This work was funded by NIH grant R01AI077610.

All procedures 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 protocols MO13M292 and MO13M370.
1. Donor Skin Harvest
<ol>
	<li>Anesthetize the donor mouse with isoflurane (induction vaporizer at 4%, maintenance at 1 - 2% through the mouse cone). Use the toe pi...

<ol>
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J., 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+the+Mouse.">Orthotopic Hind Limb Transplantation in the Mouse.</a> J Vis Exp. , (2016).</li><li>Oh, 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=A+Novel+Microsurgical+Model+for+Heterotopic,+En+Bloc+Chest+Wall,+Thymus,+and+Heart+Transplantation+in+Mice.">A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice.</a> J Vis Exp. , (2016).</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. , e50753 (2014).</li><li>Jones, T. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviour+and+fate+of+skin+autografts+and+skin+homografts+in+rabbits:+A+report+to+the+War+Wounds+Committee+of+the+Medical+Research+Council.">The behaviour and fate of skin autografts and skin homografts in rabbits: A report to the War Wounds Committee of the Medical Research Council.</a> J Anat. 78, 176-199 (1944).</li><li>Billingham, R. E., Brent, L., Medawar, P. B., Sparrow, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+studies+on+tissue+transplantation+immunity.+I.+The+survival+times+of+skin+homografts+exchanged+between+members+of+different+inbred+strains+of+mice.">Quantitative studies on tissue transplantation immunity. I. The survival times of skin homografts exchanged between members of different inbred strains of mice.</a> Proc R Soc Lond B Biol Sci. 143, 43-58 (1954).</li><li>Garrod, K. R., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+skin+transplantation.">Murine skin transplantation.</a> J Vis Exp. , (2008).</li><li>Schmaler, M., Broggi, M. A., Rossi, S. W. <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+tail+skin+to+study+allogeneic+CD4+T+cell+responses+in+mice.">Transplantation of tail skin to study allogeneic CD4 T cell responses in mice.</a> J Vis Exp. , e51724 (2014).</li><li>Bergstresser, P. R., Toews, G. B., Gilliam, J. N., Streilein, J. 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Since its introduction by Medawar, first in human studies and then in rabbits and mice, skin transplantation has been an invaluable model for the study of allogeneic immune responses6,7. In this manuscript, we present a model of large-scale, non-vascularized, full-thickness skin transplantation using the upper and lower back skin. Various alternative methods, including using the tail skin or ear skin of the mouse as the graft tissue source, have been reported to date8,9. These models present the dis...

Organ transplantation is the treatment of choice for patients with end-stage organ failure, and outcomes have improved remarkably with advances in surgical procedures and immunosuppression protocols. However, long-term immunosuppression is associated with significant side effects, and the development of new strategies that promote tolerance remains the goal of modern transplantation research.
Numerous animal models have been developed for basic research in transplantation, to study the mechanisms of allograft rejection and to test immunosuppression approaches for preventing graft rejection and for promoting long-term tolerance1-3. Mouse models have become the mainstay of immunological research due to the exclusive and vast availability of diagnostic and therapeutic antibodies and well-defined inbred and transgenic strains. Skin transplantation is a simple procedure that does not require special microsurgical skills and can be easily monitored postoperatively. Taken together, mouse skin transplantation has been an exceptional tool to study many aspects involved in the alloimmune response, including antigen delivery, cell trafficking, and tissue destruction during graft rejection4,5.
Here, we show the step-by-step procedure for full-thickness skin transplantation using the mouse model, and we describe the important steps necessary for a successful engraftment of the transplanted skin.

<table border="0" cellpadding="0" cellspacing="0" width="923">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Straight micro forceps&#160;</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:136px;">F4017</td>
			<td style="width:415px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Curved micro forceps&#160;</td>
			<td style="width:125px;">Aesculap</td>
			<td style="width:136px;">BD333R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Curved Stevens tenotomy scissors&#160;</td>
			<td style="width:125px;">Aesculap</td>
			<td style="width:136px;">BC905R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mayo dissecting&#160; scissors</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:136px;">S3146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Micro needle holder</td>
			<td style="width:125px;">Aesculap</td>
			<td style="width:136px;">BM563R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sterile gauze</td>
			<td style="width:125px;">Covidien</td>
			<td style="width:136px;">441218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">6-0 Nylon suture&#160;</td>
			<td style="width:125px;">MWI</td>
			<td style="width:136px;">31849</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Plastic Strips Band-Aid</td>
			<td style="width:125px;">Johnson &#38; Johnson</td>
			<td style="width:136px;"></td>
			<td>Obtained from pharmacy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">10 cm Petri dish</td>
			<td style="width:125px;">Fisherbrand</td>
			<td style="width:136px;">FB0875712</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">PBS</td>
			<td style="width:125px;">Quality Biological</td>
			<td style="width:136px;">119069131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Buprenorphine</td>
			<td style="width:125px;"></td>
			<td style="width:136px;"></td>
			<td>DEA Number required; Obtained from hospital pharmacy</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Enrofloxacin</td>
			<td style="width:125px;">Bayer Health Care</td>
			<td style="width:136px;">186599</td>
			<td></td>
		</tr>
	</tbody>
</table>

murine full-thickness skin transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

The placement of the bandage on the recipient mouse is an important step of the procedure. The skin graft is positioned on the recipient trunk, between the shoulder, hip, and spine (Figure 1). The bandage is made with folded gauze and the combination of two plastic adhesive bandages. The recipient mouse is placed with the graft down over the gauze on the center of the bandage. Using two curved micro forceps, the lower end of the bandage is pulled first, and then the top o...

