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Editorial Views  |   August 2004
Anesthesia-induced Developmental Neuroapoptosis: Does It Happen in Humans?
Author Affiliations & Notes
  • John W. Olney, M.D.
    *
  • Chainllie Young, M.D., Ph.D.
    *
  • David F. Wozniak, Ph.D.
    *
  • Chrysanthy Ikonomidou, M.D., Ph.D.
  • Vesna Jevtovic-Todorovic, M.D., Ph.D.
  • *Department of Psychiatry, Washington University School of Medicine, St. Louis, Missouri. †Department of Pediatric Neurology, Children’s Hospital, Charite-Virchow Clinics, Humboldt University, Berlin, Germany. ‡Department of Anesthesiology, University of Virginia Health System, Charlottsville, Virginia.
Article Information
Editorial Views
Editorial Views   |   August 2004
Anesthesia-induced Developmental Neuroapoptosis: Does It Happen in Humans?
Anesthesiology 8 2004, Vol.101, 273-275. doi:
Anesthesiology 8 2004, Vol.101, 273-275. doi:
RECENTLY, we reported that transient exposure of infant rats or mice to specific classes of drugs, including those that block N  -methyl-d-aspartate glutamate receptors, those that activate γ-aminobutyric acidA(GABAA) receptors, and ethanol (which has both N  -methyl-d- aspartate antagonist and GABAmimetic properties), triggers widespread apoptotic neurodegeneration in the developing brain.1–6 Because ethanol acts by a combination of N  -methyl-d-aspartate/GABAAmutually reinforcing mechanisms, it triggers a particularly robust neuroapoptotic response,2,5 which we propose can explain the neurodevelopmental disturbances associated with the human fetal alcohol syndrome. Most general anesthetics used in pediatric and obstetric medicine have either N  -methyl-d-aspartate antagonist or GABAmimetic properties. Anesthetic cocktails containing drugs from both of these categories are, like ethanol, particularly effective in triggering neuroapoptosis in the developing rodent brain.7 The mechanisms and intracellular pathways that mediate this apoptosis response are depicted in figure 1. The window of vulnerability to these agents coincides with the developmental period of synaptogenesis, also known as the brain growth spurt period, which in mice and rats occurs primarily postnatally but in humans extends from about midgestation to several years after birth.8 Anand and Soriano discuss these findings and conclude that they are “certainly sound, but it may be premature to apply them to clinical settings.”9 
Fig. 1. The mechanism of cell death triggered by ethanol and related drugs involves translocation of Bax protein to mitochondrial membranes,  15 where it disrupts membrane permeability, allowing extramitochondrial leakage of cytochrome c, followed by a sequence of changes culminating in activation of caspase-3.  16,17 The specific steps in the upstream pathways through which the signal is relayed from the cell surface γ-aminobutyric acidA(GABAA) and  N  -methyl-d-aspartate (NMDA) receptors to Bax protein remain to be identified. We propose that the steps causing the cell to become abnormally inhibited precede and are separate from, but interactive with, the series of steps leading to Bax translocation (represented as a row of dominos). When the first domino is toppled, the reaction progresses down the line until it arrives at Bax protein and triggers Bax translocation to the mitochondrial membrane. If methods can be developed for stabilizing one or more dominos, thereby arresting the chain reaction before the Bax protein step, the suicide signal will not be activated. Arresting this chain reaction is an important research goal, as it may allow anesthetic drugs to put neurons to sleep without accidentally triggering a cell suicide signal. 
