The Effect Of CELSR1 On The Prevention,Neurogenesis And Angiogenesis Of Cerebral Ischemic Injury

BackgroundStroke is one of the most common diseases in China,and its morbidity and mortality are very high.According to the cause of the disease can be divided into hemorrhagic stroke and ischemic stroke.The incidence of cerebral ischemia has reached more than 80%.At present,there are many clinical treatments for cerebral ischemia,such as neuroprotective agents,herbal medicines,and thrombolytic drugs that reduce neuronal death.However,the weakness of these treatments are low efficacy and side effects.After cerebral ischemia,the brain will have a self-repair process,and the incomplete self-repair will lead to sequelae of cerebral ischemia.The process of[self-repair includes neurogenesis and angiogenesis.Therefore,in order to restore brain function,we can stimulates brain-derived neurogenesis and angiogenesis,differentiated neurons will be integrated into the neural network.It is a hot research field in the treatment of stroke.In 2009,Japanese scientist Yoshiji Yamada and his laboratory members screened 100 single nucleotide polymorphisms(SNPs)associated with ischemia-induced stroke.Three of the SNPs(rs1671021 for LLGL2,rs9615362 for CELSR1 and rs753307 for RUVBL2)were significantly associated with ischemic stroke(P<0.05).Therefore,it is speculated that CELSR1 is one of the sensitive genes of Japanese stroke,and the same result was found in the Portuguese and C.hinese population.CELSR1 is a member of the cadherin superfamily and is the core protein of the non-canonical Wnt/PCP signaling pathway.CELSR1 distributes the proliferation of neural stem cells,including the ventricles of the embryonic development period and the adult hippocampus brain regions.It can participate in the Wnt/PCP pathway together with Fzd3,Fzd6,Dvl1,Dvl2 and Vang12 to regulate the proliferation,differentiation and apoptosis of living cells.It has been proved that Wnt/PCP signaling pathway can play an important role in neurogenesis and angiogenesis.Cerebral ischemia can stimulate endogenous neurogenesis and angiogenesis.It is reported that CELSR1 regulates the orientation of neuronal dendritic initiation sites during adult hippocampal neurogenesis.Subsequently,it was demonstrated that CELSR1 can positively regulate the migration of endothelial cells and promote the angiogenesis.This suggests that CELSR1 plays a role in the process of neurogenesis and angiogenesis.However,it has not been reported whether CELSR1 exerts a certain regulatory effect on neurogenesis and angiogenesis during cerebral ischemia.In order to detect the effect of CELSR1 on the process of post-ischemic nerve repair,this study used lentiviral injection technology to silence with the expression of CELSR1 in the SVZ and DG.In addition,this study used the MCAO model of rats for further study the effects of CELSR1 on self-repair,neurogenesis and angiogenesis after cerebral ischemic injury using techniques such as viral interference,molecular biology,cell biology and animal behavioral testing.Objective:1.The effect of CELSR1 on neurogenesis in cerebral ischemic injury.2.The effect of CELSR1 on angiogenesis in cerebral ischemic injury.3.To investigate the role of CELSR1 in the treatment of cerebral ischemic injury and mechanisms.Methods:1.MCAO modelThe nylon filament was inserted into the middle artery of the right brain of SD rats,resulting in middle cerebral artery occlusion(MCAO),and was reperfused for 24 hours by ischemia for 2 hours.2.Lentivirus microinjectionRats were anesthetized by intraperitoneal injection of chloral hydrate,and the rats were fixed on a brain stereotaxic instrument to adjust the parameters.skin was cut the,the skull was exposed,and the lentivirus was injected into the brain.3.BrdU labeling and IHCIntraperitoneal injection of BrdU was performed before the rats were sacrificed.After 2 hours,the brain was perfused.After re-fixation,the rat brain was cut into 40 micron slices from front to back,and the designated brain regions were collected for immunofluorescence histochemical staining.Antibody source properties and concentrations used were:BrdU(Sheep,1:500);CELSR1(Rb,1:500);Caspase-3(Rb,1:500);Ki67(Ms,1:100);Nestin(Ms,1:500);CD31(Rb,1:50).4.Real-time PCRTissue RNA was extracted from rat SVZ,DH,ischemic core,striatum,ischemic penumbra and cortical regions and reverse transcribed into cDNA.The expression level of CELSR1 was detected using q-PCR.5.Calculating the ischemic volumeThe brain was cut into 2 mm thick brain slices,and the brain slices were placed in a 2%TTC and incubated at 37? for 30 min.The slices were scanned into a picture with a scanner.Photoshop software was used to calculate the volume of cerebral ischemia.6.Protein extraction and SDS-PAGETissue protein is extracted.The expression level of the target protein was examined by Western blot.7.Virus transfectionThe lentivirus was added to the cultured 293 cells for 24 hours and 36 hours,and photographed under a fluorescence microscope.After 48 hours of culture,the RNA was extracted and reverse transcribed into cDNA q-PCR.8.Neurological deficit scoreNeurological deficit scores were performed according to the five-point method of Longa and Bedersond.Scoring criteria:0',no obvious symptoms of injury;1',the contralateral forelimbs of the damaged brain area could not be fully extended;2',turning to the opposite side;3',dumping to the opposite side;4',sputum,unconscious.Results:1.The level of CELSR1 increased significantly in the ischemic SVZ and DGRats were divided into sham operation group and operation group,and the model of MCAO was established.The tissues of SVZ,DH,striatum,ischemic