Effects Of Transcription Factor FEZF2 Reprogramming Cortical Projection Neurons On Motor Function In Mice With Cerebral Infarction

Objective: We selected the Ca MKIIα-Cre ER transgenic tool mice,which can regularly make virus specificity infect excitatory neuron of cortex.Endothelin intracranial injection is used to establish a stable motor cortex cerebral infarction mice model,and we observe the reshaping of the contralateral cortex neurons after cerebral infarction.To observe the recovery of motor function in cerebral infarction mice model,the callosal projection neurons were reprogrammed into corticofugal neurons by implanting transcription factor Fezf2 into the contralateral cortex of the cerebral infarction mice model.Methods: 1.Detection of neuronal plasticity in contralateral cortex of the brain after cerebral infarction: 6 to 10 months old mice were randomly divided into two groups: sham-operated group(n=12)and experimental group(n=12).Focal cerebral infarction mice model was established by intracranial injection of endothelin.The sham group received only PBS injection.GAP-43 immunofluorescence staining was used to detect the expression of GAP-43 in contralateral cortex of brain in 0,5,15 and 20 days after cerebral infarction(n=3at each time point).2.Reprogramming of cortical projection neurons in the contralateral cortex of the brain after cerebral infarction: 6 to 10 months old Ca MKIIα-Cre ER transgenic tool mice were randomly divided into two groups after intracranial injection of endothelin: Adeno-associated virus empty vector control group(n=12)and Adeno-associated virus Fezf2 overexpression group(n=12).On the 6th day after the injection of endothelin,the virus was injected into the contralateral cortex of brain and the tamoxifen oil solution was injected intraperitoneally for 5 consecutive days in 4 days after the full expression of the virus.Immunofluorescence staining was performed on the brain and spinal cord at 14 and 28 days after virus injection to detect molecular markers and projection pathways of reprogrammed neurons(n=3 at each time point).3.Behavioral test to evaluate the motor recovery of cerebral infarction mice model:6 to 10 months old male Ca MKIIα-Cre ER transgenic tool mice were chosen as a test subject,then exercise behavior training was conducted for three consecutive days,including the grid walking test,the rotarod test and the balance beam test.Behavioral data were recorded the day before cerebral infarction.Focal cerebral infarction mice model was established by intracranial injection of endothelin.The sham group received only PBS injection.The successful mice were randomly divided into three groups: Cerebral ischemia in the control group(n =8,only injection of endothelin),adeno-associated virus empty vector in the control group(n = 8,injection of endothelin and empty vector virus),adeno-associated virus Fezf2 overexpression group(n = 8,injection of endothelin and overexpression virus).At the same time combined with sham group(n = 6,only inject PBS),we finally choose these four groups of mice for formal experiment.On the 6th day after the surgery,we injected adeno-associated virus into the contralateral cortex of the empty adeno-associated virus group and the Fezf2 overexpression group.After the virus was fully expressed in the brain for 4 days,mice were intraperitoneally injected with tamoxifen oil solution to initiate Cre nuclear expression for 5consecutive days.The changes of motor ability of mice in each group on the 5th,15 th,25th,35 th and 45 th days after cerebral infarction were measured by motor behavior test including grid walking test,rotarod test and balance beam test.Results: The expression of GAP-43 in the contralateral cortex of brain began to increase from day 5 after cerebral infarction,and maintained until day 20 after cerebral infarction;After the injection of Fezf2 overexpressed virus into the contralateral cortex of brain after cerebral infarction,the expression of callosal projection neurons marker CUX1 did not change on the 14 th and 28 th day after the injection of overexpressed virus,while the expression of corticofugal projection neuron marker CRYM increased on the 14 th and 28 th day;Compared with the empty vector virus group,on the 28 th day after the injection of overexpressed virus,the fibers of neurons in the contralateral cortex which project to the corpus callosum and the ipsilateral striatum decreased,and fibrous projections are seen in the subcortical anterior horn of the cervix;In the behavior test,the grid walking test found that grid walking rate in the Fezf2 overexpression group was lower than that in the empty vector of AAV control group from the 29 th day after virus injection,while the balance beam test and the rotarod test did not find any difference between these two groups.Conclusion: 1.On the 5th day after cerebral infarction,the expression of GAP-43 increased in the contralateral cortex of the brain,and neural remodeling occurred.After cerebral infarction,the neurons in the contralateral cortex of the brain can be regulated 2.On the 14 th and 28 th days of overexpression of Fezf2 in the contralateral cortex layer Ⅱ/Ⅲ after cerebral infarction,Neurons are reprogrammed to acquire new molecular identities,the expression of corticofugal projection neuron marker CRYM was increased.The reprogrammed neuronal projection path is altered,and the fibers project to the cervical pulp in the subcortical region.3.After 29 days of Fezf2 overexpression in layer II/III cortex on the contralateral side of cerebral infarction,the fine motor ability of upper limbs of cerebral infarction mice was recovered to some extent.