Mechanism Of Exogenous Recombinant Gas6 In Improving Hemorrhage Transformation In Stroke Rats

Background:Hemorrhagic transformation(HT)is a common complication of ischemic stroke,which is closely related to the poor prognosis of stroke.The mechanism of HT after stroke is complicated,and it is known that disruption of the blood-brain barrier(BBB)plays an important role in this process.Axl is considered to be strongly associated with BBB function,and growth arrest-specific gene 6(Gas6)is the primary ligand of Axl.So far,some studies have found that the activation of Gas6/Axl pathway could protect several organs from ischemia reperfusion injury,however,its protective effect in HT has not been reported yet.Thus,we treat hyperglycemic focal cerebral ischemia reperfusion rats with recombinant Gas6(rGas6),aiming to explore the role of Gas6 and its downsteam pathway in protecting BBB function and reducing HT after stroke.Methods:A total of 108 male Sprague-Dawley rats(250-280g)were used in this study.Animals were randomly divided into three groups: sham group,HT group,and rGas6 treatment group.Each group was intraperitoneally injected with 50% high glucose(6ml/kg),15 minutes before middle cerebral artery occlusion,to induce acute hyperglycemia.The suture was removed 90 min after occlusion.PBS or rGas6 dissolved in PBS(20 ug/kg,0.25 μg/μl)was delivered into the nares of rats in HT group and rGas6 treatment group,respectively,60 min after occlusion.Rats in sham group underwent the same surgical procedures without insertion of the suture.After reperfusion for 24 h,modified Garcia neurological score,corner turn test,and forelimb placing test were used to evaluate neurological functions;TTC staining was used to assess cerebral infarct volume;hemoglobin content was used to assess intracerebral hemorrhage;brain water content was used to reflect the severity of brain edema;extravasation of Evan’s blue dye was used to measure the permeability of BBB;western blot was used to detect the expression of Axl and p-Axl.Results:(1)Neurological function: Compared with the sham group,rats in the HT group and the rGas6 treatment group shown significantly worse neurological function,including modified Garcia neurological score,forelimb placing test,and corner turn test.However,compared with the HT group,the neurological function was significantly improved in the rGas6 treatment group.(2)Cerebral infarct volume: Compared with the sham group,infarct volume in the HT group and the rGas6 treatment group were significantly increased.Meanwhile,treatment with rGas6 significantly reduced infarct volume compared with those in the HT group.(3)Hemorrhage volume: Compared with the sham group,hemorrhage volume in the HT group and the rGas6 treatment group were significantly increased.Meanwhile,treatment with rGas6 significantly reduced hemorrhage volume compared with those in the HT group.(4)Brain edema: Compared with the sham group,water content in the ipsilateral hemisphere were significantly increased in the HT group and the rGas6 treatment group,while rGas6 treatment reduced brain edema in the ipsilateral hemisphere compared with those in the HT group.Meanwhile,compared with the contralateral hemisphere,water content in the ipsilateral hemisphere were significantly increased in the HT group and the rGas6 treatment group.However,water content in the contralateral hemisphere and cerebellum did not change significantly between each groups.(5)BBB permeability: Compared with the sham group,the Evan’s blue content in the ipsilateral hemisphere were significantly increased in the HT group and the rGas6 treatment group,while rGas6 treatment significantly reduced the Evan’s blue content in the ipsilateral hemispheres compared with those in the HT group.Compared with the contralateral hemisphere,the content of Evan’s blue in the ipsilateral hemisphere were significantly increased in the HT group and the rGas6 treatment group.(6)Axl,p-Axl expression: Compared with the sham group,the expression of total Axl was significantly increased in the HT group and the rGas6 treatment group.However,rGas6 treatment did not remarkably change the expression of total Axl compared with those in the HT group.Compared with the sham group,the expression of phosphorylated Axl was significantly decreased in the HT group,while rGas6 treatment significantly increased the expression of phosphorylated Axl compared with the HT group.Conclusions:(1)rGas6 can improve CNS dysfunctions and HT in hyperglycemic focal cerebral ischemia reperfusion rats.(2)rGas6 attenuates HT in hyperglycemic focal cerebral ischemia reperfusion rats by inducing Axl phosphorylation to improve BBB function.