重复经颅磁刺激对卒中后睡眠剥夺模型大鼠学习记忆及海马突触可塑性的影响

Effects of repetitive transcranial magnetic stimulation on learning-memory and synaptic plasticity in hippocampus of rats with post-stroke sleep deprivation

目的探讨重复经颅磁刺激(repetitive transcranial magnetic stimulation,rTMS)对卒中后睡眠剥夺模型大鼠学习记忆及海马区突触可塑性蛋白表达的影响。方法28只SPF级8周龄健康雄性Wistar大鼠,按照随机数字表法分为对照组、假手术组、模型组和rTMS组,每组7只。模型组和rTMS组大鼠采用大脑中动脉栓塞+对氯苯丙氨酸腹腔注射法建立卒中后睡眠剥夺模型,rTMS组大鼠在建模后连续14 d进行rTMS干预;假手术组大鼠仅分离动脉但不结扎、不插线;对照组大鼠正常饲养。采用旷场实验观察大鼠自主行为,水迷宫实验观察其空间学习记忆能力,Western blot法检测海马组织酪氨酸激酶受体B(tyrosine kinase receptor type B,TrkB)含量,免疫荧光法检测海马组织脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)、即刻早期基因c-fos表达情况,光学显微镜及透射电镜观察海马区神经元形态及结构。采用SPSS 21.0软件进行统计分析,逃避潜伏期数据采用重复测量方差分析,其他数据多组间比较采用单因素方差分析,进一步两两比较采用LSD检验。结果(1)旷场实验结果表明,干预后4组大鼠穿越方格数、直立次数和总得分均差异有统计学意义(F=27.638,10.425,30.690,均P<0.001)。rTMS组大鼠穿越方格数[(72.71±10.10)个]、直立次数[(6.57±0.87)次]、总得分[(79.29±10.03)分]均高于模型组[(43.71±6.96)个,(3.43±0.65)次,(47.14±6.82)分](均P<0.05)。水迷宫实验结果表明,rTMS干预后,4组大鼠逃避潜伏期比较,交互作用不显著(F=1.108,P=0.37),时间主效应(F_(时间)=27.295,P_(时间)<0.01)和组别主效应(F_(组别)=8.691,P_(组别)<0.01)均显著,在第3天和第4天,rTMS组大鼠逃避潜伏期均低于模型组(均P<0.01)。干预后,4组大鼠穿越平台次数、目标象限游泳距离及停留时间均差异有统计学意义(F=8.569,3.308,3.547,均P<0.05),rTMS组大鼠的穿越平台次数[(2.00±0.31)次]、目标象限游泳距离[(196.95±24.57)cm]及停留时间[(17.72±1.36)s]均高于模型组[(1.57±0.30)次,(146.61±4.79)cm,(13.58±0.98)s](均P<0.05)。(2)光学显微镜和透射电镜下均显示,与正常对照组比较,模型组大鼠海马区细胞排列紊乱、细胞器完整性被破坏,经rTMS干预后,rTMS组大鼠海马区神经细胞排列及结构均有所改善。(3)免疫荧光结果显示,rTMS组大鼠海马区的c-fos(1.49±0.09)、BDNF(0.84+0.06)均高于模型组[(1.24±0.12),(0.48±0.08),均P<0.05]。Western blot结果显示,rTMS组大鼠海马组织TrkB(1.81±0.03)表达水平高于模型组(0.96±0.02)(P<0.05)。结论rTMS能改善卒中后睡眠剥夺大鼠的学习记忆能力和自主行为能力,可能与促进海马区c-fos、BDNF、TrkB表达有关。

ObjectiveTo investigate the effects of repetitive transcranial magnetic stimulation(rTMS)on learning-memory and the expression of synaptic plasticity proteins in hippocampus of rats with post-stroke sleep deprivation.MethodsA total of 28 SPF grade healthy male Wistar rats with 8-week old were randomly divided into four groups(control group,sham operation group,model group and rTMS group)according to random number table method,with 7 rats in each group.The rats in the model group and the rTMS group were treated with middle cerebral artery occlusion and p-chlorophenylalanine intraperitoneal injection to establish the post-stroke sleep deprivation model.The rats in the rTMS group were treated with rTMS intervention for consecutive 14 days after modeling.The rats in the sham operation group were only separated arteries but not ligated and inserted.The rats in control group were fed normally.The open field test(OFT)was used to observe the autonomous behavior of rats.The water maze test(WMT)was used to observe the spatial learning and memory ability of rats.The content of tyrosine kinase receptor type B(TrkB)in hippocampus was detected by Western blot.The expressions of brain-derived neurotrophic factor(BDNF)and immediate early gene c-fos in hippocampus were detected by immunofluorescence.The morphology and structure of neurons in hippocampus were observed by optical microscopy and transmission electron microscopy.SPSS 21.0 software was used for statistical analysis,and repeated measurement ANOVA was used for the escape latency data,one-way ANOVA was used for the comparison of other data among multiple groups,and LSD test was used for further pairwise comparison.Results(1)The OFT results showed that there were statistical differences in the numbers of crossing squares,upright times and total points of rats in the four groups after intervention(F=27.638,10.425,30.690,all P<0.001).The numbers of crossing squares((72.71±10.10)),upright times((6.57±0.87)times)and total points((79.29±10.03)points)of rats in rTMS group were all higher than those in model group after intervention((43.71±6.96),(3.43±0.65)times,(47.14±6.82)points)(all P<0.05).As for the escape latency of WMT among the four groups of rats,the interaction effect was not significant(F=1.108,P=0.37),and the time main effect(F_(time)=27.295,P_(time)<0.01)and group main effect(F_(group)=8.691,P_(group)<0.01)were significant after rTMS intervention.On the 3rd and 4th day,the escape latency of rTMS group rats was lower than that of the model group(both P<0.01).There were statistically significant differences in the numbers of crossing platform,swimming distance and residence time in target quadrant of rats in the four groups after intervention(F=8.569,3.308,3.547,all P<0.05).The numbers of crossing platform((2.00±0.31)times),swimming distance in target quadrant((196.95±24.57)cm)and residence time((17.72±1.36)s)of rats in rTMS group were all higher than those in model group after intervention((1.57±0.30)times,(146.61±4.79)cm,(13.58±0.98)s)(all P<0.05).(2)Optical microscopy and transmission electron microscopy showed that the hippocampal cells arranged irregularly,the organelles'integrity was destroyed in the model group compared with the normal control group.In rTMS group the arrangement and structure of nerve cells in the hippocampus were improved after rTMS intervention.(3)The immunofluorescence results showed that c-fos(1.49±0.09)and BDNF(0.84±0.06)in the hippocampus of rats in rTMS group were both higher than those in model group((1.24±0.12),(0.48±0.08))(both P<0.05).The Western blot results showed that the expression level of TrkB(1.81±0.03)in the hippocampus of rats in rTMS group was higher than that in model group(0.96±0.02)(P<0.05).ConclusionThe rTMS can improve the learning-memory ability and autonomous capacity of rats with post-stroke sleep deprivation,which may be related to promoting the expression of c-fos,BDNF and TrkB in hippocampus tissue.