迷走神经刺激对难治性癫痫大鼠海马神经炎性反应及α7nAChR表达的影响*

doi:10.12047/j.cjap.6274.2022.070

摘要/Abstract

摘要： 目的: 探讨迷走神经刺激(VNS)对难治性癫痫(IE)模型大鼠海马神经炎性反应及α7nAChR表达的影响。方法: 80只成年雄性SD大鼠,SPF级,随机分为对照组、模型组、VNS组、甲基牛扁亭(MLA)+VNS组,其中对照组与MLA+VNS组分别20只,模型组与VNS组因模型制作失败与动物死亡,分别剩下15只和14只。除对照组之外,其余各组皆通过腹腔注射皮罗卡品建立氯化锂-皮罗卡品IE大鼠模型。对照组仅分离迷走神经,不采取电刺激;模型组不采取任何干预措施;VNS组在模型制作成功后7 d采取VNS,连续4周;MLA+VNS组先侧脑室给药MLA(3.4 μg/μl,5 μl),然后给予VNS,连续4周。观察并记录各组大鼠癫痫发作的次数与持续时间的变化;然后断头处死大鼠,快速分离海马并制备10%组织匀浆,离心并提取上清液,通过分光光度法测定上清液中AChE、ChAT活性;ELISA法检测TNF-ɑ、IL-6和IL-1β表达;Western blot检测海马组织α7nAChR蛋白表达;免疫荧光染色法检测海马组织α7nAChR与小胶质细胞共表达。结果: ①通过VNS治疗4周后,大鼠癫痫发作的频率以及持续的时间都明显低于模型组(P<0.01);MLA阻断后在给予VNS,大鼠癫痫发作的频率以及持续的时间也明显低于模型组,但高于VNS组(P<0.01)。②与对照组比较,模型组大鼠海马组织ChAT表达明显下降,AChE表达明显升高(P<0.01);与模型组比较,VNS组与MLA+VNS组大鼠海马组织ChAT表达明显升高,AChE表达明显降低(P< 0.01);与VNS组比较,MLA+VNS组大鼠海马组织ChAT、AChE表达无明显变化(P>0.05)。③与对照组比较,模型组大鼠海马组织TNF-ɑ、IL-6和IL-1β表达明显升高(P<0.01);与模型组比较,VNS组大鼠海马组织TNF-ɑ、IL-6和IL-1β表达明显降低(P<0.01);与VNS组比较,MLA+VNS组大鼠海马组织TNF-ɑ、IL-6和IL-1β表达明显升高(P<0.01)。④与对照组比较,模型组大鼠海马组织以及小胶质细胞上α7nAChR表达明显降低(P<0.01);与模型组比较,VNS组大鼠海马组织以及小胶质细胞上α7nAChR表达明显上调(P<0.01);与VNS组比较,MLA+VNS组海马小胶质细胞上共表达α7nAChR数量明显减少(P<0.01)。结论: VNS对IE大鼠有明显的治疗作用,其机制可能是通过直接激活海马小胶质细胞CAP,抑制海马神经炎性反应来实现的。

关键词: 难治性癫痫, 迷走神经刺激, α7nAChR, 胆碱能抗炎, 海马

Abstract: Objective: To investigate the effects of vagus nerve stimulation(VNS) on hippocampal neuro-inflammatory and α7 nicotinic acetylcholine receptor (α7nAChR) expression in rats with intractable epilepsy (IE). Methods: Eighty adult male SD rats (SPF) were randomly divided into control group, model group, VNS group and MLA+VNS group. There were respectively 20 rats in the control group and MLA+VNS group, and because of model failure and animal death, 15 rats and 14 rats in the model group and VNS group were left respectively . Except the control group, the IE model was established in other groups. Only the vagus nerve was isolated in the control group without electrical stimulation; the model group did not take any intervention measures; the VNS group was treated for 4 weeks with VNS after the model was successful; the MLA(3.4 μg/μl, 5 μl) was given to the lateral ventricle in the MLA+VNS group, and then VNS for 4 weeks. Seizure frequency and duration in each group were observed and recorded. And then the rats were decapitated, the hippocampus were quickly separated and 10% tissue homogenate was prepared. The homogenate was centrifuged and the supernatant was extracted. The activities of AChE and ChAT in the supernatant were measured by spectrophotometry, and the levels of TNF-ɑ, IL-6 and IL-1β were detected by ELISA. The expression of α7nAChR in rat hippocampals was detected by Western blot. The expression of α7nAChR on microglias in rat hippocampals was assesed by double-labeled immunofluorescence. Results: ①After VNS for 4 weeks, the frequency and duration of seizures in rats were decreased significantly, which were lower than those of the model group (P<0.01); After treated with MLA +VNS, the frequency and duration of seizures in rats were also reduced significantly, which were lower than those of the model group, but higher than those of the VNS group (P<0.01).②Compared with the control group, the expression of ChAT in the hippocampus of rats in the model group was decreased significantly and the expression of AChE was increased significantly (P<0.01); Compared with the model group, the expressions of ChAT in the hippocampus of rats in the VNS group and MLA+VNS group were increased significantly and the expressions of AChE were decreased significantly (P<0.01); Compared with the VNS group, in the hippocampus of rats in the MLA+VNS group, the expressions of ChAT and AChE had no significant changes (P>0.05). ③Compared with the control group, the expressions of TNF-ɑ, IL-6 and IL-1β in the hippocampus of rats in the model group were increased significantly (P<0.01). Compared with the model group, the expressions of TNF-ɑ, IL-6 and IL-1β in the hippocampus of rats in the VNS group were decreased significantly (P<0.01). Compared with the VNS group, the expressions of TNF-ɑ, IL-6 and IL-1β in the hippocampus of rats in the MLA+VNS group were increased significantly(P<0.01). ④Compared with the control group, the expression of α7nAChR in hippocampus and microglia of rats in the model group was decreased significantly(P<0.01); Compared with the model group, the expression of α7nAChR in hippocampus and microglia of rats in the VNS group was up-regulated significantly (P<0.01); Compared with the VNS group, coexpression of α7nAChR on microglia wasreduced significantly in the MLA+VNS group (P<0.01). Conclusion: VNS has obvious therapeutic effect on IE rats, and its mechanism may be related to activating hippocampal microglia cholinergic anti-inflammatory pathway directly and inhibiting hippocampal neuro-inflammatory response.

Key words: intractable epilepsy, vagus nerve stimulation, α7nAChR, cholinergic anti-inflammatory pathway, hippocampus

中图分类号:

R285.5

李永格, 周舒, 姚银乐, 未小明, 张士锋. 迷走神经刺激对难治性癫痫大鼠海马神经炎性反应及α7nAChR表达的影响^*[J]. 中国应用生理学杂志, 2022, 38(4): 373-378.

LI Yong-ge, ZHOU Shu, YAO Yin-le, WEI Xiao-ming, ZHANG Shi-feng. The effects of vagus nerve stimulation on hippocampal neuro-inflammatory and α7nAChR expression in rats with intractable epilepsy[J]. CJAP, 2022, 38(4): 373-378.

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迷走神经刺激对难治性癫痫大鼠海马神经炎性反应及α7nAChR表达的影响^*

The effects of vagus nerve stimulation on hippocampal neuro-inflammatory and α7nAChR expression in rats with intractable epilepsy

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