电针对肌萎缩侧索硬化症模型小鼠皮质小胶质细胞/神经元活性影响的机制研究

关键词：肌萎缩侧索硬化症  电针干预  小胶质细胞  神经炎症  大脑皮质

Keywords:amyotrophic lateral sclerosis  electroacupuncture intervention  microglia  neuroinflammation  cerebral cortex

摘要：

      摘要 目的：观察电针干预对肌萎缩侧索硬化小鼠（amyotrophic lateral sclerosis， ALS）小胶质细胞和大脑皮质兴奋性的影响，探讨电针干预改善ALS动物模型运动功能的中枢机制。 方法：①按随机数字法将16只hSOD1G93A小鼠分为模型组和电针组，hSOD1G93A阴性小鼠为空白组，每组8只。电针组电针干预百会、双侧天柱、天枢。10min/次，5次/周，1周为1个疗程，连续3个疗程。采用后肢紧抱实验、旷场实验评估各组小鼠运动功能；ELISA法检测血清炎症因子IL-1β及IL-6的含量；免疫荧光染色法和流式细胞术观察大脑皮质Iba-1阳性细胞率及细胞表型。②21只hSOD1G93A小鼠，7只同窝野生鼠。向小鼠大脑皮质M1区注射化学遗传兴奋病毒[rAAV-CaMKIIa-hM3D（Gq）-EGFP-WPRE-hGH polyA]，待病毒表达21天后，随机选取3只SOD1G93A小鼠和1只hSOD1G93A阴性小鼠于荧光显微镜下观察病毒表达情况，将剩余SOD1G93A小鼠随机分为3组：模型组、电针组和化学遗传兴奋病毒+电针组［腹腔注射氯氮平-N-氧化物（CNO）+电针］，每组6只。采用免疫荧光法观察大脑皮质c-Fos阳性细胞率，旷场实验评估各组小鼠运动功能。 结果：与空白组比较，模型组小鼠旷场运动总距离缩短、神经学评分升高（P＜0.01），血清IL-1β、IL-6含量、大脑皮质Iba-1、c-Fos阳性细胞率均升高（P＜0.01），M1型、M2型小胶质细胞占比升高（P＜0.01，P＜0.05）。与模型组比较，电针组小鼠旷场运动总距离延长、神经学评分降低（P＜0.01，P＜0.05），血清IL-1β、IL-6含量、大脑皮质Iba-1、c-Fos阳性细胞率均降低（P＜0.01，P＜0.05），M2型小胶质细胞占比升高（P＜0.05）；病毒+电针组c-Fos阳性细胞率降低、旷场运动总距离延长。 结论：电针干预可以改善早期ALS模型小鼠运动功能，其机制可能与抑制小胶质细胞的活性，降低炎症因子表达，降低M1皮质神经元兴奋性有关。

Abstract:

      Abstract Objective： To observe the effect of electroacupuncture（EA） on microglia and cortical excitability in hSOD1G93A mice and to explore the potential central mechanisms of EA intervention to improve motor function in amyotrophic lateral sclerosis（ALS） mice. Method： The present study includes 2 parts. ①Sixteen hSOD1G93A mice were randomly divided into model group and EA group (n=8 per group). Eight hSOD1G93A-negative mice were used as the blank group. The EA group was applied to the Baihui， bilateral Tianzhu and bilateral Tianshu acupoints（10 minutes/session， 5 sessions/week， for 3 weeks）. After 3 weeks of EA， hind limb functional neurologic score and open field test were used to evaluate the changes in motor function of mice. The concentrations of IL-1β and IL-6 were detected by ELISA. Immunofluorescence staining and flow cytometry were employed to observe the rate of Iba-1 positive cells and cell phenotypes in the cerebral cortex. ②Twenty-one hSOD1G93A mice and seven wild-type mice were included. The Chemical genetic excitatory virus [rAAV-CaMKIIa-hM3D（Gq）-EGFP-WPRE-hGH polyA] was injected into the primary motor cortex （M1） area of the mice. After 21 days， four mice (3 SOD1G93A and 1 wild-type) were randomly selected for fluorescent microscopy to confirm viral expression. The remaining hSOD1G93A mice were randomly divided into 3 groups： model， EA group, and chemogenetic excitatory virus (intraperitoneal injection of clozapine-N-oxide [CNO]) combined with EA group， with 6 mice per group. The rate of c-Fos positive cells in the cerebral cortex was observed by immunofluorescence. The open field experiment was used to evaluate the changes in motor function of mice. Result： Compared with the blank group，the model group showed significantly reduced total distance in the open-field test and increased neurologic scores(P<0.01）, elevated serum IL-1β and IL-6 levels, increased cortical Iba-1 and c-Fos expression（P＜0.01）, and higher proportions of M1 and M2 microglia (P<0.01, P<0.05). Compared with the model group， the total distance of open field movement was prolonged and the neurological score decreased in the EA group（P<0.01，P<0.05）. The contents of serum IL-1β and IL-6， the expression of Iba-1、c-Fos in cerebral cortex were decreased in the EA group（P<0.01，P<0.05），with an increased proportion of M2 microglia（P<0.05）； the rate of c-Fos positive cells decreased in the virus+EA group， and the total distance of open field movement was prolonged. Conclusion： EA intervention can improve motor function in ALS mice. The underlying mechanism may be related to suppress the activity of microglia， reduce the expression of inflammatory cytokines and further to decrease the excitability of M1 cortical neurons.