penumbra and core area were extracted for q-PCR.The expression level of CELSR1 in DG and SVZ brain regions significantly increased,and the expression level of CELSR1 in the ischemic penumbra was significantly decreased,while the striatum and ischemic core region had no change,2.Protective effects of CELSR1 in the cerebral ischemia2.1 The knock-down of CELSR1 in the SVZ can increases mortality after cerebral ischemiaThe mortality after cerebral ischemia was found to increase the mortality of cerebral ischemia in rats after reducing interfering with the expression of CELSR1.2.2 The knock-down of CELSR1 in the SVZ can increases the area of ischemic injuryThe MCAO model was constructed 12 days after the injection of the CELSRI-shRNA lentivirus.Ischemia for 2 hours and reperfusion for 3 days,The brain was taken for 2%TTC staining.Scanning statistics showed that the ischemic area was significantly increased after interference with CELSR1.2.3 The knock-down of CELSR1 in the SVZ can increases neurological deficit scoresNeurological deficit scores were scored according to Longa and Bederson's 5-point system when the middle cerebral artery was occluded for 2 hours and reperfused for 24h,48h,and 72h.After statistical findings,the neurological deficit score was significantly increased after after knocking-down CELSR1.2.4 The knock-down of CELSR1 in the SVZ can increases the number of apoptotic neurons in the ischemic penumbraCaspase3 immunofluorescence staining was performed on the ischemic penumbra.After statistical analysis,the number of Caspase3-positive cells in the ischemic penumbra was significantly increased after knocking-down CELSR1.To further verify this result,we performed the TUNEL staining and obtained the same results.2.5 The knock-down of CELSR1 in the DG has no effect on neurological deficit scoresThe CELSR1-shRNA lentivirus were injected into the DG,and the MCAO model was constructed 12 days after the virus expression.Ischemia for 2 hours and reperfusion for 3 days,and the neurological deficit score was performed.It was found thatthe knock-down of CELSR1 in the DG had no effect on neurological deficit score after cerebral ischemia.2.6 The knock-down of CELSR1 in DG has no effect on neuronal apoptosis after cerebral ischemiaCaspase3 immunofluorescence staining was performed on the ischemic penumbra.After statistical analysis,it was found that the expression of CELSR1 in the DG had no significant effect on the apoptosis of neurons in cerebral ischemia compared to the control group.To further verify this result,we performed the TUNEL staining and obtained the same results.3.Effect of CELSR1 on neurogenesis in cerebral ischemia3.1 The knock-down of CELSR1 in the SVZ significantly reduced the proliferation of NSCThe MCAO model was constructed 12 days after the injection of the CELSR1-shRNA lentivirus.Ischemia for 2 hours and reperfusion for 3 days,BrdU immunofluorescence staining was performed in the SVZ brain region.It has been found that the knock-down of CELSR1 in the SVZ can reduce the proliferation of neural stem cells after cerebral ischemia.3.2 The knock-down of CELSR1 in the DG significantly reduced the proliferation of NSCThe MCAO model was constructed 12 days after the injection of the CELSR1-shRNA lentivirus.Ischemia for 2 hours and reperfusion for 3 days,BrdU immunofluorescence staining was performed in the DG brain region.It has been found that the knock-down of CELSR1 in the DG can reduce the proliferation of neural stem cells after cerebral ischemia.4.Effect of CELSR1 on angiogenesis after cerebral ischemia4.1 The knock-down of CELSR1 in the SVZ can inhibit angiogenesis after ischemiaTo observe the effect of CELSR1 on angiogenesis after cerebral ischemia in the SVZ.We injected CELSR1-shRNA lentivirus in the SVZ,and the MCAO model was constructed 12 days after the virus expression.After 3 days of reperfusion,the brain was perfused.After the fixation,the frozen sections were taken.CD31 immunofluorescence staining was performed on the ischemic penumbra.It was found that the knock-down of CELSR1 in the SVZ can inhibit angiogenesis after ischemia.4.2 The knock-down of CELSR1 in the DG has no significant effect on angiogenesis.To observe the effect of CELSR1 knock-down on angiogenesis in after cerebral ischemia.We injected CELSR1-shRNA lentivirus in the SVZ,and the MCAO model was constructed 12 days after the virus expression.After 3 days of reperfusion,the brain was perfused.After the fixation,the frozen sections were taken out for immunostaining.CD31 immunofluorescence staining was performed on the ischemic penumbra.It was found that the knock-down of CELSR1 in the DG has no significant effect on angiogenesis5.Mechanism of effect of CELSR1 on cerebral ischemic injuryTo explore the molecular mechanism of CELSR1 involved in cerebral ischemic injury.We inject the CELSR1-shRNA lentivirus nd constructed the MCAO model after 12 days.Three days after reperfusion,the ischemic penumbra and SVZ of the rat was taken and the tissue protein was extracted.Related proteins in the Wnt/PCP pathway were detected by Western blot.It has been found that the knock-down of CELSR1 can inhibit the phosphorylation level of PKC,but it has no effect on the expression of p-JNK and ?-catenin.Conclusion:1.CELSR1 promotes neurogenesis after cerebral ischemic injury2.CELSR1 promotes self-repair and angiogenesis after cerebral ischemic injury.3.Through Wnt/PKC signaling pathway,CELSR1 regulates the neurogenesis and angiogenesis after cerebral ischemia.Significance:1.The role of CELSR1 in nerve repair in cerebral ischemic injury2.The role of CELSR1 on neurogenesis and angiogenesis in cerebral ischemic injury.