The role of oxidative stress and caspase activation in ethanol-induced cell loss in the developing rat cerebellum

Microencephaly, a hallmark feature for the clinical diagnosis of Fetal Alcohol Syndrome, results from decreases in neuronal cell populations. Cerebellar Purkinje cells are most vulnerable to ethanol-induced cell loss when the exposure occurs on postnatal days 4--6. Oxidative stress and caspase-dependent apoptotic cell loss have been posited to play a role in ethanol neurotoxicity. The purpose of this dissertation was to test the hypothesis that a single, binge-like ethanol exposure given during a period of development known to be vulnerable to ethanol-induced Purkinje cell loss, would induce oxidative stress and increase caspase-3 activation within the rat cerebellum, whereas a similar exposure five days later would not induce these processes. Long Evans rat pups were matched for sex and rats within each pair received either control intubations or 5.25 g/kg ethanol delivered in two 11.9% (v/v) intubations, two hours apart. Results indicated that ethanol exposure on postnatal day 4, but not postnatal day 9 significantly increased cerebellar reactive oxygen species levels. Elevations in reactive oxygen species were only evident 12 hours after the initial ethanol exposure and occurred in the absence of decreases in total antioxidant capability and protective antioxidants such as glutathione. In addition, ethanol exposure on postnatal day 4 significantly increased both caspase-3 active subunit expression (as early as 4 hours) and activity (at 6 and 12 hours) within the cerebellum, an effect that was not seen following exposure on postnatal day 9. Increases in caspase-3 active subunit expression were dose-dependent in nature and were not prevented by pretreatment with EGCG. Immunohistochemistry revealed caspase-3 active subunit localization within the cerebellar Purkinje cells with linear density measurements demonstrating significant ethanol-induced increases 6 and 8 hours after exposure was initiated. Since increases in caspase-3 activity occurred earlier than elevations in reactive oxygen species (6 hours vs. 12 hours), it appears that caspase-3 activation is not triggered by oxidative stress. Collectively, these data suggest that ethanol-induced increases in reactive oxygen species may be a consequence of cerebellar cell pathology initiated by cell death cascades, rather than a mechanism underlying the initiation of that pathogenesis.