| ObjectiveAt present,the treatment of ischemic stroke can improve the symptoms of neurological deficit and inhibit.inflammatory response to a certain extent.However,the problem that neurons can not regenerate after nerve cell injury that still not be solved.This study attempts to observe whether NeuroD1 can transform reactive astrocytes into functional neurons by stereotactic injection of neurotranscriptional factor NeuroD1 in the brain area of middle cerebral artery injury.Electroacupuncture can improve animal behavioral disorders of ischemic stroke.Therefore,whether the combination of electroacupuncture and NeuroD1 can further optimize the treatment of ischemic stroke,increase the conversion rate of neurons,reduce the inflammatory reaction of brain injury,and further improve the effect of brain repair.To explore the possible mechanism of electroacupuncture combined with nerve transcription factor NeuroD1 in regulating ischemic stroke.MethodsNine C57BL/6 adult male mice were randomly divided into 3 days,14 days and 30 days after modeling.The symptoms of nerve defect,laser speckle cerebral blood flow imaging and TTC staining were observed in the three groups.At the same time,immunofluorescence staining was used to observe the changes of GFAP,IBA1 reactive glial cells.Nine SPF grade C57BL/6 adult male mice were randomly divided into 7-day,14-day and 30-day hGFAP::Cre/FLEX-CAG::NeuroD1-P2A-GFP virus injection groups.Immunofluorescence double staining was used to observe the transformation rate of neural transcription factor NeuroD1 in different brain regions(striatum and cortex)of normal mice.Electrophysiological patch clamp technique was used to observe whether the reprogrammed neurons had neuronal function.Thirty-five C57BL/6 adult.male mice weighing 20g-30g were randomly divided into three groups:Sham operation group,I/R+mCherry group and I/R+NeuroD1 group.Animal behavior experiments were carried out at 4 days,14 days,28 days,60 days and 90 days after intervention,including adhesive dot removal test,grid walking test,cylinder test and footprint analysis.The transformation of GFAP-labeled reactive astrocytes into NeuN-labeled neurons was observed by immunofluorescence staining,and the changes of cerebral infarction area,fluorescence intensity of reactive astrocytes and microglia,and morphological changes of inflammatory microglia were observed in the three groups.Fifty-five C57 mice were randomly divided into 4 groups:Sham group,I/R+mCherry group,I/R+NeuroD1 group,I/R+electroacupuncture(EA)+NeuroD1 group.Animal behavior experiments were carried out in 4 days,14 days,28 days,60 days and 90 days after intervention,including adhesive dot removal test,grid walking test,cylinder test and footprint analysis.The transformation of GFAP-labeled reactive astrocytes into NeuN-labeled neurons was observed by immunofluorescence staining,and the changes of cerebral infarction area of the four groups were observed,the inflammatory intensity of reactive astrocytes GFAP and microglia IBA1 were observed,and the morphological changes of inflammatory microglia under different intervention were detected.Results1.Model evaluation part:(1)The neurological deficit symptom score of MCAO model mice was 1 at 3 days,14 days and 28 days.(2)The blood flow of laser speckle was monitored after the thread was inserted.It was observed that the perfusion blood flow of the occluded side(left side)was 101.92±42.44,and the cerebral perfusion blood flow of the same area of the healthy side was 268.51±80.70.(3)A large number of neuronal losses were observed in the striatum and the cortex near the striatum in the injured side of the MCAO mice after TTC and neuronal NeuN staining.(4)The average optical intensity of reative astrocyte in the affected side and the normal side was(21.98±2.44;7.92±0.69);(19.47±3.05;6.15±1.35);(22.38±3.61;5.98±1.27)respectively at the 3d,14d,30d time points.In IBA1,the average optical intensity of microglia in the affected side of the brain and the normal brain area were(21.98±2.447.92±0,69);(19.47±3.05;6.15±1.35);(22.38±3.61;5.98±1.27)respectively at 3d,14d and 30d.2.Neural transcription factor NeuroD1 transformed astrocytes into functional neurons in normal brain regions.(1)Cortical brain area:it was observed that the transformation rate of neurons in AVV9-Flx-cre-GFP-NeuroD1 mice was 4.06±2.33%,16.85±6.75%,84.42±1.07%at 7d,14d,28d,respectively.The transformation rate of cortical astrocytes into neurons in 14 days and 30 days after virus injection was higher than that in 7d,and the difference was statistically significant(P<0.05).