The Interaction Of Nitric Oxide/Peroxynitrite And Zinc In Cerebral Ischemic Injury

Objective: The objectives of this study are to investigate the changes and colocalization of nitric oxide(NO)/ peroxynitrite(ONOO~-)and zinc in the penumbra of middle cerebral artery occlusion(MCAO)and reperfusion rats,and to study the neurologic deficit,brain infarction percentage,as well as the levels of NO/ONOO~-and zinc in the penumbra of MCAO and reperfusion rats upon NO scavenger,nitric oxide synthetase(NOS)inhibitor or zinc chelator treatment,in order to investigate the interaction of NO/ONOO~-and zinc following focal cerebral ischemia and reperfusion,and to explore the probable mechanism by which NO/ONOO~-and zinc are involved in cerebral ischemia reperfusion injury.Method: 60 male Sprague Dawley rats were randomly divided into five groups: Sham group,MCAO group,MC AO+TPEN group,MCAO+L-NAME group and MCAO+c-PTIO group.Each group was further divided into 2 subgroups according to the different reperfusion time(6 h and 24 h).There were 6 rats in each group at each reperfusion time point.TPEN or L-NAME was injected intraperitoneally at 30 min before MC AO with dose of 15 mg/kg or 1 mg/kg.C-PTIO was injected intraperitoneally at 1 h before MCAO with dose of 0.6 mg/kg.MCAO operation was performed by suture method in MCAO group,MCAO+TPEN group,MCAO+L-NAME group and MCAO+c-PTIO group.The rats underwent right MCAO for 90 min.The rectal temperature,mean arterial blood pressure(MABP)and heart rate were monitored to keep in normal range during the operation.At 6 h or 24 h after reperfusion,Longa score and Ludmila Belayev 12 score were used to evaluate neurological deficits of rats.Then the brains were removed and dyed with 2,3,5-triphenyltetrazolium chloride(TTC)to measure the infarct volume.Zinc fluorescent probe,Newport Green(NG)were used to detect the zinc level in the penumbra of the brain.Immunofluorescence staining,Western blotting analysis,NO assay kit and nitrate plus nitrite(NOx-)assay kit were used to detect NO/ONOO~-in the penumbra of the brain.Colocalization of NG and NO/ONOO~-were performed to study the relationship between zinc and NO/ONOO~-.Results:(1)There was no statistical difference in rectal temperature,MABP and heart rates among Sham,MCAO,MCAO+TPEN group,MCAO+L-NAME group and MCAO+c-PTIO group during the operation of MCAO.(2)From 6 h to 24 h after reperfusion,the number of nNOS-positive,iNOS-positive and 3-NT-positive(ONOO~-)cells in the penumbra of MCAO group rats increased significantly(P<0.05).(3)In Sham group rats,the neurological function scores(Longa and Ludmila Belayev 12 scores)were 0 and no infarction was observed.At the same reperfusion time point(6 h and 24 h),the neurological function scores and infarction percentage in MCAO+L-NAME group and MCAO+c-PTIO group rats decreased significantly compared with those of MCAO group(P<0.05).(4)The number of nNOS-positive or iNOS-positive cells showed positive correlation with the number of NG-positive cells(r1=0.937,r1=0.932,P<0.05).(5)No NG-positive cell was found in Sham group rats.From 6 h to 24 h after reperfusion,the number of NG-positive cells in the penumbra of MCAO group,MCAO+L-NAME and MC AO+c-PTIO group rats showed time-dependent increasing tendency.At the same reperfusion time point(6 h and 24 h),the NG-positive cells in the penumbra of MC AO+L-NAME group and MCAO+c-PTIO rats decreased significantly compared with those of MCAO group(P<0.05).(6)At the same reperfusion time point(6 h and 24 h),the level of NO in ischemic cerebral hemisphere of MCAO+TPEN group rats decreased significantly compared with those of MC AO group(P<0.05).(7)From 6 h to 24 h after reperfusion,the level of NO and the activity of superoxide dismustase(SOD)in ischemic cerebral hemisphere of MC AO group showed time-dependent decreasing tendency,but the number of ONOO~-(3-NT-positive cells)in the penumbra of MC AO group rats increased significantly(P<0.05).And co-staining of ONOOand TUNEL fluorescence demonstrated that ONOO~-and TUNEL were colocalized in the same cells and area in the brain penumbra of MC AO rats at 6 h,24 h after reperfusion.(8)The number of 3-NT-positive cells showed positive correlation with the number of NG-positive cells(r=0.941,P<0.05).(9)Co-staining of ONOO~-and zinc fluorescence demonstrated that ONOO~-and zinc were colocalized in the same cells and area in the brain penumbra of MC AO rats at 24 h after reperfusion.(10)Sham group showed little 3-NT-positive cells.At the same reperfusion time point(6 h and 24 h),the number of 3-NT-positive cells in the penumbra of MC AO+L-NAME group,MCAO+c-PTIO group and MCAO+TPEN group rats decreased significantly compared with those of MC AO gro up(P<0.05).Conclusion:(1)NO and ONOO~-are involved in the cerebral ischemia reperfusion injury.Inhibition of NO decreases ONOO~-production,improves neurological outcomes and reduces brain ischemic damage.(2)From 6 h to 24 h after reperfusion,both nNOS/iNOS and zinc in the penumbra of MCAO rats show time-dependent increasing tendency.There is a positive feedback loop between NO production and zinc accumulation.(3)From 6 h to 24 h after reperfusion,both ONOO~-and zinc in the penumbra of MCAO rats show time-dependent increasing tendency.Zinc and ONOO~-are colocalized in the same cells.(4)The positive feedback loop between NO and zinc may be mediated by ONOO~-in the penumbra of MCAO rats.