Name: modified heterotopic hindlimb osteomyocutaneous flap model in the rat for translational vascularized composite allotransplantation research
Uploaded: 2019-04-26T14:00:00
Description: Watch this Scientific Journal Video about Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research at JoVE.com

This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the&#160;Congressionally Directed Medical Research Program under Award No. W81XWH-13-2-0057. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense.

All animal surgeries were performed in accordance with protocols approved by the University of Louisville&#39;s Institutional Animal Care and Use Committee (IACUC-approved protocol 18198) and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals10. Four-month-old male Brown-Norway (RT1.An) and 4-month-old male Lewis (RT1.Al) rats were used as VCA donor and recipients, respectively.
1. Donor Allograft Harves...

<ol>
	<li>Gilbert Fernandez, J. J., Febres-Cordero, R. G., Simpson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Untold+Story+of+the+First+Hand+Transplant:+Dedicated+to+the+Memory+of+one+of+the+Great+Minds+of+the+Ecuadorian+Medical+Community+and+the+World.">The Untold Story of the First Hand Transplant: Dedicated to the Memory of one of the Great Minds of the Ecuadorian Medical Community and the World.</a> Journal of Reconstructive Microsurgery. , (2018).</li><li>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=Human+hand+allograft:+report+on+first+6+months.">Human hand allograft: report on first 6 months.</a> Lancet. 353 (9161), 1315-1320 (1999).</li><li>Jones, J. W., Gruber, S. A., Barker, J. H., Breidenbach, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+hand+transplantation.+One-year+follow-up.+Louisville+Hand+Transplant+Team.">Successful hand transplantation. One-year follow-up. Louisville Hand Transplant Team.</a> The New England Journal of Medicine. 343 (7), 468-473 (2000).</li><li>Devauchelle, B., 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=First+human+face+allograft:+early+report.">First human face allograft: early report.</a> Lancet. 368 (9531), 203-209 (2006).</li><li>Broyles, 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=Functional+abdominal+wall+reconstruction+using+an+innervated+abdominal+wall+vascularized+composite+tissue+allograft:+a+cadaveric+study+and+review+of+the+literature.">Functional abdominal wall reconstruction using an innervated abdominal wall vascularized composite tissue allograft: a cadaveric study and review of the literature.</a> Journal of Reconstructive Microsurgery. 31 (1), 39-44 (2015).</li><li>Kollar, B., 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=Innovations+in+reconstructive+microsurgery:+Reconstructive+transplantation.">Innovations in reconstructive microsurgery: Reconstructive transplantation.</a> Journal of Surgical Oncology. 118 (5), 800-806 (2018).</li><li>Kaufman, C. L., 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=Graft+vasculopathy+in+clinical+hand+transplantation.">Graft vasculopathy in clinical hand transplantation.</a> American Journal of Transplantation. 12 (4), 1004-1016 (2012).</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 Research Part C: Embryo Today. 96 (1), 39-50 (2012).</li><li>Kaufman, C. L., 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=Immunobiology+in+VCA.">Immunobiology in VCA.</a> Transplantation International. 29 (6), 644-654 (2016).</li><li>Committee for the Update of the Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research, Division on Earth and Life Studies, National Research Council of the National Academies. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals, 8th edition. , (2011).</li><li>Nazzal, J. A., Johnson, T. S., Gordon, C. R., Randolph, M. A., 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=Heterotopic+limb+allotransplantation+model+to+study+skin+rejection+in+the+rat.">Heterotopic limb allotransplantation model to study skin rejection in the rat.</a> Microsurgery. 24 (6), 448-453 (2004).</li><li>Ulusal, A. E., Ulusal, B. G., Hung, L. M., Wei, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+hindlimb+allotransplantation+in+rats:+an+alternative+model+for+immunological+research+in+composite-tissue+allotransplantation.">Heterotopic hindlimb allotransplantation in rats: an alternative model for immunological research in composite-tissue allotransplantation.</a> Microsurgery. 25 (5), 410-414 (2005).</li></ol>

In developing this model of VCA, several key issues were considered. First, it was important to include intact bone (tibia and fibula), bone marrow, and skin in the graft. While clinical hand transplants from adult donors do not transfer significant amounts of actively hematopoietic marrow, studies of the role of the bone marrow niche are better mirrored using an intact, vascularized bone rather than a cut long bone, which results in fibrosis of the exposed marrow. Moreover, the closed bone osteomyocutaneous flap design ...

