Name: orthotopic hind limb transplantation in the mouse
Uploaded: 2016-02-12T13:00:00
Description: Watch this Scientific Journal Video about Orthotopic Hind Limb Transplantation in the Mouse at JoVE.com

This work was supported by the Army, Navy, NIH, Air Force, VA and Health Affairs to support the AFIRM II effort, under Award No. W81XWH-13-2-0053. The U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick MD 21702-5014 is the awarding and administering acquisition office. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the Department of Defense.
The authors would like to thank Jessica Izzi, D.V.M, Caroline Garrett, D.V.M. and Julie Watson, D.V.M. for their excellent veterinary support during this study.

All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institute of Health (NIH) and were approved by the Johns Hopkins University Animal Care and Use Committee (JHUACUC). The specific procedures were performed under the approved ACUC protocol MO13M108.&#160;
1. Donor Operation
<ol>
	<li>Administer analgesia at the appropriate time point for each pharmacological formulation prior to surgery. As per the approved animal care and use protocol use 0.1 m...

<ol>
	<li>Khalifian, 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=Facial+transplantation:+the+first+9+years.">Facial transplantation: the first 9 years.</a> Lancet. , (2014).</li><li>Petruzzo, P., Dubernard, J. M. <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+allotransplantation.">The International Registry on Hand and Composite Tissue allotransplantation.</a> Clin. Transpl. , 247-253 (2011).</li><li>Shores, J. T., Brandacher, G., Lee, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hand+and+Upper+Extremity+Transplantation:+An+Update+of+Outcomes+in+the+Worldwide+Experience.">Hand and Upper Extremity Transplantation: An Update of Outcomes in the Worldwide Experience.</a> Plast. Reconstr. Surg. , (2014).</li><li>Levi, D. 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=Transplantation+of+the+abdominal+wall.">Transplantation of the abdominal wall.</a> Lancet. 361, 2173-2176 (2003).</li><li>Strome, 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=Laryngeal+transplantation+and+40-month+follow-up.">Laryngeal transplantation and 40-month follow-up.</a> N.Engl.J. Med. 344, 1676-1679 (2001).</li><li>Rose, K. G., Sesterhenn, K., Wustrow, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracheal+allotransplantation+in+man.">Tracheal allotransplantation in man.</a> Lancet. 1, 433 (1979).</li><li>Hofmann, G. O., 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=Allogeneic+vascularized+transplantation+of+human+femoral+diaphyses+and+total+knee+joints--first+clinical+experiences.">Allogeneic vascularized transplantation of human femoral diaphyses and total knee joints--first clinical experiences.</a> Transplant. Proc. 30, 2754-2761 (1998).</li><li>Hu, W., 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=A+preliminary+report+of+penile+transplantation.">A preliminary report of penile transplantation.</a> Eur. Urol. 50, 851-853 (2006).</li><li>Brannstrom, 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=Livebirth+after+uterus+transplantation.">Livebirth after uterus transplantation.</a> Lancet. , (2014).</li><li>Sarhane, K. 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=Diagnosing+skin+rejection+in+vascularized+composite+allotransplantation:+advances+and+challenges.">Diagnosing skin rejection in vascularized composite allotransplantation: advances and challenges.</a> Clin. Transplant. 28, 277-285 (2014).</li><li>Schneeberger, S., Khalifian, S., Brandacher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosuppression+and+monitoring+of+rejection+in+hand+transplantation.">Immunosuppression and monitoring of rejection in hand transplantation.</a> Tech. Hand Up. Extrem. Surg. 17, 208-214 (2013).</li><li>Lee, W. P., 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=Relative+antigenicity+of+components+of+a+vascularized+limb+allograft.">Relative antigenicity of components of a vascularized limb allograft.</a> Plast. Reconstr. Surg. 87, 401-411 (1991).</li><li>Sucher, 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=Hemiface+allotransplantation+in+the+mouse.">Hemiface allotransplantation in the mouse.</a> Plast. Reconstr. Surg. 129, 867-870 (2012).</li><li>Ibrahim, Z., 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=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.</a> J Vis Exp. , (2013).</li><li>Oberhuber, 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=Murine+cervical+heart+transplantation+model+using+a+modified+cuff+technique.">Murine cervical heart transplantation model using a modified cuff technique.</a> J Vis Exp. , (2014).</li><li>Sucher, 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>Maglione, 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=A+novel+technique+for+heterotopic+vascularized+pancreas+transplantation+in+mice+to+assess+ischemia+reperfusion+injury+and+graft+pancreatitis.">A novel technique for heterotopic vascularized pancreas transplantation in mice to assess ischemia reperfusion injury and graft pancreatitis.</a> Surgery. 141, 682-689 (2007).</li><li>Sucher, 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=Orthotopic+hind-limb+transplantation+in+rats.">Orthotopic hind-limb transplantation in rats.</a> J Vis Exp. , (2010).</li><li>Zou, Y., Brandacher, G., Margreiter, R., Steurer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cervical+heterotopic+arterialized+liver+transplantation+in+the+mouse.">Cervical heterotopic arterialized liver transplantation in the mouse.</a> J. Surg. Res. 93, 97-100 (2000).</li><li>Zhang, F., 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=Development+of+a+mouse+limb+transplantation+model.">Development of a mouse limb transplantation model.</a> Microsurgery. 19, 209-213 (1999).