RLIPostC Protects Against Cerebral Ischemia-reperfusion Injury Through Improved Synaptogenesis

Background:Cerebral ischemia(cerebral stroke,brain stroke)is an acute cerebrovascular disease characterized by neural tissue injury caused by ischemia and hypoxia in the brain and corresponding neurological dysfunction.Although the research in basic and clinical have been study the pathogenesis and treatment of cerebral ischemic injury for many years,there is still no effective treatment strategy in clinical practice.Remote limb ischemic post-conditioning(RLIPostC)has been shown its neuroprotective effects in treatment for cerebral ischemia,the molecular mechanism,however,remains unclear.Objective:To investigate the neuroprotective effects of RLIPostC on experimental cerebral ischemia-reperfusion injury in rats and the effects of RLIPostC on synaptogenesis in infarct penumbra,and to explore the molecular mechanisms involved.Methods:The cerebral ischemia-reperfusion injury model of Sprague-Dawley(SD)rats was duplicated by middle cerebral artery occlusion/reperfusion(MCAO/R).The rats were randomly divided into sham group,model control group(Model group),RLIPostC group,PI3K/Akt pathway inhibitor LY294002 group(LY group),and RLIPostC+PI3K/Akt pathway inhibitor LY294002 group(RLIPostC+LY group).RLIPostC group received 3 cycles of RLIPostC treatment immediately after reperfusion(bilateral femoral ischemia 10 min and reperfusion 10 min for each cycle).The rats in RLIPostC+LY group were administrated 25 mmol/L LY294002 in 5μl DMSO at 30 min before MCAO,and then they were subjected to MCAO,thereafter,received 3 cycles RLIPostC treatment immediately after reperfusion.The rats in LY group were administrated 25 mmol/L LY294002 at 30 min before MCAO,and the others steps were the same as RLIPostC+LY group.The neurological function was assessed by neurological deficits scores,foot fault test and Morris water maze at day 7 and 14 after MCAO.At day 14 after MCAO,recpectively;the infarct volume and brain edema were determined by cresyl violet staining and brain water content,respectively.The expression of synaptogenesis-related proteins was evaluated by western blotting,and synaptogenesis was detected by immunofluorescence staining.Results:The results showed that compared with model group,RLIPostC treatment promoted the recovery of neurological function in rats with stroke,including alleviating the symptoms of neurological deficit in rats with stroke(P<0.05),enhanced the recovery of motor coordination(P<0.05)and learning and memory abilities(P<0.05);the morphological results showed that RLIPostC treatment reduced cerebral infarction volume(RLIPostC group 35.02%±2.321%vs.Model group 50.67%±4.926%,P<0.05)and cerebral edema(80.40%±3.19%vs.86.91%±2.03%in RLIPostC group,P<0.05).Western boltting analysis showed that RLIPostC treatment increased the expression of the pre-synaptic marker protein SYN1 and the post-synaptic marker protein PSD95,and up-regulated the expression of the growth-related protein GAP43.Immunofluorescence detection of presynaptic and postsynaptic marker proteins indicated that RLIPostC treatment significantly increased the fluorescence intensity of the presynaptic marker protein Synl(fold of the sham group,RLIPostC group 1.22±0.38 vs.Model group 0.58 ± 0.42,P<0.05)and post-synaptic PSD95(fold of the sham group,RLIPostC groupl.41±0.39 vs.Model group 0.57±0.32,P<0.05);When treated with PI3K/Akt pathway inhibitor LY294002,despite RLIPostC can still improved the recovery of neurological function in rats(RLIPostC+LY group vs.Model group,P<0.05),but the protective effect was attenuated(RLIPostC+LY group vs.LY group,P<0.05).RLIPostC also had a protective effect on reducing the volume of cerebral infarction,but the protective effect was also attenuated(RLIPostC+LY group vs.Model group,P<0.05;RLIPostC+LY group vs.LY group,P<0.05),but the effect of RLIPostC on relieving cerebral edema was eliminated(RLIPostC+LY group vs.LY group,P>0.05).Western blotting results showed that RLIPostC increased synaptic-associated protein Synl,PSD95 and GAP43,but the effect is also reduced(RLIPostC+LY group vs.Model group,P<0.05;RLIPostC+LY group vs.LY group,P<0.05).Conclusions:(1)RIPostC in bilateral hind limbs initiated after cerebral ischemia-reperfusion improves the recovery of behavioral function in rats,including promoting the recovery of neurological deficit symptoms,motor coordination,learning and memory;(2)RIPostC in bilateral hind limbs initiated after cerebral ischemia-reperfusion in rats reduces the infarction volume caused by cerebral ischemia-reperfusion injury and reduced cerebral edema;(3)RIPostC in bilateral hind limbs initiated after cerebral ischemia-reperfusion up-regulates the expression of synaptic marker protein Synl and the post-synaptic marker protein PSD95 in the cerebral ischemic penumbra,and up-regulates the expression of synaptic regeneration-related protein GAP43;(4)RIPostC in bilateral hind limbs initiated after cerebral ischemia-reperfusion increases the positive fluorescence intensity of synaptic marker protein Syn1 and the post-synaptic marker protein PSD95 in the cerebral ischemic penumbra(5)The application of PI3K/Akt pathway inhibitor LY294002 attenuates the neuroprotective effects and the increased synaptogenesis of RIPostC after cerebral ischemia-reperfusion...