Mechanism of ethanol-induced Purkinje cell death in developing rat cerebellum: Its implication in apoptosis and oxidative damage

Abstract

Ethanol treatment during the brain growth spurt period has been known to induce the death of Purkinje cells. The underlying molecular mechanisms and the role of reactive oxygen species (ROS) in triggering ethano-induced Purkinje cell death are，however， largely unresolved. We underlook TUNEL staining，Eestern blotting assay and immunohistochemistry for the cleaved Forms of caspase-3 and -9 ，with calbindin D28K double immunostaining to identify apoptotic Purkinje cells. The possibility of ROS-induced Purkinje cell death was immunohistochemically determined by using anti-8-hydroxy-2'--deoxyguanosine (8-OHdG), a specific cellular marker for oxidative damage. The results show thal Purkinje cell death of PD 5 rat cerebellum following ethanol administration is mediated by the activation of caspase-3 and -9. However，unexpectedly，TUNEL staining did not reveal any positive Purkinje cells while there were some TUNEL-positive cells in the internal and external granular layer. 8-OHdG was detected in the Purkinje cell layers at 8 h，peaked al 12-24 h，but not at 30 h post-Ethanol treatment. No 8-0HdG immunoreactive cells were detected in the internal and external granular layer. The lobule-specific S-QHdG staining patterns following ethanol exposure are consistent with thal of ethanol-induced Purkinje cell loss. Thus. we suggest that ethanol-induced Purkinje cell death may not occur by the classical apoptotic pathway and oxidative damage is involved in ethanol-induced Purkinje cell death in the developing cerebellum.