Effects of mild and moderate hypothermia on apoptosis in neuronal PC12 cells

Background. There is still a possibility that mild hypothermic therapy may be useful as a neuroprotective tool during the intraoperative period, although the mechanism of cerebral protection by mild hypothermia is not well understood. We hypothesized that mild hypothermia may be protective against cerebral ischaemia by inhibiting post-ischaemia apoptosis. In this study, we used serum-deprived PC12 cells as the neuronal apoptotic model and examined the direct effects of mild and moderate hypothermia. Methods. Apoptosis was induced by depriving the cell culture medium of serum, which is one of the most representative methods to induce apoptosis, but not necrosis, in PC12 cells. Effects of mild (35 and 33∞C) and moderate (31 and 29∞C) hypothermia on apoptosis were evaluated. Cytotoxicity (lactate dehydogenase leakage) and the percentage of apoptotic cells (calculated by flow cytometry with propidium iodide) were evaluated 4 days after induction of apoptosis. As a control, cells without induction of apoptosis were incubated under the same conditions as the apoptosis group. Results. Without induction at 37∞C, cytotoxicity and the percentage of apoptotic cells were over 60 and 90%, respectively. At each temperature examined below 35∞C, significant decreases in cytotoxicity and the percentage of apoptotic cells were observed. Mean cytotoxicity at 31 and 29∞C was 50.2 (SD 4.2)% and 47.9 (4.4)%, respectively. The percentage of apoptotic cells at 31 and 29∞C was 42.5 (7.4)% and 36.5 (7.3)%, respectively. In the control group, cytotoxicity and the percentage of apoptotic cells were significantly higher at 29∞C than at 37∞C. Conclusions. Mild and moderate hypothermia (29‐35∞C) inhibited apoptosis, although hypothermia below 30∞C may induce apoptosis in intact cells.