改良的新生大鼠海马脑片缺氧缺糖模型的制备方法

An improved method of making oxygen-glucose deprivation model of hippocampal slice in vitro in neonatal rats

目的：介绍一种经济实用的海马脑片缺氧缺糖模型的制备方法。方法：选出生8～10d的SD乳鼠，分离大脑海马．切成400μm厚的脑片，转至带有Millicel微孔膜插件的培养皿中。分别培养7d、14d、20d和30d，倒置显微镜观察形态。碘化丙啶（Propidiumiodide，PI）染色后荧光显微镜下检测脑片活力。充入氮气于不同时相点观察脑片缺氧状况。结果：①培养7d和14d的脑片中所有细胞PI染色呈阴性，培养30d时脑片中部分神经元PI染色阳性，细胞变性坏死。②充入氮气时间越长．海马脑片的死亡细胞越多。⑧充入氮气1．5h后正常培养24h可观察到海马脑片CA1、CA3、DG均出现强荧光显色。结论：脑片存活时间约30d．充入氮气1．5h可建立稳定的缺氧缺糖模型。

Objective：To introduce an economical and pragmatic method of establishing oxygen-glucose deprivation model of hippocampal slice in neonatal rats in vitro,Methods：Hippocampal slices（400μ m） from 8-10-day-old rats are transferred into an incubator with insert of Millicell membrance and incubated for 7 d,14 d,20 d and 30 d,respectively.Tbe growing state of the hippocampal slices were observed under the convened microscope,and its ability to absorb propidiumiodide （PI） was detected under fluorescent microscope after the hippocampal slice was stained by PI,and the state of the hippocampal slices at different time after aerating Nitrogen gas was observed. Results： ① No PI labelled ceils were found after 7 d and 14 d. However,15% PI positive ceils were found after 30 d. ②The longer in Nitrogen gas,the more apoptosis ceils. ③Intensive fluorescence was observed in the CA1,CA3 and DG areas of the hippoeampal slice after aerating Nitrogen gas for 1.5h. Conclusion：The hippocampal slice can be alive for about 30 days. Stable oxygen-glucose deprivation model of the hippocampl slice in vitro was successfully established after aerating Nitrogen gas for 1.5 h.