Name: a modified heterotopic swine hind limb transplant model for translational vascularized composite allotransplantation (vca) research
Uploaded: 2013-10-14T08:00:00
Description: Watch this Scientific Journal Video about A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research at JoVE.com

We would like to acknowledge the following individuals for their contribution to this
project: Kakali Sarkar, PhD, Joani Christensen, BS, Kate Buretta, BS, Nance Yuan,
BS, William Lehao, MD, Johanna Grahammer, Georg Furtm&uuml;ller, MD, Erin Rada MD,
Mohammed Al-Rakan MD, Karim Sarhane MD, Saami Khalifian, BS, Mao Qi, MD, and
Angelo Leto Barone MD, VCA Laboratory, Department of Plastic and Reconstructive 
Surgery, Johns Hopkins University School of Medicine, Janis Taube, MD, Mark Fischer, 
MD, Departments of Dermatology and Pathology, Johns Hopkins University School 
of Medicine, Sue Eller, Minimally Invasive Surgery Training Center, Johns Hopkins 
University School of Medicine and Cheng-Hung Lin, MD Chang Gung Memorial 
Hospital, Linkou, Taiwan.
Funding source: Armed Forces Institute of Regenerative Medicine (DoD W81XWH-08-
2-0032)

In this video publication, all animal procedures were conducted in accordance with an animal protocol approved by the Johns Hopkins University Institutional Animal Care and Use Committee (IACUC).
1. Preoperative Planning
<ol>
	<li>We utilize MHC-defined MGH inbred miniature swine (15-20 kg) in our translational studies for limb allotransplantation wherein one male donor provides limbs for two female recipients (Figure 1). Prior to surgery, donor and recipient animals undergo...

<ol>
	<li>Barth, R. N., Rodriguez, E. D., Mundinger, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+bone+marrow-based+immunosuppression+inhibits+rejection+of+vascularized+composite+allografts+in+nonhuman+primates.">Vascularized bone marrow-based immunosuppression inhibits rejection of vascularized composite allografts in nonhuman primates.</a> Am. J. Transplant. 11 (7), 1407-1416 (2011).</li><li>Brandacher, G., Gorantla, V. S., Schneeberger, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hand/Forearm+transplantation+using+a+novel+cell-based+immunomodulatory+protocol-experience+with+five+patients.">Hand/Forearm transplantation using a novel cell-based immunomodulatory protocol-experience with five patients.</a> Am. J. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+to+composite+tissue+allografts+across+a+major+histocompatibility+barrier+in+miniature+swine.">Tolerance to composite tissue allografts across a major histocompatibility barrier in miniature swine.</a> Transplantation. 27, 514-521 (2004).</li><li>Ibrahim, Z., Busch, J., Awwad, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selected+physiologic+compatibilities+and+incompatibilities+between+human+and+porcine+organ+systems.">Selected physiologic compatibilities and incompatibilities between human and porcine organ systems.</a> Xenotransplantation. 13 (6), 488-499 (2005).</li><li>Kirk, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossing+the+bridge:+large+animal+models+in+translational+transplantation+research.">Crossing the bridge: large animal models in translational transplantation research.</a> Immunol. Rev. 19, 176-196 (2003).</li><li>Lee, W. P., Rubin, J. P., Cober, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+swine+model+in+transplantation+of+vascularized+skeletal+tissue+allografts.">Use of swine model in transplantation of vascularized skeletal tissue allografts.</a> Transplant Proc. 30, 2743-2745 (1998).</li><li>Mathes, D. W., Hwang, B., Graves, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+to+vascularized+composite+allografts+in+canine+mixed+hematopoietic+chimeras.">Tolerance to vascularized composite allografts in canine mixed hematopoietic chimeras.</a> Transplantation. 92 (12), 1301-1308 (2011).</li><li>Mathes, D. W., Randolph, M. A., Solari, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split+tolerance+to+a+composite+tissue+allotransplant+in+a+swine+model.">Split tolerance to a composite tissue allotransplant in a swine model.</a> Transplantation. 75 (1), 25-31 (2003).</li><li>Mezrich, J. D., Haller, G. W., Arn, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histocompatible+miniature+swine:+an+inbred+largeanimal+model.">Histocompatible miniature swine: an inbred largeanimal model.</a> Transplantation. 75 (6), 904-907 (2003).</li><li>Petruzzo, P., Lanzetta, M., Dubernard, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+International+Registry+on+Hand+and+Composite+Tissue+Transplantation.">The International Registry on Hand and Composite Tissue Transplantation.</a> Transplantation. 90, 1590-1594 (2010).</li><li>Sucher, R., Lin, C., Zanoun, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+hind+limb+transplantation:+a+new+composite+tissue+allotransplantation+model+using+nonsuture+supermicrosurgery.">Mouse hind limb transplantation: a new composite tissue allotransplantation model using nonsuture supermicrosurgery.</a> Transplantation. 90, 1374-1380 (2010).</li><li>Ust&#252;ner, E. T., Majzob, R. K., Ren, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swine+composite+tissue+allotransplant+model+for+preclinical+hand+transplant+studies.">Swine composite tissue allotransplant model for preclinical hand transplant studies.</a> Microsurgery. 20, 400-406 (2000).</li><li>Ust&#252;ner, E. T., Zdichavsky, M., Ren, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+composite+tissue+allograft+survival+in+a+porcine+model+with+cyclosporine/mycophenolate+mofetil+therapy.">Long-term composite tissue allograft survival in a porcine model with cyclosporine/mycophenolate mofetil therapy.</a> Transplantation. 661, 581-587 (1998).</li><li>Wachtman, G. S., Wimmers, E. G., Gorantla, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologicals+and+donor+bone+marrow+cells+for+targeted+immunomodulation+in+vascularized+composite+allotransplantation:+a+translational+trial+in+swine.">Biologicals and donor bone marrow cells for targeted immunomodulation in vascularized composite allotransplantation: a translational trial in swine.</a> Transplant Proc. 43, 3541-3544 (2011).</li></ol>

Historically, the heterotopic hind limb transplant protocol included exteriorization of a skin paddle to the ventral abdominal wall and the vessels were anastomosed in an end-to-side manner (Hettiarachty 2004). However, in our modified method, an inverted flap insetting and end-to-end anastomoses bring the skin paddle more laterally and hence facilitates immune monitoring in a standing position of the animal. The identification of the sural artery and the zones of maximum perfusion using near infrared laser angiography f...

