不同温度对谷氨酸诱发原代培养皮质神经元损伤的保护机制

Effects of Mild Hypothermia on Glutamate-induced Neurotoxicity in Primary Cultures of Cortical Neurons

目的探讨不同温度对谷氨酸诱发皮质神经元损伤的保护机制。方法体外培养新生24 h内Wistar大鼠的皮质神经元,毒性剂量的谷氨酸(200μmol·L-1)诱致皮质神经元损伤,随机分为37℃组(常温)和33℃组(亚低温),两组再根据谷氨酸持续作用时间点(10 min,3 h,6 h,24 h)分为4个亚组,标记为T1,T2,T3,T4。两组细胞均在损伤后24 h,通过激光扫描共聚焦显微镜测量细胞内Ca2+浓度([Ca2+]i)、线粒体内Ca2+浓度([Ca2+]m)及线粒体跨膜电位(ΔΨm)。结果 37℃组和33℃组的[Ca2+]i及[Ca2+]m均随损伤时间的延长而显著性增加,同时伴有ΔΨm显著下降(P<0.05);33℃组较37℃组[Ca2+]i及[Ca2+]m的增加明显减少,同时显著减低ΔΨm下降幅度(P<0.05)。结论 33℃可抑制谷氨酸诱发皮质神经元损伤时的[Ca2+]m内流,减少[Ca2+]m,维持ΔΨm。

cerebral ischemia; glutamate; different temperature; neurotoxicity; cortical neurons ABSTRACT Aim To investigate the protective effect of mild hypothermia on glutamate （Glu） induced neurotoxicity. Methods The degree of neurotoxicity induced by Glu was analyzed in two temperature groups （normothermia, 37 ℃; mild hypothermia, 33 ℃ ） of cultured newborn Wistar rat within 24 h cortical neurons. Cultured neurons in each temperature group were exposed to 200 μmol·L1 Glu for four different time courses： 1/ 6 h, 3 h, 6 h, 24 h. Confocal laser-scanning microscopy （LSM） in combination with fluorescent indicator dyes were used to investigate the effects of Glu on intraceUular calcium ion concentration（[Ca2＋]i）, mitochondrial Ca2＋ ion concentration （[Ca2＋]m） and mitochondrial membrane potential（△Ψm）. Results The [Ca2＋] and [Ca2＋]m significantly increased both in mild hypothermic group and normothermic group in the different time courses （P〈 0.05）. The ATm significantly decreased both in the two groups in the different time courses （P〈0.05）. The mild hypothermia significantly reduced the increase of Glu-induced in [Ca2＋]j （P〈0.05）, [Ca2＋]m （P〈0.05） and attenuated the decrease in △Ψm （P〈0.05）. Conclusion Mild hypothermia can inhibit the intracellular calcium influx and reduce the calcium in mitochondria, maintaining mitochondrial membrane potential.