The Research Of Cholinergic Anti-inflammatory Pathway In Remote Ischemic Postconditioning Alleviating Brain Injury After Cardiopulmonary Resuscitation

Cerebral hypoxic-ischemic injury and reperfusion injury after cardiac arrest,inducing neuronal apoptosis and necrosis,will eventually cause corresponding neurological deficits.Research showed that less than 5% of surviving patients after cardiopulmonary resuscitation have achieved satisfactory neurological recovery.Then how to reduce the neurological complications and improve the survival rate and quality of life after cardiopulmonary resuscitation is a clinical unsolved puzzle,but also one of the research focuses and hot spots.Remote ischemic postconditioning(RIPost),a new neuroprotection strategy,having aroused the concern and great research enthusiasm of the majority of scholars,can significantly reduce the ischemia-reperfusion injury of the heart and brain.And our previous studies have shown that RIPost can reduce the brain injury in a rat asphyxial cardiac arrest model,these findings provide a new way of thinking for the treatment of brain injury after cardiopulmonary resuscitation.However,the protection mechanism of RIPost has not yet fully elucidated.The inhibitory effect of RIPost on inflammatory response was confirmed by some experimental studies.Inflammation is one of the important pathogenesis of brain injury after cardiopulmonary resuscitation,and inhibit it can improve the experimental animal’s prognosis.It is believed that reducing the inflammatory response is one of the organ protection mechanisms of RIPost.Cholinergic anti-inflammatory pathway(CAP)is a newly discovered neural-immunoregulation pathway,based on vagal,acetylcholine and specific acetylcholine receptors,can effectively lessen the release of inflammatory factors when activated.In recent researches about remote ischemic conditioning,it was found that administration of ganglion blockers or vagotomy would eliminate the cerebral protective effect of ischemic conditioning,suggesting that CAP and RIPost are likely to be linked.Our pre-experiment found that RIPost not only reduced the levels of inflammatory factors such as IL-1β,IL-6 and TNF-α in the hippocampus and serum after cardiopulmonary resuscitation,but also increased the level of acetylcholine(Ach)and RIPost brain protection was significantly reduced after cervical vagal disconnection.Therefore,this study investigated whether cholinergic anti-inflammatory pathway is involved in RIPost alleviating brain injury after cardiopulmonary resuscitation.It may provide a new theoretical basis for the protective effect of RIPost and a new target for the development of neuroprotective drugs.Experiment 1 The effect of remote ischemic postconditioning on inflammatory response in rats with brain injury after cardiopulmonary resuscitationObjective To observe the effect of RIPost on inflammatory response in rats with brain injury after cardiopulmonary resuscitation.Methods Sixty-nine male Sprague-Dawley rats were randomly divided into 5 groups: sham group(n=9),control group(n=15),and three different forms of remote ischemic postconditioning groups(RIPost_A,RIPost_B and RIPost_C groups,n=15 each).The animals in sham group were only intubated,punctured and cannulated in femoral artery and vein,while in the other four groups were asphyxiated by occlusion of the tracheal tube and resuscitated 8 min later.In RIPost groups,RIPost,produced by 3 cycles of 15-min occlusion and 15-min release of the right hind by a tourniquet,were implemented immediately after restoration of spontaneous circulation(ROSC)in group RIPost_A,immediately and the 3rd hour after ROSC in group RIPost_B,immediately,the 3rd and the 6th hour in group RIPost_C.The general conditions and resuscitation success rate of each group were observed.The contents of IL-1β,TNF-α and HMGB-1 in plasma were assessed at the 1st(T1),8th(T2)and 24th(T3)hour after ROSC by ELISA,while in cerebral cortex and hippocampus were detected at the 24 th hour after ROSC.Neuron specific enolase concentration in serum was measured at the 48 th hour after ROSC by ELISA.Neurological deficit score was assessed at at 1,3,5 and 7 day after ROSC.Morris water maze test was used to quantify spatial learning and memory deficits at the 4th day after ROSC.The number of viable neurons in hippocampal CA1 region was recorded by Nissl’s staining at the 8th day after ROSC.Results The general conditions and resuscitation success rate in each group had no significant difference.Compared with group sham,the contents of IL-1β,TNF-α,HMGB-1 both in plasma and brain tissue and NSE in serum were significantly increased,NDS and the number of viable neurons in hippocampal CA1 region were decreased,the escape