The Role And Mechanism Of Lipocalin-2 On Blood Brain Barrier Disruption After Subarachnoid Hemorrhage

Background:Subarachnoid Hemorrhage(SAH)is a hemorrhagic stroke with high mortality and high disability rate.Intracranial rupture of aneurysm is the main reason of SAH,which takes for about 90%of the cases.Acute mechanical brain injury and elevated intracranial pressure with arterial hemorrhage after aneurysm rupture are followed by cerebral ischemia.Post-subarachnoid hemorrhagic ischemia,tissue injuries,extravasated blood components and the breakdown products could activate microglia and astrocytes,as well as disrupt blood-brain barrier(BBB)associated with the induction of inflammation and other cascades.Once blood-brain barrier is disrupted,brain tissues are directly exposed to harmful blood contents and immune cells,which aggravate brain injuries furthermore.The blood-brain barrier disruption after subarachnoid hemorrhage may be caused by a variety of mechanisms,including endothelial cell death and the destruction of tight junction proteins,which may involve multiple independent or interrelated molecules and signaling pathways,but the exact mechanism is still unclear.Lipocalin-2(LCN2)is a kind of acute protein secreted by activated neutrophils which has multiply function.Many studies shows that LCN2 is increased immediately after cerebral hemorrhage,and expressed in neutrophils,astrocytes,microglia and vascular endothelial cells,which released various inflammatory factors and damage the blood-brain barrier.However,the mechanisms need further investigation.Our study will explore the role and mechanism of LCN2 on the blood-brain barrier disruption after subarachnoid hemorrhage.First,we investigate the trend of BBB destruction after subarachnoid hemorrhage,the relationship between LCN2 expression and BBB disruption and the possible mechanism.Secondly,we research the mechanism of LCN2 on BBB disruption after subarachnoid hemorrhage,involving the changes of 1-phosphosphingosine receptor 2 on vascular endothelial cells.Methods:Part ?Experiment ?:The SAH model was performed using the endovascular perforation technique.Adult wild male C57BL/6 rats were randomly divided into 6 groups.the sham group and SAH groups.And SAH rats were further divided into SAH 6h group,SAH 12h group,SAH 24h group,SAH 48h group and SAH 72h group according to the duration between SAH and execution.The sham group performed the same operation as the SAH model group except that the wire plug did not puncture the vascular wall.Western blot was used to detect the changes of albumin content and neutrophil infiltration marker MPO expression in ipsilateral cerebral cortex of mice in each group at different time points,which could find out the trend of blood-brain barrier disruption after SAH.Experiment ?:Using male LCN2 knockout and wild-type C57BL/6 mice,the wild-type mice were randomly assigned to the sham group and the subarachnoid hemorrhage group,and the LCN2 knockout mice were assigned to the subarachnoid hemorrhage group.The LCN2 knockout group and wild-type group using the endovascular perforation technique to make SAH model and the sham group did not puncture the vascular wall.Immunohistochemical staining,TUNEL staining,FJC staining and western blot were used to detect the contents of albumin,neutrophil infiltration,microglia activation,oxidative stress reaction,secondary brain injury and neuron death in the cerebral cortex of mice in each group.Part ?The SAH model was performed using the endovascular perforation technique.Using male LCN2 knockout and wild-type C57BL/6 mice,the wild-type mice were randomly assigned to the sham group and the subarachnoid hemorrhage group,and the LCN2 knockout mice were assigned to the subarachnoid hemorrhage group.The damage of cerebral vascular endothelial cells and the changes of Sphingosine-1-phosphate receptor 2 after SAH were observed by immunofluorescence staining and western blot.Results:Part?(1)The content of albumin in brain tissue increased 6 hours after SAH and remained at a high level from 24 hours to 72 hours.(2)Myeloperoxidase(MPO),a marker of neutrophils in the brain parenchyma,increased 12 h after SAH in mice,peaked at 24 h,and then decreased gradually.(3)The LCN2 in the cerebral cortex of wild mice increased significantly 24 hours after SAH,while none LCN2 expression in the cortex of LCN2 knockout mice 24 hours after SAH.(4)The levels of albumin,MPO,microglial cell activation,heat shock protein and neuronal cell death in the cerebral cortex of SAH mice were significantly lower than those in the wild mice.Part ?(1)Compared with wild mice,the loss of vascular endothelial cells in LCN2 knockout mice after SAH was reduced.(2)The expression of Sphingosine-1-phosphate receptor 2 in wild mice after SAH was significantly increased,which was expresse donvascular endothelial cells and associated with endothelial cell loss,and decreased after SAH in LCN2 knockout mice.Conclusions:(1)LCN2 level in cerebral cortex was significantly increased 24 hours after SAH,and meanwhile,albumin and MPO in brain tissue also reached a peak.These indicated that the time trend of LCN2 elevation was similar to that of blood-brain barrier disruption.In LCN2 knockout mice,the blood-brain barrier disruption was alleviated after SAH,and the oxidative stress and neuronal cell death were also reduced.(2)The loss of vascular endothelial cells was reduced after SAH in LCN2 knockout mice,while the loss of LCN2 reduced the expression of Sphingosine-1-phosphate receptor 2 on endothelial cells,thus reducing the loss of endothelial cells and the disruption of the blood-brain barrier.