| Background and ObjectivesWith the application of thrombolytic therapy;percutaneous coronary intervention (PCI) and coronary artery bypass grafting(CABG) in the clinic,coronary reperfusion therapy has become established for the management of acute myocardial infarction. Although ischemic myocardium can be salvaged by timely reperfusion of the occluded artery,reperfusion may lead to detrimental consequences,which is called ischemia-reperfusion injury.The phenomenon is now known to involve extension of myocardial stunning,microvascular dysfunction and other damages over reperfusion time. Many efforts are being made to investigate the pathophysiological mechanism of myocardial ischemia-reperfusion injury and to search for strategy that will protect myocardium against ischemia-reperfusion injury.One of the approaches that have gained widespread recognition involves the use of ischemic preconditioning,which has displayed significant protective effects in myocardial ischemia-reperfusion injury through its abilities to stimulate endogenous protective mechanisms.However,ischemic preconditioning must be performed prior to the sustained ischemic.Its usefulness is limited in clinical practices.Recently,ischemic postconditioning has attracted extensive interest due to a novel strategy of attaining cardioprotection.Ischemic postconditioning refers to the brief episodes of ischemia at the onset of reperfusion following a prolonged ischemia.It may alleviate myocardial ischemia-reperfusion injury.Since ischemic postconditioning can be easily applied with precisely controlled timing,this approach appears to have a great potential for clinical application.Many studies about the reduction of ischemic-reperfusion injury by postconditioning has now been documented and reported in small animal models using such as rats,rabbits and canines.But the effects on pigs,a large animal species that has most similar heart to humans,are not clear yet.In addition,there are few experiences in closed-chest of larger animals.Furthermore,most of the previous studies have reported that ischemic postconditioning inhibits local informatory responses in the area at risk and reduces infarct size,as well as improves cardiac function after a short period of reperfusion(<3 h after reperfusion).We have little information about whether long-term cardioprotection effect is likely achieved by antiapoptotic mechanisms after prolonged reperfusion.Pig was used in our research.We established the closed-chest model of myocardial infarction in pigs.Based on the model,we observed ischemic postconditioning treatment on pig myocardial protection.Next,we explored antioxidant and inflammatory mediators in ischemic postconditioning against myocardial ischemia-reperfusion injury.Then,we observed and compared the effects of ischemic postconditioning on myocardial biochemical marker,infarct size,ultrastructural alterations of cardiomyocytes,myocardial apoptosis and apoptosis related-genes(Bcl-2,Bax) protein expression.This study may provide not only a novel approach for clinic application but also useful theoretical and practical evidence for reducing myocardial ischemia-reperfusion injury.Methods1.Establishment of closed-chest model of myocardial infarction in pigs.Healthy Diannan small-ear pigs were randomly divided into 2 groups.(1) sham-operated (Sham) group:only put the balloon up to the distal position of the left anterior descending artery(LDA),without ischemia;(2) ischemia-reperfusion(I/R) group:animals underwent 60min of LAD occlusion followed by 72 hrs of reperfusion without additional intervention. During the experiment,animals were monitored by heart rate,blood pressure,coronary angiography,and ECG.Myocardial biochemical marker of animals were tested,including creatine kinase(CK),lactic dehydrogenase(LDH),creatine kinase-MB(CK-MB),and troponin I(cTnI,).And the changes of myocardial histopathology were observed.2.The protective role of ischemic postconditioning against ischemia-reperfusion injury in pig myocardiumPigs were randomly divided into 4 groups.(1) Sham-operated(Sham) group;(2) Ischemia-reperfusion(I/R) group;(3) ischemic postconditioning(Postcond) group:treated the same as I/R group and 8 cycles of 30s R and 30s I before reperfusion.(4) ischemia(I) group:the 1/2 size of balloon were placed in remote LAD,resulting in the formation of autologous blood clot that caused myocardial ischemia.Detected myocardial biochemical marker included CK,LDH,CK-MB and cTnI.And myocardial infarct size and ultrastructure of myocardial cells were also detected.3.Anti-oxidation and regulation of inflammatory cytokines of Ischemic postconditioning treatment to pig myocardial ischemia-reperfusion injuryThe contents of serum malondialdehyde(MDA) and superoxide dismutase(SOD) were determined respectively by spectrophotometer.Using ELISA,concentrations of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6) and Interleukin-10(IL-10) were examined.4.The role and its mechanism of ischemic postconditioning against apoptosis in myocardial ischemia-reperfusion pigsThe apoptotic cells in the myocardium were determined with electron microscope