To investigate the events accounting for degeneration or recovery of neuronal cells following hypoxia and reoxygenation in the developing brain, the effects of hypoxic insults of increasing duration were studied in a model of cultured neurons from the fetal rat forebrain. Cells were kept for 6 days in controlled chemically-defined medium, then exposed for 3 or 6h to hypoxia(95% N2-5% CO2) that decreased extracellular PO2 by 75-80% below controls kept in normoxia (95% air-5% CO2). Cells were finally reoxygenated for 96h in standard conditions. As a function of time during hypoxia and reoxygenation, alterations in cell viability, assessed by the tetrazolium salt MTT, were correlated to changes in protein synthesis, as shown by the incorporation of[3H]leucine. Data are reported as percentage of changes as compared to controls maintained under standard conditions. Development of apoptosis-related features, i.e. DNA fragmentation in agarose gels, and chromatin condensation revealed by the nuclear incorporation of the fluorescent dye DAPI (4,6-diamidino-2-phenylindole), was also analysed. A 6-h hypoxia led to reduced cell viability (-36%, n=30 dishes, p<0.01) at the end of the reoxygenation period, while 23% of the neurons exhibited noticeable signs of apoptosis versus 1.5% in controls(p<0.01). Rates of protein synthesis increased by 77% (n=26,p<0.01) as soon as 1h after the onset of the hypoxic insult and then by 72% (n=26, p<0.01) at 48h following reoxygenation, to finally decrease by 43% (n=24, p<0.01) below controls at 96h, with altered cell morphology. By preventing these changes in protein synthesis, a treatment by cycloheximide during hypoxia protected the cells from damage, thus confirming a programmed process. When neurons were exposed to hypoxia for 3h, their viability increased during reoxygenation to reach 13% above controls (n=10, p<0.01) at 96h, along with enhanced rates of protein synthesis (+69%, n=10, p<0.01). Meanwhile, labeling of cell nuclei by DAPI revealed an increased number of mitotic neurons (6%versus 1% in controls, n=5). These data show that transient hypoxia for 6h followed by standard reoxygenation would activate a programmed cell death corresponding to apoptosis in neuronal cells. Conversely, short-time hypoxia (3h) would allow the neurons to re-enter the cell cycle, leading to increased cell proliferation, and thus dodging cell damage.
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Daval, JL., Bossenmeyer, C., Chihab, R. et al. OPPOSITE EFFECTS OF SHORT- AND LONG-TIME HYPOXIA ON THE OUTCOME OF CULTURED CENTRAL NEURONS AFTER REOXYGENATION † 1720. Pediatr Res 41 (Suppl 4), 289 (1997). https://doi.org/10.1203/00006450-199704001-01739
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DOI: https://doi.org/10.1203/00006450-199704001-01739