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 JoVE Biology

Murine Skin Transplantation

1, 1

1Department of Physiology and Biophysics, University of California, Irvine (UCI)

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    Summary

    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-->C57BL/6 skin transplant.

    Date Published: 1/16/2008, Issue 11; doi: 10.3791/634

    Cite this Article

    Garrod, K. R., D. Cahalan, M. Murine Skin Transplantation. J. Vis. Exp. (11), e634, doi:10.3791/634 (2008).

    Abstract

    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 <10 min to perform.

    Protocol

    Prior to the initiation of experiment:

    • Obtain IACUC approval
    • Sterilize surgical instruments and Q-tips

    I. Harvest Donor Tissue

    1. Euthanize donor mouse (in accordance with institution's SOP)
    2. Wet hair back from base of ear with alcohol
    3. Harvest donor skin by cutting ear off at base
    4. Place tissue in plate on ice
    5. 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

    II. Prepare Recipient for Transplant

    1. Anesthetize mouse using approved procedure
    2. Administer analgesic for post-op pain relief (i.e. 0.05-0.1 mg/g Bupernex ip)
    3. Place mouse in lateral position
    4. Perform toe pinch to ensure animal is sufficiently sedated
    5. Wet hair with alcohol and shave thorax
    6. Wipe area clean with alcohol swab

    III. Skin Transplant

    1. Make ~ 1 cm incision: tent skin with rat-tooth forceps and cut ~ 0.25-0.5 cm below forceps
    2. 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
    3. 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)
    4. Wrap recipient in sterile bandage (with the non-adhesive gauze segment placed over the skin graft)
    5. Ensure that bandage is not too tight (assess animal's breathing) and that arms are freely mobile
    6. Secure bandage with a single 2-0 silk suture
    7. Place animal in clean cage (under heating lamp until animal has sufficiently recovered from anesthesia)
    8. Monitor recipient 1-2/day for signs of distress and administer analgesic as needed for pain relief

    IV. Bandage Removal (6-7 days post-transplant)

    1. Anesthetize mouse using approved procedure
    2. Cut and remove silk suture
    3. Using blunt end scissors, carefully cut through bandage (making sure not to disrupt graft) and gently remove
    4. Return mouse to clean cage
    5. 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.
    6. Monitor daily for signs of rejection (usually defined as ~80% necrosis of the donor tissue)
    7. Allogeneic skin transplants will usually reject between d8-12 post-op

    V. Helpful Hints

    1. 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
    2. Always use a new razor blade for shaving recipient
    3. If analgesic is to be given ip, administer prior to transplant procedure to minimize post-op handling (minimize chance of disrupting the graft)
    4. If possible, remove gloves before applying the bandage. Gloves tend to stick the bandage and complicate application
    5. Placing a single suture at the bandage end will minimize premature removal
    6. 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.

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    Discussion

    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.

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    Disclosures

    The authors have nothing to disclose.

    Acknowledgements

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

    Materials

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

    Comments

    19 Comments

    Kym, I would really be interested to see a histologic profile of the post-op period - do you have the respective images?
    Thank you in advance,
    Peter
    Reply

    Posted by: AnonymousJanuary 22, 2008, 4:34 PM

    Dear Peter- Thanks for the great suggestion. I've contacted JoVE to see if a section can be added to the article containing histo pics from allo- and syn- transplants.
    Reply

    Posted by: AnonymousJanuary 22, 2008, 7:33 PM

    The connecting speed is too slow, it spends loog time to watch the whole video. Can we download the video?
    Reply

    Posted by: AnonymousMarch 28, 2008, 9:35 AM

    Hi Merlin. I am sorry you are having trouble.  Is it your connection that is slow or ours?  Please use www.speedtest.net to test your connection.  If the problem is on your end, we are now working on a solution to help people with slow connections.  If you would like to discuss this, please send me an email directly to support@jove.com and I'll see what may be the problem.
    Reply

