The Role And Mechanism Of Necroptosis In Secondary Injury After Experimental Spinal Cord Injury In Adult Mice

Background:Individuals paralyzed resulted from the spinal cord injury(SCI)are one of the most physically disabling and psychologically devastating conditions known to humans.1 it is evaluated that the annual incidence of SCI,not including those who die at the scene of the accident,is approximately 40 eases per million population in the U.S.or approximately 12000 new cases each year.Unfortunately,although great efforts have made to improve the outcome of patients with SCI,we at present have little effective therapy in clinic except high-dose methylprednisolone,which does not significantly improve functional recovery and has serious adverse effect.Nowadays,traumatic SCI and its devastating consequences represent one of the greatest challenges for us.So it is crucial and significant that finding a new and promising therapeutic approach.The pathophysiologic processes of traumatic SCI are complicated.Generally,it is considered to include two stages:the primary injury,including hemorrhage and cell necrosis(neurons,oligodendrocytes and endothelial cells)in the epicenter of the lesion,is an irreversible,mechanical insult;the subsequent secondary insult,including excitotoxicity,oxidative stress,ischemia,inflammation,ionic homeostasis and necrotic and apoptotic cell death,all of which lead to neuronal and supporting cell death.So it is concluded that cell death is a fundamental and core issue following spinal cord damage.It is possible that we can reduce the extent of tissue damage and demyelination through attenuation of neurons and oligodendrocytes death.Generally,cell death includes apoptosis,nercrosis and autophagy.Apoptosis is often characterized by cellular shrinkage,plasma membrane blebbing,chromatin condensation,appearance of apoptotic body and nuclear degradation.Necrosis is morphologically identified by increasing in cell volume,swelling of organelles and early ruptures of plasma membrane.Autophagy is characterized by lack of chromatin condensation,redistribution of LC3 into autophagosomes membrane and accumulation of double-membrane covered vacuoles containing cytoplasmic organelles or cytosol.Necrosis is often viewed as an accidental and unregulated cellular event.However,accumulating evidence suggests that necrosis,like apoptosis,can partly be executed by regulated mechanisms.In 2005,necrostatin-1(Nec-1)was initially synthesize by Degterev as a specific tool drug to distinguish necroptosis from other cell death processes.From then the term "necroptosis" was coined to describe a ligation of death receptor-induced,caspase-independent,highly regulated type of programmed cell death process with morphological resemblance of necrosis.RIPs are a group of specific serine/threonine kinases that exhibiting a homologous N-terminal kinase domain.and a different C-terminal kinase domain.Ripl knocked mice exhibit exaggerated apoptosis in lymphoid and adipose tissues and die a few days after birth;RIP 1-deficient T cells were reported to be protected from necroptosis triggered by CD95L,TRAIL or TNF and Z-VAD.fmk,and overexpressed RIP1 can induced necroptosis in Jurkat T cells.All above stdudies demonstrated that RIP1 play a important role in both NF-kB signaling pathway and pronecrotic signaling pathway.RIP3 has an N-terminal kinase domain and interacts with RIP1 via a C-terminal RIP homotypic interaction motif(RHIM).Contrarily to RIP1,RIP3 does not participate in NF-kB signaling pathway,but RIP3 is essential for TNFR1-induced necroptosis.Degterev et al identificated that RIP1 kinase is a specific cellular target of necrostatins in 2008.In 2009,three independent research teams identified that RIP3 kinase is required for necroptosis pathyway.The association of RIP1 and RIP3 within necrosome play an important role in the execution of necroptosis.Necrosome increases reactive oxygen species production and activates necrotic cell death.In recent years,necropotosis was reported to involved in a number of disease states(ischemia,traumatic injury,inflammatory disease,virus infection,tumor and myocardial infarction,et al).Bonapace et al found that necroptosis contributed to overcome glucocorticoid resistance for childhood acute lymphoblastic leukemia cells.Several studies have shown that necroptosis can alleviate resistance to cancer drugs mediated by P-glycoprotein and Bcl-2 in vitro.Virus-infected cells,which are resistant to apoptosis,are found to be highly sensitive to necroptosis.necroptosis has been reported to contribute to the attenuation of pathological injury and to improvefunctional outcome in animal models of cerebral ischemia,traumatic brain injury,and myocardial infarction,etc.until now,Most of prior studies about SCI focused on apoptosis and autophagy because necrosis traditionally was described as unadjustable.none of researches about necroptosis following SCI has been reported.But necrosis is maybe more important for SCI,because most of neuronal and supporting cell death following SCI are due to necrosis in an early stage.Although most of necrosis resulted from the direct mechanical damage cannot be influenced,it is promising for SCI patients even if a part of "necrosis" can be regulated.So In this study,we tested the hypothesis that necroptosis contributes to neural cells death after SCI in mice,and investigated the molecular mechanism of necroptosis in SCI,and that treatment with necrostatin-1 would reduce histopathologic and functional deficits.Section one Necroptosis,a novel type of programmed cell death,contributes to early nerve cells damage after Spinal cord injury in adult miceObjective:The aim of this study are:1.To test the hypothesis that necroptosis contributes to neural cells death after SCI in mice;2.To test the hypothesis that treatment with necrostatin-1 would