| ObjectiveTo explore the effect of electroacupuncture combined with transcription factor NeuroD1&DLX2 gene therapy to improve the motor function of MCAO(Middle cerebral artery occlusion,MCAO)model mice;Analyze the effect of electroacupuncture on the behavioral and pathological characteristics of MCAO mice in the acute phase;And the effect of electroacupuncture combined with transcription factor NeuroD 1(NeuroD 1,ND1)&DLX2 on the behavioral and pathological characteristics of MCAO mice during recovery period,and explore the mechanism of stressed astrocjytes reprogramming neurons in the injured brain area.MethodsThe first part:1.Establish a mouse model of MCAO in the acute phase,and intervene after 24 hours:divided into 4 groups:MCAO+electroacupuncture(EA)group,MCAO+hand acupuncture(Handacupuncture,HA)group,MCAO group and sham group;Through behavioral tests,it is evaluated that electroacupuncture improve the motor function of MCAO mice in the acute phase at different time points;2.Establish a mouse model of MCAO recovery period and intervene after 30 days:divided into MCAO+EA group,MCAO group and sham Group,through behavioral tests to evaluate the effect of electroacupuncture treatment on improving the motor function of MCAO mice during the recovery period at different time points;The second part:After the intervention of the acute phase of MCAO mice,take the slices and immunofluorescence staining NeuN to evaluate the infarct area;stain the microglia biomarker IB A1(ionized calcium binding adapter molecule 1,IBA1)and astrocytes Biomarker GFAP(Glial fibrillary acidic protein,GFAP)assesses the degree of activation in different damaged brain regions;and conducts morphological analysis of IBA1 to assess its morphological change trend after activation of IBA1;The third part:1.Nine normal 7-8 weeks old C57BL/6 male mice were randomly divided into 3 groups,and microinjected into the striatum.①AAV2/5-gfaABC1D-iCre+AAV2/5-CAG-Flex-DLX2-P2A-EGFP;② AAV2/5-gfaABC1D-iCre+AAV2/5-CAG-FlexND1-P2A-mcherry;③ AAV2/5-gfaABC1D-iCre+AAV2/5-CAG-Flex-DLX2-P2A-EGFP+AAV2/5-CAG-Flex-ND1-P2A-mCherry virus,perfusion slices and fluorescent staining were performed on the 7th,14th,and 28th day after virus injection.NeuN was observed at different time points the coincidence rate of transcription factor NeuroD1&DLX2 and NeuN;2.50 male mice of MCAO recovery period model were randomly divided into 5 groups by random number table method,which are sham group,MCAO+ND1+DLX2 group,MCAO+ND1+DLX2+EA group,MCAO+EA group,MCAO+GFP(Green fluorescent protein,GFP)group,through behavioral tests to evaluate different time points to improve the motor function of MCAO mice during the recovery period;the mice after the intervention were immunofluorescent stained NeuN,calculate the overlap rate with the transcription factor NeuroD1&DLX2,the evaluation of the infarct size,and the staining of GFAP to evaluate the stress degree of GFAP on the side of the lesion.Results1.The first part:1.1 Behavioral observations in the acute phase of MCAO mice in the removal of the sticky paper test,compared with the MCAO model group,the MCAO+EA group and the MCAO+HA group were different in the electroacupuncture at 7 d,14 d and 30 d,and the differences were both There is statistical significance(P<0.05);in the grid-walking test,compared with the MCAO model group for 30 days of electroacupuncture,there is a difference between the MCAO+EA group and the MCAO+HA group,and the difference is statistically significant(P<0.05),Indicating that with the increase of electroacupuncture treatment time,the advanced motor function of the affected limb of MCAO mice in the acute phase gradually improved.1.2 Behavioral observation in the MCAO recovery period mice were in the walking grid test and the climbing cup test.Compared with the MCAO model group,the MCAO+EA group had no improvement trend at different time points,indicating that electroacupuncture did not alleviate the MCAO recovery period mice.Motor function of limbs.2.The second part:2.1 Infarct size:Compared with the MCAO model group with electroacupuncture for 30 days,the difference in infarct size between MCAO+EA group and MCAO+MA group is statistically significant(P<0.05);it shows that electroacupuncture and hand acupuncture can effectively reduce the infarct size and protect the brain Damaged tissue in the area.2.2 The average fluorescence intensity of IBA1 in different damaged brain areas:30 days of electroacupuncture in the MCAO+EA group and MCAO+MA group both improved the stress and expression level of IB A1 in the damaged brain area to varying degrees.Compared with the MCAO group,in the core infarct area,the MCAO+EA group and the MCAO+MA group effectively reduced the expression level of IBA1 and the difference was statistically significant(P<0.05);in the ischemic penumbra area,MCAO+EA Both the MCAO+MA group and the MCAO+MA group have a tendency to reduce the expression level of IBA1;in the cortical area on the side of the lesion,the MCAO+EA group effectively reduces the expression level of IBA1 and the difference is statistically significant(P<0.05).The MCAO+MA group has Decrease trend.2.3 The average fluorescence intensity of GFAP in different damaged brain areas:30 days of electroacupuncture in the MCAO+EA group and MCAO+MA group have different degrees of improvement in the stress degree and expression level of GFAP in the damaged brain area.Compared with the MCAO group,in the core infarct area and the ischemic penumbra area,the MCAO+EA group and the MCAO+MA group effectively reduced the