    Posted by: AnonymousMarch 28, 2008, 11:09 AM

    Hi,nikitab. Thank your help. I am from China, I test my connection, the connection is slow from USA to my city. could you tell me how to fix this problem?
    Reply

    Posted by: AnonymousMarch 28, 2008, 10:04 PM

    If you click the pause pause/play button, the video will buffer and you should be able to play it without interruptions after some time.  Hope tihs helps!
    Reply

    Posted by: AnonymousMarch 28, 2008, 10:21 PM

    Merin, just wanted to let you know that we've upgraded the video player.  How are the videos playing now?
    Reply

    Posted by: AnonymousMay 15, 2008, 6:34 PM

     wow ive learned alot from this can you tell me more about this? is it different from human skin transplants? there is alot going on with stem cell, is this simular, this is intresting, would this maby help people that have bad skin burns?
    Reply

    Posted by: AnonymousFebruary 3, 2009, 11:16 PM

    hello this is interesting, is this simular to human skin transplants? dŒs this help people with bad skin burns?  theres alot going on with stem cell research is this simular? i have learned alot from this vidio, i wait for your answers.
    Reply

    Posted by: AnonymousFebruary 3, 2009, 11:32 PM

    Hi, Kym, I learn the technique of ear-skin graftting through the video. Well, in the articles about skin graft in mice, most of them just use the method of back-back or tail-back skin transplantation. And do you provide some articles about ear-back skin graft to me? Thank you very much.
    cai
    Reply

    Posted by: AnonymousJuly 9, 2009, 7:37 PM

    beautiful video. we use a similar technique at the Diamantina Institute, but I got some handy hints watching your technique. thanks.
    Reply

    Posted by: AnonymousAugust 5, 2009, 1:41 AM

    Are donor and recipient mice females or males?
    Reply

    Posted by: AnonymousSeptember 24, 2009, 12:54 PM

    Hi Kym. I was wondering if you or your colleges transplant embryonic skin that way, or if there has to be special protocol for that?
    Reply

    Posted by: AnonymousJanuary 21, 2010, 4:29 PM

    Hi Kim,
    we do skin grafts and we have problems with the bandage. I use band-aids (the ones sold in the regular pharmacy for cuts and bruises) and mice chew them off as soon as they weak up from anesthesia. I do not see in your materials list what kind of bandage you use. Would you please tell me where do you buy it and the catalog number? I would really appreciate.
    best regards
    sg
    Reply

    Posted by: AnonymousMay 28, 2010, 3:03 PM

    why i can't see it? is it because the subscription lock? Our school don't have this database. How can i see it ? is it mean that that i need to buy it ?
    Reply

    Posted by: jin l.October 16, 2011, 8:02 AM

    Access to this article is only available through an institutional subscription. Please contact your Librarian and include me on cc, and we will arrange a ² weeks FREE trial to the entire institution. Not only will this give you access to this article but will provide you with the opportunity to access other content.

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    Reply

    Posted by: AnonymousOctober 16, 2011, 6:40 PM

    I am very sorry that i reply so late. At first, i try to contact you through email, but it was rejected by the recipient domain.
    I am from China and our school librarian didn't have this service now, so i only to contact you, and thank you to arrange a free trial for me. Please tell me what should i do to apply the free trial, you can send the details to me. If i can't receive your email. i will post message on jove.thank you again.
    Best wishes
    jin liu
    Chengdu, China. Chengdu medical college
    Sichuan Province, Chengdu Chengdu City Jinniu District Road No. 601
    Reply

    Posted by: jin l.October 19, 2011, 9:37 PM

    Dear Jin,
    I have forwarded your response to our account manager in China. She will be in touch in due course. Thank you for your interest in JoVE.

    Best,
    Ward
    Reply

    Posted by: AnonymousOctober 20, 2011, 12:14 PM

    thank you!
    Reply

    Posted by: jin l.October 21, 2011, 9:37 AM

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