The Injury Effect And Mechanism Of Necroptosis After Subarachnoid Hemorrhage

IntroductionSubarachnoid hemorrhage(SAH)is a particularly devastating type of hemorrhagic stroke that is mostly caused by basicranial arterial aneurysm rupture leading to artery blood to enter the subarachnoid space instantaneously.With the improvement of health awareness and advance in neuroimaging,the detectable rate of unruptured intracranial aneurysm significantly elevated;benefit from the considerable progress of interventional endovascular techniques,unruptured intracranial aneurysm can be properly processed without any adverse impact on the life quality and expectancy.O n the other hand,according to a incomplete statistics,SAH induced by ruptured aneurysm results in pre-hospital death of almost one-third of the victims,while one-third dead within a week or left with severe neurological dysfunction and the remaining survived without any obvious deficits.It is obviously that SAH has already became the bottleneck of the treatment of intracranial aneurysm.Cerebral vasospasm(C VS)and early brain injury(EBI)are two prevailing theories about pathomechanism of SAH in the worldwide.The former emphasizes the overflowed blood plays an important role in causing intracranial vasospasm and ischemia in blood supplying area of the affected artery.C VS has been supported by a clinical observation of narrowing intracranial artery in cerebral angiogram of SAH patients and increasing experimental evidence from animal model of SAH.In the meanwhile,a lot of new targeting points and alternative experimental durgs has been screened,although none of them exhibit any protective effect in the clinical experiments.For instance,C lazosentan,a new selective endothelin receptor antago nist,could completely relieve vasospasm but has no help to improve prognosis of SAH in a multi-center double-blind randomized control clinical trial.On this account,researchers raised the conception of EBI which is as a consequence of various pathological factors such as reactive oxygen(ROS),cortical spreading depolarization(CSD),excitability amino acid toxicity(EAAT),inflammatory cells,inflammatory mediators occurred within 72 h after SAH.Core of this theory is the devastating effects mainly depend on the early injury network formed in the first 72 h of SAH onset,and emphasize the vital significance of blocking cascade reaction in the early stage.Whatever C VS or EBI,each mechanism has its owm focus and complement the other,both failed to get an exciting result in the clinical transformation.For neurosurgeons,the one and only terminal goal of SAH treatment is to reduce death rate and to raise living quality.From this point,neuron will be most potential candidate for this purpose.According to the classic theory of cytobiology,neurons and cardiocytes are the only two kinds of so-called permenant cells,which means both of them have a constant number from birth and cannot repair the pathological damage by the way of regeneration just like fibrocytes,epithelial cells do.It is believed that this is the most important reason for poor outcomes of cardio-cerebral diseases.Unfortunately,the solo clinical strategy of neuron protection which has been unswervingly pursued by neurosurgeons and neurologists was failed to work.Two reasons should be responsible for the result.First of all,isolate the neurons from the complex neural-vascular network but completely ignore the indispensability of steady state of integrated central nerve system(CNS)for neuronal physiological functions and surviving.Second,restricted by the traditionary view on cell death.More and more researchers had recognized the inadequacy of solo strategy of neuron protection and shifted the concern from neuron to the whole CNS.Furthermore,the development of cytobiology could make a deeper understanding of cell death.Lots of neurons suffer apoptosis and necrosis from the p athological injuries after SAH.The rate of apoptotic neurons is about 20% of the total death neurons,and necrotic neurons take the rest.Interestingly,more than 90% published articles about neuron death were focused on apoptosis,and less than 10% artic les described the neuronal necrosis without any internal mechanism investigation.Due to the definition of necrosis,unlike apoptosis,as an uncontrolled response to overwhelming stress by conventional pathological notion.Researchers chose to deliberately ignore necrotic neurons considering its ?uncontrolled? characteristics.Uncontrolled necrosis means no available target to be intervened and unusefulness for research.Decades ago,cell biologists had investigated that once tumor necrosis factor alpha(TNF-?)binding tumor necrosis factor receptor(TNFR)can mediate