Role And Mechanism Of Necroptosis In Early Brain Injury Following Subarachnoid Hemorrhage In Rats

PurposeSubarachnoid hemorrhage(SAH)is a cerebrovascular disease with high mortality.Recent studies have reported an nonapoptotic mechanism of cell death—necroptosis is involved in several central nervous system,such as ischemic stroke and multiple sclerosis.However,it is still unclear about the role of necroptosis in early brain injury(EBI)post SAH.Thus,the current study was aimed to investigate the effect of necroptosis against EBI following SAH and possibly potential mechanism.MethodsSAH model in rats was established by means of modified perforation for internal carotid artery.Primarily,male Sprague-Dawley rats were divided into seven groups according to time courses:sham group,SAH 3 h group,SAH 6 h group,SAH 12 h group,SAH 24 h group,SAH 48 h group and SAH 72 h group.Western blot was applied to detect the expression of RIPK3,MLKL and HMGB1 in the cortex tissues from each group.The distribution of RIPK3 in brain and the relationship between necrotic neural cells and RIPK3 were determined by immunofluorescence staining and propidium iodide(PI)staining at 72 h,respectively.The ultrastructural changes of necrotic neural cells were observed by transmission electron microscope(TEM).After that,rats were divided into three groups:sham group,SAH+vehicle group and SAH+GSK'872 group(selective inhibitor of RIPK3).Neurological functions,Brain water content,proteins expression,PI staining and immunofluorescence staining were estimated after surgery.ResultsAfter SAH induction,the expression of RIPK3 started to elevate at 6 h,slightly decreased at 12 h and increased again to reach peak at 72 h.Double fluorescence labeling revealed that RIPK3 was mainly expressed in neurons.Consistently,immunofluorescent staining showed that necrotic(PI-positive)neural cells were mainly neurons,which further confirmed by TEM.Notably,compared with sham group,morphological features of necroptosis were observed by TEM,such as nuclear fragmentation,mitochondria swelling,loss of plasma membrane integrity,autophagosome formation,and the appearance of translucent cytosol.Compared with SAH+vehicle group,significantly decreased brain water content and necrotic neural cells as well as improved neurological functions were detected in SAH+GSK'872 group.More importantly,GSK'872 could reduce the expression level of RIPK3 and MLKL,and suppress the cytoplasmic translocation of HMGB1.ConclusionThese data demonstrated the involvement of RIPK3-mediated necroptosis in EBI after SAH.GSK'872,a selective inhibitor of RIPK3,could effectively decreases the RIPK3-mediated necroptosis and subsequent cytoplasmic translocation of HMGB1,as well as ameliorates brain edema and neurological deficits.Inhibiting necroptosis could be a clinically promising method for SAH therapy.