Role Of Necroptosis In The Early Brain Injury After Subarachnoid Hemorrhage In Rats

Background and purposes: Subarachnoid hemorrhage(SAH)is a common cerebrovascular emergency in department of neurology and neurosurgery,largely due to the rupture of intracranial aneurysms.Despite obiviously advanced in aneurysmal surgery and neurological intensive care techniques.However,the mortality and disability in patients suffering from SAH still remain high.The current studies suggest that early brain injury may be one of the major causes of death and poor prognosis in patients with SAH.Necroptosis is a new type of cell death proposed in recent years.Evidences have shown that receptor-interacting protein 1(RIP1),RIP3 and mixed lineage kinase domain-like protein(MLKL)proteins play an important role in this type of cell death.Additionally,specific inhibitors of RIP1,RIP3 and MLKL,such as Necrostatin(Nec)-1,GSK' 872 and Necrosulfonamide(NSA),can significantly inhibit necroptosis.Moreover,RIP1/RIP3/ MLKL-mediated cell death has also widely been recognized as the most important form of necroptosis.Evidences have indicated that necroptosis is closely associated with brain injuries secondary to several common neurological diseases such as cerebral ischemia,intercerebral hemorrhage and traumatic brain injury.SAH shares many similar pathological mechanisms with above-mentioned diseases.However,to the best of our knowledge,the contribution of RIP1/RIP3/MLKL dependent necroptosis to the early brain injury following SAH has not been fully elucidated.Therefore,the present study aimed to clarify the activation of RIP1/RIP3/MLKLmediated necroptosis in rats suffering from SAH,to reveal the effect of necroptosis on early brain injury following SAH,and to explore the neuroprotective effect of specific inhibitors and its molecular mechanisms.As a result,we expected to provide a new target for the intervention of cell death after SAH,and ultimately to improve the therapeutic effect of SAH.Methods:1)SAH rat model was established by endovascular perforation.Western blot and immunofluorescence were used to detect the expression level of RIP1,RIP3 and MLKL proteins in brain tissue in the early stage of SAH(within 72 hours).Meanwhile,propidium iodide and transmission electron microscope were used to determine the type of cell death,eventually obtaining the reliable evidence for necroptosis in SAH rats.The animals were randomly divided into three groups: Sham,SAH + DMSO and SAH + Nec-1.A single dose of Nec-1 or DMSO was injected intracerebroventricularly 30 minutes after SAH induction.The severity of SAH,neurological performances,brain edema,and blood-brain barrier damage in each group were evaluated to assess the neuroprotective effect of Nec-1.In order to investigate the potential mechanisms of Nec-1,necroptosisrelated proteins,apoptosis-related proteins(caspase 3,caspase 9 and Bcl-2),NF-?B p65 subunit and phosphorylated MAPKs were measured with Western blot,using enzymelinked immunosorbent assay to determine the release of inflammatory cytokines and TUNEL staining to further verify the level of apoptosis.2)Primary rat cortical neurons were cultured to establish the SAH model in vitro.The releases of lactate dehydrogenase(LDH)in culture medium were tested after Hb stimulation,which was used to determine the time point of subsequent experiments.The cultured neurons were divided into 7 groups: DMSO,z VAD(20 ?M),NSA(2 ?M),Hb(10 ?M),Hb + z VAD,Hb + z VAD + NSA and Hb + NSA.To explore the neuroprotective effect of NSA and its underlying mechanisms,propidium iodide positive cells and LDH leakage were counted to assess cell death.In addition,flow cytometry and Western blot were also used to measure reactive oxygen species(ROS)release and expression of necroptosis-related proteins,respectively.Results: 1)In the early stage of SAH(within 72 hours),the expression levels of RIP1,RIP3 and MLKL were significantly increased in cerebral cortex and peaked at 48 hours after SAH.Immunofluorescence double labeling further confirmed that these proteins were upregulated and mainly located in neuronal cytoplasm.2)Intracerebroventricular injection of a single dose of Nec-1 could significantly ameliorate the blood-brain barrier damage,alleviating cerebral edema,decreasing necrotic cell death and promoting neurological recovery after SAH,but not reduce the severity of SAH.3)Nec-1 significantly inhibited the expression of RIP1,RIP3 and MLKL proteins,reducing the nuclear translocation of NF-?B and release of inflammatory mediators,as well as phosphorylation of ERK.However,Nec-1 had no significant effect on apoptoticactivity.4)NSA significantly inhibited MLKL expression,decreasing Hb-induced neuronal death,and increased cell viability.But it had little effect on upstream molecules RIP1 and RIP3 expression.Additionally,NSA could not reduce mitochondrial ROS accumulation.Conclusions: 1)Our results suggest that there may exist activation of RIP1/RIP3/MLKL dependent necroptosis in the cerebral cortex in early stages of SAH(within 72 hours),and the necrotic cells were primarily cortical neurons,suggesting that necroptosis may be involved in early brain injury after SAH.2)Treatment of Nec-1 significantly alleviate brain edema,improving the permeability of blood-brain barrier,decreasing necrotic cell death,and promote neurological recovery after SAH in rats,suggesting that Nec-1 reduces early brain injury following SAH.3)The neuroprotective effect of Nec-1 in early brain injury after SAH is obtained from blocking RIP1/RIP3/MLKL dependent necroptosis by inhibiting RIP1 phosphorylation,reducing neuroinflammation and inhibiting ERK activity,but independent of apoptotic activity.4)NSA is also able to significantly reduce Hb-induced neuronal necrosis.Its neuroprotective effect may be related to block the execution of necroptosis secondary to inhibit MLKL.