Name: murine full-thickness skin transplantation
Uploaded: 2017-01-02T15:00:00
Description: Watch this Scientific Journal Video about Murine Full-thickness Skin Transplantation at JoVE.com

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>
	<li>Furtmuller, G. 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. R., Shirasugi, N., Adams, A. B., Pearson, T. C., Larsen, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+identifies+selectins+that+regulate+T+cell+traffic+into+allografts.">Intravital microscopy identifies selectins that regulate T cell traffic into allografts.</a> J Clin Invest. 112, 1714-1723 (2003).</li><li>Celli, S., Albert, M. L., Bousso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+the+innate+and+adaptive+immune+responses+underlying+allograft+rejection+by+two-photon+microscopy.">Visualizing the innate and adaptive immune responses underlying allograft rejection by two-photon microscopy.</a> Nat Med. 17, 744-749 (2011).</li><li>Medawar, P. 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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unusual+numbers+and+distribution+of+Langerhans+cells+in+skin+with+unique+immunologic+properties.">Unusual numbers and distribution of Langerhans cells in skin with unique immunologic properties.</a> J Invest Dermatol. 74, 312-314 (1980).</li><li>Chen, H. D., Silvers, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Langerhans+cells+on+the+survival+of+H-Y+incompatible+skin+grafts+in+rats.">Influence of Langerhans cells on the survival of H-Y incompatible skin grafts in rats.</a> J Invest Dermatol. 81, 20-23 (1983).</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>Mayumi, H., Nomoto, K., Good, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surgical+technique+for+experimental+free+skin+grafting+in+mice.">A surgical technique for experimental free skin grafting in mice.</a> Jpn J Surg. 18, 548-557 (1988).</li><li>McFarland, H. I., Rosenberg, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+allograft+rejection.">Skin allograft rejection.</a> Curr Protoc Immunol. Chapter 4, (2009).</li><li>Lee, C. 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=Preventing+Allograft+Rejection+by+Targeting+Immune+Metabolism.">Preventing Allograft Rejection by Targeting Immune Metabolism.</a> Cell reports. 13, 760-770 (2015).</li><li>Pollizzi, K. N., Powell, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+canonical+and+metabolic+signalling+programmes+in+the+regulation+of+T+cell+responses.">Integrating canonical and metabolic signalling programmes in the regulation of T cell responses.</a> Nat Rev Immunol. 14, 435-446 (2014).</li><li>Pearce, E. L., Poffenberger, M. C., Chang, C. H., Jones, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fueling+immunity:+insights+into+metabolism+and+lymphocyte+function.">Fueling immunity: insights into metabolism and lymphocyte function.</a> Science. 342, 1242454 (2013).</li></ol>

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

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Granulocyte-dependent Autoantibody-induced Skin Blistering

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to Witebsky's postulates, one of the criteria in diagnosing a disease as autoimmune is the reproduction of the disease in experimental animals by the passive transfer of autoantibodies. For epidermolysis bullosa acquisita (EBA), a prototypic organ-specific autoimmune disease of skin and mucous membranes, several experimental models were recently established. In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human EBA 1. Full-blown widespread disease is usually seen 5-6 days after the first injection and the extent of the disease correlates with the dose of the administered collagen VII-specific IgG. The tissue damage (blister formation) in the experimental EBA is depending on the recruitment and activation of granulocytes by tissue-bound autoantibodies 2,-4. Therefore, this model allows for the dissection of the granulocyte-dependent inflammatory pathway involved in the autoimmune tissue damage, as the model reproduces only the T cell-independent phase of the efferent autoimmune response. Furthermore, its value is underlined by a number of studies demonstrating the blister-inducing potential of autoantibodies in vivo and investigating the mechanism of the blister formation in EBA 1,3,-6. Finally, this model will greatly facilitate the development of new anti-inflammatory therapies in autoantibody-induced diseases. Overall, the passive transfer animal model of EBA is an accessible and instructive disease model and will help researchers to analyze not only EBA pathogenesis but to answer fundamental biologically and clinically essential autoimmunity questions.

In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human epidermolysis bullosa acquisita (EBA)1.

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of experimental models needs to be as rigorous as that applied for human samples. Indeed, drawing reliable and accurate conclusions is critically influenced by the quality of tissue section preparation. Here, we describe a protocol for histopathological analysis of murine tissues that implements several automated steps during the procedure, from the initial preparation to the paraffin embedding of the murine samples. The reduction of methodological variables through rigorous protocol standardization from automated procedures contributes to increased overall reliability of murine pathological analysis. Specifically, this protocol describes the utilization of automated processing and embedding robotic systems, routinely used for the tissue processing and paraffin embedding of human samples, to process murine specimens of intestinal inflammation. We conclude that the reliability of histopathological examination of murine tissues is significantly increased upon introduction of standardized and automated techniques.

Lack of standardization for murine tissue processing reduces the quality of murine histopathological analysis as compared to human specimens. Here, we present a protocol to perform histopathological examination of murine inflamed and uninflamed colonic tissues to show the feasibility of robotic systems routinely used for processing and embedding&#160;human samples.

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of ...

Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation is an important method for investigating thymic epithelial cell function and T cells maturation in vivo. Here we will describe the experimental methods used within our laboratory to transplant 2&#8217;-deoxyguanosine (to deplete donor&#8217;s lymphocytes) treated embryonic thymus into the renal capsule of an athymic nude mouse. This method is both simple and efficient and does not require special skills or devices. The results obtained via this simple method showed that transplanted thymus can effectively support the recipient&#8217;s T cells production. Additionally, several key points with regards to the protocol will be further elucidated.

Renal Subcapsular Transplantation of 2&#039;-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice

We provide a simple and efficient method to transplant 2&#8217;-deoxyguanosine treated E18.5 thymus into the renal capsule of a nude mouse. This method should aide in the study of both thymic epithelial cells function and T cells maturation.

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...