Fig. 1. The mechanism of cell death triggered by ethanol and related drugs involves translocation of Bax protein to mitochondrial membranes,  15where it disrupts membrane permeability, allowing extramitochondrial leakage of cytochrome c, followed by a sequence of changes culminating in activation of caspase-3.  16,17The specific steps in the upstream pathways through which the signal is relayed from the cell surface γ-aminobutyric acidA(GABAA) and  N  -methyl-d-aspartate (NMDA) receptors to Bax protein remain to be identified. We propose that the steps causing the cell to become abnormally inhibited precede and are separate from, but interactive with, the series of steps leading to Bax translocation (represented as a row of dominos). When the first domino is toppled, the reaction progresses down the line until it arrives at Bax protein and triggers Bax translocation to the mitochondrial membrane. If methods can be developed for stabilizing one or more dominos, thereby arresting the chain reaction before the Bax protein step, the suicide signal will not be activated. Arresting this chain reaction is an important research goal, as it may allow anesthetic drugs to put neurons to sleep without accidentally triggering a cell suicide signal. 
Fig. 1. The mechanism of cell death triggered by ethanol and related drugs involves translocation of Bax protein to mitochondrial membranes,  15 where it disrupts membrane permeability, allowing extramitochondrial leakage of cytochrome c, followed by a sequence of changes culminating in activation of caspase-3.  16,17 The specific steps in the upstream pathways through which the signal is relayed from the cell surface γ-aminobutyric acidA(GABAA) and  N  -methyl-d-aspartate (NMDA) receptors to Bax protein remain to be identified. We propose that the steps causing the cell to become abnormally inhibited precede and are separate from, but interactive with, the series of steps leading to Bax translocation (represented as a row of dominos). When the first domino is toppled, the reaction progresses down the line until it arrives at Bax protein and triggers Bax translocation to the mitochondrial membrane. If methods can be developed for stabilizing one or more dominos, thereby arresting the chain reaction before the Bax protein step, the suicide signal will not be activated. Arresting this chain reaction is an important research goal, as it may allow anesthetic drugs to put neurons to sleep without accidentally triggering a cell suicide signal. 
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Anand and Soriano base much of their reasoning on a single assumption: that it requires extreme conditions for anesthetic drugs to trigger neuroapoptosis in animals. They cite a study in which Soriano et al  .10 reproduced our finding1 that ketamine, if given in repeated doses (extreme condition), triggers neuroapoptosis in the infant rat brain, but they rest their case on the additional finding that single-dose exposure to ketamine (mild condition) did not, in their hands, produce neuroapoptosis.10 In our original study, we did not test single-dose exposure to ketamine, but we subsequently have done so and have found (fig. 2) that a single subanesthetic dose of ketamine triggers a significant (fourfold) increase in neuroapoptosis in the infant rodent brain.11 Therefore, in responding to Anand and Soriano, we must challenge their assumption that it requires extreme conditions for anesthetic drugs to trigger neuroapoptosis.
Fig. 2. In this study  11 we exposed P7 infant mice to a single subcutaneous injection of saline or ketamine at 10, 20, 30, or 40 mg/kg and at 5 h posttreatment counted neurons showing caspase-3 activation in the brain. We found a dose-dependent increase in apoptotic neurodegeneration in the ketamine  versus  saline pups, which was statistically significant at 20, 30, and 40 mg/kg, but marginally nonsignificant at 10 mg/kg. The brain sections depicted here are from the caudate nucleus and are representative of the rate of neuroapoptosis (neurons showing caspase-3 activation) following saline or ketamine at 40 mg/kg. At this dose, the number of apoptotic neurons in the caudate nucleus of ketamine-treated pups was 4.3 times higher than in litter-matched controls, a difference significant at the  P  > 0.0001 level. In separate groups of infant mice we obtained arterial blood samples by cardiac puncture at periodic intervals (0, 15, 30, 60, 120, 180, or 240 min) following ketamine at 40 mg/kg. Throughout this period, blood gases remained within normal limits, including arterial oxygen saturation, which fluctuated from a low of 97 percent to a high of 99 percent. 
