Anesthesia-induced Developmental Neuroapoptosis

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.1–6Because ethanol acts by a combination of N -methyl-d-aspartate/GABAAmutually reinforcing mechanisms, it triggers a particularly robust neuroapoptotic response,2,5which 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.7The 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.8Anand and Soriano discuss these findings and conclude that they are “certainly sound, but it may be premature to apply them to clinical settings.”9Anand 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 .10reproduced our finding1that 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.10In 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.11Therefore, in responding to Anand and Soriano, we must challenge their assumption that it requires extreme conditions for anesthetic drugs to trigger neuroapoptosis.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.11In 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.12–14In 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.3,5,13,14Anand 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.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. olneyj@wustl.edu