To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.
Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.
Cardiac transplantation is the treatment of choice for end-stage heart failure. Both technical advancements and pharmacological innovations have propelled the field to early graft acceptance rates above 90%.12,13 Despite this, 60-80% 5-year graft survival is at a standstill and chronic rejection, characterized by transplant vasculopathy, remains inevitable.14-16 Furthermore, patients are subjected to multiple surgical procedures and lifelong immunosuppression, which are associated with chest wall deformities and medical sequelae and toxicities, respectively. The need for innovative approaches to extend allograft survival, minimize the immunosuppressive requirements, and offer reconstructive options for anatomical deformities is pressing.
Vascularized composite allotransplantation offers a unique strategy for improving heart transplant outcomes both from an immunological aspect as well as a reconstructive perspective.17 Vascularized composite allografts are also unique in a way that they have an inherent source of donor-derived hematopoietic stem cells which has shown a favorable ability to reduce immunosuppression and induce and sustain mixed chimerism.1-8 Additionally, co-transplantation of the thymus has shown to prolong survival of both, solid organ transplants and vascularized composite allografts.2,9-11 Combining these strategies with heart transplantation offers a novel solution to the aforementioned challenges facing heart transplantation.18
Murine models serve as excellent platforms for mechanistic in vivo investigation because of the availability of antibodies and well-defined inbred and knockout strains.19-21 Although heart transplantation in mice is commonly studied using a heterotopic intraabdominal microsurgical suture transplant model22-25, a heterotopic, cervical, non-suture cuff technique model has shown to be extremely replicable, reliable, and carries fewer rates of thrombosis.19,26,27 The goal of this study is to develop a heterotopic en bloc osteomyocutaneous chest wall, thymus, and heart transplant technique in mice to study the immunological mechanisms of combined solid organ and vascularized composite allotransplantation using a cervical non-suture cuff technique. This cluster allograft is perfused through the anastomosis of the donor descending aorta to the right common carotid artery and the donor pulmonary artery to the right external jugular vein. Preservation of the internal thoracic vessels and associated thymus branches is paramount to perfusing the chest wall (sternum, ribs, muscles, and skin) and thymus.
All operative procedures were completed in compliance with Johns Hopkins University and the United States Department of Agriculture and Public Health Service requirements. This protocol follows the Johns Hopkins University Animal Care and Use Committee, institutional review board approved guidelines (protocol number M013M490). Final survival data was recorded for the surgical procedures described below. Both donor and recipient animals receive pre-emptive anesthesia using buprenorphine at 0.1 mg/kg s.c. one hour prior to surgery and in the recipient animal buprenorphine is re-administered at the same dose after transplant and re-dosed as needed in the first 48 hours after surgery.
1. Donor Allograft Recovery
Note: Begin the donor portion of the transplant 40 min earlier than the recipient transplant to minimize recipient anesthesia time and to facilitate a simultaneous end time or slightly earlier end time versus the recipient preparation.
2. Recipient Preparation
Note: To minimize recipient anesthesia time, begin the recipient preparation at a separate operative station approximately 40 min prior to completion of the donor allograft harvest.
3. Allograft Inset
4. Postoperative Care
Syngeneic C57BL/6 transplants achieved long term survival. The design of the allograft (Figure 1) proved to be successful from an animal survival perspective and the ability to evaluate ongoing allograft survival. This was demonstrated through overlying skin remaining viable, active ongoing allograft hair growth, and heartbeats were able to be evaluated with visualization and finger palpation. Survival data is represented in Figure 2 for syngeneic transplanted mice. The mean survival time was greater than 109 days. Based on the survival data, it is reasonable to infer that the technical aspect of the transplanted allograft is designed to perfuse the entirety of the chest wall, thymus, and heart. Furthermore, the ability of the syngeneic animals to survive long term further supports that this mouse model is not only feasible but can be replicated. This proof-of-concept en bloc chest wall, thymus, and heart transplant validates the murine model to study combined solid organ and vascularized composite allotransplantation.
Figure 1. Intraoperative photos. (A) The chest wall, thymus, and heart allograft is successfully recovered, trimmed, and visualized ex vivo from the posterior aspect. The bilateral internal thoracic vessels are preserved. (B) The recipient external jugular vein (arrow) and common carotid artery (arrowhead) are everted fixed over polyimide cuffs in preparation for vascular anastomosis. (C) Allograft vascular anastomoses are completed. The arrow shows the anastomosis between the donor pulmonary artery and the recipient external jugular vein. The arrowhead shows the anastomosis between the donor descending aorta and the recipient common carotid artery. The asterisk identifies the thymus and the reflected chest wall is visualized overlying the thymus. (d) A higher magnification shows the microvascular-cuff anastomoses. (e) Complete allograft inset. Please click here to view a larger version of this figure.
Figure 2. En bloc chest wall, thymus, and heart allograft survival. Kaplan-Meier survival curves of the en bloc chest wall, thymus, and heart allotransplant in syngeneic C57BL/6 mice (n = 3; mean survival time was greater than 109 days). Please click here to view a larger version of this figure.
There are a multitude of phenomena that factor into the immunological investigation of allotransplantation, which include but are not limited to mechanisms of acute and chronic rejection, direct and indirect antigen presentation, recipient sensitization, or the induction of mixed chimerism.19 Animal models have become the gold standard for the study of transplant immunology, and mouse models are popularly implemented due to their low cost, availability of transgenic and gene knockout mice, commercially available monoclonal antibodies, relative decreased veterinary and housing demands, and the ease of replication. To date, multiple heart transplant models have been designed to study solid organ transplantation.19,22-27 Similarly, a plethora of mouse models have been developed to study vascularized composite allotransplantation.28 However, the study of combined solid organ and vascularized composite allotransplantation is limited, and techniques have yet to be established in mice. The en bloc chest wall, thymus, and heart transplant murine model presented here is a reliable and replicable tool to study the effects and immunological mechanisms of combined solid organ and vascularized composite allotransplantation.