Fig. 2. In this study  11we exposed P7 infant mice to a single subcutaneous injection of saline or ketamine at 10, 20, 30, or 40 mg/kg and at 5 h posttreatment counted neurons showing caspase-3 activation in the brain. We found a dose-dependent increase in apoptotic neurodegeneration in the ketamine  versus  saline pups, which was statistically significant at 20, 30, and 40 mg/kg, but marginally nonsignificant at 10 mg/kg. The brain sections depicted here are from the caudate nucleus and are representative of the rate of neuroapoptosis (neurons showing caspase-3 activation) following saline or ketamine at 40 mg/kg. At this dose, the number of apoptotic neurons in the caudate nucleus of ketamine-treated pups was 4.3 times higher than in litter-matched controls, a difference significant at the  P  > 0.0001 level. In separate groups of infant mice we obtained arterial blood samples by cardiac puncture at periodic intervals (0, 15, 30, 60, 120, 180, or 240 min) following ketamine at 40 mg/kg. Throughout this period, blood gases remained within normal limits, including arterial oxygen saturation, which fluctuated from a low of 97 percent to a high of 99 percent. 
Fig. 2. In this study  11 we exposed P7 infant mice to a single subcutaneous injection of saline or ketamine at 10, 20, 30, or 40 mg/kg and at 5 h posttreatment counted neurons showing caspase-3 activation in the brain. We found a dose-dependent increase in apoptotic neurodegeneration in the ketamine  versus  saline pups, which was statistically significant at 20, 30, and 40 mg/kg, but marginally nonsignificant at 10 mg/kg. The brain sections depicted here are from the caudate nucleus and are representative of the rate of neuroapoptosis (neurons showing caspase-3 activation) following saline or ketamine at 40 mg/kg. At this dose, the number of apoptotic neurons in the caudate nucleus of ketamine-treated pups was 4.3 times higher than in litter-matched controls, a difference significant at the  P  > 0.0001 level. In separate groups of infant mice we obtained arterial blood samples by cardiac puncture at periodic intervals (0, 15, 30, 60, 120, 180, or 240 min) following ketamine at 40 mg/kg. Throughout this period, blood gases remained within normal limits, including arterial oxygen saturation, which fluctuated from a low of 97 percent to a high of 99 percent. 
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The discrepancy between our observations and those by Soriano et al.  can be explained, we believe, by differences in methodology. To detect and quantify increases in neuroapoptosis, they applied a silver staining method 24 h after ketamine treatment. Although this is a protocol we previously described for mapping massive patterns of neurodegeneration 24 h after high-dose treatments, we currently recommend a different method (immunohistochemical staining for caspase-3 activation) as the preferred method for quantifying subtle increases in neuroapoptosis that occur at approximately 4–5 h after low-dose treatments. Certain populations of neurons (for example, those in the caudate nucleus) are extremely sensitive to ketamine, and these neurons begin showing cell death commitment (caspase-3 activation) within 3 h following a single dose of ketamine. Silver staining will also detect these neurons if applied at 7–8 h but not if applied at 24 h because by then these early-dying neurons have degenerated into nebulous debris. Although it will be important for others to test the reproducibility of our findings, we will assume for the purposes of the present discussion that the findings are reproducible.
