| Purpose organ transplantation, such as transplantation of hearts, livers, kidneys and lungs, has become one of the common and effective therapeutic options for irreversible organ failure. Due to advancement of surgical techniques, effective use of immunosuppressive agents and improvement of postoperative care, graft survival rate and favorable prognosis have improved gradually. However, the problem of organ shortage has become prominent. The fact that isolated donor hearts cannot be preserved for a long time narrows the source of the organ, which has become the bottleneck for heart transplantation. Thus research centers worldwide have focused on studying methods for extending preservation time of donor hearts so as to expand the source of hearts. Based on recent research, methods for heart preservation include cold preservation, preservation in cold and high pressured oxygen, continuous perfusion preservation, intermittent perfusion preservation and preservation in very low temperature. The above methods can extend preservation time of donor hearts to a certain extent. In these methods, donor hearts are preserved in cardioplegic solution which works as a media for in vitro organ preservation. But as a matter of fact, due to absence of blood and oxygen-a major impediment for ATP generation, sodium, potassium and mercury in cardiomyocytes become inactive, which causes imbalance of osmolality of cells and as a result, there will be edema and irreversible damage on myocardial fibers. Thus, no breakthrough improvement can be made on isolated heart preservation. For now, it is agreed that 4-6 hours is the best we can do to preserve isolated hearts. Graft survival and favorable prognosis can be improved if we can find an effective method to preserve isolated hearts for a long time. And it will definitely bring new hope to patients with heart disease. In this study, a new preservation method was used and protective effect of the method on mice hearts subjected to cold ischemia and reperfusion was studied. Finally, the mechanism of the method will be looked into.Method C57BL/6 male mice were used to establish model of mice cervical heterotopic heart transplantation.48 donor mice,4-6 weeks old, were randomly divided into 4 equal groups(n=12). They were subjected to naive operation (Group A), sham operation (Group B), standard control (Group C) and experimental control (Group D) respectively.36 recipient mice,8-10 weeks old, were randomly divided into 3 equal groups (n=12). They were subjected to sham operation, standard control and experimental control respectively.Operation on donor mice:4-6 weeks old donor mice were anesthetized and subjected to injection by lml (100u/ml) of 4℃ heparin saline through inferior vena cava. Chest wall and diaphragm were opened to see the heart and hyperkalemia cardioplegic solution were injected through inferior vena cava slowly till the heart stopped beating. Hearts and lungs were removed and put into a culture dish filled with 4℃ heparin saline.Operation on donor hearts:under microscope, donor hearts were prepared by ligaturing cardiovascular except pulmonary artery and aorta. Microsurgical forceps was used to enter LA and RA by cutting through left and right atrial appendage. Blunt dissection of inter-atrial septum was applied to connect LA and RA. Tubes were put to connect the aorta stump and slow retrograde perfusion of solution was applied (Group A, B and C:HTK solution; Group D:KH solution). Perfusion pressure was set as 30cmH2O and perfusion time was around 2-3 minutes. After the above operation, donor hearts in Group A were sent for test while heart transplantation was performed for Group B. Donor hearts in Group C were preserved for 24 h in 15ml of 4℃ HTK solution. For donor hearts in Group D, tubes for perfusion were kept to connect the aorta stump so that the aorta stump was kept open. Then donor hearts were put into a high pressured gas (P02:3.2x101.3 kPa+PCO:0.8×101.3 kPa=4×101.3 kPa) bottle and preserved in a refrigerator at 4℃ for 24 h. Pressure change in the bottle was carefully observed and gas was added in time.Establishing model of mice cervical heterotopic heart transplantation:8-10 weeks old mice were taken as recipient mice. After satisfactory anesthetization, cervical skin was cut from the middle of the collarbone up to the right below the jaw to see and separate right internal jugular vein and common carotid artery. The far ends of the vein and artery were ligatured and the near ends were closed with micro vascular clips. Using the improved cuff method, vein stump was put into venous cannula and then fixed at the cannula wall. Heparin saline was used to clean vena cava and remove residual blood. The same method applied to artery. When the recipient mice was ready to receive transplantation, donor hearts preserved with different methods were put into culture dish with saline and ice and prepared. Saline was used to clean and gas was removed from left and right atrial appendage, pulmonary artery and aorta. Cut-off at the left and right atrial appendage was ligatured. Arterial cannula was removed from aorta stump and pulmonary artery and aorta were trimmed to appropriate length. Under microscope, common carotid artery with cannula of the recipient mice was put to connect aorta of donor hearts while internal jugular vein was put to connect pulmonary artery. Then aortic wall and pulmonary artery wall were ligatured and fixed on the cannula wall. After the operation, the near the heart end of the artery and vein was released to observe if bleeding and obstruction happened and to manage them accordingly.2 h observation was required and resuscitation was recorded. Re-beating rate of donor heart was recorded according to the beating state. After the observation, close the skin and put recipient mice in warm and separated cage.Testing after transplantation:Mice in Group A were killed after hearts were taken out and test samples were taken. Mice in the other groups were killed 24 h after transplantation and test samples were taken. Blood tests were done to observe Troponin I (Tnl) change in concentration. Using hematoxylin and eosin (HE) staining, myocardial tissues were taken to compare myocardial cells arrangement and edema, change of tissues around coronary artery and inflammatory cell infiltration. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) was used to identify apoptotic myocardial cells and apoptotic cell count was done under high magnification to calculate apoptotic index and the degree of apoptosis was compared. The expression of B cell lymphoma/leukemia-2 (Bcl-2) was observed by Western blotting (WB) to deduct possible reasons for apoptosis change.Results 2 h after successful establishment of model, it could be seen from the microscope that donor hearts in Group B were beating strongly with sinus rhythm and that ventricular wall was soft and resilient. Also coronary was clear without obvious bleeding or congestion. In Group C, hearts were presenting dark red color with stiff ventricular wall. Most of the hearts were not beating or beating partially. Arrhythmia was presented. Congestion in the coronary and diffuse hemorrhage around the coronary were found. In Group D, hearts were presenting red color with fewer resiliencies. Ventricular wall was slightly stiff. Sinus rhythm was presented but cardiac contractility was weaker than Group B. Blood flowed smoothly in the coronary and a few bleeding was found scattering around the coronary. In Group B, C and D,10,2 and 9 hearts were rebeating, representing a rebeating rate of 83.3%,16.7% and 75.0% respectively.In terms of rebeating rate, the difference between Group D and B was not significant (P>0.0167) while Group D was significantly higher than Group C (P<0.0167). The concentration of cTnI of the four groups were (0.28±0.02) ng/ml in Group A. (0.37±0.03) ng/ml in Group B,(2.02±0.05) ng/ml in Group C and (1.59±0.04) ng/ml in Group D. The difference among groups was significant (F= 6404.74, P<0.01). Group B was slightly higher than Group A (P<0.001) while Group D was significantly higher than Group B (P<0.001) but lower than Group C (P<0.001). The difference was significant.HE staining of the four groups was observed under optical microscope. In Group A and B, myocardial cells were found in normal order and no obvious edema were presented. In Group B, neutrophils could be found scattering among cells. In Group C, myocardial cells were in disordered arrangement with obvious edema. Numerous erythrocytes and neutrophils are found among cells. In Group D, slightly disordered arrangement of myocardial cells and slight edema were presented. A few inflammatory cells could be found among cells but they were much less than that in Group C. Erythrocytes was not found. Tunel testing showed that myocardial cells in Group A were stained equally and slightly and that abnormal karyotypes were not found. In Group B, myocardial cells were equally stained with a few apoptotic green cells while numerous of them were found in Group C. In Group D, some apoptotic green cells were found but that was significantly less than Group C. The index for apoptosis was 1.08±0.15、3.96±0.41、15.83±1.33 、4.21±0.28 for the four groups respectively and the difference was significant (F=1003.67, P<0.01). Compared to Group A and B, Group C and D had a higher apoptosis index, which was significant (P<0.05). The index for Group D was lower than that in Group C, which was also significant (P<0.05). The difference between Group D and B was not significant. (P=0.44>0.05)The results of Western-blot showed that the expression of Bcl-2 were (0.14±0.01), (0.18±0.01), (0.62±0.05), (0.71±0.05) for the four groups respectively. The difference among groups was significant (F=750.79, P<0.001). The difference between each pair of groups was also significant (P<0.01). Bcl-2 expression was the highest in Group D.Conclusions The results have suggested that high pressured CO preservation is an effective method for heart preservation. This method can effectively extend preservation time of isolated mice hearts, reduce cold ischemia and reperfusion injury and improve resuscitation rate. The potential mechanism of reducing cold ischemia and reperfusion injury probably lies in the following three areas. Firstly, necessary oxygen for myocardial cells is provided by high pressured oxygen in the process of cold preservation. This can partially alleviate hypoxia and strengthen aerobic respiration. As a result, generation and supply of energy will be increased, which guarantees myocardial cell’s survival for a longer time. Secondly, CO can restrain the activity of some enzymes, which reduces myocardial cell’s consumption of energy during cold preservation and reduces the need for energy substances required for long-term survival of cells. At the same time, by increasing Bcl-2, the activation and development of apoptosis of myocardial cells after reperfusion will be restrained and as such dysfunction of hearts will be reduced and resuscitation rate will be increased. Thirdly, the improved KH solution has played an inevitable role in dry preservation. KH solution forms a high penetration environment, which transfers water from inside the myocardial cells to outside. As a result, myocardial cells are shrank in size and are in the state of comparatively short of water. This, on one hand, increases tolerance of high pressure for cells, and on the other hand, reduces activity of some enzyme and thus reduces consumption of energy and extents survival time of cells so that reperfusion injury will be further reduced. With the joint effects of the above three, high pressured CO preservation alleviates cold ischemia and reperfusion injury and extents preservation time. More study should be done to reveal the actual mechanism of high pressured CO preservation. |
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