| Purpose:Exercise training has been proven to be an effective therapy to promote stroke recovery by inhibiting the microglia-initiated inflammatory response.Yet few studies have focused on the phenotypic changes in microglia cells caused by exercise training.The aim of the present study was to evaluate the effects of treadmill exercise on neurobehavioral performance and microglia polarization in rats after cerebral ischemia-reperfusion injury,so as to investigate whether the protective effect of treadmill exercise against cerebral ischemia-reperfusion injury is associated with microglia polarization.This study provides laboratory evidence for the application of exercise training in stroke rehabilitation.Methods:Male Sprague-Dawley(SD)rats were used in this study and randomly assigned into 3 groups:Sham,MCAO and Exercise.There were 3 subgroups of Sham and MCAO groups:24h,4d and 7d post-surgery.The Exercise group was divided into two subgroups:4d and 7d post-surgery.The MCAO and Exercise groups received right middle cerebral artery occlusion(MCAO)surgery,following the Zea-Longa test 24h after the reperfusion.Only the rats with a score of 1-3 were included in this study.The sham group underwent a sham operation.Treadmill exercise started at 24h post-surgery and took 30min every day,lasting for 3d or 6d.The speed was maintained at 5 m/min in the first 10min,then increased to 9 m/min in the next 10min and 12 m/min in the last10min.A modified neurological severity score(m NSS)was used to evaluate neurological deficits between MCAO and Exercise groups before and after the intervention.At 24h,4d and 7d post-surgery,quantitative Real-Time Polymerase Chain Reaction Analysis(q RT-PCR)and Luminex was employed to determine the expression of interleukin(IL)-4 in the penumbra.Luminex was to detect the expression of IL-13,M1 microglia markers(IL-1α,IL-1β,IL-6,and MCP-1)and M2 microglia marker(IL-10)in the penumbra.Western blotting was used to identify the levels of p-JAK1 and p-STAT6 in the penumbra.Double immunofluorescence was conducted to identify CD68/Iba1 and Arg-1/Iba1 positive cells of the cortex and striatum in the penumbra.Results:1.Effects of treadmill exercise on neurological scores after CI/RPThere is no significant difference in m NSS scores between the MCAO and Exercise groups at 4d after surgery(P=0.293).The m NSS scores of the Exercise group were significantly lower than those of the MCAO group at 7d after operation(P<0.001),mainly in the motor(P=0.004)and balance(P=0.032)subitems.2.Effect of treadmill exercise on M1/M2 microglia polarization after CI/RPCD68/Iba1 immunofluorescence staining showed that compared to the Sham group,CD68~+microglia in the cortex and striatum of the MCAO group increased at24h,4d and 7d after the operation(P<0.05),as well as CD68~+microglia in the cortex of the Exercise group at 4d(P=0.001)and 7d(P=0.033)post-surgery,while CD68~+microglia in the striatum showed no significant difference at two time points(P>0.05).In comparison with the MCAO group,CD68~+microglia in the striatum of the Exercise group reduced significantly at 4d after the surgery(P=0.029).On day 7 after the operation,CD68~+microglia in the cortex and striatum of the Exercise group decreased markedly(P<0.001).The results of Arg-1/Iba1 immunofluorescence staining showed that compared to the Sham group,Arg-1~+microglia in the striatum at 24 h(P=0.003),Arg-1~+microglia in the cortex and striatum at 4d(P<0.001)and 7d(P<0.05)post-surgery significantly elevated in the MCAO group,as well as in the Exercise group(P<0.001).There is remarkable increase of Arg-1~+microglia in the cortex(P=0.035)and striatum(P=0.006)in the Exercise group compared to the MCAO group at 7d after the surgery.3.Effects of treadmill exercise on the levels of M1/M2 microglia markers after CI/RPBased on the results from Luminex,among the M1 microglia markers,the levels of IL-1α,IL-6 and MCP-1 were higher in the MCAO group than those in the Sham group at 24h after surgery(P=0.013 for IL-1α,P<0.001 for IL-6,and P=0.036 for MCP-1).At 4d post-surgery,only IL-1βsignificantly increased(P=0.027),while at7d,only IL-6 showed great elevation(P=0.001).There is significant difference in IL-1α,IL-1β,IL-6,and MCP-1 expression between the Exercise and MCAO group at 7d after the surgery(P<0.05).According to the comparison of levels of M2 microglia marker IL-10 in Sham,MCAO and Exercise groups,we found that the expression of IL-10 in MCAO group increased significantly at 4d(P<0.001),and decreased at 7d(P=0.004)post-surgery compared with Sham group.The expression of IL-10 in Exercise group was significantly increased on day 7 post-operation(P=0.002).4.Effect of treadmill exercise on IL-4 expression after CI/RPAccording to the results of IL-4 from Luminex,there is significant difference between the Sham and MCAO groups only at 24h after surgery(P=0.048).Compared with the MCAO group,the Exercise group significantly increase the expression of IL-4 at 7d after surgery(P=0.004).The results of q RT-PCR also showed that the expression of IL-4 in the Exercise group was higher than that in the MCAO group at4d and 7d after surgery(P<0.01).5.Effects of treadmill exercise on JAK1/STAT6 pathway after CI/RPAfter the analysis of the results of p-JAK1 and p-STAT6,we found that p-JAK1showed significant difference at 24h(P=0.005)and 4d(P=0.001)and p-STAT6showed significant difference at 24h,4d and 7d after surgery(P<0.01)between the MCAO and Sham groups.Besides,the level of p-JAK1 was higher at 4d and 7d in the Exercise group than the MCAO group(P=0.001)and the expression of p-STAT6 was significantly elevated at 7d(P<0.001)post-surgery in the Exercise group compared to the MCAO group.Conclusions:Treadmill exercise could ameliorate cerebral ischemia-reperfusion injury by enhancing IL-4 expression to promote M2 microglia polarization,possibly via the JAK1-STAT6 pathway. |
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