Anand and Soriano have suggested that the apoptotic neurodegeneration we have reported in infant rodents treated with anesthetic drugs is caused by hypoxia/ischemia that putatively occurs because cardiorespiratory functions are not being adequately monitored and controlled. However, in infant mice we have measured arterial blood gases at periodic intervals after administration of ketamine at a dose that triggers neuroapoptosis (fig. 2) and have found that all blood gas values, including arterial oxygen saturation, remain in a normal range throughout the posttreatment observation period.11 In addition, a flaw in the hypoxia/ischemia argument is that when one intentionally induces hypoxic/ischemic neurodegeneration in the infant rodent brain, the acute cell death that ensues is not apoptotic. It is excitotoxic, and ultrastructurally does not resemble apoptosis.12–14 In contrast, the acute cell death response to ethanol or anesthetic or antiepileptic drugs is decidedly apoptotic and ultrastructurally does not resemble the acute excitotoxic cell death response that typifies hypoxia/ischemia.3,5,13,14 
Anand and Soriano propose that the adverse effects we attribute to anesthesia exposure in infant rodents can be explained by a disturbance in nutritional status. This is not a tenable argument. At the beginning of the experiment, our infant rodents have a belly full of milk (clearly visible through the abdominal wall). In a typical experiment, we treat rat or mouse pups subcutaneously, the controls with saline and the experimentals with ethanol or an anesthetic drug, and all animals are sacrificed 4 to 8 h later for histologic evaluation of the brains. Both controls and experimentals are exposed to the same degree of nutritional and maternal deprivation; both are removed from the maternal cage and are maintained at normal body temperature in a compartment separate from their mother for the duration of the experiment. There is no rational basis for invoking nutritional factors to explain the robust pattern of neuroapoptosis that consistently shows up in the experimental brains and not in the controls.
Anand and Soriano suggest that because the life span is much longer in humans than in rodents, it might require a much longer exposure time (weeks) for a toxic chemical to induce neuroapoptosis in the developing human brain compared to a brief period (hours) in the rodent brain. They cite a theoretical treatise, but no evidence, to support this conjecture. The life span argument in its fully developed form is as follows: Disruption of synaptogenesis is the proposed mechanism by which anesthetic drugs trigger neuroapoptosis. In the rodent, synaptogenesis is completed within a period of weeks, whereas in the human it is completed within a period of several years. Neurons are programmed to commit suicide if their synaptic mission is thwarted to some critical degree. It may be argued that for the rodent neuron, 2 h of disruption exceeds the critical limit, whereas for the human neuron the critical limit may be much longer. As there are other species, including nonhuman primates, that have more prolonged synaptogenesis periods, this hypothesis should be tested in such species, the sooner the better.
This brings us to the very difficult question, how can we know whether anesthetic drugs do or do not trigger neuroapoptosis in the developing human brain? Rodent data provide an imprecise basis at best, and an irrelevant basis at worst, for evaluating human risk. An important next step, therefore, would be to conduct well designed nonhuman primate studies. Serious consideration should also be given to conducting autopsy studies in which the brains of human neonates who have died on the surgical table after prolonged anesthesia would be compared with the brains of neonates who have died from other causes in the absence of anesthesia. Obtaining the brains immediately after death would be important to ensure successful application of special stains and related assays for diagnosing acute neuroapoptosis.
Department of Psychiatry, Washington University School of Medicine, St. Louis, Missouri. Department of Pediatric Neurology, Children’s Hospital, Charite-Virchow Clinics, Humboldt University, Berlin, Germany. Department of Anesthesiology, University of Virginia Health System, Charlottsville, Virginia.
