离体培养大鼠海马脑片的形态学与细胞反应性研究

A study for morphology and the reactivity of rats hippocampal slice in vitro culture

目的:探索离体大鼠海马脑片培养方法,观察培养脑片的形态变化和细胞反应性。方法:选择生后6～9d的Wistar鼠,分离海马,切成400μm厚的脑片,转移至带有微孔膜插件的6孔培养板内,入37%的CO_2培养箱中进行培养。通过肉眼、倒置相差显微镜观察培养海马脑片的形态学变化;用免疫组化染色检测脑片神经细胞内Fos蛋白表达变化,以判断培养脑片有无对外界伤害性刺激的应激反应能力;用膜片钳技术检测神经细胞的电生理活动,以判断培养海马脑片的活性和生理功能。结果:(1)随着体外培养时间延长,培养脑片内神经细胞数目增多,而脑片明显变薄,至培养4周时厚度约150μm厚。(2)致癫痫样发作药物匹罗卡品作用于脑片后,导致培养海马脑片CA1区细胞内Fos蛋白表达增高。(3)利用膜片钳技术,在培养1、2、3、4周的4个时点对海马脑片CA1区锥体细胞作全细胞记录,皆记录到细胞电流变化图形。结论:本方法在体外培养的海马脑片至少可存活4周,且具备良好的活性和一定的生理功能。

Objective：To explore the method of culturing rat hippocampal slice in vitro and to observe the changes of neurons＇ morphology and reactivity in cultured slice. Methods：Organotypic hippocampal slices were prepared by taking brains from 6-9 day old wistar rats. After the hipp^ampi was isolated and chopped into 400 ~ m thickness,slices were transferred to Millipore inserts and placed in six-well trays in CO2 incubators at 37℃. Then the cultured hippoeampal slices were observed for the changes of morphology by macroscopic observation and using inverted phase contrast microscope;Expression of Fos protein in cultured hippocampal slice was detected by immunocytochemistry for observation of the internal response of hippocampal neurons to the damage;The electrophysiological activity of hippocampal neurons were detected by using patch clamp technique. Results：（1）The number of neurons in cultured hippocampal slices was increased,and the thickness of cultured hippocampal slices was decreased markedly along with the culturing period in vitro. After being cultured for 4 weeks,the thickness of hippocampal slices was decreased to about 150μm.（2）With the treatment of pilocarpine to induce seizure-like activity,the number of neurons which expressing Fos protein was increased in CA1 area of the cul（ured hippocampal slice.（3）With the application of pathch clamp technique,the changes of the ion currents of pyramidal neurons in CA1 area were recorded by the whole-cell recording after culturing for 1 week,2 weeks,3 weeks and 4 weeks respectively. Conclusion：Cultured hippocampal slice in vitro could retain satisfactory liveness and function for at least 4 weeks.