Resolvin D2 Protects Against Brain Injury After Cerebral Ischemia/reperfusion And Its Mechanism Research

Part?: Resolvin D2 protects against brain injury after cerebral ischemia/reperfusionObjective To investigate the expression of endogenous Resolvin D2 in brain following Cerebral ischemia/reperfusion in rats and to study different doses of exogenous Resolvin D2 rotects against brain injury after cerebral ischemia/reperfusion.Methods 1.Animal groups : 36 health male Sprague-dawley(SD)rats were assigned randomly into 6 groups of 6 rats each,a sham group and five MCAO/R groups arranged by time: 12,24,48,72 and 96 hours after MCAO/R.Observe the change of endogenous RvD2 expression in different time points.72 adult male SD rats were divided into six groups(72h): sham group,MCAO/R group,MCAO/R+ vehicle group,MCAO/R + RvD2(25ng/kg)group,MCAO/R + RvD2(50ng/kg)group and MCAO/R + RvD2(100ng/kg)group(n=6 per group).2.Animal model making : Under an operating microscope,focal cerebral ischemia was achieved by right-sided endovascular MCAO.In brief,the right common,external,and internal carotid arteries(CCA,ECA,and ICA)were revealed via a midline cervical incision.Then,a piece of 4-0-monofilament nylon suture with blunted tip coated with poly-lysine was inserted through the right CCA and advanced it along the ICA until the tip occluded the proximal stem of the middle cerebral artery(MCA).Rectal temperature was maintained between 36.5 and 37.5 ? with a heating pad.After 2 h of ischemia,the filament was withdrawn to allow reperfusion.3.Detection methods and indicators: The cerebral cortical penumbra and the analogous contralateral region were harvested for western blot and immunofluorescence analysis.The brain cortex of six rats in each group was extracted and used for double immunofluorescence analysis.At 12,24,48,72 and 96 h after reperfusion,behavioral activity was examined in all groups,and blood samples were used to detect the concentration of RvD2?IL-6 and TNF-?.The ischemic penumbra was determined by TTC;The brain cortexes of six rats were extracted for terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL)staining,fluoro-jade B staining analysis.The index of brain edema was evaluated using the wet/dry method.Results 1.The results revealed that the endogenous RvD2 levels were decreased in MCAO/R group compared with sham group(P < 0.05).After I/R,48 h,endogenous RvD2 expression peak(P < 0.01);72h,endogenous RvD2 expression significantly decreased(P < 0.05).2.The levels of IL-6 and TNF-? in infarct-side brain tissue were significantly increased after MCAO/R(72 h),which were significantly decreased with the RvD2 50 ng/kg treatment(P < 0.05).3.The infarct area was observed by TTC staining and showed that the infarct area significantly decreased with the RvD2 50 ng/kg treatment.4.Compared with the sham group,neurological score in the MCAO/R group was significant higher,suggesting a remarkable neurological defect induction by I/R model(P < 0.01).However,neurological score in the MCAO/R + RvD2(50 and 100 ng/kg)groups were obviously lower than that in the MCAO/R groupl(P < 0.001).5.Brain water content was found to be significantly lower in infarct-side brain tissue samples of RvD2 treatment group than that in rats subjected to the vehicle group(P < 0.05).6.Compared with the sham group,TUNEL-positive neurons were significantly increased in MCAO/R group(P < 0.01),at 72 hours after MCAO/R +RvD2(50 ng/kg),TUNEL-positive neurons were most obviously decreased compared with the MCAO/R+ RvD2(25 ng/kg)or RvD2(100 ng/kg)group(P < 0.05).Consistently,the apoptotic index of BMVECs showed the same trend.Meanwhile,FJB-positive cells in MCAO/R group were obviously increased compared with the sham group,while the number of FJB-positive brain cells were dramatically decreased in RvD2(50 and 100 ng/kg)group at 72 h after I/R(P < 0.05).Conclusions 1.MCAO/R stimulus led to a significant decrease in endogenous production of RvD2.2.Exogenous RvD2 exerted rescue effects on MCAO/R-induced neuron and BMVEC death,exogenous supply of RvD2 reversed MCAO/R-induced brain injury.Part II: Experimental study of the effect of Resolvin D2 in the regulation of GPR18-ERK1/2-NOS on brain injury after cerebral ischemia/reperfusion and its possible mechanismObjective To observe the effect of Resolvin D2 in the regulation of GPR18-ERK1/2-NOS on brain injury after cerebral ischemia/reperfusion and its possible mechanism.Methods 1.Animal groups : 30 adult male SD rats were divided into five groups: sham group,MCAO/R group,MCAO/R + vehicle group,MCAO/R +RvD2(50 ng/kg)group and MCAO/R+ RvD2(50 ng/kg)+O-1918 group.2.Animal model making:Under an operating microscope,focal cerebral ischemia was achieved by right-sided endovascular MCAO.In brief,the right common,external,and internal carotid arteries(CCA,ECA,and ICA)were revealed via a midline cervical incision.Then,a piece of 4-0-monofilament nylon suture with blunted tip coated with poly-lysine was inserted through the right CCA and advanced it along the ICA until the tip occluded the proximal stem of the middle cerebral artery(MCA).Rectal temperature was maintained between 36.5 and 37.5 ? with a heating pad.After 2 h of ischemia,the filament was withdrawn to allow reperfusion.At 72 h after reperfusion,the brain tissue samples were obtained for assays.3.Detection methods and indicators:72 h after reperfusion,the rats were euthanized,and the brains were removed quickly and frozen 10 min.The brains were subsequently sectioned coronally into 2-mm thick slices starting from the