| Objective: In 1990,Kitagawa et al confirmed that brain ischemic preconditioning could alleviate brain ischemia/reperfusion injury by inducing brain ischemic tolerance.Subsequently,some scholars have put forward the remote organ ischemic preconditioning,which provides a new way for ischemic preconditioning against ischemia/reperfusion(I/R)injury.In recent years,our previous studies confirmed that limb ischemic preconditioning(LIP)could reduce I/R injury of hippocampal CA1 region in rats.Although both the brain ischemic preconditioning and LIP can alleviate brain I/R injury,LIP has better safety and operability considering of the clinical application.Furthermore,LIP is easily accepted by patients.However,the mechanisms of neuroprotection induced by LIP are not entirely clear.In the 50 s of the last century,the Belgian scientist de Duve observed the structure of autophagy firstly by transmission electron microscopy,and raised the concept of "autophagy" at the International Conference on lysosomes in1963.The researchers found that there was a phenomenon of activation of autophagy in the process of brain I/R injury.A large number of studies found that moderate activation of autophagy may produce the protective effect on brain I/R injury.In 2010,Sheng et al confirmed that both focal brain ischemia and brain ischemic preconditioning in rats can induce autophagy.Compared with the ischemia injury,autophagy induced by ischemic preconditioning lasted longer and large intensity,and effectively reduced the focal brain ischemia injury.Pretreatment with autophagy inhibitor bafilomycin A1 or3-MA inhibited the neuroprotection of brain ischemic preconditioning by up-regulating the expression of LC3-II,Beclin1 and HSP 70,and down-regulating the expression of p62.On the contrary,the activator of autophagy rapamycin mimiced brain ischemic preconditioning and induce theautophagy by up-regulating the expression of LC3-II and Beclin1,which reduced ischemic injury.In 2011,Yan et al demonstrated that hyperbaric oxygen preconditioning alleviated ischemic injury and play an important role of neuroprotection by the activation of autophagy in the model of focal brain ischemia in rats.Administration of autophagy inhibitor 3-MA decreased the brain protective effects.Later,Jiang et al found that autophagy activated by metformin could alleviate brain ischemia injury,which was eliminated after using AMPK inhibitor compound C or autophagy inhibitor 3-MA.These results confirmed that metformin pretreatment induced the activation of autophagy through AMPK signaling pathway,which plays the protective role in brain ischemia.These studies showed that the moderate activation of autophagy play the protective role in brain ischemic model.Similarly,multiply preconditioning methods could alleviate the ischemia/reperfusion injury and protect brain by activating autophagy.Therefore,as an endogenous protective mechanism,autophagy provides a new way to LIP about the induction of brain ischemic tolerance.Based on these above studies,our present study used 4-vessel occlusion global brain ischemia model,and observed the expression of the autophagosome and autophagy marker protein LC3 in brain ischemia/reperfusion injury and in brain ischemic tolerance induced by LIP,and discussed the effect of autophagy in brain ischemic tolerance induced by LIP.The solution of these problems will provide the experimental basis for the neuroprotection of LIP,and new ideas for clinical prevention and treatment of acute brain ischemic disease.Methods and results:1 The expression of autophagy in the rat brain ischemia/ reperfusion injury.1.1 The expression of autophagysome in the rat brain ischemia/ reperfusion injuryAnimal grouping and methods: 21 male Wistar rats whose bilateral vertebral arteries were occluded permanently after two days recovery were randomly divided into two groups:(1)sham group of brain ischemia(n=3):The rats were only the exposure of bilateral common carotid arteries,without blocking blood flow.(2)brain ischemia(BI)group(n=18): The rats were clamped bilateral common carotid arteries for 8 min,and recovered blood flow,then divided for BI 0 h,12 h,1 d,3 d,5 d and 7 d groups according to different reperfusion time.The rats were sampled respectively at the corresponding time points after the sham operations of brain ischemia,or brain ischemia in each group.These rats were used for transmission electron microscopy examination.The results of transmission electron microscopy showed that the structure of neurons of hippocampal CA1 in sham group of brain ischemia were normal,including intact nuclear membrane,clear nuclear shape,chromatin distribution in the cytoplasm,abundant organelles,normal morphology,clear structure,no autophagosome.The structure of neurons of hippocampal CA1 in BI groups were abnormal with reperfusion time elapsing,the cytoplasm was swollen and mitochondrial were edematous,and their cristae and membrane were syncretic,endoplasmic reticulum and Golgi were expansive,meanwhile,other included ribosome disattachment from rough endoplasmic reticulum,chromatin condensation,autophagosomes appear occasionally.Until 3 d after reperfusion,some autophagosomes can be seen in neurons,decreased significantly organelles,irregular karyotype,mitochondrial membrane separated layer,mitochondrial appeared densification and malformation.Meanwhile,rough endoplasmic reticulum appeared expansion and degranulation.At 5 d and 7 d after reperfusion,the neurons were found nuclear condensation and fragmentation,and cytoplasm with no autophagosomes appearance.These above results indicated that the rat hippocampal CA1 neurons were damaged and activated autophagy after brain ischemia/reperfusion,the neurons can be seen the autophagosome from 12 h to 3 d after reperfusion.As time goes on,autophagy expression gradually decreased,even disappeared,the neurons appeared ultimately delayed neuronal death.1.2 The expression of autophagy surface marker protein LC3 in the rat brain ischemia/reperfusion injuryAnimal grouping and methods: 35 male Wistar rats whose bilateral vertebral arteries were occluded permanently after two days recovery were randomly divided into two groups:(1)sham group of brain ischemia(n=5):The rats were only the exposure of bilateral common carotid arteries without blocking blood flow.