To further advance the field of transplantation, the promise of prolonging allograft survival and minimization of immunosuppression through novel treatment modalities must be pursued. One such approach is through the induction of mixed chimerism (partial engraftment of donor hematopoietic cells in the recipient), which can lead to immunosuppression-free donor specific tolerance, even if in some instances chimerism is not sustained.29,30 Bone marrow transfusion/transplantation paired with solid organ31 or vascularized composite allotransplanation32,33 requires extensive preconditioning posing a significant challenge. Vascularized bone marrow, as part of a vascularized composite allograft construct, can circumvent this problem. Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and is considered to be superior to cellular bone marrow transplantation alone in the induction of tolerance and reduction of immunosuppression requirements.34-36 Moreover, the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism of donor origin ultimately playing a supportive role in the induction and maintenance of chimerism.2,9 The aforementioned strategies of prolonging allograft survival and minimizing immunosuppression was the foundation to conceptualize a combined solid organ, thymus, and vascularized composite allograft mouse model.
The heterotopic en bloc chest wall, thymus, and heart transplantation is an amalgamation of multiple historical animal models. Heterotopic cervical heart transplantation using a non-suture cuff in mice has been well established and considered less technically demanding than a heterotopic abdominal microvascular heart transplant.19 In fact, the cuff technique has been implemented in multiple other animal transplant models.37-44 Heterotopic sternal transplantation in rats was introduced in 1999 by Santiago et al. as an alternative method to study vascularized bone marrow transplantation.1 They were able to show long term peripheral chimerism, tolerance, and survival following cessation of immunosuppression on postoperative day 30.1 Bozkurt et al. subsequently developed a rat model in 2013 to incorporate the thymus and the full extent of the osteomyocutaneous portion of the chest wall. It should be noted, however, that this model differs from our model in multiple aspects. This includes: (1) their model being devoid of any solid organs, (2) being completed in rats using traditional microsurgical techniques, (3) ligation of the internal thoracic vessels during donor harvest, (4) implementation of a unilateral, single pedicle via a common carotid artery and external jugular vein, and (5) transplantation of the allograft into the inguinal region.2 Nevertheless, their model was able to demonstrate that the thymus of donor-origin plays a significant role not only for chimerism augmentation but also for chimerism maintenance as compared with vascularized bone marrow transplantation alone.2 Subsequent swine models of co-transplantation of the thymus and heart have shown a superior effect on heart allograft survival.10,11 The advantages of each the animal models, but lack of mechanistic in vivo studies pertaining to combined solid organ, thymus, and vascularized composite allotransplantation prompted our group to design this model.
The experience executing this novel model exhibited certain lessons requiring our team to institute modifications in order to achieve better animal survival. This model was attempted with one operator, which ultimately prolonged the operative and anesthesia time to over 3-4 hr and also prolonged cold ischemia time. Animals would not awake following termination of the procedure. Implementation of a two-team approach cut the total operative and anesthesia time to 90 min. This reflected in 60 min anesthesia time of the recipient mouse, and 0-10 min allograft cold ischemia time. During allograft reperfusion, the mouse is susceptible to hemorrhage, which can limit its survivability in the immediate peri-operative. We advocate meticulous inspection of the allograft ex vivo for potential sources of hemorrhage, as well as gentle release of the bulldog microvascular clamp during allograft reperfusion. By placing the allograft in a reflected position it is easier to identify specific sites of bleeding. Furthermore, this graft inset position fosters the most ergonomic lay of vessels minimizes the risk of vessel kinking. Lastly, with the first 48 hr of recovery, the recipient mouse’s right upper extremity range of motion may be hindered with regards to climbing the cage to obtain food and water. Therefore, we recommend placement of gelatin nutritional sources along the cage to facilitate nutritional intake. Typically by postoperative day 3, full range of motion is returned within the right upper extremity.
Although there are limitations to this model, which include the need for technical skill in microsurgery, availability of two simultaneous microscopes, and the requirement of a two-team approach, it has nevertheless shown to be a successful approach to perform mechanistic immunological studies related to combined solid organ and vascularized composite allotransplantation. Its broader application may further contribute to develop novel immunosuppressive protocols, studying mechanics of acute and chronic rejection, and implementation of potential strategies to induce and sustain chimerism, and prolong allograft survival.
The authors have nothing to disclose.
This work was funded by the American Association of Plastic Surgeons 2014 Academic Scholar Award.
Euro-Collins Solution | The solution is not commercially purchased but rather prepared in the laboratory. To make a 500ml solution add the ingredient listed below to a 330ml of double distilled water. Mix well, and then fill in the rest of the 170ml of double distilled water into the solution to a final volume of 500ml. Ingredients: 1.02g KH2PO4, 3.66g K2HPO4, 0.56g KCl, 0.42g NaHCO3, and 17.52g of glucose. |
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Suture | Ethilon | MWI 72667 | 6-0 Ethilon |
Suture | Microsurgery Instruments, Inc. | 10-0 Nylon | 10-0 Nylon |
Polyimide Cuff Vein (21G) | Vention Medical | 141-0043 | http://www.ventionmedical.com/products-and-services/polyimide-tubing/ |
Polyimide Cuff Artery (24G) | Vention Medical | 141-0027 | http://www.ventionmedical.com/products-and-services/polyimide-tubing/ |
Soft plastic tip catheter | Terumo | SR*OX2419CA | 24G x 3/4" |
Microsurgical dilator | S&T | D-5a.1 | Dilator, 11cm, FH, 0.1mm AT10d |