Name: Murine Skin Transplantation (Video) | JoVE Journal
Uploaded: 2008-01-16T10:34:00.000+00:00
Duration: 6 min 42 s
Description: Learn skin transplantation techniques for immunology research using the BALB/c to C57BL/6 mouse model. This method enables researchers to study allogeneic responses and graft rejection mechanisms in vivo.

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5 minutes

NIH Immunology Training Grant T32 AI 60573 (KRG) and NIH grant GM 41514 (MDC)

Prior to the initiation of experiment:
<ul>
<li>Obtain IACUC approval</li>
<li>Sterilize surgical instruments and Q-tips</li>
</ul>
I. Harvest Donor Tissue
<ol>
<li>Euthanize donor mouse (in accordance with institution's SOP)</li>
<li>Wet hair back from base of ear with alcohol</li>
<li>Harvest donor skin by cutting ear off at base</li>
<li>Place tissue in plate on ice</li>
<li>Split ear: pinch ventral and dorsal sides of tissue at base of ear with rat-tooth forceps; gently pull apart tissue; float dorsal tissue, internal surface down, in PBS on ice; discard collagenous ventral flap</li>
</ol>
II. Prepare Recipient for Transplant
<ol>
<li>Anesthetize mouse using approved procedure</li>
<li>Administer analgesic for post-op pain relief (i.e. 0.05-0.1 mg/g Bupernex ip)</li>
<li>Place mouse in lateral position</li>
<li>Perform toe pinch to ensure animal is sufficiently sedated</li>
<li>Wet hair with alcohol and shave thorax</li>
<li>Wipe area clean with alcohol swab</li>
</ol>
III. Skin Transplant
<ol>
<li>Make ~ 1 cm incision: tent skin with rat-tooth forceps and cut ~ 0.25-0.5 cm below forceps</li>
<li>Remove donor skin from PBS, place atop graft bed (with internal surface of ear skin facing graft bed), and remove excess PBS with sterile Q-tip</li>
<li>Carefully trim away excess donor skin so that transplant lies flatly within graft bed (making sure that graft edges are not curled under and that graft does not hangover onto recipient's trunk skin)</li>
<li>Wrap recipient in sterile bandage (with the non-adhesive gauze segment placed over the skin graft)</li>
<li>Ensure that bandage is not too tight (assess animal's breathing) and that arms are freely mobile</li>
<li>Secure bandage with a single 2-0 silk suture</li>
<li>Place animal in clean cage (under heating lamp until animal has sufficiently recovered from anesthesia)</li>
<li>Monitor recipient 1-2/day for signs of distress and administer analgesic as needed for pain relief</li>
</ol>
IV. Bandage Removal (6-7 days post-transplant)
<ol>
<li>Anesthetize mouse using approved procedure</li>
<li>Cut and remove silk suture</li>
<li>Using blunt end scissors, carefully cut through bandage (making sure not to disrupt graft) and gently remove</li>
<li>Return mouse to clean cage</li>
<li>Check graft ~8 hrs after bandage removal for signs of scabbing or contraction. If present, graft may have failed to vascularize and should be considered a technical failure.</li>
<li>Monitor daily for signs of rejection (usually defined as ~80% necrosis of the donor tissue)</li>
<li>Allogeneic skin transplants will usually reject between d8-12 post-op</li>
</ol>
V. Helpful Hints
<ol>
<li>Harvesting donor tissue close to the base of the ear will: 1) maximize graft size, and 2) provide an uneven ventral overhang that will facilitate splitting</li>
<li>Always use a new razor blade for shaving recipient</li>
<li>If analgesic is to be given ip, administer prior to transplant procedure to minimize post-op handling (minimize chance of disrupting the graft)</li>
<li>If possible, remove gloves before applying the bandage. Gloves tend to stick the bandage and complicate application</li>
<li>Placing a single suture at the bandage end will minimize premature removal</li>
<li>Wrap bandage as tight as permitted and assess breathing BEFORE placing suture. If bandage is too tight, animal will stop breathing. If bandage is too loose, animal will remove it.</li>
</ol>

The authors have nothing to disclose.

As demonstrated in this video-article, the skin transplant model is a quick and easy method for monitoring allogeneic T cell responses. Allogeneic skin transplantation has a wide range of applications and has been utilized to assay the efficiency of a variety of immunosuppressive agents and tolerance induction protocols, as well as to investigate the biological effects of various blocking antibodies.

