Necrostatin-1 Protects The Survival Of Oligodendrocyte Precursor Cell Following Cerebral Ischemia Reperfusion Injury

Objective:Recent study had demonstrated that the necroptosis inhibitor,Necrostatin-1,could inhibit the necropotosis of neuron after transient cerebral ischemia.The present study aimed to investigate the effect of Necrostatin-1,on the survival of OPC,following oxygen-glucose deprivation(OGD)in vitro and middle cerebral artery occlusion(MCAO)in vivo,and on white matter injury and long-term functional recovery following MCAO in mice.Methods:1.In vitro experiment:The culture of primary mouse OPC were subjected to OGD for 2 h.The necroptosis inhibitor Necrostatin-1,the broad-spectrum caspase inhibitor Z-VAD-fmk,and the inactive control dimethyl sulfoxide(DMSO)were added 1 h prior to stimulation and maintained during the stimulation and post-stimulation periods.Twenty-four hours after the insult,cell viability was assessed using propidium iodide(PI)and Calcein-AM(C-AM)staining as well as 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide(MTT)and lactate dehydrogenase(LDH)assays.Total protein from OGD-challenged OPC was also extracted 6 h,12 h,and 24 h after OGD.We observed the expression level of receptor-interacting protein kinase 1(RIPK1),receptor-interacting protein kinase 3(RIPK3),mixed lineage kinase domain-like protein(MLKL)and phosphorylated mixed lineage kinase domain-like protein(P-MLKL)through Western blot.2.In vivo experiment:Male adult ICR mices(25-30g)were subjected to 60-min middle cerebral artery occlusion(MCAO)and 24 h of reperfusion.Necrostatin-1 and DMSO(0.04 mg/kg)were injected intracerebroventricularly beginning 1 h before the onset of MCAO.The total protein from the subventricular zone(SVZ)and corpus callosum(CC)regions was extracted using RIPA lysis buffer after 24 h of MCAO.We also detected the expression level of RIPK1,RIPK3,MLKL and P-MLKL through Western blot in SVZ and CC regions following MCAO.We also observed the survival of OPC in SVZ and CC regions following MCAO through the immunofluorescence staining,and the Luxol Fast Blue(LFB)staining was performed to examine the white matter injury in vivo following MCAO.We evaluated the long-term neurological function of mice using the Morris water maze test and foot-fault test after MCAO.Results:1.In vitro experiment(1)We labeled the dead and live cells using the nucleic acid dyes PI and C-AM.Our results indicated that OGD insults significantly increased PI-positive OPC(P<0.001)and that Necrostatin-1 significantly reduced PI-positive OPC when compared with OGD group(P<0.001).We then conducted an MTT assay in order to determine cell viability following OGD insults.OPC viability was significantly reduced following OGD(P<0.001).Furthermore,treatment with Necrostatin-1 increased the viability of OPC(P<0.05).Finally,we conducted an LDH assay in order to obtain an indirect measure of membrane leakage.The results of this assay indicated that OGD significantly increased LDH release from OPC(P<0.001).Further analysis revealed that Necrostatin-1 significantly inhibited this LDH release(P<0.001).However,our results also demonstrated that the pan-caspase inhibitor Z-VAD-fmk did not significantly reduce necrosis of OPC following OGD insults relative to OGD treatment alone(P>0.05).(2)Our results indicated that RIPK1 expression was significantly upregulated 6 h following OGD(P<0.05),while RIPK3 expression was also significantly upregulated 12 h following OGD(P<0.01).We further observed significant upregulation of MLKL protein expression,a downstream target of RIPK3,24 h following OGD(P<0.05).The main component of MLKL protein upregulation was P-MLKL,an executioner of necroptosis(P<0.05).(3)Necrostatin-1 significantly inhibited the expression of RIPK1(P<0.05)and RIPK3(P<0.01)in OPC following OGD.Our results further indicated that Necrostatin-1 significantly inhibited the expression of MLKL(P<0.05)and P-MLKL(P<0.05)in OPC following OGD.2.In vivo experiment(1)Our results indicated that RIPK1 and RIPK3 levels were significantly upregulated 24 h after MCAO,especially in the SVZ and CC(P<0.05).MLKL expression was also significantly increased in the SVZ and CC following MCAO(P<0.05),when compared with sham control(P<0.05).We also observed upregulation of the necroptosis executioner P-MLKL in the SVZ and CC following MCAO relative to sham control(P<0.05).(2)Our results indicated that Necrostatin-1 significantly inhibited the expression of RIPK1 in the ipsilateral SVZ and CC(P<0.05),and inhibited RIPK3 in the SVZ(P<0.05)and CC(P<0.01)24 h after MCAO.MLKL expression was also significantly decreased in the ipsilateral SVZ and CC following MCAO after administration of Necrostatin-1 compared to vehicle administration(P<0.05).We also observed that Necrostatin-1 inhibited the expression of the necroptosis executioner P-MLKL in the ipsilateral SVZ and CC following MCAO,compared to vehicle administration(P<0.05).(3)We labeled OPC using NG2(the marker of OPC).Our results indicated that compared to the vehicle,Necrostatin-1 significantly increased the number of NG2-positive OPC in the ipsilateral CC(P<0.05).The white matter injury was stained with Luxol Fast Blue(LFB).The density of LFB-positive myelin in the ipsilateral CC was increased in the Necrostatin-1 group as compared to the vehicle group(P<0.05).(4)We assessed mouse behavior using the Morris water maze test and foot-fault test.In the Morris water maze test,the latency to escape onto the hidden platform in the Necrostatin-1 group was significantly less than that observed for the vehicle group(P<0.05).When the platform was removed,the number of times that the mouse crossed the target region was significantly increased in the Necrostatin-1 group compared with that of the vehicle group(P<0.05).In probe trials,the time spent in the target quadrant was markedly longer in the Necrostatin-1 group than in the vehicle group(P<0.01).In the foot-fault test,the number of left side-right side foot-faults was significantly reduced in the Necrostatin-1 group relative to that of the vehicle group(P<0.01).Conclusion:Ischemic injury induced the necroptosis of OPC,which was dependent on the activation of RIPK1,RIPK3,MLKL and P-MLKL following cerebral ischemia.Our results demonstrated that the RIPK1 inhibitor Necrostatin-1 could inhibit the expression of RIPK1,RIPK3,MLKL and P-MLKL,thus Necrostatin-1 may preserve the survival of OPC and improve the recovery of white matter injury and long-term neurological function,suggesting a role for its use in therapeutic intervention following white matter injury due to ischemic stroke.