</li><li>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=Upper-extremity+transplantation+using+a+cell-based+protocol+to+minimize+immunosuppression.">Upper-extremity transplantation using a cell-based protocol to minimize immunosuppression.</a> Ann. Surg. 257, 345-351 (2013).</li><li>Azari, K., Brandacher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+composite+allotransplantation.">Vascularized composite allotransplantation.</a> Curr Opin Organ Transplant. 18, 631-632 (2013).</li><li>Pomahac, B., Gobble, R. M., Schneeberger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facial+and+hand+allotransplantation.">Facial and hand allotransplantation.</a> Cold Spring Harb. Perspect. Med. 4, (2014).</li><li>Chong, A. S., Alegre, M. L., Miller, M. L., Fairchild, 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=Lessons+and+limits+of+mouse+models.">Lessons and limits of mouse models.</a> Cold Spring Harb. Perspect. Med. 3, a015495 (2013).</li><li>Lin, C. H., 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+neck+as+a+preferred+recipient+site+for+vascularized+composite+allotransplantation+in+the+mouse.">The neck as a preferred recipient site for vascularized composite allotransplantation in the mouse.</a> Plast. Reconstr. Surg. 133, 133e-141e (2014).</li><li>Shapiro, R. I., Cerra, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+reimplantation+and+transplantation+of+a+complex+organ:+the+rat+hind+limb.">A model for reimplantation and transplantation of a complex organ: the rat hind limb.</a> J. Surg. Res. 24, 501-506 (1978).</li><li>Leto Barone, A. 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=The+gracilis+myocutaneous+free+flap+in+swine:+an+advantageous+preclinical+model+for+vascularized+composite+allograft+transplantation+research.">The gracilis myocutaneous free flap in swine: an advantageous preclinical model for vascularized composite allograft transplantation research.</a> Microsurgery. 33, 51-55 (2013).</li><li>Mathes, D. W., 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=A+preclinical+canine+model+for+composite+tissue+transplantation.">A preclinical canine model for composite tissue transplantation.</a> J. Reconstr. Microsurg. 26, 201-207 (2010).</li><li>Barth, R. N., 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=Prolonged+survival+of+composite+facial+allografts+in+non-human+primates+associated+with+posttransplant+lymphoproliferative+disorder.">Prolonged survival of composite facial allografts in non-human primates associated with posttransplant lymphoproliferative disorder.</a> Transplantation. 88, 1242-1250 (2009).</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, 39-50 (2012).</li><li>Foster, R. D., Liu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+hindlimb+transplantation+in+the+mouse.">Orthotopic hindlimb transplantation in the mouse.</a> J. Reconstr. Microsurg. 19, 49 (2002).</li><li>Tung, T. H., Mohanakumar, T., Mackinnon, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Mouse+Model+for+Heterotopic+Limb+and+Composite-Tissue+Transplantation.">Development of a Mouse Model for Heterotopic Limb and Composite-Tissue Transplantation.</a> J. Reconstr. Microsurg. 17, 267-274 (2001).</li><li>Zhang, F., Shi, D. Y., Kryger, Z., Moon, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+mouse+limb+transplantation+model.">Development of a mouse limb transplantation model.</a> Microsurgery. 19 (5), 209-213 (1999).</li><li>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=Atypical+acute+rejection+after+hand+transplantation.">Atypical acute rejection after hand transplantation.</a> Am. J. Transplant. 8, 688-696 (2008).</li><li>Mathes, D. W., 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=Stable+mixed+hematopoietic+chimerism+permits+tolerance+of+vascularized+composite+allografts+across+a+full+major+histocompatibility+mismatch+in+swine.">Stable mixed hematopoietic chimerism permits tolerance of vascularized composite allografts across a full major histocompatibility mismatch in swine.</a> Transpl. Int. 27, 1086-1096 (2014).</li><li>Yamada, Y., 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+CTLA4Ig+for+induction+of+mixed+chimerism+and+renal+allograft+tolerance+in+nonhuman+primates.">Use of CTLA4Ig for induction of mixed chimerism and renal allograft tolerance in nonhuman primates.</a> Am. J. Transplant. 14, 2704-2712 (2014).</li><li>Sachs, D. H., Kawai, T., Sykes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+tolerance+through+mixed+chimerism.">Induction of tolerance through mixed chimerism.</a> Cold Spring Harb. Perspect. Med. 4, a015529 (2014).</li><li>Kawai, T., 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=HLA-mismatched+renal+transplantation+without+maintenance+immunosuppression.">HLA-mismatched renal transplantation without maintenance immunosuppression.</a> N. Engl. J. Med. 358, 353-361 (2008).</li><li>Kawai, T., Sachs, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+induction:+hematopoietic+chimerism.">Tolerance induction: hematopoietic chimerism.</a> Curr Opin Organ Transplant. 18, 402-407 (2013).</li><li>Kawai, T., Sachs, D. H., Sykes, M., Cosimi, A. .. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA-mismatched+renal+transplantation+without+maintenance+immunosuppression.">HLA-mismatched renal transplantation without maintenance immunosuppression.</a> N. Engl. J. 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Vascularized Composite Allotransplantation, such as upper extremity and face transplantation for reconstruction of devastating tissue defects, has evolved as a valid treatment option for patients not amendable to conventional reconstructive procedures. Technical advances in the field of reconstructive microsurgery as well as a vast experience with potent immunosuppressive and immune modulatory therapies in solid organ transplantation, now enables long-term allograft survival in this unique patient population 3,21</s...

