The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.
Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.
Cardiac transplantation represents the therapy of choice for patients suffering from different end-stage heart diseases. Advances in surgical techniques, more effective prophylaxis of infections, and novel immunosuppressive regimens resulted in markedly improved outcome of organ transplantation6. However, long-term graft survival has not improved rigorously over the past years7. Chronic rejection, characterized by transplant arteriosclerosis continues to be a major obstacle to long-term graft survival8-11.
The model of heterotopic heart transplantation in mice provides an important and valid tool for analysis of immunological mechanism during acute as well as chronic rejection12-15.
To date the most frequent transplant model is still the abdominal mouse heart transplantation using the suture technique. The ascending aorta of the donor heart is anastomosed to the abdominal aorta and the pulmonary artery is anastomosed to the recipient’s inferior vena cava. Much of the microsurgical difficulty of the suture model is based on the small size of the vessels, which are sutured16,17.
In contrast to the suture model the heart is placed in the neck region of the recipient where the external jugular vein is anastomosed to the pulmonary artery and the common carotid artery the aorta of the donor.
The rationale of the development of the cervical heart transplantation model using the cuff technique was to have an animal model, which allows achieving high success rates with basic microsurgical skills that will allow broad application of this model. The major advantages of this method are the significantly less occurring anastomosis related complications like hemorrhages and thrombosis when compared to the suture model18.
Animals are housed in a barrier pathogen free facility. All animals receive human care in compliance with the “Principals of Laboratory Animal Care” formulated by the National Society for Medical Research” and the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication no. 86-23, revised 1985). All experiments are approved by the Austrian Ministry of Education, Science and Culture.
1. Recipient Preparation
2. Heart Procurement
3. Implantation
4. Postoperative Care, Endpoint
Figure 2 shows representative survival data of cardiac allografts from BALB/c donors after transplantation into fully MHC mismatched C57BL/6 recipients. Hearts are rejected on day 7 ± 1 after transplantation. Finger palpation of the transplanted allograft is a sensitive method to detect the time dependent course of allograft rejection. All syngeneic heart transplants performed in our series survived indefinitely (>150 days).
Figure 1: Intraoperative pictures. (a) Operative field during recipient preparation. (b) Heart graft after procurement. (c) The heart graft placed in the recipient neck region in an upside down position. (d+e) The donor site prior to implantation.( f) Schematic diagram illustrating the recipient vessel, the donor vessel and the cuff
Figure 2: Cardiac allograft survival. Kaplan-Meier plot displaying the survival of cardiac allograft from BALB/c mice after grafting into fully MHC- mismatched BL6 recipients (n = 7; mean survival time = 7 days) as well as a syngeneic control group (n = 7; graft survival >150 days). The day of rejection was defined as the day of cessation of heart beat.
Rejection of vascularized allografts entails a plethora of different steps which can only be insufficiently assessed using in vitro models. Phenomena like the sensitization of recipients, antigen processing in secondary lymphoid organs but also the differentiation and proliferation of immune-competent cells are better characterized in vivo. Animal models are thereby the ideal tool for translational research16. Mouse models still represent the gold standard in basic transplant and immunologic research since a broad range of transgenic and gene knockout mice are available, and a large number of immunologic and diagnostic tools have been developed exclusively for mice18. The murine cervical heart transplantation model offers a unique tool to address questions and perform mechanistic studies related to ischemia reperfusion injury, immunosuppression, acute and chronic rejection and tolerance induction1-3, 17, 22.
The mouse abdominal heart transplantation model using a suture technique was first described by Corry et al. in 197319. Technical challenges associated with the suture- anastomosis of small vessels, however, limited its widespread use.
In 1991, Matsuura et al. introduced the cervical heart transplantation model using a non-suture cuff technique in mice16. Since its introduction by Zimmerman et al., and Kamada and Calne “the cuff technique” has been used for microvascular anastomoses in several different murine models of organ transplantation20-22.
We have developed a modified non-suture cuff technique for revascularization and have completed over 1,000 successful transplants in the past several years, with success rates >95%. Several different strain combinations and treatment regimens were used. Applying the technique described here, the total operation time can be reduced to approximately 45 min. Importantly the total ischemia time can be kept at 20 min with an implantation time of less than 7 min, which in contrast is usually more than 15 min when using the suture technique23. Compared with the mouse abdominal heart transplant model, cervical heart transplant has several advantages, such as minor postoperative stress and high surgical success rate. The beating of the heart graft can easily be monitored owing to the superficial position. Complications associated with suture anastomosis, including bleeding and thrombosis are significantly less common when using the cuff technique18.
The following modifications of this technique proposed by our group led to an improved success rate and to a dramatic reduction of the operation time:
One limitation that this method might have is that it may be difficult to obtain appropriate cuffs. We therefore used commercially available polyethylene cuffs from Rivertech medical (Chattanooga, TN).
Main trouble shooting during the procedure were as follows.
Blood flow disorder: Flush donor heart until the color becomes white in color. Flush without too much pressure as excessive pressure damages the graft.
Despite the fact that the heterotopic heart transplantation in mice is technically challenging, it remains the gold standard for any mechanistic in vivo studies related to transplant immunology. Our modified approach allows achieving high success rates with basic microsurgical skills that will allow broad application of this model. This video aims to help and guide other researches to establish the model in their laboratories.
The authors have nothing to disclose.
The authors declare that they have no competing financial interests.
Yasargil Clip Mini Permanent 7mm | Aesculap | FE720K | |
Micro vessel clip | S & T | B1 00396 V | |
Microscissor | FST | 14075-11 | |
Vesseldilatator | S & T | D-5a.2 00125 | |
Microforceps | FST | Dumont 11271- 30 | |
Clipapplicator | S & T | CAF-4 00072 | |
Microvessel clip | S & T | B1 00396 V |