expression level of GFAP and the difference was statistically significant(P<0.05);in the cortex on the lesion side Area,MCAO+EA group and MCAO+MA group all have a tendency to reduce the expression level of GFAP.2.4 Morphological analysis of IBA1 in the core infarct area:30 days of electroacupuncture in the MCAO+EA group and MCAO+MA group significantly alleviated the morphological stress changes of IBA1.Compared with the MCAO group,the MCAO+EA group and the MCAO+MA group effectively reduced the number of IBA1 aggregated cells,significantly increased the number of protruding branches and the length of the branches,and the differences were statistically significant(P<0.05).2.5 Morphological analysis of IBA1 in ischemic penumbra:30 days of electroacupuncture in MCAO+EA group and MCAO+MA group all alleviated the morphological stress changes of IBA1 to varying degrees.Compared with the MCAO group,the MCAO+EA group and MCAO+MA group effectively reduced the number of IBA1 aggregated cells,significantly increased the number of protruding branches,and the difference was statistically significant(P<0.05);the MCAO+EA group significantly increased the branch length and the difference was statistically significant(P<0.05),and there was an increasing trend in the MCAO+MA group.2.6 Morphological analysis of IBA1 in the cortical area of the lesion:30 days of electroacupuncture in the MCAO+EA group and MCAO+MA group all relieved the morphological stress changes of IB A1 to varying degrees.Compared with the MCAO group,the MCAO+MA group effectively reduced the number of IBA1 aggregated cells and the difference was statistically significant(P<0.05),the MCAO+EA group had a decreasing trend;the MCAO+EA group and the MCAO+MA group effectively increased significantly the number of protruding branches and the length of the branches were statistically significant(P<0.05).3.The third part:3.1 After microinjection of transcription factor ND1+DLX2 into the brain area of normal mice,the conversion of stress glial cells into functional neurons was observed.The conversion rate of stress astrocytes in the brain area after 7 days was about 8%.The 14-day conversion rate was 46%,and the 30-day conversion rate was 65%;3.2 Behavioral observation results:In the walking grid test,the difference was statistically significant in the MCAO+ND1+DLX2 group compared with the MCAO+GFP group for 14 days(P<0.05);the intervention for 30 days was similar to the MCAO+GFP group Compared with the MCAO+ND1+DLX2 group and the MCAO+ND1+DLX2+EA group,the difference was statistically significant(P<0.05);the cup climbing test showed that the MCAO+ND1+DLX2 group was compared with the MCAO+GFP group after 30 days of intervention the difference between MCAO+ND1+DLX2+EA group and MCAO+EA group was statistically significant(P<0.05);compared with MCAO+GFP group at 60 days of intervention,MCAO+ND1+DLX2 group,MCAO+ND1+DLX2+ The difference in the EA group was statistically significant(P<0.05).Both ND1+DLX2 and combined electroacupuncture can reduce the rate of walking on the grid and the dragging rate of the cup during the MCAO recovery period.3.3 Infarct area of mice in MCAO recovery period:Compared with MCAO+GFP group after 60 days of intervention,the difference in infarct area of MCAO+ND1+DLX2 is statistically significant(P<0.05);it shows that ND1+DLX2 can effectively reduce the infarct area than the empty group,Protect the damaged tissues of the brain area.3.4 Statistical results of GFAP expression levels on the lesion side of MCAO mice during the recovery period:Compared with the MCAO+GFP group,the differences in the MCAO+ND1+DLX2 group were statistically significant(P<0.05),and the ND1+DLX2 intervention was better than the no-load group The average fluorescence intensity of astrocytes on the side of the lesion in MCAO mice has a significant decrease.3.5 The conversion rate of ND1+DLX2 in the striatum of MCAO mice during the recovery period from stress astrocytes into functional neurons:14 days after injection,the conversion rate reached about 39%;30 days after injection The conversion rate was 53%.After 60 days of injection,the conversion rate was still around 53%.Conclusions1.Acupuncture can effectively improve the motor function of mice in the acute phase of MCAO,reduce the area of cerebral infarction damage,and protect the brain tissue;reduce the stress inflammatory response of glial cells in different damaged brain regions;at the same time improve the microglia cells in the damaged brain regions The morphology is abnormal;the effect of acupuncture on MCAO mice in the recovery phase is worse than that in the acute phase,so acupuncture should be involved in the treatment of ischemic stroke in the early stage,which is beneficial to the recovery and treatment in the later stage.2.The neural transcription factor ND1+DLX2 effectively transforms the stress-induced astrocytes in the striatum of normal mice into neurons.The in situ transformation technology in the brain provides more options and possibilities for the treatment of diseases,which will be the future It is applied to the clinic to accumulate a solid theoretical and technical basis.3.The nerve transcription factor ND1+DLX2 can effectively improve the motor function of the affected limb in MCAO mice during the recovery period,and significantly reduce the area of cerebral infarction damage;effectively reduce the stress inflammatory response of astrocytes on the side of the infarct;Astrocytes are transformed into neurons. |
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