ANALYSIS OF NEURONAL DEATH IN THE CENTRAL-NERVOUS-SYSTEM USING A NEW APOPTOSIS MODEL

To examine in detail neural apoptosis in the central nervous system (C NS) for the establishment of new therapies, we have developed an exper imental in vitro model of neuronal death and analyzed the mechanism of apoptosis. Septal nuclei were dissected from embryonic brains (E16) o f Wistar rats and cultured in chemically defined medium. Highly enrich ed neurons were obtained from the cultures at 4 days. Exposure to heat shock (43.0 degrees C, 60 min) between 24 and 36 h later, resulted in the death of approximately 70% of cells. Morphologically, dying neuro ns showed disruption of neurites, nuclear condensation, multiple nucle ar fragments, condensation of cytoplasm and multiple cellular fragment ation. Agarose electrophoresis of chromosomal DNA revealed a typical l adder-pattern of fragmentation. Following heat treatment, incubation a t 37 degrees C was necessary to detect DNA fragmentation. Quantitative analysis by in situ terminal deoxynucleotidyl transferase assay, reve aled that the percentage of apoptotic cells markedly increased 8 h aft er heat treatment, and continued to gradually increase up until 30 h. Neuronal death and DNA fragmentation were prevented by inhibiting RNA and protein synthesis. Cell death and the DNA cleavage were also inhib ited by cultivation in calcium-free medium. The addition of homogenous basic fibroblast growth factor to the medium markedly enhanced cell s urvival under these pathogenic conditions. These results suggest that neural cell death after mild heat treatment has apoptotic characterist ics and may be useful for analyzing the mechanism of apoptosis. The cl inical application of drugs acting against molecular components and as well as neurotrophic factors, may in the future prevent apoptosis in neural disease.