Transient hypoxia may lead to neuronal proliferation in the developing mammalian brain: From apoptosis to cell cycle completion

Cerebral hypoxia/ischemia was shown to induce delayed, apoptotic neuronal d eath occurring through biochemical pathways potentially sharing common even ts with cell proliferation. This study was designed to test the hypothesis that a sublethal hypoxia may promote mitotic activity in developing central neurons. After six days in vitro, cultured neurons from the forebrain of i ii-day-old rat embryos were exposed to hypoxia (95% N-2/5% CO2) for 3 h and re-oxygenated for up to 96 h. Controls were kept in normoxia. As a functio n of time, cell viability was measured by diphenyltetrazolium bromide, and rates of DNA and protein synthesis were monitored using [H-3]thymidine and [H-3]leucine, respectively. Morphological features of apoptosis, necrosis a nd mitosis were scored under fluorescence microscopy after nuclear staining with 4,6-diamidino-2-phenylindole, and the expression profile of prolifera ting cell nuclear antigen, a cofactor for DNA polymerase, was analysed by i mmunohistochemistry. Data were compared to those obtained after transient h ypoxia for 6 h followed by re-oxygenation for 96 h and which was shown to i nduce apoptosis. Whereas a 6-h insult reduced cell viability, with 23% of t he neurons exhibiting apoptosis by the end of re-oxygenation, a 3-h hypoxia led to a cycloheximide-sensitive increase in the final number of living ne urons compared to controls (13%, P < 0.01), with no signs of apoptosis, sig nificantly increased thymidine incorporation into acid-precipitable fractio n, and persistent over-expression of proliferating cell nuclear antigen. Ac cordingly, final score of mitotic nuclei was significantly enhanced. In add ition, the cell cycle inhibitor olomoucine (50 mu M) prevented apoptosis co nsecutive to a 6-h hypoxia, but impaired the stimulatory effects of a 3-h i nsult. These findings support the conclusion that some neurons exposed to subletha l hypoxia may dodge apoptotic death by fully achieving the cell cycle. (C) 1999 IBRO. Published by Elsevier Science Ltd.