| BackgroundStroke is the second most common cause of disability and death in the world,and it is the first major cause of disability and death in China.Patients with cerebral infarction accounts for about 70% of the total number of patients with stroke.Stroke survivors are often accompanied by varying degrees of dysfunction,of which motor dysfunction(MD)is a common dysfunction after acute cerebral infarction(ACI).MD after ACI not only severely limits the ability of patients and reduces the quality of life,but also requires long-term care and rehabilitation,resulting in a heavy burden on patients,families and society.ACI not only causes damage to the infarct area and its adjacent brain tissue,but also causes secondary damage to the relevant brain tissue away from the infarcted,non-injured blood supply area,but the nerve fibers are in relation to the infarcted.This secondary damage is called as remote damage after ACI.Recent studies have found that remote damage after ACI may impede neural function repair and are closely related to MD.It is a potential cause of high disability and poor prognosis in ACI.At present,electroacupuncture(EA)is widely used clinically to treat MD after ACI,however,its curative effect and mechanism are not yet clear.How about the relationship between remote damage and neurological deficit in ACI? What is the effect and mechanism of EA on the remote damage after ACI? These new scientific issues are not yet clear at home and abroad and need further study.ObjectiveThe purpose of this study was to observe the efficacy and safety of EA at Baihui(GV 20)and Dazhui(GV 14)acupoints on neurological rehabilitation in patients with MD after ACI.And based on Nogo-A / RhoA signaling pathway,the effect of EA on axon regeneration in distal part of infarction in rat withmiddle cerebral artery occlusion(MCAO)was also studied.In other words,the aim of this study was to study the possible mechanism of EA in reducing ACI-induced neurological deficit,promoting nerve regeneration and accelerating the recovery of nerve function,so that to provide evidence-based medical evidence and theoretical basis for clinical application of EA in treating ACI.MethodsClinical study section:92 patients with motor dysfunction after ACI,who were hospitalized in Foshan Hospital of Traditional Chinese Medicine from January 2017 to February2018 and were eligible for inclusion in this study,were divided into the control group and the EA group by using SAS statistical software in accordance with 1: 1 simple randomized grouping,46 cases in each group.The control group was given basic treatment,including routine medical treatment,routine rehabilitation training and conventional body acupuncture treatment.The EA group was additionally given EA at Baihui and Dazhui acupoints on the basis of the control group.The two groups were treated for 2 weeks.Before treatment and after 2 weeks of treatment,the Simplified Fugl-Meyer Motor Function Assessment(FMA),National Institutes of Health Stroke Scale(NIHSS),Modified Barthel Index(MBI),Stroke Special Quality of Life Scale(SS-QOL)and Modified Rankin Scale(mRS)were respectively used to evaluate the motor function,neurological function,activities of daily living(ADL),quality of life and degree of disability.At the end of 90 days of follow-up,MBI scale and mRS scale were respectively assessed.In addition,the occurrence of adverse events in the whole experiment was observed,and the appropriate treatment and record were also made.Mechanism study section:130 healthy male SPF grade Sprague-Dawley rats(weighing 250~300g,10~12 weeks old)were fed for 1 week,then 27 rats were randomly assigned to the sham-operated group according to the random number table,and the remaining rats were used to make the MCAO model according to Zea-longa improved thread method.54 rats with MCAO were randomly divided into the model group and the EA group according to the random number table,27 rats in each group.Then each group was subdivided into three subgroups according to different time points(the first day,the seventh day and the fourteenth day after theoperation).The rats in the sham-operated group only exposed subcutaneous tissue,muscle,fascia,right common carotid artery,external carotid artery and internal carotid artery,but without operating cerebral artery occlusion,then routinely fed without any treatment.The rats in the model group were also routinely fed without any treatment.The rats in the EA group were treated by EA at Baihui(GV 20)and Dazhui(GV 14)acupoints for 14 days,one time each day for 15 minutes per session.And the first treatment of EA began when the rats awake after the operation.6 rats in each subgroup were randomly selected and were assessed by neurological deficit score.3 rats in each subgroup were randomly selected to detect cerebral infarction volume by TTC staining.Another 