NMDA对新生大鼠皮层神经元的兴奋毒作用

NMDA-induced neurotoxicity on primary cultured rat cortical neurons

目的:建立NMDA诱导原代培养皮层神经元兴奋毒损伤模型,探讨NMDA对NMDA受体过度活化诱导兴奋性神经毒的可能途径。方法:原代培养新生大鼠大脑皮层神经元,通过倒置显微镜形态学观察、细胞活力检测(MTT及LDH释放的检测)及胞内Ca2+的动态测定,探索NMDA诱导毒性作用的适当浓度及时间。通过对ROS、NO检测,分析NMDA诱导毒性作用于线粒体的损伤情况。结果:NMDA(100μmol/L/2 h)引起皮层神经元形态学改变,且引起神经元细胞活力时间和浓度依赖性的下降,由同时伴随LDH释放增加(P<0.05),ROS和NO的生成量明显增加(P<0.05),皮层神经元内Ca2+的快速升高,并维持在高水平。结论:NMDA诱发皮层神经元明显的细胞毒性作用,提示NMDA过度活化NMDA受体后通过神经元膜内Ca2+超载造成ROS和NO的生成量增加,导致皮层神经元产生毒性损伤。

Objective： To study the damage effects of NMDA-induced neurotoxicity on primary cultured cortical neurons from new born rat（ 1 - 3 d）. Methods：Cortical neurons from neonatal rat were primarily cultured. An appropriate concentration of NMDA and the time it works were explored by monitoring morphological changes under contrast phase microscope, MTY, LDH release in the medium, and Ca2＋ concentration in the cytoplasm so as to build an appropriate cellular model in cortical neurons in rat. ROS and NO were detected to evaluate the damage of mitochondria in the model. Resuits ：The administration of NMDA （ 100μmol/L, 2 h） in the cultures caused pathological changes of morphology and cell viability decrease dose-dependently and time-dependently. NMDA caused cell viability decrease by about 47% and LDH release increase by about 66%, compared with control （P 〈 0.05 ）. Activation of NMDAR triggered the rise of Ca2~ in cytoplasm and the maintenance of the highest level during the observation. Excessive ROS and NO were detected in the model（ P 〈 0.05 ）. Condusion：NMDA （ 100 μmol/L, 2 h） triggers obvious neuronal loss and is considered as an appropriate concentration and duration to cause excitatory neurotoxicity. Mitochondria damaged in the model.