Name: protocol to create chronic wounds in diabetic mice
Uploaded: 2019-09-25T13:30:00
Description: Watch this Scientific Journal Video about Protocol to Create Chronic Wounds in Diabetic Mice at JoVE.com

The authors have no acknowledgements.

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
<ol>
	<li>Use diabetic and obese B6.BKS(D)-Leprdb/J mice for the chronic wound model. Purchasing options include either heterozygotes for breeding or homozygotes directly for experiments.</li>
	<li>Breed heterozygote males and females to produce offspring. Only...

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...

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 secrete proteolytic enzymes2. Activated platelets secrete a variety of growth factors to stimulate the keratinocytes at the wound edge to re-epithelialize the wounded area. Monocytes recruited to the wound site differentiate into macrophages which phagocytose bacteria and dead neutrophils and secrete additional factors to maintain keratinocyte proliferative and pro-migratory signals. In the proliferation phase, while re-epithelialization continues, new granulation tissue composed of fibroblasts, monocytes/macrophages, lymphocytes, and endothelial cells continue the rebuilding process2. Angiogenesis is stimulated by promoting endothelial cell proliferation and migration, resulting in new vessel development. Epithelialization and remodeling of the extracellular matrix construct a barrier against the environment. As the wound heals and granulation tissue evolves into a scar, apoptosis eliminates inflammatory cells, fibroblasts, and endothelial cells without causing additional tissue damage. The tensile strength of the tissue is enhanced by fibroblasts remodeling various components of the extracellular matrix, like collagen, so that the newly formed tissue is almost as strong and flexible as unwounded skin2.
Any deviation from this highly concerted progression towards wound closure leads to impaired and/or chronic wounds3. Chronic wounds are characterized by increased oxidative stress, chronic inflammation, damaged microvasculature, and abnormal collagen matrix in the wound4. Oxidative stress, especially in the wound, can delay wound closure2,5. When, in the first stage of wound healing, the inflammatory phase becomes unregulated, the host tissue assumes extensive damage due to a continuous influx of inflammatory cells5 that release cytotoxic enzymes, an increase in free oxygen radicals, and unregulated inflammatory mediators, resulting in cell death6,7.
In this destructive microenvironment, biofilm-forming bacteria take advantage of host nutrients and contribute to the damage of the host tissue2. These biofilms are difficult to control and remove because the hydrated extracellular polymeric substances composed of proteins, DNA, RNA, and polysaccharides allows bacteria harbored within to be tolerant to conventional antibiotic therapies and evade the host&#39;s innate and adaptive immune response2,8,9.
Studying chronic wounds is crucial because they impact ~6.5 million people and cost ~$40 billion per year in the US alone10. Patients with diabetes have increased risks for developing chronic wounds that require amputation in order to contain the spread of infection. These patients have a 50% mortality risk within 5 years of amputation that is attributed to the pathophysiology mechanism of diabetes11. The relationship between the host&#39;s immune system and the microbiome in wound healing is a vital topic of ongoing research because consequences of chronic wounds, if unresolved, include amputation and death12.
Although a significant effort has been invested in understanding how chronic wounds develop in humans, it is still unclear how and why chronic wounds form. Experiments to study the mechanisms of impaired healing is difficult to conduct in humans, and wound healing specialists only see patients with chronic wounds that have already reached chronicity for weeks to months. Thus, specialists are unable to study what processes went wrong that lead the wound to develop to become chronic2. There is a lack of animal models that recapitulate the complexity of human chronic wounds. Until our model was developed, no model for chronic wound studies existed.
The chronic wound model was developed in mice that have a mutation in the leptin receptor (db/db-/-)13. These mice are obese, diabetic, and have impaired healing but do not develop chronic wounds14. Blood glucose levels average around 200 mg/dL, but can be as high as 400 mg/dL15. When high levels of oxidative stress (OS) in the wound tissue are induced immediately after wounding, the wound becomes chronic16. The db/db-/- wounds are considered chronic by 20 days and remain open for 60 days or more. Biofilm produced by bacteria can be seen developing beginning three days after wounding; a mature biofilm can be seen 20 days after wounding and persists until either wound closure. The biofilm-forming bacteria we find in these mice are also found in human diabetic chronic wounds.
Oxidative stress is induced by treating the wounds with two inhibitors of antioxidant enzymes, catalase and glutathione peroxidase, two enzymes with the capacity to break down hydrogen peroxide. Hydrogen peroxide is a reactive oxygen species and can cause cellular damage through the oxidation of proteins, lipids, and DNA. Catalase catalyzes the decomposition of hydrogen peroxide into less harmful chemicals oxygen and water. 3-Amino-1,2,4-triazole (ATZ) inhibits catalase by binding specifically and covalently to the active center of the enzyme, inactivating it17,18,19. ATZ has been used to study the effects of oxidative stress both in vitro and in vivo through the inhibition of catalase20,21,22,23,24. Glutathione peroxidase catalyzes the reduction of hydrogen peroxide through the antioxidant, glutathione, and is an important enzyme that protects the cell against oxidative stress25. Mercaptosuccinic acid (MSA) inhibits glutathione peroxidase by binding to the selenocysteine active site of the enzyme with thiol, inactivating it26. MSA has been used to study the effects of oxidative stress in vitro and in vivo as well20,27,28.
This novel model of chronic wounds is a powerful model to study because it shares many of the same features observed in human diabetic chronic wounds, including prolonged inflammation from increased OS and natural biofilm formation from skin microbiome. The wounds have impaired dermal-epidermal interaction, abnormal matrix deposition, poor angiogenesis and damaged vasculature. Chronic wounds will develop in both male and female mice, so both sexes can be used to study chronic wounds. Therefore, the chronic wound model can contribute significantly to advance fundamental understanding of how such wounds begin. Using this chronic wound model can provide answers to fundamental questions about how chronicity is initiated/achieved through contributions from the physiology of impaired wound healing and the microbiome of the host.

