Abstract 2004 Poster Session II, Sunday, 5/2 (poster 239)

We previously demonstrated that transient hypoxia for 6h (95% N2-5% CO2, 37°C) leads to delayed apoptotic death in cultured neurons from fetal rat forebrain. While vital changes are linked to sequential alterations in protein synthesis, we showed that preconditioning neurons by a brief hypoxia prevents cell injury (Pediatr Res, 1998, 43:46A). To test whether hypothermia also protects neurons in the same model and compare biochemical mechanisms with those involved in preconditioning, cultured neurons after 6 days in vitro were divided into three distinct groups, along with their matched controls. In the H6 group, neuronal cultures were submitted to hypoxia for 6h. Other culture dishes were exposed to the same hypoxic insult but were either placed in hypothermia (32°C) concomitantly to hypoxia (HH6 group) or preconditioned the proceeding day by 1h hypoxia (PH6 group). As a function of time, cell viability was measured by the tetrazolium salt MTT, DNA integrity was analysed using the flourescent dye DAPI, while DNA synthesis was assessed by [3H]thymidine incorporation. Synthesis of total proteins was monitored by [3H]leucine incorporation, and the expression profile of PCNA, a cofactor for DNA polymerase, was analysed by immunohistochemistry. At 96h post-reoxygenation, cell viability was decreased by 36% (n=15) in H6 group, but was significantly enhanced compared to normoxic controls in both PH6 and HH6 groups (14% and 13%, respectively, n=15). Whereas 23% of apoptotic neurons were detected by DAPI in the H6 group, other culture sets did not exhibit more apoptosis than controls. However, a significant stimulation of mitotic activity, starting from 72h post-reoxygenation, was measured in PH6 neurons, whereas HH6 group showed only transient augmentation of mitotic neurons between 24h and 48h (4.8% vs 1.2% in controls), and a significantly reduced number of necrotic neurons (2.5% vs 6.2% which was unaffected in PH6. After a biphasic increase in total protein synthesis in the H6 group, rates of leucine incorporation were reduced by 43% (n=12) at 96h post-reoxygenation. No biphasic changes in protein synthesis could be detected in the HH6 group, whereas PH6 neurons showed a significant stimulation of leucine incorporation (21%, n=10) at 48h. Final incorporation rates remained in the control range at 96h in HH6 and PH6 groups (+13%, +11%). Finally, immunohistochemical analysis showed overexpression of PCNA, starting from 24h post-reoxygenation, specifically in the PH6 group. Our data suggest that, while preconditioned neurons from the developing brain are more resistant to further hypoxia through activation of anti-apoptotic gene products, including those stimulating the cell cycle, protective effects of hypothermia would not involve changes in protein expression, but certainly reflect a direct consequence of the globally reduced metabolic activity consecutive to low temperature that impairs active cell process.