survival,apoptosis or necrosis dependents on self-conditions of cells.Professor Yuan Junying from Havard medical school identified a small molecular necrostatin-1,a potent and selective blocker of cell death caused by TNF-? stimulation in vitro,from ~15,000 compounds with the help of chemical library screening.Simultaneously,Prof.Yuan termed this cell death pattern ?necroptosis? which can be induced by TNF-? but interrupted by necrostatin-1.The establishment of necroptosis challenges classic pathological theory about cell death,and reveals an open world how does the organism regulate the life and death of cells? Moreover,recent evidence suggests that necrotpsis plays an most important role in necrotizing pancreatitis,necrotizing enterocolitis,glomerular and renal tubular necrosis,myocardial infarction and cerebral infarction which characteristic by necrotic pathological injury.All of these raises the possibility of entirely new theropy and predicts an inestimable benefit for clinic.In conclusion,this study is to evaluate the role of necroptosis in SAH and find out the key signal proteins,to reveal the pathological effects on the neuron death after SAH,which may lay a foundation for further study to explore a more effective method and strategy for clinical treatment.Material and Methods1.SAH model of C57BL/6J mice was induced by cerebral middle artery puncture.2.Compared the different level of neurological score,neuron death,brain water content,expression of inflammation factors,lactate dehydrogenase(LDH)in cerebrospinal fluid,activation of glia cells,survival rate and hippocampal atrophy between RIP3-/-,MLKL-/-,CypD-/-and WT mice.3.Isolated and primary cultured hippocampal neurons(PCHN s)from embryonic day 17.5 mouse pups treated with TNF-?,z-VAD-fmk(z-VAD),smac mimetics(SM),autologous blood,oxyhemoglobin,ferrous chloride(FeC l2)or ferric chloride(FeC l3)to construct a PCHN s model of necroptosis in vitro and to detected the level of RIP3,MLK L,LDH and cell death rate.4.With the aid of organelles fractionation,mitochondrial Inner and outer membrane separation,investigate distribution of activated MLKL.5.C larified the relationship between MLKL,C yp D and mitochondrial p ermeable transition pore(mPTP)and found out the interaction between MLK L,C yp D at the level o f protein modification through co-immunoprecipitation(co-IP).6.Western blot(WB)was used to analysis RIP3,MLK L protein level in surgical specimens of SAH patients who had suffered from an emergency intracranial decompression.Results1.For the first time,we found that devastating necroptosis occurred in granule neurons of dentate gyrus.Neurons in C A1 and CA3 had a moderate susceptibility to necroptos is.Surprisingly,cortical neurons contacting directly with hemotoma on skull base were insensitivity to necroptosis.RIP3 knockout was effective in protecting granule neuron from injury of necroptosis,alleviating edema,improving neurological scores,lowing animal?s death rate and attenuating hippocampal atrophy.2.Necroptosis of PCHNs could be induced by oxyhemoglobin strongly,and a moderate susceptibility to the combined treatment of TNF-?+z-VAD+Smac mimetics(TZS),but non response for autologous blood,FeCl2 or FeCl3.3.A up-regulated protein level of MLK L?GLUD?GLUL?PYGL?ox-CaMK? after SAH.MLK L plays as a mainly mediator of necroptosis by interacting with C yp D to interrupt the function of mitochondria.4.MLK L can enhance the binding of C yp D and ANT to open the mitochondrial permeable transition pore.5.Magnetic resonance imaging(MRI)scanning showed that RIP3,MLK L or C ypD knockout mice had a less hippocampal atrophy in 30 d after SAH compared with WT mice.Correspondingly,RIP3,MLK L or C yp D knockout mice performed better in morris water maze test and had a lower death rate.The protein expression of p-RIP1?RIP1?p-RIP3?RIP3?p-MLKL and MLKL of surgical specimens increased obviously.Conclusion1.This study found firstly that severe and special necroptosis in the granule neruons of dentate gyrus which has a pathological significance for disease progresses and prognosis of SAH.2.The phenomenon that oxyhemoglobin inducing activity of necroptosis in PCHNs implicates the oxyhemoglobin may be an important injurious medium after SAH.3.The activation of necroptosis after SAH dependents on the RIP1-RIP3-MLK L signaling pathway,and translocation of MLK L to mitochondria plays a vital role in the lethal effect on neurons.4.For the first time,experimental evidence have proved the interaction between MLKL and CypD,and clarified mitochondira is the damage target in the process.5.Put forward a new idea that protection of neuron from necroptosis which had been ignored for a long time may be a crucial strategy for the clinical treatment.