Expression Of EphrinB3 In Brain Tissue In Early Stage Of Subarachnoid Hemorrhage And Protection Of Blood-brain Barrier Integrity

Objective: To explore the expression changes of Ephrin B3,MMP9 and occludin in the brain tissue of early brain injury(EBI)in rats with subarachnoid hemorrhage(SAH);And to explore whether Ephrin B3 participates in the early brain injury of subarachnoid hemorrhage by protecting the integrity of blood-brain barrier.Methods:The SAH model of rats was established by intravascular puncture with mature technology,and the rats with SAH score ≥ 9 were selected for experiment.Western blot(WB)was used to detect the expression of Ephrin B3,occludin and MMP9 in the brain tissue of puncture side in the early stage after SAH;Immunofluorescence(IF)double labeling method was used to detect the expression of Ephrin B3 protein in astrocytes and brain microvascular endothelial cells(BMEC)after SAH,and the brain samples were taken at the time point when the expression of the above proteins was most obvious.Dry wet weight method was used to measure the brain water content(BWC)and observe the severity of brain edema of rats after SAH.The neurological function of early SAH rats was evaluated by modified Garcia and balance beam test,and the mortality of SAH rats was statistically calculated.After administration of Ephrin B3 si RNA,the changes of neural function,water content of each part of brain tissue and the expression of Ephrin B3,occludin and MMP9 were evaluated.Results: After subarachnoid hemorrhage in rats,it was observed in neuroethology that in the balance beam and modified Garcia test,the nerve function was damaged at6 h,12h,24 h and 72 h after SAH,which was significantly different from that in sham group(P < 0.05);In the determination of brain water content,it was found that the BWC of left and right cerebral hemisphere and cerebellum of rats at 6h,12 h,24h and72 h after SAH was significantly higher than that of sham group(P < 0.05),while the BWC of brain stem had no significant difference compared with sham group(P >0.05).Western blot was used to detect the expression levels of Ephrin B3,occludin and MMP9 at different time points after SAH.It was found that the expression of Ephrin B3 protein in rat brain increased gradually at 6h,12 h,24h and 72 h after SAH;The expression of MMP9 protein peaked at 24 hours after SAH and decreased until72 hours after SAH;Occludin protein showed an obvious downward trend at 3hours after SAH.The expressions of Ephrin B3 and MMP9 protein at 12 h,24h and 72 h after SAH were significantly higher than those in sham group(P < 0.05);The expression level of occludin protein at 6h,12 h,24h and 72 h after SAH was significantly lower than that in sham group(P < 0.05).Ephrin B3 protein was expressed on astrocytes and brain microvascular endothelial cells(BMEC)by immunofluorescence double labeling at 24 hours after SAH.After transfection of si RNA and silencing Ephrin B3 gene,it was observed that the neurological function of rats was more seriously damaged,the brain water content increased,the expression of Ephrin B3 and occludin protein decreased,and the expression of MMP9 protein increased at 24 hours after SAH.Conclusion: 1.Ephrin B3,MMP9 and occludin were dynamically expressed in the early stage of subarachnoid hemorrhage.2.Ephrin B3 may reduce the damage of blood-brain barrier by regulating the expression of MMP9 and occludin,and participate in the early brain injury of subarachnoid hemorrhage.