Study On The Mechanism Of Enhanced External Counterpulsation Improve The Cardiac Function Of Beagles After Cardiopulmonary Resuscitation

Background:In the United States,approximately 300,000 people have an out-of-hospital cardiac arrest each year,and approximately 30–40%of those cases achieve return of spontaneous circulation?ROSC?,with only 7%of patients surviving until hospital discharge.Most patients die from post-cardiac arrest syndrome,which is a complex constellation of multi-organ failure,comprising brain injury,systemic ischemia-reperfusion response,and post-cardiac arrest myocardial dysfunction.Myocardial dysfunction is an important cause of post-resuscitation circulatory failure that may lead to early mortality after ROSC.It is defined as a reversible global dysfunction due to a stunned myocardium in the absence of coronary occlusion and microcirculation disorder of the coronary artery.Hemodynamics is very complicated after ROSC,and although a stable circula-tion may be indicated,the ischemia,hypoxia,and metabolic state of tissues may not truly be represented.Clinical and experimental studies have found that,in some situations,persistent tissue hypoxia may lead to organ damage despite the restoration of global hemodynamic parameters?heart rate,mean artery pressure,and cardiac index?to their“normal''levels.Clinical interventions to improve microcirculatory blood flow are still very limited.Enhanced external counterpulsation?EECP?,which was introduced in the American Heart Association/American College of Cardiology Guideline of Coronary Artery Disease in 2002,has been proven to be an effective,safe,and economical therapy for the management of ischemic cardiovascular and cerebrovascular diseases.The device for EECP consists of inflatable cuffs that encircle the calf,thigh,and upper thigh.The cuffs squeeze sequentially from low to high during diastole and then rapidly,and simultaneously,deflate at the onset of systole.The mechanism regulating the device is based on an electrocardiogram.The arterial hemodynamics that are generated by the device simulate those of the intra-aortic balloon pump with the generation of a retrograde arterial wave pulse.However,unlike an intra-aortic balloon pump,a retrograde venous pulse is also generated,which increases venous return.The retrograde arterial wave pulse causes augmented coronary flow,whereas the improved venous return helps to improve cardiac output and decrease oxygen consumption?VO2?.The systolic deflation/diastolic inflation sequence of EECP leads to systolic unloading and diastolic augmentation,resulting in a pulsatile increase in blood flow.EECP has been proven to be able to augment shear stress of blood flow,and thus improve the function of vascular endothelial cells,which in turn improves microcirculation.In a recent study,we found that EECP treatment after ROSC improved neurological function in beagle dogs,increased the release of vascular endothelial-derived relaxing factor NO-1,and increased brain microcirculation blood flow.However,whether EECP can improve myocardial blood flow?MBF?after cardiopul-monary resuscitation?CPR?is still unclear.The PiCCO2 is a device for continuous cardiac output measurement combined with monitoring of cardiac preload volume,extravascular lung water?EVLW?,and arterial and central venous oxygen saturation?ScvO2?.The PULSION PiCCO2computes the cardiac output continuously,utilizing an improved arterial pulse contour analysis algorithm.The pulse contour cardiac output?PCCO?is calibrated by means of a transpulmonary thermo-dilution measurement.A cold or room-temperate bolus?e.g.,normal saline 0.9%?is injected through a central venous catheter.A thermod-ilution curve is recorded by an arterial thermodilution catheter,which is also used for pressure monitoring.In addition to PCCO calibration,transpulmonary thermodilution also yields cardiac preload by means of global end-diastolic volume?GEDV?and estimation of both intrathoracic blood volume?ITBV?and EVLW.Furthermore,the PiCCO₂ continuously measures ScvO2after calibration with blood gas analysis results and can continuously calculate oxygen delivery?DO₂?and VO₂.Therefore,in addition to monitoring hemodynamics,PiCCO₂ can also reflect tissue perfusion and is an ideal monitoring tool after ROSC.The effects of EECP on hemodynamics are complex,and the PiCCO₂ system is a comprehensive tool for assessing hemodynamics.For the first time,we used the PiCCO₂ system to monitor the effects of EECP on the systemic hemodynamics of dogs after ROSC.We aimed to investigate the influence of EECP on systemic hemodynamics,MBF,and tissue oxygenation of beagle dogs after ROSC.Objective:The aim