Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Summary

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

Abstract

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Introduction

There are many murine model systems available to examine wound healing processes, each possessing specific advantages and limitations^1,2. The following methods present two murine wound models, each of which addresses a particular aspect of the wound healing response, and which can be used to identify the cause and effect of perturbations in the response to injury. The process of wound healing occurs in distinct phases. The first phase is inflammatory, characterized by the rapid influx of platelets, neutrophils, and monocytes/macrophages, as well as the production of proinflammatory cytokines and chemokines. Fo....

Protocol

All animal studies described here were approved by the Brown University Institutional Animal Care and Use Committee and carried out in accordance with the Guide for the Care and Use of Animals of the National Institutes of Health.  NOTE: in the video, the surgical drape has been omitted for demonstration purposes. 

1. Subcutaneous implantation of PVA sponges

1. Use scissors to cut sheets of PVA sponge into 8 mm x 8 mm x 4 mm pieces. Rehydrate the pieces of PVA sponge by submerging them in sterile 1x PBS in a beaker.
2. Autoclave the sponges in 1x PBS to sterilize, and cool completely. Store autoclaved sponges in st....

Results

Systemic inflammatory response following PVA sponge implantation
The PVA sponge implantation surgery generated a systemic inflammatory response, as demonstrated by the induction of IL-6 in the plasma 1 day after wounding (Figure 2A). Other proinflammatory cytokines including TNF-α and IL-1β, as well as an array of chemokines including CCL2 and CXCL1 were induced systemically in the first 7 days post-PVA sponge implantation, and have been described elsewhere

Discussion

This article describes two tractable murine wound models that allow for the assessment of the acute wound healing response. The first method involves the surgical implantation of PVA sponges in the dorsal subcutaneous space. This approach offers a distinct advantage over biopsy-based wound models for studying the cellular wound healing response due to the large number of cells and quantity of wound fluids obtained from the isolated sponges. For the successful execution of this procedure, maintaining a sterile surgical fi.......

Materials

Name	Company	Catalog Number	Comments
10x Phosphate Buffered Saline	Fisher Scientific	BP3991
15 mL centrifuge tubes, Olympus	Genesee	28-103
1x HBSS (+Calcium, +Magnesium, –Phenol Red)	ThermoFisher Scientific	14025076
5ml Syringe	BD	309646
Anti-mouse CD45.2-APC Fire750	BioLegend	109852	Clone 104
Anti-mouse F4/80-eFluor660	ThermoFisher Scientific	50-4801-82	Clone BM8
Anti-mouse Ly6C-FITC	BD Biosciences	553104	Clone AL-21
Anti-mouse Ly6G-PerCP-eFluor710	ThermoFisher Scientific	46-9668-82	Clone 1A8-Ly6g
Anti-mouse Siglec-F-APC-R700	BD Biosciences	565183	Clone E50-2440
Autoclip Stainless Steel Wound Clip Applier	Braintree Scientific	NC9021392
Autoclip Stainless Steel Wound Clips, 9mm	Braintree Scientific	NC9334081
Blender Bag, 80mL	Fisher Scientific	14258201
Culture Tube, 16mL, 17x100	Genesee Scientific	21-130
Fetal Bovine Serum - Standard	ThermoFisher Scientific	10437028
Fixable Viability Dye eFluor506	ThermoFisher Scientific	65-0866-14
Hepes Solution, 1M	Genesee Scientific	25-534
ImageJ Software	NIH
Penicillin-Streptomycin (5000 U/mL)	ThermoFisher Scientific	15070-063
Polyvinyl alcohol sponge - large pore size	Ivalon/PVA Unlimited		www.sponge-pva.com
Povidone-iodine solution, 10%	Fisher Scientific	3955-16
Spray barrier film, Cavilon	3M	3346E
Stomacher 80 Biomaster, 110V	Seward	0080/000/AJ