References
Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vockler J, Dikranian K, Stefovska V, Turski L, Olney JW: Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 1999; 283:70–4Ikonomidou, C Bosch, F Miksa, M Bittigau, P Vockler, J Dikranian, K Stefovska, V Turski, L Olney, JW
Ikonomidou C, Bittigau P, Ishimaru MJ, Wozniak DF, Koch C, Genz K, Price MT, Stefovska V, Horster F, Tenkova T, Dikranian K, Olney JW: Ethanol-induced apoptotic neurodegeneration and fetal alcohol syndrome. Science 2000; 287:1056–60Ikonomidou, C Bittigau, P Ishimaru, MJ Wozniak, DF Koch, C Genz, K Price, MT Stefovska, V Horster, F Tenkova, T Dikranian, K Olney, JW
Dikranian K, Ishimaru MJ, Tenkova T, Labruyere J, Qin YQ, Ikonomidou C, Olney JW: Apoptosis in the in vivo  mammalian forebrain. Neurobiol Dis 2001; 8:359–79Dikranian, K Ishimaru, MJ Tenkova, T Labruyere, J Qin, YQ Ikonomidou, C Olney, JW
Bittigau P, Sifringer M, Genz K, Reith E, Pospischil D, Govindarajalu S, Dzietko M, Pesditschek S, Mai I, Dikranian K, Olney JW, Ikonomidou C: Antiepileptic drugs and apoptotic neurodegeneration in the developing brain. Proc Nat Acad Sci U S A 2002; 99:15089–94Bittigau, P Sifringer, M Genz, K Reith, E Pospischil, D Govindarajalu, S Dzietko, M Pesditschek, S Mai, I Dikranian, K Olney, JW Ikonomidou, C
Olney JW, Tenkova T, Dikranian K, Qin YQ, Labruyere J, Ikonomidou C: Ethanol-induced apoptotic neurodegeneration in the developing C57BL/6 mouse brain. Dev Brain Res 2002; 133:115–26Olney, JW Tenkova, T Dikranian, K Qin, YQ Labruyere, J Ikonomidou, C
Olney JW, Wozniak DF, Jevtovic-Todorovic V, Farber NB, Bittigau P, Ikonomidou C: Drug-induced neurodegeneration in the developing brain. Brain Pathol 2002; 12:1–11Olney, JW Wozniak, DF Jevtovic-Todorovic, V Farber, NB Bittigau, P Ikonomidou, C
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 brain and persistent learning deficits. J Neurosci 2003; 23:876–82Jevtovic-Todorovic, V Hartman, RE Izumi, Y Benshoff, ND Dikranian, K Zorumski, CF Olney, JW Wozniak, DF
Dobbing J, Sands J: Comparative aspects of the brain growth spurt. Early Hum Dev, 1979; 3:79–83Dobbing, J Sands, J
Anand KJS, Soriano SG: Anesthetic agents and the immature brain: Are these toxic or therapeutic? Anesthesiology 2004; 101:527–30Anand, KJS Soriano, SG
Hayashi H, Dikkes P, Soriano SG: Repeated administration of ketamine may lead to neuronal degeneration in the developing rat brain. Paediatr Anaesth 2002; 12:770–4Hayashi, H Dikkes, P Soriano, SG
Young C, Tenkova T, Wang H, Qin Y, Labruyere J, Jevtovic-Todorovic V, Olney JW: A single sedating dose of ketamine causes neuronal apoptosis in developing mouse brain. Program No. 748.6. 2003 Abstract Viewer/Itinerary Planner  . Washington, DC: Society for Neuroscience, 2003Young, C Tenkova, T Wang, H Qin, Y Labruyere, J Jevtovic-Todorovic, V Olney, JW Washington, DC Society for Neuroscience
Ikonomidou C, Price MT, Mosinger JL, Frierdich G, Labruyere J, Shahid Salles K, Olney JW: Hypobaric-ischemic conditions produce glutamate-like cytopathology in infant rat brain. J Neurosci 1989; 9:1693–700Ikonomidou, C Price, MT Mosinger, JL Frierdich, G Labruyere, J Shahid Salles, K Olney, JW
Ishimaru MJ, Ikonomidou C, Tenkova TI, Der TC, Dikranian K, Sesma MA, Olney JW: Distinguishing excitotoxic from apoptotic neurodegeneration in the developing rat brain. J Comp Neurol 1999; 408:461–76Ishimaru, MJ Ikonomidou, C Tenkova, TI Der, TC Dikranian, K Sesma, MA Olney, JW
Young C, Tenkova T, Dikranian K, Labruyere J, Olney JW: Excitotoxic versus apoptotic mechanisms of neuronal cell death in perinatal hypoxia/ischemia. Curr Mol Med 2004; 4:77–85Young, C Tenkova, T Dikranian, K Labruyere, J Olney, JW
Young C, Klocke B, Tenkova T, Choi J, Labruyere J, Qin Q, Holtzman D, Roth K, Olney JW: Ethanol-induced neuronal apoptosis in the in vivo  developing mouse brain is BAX dependent. Cell Death Differ 2003; 10:1148–55Young, C Klocke, B Tenkova, T Choi, J Labruyere, J Qin, Q Holtzman, D Roth, K Olney, JW
Olney JW, Tenkova T, Dikranian K, Muglia LJ, Jermakowicz WJ, D’Sa C, Roth KA: Ethanol-induced caspase-3 activation in the in vivo  developing mouse brain. Neurobiol Dis 2002; 9:205–19Olney, JW Tenkova, T Dikranian, K Muglia, LJ Jermakowicz, WJ D’Sa, C Roth, KA
