| PrefaceMyocardial ischemia - reperfusion injury is a common clinical problem associated with cardiopulmonary bypass, coronary bypass, angioplasty and throm-bolysis. The pathogenesis of myocardial ischemia - reperfusion injury has been studied as an important problem for many years. Although there are two main hypotheses, namely oxidative stress and Ca2+ overload, which have been proposed to explain the pathogenesis of ischemia - reperfusion injury, its exact mechanisms are not well defined. Tumor necrosis factor (TNF) is a proinflam-matory cytokine that has been implicated in the pathogenesis of cardiovascular diseases, including acute myocardial infarction, chronic heart failure, atherosclerosis , viral myocarditis and cardiac allgraft rejection. Myocardial TNF contributes to myocardial dysfunction and cardiomyocyte death in ischemia reperfu-sion injury. There are two forms of TNF, TNF - a and TNF -β. For the purpose of this study, TNF refers to TNF - a, which is the form associated with ischemia - reperfusion injury.Many studies have recently focused the role of TNF in ischemia - reperfusion injury and the treatment of anti - TNF, however, the exact mechanism (s) for any potential TNF - induced effects in ischemia - reperfusion injury is ( are) not well defined. The purpose of this study is to further examine the mechanism for TNF in ischemia reperfusion injury. Accordingly, in the present study we systematically examined the level of myocardial TNF, myocardial TNF mRNA and protein, Fas/Fas - L mRNA and protein, cardiac myocyte apoptosis by ter-minal deoxytransferase - mediated bio - dUTP nick - end labeling ( TUNEL) and electron microscopy in isolated reperfused heart model in rat. We also used anti -oxidant agent (astragli injection) and observed the changes of TNF. This study would provide new insights and important theratical bases preventing myo-cardial ischemia reperfusion injury by appropriate anti - TNF treatment.Material1. Animal: Thirty - four adult male Wistar rats weighing 250 - 300 g were used in the study.2. Major instruments; Olympus BC60 light microscopy, Kodak ID gel -imaging system analysoa, PTC - 100 PCR extender, comminuter, Philips CM 10 transmission electroscope, HERMLEZ383K hypothermic high -speed centrifu-ger.3. Major reagents; TNF, Fas/Fas - L immunohistochemistry kit, TNF, Fas/Fas - L in situ hybridization detecting kit, TaKaRa PCR reagent, TNF enzyme - linked immunosorbent assay reagent, astraglus injection.Methods1. Preparation of the isolated heart; The isolated, crystalloid - perfused rat heart model was used as described previously. After anesthesia induction ( Ure-thane 1g/kg intraperitoneally) and heparinization ( Heparin -sodium 500U in-traperitoneally) , hearts were excised rapidly, rinsed thoroughly in chilled (4℃ ) saline, suspended on a cannula, and then perfused retrogradely in the i-solated Langendorff model, at a constant pressure of 70 mmHg, with 37℃ oxygenated Krebs - Henseleit ( KH) buffer. Thirty - four Wistar rats were divided randomly into three groups; Control group, I/R group and astraglus group.After 15 minutes equilibration, the hearts in I/R group were perfused retrogradely with 37℃ cardioplegia (Thomas' solution) for 2 minutes, the hearts received astraglus injection in the cardioplegic solution (10 ml/100 ml,2 ml/kg) in astraglus group. The hearts in I/R group and astraglus group received 30 minutes of global ischemia and reperfused with KH solution at 37℃. The sampleswere collected at postischemia and 30, 60, 120 minutes of reperfusion respectively in I/R group and astraglus group. The hearts of control group did not undergo global ischemia and perfused with KH solution consistently at 31℃. The samples were collected at the corresponding points for the sake of contrast.2. Collection of samples: At the end of the experiments, a 3 -5 gram sample of myocardium was excised from the suspended heart and fixed in 4% paraformaldehyde (contains 0.1% DEPC) at 4℃ for 12 -24 h. The tissue was... |
