Metformin Improves Neurological Outcome Via AMPK Mediated Autophagy Activation In A Rat Model Of Cardiac Arrest And Resuscitation

BackgroundSudden cardiac arrest(CA)is one of the major causes of death and disability.The annual incidence of CA involves approximately 320,000 out-of-hospital patients and 200,000 in-hospital patients in the United States.Currently,the overall survival rate for CA patients remains low,with a rat of<15%for out-of-hospital CA and<20%for in-hospital CA.Severe energy depletion is one of the main characteristics after CA.During ischemia,the decrease of ATP synthesis leads to fall of mitochondrial membrane potential and influx of calcium to induce neuron injury.Following recovery of spontaneous circulation(ROSC),reperfusion triggers excessive generation of oxygen-free radicals that directly damage the cell membrane and promote inflammation.As brain tissue is exquisitely susceptible to ischemia injury,CA impacts most profoundly on the neurological dysfunction,which contributes significantly to mortality and morbidity among survivors achieving ROSC.Currently,there is no effective medicine intervention to reduce neuronal death and neurological deficits in CA patients.Autophagy,a highly conserved cellular catabolic process for degradation and recycling of misfolded or damaged proteins and organelles,is considered to be a cytoprotective mechanism under most circumstances,though pathologically increased autophagy may lead to cell death.A large pool of evidence has demonstrated that autophagy activation is neuroprotective in ischemic stroke.Despite the growing attention on autophagy as a novel target for ischemic brain injury,studies on agents that modulate autophagy and could be used clinically are still limited.Metformin is the first-line hypoglycemic agent for treating type 2 diabetes.Emerging clinical and experimental studies have demonstrated that metformin possesses a variety of other beneficial effects beyond its glucose-lowering effect.The UK Prospective Diabetes Study(UKPDS)has shown that chronic metformin treatment can effectively reduce stroke incidence and cardiovascular mortality.Recent animal studies have further demonstrated that chronic metformin pretreatment can obviously alleviate the damage of ischemic stroke to brain tissue.In addition,metformin is also effective in treating global cerebral ischemia,intracerebral hemorrhage,epilepsy,Parkinson's disease,and Huntington's disease.However,whether and how metformin treatment is effective in reducing brain injury following CA remains uninvestigated.AMP-activated protein kinase(AMPK)serves as a cellular energy sensor and is activated in response to a series of metabolic stresses such as hypoxia,oxidative stress,glucose deprivation.Once activated,AMPK promotes catabolic pathways to generate more ATP,and inhibits anabolic pathways,thus allowing for adaptive changes in growth and metabolism under low energy conditions.Emerging evidence indicates that AMPK can be activated by metformin in the brain,exerting neuroprotective role in response to energy depletion through maintaining cellular energy homeostasis,promoting mitochondrial biogenesis,and increasing brain-derived neurotrophic factor(BDNF)expression to promote neuronal survival.Recently,autophagy has been proposed as a downstream target of AMPK,and AMPK-induced autophagy activation protects against ischemic injury to peripheral tissue injury as well as ischemic brain injury.These studies have promoted us to hypothesize that metformin pretreatment may prime the brain to better stand ischemic damage resulting from asphyxia CA and cardiopulmonary resuscitation(CA/CPR)through promoting AMPK-mediated autophagy activation.In the present study,we tested the above hypothesis by investigating the effects of chronic metformin pretreatment on neuroprotection,AMPK activation and autophagy induction in a rat model of asphyxia CA/CPR.Our data showed that chronic metformin pretreatment significantly ameliorated CA/CPR-induced neuronal injury,profoundly facilitated functional recovery.Furthermore,AMPK-dependent autophagy activation played an important role in mediating metformin's neuroprotection.Methods1.The neuroprotection of metformin in a rat model of 9-min ACA/CPRTo investigate whether metformin treatment is ef-fective in reducing brain injury following CA,rats were