Impacts And Possible Mechanism Of VEGI On The Permeability Of Blood-Brain Barrier In The Acute Phase Of Traumatic Brain Injury

Objective: Traumatic brain injury(TBI)has become a significant public health issue worldwide due to its high motility and morbidity rate.The disruption of blood-brain barrier(BBB)after primary injury leads to a series of pathological processes,such as poor cerebral blood flow,metabolic imbalance and coagulopathy,thus giving rise to secondary brain injury,exacerbating blood-brain barrier damage,and ultimately forming a vicious cycle.Therefore,timely restoring the integrity of blood-brain barrier attracts much attention in the respect of TBI treatment.Vascular endothelial growth factor(VEGF),known as vascular permeability factor,plays a vital role in the initiation and perpetuation of BBB permeability(damage)and angiogenesis.Vascular endothelial growth inhibitor(VEGI)serve as a nature inhibitor of VEGF,and has an advantage of hindering the secretion of VEGF without interfere the normal function of endothelial cells.Therefore,our study evaluated the respective level of VEGF and VEGI at different time-points during the acute phase of TBI and investigate their influence on BBB-associated brain injury.We,then,administrated the VEGI via intracerebroventricular injection to observe the alteration of VEGF expression,the permeability of BBB,the apoptotic level of the traumatic hemisphere and neurological function after TBI.Meanwhile,we trace the alteration of VEGF downstream as well as the possible mechanism on angiogenesis related to the VEGI intervention.Methods: 1)Established the TBI model on C57BL/6 mice and screened the leakage of fibrinogen of ipsilateral side at different time-points after TBI via Western Blot analysis.2)Examined the respective expression of VEGF and VEGI of ipsilateral side at different time-points after TBI via Western Blot analysis.3)Administration the VEGI recombinant protein via intracerebroventricular injection after TBI.4)Evaluated the neurological function via modified Neurological Severity Score and foot-fault test.Selected the most pronounced time-point before and after intervention for further study.5)Further analyzed the effects of VEGI on the permeability of BBB via both Western Blot and immunofluorescence technique.6)Identified the possible impacts of VEGI on VEGFR2 and its downstream eNOS in both TBI-control group and VEGI-treated group via Western Blot analysis.7)Analyzed the apoptotic cells of ipsilateral side in both TBI-control group and VEGI-treated group via flow cytometry and Annexin V/PI Apoptosis Detection kit as well as immunofluorescence technique.8)Examined the effects of VEGI on angiogenesis-associated cells via Western Blot and immunofluorescence technique.9)Compared the impact of VEGI on the effects of endothelial cells-induced tube formation and pericytes-induced tube formation in in vitro study.10)Examined the effects of VEGI on the balance between pro-angiogenic and anti-angiogenic factors in the acute phase of TBI via Western Blot analysis.Results: 1.The most prominent disruption of BBB was at the 3rd day after TBI.Within 7 days after TBI,VEGF was increased and reached the peak level at the 3rd day after TBI,whereas VEGI reached the nadir level at the same time-point.The VEGF/VEGI ratio was correspondingly increased after TBI with peak level at the 3rd day after TBI.2.VEGI intervention significantly improved the neurological function started at the 3rd day after TBI.VEGI significantly reduced the apoptotic cells of ipsilateral side,attenuated BBB damage.VEGI inhibited the phosphorylation of the VEGFR2 and of its downstream eNOS.3.VEGI intervention significantly suppressed the post-traumatic angiogenesis,as evidenced by lowering the expression angiogenic endothelial cells and normalizing the balance between pro-angiogenic and anti-angiogenic factors.Conclusion: 1.The high expression of VEGF alone could not fully explain the timing of BBB breakdown in the acute phase of TBI.Compared the relative alteration of both VEGF and VEGI might give a clearer answer.At the 3rd day after TBI,the increased ratio of VEGF to VEGI may attribute to the prominent leakage of BBB.2.The VEGI intervention significantly improved the neurological function of TBI mice on the 3rd after TBI.Therefore,our further studies were focused this time-point.Then,we found the apoptotic cells was much lower in the VEGI-treated group than that of the control group.3.VEGI intervention suppressed the local cell apoptosis.Meanwhile it alleviated the injury of tight junctions,decreased the expression of MMP9,inhibits the phosphorylation of VEGF-VEGFR2 downstream,all of which are conducive to the reduction of BBB permeability.4.VEGI attenuated the BBB hyperpermeability after TBI,which,at least partly,due to its ability to normalize the balance between pro-angiogenic and anti-angiogenic factors,and subsequent suppression of angiogenesis.