Protocol for Cutaneous Wound Healing Assay in a Murine Model

  • Gitali Ganguli-Indra
Part of the Methods in Molecular Biology book series (MIMB, volume 1210)


Cutaneous wound healing assay is important to address many key questions including (1) migration ability of different cells; (2) communication between the different cell types such as keratinocytes, fibroblasts, and immune cells; (3) understanding the cell-autonomous and non-cell-autonomous function(s) of the different cells; and (4) gene regulation in healing processes. Wound healing studies can be used to test new treatment modalities, function of new drugs/compounds, and stem cell-based therapies on the different stages of healing and for accelerating wound healing in patients with compromised healing. In this chapter, we have described a simple step-by-step protocol to generate full-thickness cutaneous wounds in the dorsal skin of mice, followed by collecting the post-wounding biopsied materials on specific days for histological and immunohistochemical analyses and for RNA and protein extractions.

Key words

Cutaneous wound healing Therapeutic targets Transcription factors Histology Immunohistochemistry Protein Biopsy Mice, Skin 



I would like to thank Xiaobo Liang and Shreya Bhattacharya for the images. I would like to also thank Arup K. Indra for critical reading of the review article. This work was supported by NIH grant 5R01AR056008-02.


  1. 1.
    Fuchs E, Raghavan S (2002) Getting under the skin of epidermal morphogenesis. Nat Rev Genet 3:199–209PubMedCrossRefGoogle Scholar
  2. 2.
    Lippens S, Denecker G, Ovaere P, Vandenabeele P, Declercq W (2005) Death penalty for keratinocytes: apoptosis versus cornification. Cell Death Differ 12(Suppl 2):1497–1508PubMedCrossRefGoogle Scholar
  3. 3.
    Proksch E, Brandner JM, Jensen JM (2008) The skin: an indispensable barrier. Exp Dermatol 17(12):1063–1072PubMedCrossRefGoogle Scholar
  4. 4.
    Blanpain C, Fuchs E (2009) Epidermal homeostasis: a balancing act of stem cells in the skin. Nat Rev Mol Cell Biol 10:207–217PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Wong VW, Gurtner GC, Longaker MT (2013) Wound healing: a paradigm for regeneration. Mayo Clin Proc 88:1022–1031PubMedCrossRefGoogle Scholar
  6. 6.
    Zaja-Milatovic S, Richmond A (2008) CXC chemokines and their receptors: a case for a significant biological role in cutaneous wound healing. Histol Histopathol 23:1399–1407PubMedCentralPubMedGoogle Scholar
  7. 7.
    Guo S, Dipietro LA (2010) Factors affecting wound healing. J Dent Res 89:219–229PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Bellavia G, Fasanaro P, Melchionna R, Capogrossi MC, Napolitano M (2014) Transcriptional control of skin reepithelialization. J Dermatol Sci 73(1):3–9PubMedCrossRefGoogle Scholar
  9. 9.
    Greaves NS, Ashcroft KJ, Baguneid M, Bayat A (2013) Current understanding of molecular and cellular mechanisms in fibroplasia and angiogenesis during acute wound healing. J Dermatol Sci 72(3):206–217PubMedCrossRefGoogle Scholar
  10. 10.
    Patel GK, Wilson CH, Harding KG, Finlay AY, Bowden PE (2006) Numerous keratinocyte subtypes involved in wound re-epithelialization. J Invest Dermatol 126:497–502PubMedCrossRefGoogle Scholar
  11. 11.
    Scheid A, Meuli M, Gassmann M, Wenger RH (2000) Genetically modified mouse models in studies on cutaneous wound healing. Exp Physiol 85:687–704PubMedCrossRefGoogle Scholar
  12. 12.
    Brakenhielm E, Cao R, Cao Y (2001) Suppression of angiogenesis, tumor growth, and wound healing by resveratrol, a natural compound in red wine and grapes. FASEB J 15:1798–1800PubMedGoogle Scholar
  13. 13.
    Kung HN, Yang MJ, Chang CF, Chau YP, Lu KS (2008) In vitro and in vivo wound healing-promoting activities of beta-lapachone. Am J Physiol Cell Physiol 295:C931–C943PubMedCrossRefGoogle Scholar
  14. 14.
    Negrao R, Costa R, Duarte D, Gomes TT, Coelho P, Guimaraes JT, Guardao L, Azevedo I, Soares R (2012) Xanthohumol-supplemented beer modulates angiogenesis and inflammation in a skin wound healing model. Involvement of local adipocytes. J Cell Biochem 113:100–109PubMedCrossRefGoogle Scholar
  15. 15.