frontal pole.Slices were then immersed in 10 ml of 2.0% TTC.The ischemic penumbra was determined by TTC.The cerebral cortical penumbra and the analogous contralateral region were harvested for western blot and immunofluorescence analysis.The brain cortex of six rats in each group was extracted and used for double immunofluorescence analysis.Western blot was performed for GPR18,p-ERK1/2,ZO-1,n NOS,e NOS.Immunofluorescence staining was performed for GPR18,double immunofluorescence analysis was performed for GPR18,n NOS,e NOS,Neu N,and v WF.Results 1.Western blot analysis showed the protein level of GPR18 was down-regulated in MCAO/R group compared to sham group(P < 0.01),whereas,compared with the MCAO/R group,50 ng/kg RvD2 treatment could reverse the decrease of GPR18(P < 0.05)and promoted ERK1/2 phosphorylation(P < 0.01);the protein levels of neuronal NOS(n NOS)and endothelial NOS(e NOS)were significantly abated in the ischemic hemisphere after MCAO/R(72h)compared to sham group(P < 0.05).Whereas,the production of the n NOS,e NOS and ZO-1 were significantly enhanced after treated by RvD2 at 72 h after reperfusion(P < 0.01).2.Double-immunofluorescence analysis showed that GPR18 was mainly expressed in neurons and BMVECs,but astrocytes.3.TTC staining showed that infarct volume was significantly increased in the RvD2+ O-1918 group compared to the RvD2 group under I/R conditions,which indicated that the neuroprotective effects of RvD2 is dependent on GPR18.(P < 0.01).4.Western blot analysis showed that RvD2 promoted ERK1/2 phosphorylation,which was inhibited O-1918 treatment(P < 0.05),the production of the n NOS,e NOS and ZO-1 were significantly enhanced after treated by RvD2 at 72 h after reperfusion,whereas,the O-1918 treatment could partly block the function of RvD2(P < 0.05).5.Immunofluorescence staining results also showed that level of neuronal NOS(n NOS)and endothelial NOS(e NOS)were significantly abated in MCAO/R +RvD2(50 ng/kg)group compared to RvD2(50 ng/kg)+O-1918 group(P < 0.05).conclution 1.RvD2 reversed MCAO/R-induced the decrease in the protein level of GPR18 and it may be a upstream signal transduction factors of GPR18.2.GPR18 was mainly expressed in neurons and BMVECs,but astrocytes.3.The rescue effects of RvD2 were reversed by GPR 18 antagonist O-1918 after MCAO/R.4.The decreasing of ERK1/2 phosphorylation and ZO-1 expression were significantly elevated by RvD2 treated after MCAO/R,resulting that the n NOS and e NOS levels were improved at the same.Resolvin D2 protects against brain injury after cerebral ischemia/reperfusion via GPR18-ERK1/2-NOS pathway,at least partly.Part?: Resolvin D2 compared the protective effects of omega-3 fatty acids on brain injury under ischemia reperfusion and its mechanismObjective To investigate the protective effects of Resolvin D2 and omega-3 fatty acids on brain injury under ischemic reperfusion.Methods 1.Animal groups : 30 adult male SD rats were divided into five groups: sham group,sham +?-3 group,MCAO/R group,MCAO/R +?-3 group and MCAO/R + Rv D2(50 ng/kg)group.2.Animal model making : Under an operating microscope,focal cerebral ischemia was achieved by right-sided endovascular MCAO.In brief,the right common,external,and internal carotid arteries(CCA,ECA,and ICA)were revealed via a midline cervical incision.Then,a piece of 4-0-monofilament nylon suture with blunted tip coated with poly-lysine was inserted through the right CCA and advanced it along the ICA until the tip occluded the proximal stem of the middle cerebral artery(MCA).Rectal temperature was maintained between 36.5 and 37.5 ? with a heating pad.After 2 h of ischemia,the filament was withdrawn to allow reperfusion.At 12,24,48,72 and 96 h after reperfusion,the brain tissue samples were obtained for assays.3.Detection methods and indicators : At 12,24,48,72 and 96 h after reperfusion,blood samples were used to detect the concentration of Rv D2;the rats were euthanized,and the brains were removed quickly and frozen 10 min.The brains were subsequently sectioned coronally into 2-mm thick slices starting from the frontal pole.Slices were then immersed in 10 ml of 2.0% TTC.The ischemic penumbra was determined by TTC.The cerebral cortical penumbra and the analogous contralateral region were harvested for western blot.Results 1.TTC staining was performed to observe the infract area of the ischemia hemisphere,and found that the volume of ischemia hemisphere was significantly decreased after the Rv D2(P < 0.01)or ?-3 treatment(P < 0.05)compared to vehicle group,while the Rv D2 group seem to be more effective compared to ?-3 treatmen group(P < 0.05).2.ELISA showed that the endogenous Rv D2 levels were decreased in MCAO/R+Rv D2 group(P < 0.01)or MCAO/R +?-3 group(P < 0.05)compared with MCAO/R group,while the ?-3 group seem significantly decreased compared to Rv D2 treatmen group(P < 0.05).3.Because of LOX activity was involved in converting ?-3 into Rv D2,we determined the phosphorylation of 5-LOX in the brain by western blot.Phosphorylated 5-LOX in brain tissue was much lower than that in the sham group after MCAO/R,which is in accordance with the change of Rv D2 production after MCAO/R as shown in experiment 1(P < 0.05).Conclusions 1.Our findings suggested that Resolvin D2 seem to be more effective compared to omega-3 fatty acids on brain injury under ischemic reperfusion.2.MCAO/R-induced decrease in 5-LOX phosphorylation and subsequent Rv D2 generation resulting that Resolvin D2 seem to be more effective compared to omega-3 fatty acids on brain injury under ischemic reperfusion.