The late nineties heralded the pioneering days of reconstructive transplantation with the first successful hand transplant performed in France in 1998. Since then, the use of VCAs for reconstruction of devastating tissue defects has been successfully employed in a wide spectrum of patients. To date, the world counts 76 recipients of 112 upper extremities as well as 31 faces 1-3. In addition, several other types of VCAs such as abdominal wall 4, larynx 5, trachea 6, vascularized joints 7, and even penis 8 have been performed. Furthermore, the live birth of a baby was recently reported after uterus transplantation 9. This growing world experience is indicative for how reconstructive transplantation has become a valid therapeutic option for patients suffering of significant functional tissue defects not amendable to conventional reconstructive and restorative surgery and treatment. 
While the idea of replacing "like with like" sparked clinical enthusiasm, initial skepticism still prevails with regards to side effects of conventional high-dose immunosuppression required to maintain allografts and their function 10,11. However, as shown by seminal work of Lee et al., these composite grafts are less likely to reject than its individual components, and furthermore, some of the tissue components such as the vascularized bone compartment have fueled optimism as they might exert unique immunological effects onto the balance of immune acceptance and rejection 12. 
Our group pioneered several microsurgical animal models for solid organ transplantation, as well as vascularized composite allotransplantation 13-19. Here we describe a novel surgical procedure using a non-suture cuff technique to perform super micro-vascular anastomosis in an orthotopic mouse hind limb transplantation model. This transplant model provides a useful tool for investigating immune acceptance and rejection mechanisms, as well as the role of individual tissue components, such as the vascularized bone marrow compartment, towards tolerance induction in the immunologically versatile setting of the mouse species. Additionally, the orthotopic placement of the limb opens the possibilities for nerve regeneration and functional outcome studies, which are critically important to the setting of VCA.