randomly divided into three groups:sham-operated group,vehicle-treated group(Veh group)and metformin-treated group(Met group).Metformin was dissolved in sterile saline at a concentration of 30 mg/ml and 200 mg/kg was administered intragastrically once daily for 2 weeks,while rats in the vehicle group received equivalent volume of saline.The last gavage was performed 24 h before induction of CA/CPR model.Then rats were followed up for 7 days and survival rate,neurological function using a validated neurological deficit scale(NDS,0 = brain death,80 = normal),neuronal degenerations using Nissl staing,dendrites losses using MAP2 staining and glial activation using imrmunohistochemical staining were evaluated.2.The effects of metformin on autophagy and AMPK activation after 9-min CA/CPRIn the first section,protein was extracted from brain tissues(cerebral cortex and hippocampus)and Western blotting was performed to measure the levels of p-AMPK,AMPK,and autophagy marker proteins of LC3 and p62 at at different time points(6h,12h,24h,and 48h)after CA/CPR in rats treated with or without Met.In the second section,rats were euthanized at 24 hours after ROSC.Immunofluorescence staining was used for evaluating the expression level of autophagy in each group and electron microscopy was also used to examine autophagy.3.Inhibition of autophagy and AMPK abolished the Met-afforded neuroprotection in CA/CPR-treated rats.Rats treated with metformin were randomly allocated and the autophagy inhibitor Chloriquine(CQ,25 mg/kg,i.p.),AMPK inhibitor Compound C(Cc,100nmol/rat,i.c.v.)were injected 1h before induction of CA.The inhibitory effects of CQ on autophagy and Cc on AMPK at 24 h after CA/CPR were verified by Western blotting.Survival,neurological outcome and histological injury were evaluated.Results1.Metformin pretreatment improves animal survival and neurological recovery in post-CA/CPR rats.The 7-day survival experiment showed that 55%of the rats(11 of 20)in the vehicle group survived at day 7,which was much lower than that in the Met group(85%,17 of 20)(p<0.05).In addition,we also assessed the NDS that represent the neurological deficiency,and found that the NDS at 24,48,and 72 h after ROSC was significantly lower in the Met group than those in the vehicle group(p<0.05).No significant difference was found at 7 days although an improved trend was observed in the Met group.The surviving neurons assessed by Nissl staining were significantly reduced in the vehicle-treated CA/CPR group comparing to sham-operated post-CA/CPR rats,and this reduction was significantly attenuated by Met treatment.Similar results were observed for MAP2,a protein that is enriched in neuronal dendrites and acts as a stabilizing molecule for the dendritic cytoskeletal integrity.Extensive loss of MAP2-immunoreactive dendrites was clearly observed in hippocampus CA1 region of vehicle-treated CA/CPR rats,and this dendritic loss was significantly attenuated by Met treatment.In addition,neuroinflammation was activated in hippocampus CA1 region post CA/CPR,evidenced by significantly increased numbers of Ibal-positive microglia and GFAP-positive astrocytes in this area of vehicle-treated CA/CPR-rats.Of note,Met treatment significantly suppressed the activation of microglia and astrocytes compared with the vehicle-treated group.What's more,immunostaining of cleaved caspase 3 were conducted to determine the apoptosis of neurons at 24 h after CA/CPR.Compared to the sham group,a robust degenerative reaction was detected in the hippocampal CA1 region of the Veh group(p<0.01).And the administration of Met significantly ameliorated the neuroapoptosis induced by CA/CPR(p<0.05).2.Metformin activates AMPK and autophagy in hippocampal neurons post CA/CPR.To further study the mechanism underlying the neuroprotective effects of Met,we examined the phosphorylation levels of AMPK in hippocampal tissue to determine whether the dose of Met used in this study could activate AMPK in the brain,A significant increase in the phosphorylation of AMPK was observed at 48 h after CA/CPR(p<0.05 vs.sham).In Met-treated group,AMPK activation occurred as early as 6 h after CA/CPR and remained elevated for up to 48 h(p<0.01 vs sham).Moreover,treatment with metformin significantly augmented the CA/CPR-induced increase in the phosphorylation and activation of AMPK(p<0.05 vs