The Effect And Mechanism Of Hydrogen Therapy After Cardiac Arrest/Cardiopulmonary Resuscitation Improving Myocardial Injury And Neurological Dysfunction In An Asphyxial Rat Model Of Cardiac Arrest

Cardiac arrest CA is one of the leading causes of death worldwide,with an acute onset and a high rate of disability and death.The previous studies have reported that patients with CA developed a systemic inflammatory response due to ischemia-reperfusion damage,leading to multi-organ dysfunction after return of spontaneous circulation(ROSC),clinically manifested as post-cardiac arrest syndrome(PCAS),primarily consisting of the following four aspects:1.post-CA brain damage;2.myocardial dysfunction;3.systemic ischemia-reperfusion injury;4.primary disease caused by CA itself.Therefore,it is critical to improve the survival rate and prognosis of cardiac arrest.Investigation showed that patients wtih ROSC mainly die from cardiac and cerebral dysfunction due to systemic ischemia-reperfusion.In the early stage,45%-60%of patients with ROSC will develop severe or lethal post-arrest myocardial dysfunction(PAMD),and 68%of patients with ROSC in the mid-to late-stage will die from post-resuscitation brain injury.Cardiac myocytes and neurons have long been considered to have poor regenerative capacity.There are three main types of programmed cell death:apoptosis,autophagy,and necrosis.Autophagy is a program that precedes apoptosis and has both anti-apoptotic and pro-apoptotic properties.Depending on the changes of cellular environment,autophagy regulates cell death and acts as a "double-edged swordr" for human health.Studies have suggested that an abnormal increase in cardiomyocyte and neuronal autophagy after reperfusion may be one of the important initiating factors that can lead to structural and functional damage in the heart.If the occurrence of autophagy can be inhibited early and effectively,it is expected to reduce myocardial and cerebral ischemia-reperfusion injury after cardiac arrest and improve cardiac arrest survival and prognosis.Hydrogen molecules,a selective antioxidant,can diffuse freely into cellular components.Studies have found that hydrogen molecules might attenuate ischemia-reperfusion injury through anti-inflammatory,anti-oxidative stress or anti-apoptotic pathways.Recent studies have suggested molecular hydrogen are closely related to autophagy,and hydrogen-rich saline were found to reduce myocardial ischemia-reperfusion injury and decrease myocardial infarct size by inhibiting cardiomyocyte autophagy.In a mouse model of neonatal ischemic encephalopathy,hydrogen-rich saline reduced brain edema and reduced infarction area,however,the exact mechanism is unclear.Thus,we focused on the role and mechanisms of hydrogen molecule therapy in the cardiac function and brain injuryof asphyxia-induced cardiac arrest model in rats.Objectives1.Investigate the effect and mechanism of hydrogen therapy on myocardial injury in an asphyxial rat model of cardiac arrest。Investigate the protective effect and mechanism of hydrogen on hypoxic/reoxygenated cardiomyocytes.2.Investigate the effect and mechanism of hydrogen therapy on nerve injury in an asphyxial rat model of cardiac arrest.Methods(1)Animal experiments1.Establishment of asphyxia cardiac arrest/cardiopulmonary resuscitation model in ratsMale Wistar rats(400-450g)were anesthetized by intraperitoneal injection of sodium pentobarbital(45mg/kg).Endotracheal intubation was performed and a ventilator was connected.Cardiac arrest was induced by asphyxiation.Mean arterial pressure(MAP)<30mmHg was defined as cardiac arrest.After 10 minutes of asphyxia,CPR was started,and artificial chest compressions were performed(200 times/min).Meanwhile,epinephrine was injected intravenously(2 μg/100g,every 3 minutes).Restoration of autonomic circulation(ROSC)was defined as restoration of sinus heart rate and MAP≥60 mmIHg,which was lasting more than 5 minutes.Animals lasting more than 10min after ROSC were enrolled in the experiment.2.Grouping and administrationMale Wistar rats were randomly divided into 6 groups(Sham-Normorxia,Sham-H2,CPR-4h-Normorxia,CPR-4h-H2,CPR-72h-Normorxia,CPR-72h-H2),10 rats in each group.Rats in the nornorxia and hydrogen inhalation sham groups were not treated with asphyxiation-induced cardiac arrest and cadiopulmonary resuscitation,and were given mechanical ventilation with air inhalation and 1.3%hydrogen/air mixture for 1 hour,respectively.After ROSC,rats in the nornorxia and hydrogen inhalation groups were inhaled with air and 1.3%hydrogen/air mixture for 1h,respectively.3.Monitoring of heart rate and blood pressureHeart rate was monitored by body surface ECG,mean arterial pressure was monitored by femoral artery catheterization.During 2h after ROSC heart rate and mean aterial pressure was mointered.4.Detection of blood gas analysisAt 30min,1h,2h,and 4h after ROSC,1.5ml femoral arterial blood was collected,and blood gas analysis was performed immediately.5.Cardiac function measurementEchocardiography was used to measure Left ventricular Ejection Fraction(LVEF)and Left ventricular Fractional shortening(LVFS)at 4 and 72 hours after ROSC.6.Serum creatine kinase isoenzyme MB(CKMB)and Cardiac Troponin T(cTnT)Blood samples were taken at 4h and 72h after ROSC,centrifuged and collected at 4℃for 20 minutes at 1000 g.Supernatant was used and detected by ELISA kit.7.Myocardial/hippocampal apoptosis measurementRats were sacrificed at 4h and 72h after ROSC,and left ventricular myocardium/hippocampal neurons were taken and fixed with 4%paraformaldehyde.After fixation,paraffin embedded and sectioned.TUNEL staining was used