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants are now a clinical reality with numerous hand and face transplants performed worldwide12. Despite the fact that early and intermediate results are favorable and highly encouraging2, the requirement of chronic multidrug immunosuppression continues to limit its widespread clinical application. The advancements in murine models of VCA including super-microsurgical anastomoses and nonsuture cuff techniques13, 3 have paved the way to a better understanding of alloimmune responses in VCA. Myriads of immunomodulatory protocols have been proposed for clinical applications based on our better understanding of immune mechanisms in VCA but they need to be validated in a large animal model that would be reasonably predictive of their performance in humans7. Based on physiologic and immunologic similarities between human and porcine organ systems6, swine VCA models can be considered reliable and cost-effective alternatives to canine9 and nonhuman primate models1.
This article provides a detailed overview of the methodology used in our MHC-defined swine heterotopic hind limb transplant model, which serves as groundwork for our current and future immunomodulatory strategies aimed at inducing immune tolerance to VCA and hence broadening its clinical application. We utilize well-characterized inbred pigs bred to homozygosity at the swine leukocyte antigen locus specifically for their use in transplant-related research11. We raise a vascularized osteomyocutaneous flap based on femoral vessels. The flap contains intact vascularized bone marrow in the distal femur and proximal tibia. The anteromedial thigh skin is also included in the graft and is exteriorized to the dorsolateral aspect of the recipient animal for immune monitoring of the most immunogenic component of the VCA. Dorsolateral positioning facilitates clinical examination in standing and sitting positions and also keeps the allograft skin relatively clean.
Ustener et al. introduced one of the first large animal translational models in VCA by transplanting radial forelimb osteomyocutaneous flaps in outbred farm pigs15. The group utilized this model to demonstrate for the first time that acute rejection in VCA which included the highly immunogenic skin component could be delayed and treated with a clinically relevant strategy without significant drug-specific complications and side effects. The favorable results obtained in this study subsequently built a foundational step in designing drug regimens for human reconstructive transplantation. Although these early pig VCA models were well suited for developing protocols to prevent rejection of skin, muscle, bones, nerves and vessels they lacked specialized structures such as articular cartilage and synovial membranes of joints. Subsequent efforts were focused on including the medial digit of the animal, which necessitated full-length cast placement to prevent graft dislodgement14. Although suitable to investigate rejection of all major components of limb transplantation, one of the major limitations of this model was post-transplant ambulatory difficulty due to cast placement. Thus, heterotopic swine limb allotransplantation models, consisting of the tibia, fibula, knee joint, distal femur, surrounding muscle, and a skin paddle, were created to primarily study immunological aspects of VCA while allowing the animal to freely ambulate postoperatively with minimal morbidit&#160;8.
The development of well-characterized SLA-defined inbred pigs through the pioneering work of Dr. David H. Sachs led to a new era of translational VCA research. Utilizing a heterotopic hind limb transplant model in a minor antigen mismatch setting, Mathes et al.10 demonstrated indefinite survival of musculoskeletal components with a short course of cyclosporine treatment. The skin component survival, however, was only prolonged when compared to no treatment controls. The loss of the skin component of the graft was attributed to an isolated and highly vigorous immune response, in particular, to the epidermis. Similarly, using fully mismatched pigs with T-cell depletion, a short course of cyclosporine and cytokine mobilized donor peripheral blood mononuclear cells induced tolerance only to musculoskeletal components and the skin component was still rejected5. This phenomenon, called &#8216;split tolerance&#8217;, brought a paradigm shift in VCA research with a greater focus on the highly immunogenic skin component, which is an integral component of the majority of reconstructive transplants performed to date.
In this modified model, we utilize end-to-end anastomosis by ligating the recipient femoral artery and rotating it cephalad (Figure 1). This not only reduces ischemia time by allowing the use of a conventional coupling device but also decreases the chances of anastomotic failure. We have not observed any ischemic events following ligation of the femoral artery in our recipients indicating that collateral circulation was sufficient to provide vascularity to the native leg. Additionally, in this modified method, the externalized skin component is mobilized based on the underlying perforator vessels and is positioned laterally (Figure 1) in contrast to a ventral groin position in the traditional model10. This allows easy visualization of the graft for immune monitoring in a standing or sitting position of the animal.
Hence, a reliable and reproducible large animal model is essential to investigate tolerance induction strategies towards the skin component of VCA and to develop novel noninvasive immune monitoring strategies for better prediction of graft survival.

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a modified heterotopic swine hind limb transplant model for translational vascularized composite allotransplantation (vca) research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

Twenty-four SLA-defined swine heterotopic hind limb transplants were performed using our modified technique with a mean ischemia time of 78 min&#160;(Range: 62-94 min). Graft inset and dorsolateral skin paddle positioning were achieved without difficulty in all animals. Near infrared laser angiography showed excellent graft perfusion in all recipients. The initial twelve venous anastomoses were performed using conventional suture techniques while the last twelve venous anastomoses were performed using a vascular coupling...

Watch this Scientific Journal Video about A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research at JoVE.com

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

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