| Objectives:Ischemic stroke is a common clinical disease,and reperfusion is the most effective measure to treat ischemic stroke.How to effectively reduce cerebral ischemia reperfusion injury and promote nerve and blood vessel regeneration after cerebral ischemia is important.Glycine receptor（GlyR）is an inhibitory pentamer receptor of alpha subunits（GLRA）and beta subunits（GLRB）,with GLRA binding ligand,and GLRB playing a structural rather than ligand binding role for GlyR function.Studies have shown that the level of Glrb mRNA continues to decline 24hours and 7 days after stroke.However,the role of Glrb gene in nerve and vascular injury and regeneration after stroke is still unclear.This project is to study the expression characteristics of Glrb gene and the effects and mechanisms of Glrb over-expression on the regeneration of blood vessels and nerves in the damaged brain tissues of mice during the recovery period of ischemic reperfusion after stroke,and to reveal the potential of Glrb gene as a target for recovery from ischemic reperfusion after stroke.Methods:1.In vitro study based on cell modelThe mouse brain microvascular endothelial cells bEnd3 cells were divided into three groups:（1）The Control group,bEnd3 cells were cultured normally,transfected with blank vector pcDNA3.1-EGFP,and then cultured normally.（2）The OGD/R group,bEnd3 cells were subjected to oxygen glucose deprivation and reperfusion treatment,then transfected with blank vector pcDNA3.1-EGFP,and then cultured normally.（3）The OGD/R+Glrb group,bEnd3 cells were subjected to oxygen glucose deprivation and reperfusion treatment,then transfected with the Glrb overexpression vector pcDNA3.1-EGFP-Glrb,and then followed by normal culture.According to the experimental groupings,the normal medium of the groups 2 and 3 was replaced with glucose-free medium,and was cultured under hypoxia for 2h.After hypoxic culture,the cells were cultured in normal medium for 24 h,and then transfected with the Glrb overexpression plasmid or the control plasmid.At 72 h after transfection,（1）QRT-PCR and WB were used to detect the change of Glrb expression level,（2）CCK-8 was used to detect the proliferation activity of bEnd3 cells,（3）tube formation analysis was used to detect the change of tube forming ability,（4）immunohistochemistry was used to detect the change of Vegf gene expression in each group.2.In vivo animal studyC57BL/6 mice were divided into three groups:（1）The Control group,mice were intraventricularly injected normal saline control after sham operation.（2）The MCAO/R group,mice were intraventricularly injected pDC316-mCMV-EGFP after middle cerebral artery occlusion/reperfusion treatment.（3）The MCAO/R+Glrb group,mice were intraventricularly injected pDC316-mCMV-EGFP Glrb adenovirus after middle cerebral artery occlusion/reperfusion treatment.50mg/kg BrdU was intraperitoneally injected 7 days before operation.After MCAO/R,the animals were tested for neurological function with Longa score.The animals with a score of 2-3were divided into the MCAO/R group and the MCAO/R+Glrb group.Each group was injected with normal saline,pDC316-mCMV-EGFP,or pDC316-mCMV-EGFP-Glrb(titer≥1*1011PFU/ml)through stereotactic brain injection.All indexes were detected24 hours and 7 days after MCAO/R.