to detect myocardial apoptosis.8.Mitochondrial morphological analysis of cardiomyocytesRats were sacrificed at 4h and 72h after ROSC,left ventricular myocardial tissue was fixed with glutaraldehyde.Transmission Electron Microscope was used for semi-quantitative analysis of the mitochondrial.9.Beclin-1,LC3B and p62 protein expression in myocardial/hippocampal tissuesRats were sacrificed at 4h and 72h after ROSC.Total protein was extracted from left ventricular/hippocampal homogenate and Western blot was used to analysis the expresssion of Beclin-1,LC3B and p62 proteins.10.Immunohistochemistry of myocardial/hippocampal tissueRats were sacrificed at 4h and 72h after ROSC,left ventricular myocardium/hippocampal tissues was fixed with 4%paraformaldehyde and sectioned.LC3B protein expression was observed and photographed under fluorescence microscope and analyzed by Image J.11.Neurological Impairment Score(NDS)At 4h and 72h after ROSC,the rats were removed from the cage for single-blind NDS detection.12.Measurement of serum NSE and S100 levelsBlood samples were taken at 4h and 72h after ROSC,centrifuged and collected at 4℃for 20 minutes at 1000g.Supernatant was used and detected by ELISA kit.(2)Cell experiments1.Establishment of H9C2 Hypoxia-Reoxygenation(H/R)modelH9C2 cells were cultured at 37℃ with 95%air and 5%CO2.Under hypoxia,the culture condition was 37℃,and the gas composition was 94%N2+5%CO2+1%O2.H9C2 cells were firstly cultured in a normoxia incubator for 24 hours,then transferred to a Hypoxia incubator for 24 hours,and then to a normoxia incubator for 4 hours or 12 hours.2.Grouping and administrationThe cell experiment was divided into 6 groups:Con-Vehicle,Con-H2,H/R-4h-Vehicle,H/R-4h-H2,H/R-12h-Vehicle,H/R-12h-H2.3.Protein detection of Beclin-1,LC3B and p62 in H9C2 cellsThe expressions of Beclin-1,LC3B and p62 proteins in H9C2 total protein were detected by Western-blot.4.Immunofluorescence of H9C2 cellsThe expression of LC3B proteins in cells was detected by immunofluorescence assay.5.Ad-RFP-GFP-LC3 transfectionH9C2 cells were transfected with Ad-RFP-GFP-LC3,and H/R model was established.The number of autophagosomes(red+green)and autophagolysosomes(red)were counted.Results:1.Basic data and model data of animal experimentsIn the animal experiments,baseline and model data in the CPR+H2 group were not statistically different from those in the CPR-Normoxia group,including MAP and HR,induced CA time,ROSC time,and NE dosage.Blood gas analysis also found no significant difference between CPR-Normoxia and CPR+H2 groups.2.Hydrogen inhalation after CPR significantly improved the survival rate and cardiac function of ROSC ratsHydrogen inhalation after CPR significantly improved the survival rate and left ventricular cardiac function of rats after ROSC.3.Inhalation of hydrogen after CPR significantly improved myocardial cell death and mitochondrial damage in ROSC ratsInhalation of hydrogen reduced the levels of CK-MB and cTnT in serum of rats after ROSC,reduced myocardial cells apoptosis,and alleviated the injury of myocardial cells.Inhalation of hydrogen significantly improved mitochondrial structure and reduced the number of autophagosomes in cardiac arrest/post-cardiac arrest rat cardiomyocytes.4.Inhalation of hydrogen after CPR significantly reduced the expression of autophagy related proteins in cardiomyocytes and inhibited autophagy activationWestern blot results showed that hydrogen inhalation significantly reduced the protein expression levels of Beclin-1 and LC3B in rat left ventricular myocardium after ROSC,and significantly increased the protein expression level of p62.Meanwhile,immunohistochemical staining showed that hydrogen inhalation significantly reduced the protein expression level of LC3B in left ventricular myocardium of rats and inhibited autophagy activation.5.Hydrogen inhibited autophagy in cardiomyocytes induced by hypoxia/reoxygenationWestern blot results showed that hydrogen significantly reduced the expression of autophagy related proteins Beclin-1 and LC3B in H/R induced H9C2 cells,and increased the protein expression of p62.Immunofluorescence assay showed that hydrogen inhibited the expression of LC3B in H/R induced cardiomyocytes,and inhibited the activation of autophagy.Ad-RFP-GFP-LC3 transfection experiments showed that hydrogen inhibited the formation of autophagosomes in H/R-induced H9C2 cells,reduced the number of autophagosomes,and inhibited the activation of autophagy.6.Inhalation of hydrogen after CPR improved the neurological function of ROSC ratsNeurological Impairment Score(NDS)data showed that hydrogen inhalation improved neurological function in ROSC rats.ELISA results showed that hydrogen inhalation reduced the levels of S100 and NSE in serum of rats after ROSC.7.Inhalation of hydrogen after CPR significantly reduced the expression of autophagy related proteins in hippocampal nerve cells and inhibited activation of autophagyWestern blot results showed that hydrogen inhalation significantly reduced the expression of autophagy related proteins Beclin-1 and LC3B in the hippocampal tissues of ROSC rats,and increased the protein expression level of p62.Immunohistochemical staining showed that hydrogen inhalation significantly reduced the protein expression level of LC3B in hippocampal nerve cells of ROSC rats and inhibited autophagy activation.Conclusions1.Hydrogen therapy significantly improve the survival,the cardiac function and the nerve function in rats’ model of asphyxial CA/ROSC.2.Hydrogen therapy inhibited autophagy in cardiomyocytes and hippocampal neurons and reduce apoptosis in rats’model of asphyxial CA/ROSC.