| Objective:In this study we discuss the effects of Electro-acupuncture(EA)at Baihui(DU20)and Shenting(DU24)acupoints for the spatial learning and memory function,which whether regulates LIMK1 phosphorylation level via miR-134 and detect the change of hippocampal synaptic plasticity in cerebral ischemia/reperfusion injured rats.Methods:All SD(Sprague-Dawley)rats were randomly divided into sham operation group(Sham)and operation group.Rats of operation group were used to make models of left middle cerebral artery occluded(MCAO)using the Koizumi methord.According to the preintervention Zea-Longa neurological behavior scores and the results of MRI after MCAO,the rats were randomly divided into ischemia control group(MCAO/R),EA treatment group(MCAO/R+EA)and non-acupoint+EA group(MCAO/R+non-EA).After 24h of ischemia/re-perfusion injury,EA treatment group received electroacupuncture at Baihui(DU20)and Shenting(DU24)points for 14 days and non-acupoint+EA group received electroacupuncture at the bilateral non-acupoints for 14 days.The stimulation parameters were 2/20 Hz dense-and-disperse square wave,30 minutes each time,once a day.Morris water maze test was used to evaluate spatial learning and memory of rats in received electroacupuncture on 10th day.Using MRI to observe the change of lesion volumes.Using golgi staining to obsever the morphology of dendritic spine in the left hippocampe CA1 area.Western Blot detected the LIMK1 protein and phosphorylation levels and RT-qPCR detected the expression of miR-134.Results:(1)After ischemia/reperfusion injury,Zea-Longa scores of sham group compared to the MCAO/R group,MCAO/R+EA and MCAO/R+non-EA decreased significantly(P<0.05).The scores of MCAO/R group,MCAO/R+EA group and MCAO/R+non-EA group were no statistical significance(P>0.05);after EA treatment for 14 days,Zea-Longa scores of MCAO/R+EA group compared to the MCAO/R group decreased significantly(P<0.05),which showed that EA at DU20 and DU24 could improve the symptoms of neurological deficits in rats after cerebral ischemia/reperfusion(I/R)injury.(2)Step-down test:After ischemia/reperfusion injury,the platform residence time of rats in sham group were more than other groups(P<0.01).After EA treatment for 14 days,EA treatment significantly reduced the platform residence time compared with those of the MCAO/R group and MCAO/R+non-EA group(P<0.05);(3)Morris water maze:The escape latency of rats in sham group were less than other groups(P<0.01),and compared with the ischemia group and non-acupoint group,the escape latency was lessen(P<0.01)in EA group in navigation experiments;space probe test showed that the number of crossing the platform of rats in the sham operation group were more than other groups(P<0.01),and compared with the ischemia group and non-acupoint group the times reduced in EA group(P<0.05),which explained that EA at DU20 and DU24 could improve spatial learning and memory ability in rats after cerebral ischemia/reperfusion(I/R)injury.(4)Volume of cerebral infarction:After 24h of ischemia/reperfusion injury,T2WI showed that cerebral infarction did not appear in rats of sham group(P<0.01),The infarction of MCAO/R group,MCAO/R+EA group and MCAO/R+non-EA group were no statistical significance(P>0.05);after EA treatment for 14 days,compared with the ischemia group and non-acupoint group,the volume of cerebral infarction reduced(P<0.05)in EA group,which showed that EA at DU20 and DU24 could decreased the volume of cerebral infarction in rats with cerebral ischemia/reperfusion(I/R)injury.(5)After EA treatment for 14d,Golgi-Cox staining results show that the dendritic spine of left hippocampe CA1 in MCAO/R+EA group were significantly intact comparing with the MCAO/R group(P<0.01);while there was no significant difference in the the MCAO/R group and MCAO/R+non-EA(P>0.05).(6)After EA treatment for 14d,transmission electron microscope(TEM)results show that the number of left hippocampal synapsis in MCAO/R+EA group were significantly increase comparing with the MCAO/R group(P<0.01);while there was no significant difference in the the MCAO/R group and MCAO/R+non-EA(P>0.05);(7)The expressing of LIMK1 and P-LIMK1 protein of left hippocampe were observed in the four groups.Compared with the sham group,the expressing of LIMK1 and P-LIMK1 protein were decreased(P<0.01),which indicate cerebral ischemia could inhibit the expression of LIMK1 and P-LIMK1.However,after EA treatment for 14d,P-LIMK1 expression of left hippocampe was significantly increased comparing with the MCAO/R group(P<0.01),which may showe that EA at Baihui(DU20)and Shenting(DU24)points can promote the activation of LIMK1 and the phosphorylation of LIMK1.(8)After EA treatment for 14d,RT-qPCR results show that the expression of miR-134 in MCAO/R+EA group were significantly decreased comparing with the MCAO/R group(P<0.01);while there was no significant difference in the the MCAO/R group and MCAO/R+non-EA(P>0.05).Conclusion:EA at DU20 and DU24 points could promote the phosphorylation of LIMK1,impacting the morphology of dendritic spine via miR-134 in hippocampe CA1 neurons.The molecular mechanism may be related to promote synaptic plasticity,improving spatial learning and memory function in cerebral ischemia reperfusion injury of rats. |
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