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			<td height="42" style="height:42px;width:308px;">B6.BKS(D)-Leprdb/J&#160;</td>
			<td style="width:176px;">The Jackson Laboratory&#160;</td>
			<td style="width:107px;">00697</td>
			<td style="width:204px;">Homozygotes and heterozygotes available&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:308px;">Nair Hair Remover Lotion with Soothing Aloe and Lanolin</td>
			<td style="width:176px;">Nair</td>
			<td style="width:107px;"></td>
			<td style="width:204px;">a chemical depilatory</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:308px;">Buprenex (buprenorphine HCl)</td>
			<td style="width:176px;">Henry Stein Animal Health</td>
			<td style="width:107px;">059122</td>
			<td style="width:204px;">0.3 mg/ml, Class 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">3-Amino-1,2,4-triazole (ATZ)</td>
			<td style="width:176px;">TCI</td>
			<td style="width:107px;">A0432</td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Mercaptosuccinic acid (MSA)</td>
			<td style="width:176px;">Aldrich</td>
			<td style="width:107px;">88460</td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Phosphate buffer solution (PBS)</td>
			<td style="width:176px;"></td>
			<td style="width:107px;"></td>
			<td style="width:204px;">autoclave steriled</td>
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			<td height="42" style="height:42px;width:308px;">Isoflurane</td>
			<td style="width:176px;">Henry Schein Animal Health</td>
			<td style="width:107px;">029405</td>
			<td style="width:204px;">NDC 11695-6776-2</td>
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			<td height="42" style="height:42px;width:308px;">Oxygen</td>
			<td style="width:176px;"></td>
			<td style="width:107px;"></td>
			<td style="width:204px;">Tank must be compatible with vaporizing system</td>
		</tr>
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			<td height="21" style="height:21px;width:308px;">Isoflurane vaporizer</td>
			<td style="width:176px;">JA Baulch &#38; Associates&#160;</td>
			<td style="width:107px;"></td>
			<td style="width:204px;"></td>
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			<td height="21" style="height:21px;width:308px;">Wahl hair clipper</td>
			<td style="width:176px;">Wahl</td>
			<td style="width:107px;"></td>
			<td style="width:204px;">Lithium Ion Pro</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Acu Punch 7mm skin biopsy punches</td>
			<td style="width:176px;">Acuderm Inc.</td>
			<td style="width:107px;">P750</td>
			<td style="width:204px;"></td>
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			<td height="42" style="height:42px;width:308px;">Tegaderm&#160;</td>
			<td style="width:176px;">3M</td>
			<td style="width:107px;">Ref: 1624W</td>
			<td style="width:204px;">Transparent film dressing (6 cm x 7 cm)</td>
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			<td height="21" style="height:21px;width:308px;">Heating pad</td>
			<td style="width:176px;">Conair</td>
			<td style="width:107px;"></td>
			<td style="width:204px;">Moist Dry Heating Pad</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:308px;">Insulin syringes</td>
			<td style="width:176px;">BD</td>
			<td style="width:107px;">329461</td>
			<td style="width:204px;">0.35 mm (28G) x 12.7 mm (1/2&#34;)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">70% ethanol</td>
			<td style="width:176px;"></td>
			<td style="width:107px;"></td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Kimwipes</td>
			<td style="width:176px;"></td>
			<td style="width:107px;"></td>
			<td style="width:204px;"></td>
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			<td height="21" style="height:21px;width:308px;">Tweezers</td>
			<td style="width:176px;"></td>
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			<td height="21" style="height:21px;width:308px;">Sharp surgical scissors</td>
			<td style="width:176px;"></td>
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			<td height="21" style="height:21px;width:308px;">Thin metal spatula</td>
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			<td height="21" style="height:21px;width:308px;">Tubing</td>
			<td style="width:176px;"></td>
			<td style="width:107px;"></td>
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			<td height="21" style="height:21px;width:308px;">Mouse nose cone</td>
			<td style="width:176px;"></td>
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			<td style="width:204px;"></td>
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			<td height="21" style="height:21px;width:308px;">Gloves</td>
			<td style="width:176px;"></td>
			<td style="width:107px;"></td>
			<td style="width:204px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">small plastic containers</td>
			<td style="width:176px;"></td>
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protocol to create chronic wounds in diabetic mice

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.