latency was prolonged,the time spent in the quadrant of the former platform and number of crosses were decreased in the other four groups.Compared with group control,the contents of IL-1β,TNF-α,HMGB-1 and NSE were significantly decreased,the number of viable neurons in hippocampal CA1 region and NDS were increased,the escape latency was shortened,the time spent in the quadrant of the former platform and number of crosses were increased in the three RIPost groups.Compared with group RIPost_A,the contents of NSE and inflammatory factors at T2 and T3 in plasma were decreased,the NDS 1d and the number of viable neurons in hippocampal CA1 region were increased,and no significant changes were found in the contents of IL-1β,TNF-α and HMGB-1 at T1 in plasma or in cerebral cortex and hippocampus,the escape latency,the time spent in the quadrant of the former platform and number of crosses in group RIPost_B and RIPost_C.No significant changes were found in the parameters mentioned above between group RIPost_B and group.Conclusion Remote ischemic postconditioning reduces the inflammation response and alleviates the brain injury induced by cardiac arrest.Intensive remote ischemic postconditioning implemented 6 hours after ROSC has subdued anti-inflammatory effects and can’t enhance the neuroprotective effect.Experiment 2 The effect of remote ischemic postconditioning on the expression of Ach and α7n ACh R in rats with brain injury after cardiopulmonary resuscitationObjective To observe the effect of RIPost on the expression of Ach and α7n ACh R in rats after cardiopulmonary resuscitation,and explore the relationship between neuroprotection and CAP.Methods Twenty-six male Sprague-Dawley rats were randomly divided into 3 groups: sham group(n=6),CA group(n=10),and RIPost group(n=10).The animals in sham group were only intubated,punctured and cannulated in femoral artery and vein,while in the other two groups were asphyxiated by occlusion of the tracheal tube and resuscitated 8 min later.In RIPost group,produced by 3 cycles postconditioning,were implemented immediately and the 3rd hour after ROSC.The Ach content in plasma and brain tissue at 24 h after ROSC was detected by ELISA,and the expression of α7n ACh R in brain tissue was inspected by western blot.Immunofluorescence staining was used to detect the expression of α7n ACh R with Neu N,CD11 b and GFAP in brain tissue 24 h after ROSC.Results The α7n ACh R was expressed in neurons,microglia and astrocytes.RIPost enhanced the expression of α7n ACh R in rats after cardiopulmonary resuscitation,while reducing the expression of microglia activation marker CD11 b and astrocyte marker GFAP.RIPost also increased Ach expression in plasma and brain tissue.Conclusion RIPost affects the expression of Ach and α7n ACh R in rats after cardiopulmonary resuscitation,inhibits the activation of microglia and the proliferation of astrocytes.The neuroprotective effect of RIPost may be related to the activation of cholinergic anti-inflammatory pathway.Experiment 3 The role of α7n ACh R in RIPost reducing the inflammatory response in rats with brain injury after cardiopulmonary resuscitationObjective To investigate the role of α7n ACh R in RIPost reducing the inflammatory response and observe the effect on NF-κB and JAK2 /STAT3 pathways.Methods Forty-six male Sprague-Dawley rats were randomly divided into 5 groups: sham group(n=6),CA group(n=10),RIPost group(n=10),RIPost+MLA group(n=10)and MLA group(n=10).The animals in sham group were only intubated,punctured and cannulated in femoral artery and vein,while in the other four groups were asphyxiated by occlusion of the tracheal tube and resuscitated 8 min later.In RIPost group,produced by 3 cycles postconditioning,were implemented immediately and the 3rd hour after ROSC.In MLA group,MLA was injected intraperitoneally at the moment of ROSC.The levels of serum NSE and inflammatory factors in brain tissue were measured by ELISA.The expressions of NF-κB,p NF-κB,STAT3 and p STAT3 in brain tissue were detected by western blot and immunofluorescence staining.Neuronal apoptosis was detected by TUNEL staining.Results In RIPost group,the levels of serum NSE and brain tissue inflammatory factors,the number of apoptotic cells and phosphorylation of NF-κB were decreased,while the phosphorylation of STAT3 was increased.After intraperitoneal injection of MLA,NSE and brain tissue inflammatory factors concentration,apoptotic cells and p NF-κB increased but the content of p STAT3 decreased.Conclusion The α7n ACh R is a key target for RIPost to produce neuroprotection,which may reduce the synthesis and release of inflammatory cytokines by regulating NF-κB and STAT3.Blocking this receptor can reduce the inhibition of inflammatory response.