| ObjectiveTo investigate the regulatory effects of electroacupuncture(EA)at Baihui and Dazhui acupoints on microglia and astrocytes in early stage of ischemic stroke.To explore the effect of electroacupuncture on microglia and astrocytes in alleviating acute ischemic stroke injury.To compare the effects of EA pretreatment and EA after ischemic stroke on reducing motor sensory impairment in the acute phase of ischemic stroke,and the related mechanisms of microglia and astrocytes regulation.MethodsThis research could be divided into the following five aspects.Experiment 1:The photochemical method was used to establish the ischemic stroke mouse model of unilateral motor and sensory cortex,and the stability of the model and the damaged area were evaluated.Specific as follows:Male C57BL/6 mice were induced in laser speckle experimental observation to blood perfusion amount of the bilateral cerebral hemisphere motor area and immediately made sensorimotor cortex ischemic stroke model by photochemical method,again after 24 hours after surgery for laser speckle experiments,evaluating its observation area the change of blood perfusion.Then,mice were selected for TTC staining or NeuN,IBA1 and DAPI immunofluorescence staining were performed to observe the loss of neurons in mouse brain tissue and the range of microglia activation zone to evaluate the infarct size.Experiment 2:Behavioral tests were performed to evaluate the changes of limb movement and sensory function of the affected side(right side)of mice before and after ischemic modeling,and whether the intervention of EA at Baihui and Dazhui acupoint for 7 days before or after ischemic stroke modeling can improve the performance.The mice were divided into non-stroke control group(Control group),stroke modeling group(Stroke group),stroke model+EA treatment group(postEA group)and EA pretreatment+stroke model group(preEA group),and the grid-walking test,cylinder test and adhesive dot removal test were conducted on 1 day before modeling,and 1 day,4 day and 7 day after modeling.Experiment 3:Immunofluorescence staining was used to observe the changes of microglia,M2 subtype microglia,astrocytes and neurons in the affected cortex of ischemic stroke mice at day 1,day 4 and day 7 after stroke,and the effect of EA on them.The details are as follows:At different time points after the modeling of ischemic stroke mice,mice in each group were collected by perfusion,brain tissue sections and immunofluorescence staining,including NeuN,IBA1,GFAP and CD206.Confocal laser imaging was performed on the stained brain slices,and the changes in the number and average fluorescence intensity were counted.Experiment 4:Enzyme-linked immunosorbent assay(ELISA)was used to determine the serum levels of TNF-α and IL-10 of ischemic stroke mice at 1,4 and 7 days after modeling.The details are as follows:After the ischemic stroke mice were modeled,the eyeballs of each group were removed for blood collection at different time points.After standing at room temperature for 1.5-2 hours,the serum was extracted by centrifugation at 4℃ and frozen at-80℃ waiting for testing.The operation was carried out strictly in accordance with THE ELISA kit.After completion,it was immediately sent to the microporous plate spectrophotometer to measure the absorbance(OD)at 450nm wavelength.Calculate the standard curve and convert OD value into the actual concentration according to the curve and dilution ratio.Experiment 5:Whole cell patch clamp was used to record the action potentials of the pyramidal neurons in the motor cortex of the healthy side before and 1 day after the modeling of ischemic stroke.The details are as follows:Clamp the neuron membrane potential at-70mV and inject current into the neuron to simulate the process of neuron depolarization and action potential.The injection current ranges from-200pA to 500pA,increasing by 50pA each time.Records and releasing frequency statistics neurons action potential,the resting membrane potential,action potential threshold,action potential amplitude,wave action potential half range wide,fast action potential after hyperpolarization potential amplitude,neuron parameters such as input impedance,time constant.ResultsExperiment 1:The comparison between the ratio of blood perfusion volume in the left/right cortical motor area before modeling and the ratio of blood perfusion volume 24 hours after modeling showed that the ratio of blood perfusion volume after modeling was lower(P=0.000),and the ratio of blood perfusion volume after modeling was lower(P=0.001)after self-paired comparison before and after modeling.The results showed that the left cortical motor area ischemia was caused by the optical thrombus model.Immunofluorescence staining results showed that 1 day after ischemic stroke modeling,a large number of NeuN positive cells were lost in the left cortical motor,and a large number of IBA1+ cells migrated to the border of the core area of infarction,and almost no IBA1 positive cells were expressed in the core area of infarction.NeuN staining and IBA1 staining showed cerebral infarction in the left motorsensory cortex.The mice model of ischemic stroke was successfully established by light embolization.Experiment 2:In the evaluation of four indicators in three behavioral experiments,the performance of mice in each group was similar before modeling without statistical difference,which was comparable.