CHANGES IN RNA AND PROTEIN SYNTHESIS FOLLOWING HYPOXIA/REOXYGENATION IN CULTURED CENTRAL NEURONS. 2225

Hypoxia/ischemia inhibits protein synthesis in vulnerable brain areasin vivo. In order to investigate the relationship between disturbances of protein synthesis and the ability of neurons to recover from hypoxia, alterations in RNA and protein synthesis were investigated in neuronal cultures following acute hypoxia which decreased extracellular PO2 values by 75-80%. Neurons from the embryonic rat forebrain were cultured in hormonally-defined conditions for 6 days and their incubating medium was loaded with [³H]uridine or [³H]leucine (0.5 μCi/ml) as markers for RNA and protein synthesis, respectively. Culture dishes(n=180) were then maintained for 1 to 6h in hypoxia (95% N2 - 5% CO2) and reoxygenated for 72h under normoxia (95% air - 5% CO2). Control dishes (n=180) were kept in normoxia. At the end of hypoxia or as a function of time after reoxygenation, acid-soluble and -insoluble fractions of the cultures were analyzed for tracer incorporations using scintillation counting. In the acid-insoluble fraction, RNA synthesis peaked at 41% above control values after 1h-hypoxia, and then decreased until 6h-hypoxia to reach 27% below controls. Following reoxygenation, uridine incorporation increased by 22% as compared to controls at 24h and by 46% at 48h. At 72h post-hypoxia, uridine incorporation decreased by 22%, concomitantly with signs of cell damage reflecting delayed neuronal death. Although slightly delayed, a similar profile was shown for [³H]leucine incorporation which increased by 50% after 1h-hypoxia, and then decreased to reach -37% after 6h-hypoxia. Whereas no differences as compared to controls were noticed 24h after reoxygenation, leucine incorporation increased by 61% at 48h, and then decreased to 32% below controls at 72h. As for changes in uridine incorporation, all data were statistically significant (p<0.01). These results confirm those obtained in vivo regarding inhibition of protein synthesis and report a deferred peak of RNA and protein synthesis at 48h post-hypoxia, suggesting that hypoxia/reoxygenation leads to sequential changes in the expression of specific mRNA and proteins which might be involved in neuronal cell death and/or protection.