. Groves PM. A theory of the functional organization of the neostriatum and the neostriatal control of voluntary movement. Brain Res 1983;286:109–32[Medline]
  79. Smith AD, Bolam JP. The neuronal network of the basal ganglia as revealed by the study of synaptic connections of identified neurons. Trends Neurosci 1990;13:259–65[Web of Science][Medline]
  80. Graybiel AM. Neurotransmitters and neuromodulators in the basal ganglia. Trends Neuroscience 1990;13:244–54[Web of Science][Medline]
  81. Kotter R. Postsynaptic integration of glutamatergic and dopaminergic signals in the striatum. Prog Neurobiol 1994;44:163–96[Web of Science][Medline]
  82. Slikker W, Xu Z, Wang C. Application of a systems biology approach to developmental neurotoxicology. Reproductive Toxicol 2005;19:305–19
  83. Ben-Ari Y. Excitatory actions of GABA during development: the nature of the nurture. Nat Rev Neuroscience 2002;3:728–39[Web of Science][Medline]
  84. Bohles H, Evangeliou A, Bervoets K, Eckert I, Sewell A. Carnitine esters in metabolic disease. Eur J Pediatr 1994;153:57–61
  85. Stumpf DA. Mitochondrial disorders. Rinsho Shinkeigaku 1983;23:1046–55[Medline]
  86. Duran M, Loof N, Ketting D, Dorland L. Secondary carnitine deficiency. J Clin Chem 1990;28:359–63
  87. Makar TK, Cooper AJ, Tofel-Grehl B, Thaler HT, Blass JP. Carnitine, carnitine acyltransferase, and glutathione in Alzheimer brain. Neurochem Res 1995;6:705–11
  88. Rubio JC, de Bustos F, Molina JA, Jimenez-Jimenez FJ, Benito-Leo J, Martin MA, Campos Y, Orti-Pareja M, Carbera-Valdivia F, Aren J. Cerebrospinal fluid carnitine levels in patients with Alzheimer disease. J Neurol Sci 1998;155:192–5[Web of Science][Medline]
  89. Orth M, Schapira AH. Mitochondrial involvement in Parkinson’s disease. Neurochem Int 2002;40:533–41[Web of Science][Medline]
  90. Mawal YR, Rama Roa KV, Qureshi IA. Restoration of hepatic cytochrome c oxidase activity and expression with acetyl-L-carnitine treatment in spf mice with ornithine transcarbamylase deficiency. Biochem Phamacol 1998;55:1853–60[Web of Science][Medline]
  91. Michalak A, Butterworth RF. Ornithine transcarbamylase deficiency: pathogenesis of the cerebral disorder and new prospects for therapy. Metab Brain Dis 1997;12:171–82[Web of Science][Medline]
  92. Cory S, Adams JM. The Bcl-2 family: regulators of cellular life-or-death switch. Nat Rev Cancer 2002;2:647–56[Web of Science][Medline]
  93. Cropton M. Bax, bid and the permeabilization of the mitochondrial outer membrane in apoptosis. Curr Opin Cell Biol 2000;12:414–19[Web of Science][Medline]
  94. Reiter RJ, Tan DX, Sainz RM, Mayo JC, Lopez-Burillo S. Melatonin: reducing the toxicity and increasing the efficacy of drugs. J Pharm Pharmacol 2002;54:1299–321[Web of Science][Medline]
  95. Yoo YM, Yim SV, Kim SS, Jang HY, Lea HZ, Jwang GC, Kim JW, Kim SA, Lee AJ, Kim CJ, Chung JH, Lee KH. Melatonin suppresses NO-induced apoptosis via induction of Bcl-2 expression in PGT-β immortalized pineal cells. J Pineal Res 2002;33:146[Web of Science][Medline]
  96. Yon JH, Carter LB, Reiter RJ, Jevtovic-Todorovic V. Melatonin reduces the severity of anesthesia-induced apoptotic neurodegeneration in the developing rat brain. Neurobiol Dis 2006;21:522–30[Web of Science][Medline]
  97. Jou MJ, Peng TI, Reiter RJ, Jou SB, Wu HY, Wen ST. Visualization of the antioxidative effects of melatonin at the mitochondrial level during oxidative stress-induced apoptosis of rat brain astrocytes. J Pineal Res 2004;37:55–70[Web of Science][Medline]
  98. Reiter RJ, Acuna-Castroviejo D, Tan DX, Burkhardt S. Free radical-mediated molecular damage. Mechanisms for the protective actions of melatonin in the central nervous system. Ann N Y Acad Sci 2001;939:200–15[Web of Science][Medline]
  99. Acuna-Carstroviejo D, Martin M, Macias M, Escames G, Leon J, Khaldy H, Reiter RJ. Melatonin, mitochondria, and cellular bioenergetics. J Pineal Res 2001;30:65–74[Web of Science][Medline]
  100. Andrabi SA, Sayeed I, Siemen D, Wolf G, Horn TF. Direct inhibition of the mitochondrial permeability transition pore: a possible mechanism responsible for anti-apoptotic effects of melatonin. FASEB J 2004;18:869–71[Abstract/Free Full Text]



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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2008 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press