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Ischemia induced cell death in cultured astrocytes : evidence of apoptosis

Authors Yung, Hong Wa
Issue Date 1998
Summary Ischemia is known to induce cell death in the nervous system. Among the neural cells, astrocytes are more tolerant to ischemic insult than neurons and in a longer period of ischemia. Astrocytes would be damaged and killed. The mechanisms involved in inducing astrocytic cell damage and death remains unclear. In this study, an in vitro ischemic model using primary astrocytic culture incubated in an anaerobic chamber was employed to investigate the mode of astrocytic cell death under ischemia. Phase contrast and electron microscopy studies showed chromatin condensation, nuclear shrinkage, cytoplasmic vacuolation and mitochondria swelling accompanied ischemia induced astrocytic death. Cell viability assay indicated a dramatic cell death from 4 (~3%) to 8 hours (~95%) of ischemia. TdT-mediated dUTP nick end labeling (TUNEL) staining showed the cell death was associated with extensive DNA fragmentation. This observation was confirmed by laddered DNA patterns shown by electrophoresis in cultures after 5 hours of ischemia. The treatment with actinomycin D and cycloheximide reduced cell death by ~60% and ~70%, respectively. Their corresponding TUNEL staining were also reduced by ~80% at 6 hours of ischemia. Attempting to elucidate the molecular mechanism involved in the regulation of cell death, we studied the expression of apoptosis-related genes in the presence and absence of these metabolic inhibitors. Quantitative reverse transcription- polymerase chain reaction was utilized to quantify the expression of bcl-2 α and β, bax, and Ice with an endogenous internal control, 18s ribosomal RNA. Results showed that bcl-2 α and β, and bax transcripts were down-regulated, and Ice level was reduced from 0 to 4 hours then gradually increased to control level at 8 hours of ischemia. With actinomycin D treatment, all the genes studied were at a level lower than the control. In the presence of cycloheximide, bcl-2 α and β, levels were lower than the control, while bax and Ice were several times higher. To investigate whether other genes were induced by ischemia in injured astrocytes, random arbitrary-primed- polymerase chain reaction was utilized to investigate the gene expression profile at 4 hours of ischemia. A total of 16 genes were identified with about 85 different arbitrary primer combinations. The results strongly indicated that ischemia is capable of inducing apoptosis in cultured astrocytes. The delay of ischemia-induced cell death in astrocytes with metabolic inhibitors supported astrocyte undergoing apoptosis in ischemia involved RNA and protein synthesis, a process completely different from the passive necrosis.
Note Thesis (M.Phil.)--Hong Kong University of Science and Technology, 1998
Language English
Format Thesis
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