<table>
	<tbody>
		<tr>
			<td>Suture, 6-0 Nylon</td>
			<td>MWI</td>
			<td>31849</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture, 6-0 Polysorb</td>
			<td>MWI</td>
			<td>72667</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture, 10-0 Nylon</td>
			<td>Aero Surgical</td>
			<td>TK-107038</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyimide Tubing, Size 25</td>
			<td>Vention Medical</td>
			<td>141-0023</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyimide Tubing, Size 27</td>
			<td>Vention Medical</td>
			<td>141-0015</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvascular Clamps (Single)</td>
			<td>Synovis</td>
			<td>00396</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvascular Clamps (Double)</td>
			<td>Synovis</td>
			<td>00414</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Scissors</td>
			<td>Synovis</td>
			<td>SAS-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Forceps</td>
			<td>Synovis</td>
			<td>FRS-15 RM-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Dilators</td>
			<td>Synovis</td>
			<td>FRS-15 RM-8d.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Needledriver</td>
			<td>Synovis</td>
			<td>C-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Clamp Applicator</td>
			<td>Synovis</td>
			<td>CAF-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Flushing Needle</td>
			<td>Hamilton</td>
			<td>N/A</td>
			<td>10MM, 30&#176;, 33G</td>
		</tr>
		<tr>
			<td>Lactated Ringers Solution</td>
			<td>Fisher Scientific</td>
			<td>NC9968051</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>DEA Number required; Obtained from hosptial pharmacy.</td>
		</tr>
		<tr>
			<td>Enrofloxacin; Baytril</td>
			<td>Bayer Health Care</td>
			<td>186599</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Obtained from hosptial pharmacy</td>
		</tr>
	</tbody>
</table>

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.

Performing vascularized composite allotransplantation in a mouse model using a non-suture cuff technique allows to achieve excellent and long term graft and animal survival as shown in Figure 1. Furthermore, it represents a reliable method of obtaining reproducible outcomes of gradual allograft rejection in vascularized composite allotransplantation as documented by the images shown in Figure 2. H&#38;E histology of tissues obtained from animals undergoin...

Watch this Scientific Journal Video about Orthotopic Hind Limb Transplantation in the Mouse at JoVE.com

Orthotopic Hind Limb Transplantation in the Mouse

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 ...

