| Research backgroundNowadays, with the development of medicine, it is widely accepted that cardiac transplantation can be one of the most effective therapies for the chronic and end-stage heart failure. However, the shortage of donor hearts has been the limitation of cardiac transplantation. At present, more and more patients who are in need of cardiac transplantation go abroad for seeking donor hearts, while in fact, even the foreign hospitals can not guarantee the supply of donor hearts, which therefore can not satisfy the demand of increasing heart transplantations. As a result, the long waiting of candidates for cardiac transplantations is leading to financial burden for medical system and society. Thanks to the development of technique of cardiac transplantation, the survival rate and life quality of the patients who receive heart transplantations have been improved dramatically. However, the number of candidates for cardiac transplantation is also increasing steadily, and unfortunately the shortage of donor hearts is more evident. It has been generally believed that one of the most important reasons of the lack of donor heats is that donor heart can not be preserved for a long period.At present, the standard technique of heart preservation is cardiac arrest followed by static cold storage in a crystalloid heart preservation solution. However, this technique of heart preservation can result in cellular edema, acidosis and metabolic disorders. Therefore, this technique ensures an acceptable level of heart protection against ischemia-reperfusion injury for less than 6 hours. The timing of heart preservation more than 6 hours may lead to irreversible damage in the donor heart and therefore negative impact on survival rate on the patients who have received heart transplantations. As a result, the growing shortage of donor hearts is one of the major factors stimulating the development of new techniques of heart preservation, and it is quite necessary to seek a more advanced technique of heart preservation.Recent researches have shown that ischemia-reperfusion injury is one of the most common pathological processes in cardiac transplantations, which is the major factor of donor heart failure and postoperative complications. Consequently, alleviating ischemia-reperfusion injury is one of the keys to a successful cardiac transplantation.Recently, a great number of experimental and clinical evidences have pointed out that myocardial apoptosis is one of the most significant pathological mechanism of ischemia reperfusion injury in cardiomyocytes. What is more, myocardial apoptosis is regulated by different genes, such as Bcl-2, Bax, and the family of Caspase(Cysteine aspartase). As a result, we may relieve ischemia reperfusion injury in the donor heart by inhibiting the apoptotic process, which may provide a new dimension for the technique of heart preservation.In 1998, Seki et al. focused on cryptobiosis, which decreases the amount of water in a living body in order to adapt to an extreme environment such as dryness and low temperature. Seki et al. showed that tardigrades can survive even under ultrahigh pressure. Consequently, he suggested that this lesson learnt from nature should be applied for organ preservation. At first, Seki et al. have conducted experiments which aimed at reducing the amount of water using a PFC solution for the preservation of isolated rat donor hearts before heart transplantations and obtained quite good results, however, the reproducibility was not sufficient. However this study has demonstrated this pioneering technique of heart preservation, which is heart preservation by desiccation.Although Carbon Monoxide is regarded as a poisonous gas which is extremely fatal to human body, the protective effects of Carbon Monoxide are also becoming the focus of the researchers recently as the functions of nitric oxide as a biological regulator have been found out gradually. During the last decade, it has been shown by related studies that exogenously provided Carbon Monoxide is able to ameliorate ischemia reperfusion injury in the organ transplantation with its anti-inflammatory and anti-apoptotic effect. Therefore, Carbon Monoxide can be used as a therapeutic tool for diseases even in the clinical setting.As a result, the Japanese researchers invented a new technique of heart preservation, preservation by desiccation using high-pressure Carbon Monoxide, which combine the potential advantage of desiccation and Carbon Monoxide. In the previous study, Yoshida et al have conducted a series of experiments with isolated rat hearts preserved by desiccation with high-pressure Carbon Monoxide, and attained a significant reproducibility after cardiac transplantations. Therefore the experiments prove that preservation by desiccation using high-pressure CO is beneficial for the storage of donor heart. However, the studies also have their own limitations:Firstly, they merely reported the revival of donor hearts without further research about the underlying mechanism. Secondly, the objects of researches are limited to murine. Thirdly, the endpoints are quite subjective, including the revival of donor hearts, electrocardiogram of donor hearts and so on.Therefore, it is necessary to make further researches on the related molecular mechanisms of preservation by desiccation using high-pressure Carbon Monoxide and provide more reliable and scientific evidences.Objective of researchBased on the earlier Japanese experiments with rats, we conducted the present study on rabbits to evaluate and explore the efficacy and mechanism of anti-apoptosis potency of this new method in comparison with the traditional Histidine Tryptophan Ketoglutarate (HTK) preservation.Innovation of research(1)Nowadays in the clinical setting and experimental researches, Histidine-Tryptophan-Ketoglutarate or the University of Wisconsin solution preservation is the major method of heart preservation, while in this study, we adopt a new technique of heart preservation, preservation by desiccation using high-pressure Carbon Monoxide.