of this study was to investigate the influence of enhanced external counterpulsation?EECP?on the cardiac function of beagle dogs after prolonged ventricular fibrillation.Through the establishment of beagle dog CA-CPR model,PiCCO₂ technique was used for the first time to evaluate the hemodynamic changes of beagle dogs after ROSC,to observe the effect of EECP on hemodynamic changes of beagle dogs after ROSC,to observe whether EECP can increase myocardial blood flow and improve cardiac function after ROSC,so as to improve the prognosis,and to observe the effect of EECP on myocardial autophagy after ROSC.In vitro experiments further confirmed the role of autophagy in myocardial injury after CA/ROSC.It is expected to provide some new theoretical basis for the effect of enhanced external counterpulsation?EECP?on the improvement of cardiac function in beagle dogs after cardiopulmonary resuscitation.Methods:Twenty-four adult male beagles were randomly divided into control and EECP groups.Ventricular fibrillation was induced in the animals for 12 min,followed by 2min of cardiopulmonary resuscitation.They then received EECP therapy for 4h?EECP group?or not?control group?.The hemodynamics was monitored using the PiCCO2 system.Blood gas and hemorheology were assessed at baseline and at1,2,and 4h after return of spontaneous circulation?ROSC?.The myocardial blood flow?MBF?was quantified by18F-flurpiridaz PET myocardial perfusion imaging at baseline and 4 h after ROSC.Echocardiography was used to evaluate the cardiac function before ROSC and 4hours after ROSC.Myocardial histopathological changes were examined by myocardial electron microscope.The expression of myocardial protein was detected by Western blotting?Westernblot?and the expression of autophagy related gene mRNA was detected by q-PCR technique.The OGD?oxygenandglucosedeprivation,OGD?model of primary cardiomyocytes of neonatal rats was established in vitro,and the expression of autophagy gene in cardiomyocytes after ischemia-reperfusion was observed.To determine the effect of EECP on cardiac function of beagle dogs after cardiopulmonary resuscitation.Results:1.The basic situation of a beagle:Survival time of the animals was recorded within 24 h.Ventricular fibrillation was successfully induced in all animals,and they achieved ROSC after cardiopulmonary resuscitation.There was no significant difference in basic physiology and CPR related parameters between the two groups.Three animals in the control group?3thumb 12?and seven animals in the EECP group survived for 24 hours.The survival time of the control group was 12.2?4,24?hours,which was significantly shorter than that of the EECP group?0.026?.2.The results of blood gas analysis:the results of blood gas analysis showed that severe metabolic acidosis occurred in the control group after ROSC,which was characterized by a decrease in the concentration of HCO₃^-,a decrease in alkali residue and an increase in blood lactic acid level,while the metabolic acidosis in the EECP group was obviously slighter than that in the control group?p<0.001?.3.Hemodynamic results:There was no obvious difference in heart rate between the two groups at each time point after ROSC.Mean artery pressure was significantly higher in the EECP group than in the control group.Central venous pressure was slightly higher in the EECP group,but the difference was not significant.However,CPP was obviously higher in the EECP group.In the EECP group,the myocardial contractility of dogs was decreased after ROSC and the values of maximum rate of increase in pressure,CO,global ejection fraction,and PCCO were decreased,but the cardiac function of the animals was improved.The values of these parameters were obviously higher in the EECP group than in the control group.GEDV and ITBV of the EECP group after ROSC were significantly higher,indicating that EECP promoted blood return to the chest and heart.Systemic vascular resistance was significantly lower in the EECP group,indicating that EECP can reduce peripheral vascular resistance and increase stroke volume.EVLW and pulmonary vascular permeability index?PVPI?of the two groups were increased at each time-point after ROSC,indicating that there may be a lung injury after ROSC,but the EVLW and PVPI were significantly lower in the EECP group.ScvO₂and DO₂values at each time-point after ROSC were significantly higher in the EECP group,whereas the VO₂values were the opposite,indicating that EECP can increase DO₂and reduce tissue VO₂.This result is consistent with the previous EECP results of reducing blood lactate levels after ROSC.4.Hemorheology results:Compared with the control group,the EECP group after ROSC