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 ~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.
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'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'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.
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
2. Vivarium and Husbandry
3. Requirements for the Development of Chronic Wounds
4. Shaving and Application of Depilatory Lotion
NOTE: Remove unwanted hair on the dorsum of the mouse before wounding. The following procedure is done on live db/db-/- mice that are not under anesthesia the day before surgery. Take precautions to prevent stress and harm to the animal.
5. Reagent Setup
NOTE: The development of chronic wounds in db/db-/- mice is accomplished by treatment with specific inhibitors for catalase and glutathione peroxidase, 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MSA), respectively16. The following procedure details the dose and administration of the analgesia and inhibitors based on the weight of the mouse.
6. Surgery
NOTE: 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 bacteria microbiome that resides in the skin is crucial for the subsequent initiation and development of chronic wounds upon treatment with inhibitors of anti-oxidant enzymes. Therefore, traditional pre-surgical preparation of the site is contra-indicated.
7. Post-Surgery Treatment and Recovery
8. Data Collection, Survival Strategies, Handling the Mice After Wounding, and Additional Tips
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 oxidative stress. Histological evidence of this wound margin reveals that the tissue is necrotic and will not participate in wound healing. The biofilm-forming bacteria in the wound can later use this necrotic tissue as a source of nutrients and structural components to produce biofilm. A chronic wound is a wound that remains open, enlarged in comparison to the initial wound, contains biofilm (EPS plus pathogenic bacterial found in human chronic wounds) and takes month or years to heal depending on the amount and content of the biofilm that prevents the wound from resolving normally. In the chronic wound model, full chronicity is set to > 20 days after surgery because the wounds not treated with the inhibitors will close by this time (Figure 5). Healing typically takes > 60 days and the time depends on the primary pathogenic bacteria present in the wound. Sometimes the mice may succumb to infection when more aggressive biofilm-forming bacteria, such as Pseudomonas, are predominate in the wound. Thus, a chronic wound is defined as a wound that will not close within 20 days, takes more than 60 days to heal, and has biofilm present on the wound.
Sex differences have been found in various diabetes models, including the db/db-/- mouse model32,33. While such differences exist, we have observed sex to not be a significant factor in the development of chronic wounds. Chronic wounds in male and female mice develop to a similar extent, so both sexes can be used to study chronic wounds. Thus, utilizing this model is advantageous since human chronic wounds can be found on both male and female diabetic patients.
Figure 1. Shaving and depilatory process. (A) The mouse before shaving. (B) The skin of the mouse is shaved to remove most of the hair on the back. (C) A dollop of depilatory lotion on the tip of a finger. More is used if the mouse is bigger. (D) The back of the mouse is covered with depilatory lotion and left to react. (E) A spatula is used to scrape off some of the lotion to see if the hair has been removed. Bright pink skin without any presence of hair is indicative that the hair removal is complete. (F) The lotion on the back is removed with running water. The skin of the mouse should be slightly pink. This figure has been modified from Kim and Martins-Green31. Please click here to view a larger version of this figure.
Figure 2. Removing hair from smaller patches of dark skin. (A) The mouse has already been treated once and the skin is wet again to prevent burns. (B) Depilatory lotion is only applied onto the patch of skin that is dark and has dense hair. (C) The lotion is washed off after the reaction to reveal the dark patch of skin without hair. This figure has been modified from Kim and Martins-Green31. Please click here to view a larger version of this figure.
Figure 3. Pre-surgery set up. (A) The mice that are to be wounded are placed in small plastic containers on top of a heating pad. (B) Some of the materials used in surgery are shown. The surgical scissors need to be sharp to ensure that the skin is not crushed when cut. The transparent dressing is cut in half. This figure has been modified from Kim and Martins-Green31. Please click here to view a larger version of this figure.
