Developmental difference in the expression of neuronal damage following simulated hypoxia with cerebellar Purkinje cells

Morphological changes leading to neuronal cell death following a hypoxic insult in the brain are unclear. Cerebellar Purkinje cells (PCs) have been shown to exhibit selective vulnerability to hypoxia. In previous studies, PCs, in adult rats exposed to hypoxia, have shown a necrotic morphology, characterized by swelling of the cells, loss of membrane integrity, and lysis. In contrast, morphological changes in neonatal PCs following hypoxic injury consisted of cytoplasmic darkening and microvacuolization. This change, known previously as coagulative necrosis, has since been referred to as "dark cell degeneration" (DCD). The intent of the present study was to identify and characterize the mode of toxicity in 9-day-old and 21-day-old rat cerebellar Purkinje cells following hypoxic injury. Subsequent studies were designed to determine whether h5^oxic-induced DCD in PCs from 9-day-old rats represented an apoptotic event.

Sagittal cerebellar slice preparation was used in the hypoxic model. Treated slices were submerged in Acsf and exposed to 30' of hypoxia (95% N2, 5% CO2) followed by 60' and 120' reoxygenation periods. The handling control slices were submerged in Acsf and exposed to an oxygenated environment (95% O2, 5% CO2) followed by reoxygenation periods. A Toluidine blue stain at the light microscopic level was used to characterize the morphological differences in the younger and older PCs. To identify whether DCD occurring in 9-day-old rat PCs is related to apoptosis, the following analyses were performed: DNA fragmentation using gel electrophoresis and in situ DNA labeling (TUNEL) and chromatin condensation observed with Hoechst nuclear stain.

Purkinje cells stained with Toluidine blue exhibited 67%(±8) DCD and 10% (±2) necrosis in the 9-day-old rats compared to 70%(±12) necrosis and 30%(±5) DCD in the 21-day-old rats. PCs from 9-day-old rat slices displayed shrunken, rounded appearance with darkened nuclei and vacuolated cytoplasm, all morphological characteristics of apoptosis. In addition to morphological similarities to apoptosis, PCs from the 9-day-old rat showed with Hoechst staining, chromatin clumping. Using DNA gel electrophoresis, hypoxic-treated cerebellar slices exhibited DNA laddering upon the formation of oligonucleosomal-sized fragments of DNA (180-200bp) TUNEL assay which provides cellular specificity of the apoptotic activity in the cerebellar slice showed limited staining in the nucleus of the PCs of the treated slice.

From the present findings, brain maturity influences the morphological phenotype of neurodegeneration of the Purkinje cells in response to a hypoxic insult. Based upon the lack of TUNEL staining in the PC nuclei, the hypoxic-induced DCD in PCs does not appear to resemble a classic type of apoptosis.