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

Abstract

Cerebral hypoxia/ischemia was shown to induce delayed, apoptotic neuronal death occurring through biochemical pathways potentially sharing common events 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 14-day-old rat embryos were exposed to hypoxia (95% N 2/5% C0 2) for 3 h and re-oxygenated for up to 96 h. Controls were kept in normoxia. As a function of time, cell viability was measured by diphenyltetrazolium bromide, and rates of DNA and protein synthesis were monitored using [ 3H]thymidine and [ 3H]leucine, respectively. Morphological features of apoptosis, necrosis and mitosis were scored under fluorescence microscopy after nuclear staining with 4,6-diamidino-2-phenylindole, and the expression profile of proliferating cell nuclear antigen, a cofactor for DNA polymerase, was analysed by immunohistochemistry. Data were compared to those obtained after transient hypoxia for 6 h followed by re-oxygenation for 96 h and which was shown to induce apoptosis. Whereas a 6-h insult reduced cell viability, with 23% of the 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 neurons compared to controls (13%, P<0.01), with no signs of apoptosis, significantly increased thymidine incorporation into acid-precipitable fraction, and persistent over-expression of proliferating cell nuclear antigen. Accordingly, final score of mitotic nuclei was significantly enhanced. In addition, the cell cycle inhibitor olomoucine (50 μM) prevented apoptosis consecutive to a 6-h hypoxia, but impaired the stimulatory effects of a 3-h insult. These findings support the conclusion that some neurons exposed to sublethal hypoxia may dodge apoptotic death by fully achieving the cell cycle.