Figure 4. Making the excision wound. (A) After the mouse is under anesthesia, the back of the mouse is wiped with 70% ethanol once. (B) The skin biopsy punch is placed on the back of the mouse and pressed hard enough to leave an impression. The biopsy punch can be rotated to make a shallow incision. (C) The middle of the outlined area is pinched with tweezers and a sharp surgical scissor is used to make the initial incision. (D) The surgical scissors are maneuvered to cut along the outline made by the biopsy punch. (E) A region of skin outlined by the biopsy pump is successfully excised. (F) The transparent dressing is positioned on the back of the mouse and secured. This figure has been modified from Kim and Martins-Green31. Please click here to view a larger version of this figure.
Figure 5. Pictures of wounds. (A) The wound on a mouse at successive times after surgery as it progresses into chronicity starting on the day of surgery. Biofilm can be seen as early as day 5 and detected as early as day 3. The wound is fully chronic with strong biofilm on Day 20. (B) Examples of human chronic wounds, specifically diabetic foot ulcers. This figure has been modified from Kim and Martins-Green31. Please click here to view a larger version of this figure.
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 wounds.
The chronic wound model is also a powerful model to study various aspects of wound healing and complications such as bioburden and cachexia. Bioburden is just one of many facets of chronic wounds that can be studied in this model, as it also affects human chronic wounds. Our procedures and Animal Use Protocol clearly identify symptomology to be monitored, along with a defined monitoring schedule. Animals identified as morbid, based on IACUC approved criteria, are euthanized in order to avoid significant suffering. In addition, the Campus Veterinarian and Animal Health Technician are consulted when certain symptomologies arise and provide supportive guidance in assessment of criteria.
Critical steps within the protocol include housing the db/db-/- mice in a conventional vivarium, removing hair with depilatory lotion, and heating before isoflurane administration. The development of chronic wounds in the chronic wound model relies on non-sterile conditions and practices. These mice are not germ-free and do not grow in very clean vivariums. The microflora that resides in the skin is crucial for the subsequent initiation and development of chronic wounds upon treatment with inhibitors for anti-oxidant enzymes. These db/db-/- mice must be exposed to an environment that contains bacteria, both commensal and pathogenic. If the skin is cleaned and dis-infected with iodine or other antiseptic methods prior to surgery in order to "sterilize" the skin, the wound may not become chronic. The bacteria in the skin microbiome are necessary for the formation and development of biofilm, and delay healing and wound closure. In the clinic, the presence of biofilm in a wound further complicates the wound healing processes and increases the risk of amputation in humans if the infection in the wound is not controlled.
Removing hair with depilatory lotion is an important step to remove excess hair and allow a smooth and clean surface for the transparent dressing to adhere firmly. The depilatory lotion used in this protocol is a chemical depilatory with added aloe for minimizing burns. This product is minimal in altering the morphology of the skin as well as preserving the skin microbiome. The product listed in the Table of Materials is recommended over other hair removing products, including physical and mechanical depilatories (waxes and commercial epilators) that can further burn and tug at the skin and/or kill the skin microbiome. Even though this chemical depilatory is physically gentle, it can still irritate the skin slightly, an effect that could alter the wound healing processes. Thus, it is most effective to wait 18-24 hours before surgery to allow the skin to recover from the procedure and ensure the skin and wound are not affected by it.
These mice are extremely docile and non-responsive to stressors. They are very easy to handle without anesthesia. They are calm enough to place on a palm or on top of a bench without running away. If the base of the tail of the mouse is secured with the thumb and second finger, the mouse will not be able to run away or turn back to bite due to their large belly size. Importantly, in our experience, these mice are extremely sensitive to anesthesia, especially as it relates to loss of homeostasis. Thus, in consultation with our IACUC, it was determined that the better option is to limit anesthetic administration.
The main purpose of this model is to create a large unhealing wound on its back, so once a wound is made, holding a mouse conventionally to make IP injections of additional buprenex or other chemical treatments periodically is impossible. Holding the mouse by the dorsal skin causes the mouse a lot of discomfort and quite possibly pain if the mouse is held in such a traditional manner; thus, all injection with the mouse right side up while it is standing on all four feet. Other experiments utilizing the db/db-/- strain use these mice when they are younger and do not weigh as much, therefore the traditional way of performing an IP injection may used. Since the chronic wound model utilizes mice up to 6 months of age and these mice can weigh up to 80 g, the conventional method is not optimal and can potentially hurt the mouse. We have utilized the method described above for a mouse this obese and have not observed any adverse outcomes when injected with this method with successful aspiration.
