Effect of mild hypothermia and barbiturate for cerebral ischemia-Evaluation by using primary neuronal culture

据报道，亚低温和麻醉剂如巴比妥酸盐具有神经保护作用。然而，这种保护作用的机制尚未得到很好的阐明。我们评估了低温和戊巴比妥对大鼠皮层神经元谷氨酸神经毒性的影响。大鼠胚胎皮质神经元取自妊娠18天的定时妊娠大鼠。在无血清培养基（添加B-27和N-2的神经基础培养基）中维持6天。该培养体系获得了接近纯的神经元群体。50 μM谷氨酸暴露30分钟后，诱导低温或戊巴比妥。24小时后，通过乳酸脱氢酶渗漏试验评估神经毒性。在低温（33℃和30℃）中观察到神经保护作用，但在临床剂量戊巴比妥中没有。然而，仅在暴露于谷氨酸时诱导低体温没有显示出任何保护作用。其次，暴露于谷氨酸后的低温并没有抑制ROS的产生（通过使用ROS特异性氟化物，C-DCDHF-DA的流式细胞术测量）和caspase-3的激活（通过devd -对硝基苯胺裂解实验测量）。我们的数据表明，低温通过抑制暴露后的任何级联而不是通过抑制暴露期间谷氨酸的直接作用，对暴露于谷氨酸具有神经保护作用。然而，在本研究中，我们并没有明确低温会抑制什么级联反应。

It has been reported that mild hypothermia and anesthetics such as barbiturates are neuroprotecitve. However, the mechanism of this protective effect has not been well clarified. We evaluated the effect of hypothermia and pentobarbital on glutamate neurotoxicity in rat cortical neurons.Rat embryonic cortical neurons were obtained from timed pregnant rats at 18 days gestation. They were maintained in serum-free medium (Neurobasal medium supplemented with B-27 and N-2) for 6 days. The nearly pure neuronal population was obtained by this culture system. After exposure to 50 μM glutamate for 30 min, hypothermia or pentobarbital was induced. After 24 hours, neurotoxicity was evaluated by a leakage assay of lactic dehydogenase. Neuroprotective effect was seen in hypothermia (33 and 30 ℃), but not in clinical doses of pentobarbital. However, induction of hypothermia only during exposure to glutamate did not show any protective effect. Second, production of ROS (measured by flow cytometry with a ROS-specific fluorogen, C-DCDHF-DA) and caspase-3 activation (measured by DEVD-p-nitroaniline cleavage assays) was not inhibited by hypothermia after exposure of glutamate.Our data indicated that hypothermia is neuroprotective against exposure to glutamate by inhibiting any cascade after the exposure, but not by inhibiting direct effect of glutamate during exposure. However, we did not make clear what cascade was inhibited by hypothermia in this study.