Young C, Olney JW, Klocke BJ, Labruyere J, Qin YQ, Roth KA: Absence of caspase-3 gene does not prevent but alters time course and morphological expression of ethanol-induced apoptotic neurodegeneration. Program No. 303.6. 2002 Abstract Viewer/Itinerary Planner  . Washington, DC: Society for Neuroscience, 2002Young, C Olney, JW Klocke, BJ Labruyere, J Qin, YQ Roth, KA Washington, DC Society for Neuroscience
Fig. 1. The mechanism of cell death triggered by ethanol and related drugs involves translocation of Bax protein to mitochondrial membranes,  15 where it disrupts membrane permeability, allowing extramitochondrial leakage of cytochrome c, followed by a sequence of changes culminating in activation of caspase-3.  16,17 The specific steps in the upstream pathways through which the signal is relayed from the cell surface γ-aminobutyric acidA(GABAA) and  N  -methyl-d-aspartate (NMDA) receptors to Bax protein remain to be identified. We propose that the steps causing the cell to become abnormally inhibited precede and are separate from, but interactive with, the series of steps leading to Bax translocation (represented as a row of dominos). When the first domino is toppled, the reaction progresses down the line until it arrives at Bax protein and triggers Bax translocation to the mitochondrial membrane. If methods can be developed for stabilizing one or more dominos, thereby arresting the chain reaction before the Bax protein step, the suicide signal will not be activated. Arresting this chain reaction is an important research goal, as it may allow anesthetic drugs to put neurons to sleep without accidentally triggering a cell suicide signal. 
Fig. 1. The mechanism of cell death triggered by ethanol and related drugs involves translocation of Bax protein to mitochondrial membranes,  15where it disrupts membrane permeability, allowing extramitochondrial leakage of cytochrome c, followed by a sequence of changes culminating in activation of caspase-3.  16,17The specific steps in the upstream pathways through which the signal is relayed from the cell surface γ-aminobutyric acidA(GABAA) and  N  -methyl-d-aspartate (NMDA) receptors to Bax protein remain to be identified. We propose that the steps causing the cell to become abnormally inhibited precede and are separate from, but interactive with, the series of steps leading to Bax translocation (represented as a row of dominos). When the first domino is toppled, the reaction progresses down the line until it arrives at Bax protein and triggers Bax translocation to the mitochondrial membrane. If methods can be developed for stabilizing one or more dominos, thereby arresting the chain reaction before the Bax protein step, the suicide signal will not be activated. Arresting this chain reaction is an important research goal, as it may allow anesthetic drugs to put neurons to sleep without accidentally triggering a cell suicide signal. 
Fig. 1. The mechanism of cell death triggered by ethanol and related drugs involves translocation of Bax protein to mitochondrial membranes,  15 where it disrupts membrane permeability, allowing extramitochondrial leakage of cytochrome c, followed by a sequence of changes culminating in activation of caspase-3.  16,17 The specific steps in the upstream pathways through which the signal is relayed from the cell surface γ-aminobutyric acidA(GABAA) and  N  -methyl-d-aspartate (NMDA) receptors to Bax protein remain to be identified. We propose that the steps causing the cell to become abnormally inhibited precede and are separate from, but interactive with, the series of steps leading to Bax translocation (represented as a row of dominos). When the first domino is toppled, the reaction progresses down the line until it arrives at Bax protein and triggers Bax translocation to the mitochondrial membrane. If methods can be developed for stabilizing one or more dominos, thereby arresting the chain reaction before the Bax protein step, the suicide signal will not be activated. Arresting this chain reaction is an important research goal, as it may allow anesthetic drugs to put neurons to sleep without accidentally triggering a cell suicide signal. 
