Protocol to Create Chronic Wounds in Diabetic Mice - an Update to the Published Protocol

Abstract

Despite considerable efforts to treat chronic wounds, success has been limited. The cellular and molecular processes that disrupt normal healing and contribute to the initiation and development of chronic wounds are not well understood. As a result, developing effective treatments for these wounds has proven challenging. This is primarily because of the lack of animal models that mimic chronic wounds in humans and because experimentation in humans with chronic wounds is limited. Previously, we have published a mouse model to study chronic wounds that share many characteristics with diabetic chronic wounds in humans. This model includes the presence of biofilms formed naturally from bacteria found in skin microbiota and the environment without the need to introduce external bacteria into the wounds. Therefore, this model can potentially advance the fundamental understanding of how wounds become chronic. Here, we present an update to the original protocol for developing this mouse model. We have concentrated on decreasing the mortality of mice from ~30% to ~5%. We also describe how this model has been used to understand the initiation and progression of wound chronicity by using next-generation transcriptomic approaches, elucidating bacterial population dynamics during infection and biofilm development, and demonstrating the value of testing the efficacy of treatments, including small molecules. So far, this is the only chronic wound model that does not heal without intervention.

Introduction

This is an update to an existing article. Please click here to see the original version.

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Protocol

UPDATES:

This is an update to the protocol presented previously¹

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

Add the following Note to Step 1.1:
NOTE: If homozygous diabetic mice are directly purchased from suppliers, house the mice in a conventional vivarium to allow colonization of the skin with normal ski....

Results

Figure 1: Genotyping for identifying heterozygotes to create breeders. An example gel shows the results of 8 unknown mouse samples. Sample 2 and 5 are heterozygotes as they contain 3 bands (610 bp, 406 bp, and 264 bp). Samples 1, 3, 4, 6,.......

Discussion

Findings using this model of chronic wounds:  

This model has been used to perform several studies in an attempt to understand the cell and molecular mechanisms of chronic wound initiation and progression. It has been shown that by inhibiting antioxidant enzymes, chronic wounds can be created in the db/db mice. The wounds initially contained a polymicrobial community that, with time, was selected for specific biofilm-forming bacteria. The chronicity was rev.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mm ear punch	Fisherbrand	13812201	2mm hole diameter
3-Amino-1,2,4-triazole (ATZ)	TCI	A0432
70% ethanol
Acu Punch 7mm skin biopsy punches	Acuderm Inc.	P750
agarose
B6.BKS(D)-Leprdb/J	The Jackson Laboratory	00697	Homozygotes and heterozygotes available
Buprenex (buprenorphine HCl)	Henry Stein Animal Health	059122	0.3 mg/ml, Class 3
Gel Loading Dye, Purple (6X)	New England Biolabs	B7024S
Gloves
Hair Remover Lotion with Soothing Aloe and Lanolin	Nair		a chemical depilatory
Heat Therapy (water) Pump HTP-1500	Adroit MedicalSystems
Heating pad	Conair		Moist Dry Heating Pad
Hose Adapters	Adroit MedicalSystems
Insulin syringes	BD	329461	0.35 mm (28G) x 12.7 mm (1/2")
Isoflurane	Henry Schein Animal Health	029405	NDC 11695-6776-2
Isoflurane vaporizer	JA Baulch & Associates
Kimwipes
Maxi-Therm Hyper-Hypothermia Blanket	Cincinnati Sub-Zero	Cat No. 247	22”x30” Re-usable HeavyDuty Pad
Mercaptosuccinic acid (MSA)	Aldrich	88460
Mouse nose cone
Oxygen			Tank must be compatible with vaporizing system
Phosphate buffer solution (PBS)			autoclave steriled
Sharp surgical scissors
small plastic containers
Sodium hydroxide
Taq 2X Master Mix	New England Biolabs	M0270L
Tegaderm	3M	Ref: 1624W	Transparent film dressing (6 cm x 7 cm)
Thin metal spatula
Tri-HCl
Tubing
Tweezers
Wahl hair clipper	Wahl		Lithium Ion Pro