| Objective By constructing the non-heart-beating donors (NHBD) models induced by rapid exsanguination, we made a comprehensive analysis of the cardiac energy metabolism, function, structure and cell apoptosis during warm ischemia. And then we investigated the effects of normal saline in rat heart from non-heart-beating donors during agonal period, thereby providing rational clinical application.MethodsSprague-Dawley (SD) male rats (n=296) were randomized into 6 groups: A, B, C, D, E and F. Each group was randomly divided into 8 subgroups: 1, 2, 3, 4, 5, 6, 7 and 8. Group 1 (control group): Hearts were harvested immediately. Group 2: Hearts were left in situ for 30 minutes after cardiac arrest was induced by rapid exsanguination. Group 3: Hearts were left in situ for 45 minutes after cardiac arrest was induced by rapid exsanguination. Group 4: Hearts were left in situ for 30 minutes after cardiac arrest was induced by rapid exsanguination and then were excised and immersed in a 4℃St. Thomas Hospital solutions 1 (STH-1) for 1.5 hours. Group 5: Rats were transfused normal saline between the beginning of exsanguination and cardiac arrest happened and then the hearts were left in situ for 30 minutes before the hearts were excised and immersed in a 4℃solutions for 1.5 hours. Group 6: Rats were transfused normal saline for 30 minutes after cardiac arrest happened and then the hearts were excised and immersed in a 4℃solutions for 1.5 hours. Group 7: Rats were transfused normal saline between the beginning of exsanguination and the beginning of excising hearts and then the hearts were immersed in a 4℃STH-1 solution for 1.5 hours. Group 8: Hearts were left in situ for 45 minutes after cardiac arrest was induced by rapid exsanguination and then were excised and immersed in a 4~CSTH-1 solution for 1.5 hours. Group A (n=96): The heterotopic NHBD heart transplantation model was established by using cuff technique for prolonged survival observation. Group B (n-48): The High-performance liquid chromatography (HPLC) was used to detect the change of energy metabolism with ATP, ADP, AMP, TAN and EC. Group C (n=48): The hearts were harvested to determine the percentage of water content. Group D (n=48): The myocardial function was recorded and evaluated with HR(bpm), LVP(mmHg), ~dp/dt(mmHglms) and CF by an isolated non-working-heart model. Group E (n=8): H-600 transmission electron microscopy was used to detect the ultrastructure of myocardial cell. Group F (n=48): Apoptotic myocytes were detected with terminal deoxynucleotide transferase-mediated deoxyuridine-biofin nick end labeling (TUNEL) method and the expressions of Bcl-2 protein, Bax protein and Caspase-3 protein were detected by immunohistochemistry analysis.Results There was no significant difference in donor heart survival between Group 1 and Group 2 (P＞0.05). Energy metabolism (ATP, ADP, TAN and EC) and myocardial function had significant difference between Group 1 and Group 2 (P＜0.05). The occurrence of apoptosis was significantly higher in Group 2 than in Group 1(P＜0.001). The expression of Bcl-2 protein was significantly lower in Group 2 than in Group 1 (P=0.006), while the expression of Bax protein and Caspase-3 protein was higher in Group 2 (P＜0.05). Hematoxylin-eosin staining indicated that myocardial alteration was not significant in Group 2. The ultrastructial changes of myocardium including the pathological changes of myofibril, mitochondria and sarcplasmic reticulum in Group 2 was slight. The donor hearts of Group 3 could not be reanimated after heterotopic transplantation or after perfusion. ATP,ADP,TAN and EC were significantly lower in Group 3 than in Group 2 (P＜0.001). The occurrence of apoptosis was higher in Group 3 than in Group 2(P=0.038). The expression of Bcl-2 protein was significantly lower in Group 3 than in Group 2 (P=0.006), while the expression of Bax protein and Caspase-3 protein was higher in Group 3(P＜0.05). Hematoxylin and eosin staining indicated that myocardial necrosis occurred in Group 3. Electron microscopy showed severe mitochondrial swelling and formed, disruption and dissolution of myofilaments. Glycogen granules in myocardial cells decreased severely. There is no difference in cardiac energy metabolism, function, structure and donor heart survival among Group 4, Group 5 and Group 6. The difference of donor heart survival and the percentage of water content between Group 1 and Group 7 had no statistical difference (P＞0.05). There is no statistically significant difference in LVP and -dp/dtmax between Group 1 and Group 7 after 10 min, 20 min and 30 min perfusion, respectively. There is also no statistically significant difference in CF between Group 1 and Group 7 after 30 min perfusion. The donor heart survival between Group 7 and Group 4 had significant difference (P＜0.001), also the level of energy metabolism and function in Group 7 were higher than that in Group 4 (P＜0.05). The occurrence of apoptosis was higher in Group 4 than in Group 7(P=0.013). The expression of Bax protein was significantly lower in Group 7 than in Group 4 (P=0.005), while the expression of Bcl-2 protein and Caspase-3 protein had no difference between Group 4 and Group 7 (P＞0.05). The ultrastructial changes of myocardium including the pathological changes of myofibril, mitochondria and sarcplasmic reticulum in Group 7 was slighter than Group 4. The donor hearts of Group 8 could not be reanimated after heterotopic transplantation or after perfusion. The percentage of water content was significantly higher in Group 8 than in the Group 1(P=0.044).Conclusions It was clear that warm ischema injury the NHBD cardiac energy metabolism, function and structure, and also induce cell apoptosis. The results of this study suggest that normal saline could exert protective effects on myocardium during warm ischemia, although it was limited. |
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