(1)Grid-walking test:One day after modeling,the experimental results showed that the forelimb error rate of stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000)were significantly higher than those of control group.There was no significant difference between the stroke group and the EA group(P=0.217).The forelimb error rate of stroke group(P=0.000)and postEA group(P=0.000)was higher than that of preEA group.After 4 days of modeling,the forelimb error rates of stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000)were significantly higher than those of control group.The foot error rate of stroke group was higher than that of postEA group(P=0.001)and preEA group(P=0.000).The forelimb error rate of postEA group was higher than that of preEA group(P=0.000).After 7 days of modeling,the forelimb error rates of stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000)were significantly higher than those of control group.The foot error rate of stroke group was higher than that of postEA group(P=0.000)and preEA group(P=0.000).The foot error rate of postEA group was higher than that of preEA group(P=0.023).(2)Cylinder test:Drag rate of affected forelimbAfter 1 day of modeling,the experimental results showed that the dragging rate of the affected forelimb in the stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000)were significantly higher than those in the control group.There was no significant difference between the stroke group and the postEA group(P=0.091).The dragging rate of the affected forelimb in the stroke group(P=0.000)and the postEA group(P=0.012)was higher than that in the preEA group.After 4 days of modeling,the experimental results showed that the dragging rate of the affected forelimb in the stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.002)were significantly higher than those in the control group.The dragging rate of stroke group was higher than that of postEA group(P=0.005)and preEA group(P=0.000).The dragging rate of postEA group was higher than that of preEA group(P=0.044).After 7 days of modeling,the experimental results showed that the dragging rate of the affected forelimb in the stroke group(P=0.000)and the EA 1group(P=0.010)were significantly higher than those in the control group,and there was no significant difference between the control group and the preEA group(P=0.185).The dragging rate of stroke group was higher than that of postEA group(P=0.000)and preEA group(P=0.000).There was no significant difference between postEA group and preEA group(P=0.185).Utilization rate of the affected forelimb(right)After 1 day of modeling,the utilization rate of affected forelimb in stroke group(P=0.000)and postEA group(P=0.016)was lower than that in control group.There was no significant difference between the control group and the preEA group(P=0.348).There was no significant difference between the stroke group and the postEA group(P=0.111).The utilization rate of affected limb in the stroke group was lower than that in the preEA group(P=0.003).After 4 days of modeling,the experimental results showed that there was no significant difference in the utilization rate of the affected limb between the control group and the postEA group(P=0.921)and the preEA group(P=0.275).The stroke group was lower than the control group(P=0.017)and the postEA group(P=0.022).There was no significant difference in the utilization rate of affected limb between the stroke group and the preEA group(P=0.177),and the postEA group was similar to the preEA group(P=0.320).After 7 days of modeling,the utilization rate of affected limb in the stroke group was lower than that in the control group(P=0.019).There was no significant difference between the control group and postEA group(P=0.408)and preEA group(P=0.354).There was no significant difference in limb utilization rate between the stroke group and postEA group(P=0.116)and preEA group(P=0.140).postEA group was similar to preEA group(P=0.921).(3)Adhesive dot removal test:After 1 day of modeling,the experimental results showed that the perception time of stroke group(P=0.000),postEA group(P=0.001)and preEA group(P=0.019)were significantly longer than those of the control group.There was no significant difference between the stroke group and the postEA group(P=0.381).The perception time of stroke group was longer than that of preEA group(P=0.032).There was no significant difference between postEA group and preEA group(P=0.192).After 4 days of modeling,the experimental results showed that the perception time of the control group was less than that of the stroke group(P=0.000),and there was no statistical difference between the control group and the postEA group(P=0.076)and the preEA group(P=0.443).Compared with postEA group(P=0.003)and preEA group(P=0.000),stroke group took more time to perceive.There was no significant difference between postEA group and preEA group(P=0.304).After 7 days of modeling,the experimental results showed that the perception time of the control group was less than that of the stroke group(P=0.000),and there was no statistical difference between the control group and the postEA group(P=0.054)and the preEA group(P=0.462).Compared with postEA group(P=0.010)and preEA group(P=0.000),stroke group took more time to perceive.There was no significant difference between postEA group and preEA group(P=0.224).Experiment 3:In the control group,there was no obvious activation of microglia at 1,4 