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 thorough clinical evaluation by a veterinarian.</li>
	<li>In order to minimize anesthesia and ischemia time, donor and recipient procedures are conducted simultaneously on three operating tables in one dedicated large animal operating room. Teamwork and coordination among surgical teams as well as the veterinary staff are critical for overall success of this procedure. The surgical plan is discussed with the veterinary team in a formal meeting 1-2 weeks prior to the day of surgery.</li>
	<li>A tunneled central venous catheter (7 Fr double lumen Hickman catheter) is placed in an external or internal jugular vein of each recipient animal prior to transplant for immunosuppressive drug level monitoring and intravenous administration of immunosuppressive and/or immunomodulatory drugs. These catheters are placed under direct vision through a venous cut-down technique and tunneled posterior to exit high on the dorsal neck and secured using monofilament nonabsorbable sutures and adhesive bandage wrap around the neck.</li>
	<li>Keep animals NPO (no solid food but are allowed water ad lib) at least 12 hr before surgery.</li>
	<li>On the day of surgery, sedate animals with ketamine 20 mg/kg and xylazine 2 mg/kg IM.</li>
	<li>Place an appropriately sized endotracheal tube and connect it to the anesthesia machine and ventilator (maintained on isoflurane 0.5-3%)</li>
	<li>Place an intravenous catheter in a marginal ear vein and administer weight based 0.9% saline throughout the procedure.</li>
	<li>Administer pre-emptive analgesia (buprenorphine 0.02 mg/kg IV).</li>
	<li>Administer prophylactic antibiotic (cefazolin 20-25 mg/kg IV) prior to the initial incision to both donor and recipient animals.</li>
	<li>Use sterile technique in all procedures for both recipients and donors. This includes the use of autoclaved or gas sterilized instruments, masks, head caps, sterile surgical gowns and sterile gloves. Shave the area of the incision with a clipper and disinfect with alternating Betadine solution and alcohol.</li>
</ol>
Intraoperative Monitoring:
<ol>
	<li>After induction and intubation, maintain anesthesia with an inhalation mixture 0.5-3% isoflurane in oxygen.</li>
	<li>Monitor heart rate, pulse oximetry, ECG, end-tidal CO2 and rectal temperature continuously and record every 15 min.</li>
	<li>Prevent hypothermia with a Bair Hugger&#160;warm forced-air blanket.</li>
</ol>
Osteomyocutaneous Limb Allograft Harvest from Donor Pig:
<ol>
	<li>Place the donor animal in a supine position on the operating table and drape in a sterile manner.</li>
	<li>Use near-infra red laser angiography to identify perforator vessels to the anteromedial skin. Inject indocyanine green at approximately 0.06 mg/kg diluted in 2-5 ml of normal saline through peripheral vascular access. Approximately 10 sec later, perform 30 sec-2 min of near infrared recordings. Use the video from these recordings to identify perforators. Based on perforators, mark an area of skin (approximately 100 cm2) for monitoring.</li>
	<li>Use a scalpel to make a groin skin incision in continuity with markings for the skin paddle (Figure 2).</li>
	<li>Identify femoral vessels and isolate them distally to the level of the suprageniculate branches and proximally to the level of the inguinal ligament.</li>
	<li>Dissect the skin and subcutaneous tissue while preserving the area of the skin paddle. Suture the edges of the skin to underlying muscles to avoid contraction during flap harvest.</li>
	<li>Perform an osteotomy just above the tarsus using a reciprocating saw (Electric Pen Drive, Synthes, West Chester PA).</li>
	<li>Divide the muscles in the proximal thigh using electrocautery.</li>
	<li>Divide the femur at mid-shaft leaving an osteomyocutaneous flap on a vascular pedicle. The flap contains the skin paddle, underlying thigh muscles, knee joint, distal femur and proximal tibia which includes intact vascularized bone marrow with its unique immunologic features.</li>
	<li>Use bone wax to achieve hemostasis in the marrow spaces. Smooth bone edges using ronjeurs in preparation for the recipient.</li>
	<li>Keep the graft perfused on its vascular pedicle until the recipient&#8217;s femoral vessels are prepared (Figure 3).</li>
	<li>Once the recipient dissection is complete, ligate the donor femoral vessels as proximal as possible and infuse the graft with histidine-tryptophan-ketoglutarate (HTK, Custodiol, Newtown PA) prior to transplantation. 
	NOTE: The donor limb is transplanted on the contralateral side of the recipient i.e. the left donor limb for the right side of the recipient and the right donor limb for the left side of the recipient).</li>
	<li>Euthanize the donor pig immediately after harvesting the second hind limb using 100 mg/kg IV sodium pentobarbital. Death is confirmed by the veterinary staff per established protocol.</li>
</ol>
4. Recipient Procedure (Hind Limb Allotransplantation)
<ol>
	<li>Start the recipient induction and intubation immediately after the initial donor incision to allow preparation of recipient vessels while the donor limb is being harvested. This eliminates unnecessary wait time and reduces the total anesthesia time for the donor and recipients.</li>
	<li>Position the animal supine with a 30&#176; rotation on the operating side to allow exposure of the dorsolateral side.</li>
	<li>Make a groin incision and expose the femoral vessels; isolate them distally to the level of the suprageniculate branches and proximally to the level of the inguinal ligament.</li>
	<li>Create a subcutaneous pocket from the groin to the dorsolateral abdominal wall for the graft inset (Figure 4).</li>
	<li>Ligate the donor vessels and flush the graft with HTK.</li>
	<li>Perform the graft inset in an inverted manner: Have the skin paddle facing dorsolateral to allow easy immune monitoring in a standing position and the cut ends of the femoral vessels facing caudally to allow end-to-end vascular anastomoses without any tension on the vessels (Figure 5).</li>
	<li>If available, use a vascular coupling device (Synovis, St Paul MN) for venous anastomoses (size 2.5-3.5 is suitable for 10-20 kg animals). This significantly reduces ischemia time when compared to conventional methods.</li>
	<li>Perform arterial anastomoses using a conventional suture technique with 9-0 suture (Synovis, St Paul MN)</li>
	<li>After vascular patency is confirmed, secure the graft in place with 3-0 PDS sutures (Figure 5). The muscles of the graft are sutured to the abdominal wall muscles. The graft muscles should not be sutured to the recipient thigh muscles since it can be deleterious when the animal moves around the cage.</li>
	<li>Exteriorize the skin paddle on the dorsolateral side for immune monitoring (Figure 6) and suture it to the adjacent skin using 3-0 Vicryl subcutaneous sutures and 4-0 monofilament absorbable interrupted skin sutures.</li>
	<li>Close the groin skin in layers using 3-0 Vicryl subcutaneous sutures and 4-0 monofilament absorbable interrupted skin sutures.</li>
	<li>Place a 50-100 mcg/hr fentanyl patch for post-operative analgesia.</li>
	<li>After hind limb allograft transplantation, return the animal to its pen once awake and breathing comfortably on its own.</li>
</ol>