Previously, injectable anesthesia such as ketamine and xylazine, were used; however, they proved difficult to use with db/db-/- mice. With a total operation time of less than 5 minutes, the long induction and recovery time was not necessary for the purposes of the experiment. Isoflurane was determined to be the better choice of anesthesia for this procedure due to easy administration and titration, rapid onset and recovery, and adequate anesthetic depth. Also, isoflurane causes minimal cardiac depression and maintains BP very well34. So, a major change in the procedure for the chronic wound model was to use isoflurane as the preferred anesthesia.
The heating before surgery is important for preventing mortality in the 2-3 days following surgery. These mice have significantly lower core body temperature35 and are not able to control their core body temperature effectively due to their genetic manipulation, so an external heating source is provided before the surgery to protect against the additional drop in core body temperature induced by anesthesia. We have assessed the need for a heating pad before, during, and after surgery. It is important to note that db/db-/- mice have unusual physiological responses. Empirically, we have found these mice to be most effectively protected with pre- and post-surgical heat support. By substituting isoflurane as the anesthetic, we measured and determined that the core body temperature did not drop significantly during the less than five minutes of surgery. While we have found the pre- and post- surgical heat support to be critical for these mice, we have not found surgical heat support to have an effect. It should be noted that the IACUC Chair provided guidance and monitored our tests related to temperature and anesthetic effects. As we are communicating this method, we find it important to indicate what is necessary for the success of the procedure.
A limitation of utilizing this method to study biofilm development is the fact that the bacteria present on the wound and producing the biofilm are not controlled. If a specific biofilm-forming bacterium is to be studied, this model may be useful if the native microbiome can be abolished before wounding either through iodine or other antiseptic methods. In response to excessive levels of oxidative stress, key pathogenic bacteria in the skin microbiome are stimulated to initiate biofilm formation. In our chronic wounds, biofilm-forming bacteria include, but are not limited to, Pseudomonas aeruginosa36,37,38,39, Enterobacter cloacae37,38,39, and various Staphylococcus40,41 and Corynebacterium species41,42,43,44,45, all of which can be found in human chronic wounds. Several chronic wound microbiome studies have been conducted on human chronic wounds for the bacteria39,40,41,43,44,45, and fungi46,47 community, including longitudinal surveys associated with poor healing48,49. Longitudinal studies of this nature can also be followed through with the chronic wound model.
It is important to acknowledge that different results may be obtained due to differences in vivaria conditions, supplies, and equipment, vendors, and source colonies for db/db-/- mice. To minimize such differences, provided in the protocol is the exact mice variety and source that is used for the chronic wound experiment. For the husbandry of these mice, the exact bedding and food brands have been provided in the Table of Materials to limit variability. In our experiments, we find that high oxidative stress is necessary and sufficient to create chronic wounds in these mice, as long as the mice are housed in a conventional vivarium and exposed to bacteria. Bacteria populations and communities may differ with vivaria; however, as long as a germ-free facility is not used to house the mice, they should have enough bacteria, both commensal and pathogenic, to reside on the hair and skin.
This method of creating chronic wounds in this protocol is significant to study chronic wounds and impaired wound healing because only oxidative stress levels are significantly altered experimentally. Oxidative stress is required for normal wound healing processes2. It is important for timely regulation and a crucial component in the functionality of cells necessary for wound healing5. However, when the levels of oxidative stress are not controlled, reactive oxygen species can damage endothelia cells, inhibit keratinocyte functionality, and delay wound closure2,5. Human chronic wounds have high levels of oxidative stress50. The mouse model has high blood glucose and already increased levels of oxidative stress due to its morbidity. These characteristics are shared with humans living with diabetes and provide a microenvironment conducive for chronicity after sustaining injury50. Humans also host diverse and complex microbiota in many locations on the body, including the skin, so a complex, but natural, microbiome is allowed to develop on the skin of the mouse.
The authors have nothing to disclose.
The authors have no acknowledgements.
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 |