| Objective:To observe the spatial and variation regularity of chemical chemotactic factors CXCL12, CXCR4, histone H3 and histone H4 acetylation level in rats with Transient MCA ischemia-reperfusion injury (Transient Middle cerebral artery occlusion, tMCAO). By intravenous injection of broad-spectrum histone acetylation enzyme inhibitors trichostatin-A, (TSA) after ischemic damage to observe its protective effect to tMCAO rats and to explore the epigenetic regulatory mechanism of CXCL12-CXCR4 signaling pathways to brain ischemia reperfusion injury.Methods:1. Healthy male SD rats were randomly divided into 4 groups:I/R group, TSA group, vehicle (DMSO) group and sham operation group (Sham). Injury models with 90-minute ischemia and reperfusion of left middle cerebral artery were prepared in I/R group rats following Zea-Longa suture method. Vascular was isolated and suture was put into extracranial segment of internal carotid artery instead of middle cerebral artery in Sham group rats. TSA group was divided into three subgroups: TSA1 group (TSA1)-with tail vein injection of TSA0.03Mg/Kg immediately after preparation of MCAO model of ischemia; TSA2 group (TSA2)-with tail vein injection of TSA0.1Mg/Kg immediately after preparation of MCAO model of ischemia; TSA3 group (TSA3)-with tail vein injection of TSA0.3Mg/Kg immediately after preparation of MCAO model of ischemia. DMSO group (DMSO):with tail vein injection of 1% DMSO lml immediately after preparation of MCAO model of ischemia. 24h after reperfusion, the neurological deficit score were evaluated, and then rats were sacrificed to measure infarct volume by TTC staining.2. Healthy male SD rats were randomly divided into I/R group and Sham group, Injury models with 90-minute ischemia and reperfusion of left middle cerebral artery were prepared in I/R group rats following Zea-Longa suture method. Vascular was isolated and suture was put into extracranial segment of internal carotid artery instead of middle cerebral artery in Sham group rats. After reperfusion for 6h,24h,72h and 7 days respectively, the neurological deficit score were evaluated, then rats were sacrificed to test the protein expression of CXCL12, CXCR4, ace-H3 and ace-H4.3. Healthy male SD rats were randomly divided into 4 groups:I/R group, TSA group, vehicle (DMSO) group and sham operation group (Sham). Immediately after suture was put into the vessel, TSA0.1Mg/Kg was injected into tail vein in TSA group,1%DMSOlml was injected into tail vein in DMSO group, saline 1 ml was injected into tail vein in I/R and Sham groups. After reperfusion for 6h,24h,72h and 7 days respectively, the neurological deficit score was evaluated, then rats were sacrificed for a series of morphological and molecular biological tests.Results:1. neurological deficit score and infarct volume:compared with Sham group, there were different levels of neurobiological defects in TSA group and I/R group, the neurological deficit scores were between 2 and 5 points, and sham group had no neurological deficit. Compared with I/R group, neurological deficit score was no significant improved in TSA0.3mg/kg group. Although infarct volume was decreased in TSA0.3mg/kg group (22%decrease compared with tMCAO group), but the difference was not statistically significant. Neurological deficit score and infarct volume was significantly reduced in TSA0.03mg/kg and 0.1mg/kg groups, compared with tMCAO group, the infarct volume of TSA0.03mg/kg and 0.1mg/kg group was decreased by 43.5%and 42.8% respectively, and there was no significant differences between TSA0.03mg/kg and TSA0.1mg/kg groups.2. Immunohistochemical test of CXCL12, CXCR4. Compared with sham group, there was decreased CXCL12 immunoreactive expression and not significantly changed CXCR4 immunoreactive expression in ischemic cortex of tMCAO rats. Compared with I/R group, immunoreactive expression of CXCL12 and CXCR4 was significantly increased in TSA group. By double-label technique of immunofluorescence, the expression of CXCL12, CXCR4 was found highly matched with that of NeuN. Though no match was found between CXCL12, CXCR4 and GFAP, there was overlap between their expressional region.3. Changes in CXCL12, CXCR4, ace-H3 and ace-H4 protein and mRNA expression in ischemic side of cerebral cortex in TMCAO rats. Compared with sham group, protein and mRNA expression of CXCL12, ace-H3 and ace-H4 was significantly decreased in I/R group and lasted 7 days after reperfusion. But protein and mRNA expression of CXCR4 did not change significantly. The protein and mRNA expression of CXCL12 increased in first 24 hours after operation in TSA goups, but after 72 hours there was no significant difference between TSA and sham groups. Expression level of CXCR4 protein and mRNA in TSA Group was significantly higher than that of sham group and lasted for 7 days after reperfusion. Expression of ace-H3 protein in TSA Group was lower than that of sham group, but significantly higher than that of I/R group. There was no significant difference in protein expression of ace-H4 between TSA Group and sham group.Conclusion:1. Single intravenous injection of 0.03mg/kg and 0.1mg/kg of deacetylase inhibitor TSA after on tMCAO ischemia exerted significant brain protective effect, and the effect was not dose-dependent. But injection of 0. 1mg/kg TSA was not effective.2. Acetylation level of histone H3, H4 decreased after ischemia reperfusion injury in tMCAO mice, accompanied by decreased expression of CXCL12. TSA attenuated the decline of CXCL12, ace-H3 and ace-H4, and increased the expression of CXCR4 significantly. These indicates that deacetylase inhibitor TSA can exert protective effect to ischemia brain through CXCL12-CXCR4 signaling pathway.3. CXCL12-CXCR4 immunoreactive expression was highly matched with that of NeuN, suggesting that in the mature central nervous system, the protective effect of CXCL12-CXCR4 signaling pathway on ischemic brain is completed in the neuronal cells. |
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