3 rats in each subgroup were randomly selected to observe the pathological changes of brain tissue via HE staining.And immunofluorescence staining was used to detect the expression of GAP-43 and APP in striatum and pontine areas of rats.In each subgroup,3 rats were selected randomly,western blot method(WB)and real-time polymerase chain reaction method(RT-PCR)were respectively used to detect the expression of Nogo-A,NgR,RhoA,ROCK2 proteins and their mRNAs.ResultsClinical study section:A total of 92 patients with MD after ACI were enrolled in this study.Among them,3 cases were exfoliated in the EA group(1 were withdrawn from the trial because of fainting needle,2 were exfoliated due to inadequate treatment),4 patients in the control group were exfoliated because of inadequate treatment.The final 85 patients completed the study,of which 43 cases were in the EA group and 42 cases in the control group.The EA group: 31 males and 12 females,aged 40 to 79 years,duration of 0.1 to 14 days,hypertension in 38 cases,type2 diabetes in 15 cases,hyperlipidemia in 10 cases,heart disease in 6 cases.The control group: 26 males and 16 females,aged 36 to 80 years,duration of0.1 to 12 days,hypertension in 34 cases,type 2 diabetes in 13 cases,hyperlipidemia in 12 cases,heart disease in 8 cases.1.The changes of motor function: Compared with pre-treatment,the FMA score of the two groups respectively increased at the end of 2 weeks of treatment,and the difference was significant,respectively(P <0.01).Compared with the control group,there was no significant difference in FMA scores between the EA group and the control group before treatment(P >0.05),the FMA score of the EA group was significantly better than that of the control group at the end of 2 weeks of treatment(P <0.05),and there was also a significant difference on the FMA score differences of before and after treatment between the two groups(P <0.01).2.The changes of nerve function: Compared with the pre-treatment,the NIHSS score of the two groups was lower than that of the same group at the end of 2 weeks of treatment,the difference was respectively significant(P<0.01).Compared with control group,there was no significant difference in NIHSS scores between the EA group and the control group before treatment(P >0.05),the NIHSS score of the EA group was significantly lower than that of the control group at the end of 2 weeks of treatment(P <0.05),and the difference of the FMA score differences of before and after treatment between both groups was also statistically significant(P <0.01).3.The changes of quality of life: Compared with pre-treatment,the SS-QOL score of the two groups respectively increased at the end of 2 weeks of treatment,and the difference was respectively significant(P <0.01).Compared with the control group,there was no significant difference in SS-QOL scores between the EA group and the control group before treatment(P >0.05),the SS-QOL score of the EA group was significantly better than that of the control group at the end of 2 weeks of treatment(P <0.01),and there was also a significant difference on the SS-QOL score differences of before and after treatment between the two groups(P <0.01).4.The changes of ADL: Compared with pre-treatment,the MBI score of both groups increased at the end of 2 weeks of treatment and 90 days of follow-up,and the differences were statistically significant(P <0.01).Compared with the control group,there was no significant difference in MBI scores between the EA group and the control group before treatment(P >0.05),the MBI score of the EA group was superior to that of the control group at the end of 2 weeks of treatment,but the difference was not significant(P >0.05).The MBI score of the EA group was superior to that of control group at the end of 90 days of follow-up,and the difference was significant(P <0.05).5.The changes of degree of disability: Compared with pre-treatment,the mRS score of both groups decreased at the end of 2 weeks of treatment and 90 days of follow-up,and the differences were statistically significant(P<0.01).Compared with the control group,there was no significant differencein mRS scores between the EA group and the control group before treatment(P >0.05),the mRS score of the EA group were lower than that of the control group at the end of 2 weeks of treatment and 90 days of follow-up,the differences were significant(P <0.01).6.Adverse events: 1 patient in the EA group had vertigo,dizziness,nausea,vomiting and other fainting symptoms in the process of receiving EA.Immediately stop EA treatment,add warmth,and relieve symptoms after bed rest.No other adverse events occurred during the trial.Mechanism study section:1.The changes of neurological deficit: Compared with the sham-operated group,the scores of neurological deficit in the model group and the EA group were significantly increased