Studying Wound Repair in the Mouse

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 H₂ O
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

Figure 1. Images of full excisional splint wounding procedure. (A‐C ) Depilation of mouse hair using rosin gum and beeswax method. (D ) Marked‐out circle on the cut site. (E ) Create an full excisional wound of 5 mm in diameter. (F ) Place and glue the splint around the wound. (G ) Cover the wound with a trimmed Tegaderm dressing.

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Figure 2. Illustration of the size of the silicon splint.

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Figure 3. Schematic diagram showing final view of a full thickness excisional splinted wound.

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Figure 4. Immunohistochemical staining of mouse skin explant for keratin 6 after culturing for 8 days.

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Figure 5. Schematic representation of the histological stained wound section with predefined labeling for measurements.

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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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Studying Wound Repair in the Mouse

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited