The Research On The Role Of VEGI In Cerebral Edema After Traumatic Brain Injury

Objective:1. In vivo study, the main objective was to observe the regulatory function of VEGI (Vascular Endothelial Growth Inhibitor) on the mouses after TBI (Traumatic Brain Injury), and to investigate the role and potential mechanism of BBB (Blood Brain Barrier) protection, cerebral edema inhibition and neuroprotection.2. In vitro study, the main objective was to observe the VEGF-induced BBB leakage and the inhibiton of the leakage and the up-regulatation of tight junction protein by VEGI. Confirm that VEGI was able to inhibit the phosphorylation of VEGFR2and consequently alleviate the VEGF-induced BBB leakage, and preliminarily discuss the mechanism of VEGI on the cell level.Methods:1. In vivo study, the SPF adult C57B1/6mice for research were randomly divided into four groups:group1(control group), group2(sham group), group3(TBI group), group4(TBI+VEGI group). Subsequently, we made the mouse fluid percussion injury model to imitate the TBI, and used the model to test the neurological and ethological function of TBI-mice by mNSS and Morris Water Maze, and detected BBB leakage, TJ-associationed protein and blood vessel by Evans Blue and immunofluorescence.2. In vitro study, the bEnd.3cells from the cerebral microvascular endothelial cell of C57mouse were used to make the BBB model in vitro, and were randomly divided into four groups:group1(control group), group2(VEGI group), group3(VEGF-A group), group4(VEGF-A+VEGI group). Using the BBB model in vitro, we tested the TEER value to measure the BBB leakage and detected the TJ-associationed protein by immunofluorescence and Western Blot. Additionally, we continuously observed the nano-scaled BBB change in morphology for24hours by HPICM (Hopping Probe Ion Conductance Microscopy).Results:1. In vitro study, the mNSS scores of mice in group3and group4after FPI were instantly increased, but had no statistically significant difference in the first12hours after FPI. However, along with the recovery of FPI-mice, the statistical difference was gradually significant. Since the24th hour after FPI, the score of TBI-mice in group3had been statistically higher than that of FPI-mice in group4(P<0.01), and this difference was inexistent in the7th day after FPI. In the MWM (Morris Water Maze) navigation test, there had been statistical difference in mice in group3or4and the group2since the3rd day after FPI, with this difference lasting while the training was over in the5th day. In spatial probe test, compared with group1or2, mice in group3and group4both had a statistically less staying time in original platform quadrant (P<0.01), and the mice in group4had a longer staying time in original platform quadrant than that in group3(P<0.01). Additionally, the mice in group3had a lower rate in Evans Blue leakage than that in group4(P<0.01). The immunofluorescence test also showed that the mice in group4had a higher expression of CLN5and a lower expression of Alb than that in group3(P<0.01).2. In vitro study, there was no significantly statistical difference in the measured bEnd.3TEER value (Ω·cm2) of the control group, VEGI group and VEGI+VEGF group, but the value of the VEGF group had a statistical lower value than that of the other three groups (P<0.01). In24-hours HPICM scanning, the control group, VEGI group and VEGI+VEGF group did not present any detectable TJ changes in morphology, however, compared with control group, changes in RMS and relative height radio were evident in VEGF group after treated with100ng/ml VEGF-A for16hours (P<0.01). The immunofluorescence test also detected that the VEGF-induced Claudin-5down-regulation was inhibited by VEGI (P<0.01). The Western Blot outcome showed that100ng/ml VEGF-A significantly decreased the expression of Claudin-5and Occlaudin, but had no statistical difference in ZO-1. Additionally, VEGI inhibited the VEGF-induced down-regulation of Claudin-5and Occlaudin, and the inhibition was performed by reducing the phosphorylation of pY1175on VEGFR2.Conclusion:1. The in vivo study showed that VEGI was able to inhibit the TBI-induced secondary cerebral edema, relieve BBB leakage, up-regulate TJ-associationed protein, protect neurofunction and consequently improve ethological function. This study provided research data for the VEGI therapy for TBI-induced cerebral edema.2. The in vitro study showed that VEGI was able to inhibit the VEGF-induced BBB leakage, up-regulate TJ-associationed protein. And it was the first time we used HPICM to detect the VEGI/VEGF-A effect on bEND.3cell in nano-scaled morphology. Additionally, we alse found that the inhibition effect of VEGI was performed by inhibition of VEGFR2phosphorylation. This study provided research data of the VEGI effect on vascular endothelial cell.