on the 1st,7th and 14 th days after operation(P <0.05).Compared with the model group,the scores of neurological deficits in the EA group were significantly improved at all time points,with significant difference(P <0.05).2.The changes of cerebral infarction volume: The volume of cerebral infarction in the model group and the EA group were increased significantly on the 1st,7th and 14 th days after operation.Compared with the model group,the volume of cerebral infarction in the EA group was the same as that of the model group on the 1th day after operation(P >0.05).However,on the 7th and14 th day after operation,the volume of cerebral infarction in the EA group were significantly reduced,and the difference was statistically significant(P <0.01).3.The pathological changes of brain tissue: Brain tissue by HE staining,the neuronal cytoplasm was reddish and the nucleus was blue.In the sham-operated group: On the 1st,7th and 14 th days after operation,the cortex and gray matter of the rat brain tissue were clearly demarcated,no edema and necrosis area were found.And the nerve cells were arranged neatly and the distribution of nerve cells was uniform.The membrane of nerve cells was intact,the nucleus and nucleus ren were clearly.In the model group: On the 1st,7th day after operation,the neurons and glial cells in the gray matter area of the rats were multifocal liquefaction and necrosis,the interstitial edema were found.And the number of the neurons decreased significantly,the nucleus of the remaining neurons were concentrated,and the atrophy of the cytoplasm were also concentrated,and the microglial cells were proliferation.On the14 th day after operation,mild necrosis of the nerve fibers in the gray matter area of the rat brain were found,and the interstitial edema were alleviation,the number of nerve cells were decreased,while the proliferation of microglial cells were observed.In the EA group: On the 1st and 7th day after operation,the brain tissue of rats showed mild liquefaction degeneration,mild interstitial edema,decreased number of nerve cells,uneven distribution and proliferation of microglial cells;On the 14 th day after operation,the distribution of nerve cells was more uniform,cell degeneration,less necrosis and microgliall cells proliferated extensively.At different time points after operation,the extent of brain tissue damage and nerve fiber necrosis in the model group were more serious than that in the EA group.4.The expression of APP:(1)In the rat striatum area: On the 1st,7th and 14 th day after operation,the expressions of APP in each group were found.At different time points,the expressions of APP in the sham-operated group was similar.The expressions of APP in the model group and the EA group were most obvious on the 1st day after operation.Compared with the sham-operated group,the expressions of APP in the EA group and the model group was significantly increased on the 1st,7th and 14 th day after operation(P <0.01).Compared with the model group,the expressions of APP in the EA group were significantly decreased on the 7th and 14 th day after operation(P <0.01),however,on the 1st day after operation,the expression of APP in the EA group was similar to that in model group(P >0.05).In the rat pontine area: The expressions of APP in each group were decreased on the 1st,7th and 14 th day after operation,and the expressions of APP in the model group and the EA group were most obvious on the 1st day after operation.Compared with the sham-operated group,the expressions of APP in the EA group and the model group were significantly increased on the 1st,7th and 14 th day after operation(P<0.05).Compared with the model group,the expressions of APP in the EA group were significantly lower than that in the model group on the 1st and 7th day after operation(P <0.05),however,On the 14 th day after operation,the expression of APP in the EA group was similar to that in model group(P >0.05).5.The expression of GAP-43:(1)In the rat striatum area: On the 1st,7th and 14 th day after operation,the expressions of GAP-43 in each group were observed.The expressions of GAP-43 in the model group and the EA group were most obvious on the 1st day after operation.Compared with the sham-operatedgroup,the expressions of GAP-43 in the EA group and the model group was significantly increased on the 1st,7th and 14 th day after operation(P <0.05).Compared with the model group,the expressions of GAP-43 in the EA group was similar to that in model group on the 1st,and 14 th day after operation(P >0.05),but the expression of GAP-43 in the EA group was more obvious than that in the model group(P <0.05).