摘要
目的 观察红藻氨酸(KA)腹腔注射成年大鼠所致的癫痫持续状态后海马神经元损伤是否是选择性的，迟发性以及谷氨酸受体-1mRNA、谷氨酸受体－2mRNA表达的变化，以研究谷氨酸受体表达变化在海马神经元死亡机制中的作用。
方法 取健康成年雄性SD大鼠(175～200g)为研究对象。分为实验组和对照组。实验组用KA(15mg／kg)腹腔注射制作癫痫持续状态，注射后观察行为变化6小时，只有表现持续的癫痫发作达1个小时的被认为是癫痫持续状态，用于此研究。然后分别在KA注射后6小时、12小时、16小时、20小时、24小时、1周断头处死。对照组腹腔内注射生理盐水(3ml／kg)，于注射后24小时、1周断头处死。所有的大鼠取脑石蜡包埋，取部分海马切片作尼氏染色及谷氨酸受体-1和谷氨酸受体-2mRNA的原位杂交染色，观察神经元损伤及谷氨酸受体表达变化。
结果 海马水平的尼氏染色显示在癫痫持续状态后24小时方可见到部分的CA1和CA3区锥体细胞的变性和损失。在癫痫持续状态后1周，在CA1、CA3区的锥体细胞中重度的神经元损失。齿状回的颗粒细胞在癫痫持续状态后未见到组织学上可见的神经元变性和损失。原位杂交结果显示，癫痫持续状态在CA1、CA3神经元损伤前(即KA注射后24小时前)诱导了谷氨酸受体-2(非谷氨酸受体-1)mRNA的下调。在CA1、CA3的谷氨酸受体-1和谷氨酸受体-2mRNA的表达在24小时均下降。齿状回的谷氨酸受体-1和谷氨酸受体-2mRNA的在KA注射后未见到表达的明显变化。
结论 我们的研究显示，成年大鼠癫痫持续状态后海马神经元发生的是选择性的、迟发性的损伤，而AMPA受体的亚单位谷氨酸受体-2的下调形成了Ca~(2+)渗透性的AMPA受体，增加了内源性谷氨酸的兴奋毒性，可能参与了迟发性神经元损伤的机制。
Objective To examine hippocampal neuronal death induced by status epilepticus and to test the role of the change of glutamate receptor-1, glutamate receptor-2 mRNA expression in mechanisms of hippocampal neuronal death.
Methods Seizures were induced in adult, male Sprague-Dawley rats (175~200g) by injection of KA (15mg/kg, i.p.). Animals were monitored behaviorally for seizures for 6h after injection. Only rats displaying status epilepticus, defined as continual seizures for at least Ih, were used in this study. Normal control group were induced in same rats as seizure group by injection of Saline (3ml/kg, i.p.). Then animals were sacrificed at 6h, 12h, 16h, 20h, 24h, and Iw after injection of KA or 24h, Iw after injection of Saline. Brains were removed and wrapped. Neuronal damage was evaluated with Nissl-stains. Glutamate receptor-1 and glutamate receptor-2 mRNA expression were assessed by in situ hybridization in order to examine the relationship glutamate receptor mRNA expression and the deladyed hippocampal neuronal death.
Results Nissl-stained sections at the level of the hippocampus revealed some neuronal degeneration and loss in the CA1, CA3 pyramidal cell only at 24h after status epilepticus, rats showed moderate to severe neuronal loss in the CA1, CA3 pyramidal cell at 1 week after status epilepticus. There was no histological detectable degeneration in the granule cell layer of the dentate gyrus after status epilepticus. In situ hybridization showed status epilepticus induced down-regulation GluR2 (but not GluRl) mRNA in CA1, CA3 before neuronal degeneration (i.e. before 24h after injection of KA). The expression of GluRl and GluR2 mRNA was both decreased at 24h after injection of KA and unchanged in the dentate gyrus after status epilepticus.
Conclusion Our study show KA-induced status epilepticus lead to selective and delayed hippocampal neuronal death. Down-regulateon of glutamate receptor 2 mRNA could lead to formation of GluR2-lacking, Ca2+-permeable AMPA receptors and increased toxicity of endogenous glutamate, which may be involved in the mechanisms of the
delayed neuronal death.
引文
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