| Objective: Selecting mouse strains with viruses specifically infecting astrocytes,establishing the stable model of cortical cerebral infarction in mice and and observing the proliferation of reactive astrocytes after cerebral infarction,reprogramming astrocytes into neurons by implanting two transcription factors(TFs)Pax6 and Ngn2 simultaneously.Methods: 1.Selection of cerebral infarction model in mouse:(1)The model of cerebral ischemia mouse was established by suture-occluded methodin:22 C57 mice were randomly divided into sham operation group(n=10),operation group(n=12),the motor function of sham operation group mice and the operation group mice were assessed at the 1st,3rd,5th,7th,9th,20 th days after operation and TTC staining of brain slices was performed at the 1st and 7th days after operation.(2)The model of focal cerebral ischemia mouse was established by injecting of ET-1 into the cortex: 24 C57 mice were randomly divided into control group(n=12,injecting saline),experimental group(n=12,injecting ET-1),the motor function of control group mice and experimental group mice were assessed at the 7th,9th,20 th and 30 th days after operation and Nissl staining was performed on 7th,28 th days after operation.2.Proliferation of reactive astrocytes after cerebral infarction: 20 C57 mice were randomly divided into sham operation group(n=10,injecting saline),operation group(n=10,injecting ET-1),the reactive astrocytes were observed and counted by GFAP immunofluorescence staining at the time points of 2nd,6th,10 th and 14 th day after operation.3.Selection of mous strain in vivo experiments:(1)20 C57 mice were randomly divided into empty vector adenovirus(AAV)group(n=10)and target AAV group(n=10),observating and analysing infection efficacy of virus in cerebral infarction cortex.(2)6 GFAP-Cre mice were randomly divided into empty vector AAV group(n=3)and target AAV group(n=3),observating and analysing infection efficacy of virus in cerebral infarction cortex.4.Observing the results of reprogramming astrocytes after implanting Pax6 and Ngn2 TFs:(1)In vitro experiments: the primary culture of astrocytes were obtained from the cerebral cortex of 3-day-old neonatal C57 mice,the astrocytes were divided into empty vector LV group and target LV group,analysing and comparing the virus-infected astrocytes’ morphological changes by fluorescence microscope.(2)In vivo experiments: Pax6 and Ngn2 TFs were implanted into the cortex of GFAP-Cre mice with cerebral infarction,then the virus-infected reactive astrocytes’ morphological changes were analyzed and compared by fluorescence microscope.Results: 1.(1)Comparison of motor function between Suture-occluded method and ET-1 method in C57 mouse cerebral infarction model: 1)Suture-occluded method: the motor function assessment of cerebral ischemia mice showed that the foot-fault rate of mice in the operation group were highest at 3rd day after operation in foot-fault test,and the foot-fault rate at 1st,5th,7th,9th and 20 th days were also higher than that of sham operation group after operation(***P<0.001).In the rotarod test,the mice in the operation group had the shortest staying time at 3rd day after operation and the staying time at 1st,5th,7th and 9th days after operation were also shorter than that in the sham operation group(**P<0.001),but the difference was not statistically significant at 20 th day after operation.In the grip test,mice’s grip in the operation group were the smallest at 1st day after operation,and were also smaller at 3rd,5th,7th and 9th days than that in the sham operation group,but there was no significant difference at 20 th day after operation(***P<0.001).2)ET-1 method:the motor function assessment of cerebral ischemia mice showed that the foot-fault rate of mice in the experimental group were highest at 9th day after operation in foot-faut test,and the foot-fault rate at 7th,20 th and 30 th days were also higher than that of control group(***P<0.001).In the rotarod test,the mice in the experimental group had the shortest staying time at 9th day after operation and the staying time at 7th,20 th and 30 th days were also shorter than that in the control group after operation(***P<0.001).In the grip test,mice’s grip in the experimental group were the smallest at 9th day after operation,and were also smaller at 7th,20 th and 30 th days than that in the control group,the difference was statistically significant.Finally,we chose the ET-1 method to establish the model of cerebral infarction in mice.(2)Comparison of infarction after cerebral infarction in mice: 1)The results of TTC staining showed that suture-occluded method caused a large range of infarction,affecting other non-cortex brain areas.2)The results of Nissl staining showed that ET-1 could cause cerebral ischemic injury limite in the motor function area of cortex.Finally,we chose ET-1 method to establish the cerebral infarction model in mouse.2.The results of GFAP immunofluorescence staining of brain slices showed that a large number of reactive astrocytes proliferated after cerebral ischemia and the peak was reached on the 6th day(*** P<0.001,*P<0.05).According to the peak of reactive astrocytes proliferation,the time of virus injection was chosen to make the virus infect more reactive astrocytes,increasing the effectiveness that virus infecting reactive astrocytes.3.After the AAV were injected into C57 mice cerebral infarction model cortex and GFAP-Cre mice cerebral infarction model cortex,founding that AAV infected neurons not astrocytes in C57 mice cerebral infarction model;while AAV specifically infected astrocytes in GFAP-Cre mice cerebral infarction model.Therefore,we selected the mouse strain that virus could specifically infect astrocytes—GFAP-Cre gene mice as our experimental mice.4.(1)After Pax6 and Ngn2 TFs were implanted into astrocytes in vitro,neuron-like cells were found.(2)After implanting the Pax6 and Ngn2 TFs into the cortexs of GFAP-Cre mice,neuron-like cells were generated.Conclusion:1.The method of injecting ET-1 into the cortex can establish a stable cortical cerebral infarction mouse model,laying the foundation for in vivo experiment of this study.2.Astrocytes proliferate massively after cerebral infarction and reach a peak on the 6th day after cerebral infarction,providing the material basis for reprogramming reactive astrocytes into neurons.3.The virus specifically infect astrocytes in cortex of GFAP-Cre mice cerebral infarction model,choosing GFAP-Cre gene mice could provide guarantee for reprogramming efficiency.4.Pax6 and Ngn2 TFs could reprogram astrocytes which cultured in vitro into neuron-like cells;Pax6 and Ngn2 TFs could reprogram reactive astrocytes into neuron-like cells in GFAP-Cre mice cerebral infarction model. |
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