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Fig. 2. In this study  11 we exposed P7 infant mice to a single subcutaneous injection of saline or ketamine at 10, 20, 30, or 40 mg/kg and at 5 h posttreatment counted neurons showing caspase-3 activation in the brain. We found a dose-dependent increase in apoptotic neurodegeneration in the ketamine  versus  saline pups, which was statistically significant at 20, 30, and 40 mg/kg, but marginally nonsignificant at 10 mg/kg. The brain sections depicted here are from the caudate nucleus and are representative of the rate of neuroapoptosis (neurons showing caspase-3 activation) following saline or ketamine at 40 mg/kg. At this dose, the number of apoptotic neurons in the caudate nucleus of ketamine-treated pups was 4.3 times higher than in litter-matched controls, a difference significant at the  P  > 0.0001 level. In separate groups of infant mice we obtained arterial blood samples by cardiac puncture at periodic intervals (0, 15, 30, 60, 120, 180, or 240 min) following ketamine at 40 mg/kg. Throughout this period, blood gases remained within normal limits, including arterial oxygen saturation, which fluctuated from a low of 97 percent to a high of 99 percent. 
Fig. 2. In this study  11we exposed P7 infant mice to a single subcutaneous injection of saline or ketamine at 10, 20, 30, or 40 mg/kg and at 5 h posttreatment counted neurons showing caspase-3 activation in the brain. We found a dose-dependent increase in apoptotic neurodegeneration in the ketamine  versus  saline pups, which was statistically significant at 20, 30, and 40 mg/kg, but marginally nonsignificant at 10 mg/kg. The brain sections depicted here are from the caudate nucleus and are representative of the rate of neuroapoptosis (neurons showing caspase-3 activation) following saline or ketamine at 40 mg/kg. At this dose, the number of apoptotic neurons in the caudate nucleus of ketamine-treated pups was 4.3 times higher than in litter-matched controls, a difference significant at the  P  > 0.0001 level. In separate groups of infant mice we obtained arterial blood samples by cardiac puncture at periodic intervals (0, 15, 30, 60, 120, 180, or 240 min) following ketamine at 40 mg/kg. Throughout this period, blood gases remained within normal limits, including arterial oxygen saturation, which fluctuated from a low of 97 percent to a high of 99 percent. 
Fig. 2. In this study  11 we exposed P7 infant mice to a single subcutaneous injection of saline or ketamine at 10, 20, 30, or 40 mg/kg and at 5 h posttreatment counted neurons showing caspase-3 activation in the brain. We found a dose-dependent increase in apoptotic neurodegeneration in the ketamine  versus  saline pups, which was statistically significant at 20, 30, and 40 mg/kg, but marginally nonsignificant at 10 mg/kg. The brain sections depicted here are from the caudate nucleus and are representative of the rate of neuroapoptosis (neurons showing caspase-3 activation) following saline or ketamine at 40 mg/kg. At this dose, the number of apoptotic neurons in the caudate nucleus of ketamine-treated pups was 4.3 times higher than in litter-matched controls, a difference significant at the  P  > 0.0001 level. In separate groups of infant mice we obtained arterial blood samples by cardiac puncture at periodic intervals (0, 15, 30, 60, 120, 180, or 240 min) following ketamine at 40 mg/kg. Throughout this period, blood gases remained within normal limits, including arterial oxygen saturation, which fluctuated from a low of 97 percent to a high of 99 percent. 
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