reduce histopathologic and functional deficits;.Methods:1.Female ICR mice were randomized equally into three groups:(a)sham group that underwent sham surgery;(b)vehicle-treated group that underwent surgery for SCI induction and was given Dimethyl sulfoxide(DMSO)by intrathecal injection;(c)necrostatin-1-treated group that underwent surgery for SCI induction and was given necrostatin-1 by intrathecal injection.2.Establishing the model of SCI in mouse:After intraperitoneal anesthesia with pentobarbital sodium(50mg/kg),0.1ml of 2%lidocaine was injected around the incision site to provide local anesthesia.A 2.5 cm skin incision along the midline of the back was made in each mouse.The subsequent operations were performed with an operating microscope.The paravertebral muscles of the thoracic level(T8-T10)vertebrae were dissected out.Laminectomy was performed with Mouse Laminectomy Forceps at the T9 level.Extradural compression with a vascular clip(10g force)was performed for 1 minute around the exposed spinal cord.Intrathecal Injections were performed with 4μl of 4mM necrostatin-1 or vehicle(Dimethyl sulfoxide,DMSO)at 5 minutes after clipped injury by inserting a 33-gauge needle connected to a 5μl Hamilton syringe with each injection lasting for 5 min.After injection,Muscles and skin were sutured in layers.3.After mice were sacrificed at 24hours post SCI,the detection of propidium iodide(PI)-Positive Cells was performed using in PI vivo labeling.4.After mice were sacrificed at 24hours post SCI,morphological analyses were performed by hematoxylin and eosin(HE)staining and nissl staining.5.After mice were sacrificed at 24hours post SCI,the samples were evaluated for apoptosis by the in situ TUNEL assay.6.After mice were sacrificed at 24hours post SCI,the expression of caspase-3 was assessed by western blot.7.Locomotor behavior of hindlimb was evaluated by BMS(Basso mouse scale)score at 1,3,5,7,14 and 28 days post injury.Results:Compared to DMSO-treated mice,necrostatin-1 treated mice decreased the number of cells with plasmalemma permeability(PI positive)in injured spinal cord(P<0.05),alleviated tissue damage and reserved more surviving neuron at 24hour after SCI(P<0.05),improved functional recovery from Day 7 to Day 28(P<0.05).Necrostatin-1 did not reduce the expression of caspase-3 and the number of TUNEL positive cells at 24hour after SCI(P>0.05).Conclusions:Necroptosis contributes to necroptotic cell death and influenced functional outcome after SCI in adult mice.The inhibition of necroptosis by necrostatin-1 may have therapeutic potential for patients with SCI.Section two The role of RIP1/RIP3 pathway on necroptosis after spinal cord injury in adult miceObjective:The aim of this study are:1.To test the hypothesis that RIP1/RIP3 pathway contributes to necroptosis of neural cells after SCI in mice;2.To test the hypothesis that necrostatin-1 reduced RIP 1-RIP3 interaction,alleviated reactive oxygen species production,protected the ultrastructure of neuronal mitochondria.Methods:1.Female ICR mice were randomized equally into four groups:(a)sham group that underwent sham surgery;(b)SCI group that only underwent surgery for SCI induction;(c)vehicle-treated group that underwent surgery for SCI induction and was given Dimethyl sulfoxide(DMSO)by intrathecal injection;(d)necrostatin-1-treated group that underwent surgery for SCI induction and was given necrostatin-1 by intrathecal injection.2.Establishing the model of SCI in mouse:After intraperitoneal anesthesia with pentobarbital sodium(50mg/kg),0.1ml of 2%lidocaine was injected around the incision site to provide local anesthesia.A 2.5 cm skin incision along the midline of the back was made in each mouse.The subsequent operations were performed with an operating microscope.The paravertebral muscles of the thoracic level(T8-T10)vertebrae were dissected out.Laminectomy was performed with Mouse Laminectomy Forceps at the T9 level.Extradural compression with a vascular clip(10g force)was performed for 1 minute around the exposed spinal cord.Intrathecal Injections were performed with 4μl of 4mM necrostatin-1 or vehicle(Dimethyl sulfoxide,DMSO)at 5 minutes after clipped injury by inserting a 33-gauge needle connected to a 5μl Hamilton syringe with each injection lasting for 5 min.After injection,Muscles and skin were sutured in layers.3.The expression of RIP 1 and RIP3 was assessed after experimental SCI at 6h,12h,24h and 48h by western blot.4.The expression of RIP1 and RIP3 was assessed in vehicle-treated group and Necl-treated group at 24h by western blot.5.The interaction of RIP 1 and RIP3 was assessed at 24h by immunoprecipitation6.The level of NO,iNOS,GSH/GSSG,MDA and ROS was assessed according to the kit instructions..7.The ultrastructure of neuronal mitochondria was observed by electron microscope.Results:RIP1 proteins were abundantly expressed at different time intervals after SCI,and RIP3 proteins were expressed and increased after SCI(P<0.05);Neither the expression of RIP1 nor RIP3 was changed by Nec-1 at 24h;The interaction of RIP1 and RIP3 was enhanced at 24h after SCI,and Nec-1 can significantly inhibit the interaction;Nec-1 treatment following SCI prevents early nitric oxide(NO)accumulation;Nec-1 treatment after SCI prevents early inducible nitric oxide synthase(iNOS)up-regulation;Nec-1 treatment after SCI prevents early glutathione oxidation in spinal cord.Nec-1 treatment after SCI prevents early ATP depletion;Nec-1 treatment after SCI prevents early MDA production;Nec-1 treatment after SCI decreases early production of ROS;Nec-1 protected the ultrastructure of neuronal mitochondria at 24h after SCI.Conclusions:RIP1/RIP3 pathway contributes to necroptosis of neural cells after SCI in mice;Necrostatin-1 reduced RIP1-RIP3 interaction,alleviated reactive oxygen species production,protected the ultrastructure of neuronal mitochondria.