(2)In the rat pontine area: On the 1st,7th and14 th day after operation,the expressions of GAP-43 in each group were found.Compared with the sham-operated group,the expressions of GAP-43 in the EA group and the model group were significantly increased on the 1st,7th and14 th day after operation(P <0.01).Compared with the model group,the expressions of GAP-43 in the EA group were significantly higher than that in the model group on the 7th and 14 th day after operation(P <0.01),however,On the 1st day after operation,the expression of GAP-43 in the EA group was similar to that in the model group(P >0.05).6.The expression of Nogo-A protein:(1)In the rat striatum area: Compared with the sham-operated group,the expression of Nogo-A protein in model group was significantly increased on the 1st,7th and 14 th day after operation(P<0.01).Compared with the model group,the expression of Nogo-A protein in EA group was decreased on the 1st,7th and 14 th day after operation(P <0.05)(2)In the rat pontine area: Compared with the sham-operated group,the expression of Nogo-A protein in the model group was significantly increased on the 1st,7th and 14 th day after operation(P <0.05).Compared with the model group,on the 1st,7th and 14 th day after operation,the expression of Nogo-A protein in the EA group was significantly decreased(P <0.05).7.The expression of NgR protein:(1)In the rat striatum area: Compared with the sham-operated group,the expression of NgR protein in the model group was significantly increased on the 1st day after operation(P <0.01).On the7 th and 14 th day after operation,the expression of NgR protein in the model group was similar to that in the sham-operated group(P >0.05).Compared with the model group,the expression of NgR protein in EA group was decreased on the 1st,7th and 14 th day after operation(P <0.01).(2)In the rat pontine area: Compared with the sham-operated group,the expression of NgR protein in the model group were significantly increased on the 7th and 14 th day after operation(P <0.05),however,the expression of NgR protein in the model group were similar to that in the sham-operated group on the 1st day after operation(P >0.05).Compared with the model group,on the 1st,7th and 14 th day after operation,the expressions of NgR protein in the EA group was significantly decreased(P <0.05).8.The expression of RhoA protein:(1)In the rat striatum area: Compared with the sham-operated group,the expression of RhoA protein in the model group was significantly increased on the 1st,7th and 14 th day after operation(P<0.01).Compared with the model group,the expression of RhoA protein in EA group was decreased on the 1st,7th and 14 th day after operation(P <0.01).(2)In the rat pontine area: Compared with the sham-operated group,the expression of RhoA protein in the model group were significantly increased on the 1st,7th and 14 th day after operation(P <0.01).Compared with the model group,the expressions of RhoA protein in the EA group were decreased on the7 th and 14 th day after operation(P <0.05),while the expression of RhoA protein in the model group was similar to that in the EA group on the 1st day after operation(P >0.05).9.The expression of ROCK2 protein:(1)In the rat striatum area: Compared with the sham-operated group,the expression of ROCK2 protein in the model group were similar on the 1st,7th and 14 th day after operation(P >0.05).Compared with the model group,the expression of ROCK2 protein in EA group was decreased on the 1st,7th and 14 th day after operation(P <0.05).(2)In the rat pontine area: Compared with the sham-operated group,the expression of ROCK2 protein in the model group were significantly increased on the 7th and 14 th day after operation(P <0.05),while the expression of ROCK2 protein in the model group were similar to that in sham-operated group on the 1st day after operation(P >0.05).Compared with the model group,on the 7th and 14 th day after operation,the expressions of ROCK2 protein in the EA group were significantly decreased(P <0.01).The expressions of ROCK2 protein in the EA group were similar to that in the model group on the 1st day after operation(P >0.05).10.The expression of Nogo-A mRNA:(1)In the rat striatum area: Compared with the sham-operated group,the expression of Nogo-A mRNA in model group was significantly increased on the 7th day after operation(P <0.05).On the1 st and 14 th day after operation,the expression of Nogo-A mRNA in the model group was similar to that in the sham-operated group(P> 0.05).Compared with the model group,the expression of Nogo-A mRNA in EA group was decreased onthe 1st,7th and 14 th day after operation(P <0.05).