| PrefacePersistent myocardial ischemia is known to result in cell injury and death. Reperfusion is very important for myocardium,but attachment of leukocyte-endothelial cell,aggregation of platelet-leukocyte and transendothelial leukocyte migration can result in cell death after reperfusion of ischemia myocardium,in addition to activation of complement and production of active oxygen free radicals also leads to severer myocardial damage,namely myocardial ischemia reperfusion injury.During the open heart operation,the coronary flow is blocked,which leads myocardium to be ischemic and hypoxemic.By the end of the operation,the coronary flow is recovered which leads to new myocardial injury,namely reperfusion injury.It is a key task in the field of cardiac surgery how to reduce myocardial ischemia reperfusion injury.The modified hyperpotassium cardioplegia was developed since Melrose created the hyperpotassic cardioplegia in 1955,which improved the effect of myocardial protection and decreased operative mortality.It is out of place or lacks sound academic and experimental basis to protect the immature myocardium using the cardioplegia suitable to mature myocardium.Recently the research on immature myocardial protection is being as a hotspot in myocardial protection.In this study,to investigate the protective mechanism of cold plasma cardioplegia on immature myocardium,we measured myocardial tumor necrosis factor(TNF) mRNA,Bcl-2 protein and cardiomyocyte apoptosis.MethodsPart one:Twenty healthy infant rabbits(age 14—26 days) weighting 350—580g were used in this study.The animal was placed in the left side position after anesthetized. Two-dimensional images and M-mode tracings were recorded from the long-axis view at the papillary muscle level.LV end-diastolic diameter(EDD),diastolic interventricular septum and pssterior wall thickness(ⅣSd,LVPWd) ejection fraction (EF),fraction shortening(FS) were measured and calculated,averaging 3 cardiac cycles. Mitral valve orifice flow velocity was recorded by Doppler flow imaging from the apical four-chamber view.Peak early velocity(E) and late filling wave(A) were measured.DTI was applied in the pulsed wave Doppler mode to allow for a spectral display of the mitral annulus velocities at its septal and lateral corners.Part two:Preparation for the isolated heart and Collection of samples:.After anesthesia by injection intramuscularly and heparinization by heparin-sodium 150 u/kg intravenously, the infant rabbits heart were excised rapidly,rinsed thoroughly in chilled 4℃saline and then suspended on a cannula,and the perfused retrogradely in the isolated Langendroff model with 37℃oxygenated K-H buffer solution at a constant pressure of 60 cm H2O for 10 minutes and 10 minutes coronary outflow solution was collected. Four control ones were stopped perfusion.After that,the isolated heart was s cooled down to 10℃,and 4℃cardioplegia(cold blood or plasma cardioplegia) was perfused at 15 ml/kg.After 30 minutes of hypothermic arrest,it is rewarmed and the K-H solution was perfused again.After 10 minutes equilibration,the time from the beginning of perfusion to heart beating and heart rate was recorded,10 minutes coronary outflow solution was collected.The rebeating time of arrested heart,heart rate, coronary outflow and myocardial water content in group P and in group B were respectively measured.Part three:We used immunohistochemical study to visualize the presence of TNF,in situ hybridization to visualize the presence and localization of TNF,TUNEL to observe apoptosis,reverse transcription-polymerase chain reaction(RT-PCR) to detect mRNA of Bcl-2.Results1.The values of EF,FS,E,A,E/A,Ea,Aa,Ea/Aa and Tei index respectively were 72.2±3.5%,37.5±3.0%,58.5±6.4cm/s,72.6±5.7cm/s,0.81±0.07,6.8±1.5cm/s, 8.5±2.3cm/s,0.84±0.24,0.31±0.05.2.The time arrested heart by the cold plasma cardioplegia or cold blood was respectively 40.5±2.2 sec or 80.0±2.0 sec,significant difference(P＜0.05 ).3.The rebeating time of arrested heart,heart rate,coronary outflow and myocardial water content in group P were respectively 46.4±8.7 sec,142.4±22.7bpm, 12.3±2.4 ml/min and 73%±5%,but in group B they were respectively 88.6±10.2sec, 122.8±14.7bpm,6.4±2.2 ml/min and 82%±14%.There was significant difference between group P and group B(P＜0.05).4.Immunostaining of TNF protein was not detected in the hearts without I/R injury.There was more obvious immunostaining in group B than in group P in the hearts undergoing I/R.The expression of TNF mRNA was not detected in the hearts without I/R injury.In situ hybridization of TNF was localized to cardiac myocytes.5.Apoptosis in cardiomyocytes was not observed by TUNEL in the control hearts. In contrast,there were a few apoptosis cardiomyocytes observed by TUNEL after I/R, and TUNEL staining was positive in these cells.6.Bcl-2 mRNA relative content in cardiomyocytes in control group,group P and group B were respectively 0.921±0.015,0.712±0.021 and 0.504±0.028,with significant difference(P＜0.01 or P＜0.05).Conclusions1.Left ventricular function could be evaluated in infant rabbit using High-frequency Echocardiography.Left ventricular diastolic function was abnormal, but systolic function was normal in infant rabbit.2.Cold plasma cardioplegia could arrest promptly beating heart at diastole.3.Cold plasma cardioplegia was able to increase recovery of coronary outflow and heart rate and to decrease myocardial edema after I/R.4.Cold plasma cardioplegia could restrain TNF expression in cardiomyocyte,and enhance Bcl-2 mRNA expression,which restrains apoptosis. |
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