(2)Striatal brain area:the transformation rate of striatal neurons in AVV9-Flx-cre-GFP-NeuroD1 mice was 5.24±2.11%,31.48±11.10%,85.18±0.47%at 7d,14d,28d,respectively.The transformation rate of striatal astrocytes into neurons after virus infection for 14 days and 30 days was higher than that at 7 days,and the difference was statistically significant(P<0.05).(3)Whole-cell patch-clamp recording:The voltage changed linearly with the increase of the injection current,indicating that the infected cells have not been reprogrammed as neurons,were still astrocytes that did not express sodium ion channels at 7 days;Striatum-labeled cells have sodium and potassium current signals and action potentials after 21 days of NeuroD1 injection,indicating that NeuroD1 reprogrammed cells express sodium channels and have neuronal characteristics and function.3.NeuroD1 transformed reactive astrocytes into functional neurons in MCAO mice(1)Animal behaviorThe length of time for removing sticky paper from the adhesive dot removal test,the rate of stepping out of the affected limb in the grid walking test,the drag rate of the affected limb in the cylinder test,and the step length of the affected limbs of the footprint analysis.All three groups showed statistically significant differences(P<0.05).Further observe the differences in the improvement of limb movement function in different groups at each time point.28,60,and 90 days after NeuroD1 virus injection,the length of sticky paper removal,foot-slips rate and the drag rate were reduced in the NeuroD1 treatment group.Compared with the model group,there were statistical differences(P<0.01);Footprint analysis was performed at 28 days and 60 days after virus injection.Compared with the mCherry group,the NeuroD1 treatment group could better increase the step lengths of the lateral forelimbs and hindlimbs of MCAO mice(all P<0.05).(2)NeuroD1 transformed reactive astrocytes into functional neurons on MCAO mouseNeuroD1 converted cortical and striatum-reactive astrocytes into neurons after 7 days.The conversion rates were 9.1%and 10%,respectively;The conversion rate of cortical and striatum-reactive astrocytes into neurons was approximately 80.86%and 82.08%at 30 days,and the control group was approximately 11.76%and 11.96%.The NeuroD1 treatment has higher neuron conversion rate in the cortical and striatum than that in the control group(P<0.01).(3)NeuroD1 reduced cerebral infarction area.in MCAO miceThe results showed that there was a statistically significant difference in the area of cerebral infarction among the three groups(P<0.01)at 30 days and 120 days,and the difference between NeuroD1 and the mCherry group was statistically significant(P<0.05).(4)Effect of NeuroD1 on inflammatory glial cells in MCAO miceThe results showed that the fluorescence intensity of astrocytes and microglia in the three groups of MCAO mice were statistically significant after 30 days and 120 days of NeuroD1 treatment(P<0.01).The fluorescence intensity of reactive astrocytes and microglia in the NeuroD1 treatment group was weaker than that in the model group(both P<0.01).(5)Effect of NeuroD1 on morphological structure of inflammatory microglia in MCAO miceThree groups showed statistically significant differences in the number of microglia,the average number of terminals,and the length of the protrusions at 30 days and 120 days(both P<0.01).Compared with the model group,the NeuroD1 treatment group could reduce the number of reactive microglia,increase the average number of microglia ends and the length of the protrusions,which were statistically significant(P<0.05).4.Electroacupuncture combined with NeuroD1 reprograms reactive astrocytes into neurons to repair ischemic stroke(1)Animal behaviorThe length of time for removing sticky paper from the adhesive dot removal test,foot-slips rate,the paw dragging rate,and the step length analysis of the affected side showed that the overall difference was statistically significant(P<0.05).The results showed that the length of removal,the rate of slipping and the rate of dragging were reduced in electroacupuncture(EA)+NeuroD1 group at 14 days,28 days,60 days,and 90 days,which were statistically different compared with the model group(P<0.01);footprint analysis was performed at 28 days,60 days,and 90 days after EA+NeuroD1 treatment.Compared with the model group,the EA+NeuroD1 group can better increase the step length of the forelimb and hind limb,and the differences were statistically significant,at 28 days,60 days,and 90 days(all P<0.05).The NeuroD1 +EA group had a lower foot-slips rate compared with the NeuroD1 treatment group a lone,which was statistically different at 14 days and 28 days(P<0.05).EA+NeuroD1 group could significantly increase the step length of the affected limb,compared with the NeuroD1 group at 90 days(P<0.05).