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

亚低温联合巴比妥治疗脑缺血的效果——原代神经元培养评价

• 批准号: 13671556
• 负责人: MORIMOTO Yuji
• 金额: $ 2.18万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671556/
• 关键词: Cell culture ; cortical neuron; Cell death apoptosis; Hypothermia ; mild hypothermia; Barbiturate ; pentobarbital; Caspase; Reactive oxygen species; Cerebral ischemia; Glutamate ; glutamate neurotoxocity; 神経細胞培養; グルタミン酸

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.

据报道，轻度体温过低和麻醉药（例如巴比妥酸盐）是神经前牙的。但是，这种保护作用的机制尚未得到充分阐明。我们评估了低温和戊巴比妥对大鼠皮质神经元中谷氨酸神经毒性的影响。在妊娠18天时，从定时怀孕大鼠那里获得了RAT胚胎皮质神经元。它们在无血清培养基（补充了B-27和N-2的神经质量培养基中）6天。该培养系统获得了几乎纯净的神经元种群。暴露于50μM谷氨酸30分钟后，诱导体温过低或戊巴比妥。 24小时后，通过乳酸脱氢酶的泄漏测定法评估了神经毒性。在体温过低（33和30℃）中观察到神经保护作用，但在pentobarbital的临床剂量中却没有。但是，仅在暴露于谷氨酸期间的体温过低诱导没有显示任何受保护作用。其次，ROS的产生（通过流式细胞术用ROS特异性荧光，C-DCDHF-DA）和caspase-3激活（通过DEVD-P-PNORONILINE裂解测定测量（通过DEVD-P-硝基氨基裂解测定法测量）并未受到谷氨酸暴露后的暴露于肠胃外的侵蚀性，但没有任何case液，而是通过神经疗法而抑制。通过抑制暴露期间谷氨酸的直接作用。但是，在这项研究中，我们尚未清楚地体温过低会抑制哪种级联反应。