经皮三叉神经电刺激对慢性癫痫大鼠癫痫状态所致海马神经元损伤的保护作用及其机制的探索

发布时间：2018-06-13 12:05

本文选题：癫痫 + 经皮三叉神经电刺激　；参考：《安徽医科大学》2014年硕士论文

【摘要】：目的： 1.观察经皮三叉神经电刺激（TcTNS）对匹罗卡品诱发的癫痫持续状态（SE）所致海马神经元损伤的保护作用。 2.观察观察经皮三叉神经电刺激（TcTNS）对匹罗卡品诱发的慢性癫痫模型大鼠海马神经元GAD65/67表达的影响，以探讨经皮三叉神经电刺激治疗癫痫及脑保护作用的可能机制。资料和方法：实验一：选用健康成年雄性SD大鼠，采用随机数字表法将大鼠随机分为二组，匹罗卡品模型组和对照组，模型组用匹罗卡品诱导癫痫点燃模型，对照组注射等量的生理盐水，经过行为学观察两周后，具有慢性癫痫自发性再发作的为模型制备成功大鼠，再将模型制备成功大鼠采用随机数字表法分为两组，治疗组和模型组，分别给予三叉神经电刺激和假刺激一个月（刺激参数为频率140Hz,电流10mA,脉宽0.5ms，正向脉冲刺激30s间歇5min，连续刺激1h，假刺激组各项参数均为0。刺激时间定为每天12:00-16:00）后终止刺激，再次注射皮罗卡品诱导一次癫痫持续状态（SE）并于SE后24h,48h,72h处死大鼠并取材，对照组亦在相应时间点取材，采用TUNNEL和NISSEL染色观察各组大鼠海马神经元细胞的凋亡和坏死情况。组间比较采用配对t检验，以p㩳0.05定义为差别有统计学意义。实验二：选用健康成年雄性SD大鼠，采用同实验一的方法建立癫痫点燃模型，将慢性癫痫模型制备成功者用随机数字表法分为模型组与治疗组，分别给予三叉神经电刺激与假刺激一个月（刺激参数及时间同实验一），于停止刺激后24h，72h，一周，二周，四周处死大鼠并取材，对照组亦于相应时间点取材。采用免疫组化的方法研究大鼠海马及皮层GAD65、67的表达情况，组间比较采用配对t检验，组内比较采用单因素方差分析，以p㩳0.05定义为差别有统计学意义。结果： 1．在实验一中： 1.1TUNNEL染色：SE后各时间点治疗组和模型组均可见TUNNEL阳性细胞，主要分布在海马的CA1和CA3区，SE后24h，48h，72h治疗组CA3区的TUNNEL阳性细胞较模型组显著减少（p＜0.05），以72小时尤为明显（p＜0.01）正常对照组可见少量TUNNEL阳性细胞。 1.2NISSEL染色：正常对照组显示海马CA1和CA3区神经元细胞排列整齐紧密，没有明显的神经元缺失，细胞核呈圆形或椭圆形，胞浆内尼氏小体丰富。模型组各时间点可见不同程度的神经元丢失，部分神经元胞体皱缩，胞浆深染，核固缩，锥体细胞存活数较正常细胞减少，尤以72小时最为明显（p＜0.01）。经皮三叉神经刺激治疗组各时间点大鼠海马CA1，，CA3区神经元细胞尚清晰完整，部分细胞出现染色质凝集，神经元丢失情况较模型组显著下降，即治疗组Nissl染色细胞数显著多于模型组（p＜0.05）。 2.在实验二中： 2.1：海马区及皮层均有GAD65的阳性细胞表达，尤以海马的CA3区表达最为明显，主要在胞核中表达，阳性细胞为棕红色颗粒，经皮三叉神经刺激治疗组较假刺激组相比，各时间点治疗组阳性细胞表达均高于模型组（p＜0.05），治疗组GAD65的表达于停止刺激后72小时-7天达到高峰，较模型组有极显著差异（p＜0.01）。后GAD65的表达下降，至28天接近正常表达水平。 2.2：海马区及皮层均可见GAD67阳性细胞，在海马的CA3区及DG区表达最为明显，主要在胞浆中表达，与模型组各时间点相比较，治疗组的表达远大于模型组（p＜0.05）。组内比较发现，GAD67无明显的时间变化趋势，至停止刺激后28天，治疗组的GAD67的表达仍大于正常水平。结论： 1.经皮三叉神经电刺激治疗可以减少癫痫大鼠海马神经元的损伤，从而发挥对癫痫大鼠海马神经元的保护作用。 2.经皮三叉神经电刺激可使癫痫大鼠脑内抑制性神经递质的标志物GAD65/67表达上调，,抑制性递质的增强可能为三叉神经电刺激治疗癫痫及发挥脑保护作用的机制之一。
[Abstract]:Purpose :

1 . To observe the protective effect of transcutaneous trigeminal nerve stimulation ( TcN ) on hippocampal neuronal injury induced by pilocarpine ( SE ) .

2 . To observe the effect of percutaneous trigeminal nerve stimulation ( TcN ) on the expression of GAD65 / 67 in rat hippocampal neurons induced by pilocarpine , and to investigate the possible mechanism of percutaneous trigeminal nerve stimulation in the treatment of epilepsy and brain protection .

Information and methods :

Experiment 1 :

Adult male SD rats were randomly divided into two groups : two groups , pilocarpine model group and control group . The model group was divided into two groups by pilocarpine . After two weeks of behavioral study , the rats were divided into two groups : treatment group and model group .

Experiment 2 :

The rats were randomly divided into three groups : model group and treatment group . The rats were sacrificed at 24 h , 72 h , one week , two weeks and four weeks after cessation of stimulation . The expression of GAD65 and 67 in hippocampus and cortex of rats was studied by immunohistochemistry .

Results :

1 . In Experiment 1 :

1 . After SE treatment group and model group , positive cells were observed , and the number of positive cells was significantly decreased at 24 h , 48 h and 72 h after SE ( p < 0 . 05 ) , and a small number of positive cells were found in the normal control group at 72 hours ( p < 0.01 ) .

1 . 2NISSEL staining : The normal control group showed that the neuronal cells in the CA1 and CA regions of the hippocampus were orderly and compact , no obvious neuronal loss was found , the nuclei were round or oval , and the Nissl in the cytoplasm was abundant .

2 . In Experiment 2 :

2.1 : The expression of GAD65 positive cells in the hippocampus and cortex was the most obvious , especially in the CA 3 region of hippocampus . The positive cells were higher than those in the model group ( p < 0.05 ) . The expression of GAD65 in the treatment group was higher than that in the model group ( p < 0.05 ) . The expression of GAD65 in the treatment group decreased from 72 hours to 7 days after cessation of stimulation , and the expression of GAD65 decreased to close to normal expression level 28 days .

2.2 : The expression of GAD67 positive cells was found in the hippocampus and in the cortex . The expression of GAD67 in hippocampus was much higher than that in model group ( p < 0.05 ) . Compared with model group ( p < 0.05 ) , the expression of GAD67 was much larger than that in model group ( p < 0.05 ) .

Conclusion :

1 . Percutaneous trigeminal nerve stimulation can reduce the damage of hippocampal neurons of epileptic rats , and play an important role in the protection of hippocampal neurons in epileptic rats .

2 . The expression of GAD65 / 67 is up - regulated by percutaneous trigeminal nerve stimulation , and the enhancement of inhibitory neurotransmitter may be one of the mechanisms of trigeminal nerve stimulation in the treatment of epilepsy and brain protection .
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.1

【参考文献】