Hypoxia induces an excitotoxic-type of dark cell degeneration in cerebellar Purkinje neurons

Abstract

In the rat cerebellar slice preparation, exposure to hypoxia elicited by a 30 min exposure to artificial cerebrospinal fluid continuously gassed with 95% N(2): 5% CO(2) induced a characteristic type of toxicity of Purkinje cells (PCs) resembling excitotoxic-mediated dark cell degeneration (DCD). Morphologically, PCs exhibited marked rounded appearance with cytoplasmic darkening, nuclear condensation and cytoplasmic vacuoles. Using gel electrophoresis, genomic DNA obtained from the cerebellar slice exhibited fragmentation. However, PCs failed to exhibit apoptotic bodies or evidence of phagocytosis, spherical- or crescent-shaped chromatin aggregations or TUNEL-positive staining. Ultrastructural analyses of granule cells revealed the presence of apoptotic bodies and discrete spherical collection of chromatin clumping as well as phagocytosis suggesting that the oligonucleosomal-sized DNA fragments primarily were derived from granule cells. PC-elicited toxicity was attenuated significantly in the presence of the competitive AMPA and NMDA antagonists CNQX and APV, respectively. The present study extends the involvement of excitotoxic processes in mediating hypoxic-induced toxicity of PCs in postnatal rats and suggests, in contrast to DCD elicited by direct application of excitotoxic agents, that DCD associated with acute hypoxic insults in PCs does not resemble classical apoptosis.