基于Notch1通路探讨不同频率重复经颅磁刺激改善大脑中动脉闭塞大鼠学习记忆能力的机制

Exploring the mechanism of different frequencies of rTMS in improving learning and memory abilities in middle cerebral artery occlusion rats based on Notch1 pathway

目的:观察不同频率重复经颅磁刺激(repetitive transcranial magnetic stimulation,rTMS)对大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)大鼠学习记忆能力的影响及探讨其作用机制。方法:将造模成功的MCAO模型大鼠随机均分为模型组、低频组、高频组,同时另设假手术组,每组12只。低频组和高频组均予采用CCY-I型经颅磁刺激仪刺激,2次/日,连续14d;低频组:1Hz,部位:大鼠颅部左侧前额叶;高频组频率:10Hz,部位:大鼠颅部右侧前额叶。模型组和假手术组不予治疗仅同等情况下抓取束缚后归笼。神经行为学评分在术后第1天和第14天进行;水迷宫测试于术后3—7天进行;TTC染色、PCR及Western blot检测关键基因(Notch1、Hes1、Hes5)的mRNA及蛋白表达均在术后第14天干预结束后进行。结果:(1)神经功能缺损评分:与模型组相比,干预治疗14d后,高频组、低频组两组神经功能评分出现降低的情况(P<0.05),模型组与高频组之间无明显差异(P>0.05)。(2)Morris水迷宫实验:定向航行实验平均逃避潜伏期:低频组、高频组、假手术组均相比于同组前一天缩短,具有显著性差异(P<0.05);每天逃避潜伏期比较,高频组较模型组短,低频组较高频组短,具有显著性差异(P<0.05)。空间探索实验穿越平台次数:低频组和假手术组比较、高频组和模型组比较无显著性差异(P>0.05);高频组、模型组次数比假手术组少,低频组比模型组、高频组次数多,具有显著性差异(P<0.05)。(3)TTC染色:低频组、高频组梗死灶面积小于模型组。(4)PCR检测及Western Blot检测:高频组、低频组与模型组相比,Notch1、Hes1、Hes5的mRNA及蛋白表达量升高,具有显著性差异(P<0.05);低频组与高频组相比,Notch1、Hes1、Hes5的mRNA及蛋白表达无显著性差异(P>0.05)。结论:高/低频rTMS均能改善MCAO大鼠的学习记忆能力,且可能都予激活提高海马组织中的Notch1通路表达,提高海马突触可塑性有关。

Objective:To observe the effects of repetitive transcranial magnetic stimulation(rTMS)at different frequencies on learning and memory abilities in rats during the early recovery phase of middle cerebral artery occlusion(MCAO)and to explore the underlying mechanisms.Method:Successfully modelled MCAO rats were randomly divided into the model group,low-frequency group,high-frequency group,and sham-operated group was set up at the same time,with 12 rats in each group.The low-frequency group and high-frequency group were stimulated with a CCY-I transcranial magnetic stimulator 2 times/day for 14d;the low-frequency group was treated with 1Hz at the left prefrontal lobe of the rat's skull,and the high-frequency group was treated with 10Hz at the right prefrontal lobe.The model group and the sham-operated group were not treated but were subjected to the same conditions of handling and restraint before returning to their cages.Neurobehavioral scores were recorded on the 1st and 14th day after the operation;the Morris water maze test was conducted on day 3—7 after the operation;TTC staining,PCR and Western blot to detect the mRNA and protein expression of key genes(Notch1,Hes1,Hes5)were performed at the end of the intervention on the 14th postoperative day.Result:①Neurological deficit scores:After 14 days of intervention,both the high-frequency and low-frequency groups showed a reduction in neurofunctional compared to the model group(P<0.05),with no significant difference between the model group and high-frequency group(P>0.05).②Morris water maze experiment:The average escape latency during the directional navigation experiment was significantly reduced day by day in the low frequency group,high frequency group and sham operation group(P<0.05).The escape latency was shorter in the high-frequency group compared to the model group,and shorter in the low-frequency group compared to the high-frequency group(P<0.05).The number of platform crossings in the spatial exploration experiment showed no significant difference between low frequency group and sham operation group,high frequency group and model group(P>0.05).The high-frequency group and the model group had fewer crossing than the sham operation group,and the low-frequency group had more than the model group and high-frequency group(P<0.05).③TTC staining:The infarct size in the low-frequency group and high-frequency group was significantly smaller than in the model group.④PCR and Western blot:Compared to the model group,the high frequency group and the low frequency group had significantly increased mRNA and protein expression of Notch1,Hes1 and Hes5(P<0.05).There was no significant difference in the mRNA and protein expression of Notch1,Hes1 and Hes5 between the low frequency group and high frequency group(P>0.05).Conclusion:Both high and low frequency rTMS can improve the learning and memory abilities in MCAO rats,potentially by activating and increasing the expression of the Notch1 pathway in hippocampal tissues,which improves the synaptic plasticity of hippocampus.