(2)In the previous Japanese studies, the objects of research are limited to murine, and endpoints in the studies are quite subjective, which are the revival rate of donor hearts, the cardiogram of donor hearts and so on. While in this present study, we test the feasibility of preservation by desiccation using high-pressure Carbon Monoxide on rabbits and explore the efficacy and mechanism of anti-apoptosis potency of this new method in comparison with the traditional Histidine-Tryptophan-Ketoglutarate preservation, which can provide scientific and reliable evidences for this pioneering technique of heart preservation.Method of research(1)48 New Zealand rabbits weighting 2.0-2.5kg were divided into three groups randomly, namely, Firstly, Naive group (n=16):The donor hearts were transplanted immediately after they were being extracted. Secondly, Histidine Tryptophan Ketoglutarate group(n=16):The donor hearts were extracted and steeped in4℃ Histidine Tryptophan Ketoglutarate cardioplegic solution for 18 hours and then were transplanted.Thirdly, Desiccation using high-pressure Carbon Monoxide group(n=16):After the donor heart was extracted, a modified Krebs-Henseleit (KH) solution(As for the KH solution, we dissolved glucose at three times as high as the normal levels) was injected into the isolated rabbit hearts chamber through the aorta and exposed to a gas mixture(P02=3200hPa, PCO=800hPa) in the chamber (Fig 1) before they were being preserved in a refrigerator at 4℃ for 18 h and then were transplanted(2)Rabbit heterotopic abdominal cardiac transplantation models were established. Firstly, the recipient rabbit was anesthetized and prepared for celiotomy, and then a midline celiotomy was performed. A 7cm midline abdominal incision was made from the middle of the abdomen to the symphysial surface of the pelvis. Secondly, we exposed the recipient’s abdominal aorta and caudal vena cava. Thirdly, after preparing the recipient rabbit, the preserved heart was transferred from the chamber to a refrigerator and temporarily immersed in normal saline and end-to-side anastomosis of the donor’s aorta to recipient’s aorta and donor’s pulmonary artery to recipient’s caudal vena cava was performed. Loupe magnification and high-quality lighting could facilitate the anastomosis. We could evaluate the anastomosis for leakage by partially opening the cross clamp, If no leakage was observed, the cross clamp was released completely. The transplanted heart started beating within the next minute or two.(3)After perfuse the donor hearts for one hour, serum myocardial enzyme, including Creatine Kinase, Creatine Kinase-MB, lactate dehydrogenase and Aspartate transaminase from three groups were tested. Secondly, the protein was extracted from cardiomyocytes in the left ventricle from three groups and the expressions of caspase-3, Bcl-2 and Bax were detected by using western blot technique. Thirdly, apoptosis indexes of eardiomyocytes from three groups were evaluated by Transferased UTP nick end labelling technique. And finally, the pathological changes were evaluated by Hematoxylin-Eosin staining technique.Results of research(1)Compared with Naive group, the amount of serum myocardial enzyme, including Creatine Kinase, Creatine Kinase-MB, lactate dehydrogenase and Aspartate transaminase significantly increased (P< 0.05, n=8) in Histidine Tryptophan Ketoglutarate group and Desiccation using high-pressure Carbon Monoxide group (P < 0.05, n=8). While in comparison with Histidine Tryptophan Ketoglutarate group, the amount of serum myocardial enzyme, including Creatine Kinase, Creatine Kinase-MB, lactate dehydrogenase and Aspartate transaminase significantly reduced in Desiccation using high-pressure Carbon Monoxide group(P< 0.05, n=8).(2)Compared with Naive group, the ratio of Bcl-2 and Bax protein expression significantly decreased (0.89±0.76 vs 0.68±0.24 vs 0.57±0.01, P< 0.05, n=8), and Caspase-3 protein expression significantly increased in Histidine Tryptophan Ketoglutarate group and Desiccation using high-pressure Carbon Monoxide group (0.41±0.03 vs 0.66±0.04 vs 0.63±0.112, P< 0.05, n=8). While in comparison with Histidine Tryptophan Ketoglutarate group, the ratio of Bcl-2/Bax protein expression significantly raised and Caspase-3 protein expression significantly reduced in Desiccation using high-pressure Carbon Monoxide group(P< 0.05, n=8).(3)Compared with Naive group (2.25±1.04)%, the up-regulation of apoptosis index was significantly observed in in Histidine Tryptophan Ketoglutarate group(33.00±6.91)% and Desiccation using high-pressure Carbon Monoxide group(5.75±3.01)%(P< 0.05, n=8). While in comparison with Histidine Tryptophan Ketoglutarate group, the reduction of apoptosis index was significantly observed in Desiccation using high-pressure Carbon Monoxide group.(4)Hematoxylin-Eosin staining revealed no obvious structural abnormalities in the naive group. Myocardial cell were arranged in three to four discrete, tightly packed layers, and their nuclei were round, large, and lightly stained. In contrast, compared with Desiccation using high-pressure Carbon Monoxide group, obvious histopathological damage was observed in Histidine Tryptophan Ketoglutarate group. The myofibers were disarrayed and the number of myocardial cells was decreased. Additionally, pyknotic nuclei were observed in the remaining cells, some of which exhibited shrunken or irregular morphology.Conclusion of researchCompared with traditional Histidine Tryptophan Ketoglutarate preservation, Preservation by desiccation using high-pressure Carbon Monoxide could alleviate rabbits’ myocardial histopathology and apoptosis induced by ischemia reperfusion injury through adjusting the ratio of Bcl-2/Bax protein expression, thus resulting in the reduction of expression of Caspase-3. However, the underlying signaling mechanism how this kind of storage regulate the ratio of Bcl-2/Bax and the expression of Caspase-3 is not well clarified, therefore, further study is needed to focus on it. |
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