Experimental Study Of Beating Heart Transplantation

In the experiment,the models of beating heart transplantation designed by ourselves(continuous perfusion of donor heart with oxygenated blood in the beating state)and arrested heart transplantation with modified Heron's technique (myocardial preservation by hypothermy potassium arrest)were established in order to study the effect of beating heart transplantation[avoiding ischemical reperfusion injury(IRI)model]on many aspects of transplanted hearts,such as myocardial preservation,immunogenicity and cardiac allograft vasculopathy (CAV),comparing with arrested heart transplantation(IRI model),which may provide scientific basis for the theoretical and practical development in organ transplantation.The conclusion was that,because of avoiding IRI,beating heart transplantation effectively prevented myocardial apoptosis,therefore improved the quality of preserved heart;greatly reduced the expressing level of MHCⅡand B7-2 mRNA and absolutely kept B7-1 mRNA and protein from expressing in transplanted heart,therefore reduced the immunogenicity of transplanted heart,which were benefit for palliating acute rejection;effectively protected donor's cardiac coronary endothelial function and eNOS/NO pathway from injury,which were benefit for CAV prevention.However,because of IRI,arrested heart transplantation induced donor's myocardial apoptosis significantly,improved the immunogenicity of transplanted heart greatly and CAV progressed acceleratively. PARTⅠEstablishment of beating heart allotransplantation in heterotopic modelObjective:To establish the model of beating heart transplantation in order to study the effect of continuous perfusion of donor heart with oxygenated blood in the beating state[avoiding ischemical reperfusion injury(IRI)]on many aspects of the transplanted heart,comparing with traditional preservation methods. Methods:The model of substituent of cardiopulmonary bypass unit with living rat,which was devised by ourselves,helped to oxygenate venous blood, and send the oxygenated blood to the donor's heart to keep it bright red and beating rhythmically.Then the donor's heart were transplanted to the recipient with the method of double vessel cannula anastomosis.In this way,the model of cervical beating heart transplantation in rats was established easily.Results:53 consecutive transplantations had been performed by double surgeons,50 of which were successful,with achievement ratio of 94.3%.The total time of surgical procedure was 90 to 120 min for each case.The mean time was respectively 18 min for preparing the recipient's cervical vessel cannulas,33 min for selecting the donor's heart,8 min for heart transplantation in cervical part and 15 min for extracorporeal perfusion.Conclusion:The model of substituent of cardiopulmonary bypass unit with living rat helped to establish the model of beating heart transplantation,which was a simplified,economical,practicable and high reproducible model without IRI,could be operated by surgeons without cardiopulmonary bypass unit and minimal microvascular technique,and applied to various transplantation studies. PARTⅡEstablishment of rattish arrested heart transplantation model with modified Heron's techniqueObjective:To modify Heron's technique for the model of heart transplantation in rats in order to establish a more reasonable and practicable model so that it fit for the requirement of the experiment.Methods:Heron's technique was improved in the aspects that the donor's ascending aorta and right pulmonary artery were respectively anastomosed to the recipient's right common carotid arterial cannula and external jugular venous cannula in the way of cerclage with a 5-0 silk suture and left pulmonary artery was closed.Then the model of rattish cervical heart transplantation was established with modified Heron's technique. Results:52 consecutive transplantations had been performed by single surgeon, 50 of which were successful,with achievement ratio of 96.2%.There was no anastomosis leakage,recirculation disorder and recipient's death after operation. The total time of surgical procedure was 50 to 60 min for each case.The mean time was respectively(18±3)min for preparing the recipient's cervical vessel cannulas,(10±1)min for selecting the donor's heart,(6±1)min for heart transplantation in cervical part and(16±1)min for cold ischemia of donor's heart. Conclusion:The modified Heron's technique was a more rensonable and practicable method to establish a reliable and high reproducible model of heart transplantation that could be operated by surgeons without minimal microvascular technique and applied to various transplantation studies. PARTⅢThe effect of continuous perfusion of donor heart with oxygenated blood in the beating state on myocardial apoptosis of the transplanted heartObjective:To study the effect of continuous perfusion of donor heart with oxygenated blood in the beating state on myocardial apoptosis of the transplanted heart.Methods:All cases were randomly divided into 3 group:control group(no transplant),beating group and arrested group.Specimens were collected on 3rd and 7th day postoperation.The variance of serumal MDA and T-SOD was respectively tested by thiomalonylurea method and xanthinoxidase method; the expressing levels of Bcl-2 and Bax protein were detected by immunohistochemical method;myocardial apoptosis in situ was test by TUNEL