高频重复经颅磁刺激促进脑缺血大鼠神经功能恢复的作用与机制

Effects and mechanism of high frequency repetitive transcranial magnetic stimulation in promoting recovery of neurological function in cerebral ischemic rats

目的探讨高频重复经颅磁刺激(HF-rTMS)促进脑梗死大鼠脑神经功能恢复的作用及机制。方法选择230~250 g的雄性大鼠构建短暂性大脑中动脉闭塞(tMCAO)模型。根据改良神经功能缺损评分(mNSS)选择7~12分的大鼠进行研究,采用随机数字表法分为经颅磁刺激组(rTMS组)、对照组(tMCAO组)和假手术组(Sham组)。rTMS组大鼠于术后第1天开始接受为期28 d的10 Hz rTMS的治疗,tMCAO组和Sham组分别予以假刺激治疗。各组大鼠于术后第1、3、7、14、21、28天分别进行mNSS评分、圆柱桶试验和胶布移除试验的行为学功能评估,采用TTC染色检测术后各组大鼠的脑梗死体积,采用ELISA方法检测各组大鼠脑脊液中白细胞介素(IL)-1β、肿瘤坏死因子(TNF-α)、IL-4和IL-10水平,并对rTMS组和tMCAO组的脑梗死大鼠的梗死灶周围皮质组织进行RNA测序分析。结果与tMCAO组相比,rTMS组在术后第14、21和28天的mNSS评分和胶布移除时间显著减少,在术后第7、14、21和28 d的前肢不对称使用评分显著减少,差异均有统计学意义(P均<0.05)。与tMCAO组相比,rTMS组的梗死体积在治疗后第14天[(45.33±5.89)%vs.(35.98±3.57)%]和第28天[(33.58±4.17)%vs.(23.00±5.29)]显著减小,差异均有统计学意义(P均<0.05)。ELISA检测显示在术后第28天,与tMCAO组相比,rTMS组IL-1β[(9.95±1.27)pg/mL vs.(6.90±0.60)pg/mL]和TNF-α[(103.30±13.35)pg/mL vs.(70.05±9.63)pg/mL]水平均显著减少,而IL-10水平显著增加[(17.32±3.07)pg/mL vs.(27.31±4.14)pg/mL],差异均有统计学意义(F=15.33、16.25、16.04,P均<0.05);IL-4的水平[(66.07±7.15)pg/mL vs.(78.41±9.85)pg/mL]增加,差异无统计学意义(F=18.91,P>0.05)。进一步RNA测序分析发现rTMS组和tMCAO组之间共有79个显著差异表达基因(DEGs)。KEGG分析显示DEGs主要富集在炎症相关通路:AGE-RAGE信号通路、Toll样受体信号通路、NOD样受体信号通路。基因集富集分析(GSEA)显示这3条通路在rTMS组中均受抑制。结论HF-rTMS能通过减轻脑梗死大鼠的神经炎症反应而促进神经功能的恢复,其机制可能与AGE-RAGE信号通路、Toll样受体信号通路和NOD样受体信号通路相关。

Objective To investigate the effects and mechanism of high frequency repetitive transcranial magnetic stimulation(HF-rTMS)on neurological function recovery in rats with ischemic cerebral infarction.Methods Transient middle cerebral artery occlusion(tMCAO)model was constructed in 230-250 g male rats.According to the modified Neurological Severity Score(mNSS),rats with 7 to 12 points were selected and divided into transcranial magnetic stimulation group(rTMS group),control group(tMCAO group),and Sham group(Sham group)by random number table method.The rats in the rTMS group were treated with 10 Hz rTMS for 28 days from the first day after surgery,and the tMCAO group and the Sham group were treated with sham stimulation,respectively.The mNSS score,cylinder test and adhesive removal test were performed at day 1,3,7,14,21 and 28 after surgery to evaluate behavioral function.TTC staining was used to detect cerebral infarction volume.The content of interleukin(IL)-1β,tumor necrosis factor-α(TNF-α),IL-4 and IL-10 in cerebrospinal fluid of rats were measured by ELISA.The peri-infarct cortical tissues from the rTMS and tMCAO group were taken for RNA sequencing analysis.Results Compared with the tMCAO group,mNSS scores and adhesive removal time were significantly reduced at 14,21 and 28 days post-stroke,and the forelimb use asymmetry score were significantly reduced at 7,14,21 and 28 days post-stroke in rTMS group,the differences were statistically significant(all P<0.05).Compared with tMCAO group,the infarct volume in rTMS group were significantly reduced at 14 days[(45.33±5.89)%vs.(35.98±3.57)%]and 28 days[(33.58±4.17)%vs.(23.00±5.29)%]post-stroke,the differences were statistically significant(all P<0.05).Compared with tMCAO group,ELISA showed that IL-1β[(9.95±1.27)pg/mL vs.(6.90±0.60)pg/mL]and TNF-α[(103.30±13.35)pg/mL vs.(70.05±9.63)pg/mL]were significantly decreased,and IL-10[(17.32±3.07)pg/mL vs.(27.31±4.14)pg/mL]were increased in rTMS group,the differences were statistically significant(F=15.33,16.25,16.04,all P<0.05).While IL-4[(78.41±9.85)pg/mL vs.(66.07±7.15)pg/mL]were increased in rTMS group compared with control group,there was no statistically significant difference(F=18.91,P>0.05).Further RNA sequencing analysis revealed a total of 79 differentially expressed genes(DEGs)between the rTMS group and the tMCAO group.KEGG analysis showed that DEGs were mainly enriched in inflammation-related pathways,such as AGE-RAGE signaling pathway,Toll-like receptor signaling pathway,and NOD-like receptor signaling pathway.Gene set enrichment analysis(GSEA)showed that these three pathways were inhibited in the rTMS group.Conclusion HF-rTMS could promote the neurological function recovery in rats with ischemic stroke by alleviating neuroinflammatory response,and its mechanism might be related to AGE-RAGE signaling pathway,Toll-like receptor signaling pathway and NOD-like receptor signaling pathway.