Protocol to Create Chronic Wounds in Diabetic Mice

Summary

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Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

Abstract

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db^-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Introduction

Wound healing involves complex cellular and molecular processes that are temporally and spatially regulated, organized in sequential and overlapping stages that involve many different cell types including but not limited to the immune response and the vascular system¹. Immediately after the skin sustains an injury, factors and blood cells aggregate to the wound site and initiate the coagulation cascade to form a clot. After homeostasis is achieved, the blood vessels dilate to let into the wound site oxygen, nutrients, enzymes, antibodies and chemotactic factors that chemoattract polymorphonucleocytes to clear the wound bed of foreign debris and....

Protocol

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

1. Animal

1. Use diabetic and obese B6.BKS(D)-Lepr^db/J mice for the chronic wound model. Purchasing options include either heterozygotes for breeding or homozygotes directly for experiments.
2. Breed heterozygote males and females to produce offspring. Only a quarter of the litter, statistically, will grow to become diabetic and obese (db/db^-/-).
3. Wean and house db/db

Results

Figure 5 depicts an example of a wound without treatment of inhibitors progressing towards wound closure and a wound with treatment of inhibitors progressing towards chronicity. The transparent dressing has been left in place on the chronic wound so that biofilm and fluid accumulation can be seen.

Chronic wound initiation takes place in less than 6 hours and the wound margin is visibly altered from .......

Discussion

Once chronic wounds are created on the mice, the model can be used to study impaired wound healing processes involved in the initiation of chronicity. The model can also be used to test the efficacy of a wide range of chemicals and drugs that can reverse chronic wound development and impaired healing and lead to wound closure and healing. Different time points after the onset of chronicity can be studied: e.g., days 1-5 after wounding for early onset of chronicity and days 20 and beyond for full strength chronic.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
B6.BKS(D)-Leprdb/J	The Jackson Laboratory	00697	Homozygotes and heterozygotes available
Nair Hair Remover Lotion with Soothing Aloe and Lanolin	Nair		a chemical depilatory
Buprenex (buprenorphine HCl)	Henry Stein Animal Health	059122	0.3 mg/ml, Class 3
3-Amino-1,2,4-triazole (ATZ)	TCI	A0432
Mercaptosuccinic acid (MSA)	Aldrich	88460
Phosphate buffer solution (PBS)			autoclave steriled
Isoflurane	Henry Schein Animal Health	029405	NDC 11695-6776-2
Oxygen			Tank must be compatible with vaporizing system
Isoflurane vaporizer	JA Baulch & Associates
Wahl hair clipper	Wahl		Lithium Ion Pro
Acu Punch 7mm skin biopsy punches	Acuderm Inc.	P750
Tegaderm	3M	Ref: 1624W	Transparent film dressing (6 cm x 7 cm)
Heating pad	Conair		Moist Dry Heating Pad
Insulin syringes	BD	329461	0.35 mm (28G) x 12.7 mm (1/2")
70% ethanol
Kimwipes
Tweezers
Sharp surgical scissors
Thin metal spatula
Tubing
Mouse nose cone
Gloves
small plastic containers