The overall goal of this instructional video is to introduce a murine model of Orthotopic Hind Limb Transplantation using a non-suture cuff technique for super-microvascular anastomosis. Animal models are paramount to the understanding and treatment of human disease and have paved the way for reconstructive transplantation to become a clinical reality over the past two decades. This particular model can help answer key questions related to the path of physiology of an alloimmune response specific to vascularized composite allografts.The main advantage of the technique is that the non-suture cuff technique significantly reduces the complexity and increases the success rate of this super-microvasular procedure. After confirming the appropriate level of sedation by toe pinch shave the hind limb and the groin of the donor animal. Next disinfect the skin with 10%povidone iodine and place the mouse under a disecting microscope.Using scissors begin the donor limb harvest with a groin skin incision proximal to the mid thigh area. Circumstantially connecting the incision to demarcate the hind limb from the rest of the rest of the body. Identify and dissect the femoral artery, vein, and nerve.Then use forceps and microscissors to separate the three structures. Preventing microtrauma to the vessel wall by using a no touch technique is essential for ensuring the success of the procedure. Once the vascular pedicle has been dissected use the microscissors to divide the vessels at the inguinal ligament.Continue the division of the individual ventral and dorsal muscle groups approximately at the mid thigh to separate the graft from the donor animal. Then, transect the femur cutting at the middle of the femoral shaft. Using a syringe equipped with a 33 gauge needle, flush the harvested limb with two ml of heparinized four degree Celsius saline.Then, place individual polyimide cuffs on the femoral vein and artery. And store the graft in wet cotton gauze in a petri dish at four degrees Celsius until inset. Mounting the pesis onto the polyimide cuffs is the single most critical step of the procedure.As malnotation may impair the bloodflow and improper tish pressmon may expose some antibiotic tissues increasing the likelihood of thrombosis formation. To remove the recipient hind limb, inject 0.3 ML of warm saline intraperitoneally. Make a circumferential incision around the hind limb and identify and dissect the femoral artery, vein, and nerve as just demonstrated.Once the vascular pedicle has been dissected clamp the femoral vessels at the inguinal ligament. And cut the vessels distally at the superficial epigastric artery. Continue to divide the individual ventral and dorsal muscle groups approximately at the mid thigh to separate the native hind limb from the recipient animal.Then transect the femur in the middle of the femoral shaft. Then using a 20 gauge spinal needle as a intramedullary rod and align the femur bones of the recipient and the graft in the native hind limb position. Connect the donor and recipient femur bones.Use absorbable suture material to coapt the ventral and dorsal muscle groups. Performing this alignment accurately and quickly prevents extended blood loss from the bone cavity. To connect the femoral vessels, pull the recipient side of the vessel over the polyimide cuffs.And use a 10-0 nylon suture to fix the recipient vessel onto the cuff with a circumferential tie. When the vessel has been stabilized release the clamps. And visually verify that the cuffs are not misrotated.Visually verify homeostasis and use electrocautery to stop any source of bleeding. Then close the skin with non-absorbable nylon sutures. And monitor the animal until it is fully recovered.Performing vascularized composite allotransplantation in a mouse model using this non-suture cuff technique facilitates the achievement of excellent and long-term graft and animal survival. Moreover, this technique represents a reliable method for obtaining reproduceable outcomes of gradual allograft rejection and a vascularized composite allotransplantation model as documented by the images. Indeed, histological analysis of tissues obtained from animals undergoing rejection further underscores the reproducible dynamics of allograft rejection in this murine model.Once mastered, this technique can be completed in 90 to 120 minutes, if it is performed properly. While attempting this procedure it's important to remember to pay the utmost attention to the handling of the tissue and to avoid any sort of bleeding. After its development, this technique can pave the way for researchers in the field of reconstructive transplantation.To explore basic mechanistic questions related to the alloimmunity of vascularized composite allografts. After watching this video, you should have a good understanding of how to perform all the critical steps of an Orthotopic Hind Limb transplantation in the mouse.

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Research

JoVE Journal

Medicine

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

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 ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

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 ...

Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. However, lung transplantation is limited by a shortage of available donor organs. As such, there is high waitlist mortality. Ex situ lung perfusion (ESLP) has increased donor lung utilization rates in some centers by 15%-20%. ESLP has been applied as a method to assess and recondition marginal donor lungs and has demonstrated acceptable short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs. Large animal (in vivo) transplantation models are required to validate ongoing in vitro research findings. Anatomic and physiologic differences between humans and pigs pose significant technical and anesthetic challenges. An easily reproducible transplant model would permit the&#160;in vivo&#160;validation of current ESLP strategies and the preclinical evaluation of various interventions designed to improve donor lung function. This protocol describes a porcine model of orthotopic left lung allotransplantation. This includes anesthetic and surgical techniques, a customized surgical checklist, troubleshooting, modifications, and the benefits and limitations of the approach.

This protocol describes a juvenile porcine model of orthotopic left lung allotransplantation designed for use with ESLP research. Focus is made on anesthetic and surgical techniques, as well as critical steps and troubleshooting.

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. ...