(2)In the rat pontine area: Compared with the sham-operated group,the expression of Nogo-A mRNA in the model group was significantly increased on the 1st and 7th day after operation(P <0.05),while the expression of Nogo-A mRNA in the model group was similar to that in the sham-operated group on the 14 th day after operation(P > 0.05).Compared with the model group,on the 1st,7the and 14 th day after operation,the expression of Nogo-A mRNA in the EA group was significantly decreased(P <0.05).11.The expression of NgR mRNA:(1)In the rat striatum area: Compared with the sham-operated group,the expression of NgR mRNA in model group were significantly increased on the 1st,7th and 14 th day after operation(P <0.01).Compared with the model group,the expression of NgR mRNA in EA group was decreased on the 1st and 14 th day after operation(P <0.01).On the 7th day after operation,the expressions of NgR mRNA in the EA group was similar with that in the model group(P >0.05).(2)In the rat pontine area: Compared with the sham-operated group,the expression of NgR mRNA in the model group were significantly increased on the 1st and 7th day after operation(P <0.01),while the expression of NgR mRNA in the model group was similar with that in the sham-operated group on the 14 th day after operation(P >0.05).Compared with the model group,on the 1st,7th and 14 th day after operation,the expressions of NgR mRNA in the EA group was significantly decreased(P <0.01).12.The expression of RhoA mRNA:(1)In the rat striatum area: Compared with the sham-operated group,the expression of RhoA mRNA in the model group was significantly increased on the 1st,7th and 14 th day after operation(P<0.05).Compared with the model group,the expression of RhoA mRNA in EA group was decreased on the 7th and 14 th day after operation(P <0.05),while the expression of RhoA mRNA in EA group was similar to that in the model group on the 1st day after operation(P >0.05).However,on the 14 th day after operation,the expression of RhoA mRNA in the EA group was similar to that in the model group(P >0.05).(2)In the rat pontine area: Compared with the sham-operated group,the expression of RhoA protein in the model group were significantly increased on the 1st,7th and 14 th day after operation(P <0.05).Compared with the model group,on the 1st,7th and 14 th day after operation,the expressions of RhoA mRNA in the EA group were obviously decreased(P <0.05).13.The expression of ROCK2 mRNA:(1)In the rat striatum area: Comparedwith the sham-operated group,the expression of ROCK2 mRNA in the model group were significantly increased on the 1st,7th and 14 th day after operation(P<0.05).Compared with the model group,the expression of ROCK2 mRNA in the EA group was significantly decreased on the 7th and 14 th day after operation(P <0.01),while the expression of ROCK2 mRNA in the EA group was similar with that of the model group on the 1st day after operation(P >0.05).(2)In the rat pontine area: Compared with the sham-operated group,the expression of ROCK2 mRNA in the model group were significantly increased on the 1st,7th and 14 th day after operation(P <0.01).Compared with the model group,on the7 th and 14 th day after operation,the expressions of ROCK2 mRNA in the EA group were significantly decreased(P <0.05),while the expression of ROCK2 mRNA in the EA group was similar to that in the model group on the 1st day after operation(P >0.05).Conclusions1.Conventional therapy,conventional therapy plus EA,both of which are effective for motor dysfunction after ACI,can both improve motor function,promote the recovery of nerve function,improve the living activity and quality of life,and reduce the degree of disability.2.After 2 weeks of treatment,EA plus conventional therapy for motor dysfunction after ACI is superior to conventional therapy in improving motor function and neurological function,promoting quality of life,and reducing the disability.However,the effect of EA plus conventional therapy on ADL is similar to that of the conventional therapy,the late result of EA plus conventional therapy on ADL is superior to conventional therapy.3.EA can improve the neurological deficit and reduce the infarct size in MCAO rats.4.The secondary damage of the axons occurred in the striatum and pontine which are in distal part of infarction in rat with MCAO,this kind of secondary damage plays an important role in neurological deficits.And EA can down-regulate the expression of APP in the striatum and pontine of rat.5.The axonal regeneration occurred in the striatum and pontine of MCAO rats,and EA can up-regulate the expression of GAP-43 in the striatum and pontine of rat with MCAO.6.The expressions of Nogo-A/RhoA signal pathway related proteins and their mRNAs were significantly enhanced in the striatum and pontine of ratswith MCAO,but EA can down-regulate their expressions.7.The underlying mechanism of EA can promote the recovery of neural function may be relations with that : EA can down-regulated the expressions of Nogo-A / RhoA signal pathway related proteins and their mRNAs in the striatum and pontine,inhibit axonal injury and promote nerve regeneration,so to reduce the secondary damage in distal part of infarction. |
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