(2)BI group(n=30): The rats were clamped bilateral common carotid arteries for 8 min,and recovered blood flow,then divided for BI 0 h,1 h,6 h,12 h,24 h and 48 h group according to different reperfusion time.The rats in each group were scacrificed by decapitation at the corresponding time points after the sham operations of brain ischemia,or brain ischemia in each group.The expression of LC3 was detected by Western blot.Western blot results showed that the weak LC3-II band was detected in the hippocampal CA1 area of the sham group of brain ischemia,which confirmed that low level of autophagy was activated in hippocampal CA1 area normally.Compared to sham group of brain ischemia,the expression of LC3-I in hippocampus CA1 area of BI groups not changed obviously,but the expression of LC3-II increased gradually,reached the peak at 12 h(P < 0.05)after reperfusion,then decreased gradually.The results show that with the reperfusion time elapsing,the expression of LC3-II in hippocampal CA1 area of BI groups showed a dynamic change,and reached peak at 12 h,then decreased gradually,suggesting that autophagy was induced by the brain ischemia/reperfusion injury.2 The role of autophagy in LIP induced brain ischemic tolerance2.1 The expression of autophagosomes in LIP induced brain ischemic toleranceAnimal grouping and methods: 12 male Wistar rats whose bilateral vertebral arteries were occluded permanently after two days recovery were randomly divided into two groups:(1)sham group(n=3): The rats were only the exposure of bilateral common carotid arteries without blocking blood flow.(2)BI group(n=3): The rats were clamped bilateral common carotid arteries for 8 min,and recovered blood flow.(3)LIP sham + BI group(n=3): The ratswere clamped bilateral common carotid arteries for 8 min after exposure to bilateral femoral artery 50 min,and recovered blood flow.(4)the LIP+ BI group(n=3): The bilateral femoral arteries were clamped for 10 min,followed by reperfusion for 10 min,and repeated for a total of three times.Then,the bilateral common carotid arteries were clamped for 8 min,and recovered blood flow.All the animals were sampled at 3 d after the sham operation or the last time of ischemia.These rats were used for transmission electron microscopy examination.The results of transmission electron microscopy showed the structure of neurons of the hippocampal CA1 were normal in sham group,including intact nuclear membrane,clear nuclear shape,chromatin distribution in the cytoplasm,abundant organelles,normal morphology,clear structure,no autophagosome.The neurons of hippocampal CA1 area of rats in BI group found some autophagosomes,decreased significantly organelles,irregular karyotype,mitochondrial appeared vacuolation and malformation.Compared with the BI group,in LIP + BI group,the ischemia/reperfusion injury was reduced significantly,mitochondria were normal,organelles were abundant,nuclear membrane integrity,karyotype rules,autophagosomes increased,whereas the ischemia/reperfusion injury in LIP sham + BI group was no significant difference.These results suggested that LIP plays a protective role by up-regulating the expression of autophagosomes in the brain ischemia.2.2 The expression of autophagy surface marker protein LC3 in LIP induced brain ischemic toleranceAnimal grouping and methods: 20 male Wistar rats whose bilateral vertebral arteries were occluded permanently after two days recovery were randomly divided into two groups:(1)sham group(n=5): The rats were only the exposure of bilateral common carotid arteries without blocking blood flow.(2)BI group(n=5): The rats were clamped bilateral common carotid arteries for 8 min,and recovered blood flow.(3)the LIP sham + BI group(n=5): The rats were clamped bilateral common carotid arteries for 8 min after exposure to bilateral femoral artery 50 min,and recovered blood flow.(4)the LIP + BIgroup(n=5): The bilateral femoral arteries were clamped for 10 min,followed by reperfusion for 10 min,and repeated three times.Then,the bilateral common carotid arteries were clamped for 8 min,and recovered blood flow.All the animals were scacrificed by decapitation at 12 h after the sham operation or the last time of ischemia,and the expression of LC3 was detected by Western blot.Western blot results showed that the expression of LC3-Ⅰ has no significant changes in each group in CA1 region of hippocampus.The weak LC3-II band was detected in the CA1 region of hippocampus in the sham group.Compared with the sham group,the expression of LC3-II increased evidently in CA1 region in BI group,and the ratio of LC3-II/LC3-I was increased significantly(P<0.05).Compared with BI group,the expression of LC3-II increased significantly in the LIP + BI group(P<0.05),and the expression of LC3-II has no significant changes in the LIP sham + BI group.These results suggested that LIP plays an important neuroprotection by up-regulating the expression of the surface marker protein LC3-II in the brain ischemia.Conclusion:1 After brain ischemia in rats,with the prolongation of reperfusion time,the expression of LC3 and autophagosomes showed a dynamic change,increased in the early stage,and reached the peak.Then decreased gradually,and the cells appeared ultimately delayed neuronal death.2 LIP can up-regulate the expression of autophagosomes and LC3 in the CA1 subfield of hippocampus of rats,activate autophagy further,improve the ability of neurons to fight against hypoxia and ischemia,and reduce the brain ischemia/ reperfusion injury. |
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