Study On The Effect Of Opposing Needling On Yangming Meridian For Motor Dysfunction And The Integrity Of Neurovascular Unit In Stroke Rats

Objective:In this study,we observed the effect of opposing needling on Yangming meridian for neurological function and motor function of the left middle cerebral artery occlusion(MCAO)rats and the integrity of neurovascular unit,in order to investigate the molecular mechanisms of opposing needling on Yangming meridian in the treatment of motor dysfunction after stroke.Methods:Forty-two male Sprague-Dawley(SD)rats,270-290 g,were randomly separated into sham operated(SO)group(n = 12)and MCAO model group(n = 30).At 24 h after the establishment of MCAO model,the success of MCAO model was determined by Zea-Longa neurological function score,3 rats in SO group and 3 rats in MCAO model group were randomly selected for TTC staining to further verify the model.And then the successful model rats were randomly assigned into model group,opposing needling on acupoint(ON)group and opposing needling on non-acupoint(NA)group(n=9/group).Two days after the establishment of MCAO model,rats of ON group and NA group received electroacupuncture(EA)treatment(dilatational wave,1-10 Hz,30 min/day)for 7 days.The acupoints of ON group were Quchi(LI11)and Zusanli(ST36)of left limbs,and NA group were located at 5 mm far from the lateral of Quchi(LI11)and Zusanli(ST36).Before and after MCAO model establishment,the modified neurological severity score(m NSS)was used to examine the nerve function of all rats,beam balance test,suspension test and Cat Walk gait analysis system were for motor function.After the functional examination,all rats were sacrificed,the brain tissue was collected.The expression of Neu N protein in motor cortex and striatum of cerebral ischemia side was detected by immunofluorescence technique.The expression of CD31,GFAP and Iba1 protein was detected by immunohistochemistry.The expression of MBP,ZO-1 and Occludin was detected by Western-blot.Results:1.Results of neurological function score: Before treatment,m NSS scores in the model group,ON group and NA group were higher than SO group,suggesting that the establishment of MCAO model was successful.After treatment,compared with model group and NA group,m NSS score in ON group was significantly lower(P(27)0.05).2.Results of beam balance test: Before treatment,compared with SO group,the beam balance test scores were higher in model group,ON group and NA group,all of which showed decreased motor function.After treatment,compared with model group and NA group,the beam balance score of ON group was lower,and the difference was statistically significant(P(27)0.05).3.Results of suspension test: Before treatment,compared with SO group,the suspension test scores were lower in model group,ON group and NA group,all of which showed motor dysfunction of different degrees.After treatment,compared with model group and NA group,the suspension test score of ON group was increased,but the difference was not statistically significant(P>0.05).4.Results of Cat Walk gait analysis: Before treatment,compared with SO group,run average speed was slower,run duration was longer,print area and max contact area of right forepaw(RF)and right hindpaw(RH)was smaller,stride length(RF,RH)was shorter,swing speed(RF,RH)was slower,swing duration(RF,RH)was longer in model group,ON group and NA group,which suggested that MCAO rats had different degree of motor dysfunction.After treatment,compared with the model group and NA group,the run average speed was higher,run duration was shorter,and the other parameters were significantly improved in ON group(P(27)0.05).5.Immunofluorescence staining results: Compared with SO group,the positive expression of Neu N in model group was significantly decreased(P(27)0.05).Compared with the model group and NA group,the expression of Neu N positive cells in ON group was significantly increased(P(27)0.05).6.Immunohistochemical staining results: Compared with SO group,the positive expression of CD31,GFAP and Iba1 in model group was increased(P(27)0.05).Compared with model group and NA group,the expression of CD31 was significantly increased in ON group,and the expression of GFAP and Iba1 was significantly decreased in ON group(P(27)0.05).7.Western-blot results: Compared to SO group,the expression of MBP,ZO-1and Occludin protein in model group was decreased(P(27)0.05).Compared to model group and NA group,the expression of MBP,ZO-1 and Occludin protein in ON group was significantly increased(P(27)0.05).Conclusion:1.Opposing needling on Yangming meridian may be beneficial to improve the neurological deficit symptoms.2.Opposing needling on Yangming meridian could alleviate motor dysfunction of MCAO rats.3.Opposing needling on Yangming meridian could regulate the expression of the important components of the neurovascular units in the ischemic motor cortex and striatum region of MCAO rats,reduce the injury of neurons,promote the regeneration of vascular endothelial cells,reduce the destruction of tight junction,inhibit the activation and proliferation of astrocytes,inhibit the activation of microglia cells,and reduce the injury of oligodendrocytes,so as to facilitate the repair of the neurovascular unit.4.By regulating the integrity of neurovascular unit,which may be one of the mechanisms of opposing needling on Yangming meridian to alleviate motor dysfunction after stroke.