title: 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% N2/5% CO2) 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 microM) 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.
authors.affiliation: INSERM U.272, Université Henri Poincaré, Nancy, France.
authors.forename: C
authors.initials: C
authors.lastname: Bossenmeyer-Pourié
authors.forename: R
authors.initials: R
authors.lastname: Chihab
authors.forename: H
authors.initials: H
authors.lastname: Schroeder
authors.forename: J L
authors.initials: JL
authors.lastname: Daval
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001921:Brain / Q000254:growth & development* / Q000473:pathology*; D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D003470:Culture Media; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D004247:DNA / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D002534:Hypoxia, Brain / Q000473:pathology*; D007150:Immunohistochemistry; D007701:Kinetin; D009419:Nerve Tissue Proteins / Q000096:biosynthesis; D009474:Neurons / Q000473:pathology*; D011247:Pregnancy; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis; D011687:Purines / Q000494:pharmacology; D051381:Rats; D015427:Reperfusion Injury / Q000473:pathology
chemical_list: D003470:Culture Media; D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D018809:Proliferating Cell Nuclear Antigen; D011687:Purines; C090046:olomoucine; D004247:DNA; D018844:Cyclin-Dependent Kinases; D007701:Kinetin