（1）Longa score was used to detect the neurological function deficit;（2）TTC staining was used to detect the area of cerebral ischemia;（3）H&E staining was used to detect the pathological changes of cerebral tissue on the ischemic side and the damages were quantitatively evaluated based on histopathological scores;（4）QRT-PCR and immunohistochemistry were used to detect the expression of Glrb gene,vascular regeneration regulator CD34 and Vegf gene,nerve regeneration regulator Bdnf and Gap-43 gene in the damaged brain tissue,（5）BrdU immunohistochemistry was used to detect the regeneration of nerve cells in the damaged brain tissue.The differences of Glrb gene expression among groups and the effects and mechanisms of Glrb gene overexpression on the neurogenesis and angiogenesis of MCAO/R mice during the recovery period were compared and analyzed.Results:1.In vitro study based on cell model1.1 Glrb mRNA and protein expression in bEnd3 cells in different groups of mice.The Glrb mRNA and protein levels in the OGD/R group were decreased,compared with the Control group（42.53±1.15%vs 100.00±0.00%,0.62±0.03 vs0.45±0.02,P=0.0001,0.0003）.They were significantly higher in the OGD/R+Glrb group than those in the OGD/R group（93.96±12.07%vs 42.53±1.15%,0.62±0.02vs 0.45±0.02,P=0.0003）.There was no significant difference in the Glrb mRNA and protein levels between the OGD/R+Glrb group and Control group.The results suggested that the expression of Glrb gene in bEnd3 cells decreased after oxygen glucose deprivation and recovery（OGD/R）,and transfection of pcDNA3.1-EGFP Glrb plasmid reversed the decrease of Glrb gene expression.1.2 Functional assessment of cerebral microvascular endothelial bEnd3 cells in different groups of mice.The cell proliferation activity（89.60±2.29%vs 100.00±6.65%,P=0.002）,the ability of cell tube formation（the number of tubes per hole 182.00±17.06 vs 307.33±28.29,the number of branches per hole 523.00±46.68 vs 846.67±31.56,P<0.05）,and the level of Vegf expression（0.33±0.06 vs 1.00±0.11,P=0.0002）were decreased significantly in the OGD/R group,compared with the Control group.They were significantly increased in the OGD/R+Glrb group,compared with those in the OGD/R group（96.77±2.04%vs 89.60±2.29%,P=0.026;the number of tubes per hole 262.00±13.45 vs 182.00±17.06,the number of branches per hole 721.00±45.83 vs 523.00±46.68,P<0.05;0.66±0.08 vs 0.33±0.06,P=0.0093）.The level of cell proliferation activity in the OGD/R+Glrb group was slightly lower than that in the Control group,with no significant difference.The results suggested that the proliferation activity,the tube formation ability,and the Vegf expression level were decreased significantly in bEnd3 cells after OGD/R.Glrb gene expression promoted significant increase in the proliferation activity,the tube formation ability,and the Vegf expression in bEnd3 cells after OGD/R.2.In vivo animal study2.1 Glrb expression in ischemic stroke animal modelsThe Glrb mRNA and protein levels in the damaged brain of the MCAO/R group were significantly lower than those of the Control group at 24 h and 7 d after MCAO/R（mRNA 24 h 0.23±0.01 vs 1.00±0.08,7 d 0.14±0.02 vs 1.00±0.04,protein 24 h 0.45±0.08 vs 1.00±0.11,7 d 0.29±0.04 vs 1.00±0.08,P<0.0001）.They were significantly higher in the MCAO/R+Glrb group than those of MCAO/R group at 24 h and 7 d after MCAO/R（mRNA 24 h 0.14±0.02 vs 0.23±0.01,7 d0.23±0.04 vs 0.51±0.06,protein24 h 0.29±0.04 vs 0.45±0.08,7 d 0.62±0.10 vs0.65±0.09,P<0.05）,but still significantly lower than those of the Control group（P<0.0001）.The Glrb mRNA level and protein levels in the MCAO/R group and MCAO/R+Glrb group detected 7 days after MCAO/R were significantly lower than those of 24 hours after MCAO/R（mRNA 24 h 0.14±0.02 vs 0.23±0.01,7 d 0.23±0.04 vs 0.51±0.06,protein24 h 0.29±0.04 vs 0.45±0.08,7 d 0.62±0.10 vs 