Studying Wound Repair in the Mouse

Nguan S. Tan1, Walter Wahli2

1 Institute of Cell and Molecular Biology, Agency for Science, Technology and Research A*STAR, Singapore, 2 Nanyang Technological University, Singapore
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo130135
Online Posting Date:  October, 2013
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Abstract

Animal models of wound healing provide vital insights into the mechanisms and pathophysiology of cutaneous wound repair and are a crucial part of clinical research into the development of new strategies and approaches to rational wound therapy. Although considerable biological variation in the wound healing response exists even among inbred animal strains, consistent surgical procedure and wound analysis can yield significant conclusions. Many different aspects of the healing process can be characterized and quantified in a reproducible, controlled environment. Here, we detail methods for full‐thickness excisional and incisional wounding and for analysis of wound biopsies. Curr. Protoc. Mouse Biol. 3:171‐185 © 2013 by John Wiley & Sons, Inc.

Keywords: wound repair; wound biopsy; histology; IHC; histomorphometric; wound models

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Wound Healing: Full‐Thickness Excisional Wounding
  • Support Protocol 1: Wound Healing: Full‐Thickness Excisional Splinted Wounds
  • Support Protocol 2: Wound Healing: Incisional Wounds
  • Support Protocol 3: Wound Healing: Mouse Skin Explant Ex Vivo Assay
  • Basic Protocol 2: Wound Healing: Wound Isolation and Basic Immunohistochemical Analysis
  • Basic Protocol 3: Histomorphometric Analysis of Wound Sections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Wound Healing: Full‐Thickness Excisional Wounding

  Materials
  • 6‐ to 8‐week‐old male C57BL/6 mice
  • Anesthetic solution (see recipe)
  • Depilating cream (Veet, cat. no. 20070747)
  • Rosin gum (Sigma, cat. no. R‐3755)
  • Beeswax, bleached white (Aldrich, cat. no. 24322‐1)
  • 70% ethanol
  • 1‐ml insulin syringe with 31‐G short needle (BD, cat. no. REF 328820)
  • “Wella Contura” hair clipper (IMS, Industrial Medical Supplies, Congleton, Cheshire, U.K.)
  • Microwave oven
  • Permanent marker (Staedtler, cat. no. 342‐9; http://www.staedtler.com/)
  • Infrared heating lamp
  • Surgical scissors and forceps
  • Disposable biopsy punch, 5 mm diameter (Stiefel; http://www.stiefel.com; optional)
  • Tegaderm transparent occlusive dressing (2‐3/8 in. × 2‐3/4 in.; available from 3M)
  • Additional reagents and equipment for injection of mice (Donovan and Brown, )

Support Protocol 1: Wound Healing: Full‐Thickness Excisional Splinted Wounds

  Materials
  • 6‐ to 8‐week‐old male C57BL/6 mice
  • 70% ethanol
  • Superglue
  • Silicon disc, 0.5 mm thick (Grace Bio‐labs; http://www.gracebio.com/)
  • Scissors (or penknife) and forceps
  • Disposable biopsy punch, 6 mm diameter (Stiefel; http://www.stiefel.com)
  • 6‐0 nylon sutures (Ethicon)
  • Needle (3/8 circle, Ethicon)
  • Needle holder
  • Tegaderm transparent occlusive dressing (2‐3/8 in. × 2‐3/4 in.; available from 3M)
  • Additional reagents and equipment for full‐thickness excisional wounding ( protocol 1)

Support Protocol 2: Wound Healing: Incisional Wounds

  Materials
  • 6‐ to 8‐week‐old male C57BL/6 mice
  • Sterile scalpels
  • Fine blunt forceps
  • Tegaderm transparent occlusive dressing (2‐3/8 in. × 2‐3/4 in.; available from 3M)