and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescence in nick end(TUNEL) method,and apoptosis-related protein molecules(Bax and Bcl-2) were studied by immunohistochemical and Western blotting analysis.Results1.Establishment of closed-chest model of myocardial infarction in pigs.After LDA distal occluded by balloon catheter,there were no blood flow showed by coronary angiography.Typical changes in ECG happened such as the elevation of the T-wave,as a first sign,and later ST-elevation.After reperfusion of 2hrs,24hrs,48hrs,and 72hrs,CK,CK-MB,cTnI,LDH myocardial biochemical markers were significantly higher than those before ischemia.Pathological features were consistent with myocardial infarction.By coronary angiography,ECG,myocardial enzymes,methods of pathological examination,it were confirmed that closed chest model of myocardial infarction for pigs was successfully established.2.The protective role of ischemic postconditioning against ischemia-reperfusion injury in pig myocardium(1) Changes in myocardial biochemical markers:compared to I/R group,CK-MB,cTnI, CK,LDH indicators in Postcond group reduced significantly(P<0.05) after reperfusion of 24hrs,48hrs and 72hrs.But index of I group was significantly increased at corresponding time(P<0.05).(2) Changes in myocardial infarction size:there was no myocardial infarction in Sham group.The myocardial infarction size of I/R group,Postcond group and I group were 23.26±3.13%,10.89±2.02%,and 33.3±12.2%,respectively.Infarct size was significantly reduced in Postcond group than in I/R group(23.26%±3.13%Vs.10.89%±2.02%, p<0.05).(3) Ultrastructural alterations of cardiomyocytes:ischemia postconditioning treatment could effectively attenuate ultrastructural damages.The results suggested that ischemia postconditioning treatment was obviously against myocardial ischemia reperfusion injury.3.Anti-oxidation and regulation of inflammatory cytokines of Ischemic postconditioning treatment to pig myocardial ischemia-reperfusion injurySOD activity was significantly increased in Postcond group than in I/R group after reperfusion of 2hrs and 24hrs.But,MDA levels were significantly decreased(P<0.05). Meanwhile,concentration of TNF-αand IL-6 in Postcond group were significantly lower (P<0.05) after reperfusion of 2hrs and 24hrs,and IL-10 concentration became significantly higher(P<0.05).4.The role and its mechanism of ischemic postconditioning against apoptosis in myocardial ischemia-reperfusion pigs(1) Changes in pig myocardial cells:with electron microscope and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescence in nick end(TUNEL) method, we found apoptotic cells in I group,I/R group,and Postcond group,except in Sham group. Apoptotic index(AI) of infarcted zone,border zone,and non-infarcted zone were significantly lower in Postcond group than those in I/R group(P<0.05).AI of infarcted and non-infarct zone in I group were significantly higher than those in I/R group(P<0.05).(2) Changes in pig myocardial cells Bcl-2 and Bax protein expressions:there were a certain amount of Bcl-2 and Bax protein expressions in Sham group myocardial cells.Bcl -2 and Bax protein expressions in I/R myocardial infarction zone,border zone and the non-infarct area increased greatly than that in Sham group(p<0.05),as well as the Bax/Bcl-2 ratio.And compared to I/R group,there were significantly reduction in Bax protein expressions of infarction zone,border zone and the non-infarcted area in Postcond group(p<0.05).There were also significantly increase of Bcl-2 protein expression in the border zone(p<0.05),and Bax/Bcl-2 ratio decline in the border zone and non-infarcted area(p<0.05).Conclusion1.Closed-chest model of pig myocardial infarction is an ideal animal model for the research of ischemic postconditioning. 2.Ischemic postconditioning can reduce the releasing of the myocardial biochemical marker;limit the infarct size and attenuate damages of myocardial cell ultrastructure after ischemia-reperfusion injury.Ischemic postconditioning can provide a long-term protection after porcine myocardial ischemia-reperfusion injury.3.The mechanisms of ischemic postconditioning resisting pig myocardial ischemia/reperfusion injury relate to anti-oxidation which may be achieved by scavenging oxygen free radicals and inhibiting lipid peroxidation,increasing antioxidant SOD levels.4.Ischemic postconditioning reduces the production of proinflammatory cytokine TNF-α, IL-6 after myocardial ischemia reperfusion injury;increases the level of anti-inflammatory cytokine IL-10 and maybe play a myocardial protection effect by regulating the balance of inflammatory cytokines.5.The mechanisms of ischemic postconditioning against pig myocardial ischemia-reperfusion injury relate to anti-apoptosis,which may be expressed by the regulating of apoptosis-related gene Bax and Bcl-2 protein expressions.6.The research provided the theoretical and experimental basis for the clinical application of ischemic postconditioning and presented the new target for the prevention of myocardial ischemia-reperfusion injury.It has the extensive application perspective as well as important value and significance...
|
PDF Full Text Request