and 7 days after modeling,and only a few astrocytes were activated.One day after the stroke model was established,the average fluorescence intensity of IBA1 in the stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000)was significantly higher than that in the control group.There was no significant difference between the stroke group and the postEA group.The mean fluorescence intensity of IBA1 in preEA group was higher than that in stroke group(P=0.001)and postEA group(P=0.008).The average fluorescence intensity of CD206 in stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000)was significantly higher than that in control group.There was no significant difference between the stroke group and the postEA group(P=0.988).The mean fluorescence intensity of CD206 in preEA group was higher than that in stroke group(P=0.039)and postEA group(P=0.034).The average fluorescence intensity of GFAP in the control group was lower than that in the stroke group(P=0.012),postEA group(P=0.001)and preEA group(P=0.000).The average fluorescence intensity of GFAP in stroke group was lower than that in postEA group(P=0.013)and preEA group(P=0.002).The average fluorescence intensity of GFAP in postEA group was lower than that in preEA group(P=0.026).The number of IBA1+ cells in stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000)was significantly higher than that in control group.There was no significant difference in IBA1+ cell count between the stroke group and the EA group(P=0.183).The number of IBA1+ cells in preEA group was higher than that in stroke group(P=0.000)and postEA group(P=0.000).The number of CD206+cells in stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000)was significantly higher than that in control group.The number of CD206+cells in stroke group was lower than that in postEA group(P=0.049)and preEA group(P=0.000).The number of CD206+ cells in postEA group was less than that in preEA group(P=0.000).The ratio of CD206+ cells to IBA1+ cells in stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000)was significantly higher than that in control group.There was no significant difference in the ratio between the stroke group and the postEA group(P=0.253).The ratio of stroke group was lower than that of preEA group(P=0.022).There was no significant difference between postEA group and preEA group(P=0.173).At 4 days after stroke modeling,the mean fluorescence intensity of IBA1 in the stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000)was significantly higher than that in the control group.The mean fluorescence intensity of IBA1 in stroke group was lower than that in postEA group(P=0.014)and preEA group(P=0.000).The mean fluorescence intensity of IBA1 in preEA group was higher than that in postEA group(P=0.002).The average fluorescence intensity of CD206 in stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000)was significantly higher than that in control group.The average fluorescence intensity of CD206 in stroke group was lower than that in postEA group(P=0.030)and preEA group(P=0.000).The average fluorescence intensity of CD206 in preEA group was higher than that in postEA group(P=0.001).The average fluorescence intensity of GFAP in stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000)was significantly higher than that in control group.The average fluorescence intensity of GFAP in stroke group was lower than that in postEA group(P=0.002)and preEA group(P=0.000).The average fluorescence intensity of GFAP in preEA group was higher than that in postEA group(P=0.000).At 4 days after stroke modeling,the results showed that the mean fluorescence intensity of IBA1 in the stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000)was significantly higher than that in the control group at 7 days after the stroke model was established.The mean fluorescence intensity of IBA1 in stroke group was lower than that in postEA group(P=0.016)and preEA group(P=0.000).The mean fluorescence intensity of IB A1 in preEA group was higher than that in postEA group(P=0.000).The average fluorescence intensity of CD206 in stroke group(P=0.001),postEA group(P=0.000)and preEA group(P=0.000)was higher than that in control group.The average fluorescence intensity of CD206 in stroke group was lower than that in postEA group(P=0.012)and preEA group(P=0.000).The average fluorescence intensity of CD206 in preEA group was higher than that in postEA group(P=0.009).The average fluorescence intensity of GFAP in stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000)was higher than that in control group.The average fluorescence intensity of GFAP in the stroke group was higher than that in the postEA group(P=0.000).There was no significant difference between stroke group and preEA group(P=0.056).The average fluorescence intensity of GFAP in postEA group was lower than that in preEA group(P=0.002).Experiment 4:ELISA results showed that TNF-α concentration in the control group was lower than that in the stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000).There was no significant difference in TNF-α concentration between the stroke group and the postEA group(P=0.771).The concentration of TNF-α in preEA group was lower than that in stroke group(P=0.000)and postEA group(P=0.031).The concentration of IL-10 in the control group was higher than that in the stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.004).There was no significant difference in