Veh).These results indicate that AMPK activation occurred earlier and in an augmented amplitude in the hippocampus of metformin-treated CA/CPR rats compared to Veh-treated group.To examine the induction of autophagy after CA/CPR,we measured the levels of autophagy marker proteins of LC3 and p62 in the hippocampal tissue.We found that CA/CPR induced a time-dependent accumulation of LC3-?,which started to increase at 12 h,and peaked between 24 h and 48 h after ROSC.Autophagy activation was further verified by the change of p62 protein that started to decline at 12 h and further attenuated at 24 h and 48 h after ROSC,as p62 expression is negatively correlated to the level of autophagy activation.These results demonstrate that autophagy is induced in a time-dependent manner after CA/CPR.Then,we next analyzed the changes of LC3 and p62 proteins in hippocampal tissue at 24 h and 48 h after CA/CPR in rats treated with or without Met.A significant increase in LC3-? formation was observed in the hippocampal tissue at 24 h(p<0.05 vs sham)and 48 h(p<0.01 vs sham)after CA/CPR.Moreover,this increase was augmented in rats pretreated by Met(p<0.05 vs Veh).Accordingly,the protein level of p62 was significantly decreased in the hippocampus of CA/CPR rats(p<0.05 vs sham),and Met pretreatment augmented this decrease(p<0.05 vs Veh).Double immunostaining also showed that Met pretreatment could further enhance CA/CPR-induced activation of autophagy.Furthermore,autophagic vacuoles were clearly seen in hippocampal neurons of Veh-and Met-treated CA/CPR rats.3.Inhibition of AMPK and autophagy reversed the neuroprotection of metformin against CA/CPR-induced brain injury.Administration of Compound C significantly inhibited CA/CPR-induced AMPK phosphorylation in hippocampal and cortical brain tissue,demonstrating the inhibitory effect of Compound C on AMPK activation under our experimental conditions.Then we wondered whether blockage of AMPK pathway have impact on long-term survival protection of Met in CA/CPR.We next assessed the effect of AMPK inhibition with Cc on Met-afforded neuroprotection at 7 days after CA/CPR.Rats in the Met+Cc group showed significantly lower NDS than those in the Met+Veh group at 24,48 or 72 h after CA/CPR,indicating that metformin-induced amelioration in neurological deficits was abolished by Cc.Similarly,Nissl staining showed that Cc treatment significantly decreased the number of surviving neurons in hippocampal CA1 region compared to Veh treatment.Moreover,dentritic injury assesssed by MAP2 staining showed that significantly more dendritic damage was observed in hippocampal CAI region of Met+Cc-treated rats than that of Met+Veh-treated rats.These results suggest that inhibition of AMPK abolished the neuroprotection against CA/CPR-induced injury by chronic metformin pretreatment.By blocking the last step of autophagic flux,CQ prevented the lysosomal degradation of LC3-? and thus led to increased accumulation of LC3-II protein in both hippocampus(p<0.05 vs Veh)and cortex(p<0.05 vs Veh).Moreover,CQ treatment significantly reversed Met-induced reduction of p62 protein levels,further supporting the inhibitory role of CQ in autophagy(as p62 level is negatively correlated to the extent of autophagy activation).Then,we investigated the influence of CQ on neuroprotective effect of Met.Met+CQ group showed significantly lower NDS than those in the Met+Veh group at 24,48 or 72 h after CA/CPR,indicating that metformin-induced amelioration in neurological deficits was abolished by CQ.Moreover,Nissl staining and MAP2 staining further showed that CQ treatment significantly decreased the number of surviving neurons and aggravated dendritic injury in hippocampal CA1 region compared to Veh-treated rats.Lastly,inhibition of AMPK by Compound C reduced cerebral autophagy activation induced by metformin in the brain of CA/CPR rats.ConclusionsOur present study demonstrates for the first time that chronic metformin pretreatment confers neuroprotection in a rat model of CA/CPR,and this neuroprotective effect is associated with enhanced autophagy induction by sustained AMPK activation.Since metformin is widely used for the treatment of diabetes and other metabolic disorders,our data support that chronic metformin administration may represent a novel and important preventive strategy to reduce CA-induced brain injury.