Effects Of Remote Ischemic Postconditioning On Mitophagy In Hippocampus Of Cardiopulmonary Resuscitation Rats

Part1:Observation of mitophagy activation in hippocampus after cardiopulmonary resuscitationObjective:This study examined the activation of mitophagy following cardiac arrest(CA)and cardiopulmonary resuscitation(CPR)and the relationship between the change with time.Methods:The male Sprague-Dawley rats were randomized into 4 groups:Sham group,CPR24h group,CPR48h group,CPR72h group.The rat model of cardiac arrest was established by asphyxiation.We employed western blot to analyze the levels of mitophagy related proteins of hippocampus,JC-1 to detect mitochondrial membrane potential(MMP)and flow cytometry to measure the rate of apoptosis of hippocampal neurons.Moreover,we also intuitively observed the occurrence of mitophagy through electron microscopy.Results:The results showed that the levels of TOMM20 and Tim23 protein were significantly decreased after CPR,which were more remarkable following 72h of CPR.However,the protein levels of dynamin related protein 1(Drp1)and cytochrome C(Cyt-c)were strongly up-regulated after CPR.Meanwhile,the hippocampal MMP decreased gradually with time after CPR.Furthermore,we more intuitively verified the activation of mitophagy through electron microscopy.In addition,the rats of apoptosis rate of hippocampus after CPR were significantly increased,which were gradually enhanced over time from 24 h until at least 72 h following CPR.Conclusion:With the enhancement of mitophagy,the apoptosis of hippocampal neurons was gradually enhanced,which suggested mitophagy may be excessive activated and aggravating brain damage after CA and CPR.Part2:Effect of inhibiting mitophagy on brain protection in rats after cardiopulmonary resuscitationObjective:This study clarified the relationship between mitophagy activation and brain injury following cardiac arrest(CA)and cardiopulmonary resuscitation(CPR)and the protective effect of Mdivi-1 on the brain.Methods:Male Sprague-Dawley rats were randomly divided into Sham group,CA/CPR group and Mdivi-1 group.The rat model was established by asphyxiation.The levels of mitophagy-related proteins,mitochondrial membrane potential(MMP)and apoptosis rate of hippocampal neurons were detected.Results:Compared with the CA/CPR group,Mdivi-1 significantly increased levels of TOMM20 and Tim23 proteins and MMP.At the same time,the apoptosis rate of hippocampus can be reduced.In addition,the NDS also showed improvements in brain function in Mdivi-1 treated rats.Conclusion:After cardiac arrest and cardiopulmonary resuscitation,hippocampal mitophagy was overactivated,aggravating brain damage.Mdivi-1 plays a neuroprotective role by inhibiting mitophagy and reducing apoptosis.Part3:Effects of remote ischemic postconditioning on the degree of mitophagy in hippocampal neuronsObjective:Remote ischemic postconditioning(RI-postC)is an effective measure to improve nerve function after cardiac arrest.However,the brain protective mechanism of RI-postC has not been fully elucidated,and whether it is related to mitophagy is unclear.In this study,we used a rat model of cardiac arrest to study the effect of RI-postC on mitochondrial autophagy and explore possible signaling pathway.Methods:Rats were randomly divided into sham group,CA/CPR group,Mdivi-1 group and RI-postC group.Animal model was established.RI-postC was performed by clamping and loosening the left femoral artery.Mdivi-1 is treated with a single intravenous injection.Level of TOMM20,TIM23,Mfn1,PINK1 and parkin were detected.Mitochondrial membrane potential and apoptosis rate were measured.Real-time PCR was used to detect relative mitochondrial DNA levels.Results:The results showed that RI-postC can inhibit the decrease of mitophagy related protein level,improve mitochondrial membrane potential and up-regulate the ratio of mt-Atp6/Rpl13 after CPR.Furthermore,RI-postC can also reduce the rate of hippocampal nerve apoptosis and improve nerve function after CPR.Moreover,RI-postC and Mdivi-1 can reduce the protein levels of PINK1 and parkin in mitochondria after CPR,while increasing their protein levels in the cytoplasm.Conclusion:These findings suggested that RI-postC can inhibit the overactivation mitophagy through the PINK 1/parkin signaling pathway,thus providing neuroprotective effects in the CA model.