0.65±0.09,P<0.05）.The results suggested that the expression of Glrb gene in the brain tissue of MCAO/R mice decreased continuously in a time-dependent manner.Infection with pDC316-mCMV-EGFP Glrb adenovirus significantly and continuously increased the expression of Glrb gene in the brain tissue of MCAO/R mice,and the degree of increase was decreased with time.2.2 The effect of Glrb on neurological function after ischemic strokeThe average scores of neurological deficit（Zea-longa scores）in the MCAO/R group and the MCAO/R+Glrb group were higher than those in the Control group at24 h and 7 d（the MCAO/R group 24 h 3.38±0.52 vs 0.00±0.00,7 d 2.25±0.50 vs0.00±0.00,the MCAO/R+Glrb group 24 h 2.50±0.53 vs 0.00±0.00,7 d 1.50±0.58 vs 0.00±0.00,P<0.0001）.The average score of neurological function deficit in the MCAO/R+Glrb group was significantly lower than that in the MCAO/R group at24 h and it was nearly significantly lower than that at 7 d（24 h 2.50±0.53 vs 3.38±0.52,P=0.001;7 d 1.50±0.58 vs 2.25±0.50,P=0.097）.The average scores of neurological deficit were significantly lower at 7 d than those at 24 h in the MCAO/R group and the MCAO/R+Glrb group（2.25±0.50 vs 3.38±0.52,1.50±0.58 vs 2.50±0.53,P=0.0006,0.0021）.The results suggested that MCAO/R treatment resulted in neural function defect,in mice.Overexpression of Glrb promoted the neural function of damaged brain tissue in MCAO/R mice.2.3 Assessment of lesion area in ischemic stroke models using TTC stainingTTC staining showed that there was no white area in the brain of the Control group,while white areas appeared in the brain of the MCAO/R group at 24 h and 7 d after MCAO/R,and a small amount of red spots appeared in the white area at 7 d.White areas were presented in the MCAO/R+Glrb group at 24 h and 7 d after MCAO/R.However,unlike MCAO/R group,there were obvious red patches in the white areas of the MCAO/R+Glrb group,and there were more red patches in the white areas of 7 d than in those of 24 h after MCAO/R.Compared with the Control group,the ischemic area of brain tissue in the MCAO/R group and the MCAO/R+Glrb group appeared significantly larger at 24 h and 7 d（the MCAO/R group 24 h 48.33±2.67 vs 0.00±0.00,7 d 40.00±2.36 vs 0.00±0.00,the MCAO/R+Glrb group 24 h 28.77±3.69 vs 0.00±0.00,7 d 18.50±2.36 vs 0.00±0.00,P<0.00001）,and the ischemic area at 7 d was smaller than those of 24 h（40.00±2.36 vs 48.33±2.67,18.50±2.36 vs 28.77±3.69,P<0.05）.Compared with the MCAO/R group,the ischemic areas of the MCAO/R+Glrb group at 24 h and7 d were smaller（24 h 28.77±3.69 vs 48.33±2.67,7 d 18.50±2.36 vs 28.77±3.69,P<0.00001）.The results suggested that MCAO/R induced cerebral ischemia in mice,and overexpression of Glrb continuously reduced the cerebral ischemic area of mice after MCAO/R.2.4 Evaluation of ischemic lesions using H&E stainingIn the Control group,neuronal pyknosis was observed at 24 hours,with a pathological score of 1,indicating that sham surgery and intracerebral injection of normal saline may cause mild damage to brain tissue.On the 7th day of the control group,the cells were arranged in order,the nuclei were large,the distribution of chromatin was uniform,the nucleoli were clear,and no degeneration,necrosis or other lesions were found.The pathological score was 0,and there was no obvious damage,indicating that the sham operation and intracerebral injection of normal saline had no obvious pathological effect on the brain tissue on the 7th day.In the MCAO/R group,cells were arranged disorderly.Nuclei were fragmented,dissolved,or disappeared.Necrosis appeared and