<table><tbody><tr><td>Micro-dissecting forceps (curved)</td><td>Sigma-Aldrich</td><td>F4142</td><td></td></tr><tr><td>Rat-tooth forceps (1x2 teeth, straight)</td><td>Fine Science Tools</td><td>11053-10</td><td>n=2</td></tr><tr><td>Strabismus scissors (blunt end)</td><td>Fine Science Tools</td><td>14074-11</td><td></td></tr><tr><td>Iris forceps (curved)</td><td>Fine Science Tools</td><td>11065-07</td><td></td></tr><tr><td>Spring scissors (angled)</td><td>Fine Science Tools</td><td>15006-09</td><td></td></tr><tr><td>Halsey micro needle holder</td><td>Fine Science Tools</td><td>12500-12</td><td></td></tr><tr><td>10 cm plates</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>PBS</td><td>Cellgro</td><td>21-040-CV</td><td></td></tr><tr><td>Bupernex (Buprenorphine hydrochloride)</td><td>Reckitt Benckiser</td><td>517201</td><td></td></tr><tr><td>Insulin syringes (29 Ga)</td><td>Monoject</td><td>8881 600145</td><td></td></tr><tr><td>Q-tips</td><td>Solon Manufacturing Company</td><td>362</td><td></td></tr><tr><td>Single edge razor blades (#9)</td><td>Smith &amp;amp; Nephew Inc.</td><td>67-0238</td><td></td></tr><tr><td>Silk suture (2-0; PS-2)</td><td>Ethicon Inc.</td><td>583H</td><td></td></tr></tbody></table>

murine skin transplantation

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

Welcome to the laboratory of Dr.Michael Kalen. I'm Kim Gard. Today we'll demonstrate a skin transplantation model using the allogeneic BC to black six strain combination as one of the most stringent and least technically challenging models of tissue graft rejection.Skin transplantation is a standard technique employed to assay allogeneic responses and is commonly used to assess efficiency of various tolerance induction strategies. So let's begin. This procedure starts with harvesting the skin from our BAB seed donor mouse.First euthanize the donor in accordance with Iacuc SOP. Now wet hair back at base with alcohol, harvest skin by removing the ear as close to the base as possible and place on ice. Next, split each ear year discarding the collagenous ventral flap and float the tissue on ice.Now the donor skin is ready for transplantation. The transplant procedure begins by anesthetizing the recipient animal using approved procedures. Once the recipient is sufficiently anesthetized, remove the animal from the chamber and give bup andnext IP for pain relief.Place the recipient in a lateral position and assure the animal is fully sedated by performing toe. Pinch wet hair with alcohol and shave. Once recipient is cleanly shaven, make approximately one to two centimeter incision.Place donor scan atop the incision And remove excess fluid with sterile Q-tip. Now carefully trim away the excess donor skin wrap, animal and bandage. Make sure bandage is not too tight by assessing, breathing, and then check that the arms are freely mobile.Now secure the bandage with a single suture. Place the mouse in a clean cage with a heat lamp until the animal has recovered for the first few days. Following surgery, you should monitor the animal a couple of times a day for signs of distress and give bup necks as needed for pain.Five to seven days Following surgery, the bandages are removed. I'll be demonstrating bandage removal in a recipient from day six post-transplant. Once the mouse is fully anesthetized, remove the suture and using a blunt nose scissors cut through the bandage and remove gently, making sure not to disrupt the transplant.Return the animal to a clean cage and monitor daily for signs of rejection. Depending on the strain combination you choose, most skin transplants are rejected, which is usually defined as 80%necrosis by day eight to 12. This mouse is day eight post-op and has received bilateral transplants.On the left is a control syngeneic black six transplant, which will not be rejected. On the right is the allogeneic BAB sea transplant that will be rejected. Here's another date transplant that is in the process of being rejected.Finally, a different date transplant that is almost completely rejected. So these three examples demonstrate the normal variation in kinetics of skin transplant rejection. In the preceding experiment, we've shown you how to perform a mirroring skin transplant.This model is a quick and easy method of monitoring allogeneic responses and has a wide range of applications. For example, skin transplantation can be used to assay the efficiency of various immunosuppressive agents blocking antibodies and tolerance induction protocols. So that's it.Thanks for watching and good luck with your experiments.

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