调控RET依赖性GDNF信号通路对大鼠癫痫模型的影响

目的:探究双侧海马CA1区立体定向注射anti-GDNF抗体对匹鲁卡品诱导的大鼠癫痫模型的影响。方法:选择成年雄性SD大鼠60只,并随机分为3组,即假手术组(sham组,n=20)、癫痫模型组(model组,n=20)和GDNF抑制剂组(anti-GDNF组,n=20)。使用氯化锂-匹鲁卡品腹腔注射诱导癫痫模型,sham组只给予氯化锂,anti-GDNF组在造模前2 h给予大鼠双侧海马CA1区立体定向注射anti-GDNF抗体。在造模后1、3、7 d观察大鼠癫痫的发作频率,7 d后采用脑电图监测(EEG)测定脑电波的变化情况,通过免疫组化方法测定海马CA1区域神经元数量变化(Neu N表达水平),造模后1 d时使用western blot方法测定海马CA1区GDNF、RET和P53蛋白的表达。结果:Model组大鼠棘-慢波数量明显高于Sham组,anti-GDNF组以上指标较model组显著减少(P0.05);Model组海马CA1区神经元大量凋亡,但anti-GDNF组凋亡较model组显著减少(P0.05)。与Sham组比较,在癫痫发作后1 d,model组的GDNF、RET表达水平上调,P53表达水平下降(P0.05),而anti-GDNF组大鼠海马CA1区GDNF、RET表达较model组明显下调,P53表达水平显著上降(P0.05)。结论:双侧海马CA1区立体定向注射anti-GDNF抗体能够减少癫痫发作,并对海马神经元起到保护作用,可能与其抑制GDNF/RET/P53信号通路有关。

The Effect of RET-dependent GDNF Signaling-regulation in a Rat Model of Epilepsy

ABSTRACT Objective: To investigate the effect of bilateral stereotactic administration of anti-GDNF antibody in the hippocampal CA1 region of a rat model of pilocarpine-induced epilepsy. Methods: 60 adult male SD rats were randomly divided into the sham group (n=20), model group (n=20) and anti-GDNF group (n=20). Lithium chloride and pilocarpine were intraperitoneally injected to induce seizures. The sham group was given lithium chloride only and anti-GDNF group was given a stereotactic injection of anti-GDNF anti- body into bilateral hippocampal CA1 region 2h before seizure induction. Then video monitoring of seizure incidence of the epileptic rats was done at 1, 3, 7d and EEG monitoring was performed at 7 d. Immunohistochemical staining was used to analyze the amount of neu- rons (NeuN expression) in the hippocampal CA1 region at 7 d, and western blot was carried out to explore the expression of protein lev- els of GDNF, RET and P53 in the hippocampal CA1 region at 1 d. Results: Model group tend to have more spike-slow waves than Sham group, and anti-GDNF group tend to have less than Model group in the parameters above (P<0.05); the amount of apoptotic neurons in the hippocampal CA1 region of Model group were large, but apoptotic cells of the anti-GDNF group were comparatively less than Model group (P<0.05); compared to Sham group, protein levels of GDNF and RET were up-regulated and P53 down- regulated in Model group 1 d after seizure induction (P<0.05), however the protein levels of GDNF and RET were obviously down-regulated and P53 up-regulated in the anti-GDNF group compared to Model group (P<0.05). Conclusion: Bilateral stereotactic administration of anti-GDNF antibody in the hippocampal CA1 region could reduce the incidence of seizure and have protective effects on the hippocampal neurons, which was probably related to suppression of GDNF/RET/P53 signaling.