(2)Electroacupuncture combined with NeuroD1 transformed reactive astrocytes into functional neuronsEA+NeuroD1 transformed the cortex and striatum reactive astrocytes into neurons with a conversion rate of 6.4%and 9.2%at 7 days,respectively.After 30 days,the EA+NeuroD1 group converted reactive astrocytes of cortical and striatum into neurons with a conversion rate were 89.74%and 90.18%,the control group was 9.43%and 11.96%.EA+NeuroD1 group was higher neuronal conversion rate than the mCherry group and the NeuroD1 group(all P<0.01).(3)Electroacupuncture combined with NeuroD1 reduces cerebra1 infarct.size in MCAO miceChanges of cerebral infarction area in MCAO mice at 30 days and 120 days:the average cerebral infarction area was statistically different between the four groups(P<0.01).EA+NeuroD1 could significantly reduce cerebral infarction area compared with the model group(P<0.01).EA+NeuroD1 could reduce the area of cerebral infarction better than NeuroD1 group with statistical difference at 120 days(P<0.01).(4)Effect of electroacupuncture combined with NeuroD1 on inflammatory glial cells in MCAO miceThe results showed that the differences in fluorescence intensity of GFAP and IBA1 in the four groups of MCAO mice were statistically significant at 30 days and 120 days(all P<0.01).In the EA+NeuroD1 group,the intensity of GFAP and IBA1 were weaker than those in the model group,with statistical differences(both P<0.01).EA+NeuroD1 could reduce the fluorescence intensity of reactive IBA1 better than NeuroD1 alone at 120 days.(P<0.01).(5)Effect of electroacupuncture combined with NeuroD1 on morphological structure of inflammatory microglia in MCAO miceThe comparison of the four groups in the number of microglia,the average number of endpoints,and the length of the process of injured brain area were statistically significant differences at 30 days and 120 days(all P<0.01).EA+NeuroD1 group could decrease the number of reactive microglial cells,increasing the average number of endpoints and the length of process compared with the mCherry group P<0.05);EA+NeuroD1 group was significantly increased microglial processes compared with NeuroD1 group alone(P<0.01).Conclusion1.The sign of stable middle cerebral artery infarction model is mainly the appearance of neurological defect symptoms.Laser speckle shows that the blood flow on the infarcted side is reduced or even disappeared.TTC staining shows that the damaged brain area turns white,mainly in the cortex and striatum area,and inflammatory glial cells will continue to reactive from 3 days to a month,forming "glial scar"hindering the repair of brain damage.2.NeuroD1 neural transcription factor can reorganize astrocytes into functional neurons on the normal mice.The astrocytes form 7 days and the conversion rate is about.10%.A small number of astrocytes were transformed into neurons,with a conversion rate of 40%at 14 days.About one month after infection,the conversion rate of astrocytes into neurons was as high as 80%.It is speculated that neurotranscription factor NeuroD1 takes about 1 month to transform astrocytes into mature neurons,and has a high neuron conversion rate in the striatum and cortex.3.NeuroD1 can improve behavioral disorders such as motor,sensation and balance function in MCAO mice.Reactive astrocytes(cortex and striatum)are reorganized into functional neurons in MCAO mice,cell morphology and functions are consistent with normal neurons,and NeuroD1 can reduce the infarct area of mice with stroke,reduce the inflammatory response in the brain area and protect the injured neurons.4.Electroacupuncture at "Baihui","Dazhui","Zusanli" and"Quchi" combined with NeuroD1 can better save MCAO mice’ s motor sensation and balance function.Electroacupuncture could increase the conversion rate of NeuroD1 to reorganize reactive astrocytes into neurons,reduce the area of injured cerebral infarction,reduce the inflammatory response of reactive glial cells in the injured brain area.5.The early intervention of electroacupuncture has the advantages of optimizing the microenvironment in the brain,improving the conversion rate and survival rate of regenerating neurons,and the rehabilitation level of animal behavior disorders.Combined with NeuroD1,the astrocytes can be reprogrammed into functional neurons in situ.The combined treatment of electroacupuncture and neuroD1 can improve the short-term and long-term neurological recovery of MCAO mice and better protect the brain from injury. |
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