    Stipcevic T, Piljac A, Piljac G (2006) Enhanced healing of full-thickness burn wounds using di-rhamnolipid. Burns 32:24–34PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Squarize CH, Castilho RM, Bugge TH, Gutkind JS (2010) Accelerated wound healing by mTOR activation in genetically defined mouse models. PLoS One 5:e10643PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Chigurupati S, Mughal MR, Chan SL, Arumugam TV, Baharani A, Tang SC, Yu QS, Holloway HW, Wheeler R, Poosala S, Greig NH, Mattson MP (2010) A synthetic uric acid analog accelerates cutaneous wound healing in mice. PLoS One 5:e10044PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Cho CH, Sung HK, Kim KT, Cheon HG, Oh GT, Hong HJ, Yoo OJ, Koh GY (2006) COMP-angiopoietin-1 promotes wound healing through enhanced angiogenesis, lymphangiogenesis, and blood flow in a diabetic mouse model. Proc Natl Acad Sci U S A 103:4946–4951PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Kim SO, Lee HS, Ahn K, Park K (2013) COMP-angiopoietin-1 promotes cavernous angiogenesis in a type 2 diabetic rat model. J Korean Med Sci 28:725–730PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Emmerson E, Campbell L, Davies FC, Ross NL, Ashcroft GS, Krust A, Chambon P, Hardman MJ (2012) Insulin-like growth factor-1 promotes wound healing in estrogen-deprived mice: new insights into cutaneous IGF-1R/ERalpha cross talk. J Invest Dermatol 132:2838–2848PubMedCrossRefGoogle Scholar
  21. 21.
    Jarvinen TA, Ruoslahti E (2010) Target-seeking antifibrotic compound enhances wound healing and suppresses scar formation in mice. Proc Natl Acad Sci U S A 107:21671–21676PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Negrao R, Costa R, Duarte D, Taveira Gomes T, Mendanha M, Moura L, Vasques L, Azevedo I, Soares R (2010) Angiogenesis and inflammation signaling are targets of beer polyphenols on vascular cells. J Cell Biochem 111:1270–1279PubMedCrossRefGoogle Scholar
  23. 23.
    Banerjee J, Chan YC, Sen CK (2011) MicroRNAs in skin and wound healing. Physiol Genomics 43:543–556PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Banerjee J, Sen CK (2013) MicroRNAs in skin and wound healing. Methods Mol Biol 936:343–356PubMedCrossRefGoogle Scholar
  25. 25.
    Cotsarelis G, Sun TT, Lavker RM (1990) Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61:1329–1337PubMedCrossRefGoogle Scholar
  26. 26.
    Jensen KB, Collins CA, Nascimento E, Tan DW, Frye M, Itami S, Watt FM (2009) Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis. Cell Stem Cell 4:427–439PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Snippert HJ, Haegebarth A, Kasper M, Jaks V, van Es JH, Barker N, van de Wetering M, van den Born M, Begthel H, Vries RG, Stange DE, Toftgard R, Clevers H (2010) Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin. Science 327:1385–1389PubMedCrossRefGoogle Scholar
  28. 28.
    Ito M, Liu Y, Yang Z, Nguyen J, Liang F, Morris RJ, Cotsarelis G (2005) Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat Med 11:1351–1354PubMedCrossRefGoogle Scholar
  29. 29.
    Jaks V, Barker N, Kasper M, van Es JH, Snippert HJ, Clevers H, Toftgard R (2008) Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet 40:1291–1299PubMedCrossRefGoogle Scholar
  30. 30.
    Levy V, Lindon C, Zheng Y, Harfe BD, Morgan BA (2007) Epidermal stem cells arise from the hair follicle after wounding. FASEB J 21:1358–1366PubMedCrossRefGoogle Scholar
  31. 31.
    Nowak JA, Polak L, Pasolli HA, Fuchs E (2008) Hair follicle stem cells are specified and function in early skin morphogenesis. Cell Stem Cell 3:33–43PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Ashcroft GS, Yang X, Glick AB, Weinstein M, Letterio JL, Mizel DE, Anzano M, Greenwell-Wild T, Wahl SM, Deng C, Roberts AB (1999) Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response. Nat Cell Biol 1:260–266PubMedCrossRefGoogle Scholar
  33. 33.
    Owens P, Engelking E, Han G, Haeger SM, Wang XJ (2010) Epidermal Smad4 deletion results in aberrant wound healing. Am J Pathol 176:122–133PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Hudson LG, Newkirk KM, Chandler HL, Choi C, Fossey SL, Parent AE, Kusewitt DF (2009) Cutaneous wound reepithelialization is compromised in mice lacking functional Slug (Snai2). J Dermatol Sci 56:19–26PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Sano S, Itami S, Takeda K, Tarutani M, Yamaguchi Y, Miura H, Yoshikawa K, Akira S, Takeda J (1999) Keratinocyte-specific ablation of Stat3 exhibits impaired skin remodeling, but does not affect skin morphogenesis. EMBO J 18:4657–4668PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Hansen SL, Myers CA, Charboneau A, Young DM, Boudreau N (2003) HoxD3 accelerates wound healing in diabetic mice. Am J Pathol 163:2421–2431PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Ashcroft GS, Mills SJ (2002) Androgen receptor-mediated inhibition of cutaneous wound healing. J Clin Invest 110:615–624PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Liang X, Bhattacharya S, Bajaj G, Guha G, Wang Z, Jang HS, Leid M, Indra AK, Ganguli-Indra G (2012) Delayed cutaneous wound healing and aberrant expression of hair follicle stem cell markers in mice selectively lacking Ctip2 in epidermis. PLoS One 7:e29999PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Jaks V, Kasper M, Toftgard R (2010) The hair follicle-a stem cell zoo. Exp Cell Res 316:1422–1428PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Pharmaceutical Sciences, College of PharmacyOregon State UniversityCorvallisUSA

Personalised recommendations