local edema and bleeding were observed at 24h,and the pathological score was 2 points（moderate damage）;In the MCAO/R group,cell necrosis and local tissue necrosis were observed on the 7th day,and the pathological score was 3 points.In the MCAO/R+Glrb group,cells were disorderly arranged,nuclei were fragmented,dissolved,or disappeared,necrosis appeared,local edema and bleeding were observed at 24 h.The pathological score was 2 points（moderate damage）.The degree of damage was equivalent to that of the MCAO/R group.Neuronal pyknosis was still visible at 7 d,and the pathological score was 1point（mild damage）.The scope of damage was significantly reduced compared with that of the MCAO/R+Glrb group at 24 h or those of the MCAO/R group at 24 and 7days.The results suggested that MCAO/R caused pathological damage to mouse brain tissue,which did not show significant repair within 7 days and became more severe over time.Overexpression of Glrb did not significantly promoted repair of pathological damage at 24 hours after injury,but promoted pathological recovery of damaged brain tissue at 7 days.2.5 CD34 protein expression in the ischemic stroke modelsThe level of CD34 protein in the damaged brain of the MCAO/R group was higher than that of the Control group at 24 h and 7 d after MCAO/R（24 h 1.53±0.23vs 1.00±0.11,7 d 2.30±0.32 vs 1.00±0.20,P=0.046,<0.0001）.It was higher in the MCAO/R+Glrb group than that of the MCAO/R group at 24 h and 7 d after MCAO/R（24 h 2.53±0.27 vs 1.53±0.23,7 d 3.26±0.25 vs 2.30±0.32,P<0.001）.It was higher on the 7th day than that at 24 h in both the MCAO/R group（2.30±0.32 vs 1.53±0.23,3.26±0.25 vs 2.53±0.27,P=0.0495）and the MCAO/R+Glrb group.The results suggested that MCAO/R caused the expression of CD34 protein in mouse brain tissue to change in a time-dependent manner,and overexpression of Glrb can promote the expression of CD34 protein in damaged brain tissue of mice after MCAO/R.2.6 VEGF expression in the ischemic stroke modelsThe Vegf mRNA and protein levels in the damaged brain of the MCAO/R group were significantly higher than those of the Control group at 24 h and 7 d after MCAO/R（mRNA 24 h 1.65±0.13 vs 1.00±0.07,7 d 3.15±0.36 vs 1.00±0.05,protein 24 h 1.53±0.15 vs 1.00±0.02,7 d 1.93±0.09 vs 1.00±0.02,P=0.021,<0.0001,0.0007,<0.0001）.They were significantly higher in the MCAO/R+Glrb group than those of the MCAO/R group at 24 h and 7 d after MCAO/R（mRNA 24 h4.97±0.24 vs 1.65±0.13,7 d 4.95±0.41 vs 3.15±0.36,protein 24 h 3.58±0.21 vs1.53±0.15,7 d 4.01±0.15 vs 1.93±0.09,P<0.0001）.They were significantly higher on the 7th day than those at 24th hour in the MCAO/R group after MCAO/R（3.15±0.36 vs 1.65±0.13,1.93±0.09 vs 1.53±0.158,P=0.0027,0.033）and they were increased to the same extent on 7th day and 24th hour in the MCAO/R+Glrb group after MCAO/R with significant changes in the protein levels（4.95±0.41 vs4.97±0.24,4.01±0.15 vs 3.58±0.21,P=0.024）.The results suggested that MCAO/R caused the expression of Vegf gene in mouse brain tissue to increase continuously in a time-dependent manner.Overexpression of Glrb promoted the expression of Vegf gene in the damaged brain tissue,and the degree of promotion did not change with time.2.7 Assessment of neuronal regeneration in lesions of the ischemic stroke models using BrdU immunofluorescenceThe level of BrdU immunofluorescence in brain of the MCAO/R group was higher than that of the Control group at 24 h and 7 d（24 h 78.67±10.50 vs 51.00±15.39,7 d 114.00±8.72 vs 50.33±9.02,P=0.025,<0.0001）.It was higher in the MCAO/R+Glrb group than that