Experimental Study Of Ischemic Postconditioning Combined With Granulocyte Colony Stimulating Factor In The Treatment Of Acute Myocardial Infarction

Background: Acute myocardial infarction is the leading causes of mortality worldwide currently. The key treatments are limiting infarct size and improve coronary blood flow to the ischemic myocardium. Ischemic postconditioning(IP) can obviously reduce myocardial ischemic/reperfusion injury and limiting infarct size. Granulocyte-colony stimulating factor (G-CSF) can prevent left ventricular remodeling and improves cardiac systolic and diastolic function by stem cells mobilization, inhibiting cardiomyocyte apoptosis and inducing neovascularisation. Clinical effects of them are not satisfied at present, however. The tentative ideas of our study are improve the prognosis and reduce left ventricular remodeling further, through the treatment of IP in combination with G-CSF in AMI.PartⅠEstablishing a Model of Ischemia and Reperfusion in Animal Heart in VivoObjectives: The research is aimed at establishing a stabile and high standards model of ischemic /reperfusion in animal heart in Vivo.Methods:1. 20 New Zealand rabbits were randomly allocated to 2 groups(n=10)。2.“6 sutures and 6 knots”was used to open the chest, and the left anterior descending artery( LAD) or left ventricular artery(LVA) were occlusion for 30 min using the method of“2 sutures and 2 knots”, respectively, and 2h reperfusion is followed. 3. Blood was drawn from femoral vein to evaluate cardiac troponin I(cTnI) values with enzyme-linked immunosorbent assay (ELISA) before operation and 120 min after reperfusion in 2 groups. 4. The hearts were enucleated after reperfusion, and myocardial infarct size were determined by staining with triphenyltetrazolium chloride(TTC)dye.Results: 1. Incidence of pneumothorax and hemorrhea significantly reduced during operation in this research, and achievement ratios were 70% in the 2 groups. 2. There were nearly 50% animals without significant ECG leadⅡST section elevation after LVA occlusion. 3. There was an obvious elevation of cTnI in blood after operation (LAD group: 0.37±0.12 vs.2.31±1.59, p<0.05;LVA group: 0.32±0.14 vs. 3.49±0.74, p<0.01). 4.The infarcted size in LVA occlusion group was bigger than that of LAD group, with less variation furthermore.(LAD group: 21.51±7.87 %, LVA group: 28.95±6.59%, p<0.05 )Conclusions: Using“6 sutures and 6 knots”to open the chest would greatly reduced incidence of pneumothorax and hemorrhea, and improve achievement ratio of operation significantly. Left ventricular artery(LVA) ligation is better than LAD in establishing ischemic/reperfusion model. There maybe some limitations to judge coronary occlusion by leadⅡST segment elevation.Part 2 Treatment of Ischemic Postconditioning Combined with Granulocyte-Colony Stimulating Factor in Acute Myocardial InfarctionObjective: To evaluate the potential protective effects and feasibility of IP , G-CSF and IP in combination with G-CSF on limiting infarct size, improve left ventricular function and remodeling after AMI , and the possible mechanism by the investigation of rabbit` heart AMI model, so as to provide new ideas for the treatment of AMI.Methods: After ischemia/reperfusion(I/R)was established by the ligation of left ventricular artery ( LVA) for 30 min , male New Zealand rabbits are randomly allocated to four groups(n=15): 1.Ischemic reperfusion group(IR): LVA occlusion 30min and reperfusion only, with saline(0.4ml) subcutaneous injection 1/d×5 12h after reperfusion; 2. G-CSF group(G-CSF): LVA occlusion 30min and reperfusion only, with G-CSF(10μg/kg) subcutaneous injection 1/d×5 12h after reperfusion; 3. Ischemic Postconditioning group(IP): Four episodes of 30s reperfusion and 30s occlusion after 30 min ischemia and before 30 min reperfusion, with saline subcutaneous injection 1/d×5 12h after reperfusion; 4. IP combined with G-CSF group (IP+G-CSF): Four episodes of 30s reperfusion and 30s occlusion after 30 min ischemia and before reperfusion, with G-CSF subcutaneous injection 1/d×5 12h after reperfusion. Electrocardiogram (ECG) monitoring were continue performed during the operation. Blood was drawn from femoral vein to evaluate white blood cell count (WBC) and cardiac troponin I(cTnI) values before operation and 7d after reperfusion in each group. Ultrasound cardiography was performed 4w after the operation to evaluate left ventricular remodeling and function. Myocardial infarcted size and cardiac muscle tissues of rabbits in each group were determined by staining with triphenyltetrazolium chloride dye. Morphologic examination of cardiac muscle tissues was done using optical microscope and transmission electron microscope. Apoptosis of cardiomyocytes peripheral to lesion were measured by Terminal deoxynucleotidyl transferase(TdT)-mediated dUTP-biotin nick end labeling (TUNEL) staining.Results: The ST-segment resolutions and cardiac arrhythmia scores were significantly decreased in IP group and IP+G-CSF group compared with other groups (P<0.05). WBC(×10~9) increased obviously in groups treated with G-CSF 1w later (IR5.4±3.8, G-CSF13.8±12.9*, IP8.5±5.6, IP+G-CSF 12.3±6.7*, *p<0.05vs. anterior treatment).Values of cTnI(ng/ml) in plasm 1w after operation were 3.34±2.21(IR), 1.62±1.65* (G-CSF,p<0.05 vs. IR), 1.69±1.70* (IP,p<0.05 vs. IR) and 1.54±1.02* (IP+G-CSF,p<0.05 vs. IR ), respectively. LVEF (%) measured by Ultrasound cardiography performed 4w after the operation were 58.2±7.5 (IR),62.21±9.3*& (G-CSF,*P<0.05 vs. IR, &P<0.05 vs. G-CSF+ IP), 61.3±8.7*& (IP,*P<0.05 vs. IR, &P<0.05 vs. G-CSF+ IP) and 70.9±9.8**(IP+G-CSF, **P<0.01 vs. IR), respectively. Myocardial infarct size(%) in each group were 31.37±12.18(IR), 19.56±7.39* (G-CSF,* P<0.05 vs. IR ), 18.27±4.76*( IP ,* P<0.05 vs. IR ) and 16.42±6.03* (IP+G-CSF, *P<0.05 vs. IR ), accordingly. Morphologic examination indicated that there were milder injuries of the cardiomyocyte of rabbits in IP , GCSF and IP+GCSF groups compared with IR group. Combined group was the best. Microvessel count (/0.01mm~2) of infarcted area in each group were 1.12±2.21(IR), 3.82±1.75* (G-CSF, *P<0.05 vs.IR), 2.1±1.49(IP) and 4.1±2.01**& (IP+G-CSF,**P<0.01 vs. IR,&P<0.05 vs. IP), respectively. There were more apoptotic cells in IR group(apoptotic index, 32±4%), higher than those of G-CSF(16±2%), IP(17±2%) and combined group (15±1%).Conclusions: Ischemic postconditioning at onset of reperfusion could obviously reduce myocardial reperfusion injury and inhibiting cardiomyocyte apoptosis . G-CSF protected the heart by inhibiting cardiomyocyte apoptosis , inducing neovascularisation and improve left ventricular function after AMI. There were no obvious difference heart protective effects between IP and G-CSF in our study. The protective effects of IP in combination with G-CSF through reducing reperfusion injury, inhibiting cardiomyocyte apoptosis and inducing neovascularisation, which were better than IP or G-CSF alone(p>0.05, mostly).IP combined with G-CSF is a safe and feasible strategy in the treatment of AMI.