had significantly increased blood flow velocity,and decreased plasma viscosity,erythrocyte aggregation index,and hematocrit.Hemorheology parameters were affected by temperature,blood glucose concentration,and hemoglobin level,but there was no significant difference between the two groups after ROSC.5.Results of resting myocardial perfusion imaging:MBF in EECP group was obviously higher than in control group under resting and dobutamine stress conditions.Multiple regression analysis showed that MBF was highly correlated with the survival time of beagle dogs.The higher the MBF value,the longer the survival time of beagle dogs?p<0.001?.6.Echocardiographic results:Before VF,the left ventricular ejection fraction?LVEF?was 62?50-75?%in control group,61?52-79?%in EECP group,and 0.494 in EECP group.The EF of the control group decreased significantly at 1,2,3 and 4 hours after ROSC,which were 40?34,45?%,38?31,59?%,42?27,57?%and 41?26,55?%,respectively.The rates of EECP group were 46?38,58?%,46?39,68?%,50?45,53?%and 52?45,58?%,respectively.EECP treatment significantly improved left ventricular function in beagle dogs after ROSC.EECP increased left ventricular end-diastolic volume,but there was no significant difference in end-systolic volume between the two groups,indicating that EECP could increase left ventricular stroke volume.7.Results of myocardial electron microscopic examination:The electron microscopic results showed that in the control group,the myocardial mitochondrial membrane was destroyed,the mitochondria were swollen,deformed,dissolved or ruptured,the arrangement of cristae was obviously disordered and there were breakage,dissolution and vacuolation,glycogen granules decreased and there were more dense granules.In EECP group,the myocardial cell membrane,nuclear membrane and mitochondrial membrane were intact,the morphology of mitochondria was basically normal,the crest arranged neatly and abundant,a little broken,glycogen granules were abundant,and a few dense granules could be seen.The formation of autophagy vesicles in cardiomyocytes was observed.under 23500 times visual field,3animals in each group and 10 cells in each group were selected to calculate the number of autophagy vesicles in the cytoplasm.The number of autophagy vesicles in EECP group was significantly more than that in Control.8.The expression of myocardial protein was detected by immunoblotting?Westernblot?:EECP increased the protein level of LC3-?and decreased the protein level of p62 in beagle canine cardiomyocytes.9.The expression of autophagy related gene mRNA was detected by q-PCR:EECP increased the expression of autophagy genes Beclin-1,ATG5,ATG6,ATG7,ATG12,PINK and BNIP3.10.The results of in vitro experiment showed that the primary cultured rat cardiomyocytes could cause 52.2±0.4%cardiomyocyte death after simulated ischem-ia and hypoxia for 6 hours and reperfusion for 4 hours.the number of cardiomyocyte death decreased to 39.5±1.9%after administration of autophagy activator Rapamycin,and increased cardiomyocyte death after blocking autophagy with 3-MA,and the mortality rate was 58.8±2.9%.It is suggested that activated autophagy can promote the survival of primary rat cardiomyocytes after simulated ischemia-reperfusion injury.The key molecules of autophagy,such as ATG5,ATG7 and LC3-?,were significantly increased after administration of autophagy activator Rapamycin.Rapamycin upregulates the expression of most autophagy genes,which is the key mechanism of activating autophagy Rapamycin to protect myocardium.Conclusion:1.EECP can improve the cardiac function of beagle dogs after ROSC in many ways,increase CCP and MBF to maintain myocardial contractility,reduce peripheral vascular resistance to reduce cardiac afterload,finally increase cardiac output and improve tissue perfusion,promote venous reflux and increase cardiac preload,thus increasing SV and CO.EECP improves hemorheology and helps to improve microcirculatory blood flow in other tissues,including myocardium.EECP increases myocardial autophagy after ROSC,which is one of the mechanisms of EECP protecting cardiac function after ROSC.2.The PiCCO₂ system is an excellent hemodynamic monitoring system,which can comprehensively evaluate the hemodynamic state after ROSC,including myocardial contractility,cardiac function,peripheral vascular resistance,organ function and tissue oxygen metabolism,as well as the hemodynamic changes after ROSC and the diagnosis of post-cardiopulmonary resuscitation syndrome.3.In primary cultured rat cardiomyocytes,Rapamycin activated autophagy and reduced cardiomyocyte death after simulated ischemia and hypoxia for 6 hours and reperfusion for 4 hours.