Watch this Scientific Journal Video about Murine Full-thickness Skin Transplantation at JoVE.com

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

The overall goal of this video article is to perform a full-thickness skin transplantation, and describe the important steps necessary for a successful procedure. Murine full-thickness skin transplantation is a well-established model to study alloimmune responses and graft rejection. The procedure is easy to perform, and does not require delicate microsurgical techniques.Graft rejection, in this model, occurs in a very reproducible immunological reaction, and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed to more complex transplant models, as an alternative and uncomplicated method to assess donor specific tolerance. After anesthetizing a donor mouse according to the text protocol, use an electric razor to shave the back of the animal.And, with 10%povidone-iodine, disinfect the skin. Using scissors and blunt dissection, harvest the donor back skin from the hip to the neck at the level of the areolar connective tissue. Place the back skin under a microscope, and use fine tenotomy scissors to separate the connective tissue and the panniculus carnosus from the skin.Cut out 15 by 15 millimeter grafts from the back skin for a 10 by 10 to 15 by 15 millimeter graft bed. Store the grafts on gauze soaked with PBS in a Petri dish, and place it on ice. Anesthetize the recipient mouse according to the text protocol.Shave the side of the back of the animal, where the graft will be inserted, and then administer 0.02 milligrams per kilogram of buprenorphine for post-operative pain relief. Use 10%povidone-iodine to disinfect the area. Using scissors, and as superficially as possible, cut a 10 by 10 to 15 by 15 millimeter square of skin.Take care to preserve the panniculus carnosus and blood vessels that run superficial to it. Next, position the graft on the graft bed, avoiding folds along the edges. Then, place eight sutures on the corners of the graft, and halfway along each edge by passing the needle through the graft, and then through the panniculus carnosus of the graft bed, below the surrounding recipient skin.Place folded gauze over a doubled-up bandage, and then position the animal on the bandage, graft side down, removing the anesthetic mask to let the animal partially recover. Then, wrap the bandage around the animal. The most critical factor in the process of engraftment is revascularization of graft tissue by ingrowth of host vessels to the graft.The dissection of the panniculus carnosus and the correct placement of the bandage are the most important steps. Immediately following surgery, administer 5 milligrams per kilogram of enrofloxacin for infection prophylaxis. Place the transplanted mouse in a clean cage until it fully recovers from anesthesia.Observe the mouse for one hour postoperatively, prior to returning it to the housing facility. Monitor the mouse closely during recovery to ensure that the bandage is not restricting thorax excursion and breathing. Eight days later, observe and palpate the graft for signs of scabbing, contraction, or hardness.If any of these signs are present, the graft may not have achieved proper vascularization, and should be considered a technical failure. Monitor the graft daily for signs of rejection. Consider grafts rejected when greater than or equal to 90%of the graft tissue becomes necrotic.In complete mismatch models, full-thickness skin grafts are usually rejected in eight to 12 days. In minor mismatch models, rejection responses are slower, more variable, and the skin graft appearance is characterized by less distinct changes, such as contraction, or loss of hair. Acute graft rejection generally begins with swelling and erythema of the graft.These events are followed by graft desiccation, scab formation, and shrinkage, as seen here. Depending on the degree of MHC mismatch, and the immunosuppression protocol, rejection can occur by a subacute process marked by subtle changes, such as loss of hair, pigmentation, dermal ridges, and graft volume. In these cases, a rejected graft appears shiny, white, and hairless, with uneven edges.Using BALB/c as donors, and C57 black 6 as recipients, it was observed that inhibiting glycolysis and oxidative phosphorylation, or glutamine metabolism alone, prolonged skin graft survival. But blocking the three pathways simultaneously resulted in significantly increased graft survival. Moreover, triple metabolic therapy was more effective than conventional cyclosporine, or rapamycin.Furthermore, the histology of skin grafts from the metabolic therapy group exhibited more intact tissue alignment, and less lymphocytic inflammatory infiltrate, compared to the untreated tissue. Outcomes of organ transplantation have improved remarkably with advances in surgical procedures, and immunosuppression protocols. However, long-term immunosuppression is associated with significant side effects, and new strategies that promote tolerance remain the goal of modern transplantation research.It is well-known the importance of metabolism in regulating T-cells. By inhibiting glycolysis, oxidative phosphorylation, and glutamine metabolism, we were able to significantly increase allograft survival. This novel approach was more effective than conventional therapy, and represents a paradigm shift in the potential approach to immune regulation and transplantation.

murine-full-thickness-skin-transplantation

Research

JoVE Journal

Immunology and Infection

19.6K vistas.  Johns Hopkins University School of Medicine. The overall goal of this video article is to perform a full-thickness skin transplantation, and describe the important steps necessary for a successful procedure. Murine full-thickness skin transplantation is a well-established model to study alloimmune responses and graft rejection. The procedure is easy to perform, and does not require delicate microsurgical techniques.Graft rejection, in this model, occurs in a very reproducible immunological reaction, and is easily monitored by dir...