method; myocardial ultrastructural alteration was observed by transmission microscope. Results:The level of serumal MDA of arrested group was significantly higher than that of beating group(P＜0.05)(the top value was on 3rd day postoperation), but that of T-SOD was lower significantly than that of beating group(P＜0.05)(the valley was also on 3rd day postoperation).There was no significance about the levels of serumal MDA and T-SOD between beating group and control group(P＞0.05).The value of Bcl-2/Bax in arrested group was significantly lower than that in beating group and control group on 3rd day postoperation(P＜0.05).The apoptotic index(AI)of control group,beating group and arrested group was respectively 0.54±0.12,0.73±0.17 and 4.92±0.40;the AI of arrested group was significantly higher than that in control group and beating group(P＜0.05),but there was no significance between beating group and control group(P＞0.05).The ultrastructural change of myocardial tissues was minimal in beating group,but outstanding in arrested group.Conclusion:That donor's hearts were continuously perfused with oxygenated blood in the beating state, greatly avioded the injury of ischemical reperfusion on transplanted hearts, effectively protected anti-apoptotic factor such as Bcl-2 and inhibited apoptotic factor such as Bax,all of which prevented myocardial apoptosis.Therefore,better preservation of donor's hearts was achieved,which also further proved the feasibility about the model of beating heart transplantation.PARTⅣThe effect of continuous perfusion of donor heart with oxygenated blood in the beating state on immunogenicity of the transplanted heartObjective:To study the effect of continuous perfusion of donor heart with oxygenated blood in the beating state on immunogenicity of the transplanted heart.Methods:All cases were randomly divided into 3 group:control group (no transplant),beating group and arrested group.Specimens were collected on 7th day postoperation.The levels of expressing MHCⅡmRNA and B7-2 mRNA in the transplanted hearts were detected by Real Time PCR.B7-1 mRNA and protein were respectively detected by Reverse-Transcription PCR and Western blotting.Results:7 days post transplant,MHCⅡmRNA and B7-2 mRNA were constitutively expressed in the normal myocardial tissues of control group.The relative intensity of MHCⅡmRNA expressed in myocardial tissues of control group,beating group and arrested group was respectively 1.36±0.26,1.48±0.31 and 9.29±0.53;and that of B7-2 mRNA was respectively 1.37±0.24,1.46±0.30 and 5.45±0.43.The expressing levels of both MHCⅡmRNA and B7-2 mRNA in arrested group were significantly higher than that in beating group or control group(P＜0.05),but there was no significance between beating group and control group(P＞0.05).Both B7-1 mRNA and B7-1protein could not be detected in control group and beating group,while which could be be detected in arrested group.Conclusion:Ischemical reperfusion injury(IRI) definitely improved the immunogenicity of transplanted heart,but that donor hearts were continuously perfused with oxygenated blood in the beating state,could greatly reduce the immunogenicity of transplanted heart because of avoiding IRI,which were benefit for palliating acute rejection.PARTⅤThe effect of continuous perfusion of donor heart with oxygenated blood in the beating state on cardiac allograft vasculopathyObjective:To study the effect of continuous perfusion of donor heart with oxygenated blood in the beating state on cardiac allograft vasculopathy. Methods:All cases were randomly divided into 3 group:control group(no transplant),beating group and arrested group.Specimens were collected on 120th day postoperation.The level of serumal NO was tested with nitrate reductase method.The levels of expressing eNOS mRNA and iNOS mRNA in the myocardial tissues were detected by Reverse-Transcription PCR.Cardiac allograft vasculopathy was observed and graded by light microscope after HE staining and Elastin-Van Gieson staining.Results:120 days post transplant,the relative intensity of eNOS mRNA expressed in myocardial tissues of control group,beating group and arrested group was respectively 0.98±0.10,0.84±0.11 and 0.25±0.07;The expressing levels of eNOS mRNA in arrested group was significantly lower than that in beating group or control group(P＜0.05).iNOS mRNA could not be detected in myocardial tissues of control group and beating group,while could be detected in arrested group.The level of serumal NO in beating group and arrested group was respectively 98.70±14.30μmol/L and 78.38±10.96μmol/L,and that of arrested group was significantly lower than that in beating group(P＜0.05).The score of CAV in control group,beating group and arrested group was respectively 0,0.72±0.21 and 2.75±0.28;that of arrested group was significantly higher than that in beating group(P＜0.05).Conclusion" Ischemical reperfusion injury(IRI)induced coronary endothelial injury and dysfunction in transplanted heart,which resulted in dysregulation of eNOS pathway and decrease of NO production,and ultimately contributed to pathologic alterations in vascular structure that were observed in CAV.But that donor hearts were continuously perfused with oxygenated blood in the beating state, could greatly reduce the coronary endothelial injury and dysfunction,and protect eNOS pathway in transplanted hearts because of avoiding IRI,which were benefit for CAV prevention.