Murine Full-thickness Skin Transplantation

Summary

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-->C57BL/6 full-thickness skin transplant.

Abstract

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.

Introduction

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 tolerance^1-3. 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 rejection^4,5.

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.

Protocol

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

1. Anesthetize the donor mouse with isoflurane (induction vaporizer at 4%, maintenance at 1 - 2% through the mouse cone). Use the toe pinch withdrawal reflex to monitor the depth of anesthesia.
2. Using an electric razor, shave the back of the animal and disinfect it with 10% povidone iodine (e.g., Betadine).
3. Using sterile scissors, gloves, and aseptic technique, harvest donor back skin from the hip to the neck, with blunt dissection at the level of the areolar connective tissue.
4. Euthanize the animal by cervical dislocation after harvesting the skin graft.
5. Under a microscope, separate the connective tissue, fat tissue, and panniculus carnosus from the back skin using fine tenotomy scissors. The panniculus carnosus is a thin, transparent muscle responsible for skin twitching movements.
6. Using sterile instruments technique, cut out 15-mm x 15-mm grafts from the back skin for a 10-mm x 10-mm to 15-mm x 15-mm graft bed.
7. Store the grafts on gauzes soaked with sterile phosphate-buffered saline (PBS) in a petri dish on ice.
   NOTE: 8 - 10 grafts can be obtained from one donor mouse.

2. Recipient Skin Transplant

1. Anesthetize the recipient mouse with isoflurane (induction vaporizer at 4%, maintenance at 1 - 2% through the mouse cone). Use the toe pinch withdrawal reflex to monitor the depth of anesthesia.
2. Administer 0.02 mg/kg BW of Buprenorphine for postoperative pain relief.
3. Shave the side of the back of the animal where the graft will be inserted and disinfect with 10% povidone iodine.
4. Using scissors, cut a 10-mm x 10-mm to 15-mm x 15-mm square of skin. The defect size should be slightly larger (10%) than the graft. Cut as superficially as possible. Take care to preserve the panniculus carnosus and vessels. The panniculus carnosus can be distinguished from the underlying fascia by its mobility and the blood vessels that run superficial to it.
5. Position the graft on the graft bed, avoiding folds along the edges.
6. Place 8 sutures on the corners and on the middle of each edge. For each suture, pass the needle through the graft and then through the panniculus carnosus of the graft bed below the surrounding recipient skin.
7. Remove the anesthetic mask and let the animal recover partially from anesthesia before applying the adhesive bandage.
8. Wrap the recipient mouse in an adhesive bandage with folded gauze over the graft. Make the bandage by combining two bandages, cutting the adhesive part of one and placing the two absorbent pads together.
9. Monitor the mouse closely during recovery to ensure that the bandage is not restricting thorax excursion and breathing. Remove the bandage if the respiratory rate decreases or the animal starts to gasp or breath shallowly.

3. Postoperative Care

1. Administer enrofloxacin 5 mg/kg after surgery for infection prophylaxis.
2. Place the transplanted mouse in a clean cage over a microwavable heating pad until it fully recovers from anesthesia.
3. Observe the mouse for 1 hr postoperatively prior to returning it to the housing facility.
4. Seven days after surgery, anesthetize the mouse as in Step 2.1. Remove the bandage by cutting only through the ventral side of the bandage.
5. Observe and palpate the graft on the following day for signs of scabbing, contraction, or hardness. If present, the graft may not have achieved proper vascularization and should be considered a technical failure.
6. Monitor daily for signs of rejection. Consider grafts rejected when ≥90% of the graft tissue becomes necrotic.
7. Euthanize the rejected animals and harvest them for analysis.

Results

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...

Discussion

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 responses^6,7. 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 date^8,9. These models present the dis...

Materials

Name	Company	Catalog Number	Comments
Straight micro forceps	Sigma	F4017
Curved micro forceps	Aesculap	BD333R
Curved Stevens tenotomy scissors	Aesculap	BC905R
Mayo dissecting  scissors	Sigma	S3146
Micro needle holder	Aesculap	BM563R
Sterile gauze	Covidien	441218
6-0 Nylon suture	MWI	31849
Plastic Strips Band-Aid	Johnson & Johnson		Obtained from pharmacy
10 cm Petri dish	Fisherbrand	FB0875712
PBS	Quality Biological	119069131
Buprenorphine			DEA Number required; Obtained from hospital pharmacy
Enrofloxacin	Bayer Health Care	186599