The Neuroprotective Effects Of Remote Ischemic Treatment In A Rat Model Of Cardiopulmonary Resuscitation

Background:In preclinical and clinical experiments,distal ischemic treatment have been demonstrated to reduce nerve injury after ischemia reperfusion,thus improving neurological function.The mechanism of action of these interventions are still unclear.Many studies have indicated neuronal apoptosis to be one of the underlying mechanism of post-CA and post-CPR neuronal cell death.Therefore,attenuation of neuronal apoptosis is a key strategy to maintain neurological function in these patients.Mitochondrial permeability is mainly regulated by mitochondrial permeability transition pore(mPTP),a protein complex channel located on the inner mitochondrial membrane.Inhibition of mPTP opening can effectively reduce neuronal apoptosis during ischemia reperfusion.Moreover,mPTP plays a key role in the maintenance of mitochondrial function,and a key controller of internal and external information exchange of mitochondria.We hypothesize that distal ischemic treatment protect animals from damage following CA by reducing neuronal apoptosis,which may be regulated by mitochondrial function,particularly by inhibiting the degree of mPTP opening.Part 1:Comparison of three ways of remote ischemic treatment on cerebral protection in rats after cardiopulmonary resuscitationObject:To observe the effects of three ways of distal ischemic treatments on cerebral protection in rats after cardiopulmonary resuscitation.Methods:A rat model of distal ischemia was established by clipping the left femoral artery.Rats were divided into blank,model,pre-distal ischemic treatment,per-treatment,and post-treatment groups.Neurological deficit score was scored to evaluate neurologic function after cardiopulmonary resuscitation for 72 h.Results:The resuscitation success rate did not differ significantly between the five groups.However,the survival rate at 48 h and 72 h after surgery in the model group was lower than that in the blank group.At 24 h post-surgery,the neurologic deficit score(NDS)of the pre-treatment group was higher than that of the model group,and after 48 h,the NDS of the pre-and post-treatment groups were higher than that of the model and per-treatment groups.After 72 h,the NDS of pre-,per-and post-treatment groups were all higher than that of the model group.Conclusion:Our results show that ischemic preconditioning and post processing can improve NDS score after cardiopulmonary resuscitation,while per-treatment have less protective effect.Part 2:Effect of remote ischemic treatment on apoptosis and mitochondrial permeability in rats after cardiopulmonary resuscitationObject:To observe the effects of distal ischemic treatments on apoptosis and mitochondrial permeability in the hippocampus after cardiopulmonary resuscitation.Methods:A rat model of distal ischemia was established by clipping the left femoral artery.Rats were divided into blank,model,pre-distal ischemic treatment,per-treatment,and post-treatment groups.We employed TUNEL and flow cytometry to measure the rate of apoptosis of hippocampal neurons,the integrity of mitochondrial membrane and the degree of mitochondrial permeability transition pore(mPTP)opening.Results:The rate of apoptosis rate of hippocampal CA1 neurons in the pre-and post-treatment groups were significantly lower than that of the model group.Moreover,the integrity of the mitochondrial membrane in the pre-and post-treatment groups was higher than that in the model and per-treatment groups.Furthermore,the degree of mPTP opening was lower in the pre-and post-treatment groups than the untreated and per-treatment groups.Conclusion:Our results show that ischemic preconditioning and post processing can maintain the integrity of mitochondria,perhaps by inhibiting the opening of mPTP,and reducing apoptosis of hippocampal neurons by regulating expression of apoptosis-related proteins after CPR,to improve neurological function.Part 3:Study of mPTP blocker and ischemic postconditioning on cerebral protection after cardiopulmonary resuscitationObject:We set out to assess whether mPTP blocker cyclosporine A(CsA)or ischemic post-treatment combines with CsA will play a more potent neuroprotective role after cardiopulmonary resuscitation(CPR),also its possible mechanism.Methods:All sixty SD rats were divided into Model group,Post-group,CsA group and CsA plus Post-(PLUS)group.CsA plus post-group received both CsA and post-treatment interventions.Neurological deficit score was scored to evaluate neurologic function after CPR for 72 h.TUNEL and flow cytometry was used to measure the rate of apoptosis of hippocampal neurons and the degree of mitochondrial permeability transition pore(mPTP)opening.Results:The rate of apoptosis was significantly lower in the Post-and CsA groups than Model group,with the lowest apoptosis rate in the PLUS group;Histological evaluation both by Hematoxylin and eosin(HE)and electron microscope showed better protective effect in PLUS group.Also,the neurological deficit score in the PLUS group were significantly higher than that of the CsA group at 72 h after resuscitation.But there was no difference on mitochondrial permeability transition pore(mPTP)opening degree between CsA and PLUS groups.Conclusion:Ischemic post-treatment combines with CsA have a better neuroprotective effect after cardiopulmonary resuscitation than used alone.Inhibiting mPTP opening is one but not the only neuroprotective mechanism.Part 4:Hypertonic saline infusion suppresses apoptosis of hippocampal cells in a rat model of cardiopulmonary resuscitationObject:Whether the beneficial effect of HS on neurological function after cardiopulmonary resuscitation(CPR)in rat model of asphyxial cardiac arrest(CA)is mediated via attenuating apoptosis of neurons is not known.We studied the neuroprotective effect of HS in rats after CA and CPR,and explored the likely underlying mechanisms.Methods:Animals were randomly assigned to 4 equal groups(n=15 each)according to the different infusions administered during resuscitation:control(C),normal saline(NS),hypertonic saline(HS),and hydroxyethyl starch(HES)groups.Assessment of neurological deficit scores(NDS),western blot analysis,and histopathological examination(hematoxylin and eosin)of brain sections was performed.Apoptosis of hippocampal neurons was determined by TUNEL and flow cytometric apoptosis assays.Results:NDS at 12,24,48 and 72h post-ROSC in the HS group were significantly higher than those in the NS and HES groups(P<0.05 for both).Western blot analysis demonstrated a significant increase in Bcl-2 expression in HS,as compared to that in the NS and HES groups(P =0.008;P =0.002,respectively).However,Bax and Caspase-3 expressions in HS were significantly lower than that in the NS and HES groups(P<0.001 for both).The apoptosis rate in HS was significantly lower than that in the NS and HES groups(P<0.05 for all).Conclusion:HS treatment during resuscitation could effectively suppress neuronal cell apoptosis in hippocampal CA1 post-ROSC and improve neuronal function.