Spatiotemporal evolution of apoptotic neurodegeneration following traumatic injury to the developing rat brain.

Closed head injury to the developing rat brain causes an acute excitotoxic lesion and axonal disruption at the impact site followed by a delayed pattern of apoptotic damage at various distant sites. Using an electromagnetic impact device to deliver a precisely controlled degree of mechanical deformation to the P7 infant rat skull, we studied the distribution of distant apoptotic lesions and the sequence and time course with which these lesions evolve following relatively mild closed head injury. The first major wave of apoptotic neurodegeneration occurred at 8 h postimpact in the retrosplenial cortex and pre- and parasubiculum. The next major wave occurred in the 16- to 24-h interval and was localized to the anterior thalamic nuclei. A third wave was detected at 36 to 48 h in the mammillary nuclei. We propose that the first and second waves were triggered by injury to a specific fiber tract, the corpus callosum/cingulum bundle that conveys reciprocal connections between the anterior thalamic nuclei and retrosplenial/pre- and parasubicular neurons. This fiber tract passes through a zone of maximum mechanical strain, as measured by tagged MRI. The third wave affecting mammillary neurons occurred because the principal synaptic targets of these neurons are the anterior thalamic neurons that were destroyed in the second wave of degeneration. Prevention of these apoptotic waves of brain damage is a realistic goal in view of the long delay between the impact event and onset of apoptotic degeneration.

Potential of ketamine and midazolam, individually or in combination, to induce apoptotic neurodegeneration in the infant mouse brain.

Recently, it was reported that anesthetizing infant rats for 6 h with a combination of anesthetic drugs (midazolam, nitrous oxide, isoflurane) caused widespread apoptotic neurodegeneration in the developing brain, followed by lifelong cognitive deficits. It has also been reported that ketamine triggers neuroapoptosis in the infant rat brain if administered repeatedly over a period of 9 h. The question arises whether less extreme exposure to anesthetic drugs can also trigger neuroapoptosis in the developing brain. To address this question we administered ketamine, midazolam or ketamine plus midazolam subcutaneously at various doses to infant mice and evaluated the rate of neuroapoptosis in various brain regions following either saline or these various drug treatments. Each drug was administered as a single one-time injection in a dose range that would be considered subanesthetic, and the brains were evaluated by unbiased stereology methods 5 h following drug treatment. Neuroapoptosis was detected by immunohistochemical staining for activated caspase-3. It was found that either ketamine or midazolam caused a dose-dependent, statistically significant increase in the rate of neuroapoptosis, and the two drugs combined caused a greater increase than either drug alone. The apoptotic nature of the neurodegenerative reaction was confirmed by electron microscopy. We conclude that relatively mild exposure to ketamine, midazolam or a combination of these drugs can trigger apoptotic neurodegeneration in the developing mouse brain.

Role of caspase-3 in ethanol-induced developmental neurodegeneration.

Acute, transient exposure to ethanol causes a widespread pattern of caspase-3 activation and neuroapoptosis in the developing rodent brain. To determine whether caspase-3 activation is an essential step in ethanol-induced developmental neuroapoptosis, we treated homozygous caspase-3 knockout mice or wild-type mice on postnatal day 7 with an apoptosis-inducing dose of ethanol and examined the brains at appropriate survival times for evidence of apoptotic neurodegeneration. In caspase-3 knockout mice, the cell death process evolved more slowly than in wild-type mice, and morphological changes observed were not those typically associated with apoptosis. However, neuronal cell counts performed 2 weeks post-treatment revealed that the extent of neuron loss was similar in wild-type and caspase-3-deficient mice. We conclude that absence of functional caspase-3 alters the time course and morphological characteristics of the neurodegenerative process but does not prevent ethanol-induced neuron death.

Ethanol-induced neuroapoptosis in the developing rodent cerebellum and related brain stem structures.

For three decades since the fetal alcohol syndrome (FAS) was first described, researchers have been keenly interested in understanding the mechanism(s) by which ethanol damages or disrupts development of the human fetal brain. It has been reported repeatedly that exposure of infant rats to ethanol causes a reduction in brain mass and loss of cerebellar Purkinje cells, but the mechanisms underlying these effects have remained elusive. In a recent series of studies, we have demonstrated that exposure of infant rats or mice to ethanol on a single occasion during the synaptogenesis period of development causes neurons in many regions of the developing central nervous system to commit suicide (die by apoptosis), but the cerebellum was not among the brain regions focused upon in these studies. Here we show in infant rats and mice that one-time exposure to ethanol triggers acute neurodegeneration of Purkinje cells and other neurons in the cerebellar cortex, deep cerebellar nuclei, and two related brainstem nuclei (nucleus pontis, inferior olivary complex). We also describe the time course of neurodegeneration and window of vulnerability for each of these neuronal cell types and demonstrate that the cell death process in each case is unequivocally apoptotic. We conclude that exposure of infant rats or mice to ethanol on a single occasion during synaptogenesis can kill Purkinje cells, and many other neuronal populations at all levels of the developing neuraxis, and in each case the mechanism of cell death is apoptosis.

Ethanol-induced apoptotic neurodegeneration in the developing C57BL/6 mouse brain.

Recent studies have shown that administration of ethanol to infant rats during the synaptogenesis period (first 2 weeks after birth), triggers extensive apoptotic neurodegeneration throughout many regions of the developing brain. While synaptogenesis is largely a postnatal phenomenon in rats, it occurs prenatally (last trimester of pregnancy) in humans. Recent evidence strongly supports the interpretation that ethanol exerts its apoptogenic action by a dual mechanism--blockade of NMDA glutamate receptors and hyperactivation of GABA(A) receptors. These findings in immature rats represent a significant advance in the fetal alcohol research field, in that previous in vivo animal studies had not demonstrated an apoptogenic action of ethanol, had not documented ethanol-induced cell loss from more than a very few brain regions and had not provided penetrating insight into the mechanisms underlying ethanol's neurotoxic action. To add to the mechanistic insights recently gained, it would be desirable to examine gene-regulated aspects of ethanol-induced apoptotic neurodegeneration, using genetically altered strains of mice. The feasibility of such research must first be established by demonstrating that appropriate mouse strains are sensitive to this neurotoxic mechanism. In the present study, we demonstrate that mice of the C57BL/6 strain, a strain frequently used in transgenic and gene deletion research, are exquisitely sensitive to the mechanism by which ethanol induces apoptotic neurodegeneration during the synaptogenesis period of development.