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 ...

Watch this Scientific Journal Video about Protocol to Create Chronic Wounds in Diabetic Mice at JoVE.com

Protocol to Create Chronic Wounds in Diabetic Mice

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.

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

This mouse model can help answer questions in the medical field of wound chronicity. The major advantage of this model is that the chronic wounds in mice mimic many aspects of the chronic wounds in humans. To begin, place the mouse on a clean surface and hold it in place by pinching the base of its tail.Then prepare the hair clippers and be ready to quickly pull them away if the mouse makes any sudden movements. Shave the whole back from the neck to the tail including all around the tail. Lightly run the blade against the direction of hair growth for the most efficient cut.Do not press the clippers deep into the skin as this can bruise or cut. Next, prepare the skin for an application of depilatory lotion. Moisten the skin with a water soaked paper towel to prevent chemical burns.Press lightly with enough pressure to wet the cut hair down against the skin. Then while the skin is wet, rub a small dollop of depilatory lotion into the shaved region for 15 to 20 seconds. Once applied, let the lotion react with skin for 20 to 45 seconds.Before rinsing off the lotion, lightly wipe away some lotion at various locations to check the reaction. If the skin is pink and hairless, the reaction is complete. Then, grip the mouse to wash it off.Using the nondominant hand with the thumb press down the base of its tail against the palm and close the other fingers. Then with mouse's nose up and away from the water, let water run over the animal's back. Then quickly, but gently, rub the back of the mouse with the other hand to wash away the lotion.After removing all the depilatory cream, dry the skin with a paper towel. It is important to not apply lotion to the ears, tail, or anywhere near the face. If this should happen, immediately wipe away the lotion with a wet towel and then run water over the mouse to prevent damage to these areas.If any dark patches are noticed after using the cream, reapply the cream to those areas. The dark patches of skin will grow hair faster throughout the duration of the experiment. So clip the hair short every three to five days when necessary.A patch or two that are not over the wounding site is acceptable, but if the back of the mouse has more dark patches, do not use it for the chronic wound model. After removing the hair, house the mouse alone for at least one day before wounding because the skin will be too sensitive from the depilatory lotion. 30 minutes before the surgery, inject the mouse with 120 microliters of buprenorphine intraperitoneally in sterile PBS.All the volumes in this procedure assume a 60 gram mouse. 20 minutes before the surgery, inject the mouse with 480 microliters of ATZ intraperitoneally in sterile PBS. Split the volume on either side of the abdomen.After administering the ATZ, place the mouse in a small plastic container on top of a warm heating pad until the surgery begins. Cover the small container with a paper towel to help contain the heat. The mouse should calm down as it warms.After confirming an anesthetized state, proceed with the wounding. To begin the wounding procedure, spray a chem wipe with 70%ethanol to wipe the back of the mouse to clean the wound site and the skin surface. Do not wipe excessively or there will be a risk of killing the bacteria present on the skin.The success of the chronic wound model relies on non-sterile conditions. These mice are not germ free and are housed in a conventional vivarium. The bacterial microbiome that resides on the skin is crucial for the subsequent initiation and development of chronic wounds upon treatment with inhibitors of antioxidant enzymes.Now consider where the place the wound. Ideally, use the dorsal side of the mouse centered and away from dark patches of skin. Now perform the wounding to dressing procedure in 30 to 45 seconds.First lightly press a seven millimeter skin biopsy punch onto the desired wound site and twist the punch around just deep enough to leave an impression of the punch. Then excise the outlined skin by pulling up the center of the punch with tweezers and cutting along the outline with surgical scissors. Once completed, cover the wound firmly with half a piece of transparent film dressing.Within 10 minutes of wounding, apply a 60 microliter aliquot of MSA in sterile PBS and deposit MSA topically onto the wound. Later, six hours after the surgery, administer another does of buprenorphine. Extra doses may be given as needed.Chronic wound initiation takes place in less than six hours and the wound margin is visibly altered from oxidative stress. A chronic wound is a wound that remains open, enlarged comparing to the initial wound and contains biofilm. After 20 days, the wound is at full chronicity.Biofilm forming bacteria that dominate the wound site include Pseudomonas aeruginosa, Enterobacter cloacae, and various Staphylococcus and Corynebacterium species. These bacteria are also found in human chronic wounds. After watching this video you should have a good understanding of how to create chronic wounds in mice.Once you have established these chronic wounds, you can then use them to investigate many of cellular molecular mechanisms involved in the initiation of wound chronicity.

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