Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

Summary

This protocol describes how to generate bilateral, full-thickness excisional wounds in mice and how to subsequently monitor, harvest, and prepare the wounds for morphometric analysis. Included is an in-depth description of how to use serial histological sections to define, precisely quantify and detect morphometric defects.

Abstract

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, proliferation and remodeling. Murine wounds have a histologically well-defined and easily recognizable wound bed over which these different phases of the healing process are measurable. Within the field, it is common to use an arbitrarily defined “middle” of the wound for histological analyses. However, wounds are a three-dimensional entity and often not histologically symmetrical, supporting the need for a well-defined and robust method of quantification to detect morphometric defects with a small effect size. In this protocol, we describe the procedure for creating bilateral, full-thickness excisional wounds in mice as well as a detailed instruction on how to measure morphometric parameters using an image processing program on select serial sections. The two-dimension measurements of wound length, epidermal length, epidermal area, and wound area are used in combination with the known distance between sections to extrapolate the three-dimension epidermal area covering the wound, overall wound area, epidermal volume and wound volume. Although this detailed histological analysis is more time and resource consuming than conventional analyses, its rigor increases the likelihood of detecting novel phenotypes in an inherently complex wound healing process.

Introduction

Cutaneous wound healing is a complex biological process with sequentially overlapping phases. It requires the coordination of cellular and molecular processes that are temporally and spatially regulated in order to restore the barrier function of the damaged epithelium. In the first phase, inflammation, neutrophils and macrophages migrate into the wound, mobilizing local and systemic defenses¹. Following and overlapping the inflammatory phase is the proliferation stage. Fibroblasts begin rapidly proliferating and migrating into the granulation tissue. Keratinocytes away from the leading edge directionally proliferate towards the wound as differ....

Protocol

All experiments were completed in accordance and compliance with federal regulations and University of Iowa policy and procedures have been approved by the University of Iowa IACUC.

1. Animals and husbandry

1. Use adult mice of the desired mouse line at 8-10 weeks of age when the hair follicle stage is in telogen.
2. On the day of surgery, separate mice into clean cages and individually house to minimize wound disruption.

2. Surgery

NOTE: It is unnecessary to maintain sterile surgical conditions. While care should be taken to maintain sterility between animals, the....

Results

Figure 5 depicts the range in measured and calculated values obtained by performing morphometric analysis on wild-type wounds generated in different mouse strains by multiple surgeons and analyzed by different individuals. Wild-type mice from different strains can display statistical differences as described both in our studies and in the literature^9,10. Based on these representative results, we recommend that, within one study, mice.......

Discussion

The bilateral excisional wound model is a highly customizable procedure which can be used to study many different aspects of wound healing. Before beginning a wound healing project, investigators should perform a power analysis to determine the number of wounds needed to detect a defect of a particular effect size. Inconsistencies exist within the literature on whether individual mice or wounds should be used as biological replicates, however, a recent study showed that there is no significant correlation between two wou.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100% ethanol
70% ethanol
80% ethanol
95% ethanol
Alcohol Prep	NOVAPLUS	V9100	70% Isopropyl alcohol, sterile
Ammonium hydroxide
Biopsy pads	Cellpath	22-222-012
Black plastic sheet			Something firm yet manipulatable about the size of a sheet of paper
Brightfield microscope			With digital acquisition capabilities and a 4X objective
Cotton tipped applicators
Coverslips			22 x 60 #1
Dental wax sheets
Digital camera			Include a ruler for scale, if applicable
Dissection teasing needle (straight)
Embedding molds			22 x 22 x 12
Embedding rings	Simport Scientific Inc.	M460
Eosin Y
Glacial acetic acid
Hair clipper
Heating pad	Conair		Moist dry Heating Pad
Hematoxylin
Microtome
Microtome blades
Paint brushes
Paraffin Type 6
Paraformaldehyde
Permount
Phosphate buffer solution (PBS)
Povidone-iodine	Aplicare	82-255
Processing cassette	Simport Scientific Inc.	M490-2
Razor blades	ASR		.009 Regular Duty
Scalpel blades #10
Scalpel handle
Sharp surgical scissors			sterile for surgery
Skin biopsy punches			Size as determined by researcher
Slide boxes
Slide warmers
Superfrosted microscope slides	Fisher Scientific	22 037 246
Temperature control water bath
Tissue embedding station			Minimum of a paraffin dispenser and a cold plate
Tissue processor			Minimum of a oven with a vacuum pump
Triple antibiotic opthalmic ointment
tweezers, curved tip			sterile for surgery
tweezers, tapered tip			sterile for surgery
WypAll X60	Kimberly-Clark	34865