Protective effect of mild hypothermia and barbiturate for cerebral ischemia-Evaluation by using neuronal cells-

据报道，亚低温和巴比妥酸盐具有神经保护作用。然而，这种保护作用的机制还没有很好地阐明。一个可能的机制是抑制神经元的凋亡。以大鼠嗜铬细胞瘤PC12细胞为模型，研究了低温(29~35℃)和戊巴比妥对神经细胞凋亡的影响。我们进一步从产生ROS和激活caspase的角度探讨了其保护作用的机制。去血清诱导PC-12细胞凋亡是最具代表性的方法之一。首先，在诱导细胞凋亡4天后评价细胞毒性(用乳酸脱氢酶漏出试验评价)和细胞凋亡率(用碘化丙啶流式细胞仪计算)。其次，用ROS特异性荧光试剂C-DCDHF-DA在诱导后3或6小时用流式细胞仪检测ROS的产生。低温(29~35℃)和临床剂量的戊巴比妥可显著降低细胞毒作用和细胞凋亡率。在37℃时，诱导细胞凋亡3小时后，ROS的产生几乎是对照组的两倍。临床剂量的戊巴比妥显着减少ROS的产生，而低温不能抑制ROS的产生。Caspase-2和-3的活性不受低温和戊巴比妥的抑制，但通过诱导细胞凋亡而增强。我们的数据表明，低温(29-35℃)和临床剂量的戊巴比妥抑制诱导神经细胞的凋亡。抑制ROS的产生可能部分归因于巴比妥酸盐的这种保护作用。然而，ROS的产生和caspase的激活可能与低温保护作用的机制无关。

It has been reported that mild hypothermia and barbiturate are neuroprotecitve. However, the mechanism of this protective effect has not been well clarified. One possible mechanism is the inhibition of neuronal apoptosis. We evaluated the effect of hypothermia (29-35℃) and pentobarbital on apoptosis in neuronal cells using PC 12 cells, which is derived from rat pheochromocytoma. We further evaluated the mechanism of protective effect from the viewpoints of production of reactive oxygen species (ROS) and caspase activationApoptosis was induced by depriving serum from the medium, which is one of the most representative methods to induce apoptosis in PC 12 cells. First, cytotoxicity (evaluated by a leakage assay of lactic dehydogenase) and the percentage of apoptotic cells (calculated by flow cytometry with propidium iodide) were evaluated 4 days after induction of apoptosis. Second, production of ROS was measured by flow cytometry with a ROS-specific fluorogen, C-DCDHF-DA 3 or 6 hours after induction. Third, caspase-2 and -3 activation was measured by VDVAD p-nitroaniline and DEVD-p-nitroaniline cleavage assays 15 hours after induction.Hypothermia (29 - 35℃) and clinical dose of pentobarbital significantly decreased cytotoxicity and the percentage of apoptotic cells. At 37℃ the production of ROS has already increased almost twice compared to control 3 hours after induction of apoptosis. Clinical dose of pentobarbital significantly decreased ROS production whereas hypothermia did not inhibit it. Caspase-2 and -3 activity was not inhibited by hypothermia and pentobarbital, although the activity increased by the induction of apoptosis.Our data indicated that hypothermia (29 - 35℃) and clinical dose of pentobarbital inhibited induced apoptosis in neuronal cells. Inhibition of ROS production may be partly attributable to this protection by barbiturate. However, production of ROS and caspase activation may not be related to the mechanism of protective effect of hypothermia.