Reconstructive surgery for the catastrophic tissue loss from amputation, blast injuries, malignancies, and congenital defects are limited by the availability of tissue from the patient and the additional morbidity caused at the donor site. In some cases, such as burn victims or quadrilateral amputees, viable tissue for reconstruction is not available from the patient. In 1964, the first modern hand transplant was performed in Ecuador. While this was a technical success, immunosuppression available at the time was insufficient to prevent rejection, and the graft was lost in less than 3 weeks1. In 1998 and 1999, the first hand transplants in the modern era of immunosuppression were performed in Lyon, France2 and Louisville, Kentucky, USA3. For the first time, reconstructive surgeons could replace like with like. Face transplantation was first performed in 20054, and a number of other VCA grafts are now routinely being performed, such as abdominal wall5, uterine, and urogenital transplants6.
Unlike solid organ transplantation, most VCA techniques involve the presence of the highly antigenic donor skin. Clinical experience has determined that the acute rejection of the skin is relatively easy to control but may contribute to the chronic rejection of the underlying tissues and vessels, which do not respond well to treatment7. The vascular dysfunction associated with an alloimmune response is a more ominous obstacle for the field of VCA7. Macrovasculopathies lead to perfusion deficits, delayed healing, and proinflammatory conditions. Both confluent aggressive large-vessel vasculopathy and focal intimal hyperplasia occur in hand transplant recipients7. Additionally, microvasculopathies likely contribute to VCA complications as well and may even lead to rejection events. While both immune and nonimmune factors likely play a role in the vasculopathy of hand transplant recipients, the specific mechanisms promoting distal vessel dysfunction in VCA are not known, particularly in the context of low-grade, chronic rejection. These unanswered questions necessitate the development of an animal VCA model that will allow for the serial assessment of the graft during the clinical course of VCA rejection/maintenance and vasculopathy. Such a model will offer insights into the rejection and vasculopathy in the face of immunosuppression, infectious challenge, and/or other postoperative traumatic injury8,9.
Presented here is an allogeneic rat VCA heterotopic hindlimb osteomyocutaneous flap model. Based on previously published VCA models, this procedure is technically easy to perform, reproducible in a large number, and exhibits minimal morbidity and discomfort to the recipient animal. This model was designed to allow clinical and histopathological assessments of VCA acceptance vs. rejection, and provides an opportunity to evaluate underlying immune and nonimmune mechanisms involved in rejection.

<table border="0" cellpadding="0" cellspacing="0" style="width:697px;" width="698">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Acepromazine</td>
			<td style="width:143px;">Henry Schein</td>
			<td style="width:119px;">5700850</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adventitia Scissors</td>
			<td>ASSI&#160;</td>
			<td>SAS15R8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Approximator Clamp (Double)</td>
			<td>ASSI</td>
			<td>ABB2V, ABB22V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Approximator Clamp (single)</td>
			<td>FST</td>
			<td>00398-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clamp Applying Forceps</td>
			<td>ASSI&#160;</td>
			<td>CAF4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Scissors</td>
			<td>ASSI</td>
			<td>SDS18R8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flushing blunt needle 27G</td>
			<td>SAI</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin Sodium</td>
			<td>Sagent</td>
			<td>25021-400-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>14043-704-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Jewelers Bipolar</td>
			<td>ASSI</td>
			<td>103000BPS03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Jewelers forceps #3</td>
			<td>FST</td>
			<td>11231-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine HCl 100 mg/ml</td>
			<td>Zoetis</td>
			<td>043-304</td>
			<td>DEA License required</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lactated Ringer Solution</td>
			<td>Hospira</td>
			<td>0409-7953-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lactated Ringer Solution + 5% Dextrose</td>
			<td>Hospira</td>
			<td>0409-7953-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Meloxicam</td>
			<td>Henry Schein</td>
			<td>11695-6925-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro forceps</td>
			<td>ASSI&#160;</td>
			<td>JFAL3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro needle holder</td>
			<td>ASSI</td>
			<td>B138</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prograf (Tacrolimus) 5 mg/ml</td>
			<td>Astellas</td>
			<td>0469-3016-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suture, 10-0 Prolene</td>
			<td>Ethicon</td>
			<td>W2790</td>
			<td>or 10-0 Ethilon (2830)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suture, 4-0 Coated Vicryl</td>
			<td>Ethicon</td>
			<td>J714D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vessel Dilator Forceps</td>
			<td>ASSI</td>
			<td>D5AZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine</td>
			<td>VetOne</td>
			<td>13985-612-50</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The rat VCA heterotopic hindlimb osteomyocutaneous flap model allows for long-term allograft survival under immunosuppression. The model is reliable, reproducible, and simple to perform. The flap is well hidden in the groin area and constitutes minimal morbidity and discomfort to the animal. The skin presentation is a clinical manifestation of the allograft&#8217;s survival and rejection (Figure 1). The flap design allows for gross clinical monitoring and creates an opportunity for various i...

Watch this Scientific Journal Video about Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research at JoVE.com

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

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 ...

Research

JoVE Journal

Medicine

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

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 ...

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 ...

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 ...

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 ...

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 ...

A Model of Free Tissue Transfer: The Rat Epigastric Free Flap

Free tissue transfer has been increasingly used in clinical practice since the 1970s, allowing reconstruction of complex and otherwise untreatable defects ...

Development of a Novel Task-oriented Rehabilitation Program using a Bimanual Exoskeleton Robotic Hand

A robot-assisted hand is used for the rehabilitation of patients with impaired upper limb function, particularly for stroke patients with a loss of motor ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...

Hickman Catheter for Long-Term Vascular Access in Swine

Hickman Catheter Use for Long-Term Vascular Access in a Preclinical Swine Model

Hickman Catheter Use for Long-Term Vascular Access in a Preclinical Swine Model (Video) | JoVE 

Central venous catheters (CVCs) are invaluable devices in large animal research as they facilitate a wide range of medical applications, including blood ...