<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. Transplant. 11, 190 (2011).</li><li>Brandacher, G., Grahammer, J., Sucher, R., Lee, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+basic+and+translational+research+in+reconstructive+transplantation.">Animal models for basic and translational research in reconstructive transplantation.</a> Birth Defects Res. C Embryo Today. 96 (1), 39-50 (2012).</li><li>Brandacher, G., Lee, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hand+transplantation.">Hand transplantation.</a> Hand Clin. 27, xiii-xiv (2011).</li><li>Hettiaratchy, S., Mendely, E., Randolph, M. 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>

The authors declare that they have no competing financial interests.

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.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the Reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>HTK</td>
			<td>Custodial</td>
			<td>N/A</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		 </tr>
		 <tr>
			<td>Electric Pen Drive</td>
			<td>Synthes, Westchester PA </td>
			<td>05.001.011 </td>
			<td>Reciprocating saw</td>
		 </tr>
		 <tr>
			<td>Vascular Coupling device</td>
			<td>Synovis, Newtown PA</td>
			<td>21003B</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

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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Research

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12.4K Views.  Johns Hopkins University School of Medicine. 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.

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

A Modified Method for Heterotopic Mouse Heart Transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37&#160;&#176;C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

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.

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 ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. <span style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand ...

A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone splenectomy regardless of precipitating causes are prone to develop an overwhelming post-splenectomy infection and experience increased risks of deep venous thrombosis and malignancies. Recently, epidemiological studies indicated that splenectomy might be associated with the occurrence of cardiovascular diseases, suggesting that physiological functions of the spleen have not yet been fully recognized. Here, we introduce a mouse model of vascularized heterotopic spleen transplantation, which not only can be utilized to study the function and behavioral activity of splenic immune cell subsets in different biologic processes, but also can be a powerful tool to test the therapeutic potential of spleen transplantation in certain diseases. The main surgical steps of this model include donor spleen harvest, the removal of recipient native spleen, and spleen graft revascularization. Using congenic mouse strains (e.g., mice with CD45.1/CD45.2 backgrounds), we observed that after syngeneic transplantation, both donor-derived splenic lymphocytes and myeloid cells migrated out of the graft as early as post-operative day 1, concomitant with the influx of multiple types of recipient cells, thus generating a unique chimera. &#160;Despite relatively challenging techniques, this procedure can be performed with &#62;90% success rate. This model allows tracking the fate, longevity, and function of splenocytes during steady state and in a disease setting following a spleen transplantation, thereby offering a great opportunity to discover the distinct role for spleen-derived immune cells in different disease processes.

This protocol details the surgical steps of a mouse model of vascularized heterotopic spleen transplantation, a technically challenging model that can serve as a powerful tool in studying the fate and longevity of spleen cells, the mechanisms of distinct spleen cell populations in disease progression, and transplant immunity.

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone ...

Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current limitations in immunosuppression and functional neuromotor recovery. Many animal models have been developed for studying unique features of VCA, but here we present a robust reproducible model of orthotopic hind limb transplant in rats designed to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery. At the core of the model rests a commitment to meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and hand sewn neural coaptation of the femoral nerve and the sciatic nerve. This approach yields durable limb reconstructions that allow for longer lived animals capable of rehabilitation, resumption of daily activities, and functional testing. With short-term treatment of conventional immunosuppressive agents, allotransplanted animals survived up to 70 days post-transplant, and isotransplanted animals provide long lived controls beyond 200 days post-operatively. Evidence of neurologic functional recovery is present by 30 days post operatively. This model not only provides a useful platform for interrogating immunological questions unique to VCA and nerve regeneration, but also allows for in vivo testing of new therapeutic strategies specifically tailored for VCA.

This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery.

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current ...