of the MCAO/R group at 24 h and 7 d after MCAO/R with the changes in 7thd were significant（24 h 100.67±8.08 vs 78.67±10.50,7 d167.00±13.00 vs 114.00±8.72,P=0.0002）.It was higher on the 7th day than that at24 h in the MCAO/R group and the MCAO/R+Glrb group after MCAO/R（114.00±8.72 vs 78.67±10.50,7 d 167.00±13.00 vs 100.67±8.08,P=0.0063,0.001）.The results suggested that MCAO/R caused nerve damage in mice brain tissues.Overexpression of Glrb promoted nerve regeneration in damaged brain tissue.2.8 Bdnf expression in in the ischemic stroke modelsThe Bdnf mRNA and protein levels in the damaged brain of the MCAO/R group were significantly higher than those of the Control group at 24 h and 7 d after MCAO/R（mRNA 24 h 3.67±0.47 vs 1.02±0.21,7 d 1.80±0.14 vs 1.01±0.15,protein 24 h 2.13±0.21 vs 1.00±0.15,7 d 1.56±0.22 vs 1.00±0.04,P=0.0007,<0.0001,0.0077）.They were significantly higher in the MCAO/R+Glrb group than those of the MCAO/R group at 24 h and 7 d after MCAO/R（mRNA 24 h 8.51±1.39vs 3.67±0.47,7 d 4.51±0.37 vs 1.80±0.14,protein 24 h 4.17±0.18 vs 2.13±0.21,7 d 2.81±0.23 vs 1.56±0.22,P<0.0001,0.0006,<0.0001,<0.0001）.The extents of induction were lower on the 7th day than those at 24 h in the MCAO/R group and the MCAO/R+Glrb group after MCAO/R（mRNA 1.80±0.14 vs 3.67±0.47,4.51±0.37 vs 8.51±1.39,protein 1.56±0.22 vs 2.13±0.21,2.81±0.23 vs2.13±0.21,P=0.011,0.0002,0.007,<0.0001）.The results suggested that MCAO/R caused the expression of Bdnf gene to increase in damaged brain tissue of mice,and the degree of increase was decreased with time.Overexpression of Glrb continuously promoted the expression of Bdnf gene in MCAO/R-damaged brain tissues,and the degree of increase was decreased with time.2.9 Gap-43 expression in the ischemic stroke modelsThe Gap-43 mRNA and protein levels in the damaged brain of the MCAO/R group were significantly lower than those of the Control group at 24 h and 7 d（mRNA 24 h 0.09±0.04 vs 1.00±0.06,7 d 0.22±0.03 vs 1.00±0.05,protein 24 h0.26±0.06 vs 1.00±0.04,7 d 0.41±0.05 vs 1.00±0.10,P<0.0001）.They were significantly higher in the MCAO/R+Glrb group than those of the MCAO/R group at24 h and 7 d（mRNA 24 h 0.31±0.07 vs 0.09±0.04,7 d 0.40±0.07 vs 0.22±0.03,protein 24 h 0.56±0.07 vs 0.26±0.06,7 d 0.67±0.09 vs 0.41±0.05,P=0.0008,0.0052,0.0005,0.0017）.They were higher on the 7thday than that at 24 h in the MCAO/R group and the MCAO/R+Glrb group after MCAO/R（P<0.05）.The extents of decreases were lower on the 7thday than those at 24 h in the MCAO/R group after MCAO/R（mRNA 0.22±0.03 vs 0.09±0.04,protein 0.41±0.05 vs 0.26±0.06,P=0.038）.The extents of increases were nearly the same on the 7thday as those at 24 h in the MCAO/R+Glrb group after MCAO/R（mRNA 0.40±0.07 vs 0.31±0.07,protein0.67±0.09 vs 0.56±0.07）.The results suggested that MCAO/R reduced the expression of Gap-43 gene in the damaged brain tissue of mice,and the degree of reduction was decreased with time.Overexpression of Glrb promoted the expression of Gap-43 gene in the damaged brain tissue,and the promotion effect last for at least7 days.Conclusions:The expression of Glrb gene continues to decline after cerebral ischemia reperfusion.Overexpression of Glrb gene promoted regeneration of nerve and blood vessel,and in turn promoted the repair of cerebral ischemia reperfusion injury.This study revealed part of the mechanism of recovery after cerebral ischemia reperfusion and provided a certain theoretical basis for therapeutic strategies to promote recovery after stroke reperfusion. |
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