IL-10 concentration between the stroke group and the postEA group(P=0.106).The concentration of IL-10 in preEA group was higher than that in stroke group(P=0.000)and postEA group(P=0.029).ELISA results at 4 days after stroke modeling showed that TNF-α concentration in the control group was lower than that in the stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000).TNF-α concentration in stroke group was higher than that in postEA group(P=0.000)and preEA group(P=0.000).The concentration of TNF-α in preEA group was lower than that in postEA group(P=0.000).The concentration of IL-10 in the control group was higher than that in the stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000).The concentration of IL-10 in stroke group was lower than that in postEA group(P=0.000)and preEA group(P=0.000).The concentration of IL-10 in preEA group was higher than that in postEA group(P=0.022).ELISA results at 7 days after stroke modeling showed that TNF-α concentration in the control group was lower than that in the stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000).TNF-α concentration in stroke group was higher than that in postEA group(P=0.000)and preEA group(P=0.000).There was no significant difference between the two groups(P=0.685).The concentration of IL-10 in the control group was higher than that in the stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.000).The concentration of IL-10 in stroke group was lower than that in postEA group(P=0.011)and preEA group(P=0.000).The concentration of IL-10 in preEA group was higher than that in postEA group(P=0.037).Experiment 5:Patch-clamp recordings of action potentials(AP)in pyramidal neurons in the right motor cortex of mice 1 day after ischemic stroke modeling showed differences in excitability and passive membrane properties among the four groups.The frequency of AP in stroke group(P=0.000),postEA group(P=0.000)and preEA group(P=0.045)were significantly higher than those in control group.The frequency of action potentials in postEA group(P=0.005)and preEA group(P=0.000)was lower than that in stroke group.There was no significant difference between postEA group and preEA group(P=0.097).The results of rest potential showed that the rest potential of stroke group(P=0.000)and postEA group(P=0.000)was higher than that of control group.There was no significant difference between the control group and the preEA group(P=0.622).There was no significant difference between the stroke group and the postEA group(P=0.972).Compared with the stroke group(P=0.002)and the postEA group(P=0.000),the preEA group had lower rest potential.The results of input resistance showed that the input resistance of the stroke group(P=0.000)and the postEA group(P=0.000)increased compared with the control group.There was no significant difference between the control group and the preEA group(P=1.000).There was no significant difference between the stroke group and the postEA group(P=1.000).Compared with the stroke group(P=0.000)and the postEA group(P=0.000),the EA group had lower input resistance.The results of time constants showed that the stroke group had a greater time constant than the control group(P=0.000).There was no significant difference between the control group and postEA group(P=0.262)and preEA group(P=1.000).The time constants of postEA group(P=0.035)and preEA group(P=0.001)were smaller than those of stroke group.There was no significant difference between postEA group and preEA group(P=1.000).There were no differences in the AP threshold,AP amplitude,rapid post-hyperpolarization potential amplitude,and AP duration of pyramidal neurons in the motor area of the healthy side at 1 day after ischemic stroke modeling.Conclusion1.EA at Baihui and Dazhui acupoint can reduce the motor and sensory function damage caused by ischemic stroke in motor sensory cortex.Compared with EA after injury,EA pretreatment could recover the motor and sensory function injury earlier.2.EA pretreatment at Baihui and Dazhui acupoint can activate microglia in the infarct area and surrounding areas from 1-7 days after modeling of ischemic stroke,and increase the number and proportion of M2 subtype microglia at the edge of the infarct core area at 1 day and 4 day after modeling.After ischemic stroke injury,EA at Baihui and Dazhui acupoint can activate M2 subtype microglia in the infarct and surrounding areas at 4-7 days after modeling and the number and proportion of M2 subtype microglia at the infarct margin were increased on day 4.3.EA pretreatment or EA after injury can promote the activation of astrocytes around the infarct at day 1 and 4 after ischemic stroke modeling.EA after injury can reduce the activation level of astrocytes around the infarct on day 7 after modeling.4.EA at Baihui and Dazhui acupoint can regulate the inflammatory responses within 7 days of ischemic stroke,and EA pretreatment program is more effective compared to that with EA after stroke.5.Ischemia in the motor sensory cortex leads to abnormal increase of excitability and change of passive membrane properties of Pyramidal neurons in the contralateral motor cortex of mice.Electroacupuncture at Baihui and Dazhui acupoints can reduce this abnormally high excitability and restore passive membrane characteristics.On the first day after injury,the effect of EA pretreatment was better than that EA after injury. |
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