Support Protocol 3: Wound Healing: Mouse Skin Explant Ex Vivo Assay

  Materials
  • 6‐ to 8‐week‐old male C57BL/6 mice
  • mKer medium (see recipe)
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • 4% (w/v) paraformaldehyde (PFA) in 1× PBS
  • 100% methanol (optional)
  • Disposable biopsy punch, 4 mm diameter (Stiefel; http://www.stiefel.com)
  • 24‐well plates
  • Additional reagents and equipment for anesthesia and depilation of mice ( protocol 1), and staining and analysis of wound sections (Basic Protocols protocol 52 and protocol 63)

Basic Protocol 2: Wound Healing: Wound Isolation and Basic Immunohistochemical Analysis

  Materials
  • Mouse, wounded according to protocol 1, protocol 2, or protocol 3
  • Normal goat serum (NGS; Vector Laboratories, cat. no. S1000)
  • Anti‐cytokeratin 6 antibody (Abcam, cat. no. ab24646)
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • Biotinylated goat anti–rabbit IgG (Vector Laboratories, cat no. BA‐1000)
  • Vectastain Elite ABC kit (Vector Laboratories, cat. no. PK 6101)
  • Vectastain DAB substrate kit for peroxidase (Vector Laboratories, cat. no. SK 4100)
  • Toluidine blue: 0.5 g toluidine blue, 1 g sodium borate, 100 ml H 2O
  • Eukitt quick‐hardening mounting medium (Fluka, cat. no. 03989)
  • Fine scissors
  • SuperFrost Plus microscope slides (Thermo Scientific, cat. no. J1800AMNZ)
  • Humidified chamber: slide box with cover containing a moist paper towel
  • Staining troughs and racks (Thermo Scientific, cat. no. E105 and E102)
  • Coverslips, thickness 1 (Thermo Scientific, D10143263NR)
  • Bright‐field microscope connected to color digital camera
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, ), fixation of tissue ( protocol 4, steps 5 to 8), and sectioning of tissue (Antal et al., )

Basic Protocol 3: Histomorphometric Analysis of Wound Sections

  Materials
  • Wound sections ( protocol 5)
  • Stains:
    • SelecTech hematoxylin and eosin staining system (Leica Microsystem)
    • Gomori's Trichrome Special Stain Kit (Leica Microsystem)
  • Bright‐field microscope connected to color digital camera
  • Adobe Photoshop CS6 (Adobe Systems software)
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Figures

Videos

Literature Cited

Literature Cited
  Alonso, L. and Fuchs, E. 2006. The hair cycle. J. Cell Sci. 119:391‐393.
  Ansell, D.M., Kloepper, J.E., Thomason, H.A., Paus, R. and Hardman, M.J. 2011. Exploring the “hair growth‐wound healing connection”: Anagen phase promotes wound re‐epithelialization. J. Invest. Dermatol. 131:518‐528.
  Antal, C., Muller, S., Wendling, O., Hérault, Y., and Mark, M. 2011. Standardized post‐mortem examination and fixation procedures for mutant and treated mice. Curr. Protoc. Mouse Biol. 1:17‐53.
  Donovan, J. and Brown, P. 2006a. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10.
  Donovan, J. and Brown, P. 2006b. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
  Galiano, R.D., Michaels, J., Dobryansky, M., Levine, J.P. and Gurtner, G.C. 2004. Quantitative and reproducible murine model of excisional wound healing. Wound Repair Regen. 12:485‐492.
  Kiritsy, C.P., Lynch, A.B. and Lynch, S.E. 1993. Role of growth factors in cutaneous wound healing: A review. Crit. Rev. Oral Biol. Med. 4:729‐760.
  Sonnemann, K.J. and Bement, W.M. 2011. Wound repair: Toward understanding and integration of single‐cell and multicellular wound responses. Annu. Rev. Cell Dev. Biol. 27:237‐263.
  Tan, N.S., Icre, G., Montagner, A., Bordier‐ten‐Heggeler, B., Wahli, W. and Michalik, L. 2007. The nuclear hormone receptor peroxisome proliferator‐activated receptor beta/delta potentiates cell chemotactism, polarization, and migration. Mol. Cell Biol. 27:7161‐7175.
  Werner, S. and Grose, R. 2003. Regulation of wound healing by growth factors and cytokines. Physiol. Rev. 83:835‐870.
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