Prevention from Hypoxia-Induced Apoptosis by Preconditioning Cultured Neurons from the Developing Brain • 252

Abstract

It has been previously shown that a 6-h hypoxia leads to delayed apoptotic cell death in cultured neurons isolated from fetal rat forebrain. Conversely, exposure to hypoxia for 3h increases the number of living neurons scored by the end of a 96h-period of reoxygenation, when compared to controls maintained in normoxia (Pediatr Res, 1997,41:289A). Such vital changes are associated to selective alterations in the synthesis of specific proteins. In order to test the hypothesis that a preceding period of hypoxia may induce tolerance to the effects of a long-lasting insult, cultured neurons after 6 days in vitro were submitted to hypoxia (95% N2-5% CO2) for 6h (H6 group) or to the same hypoxia preceded the day before by a transient exposure to hypoxia for 1h (P1H6 group). As a function of time following reoxygenation, cell viability was measured by the tetrazolium salt MTT, DNA synthesis was assessed by [3H]thymidine incorporation, and DNA integrity was analyzed using the fluorescent dye DAPI. Whereas changes in the synthesis of total proteins were monitored by [3H]leucine incorporation, the expression profile of specific proteins such as Bcl-2, HSP70 as well as PCNA, a cofactor for DNA polymerase, was analyzed by immunohistochemistry. At 96h post-reoxygenation, cell viability decreased by 36% (n=15) in H6 group, while it was significantly enhanced (14%, n=15) in P1H6 group as compared to normoxic controls. The use of DAPI revealed the presence of apoptotic neurons (23%, n=15) in the H6 group, while P1H6 neurons did not exhibit any sign of apoptosis but showed a significant stimulation of their mitotic activity. In parallel, thymidine incorporation increased by 20% (n=12) in the latter group, and decreased by 12% (n=12) below basal values in H6. After a biphasic increase of total protein synthesis, the final rates of leucine incorporation were significantly reduced at 96h post-reoxygenation (43%, n=12) in H6, whereas they remained in the range of basal values in P1H6. Finally, immunohistochemical analysis showed overexpression of PCNA, Bcl-2 and HSP70 starting from 24h after reoxygenation in P1H6 group, the expression of these proteins being unaffected in H6 neurons compared to normoxic controls. These data suggest that a preceding period of brief hypoxia promotes molecular events which render developing brain neurons more resistant to severe hypoxic stress. Adaptative mechanisms may involve anti-apoptotic gene products as well as related proteins which regulate the cell cycle.