Wound Healing Kinetics of the Genetically Diabetic Mouse

With diabetes mellitus and obesity on the rise,^1,2 and the increased prevalence of complex wounds and impaired healing,^1,2 there is a crucial need to develop reliable wound repair models to identify effective therapeutic approaches.^3,4
To be clinically relevant, an experimental model should appropriately reproduce the clinical situation physiologically and pathogenically.⁴ Experimental models can be either in vitro or in vivo.
In-vitro models are necessary in wound healing because of the orchestral fashion in which different cell types and the vascular and immune systems interact. In-vitro models are necessary in wound healing to study specific conditions, mechanisms of action, and the direct influence of therapeutics on individual cell types. Numerous in-vitro studies explore individual function and response to miscellaneous factors of cells such as keratinocytes,^5,6,7–9 endothelial cells,^10–12 fibrocytes,^13–16 or molecules such as collagen,¹⁷ and many other components involved in wound healing. Additionally, in-vitro studies are often faster, cheaper, simpler, and more specific than in-vivo studies. In-vitro studies can also limit complications and variability.⁴
All findings derived from in-vitro studies have helped to elucidate important aspects of wound healing at the molecular level.
However, in-vitro studies often fail to reproduce the complexity of an organism and pathologic condition, making the in-vivo model a crucial tool for discoveries with clinical relevance to wound healing.
Researchers have studied in-vivo wound healing in a variety of species including pigs,^18,19 rabbits,^20–23 rats,^24,25 and mice.^2,4,26–32 The present review focuses on the mouse model, which has been instrumental throughout the development of the wound care field. Mice are appealing candidates for wound healing research because of their availability, low cost, and the ability to test a large number of animals in small facilities with highly reproducible results. In addition, the wide variety of knockouts and diseased mice available are important tools for the comprehension of wound healing mechanisms.
Wound repair kinetics vary among species. Mice, for example, heal mainly by contraction^2,26,27,33 due to the presence of the panniculus carnosus muscle in the subcutaneous tissue. Humans lack this muscle and thus heal less through contraction and more through re-epithelialization—except in some areas where substantial wound contraction can occur, such as the joints.²⁹
To generate a model that resembles human wound healing, researchers have developed ingenious models using wild-type mice and mice of various phenotypes including several diabetic mice, and have shown different levels of wound healing impairment. A phenotype similar to diabetes type 1 can be induced in mice by administering the cytotoxin streptozocin, which targets and inhibits proper pancreatic beta cell function.²⁹ It has been reported that these mice have high variability in glucose levels,³⁴ lower collagen synthesis resulting in reduced wound strength,^35,36 less granulation tissue formation,³⁷ altered T-cell function, and decreased macrophage phagocytosis.³⁶
Other groups use the ob/ob mouse for wound healing research.^35,38,39 The ob/ob mouse lacks the leptin gene causing high food intake and metabolic changes leading to obesity and resembles the type 2 diabetic phenotype.^40,41 The ob/ob mouse appears to display impaired reproduction, hyperphagia, decreased thermogenesis,⁴² insulin resistance with secondary insulinemia,⁴¹ and impaired wound healing with decreased collagen accumulation.^39,43,44 Topical and systemic leptin application in the leptin deficient ob/ob mouse increased wound healing, suggesting leptin has an impact in wound healing.⁴⁴
The authors’ group and others rely on the db/db strain.^{26,31,32,45–49} These genetically diabetic mice have a phenotype similar to diabetes type 2 that mimics the authors’ target patient population. A homozygous point mutation on the leptin receptor gene (LEPR) in the hypothalamus changes the metabolic and behavioral phenotype of the db/db mouse.^42,50,51 Lack of a functional central leptin receptor results in loss of a negative feedback signal critical to the normal control of food intake that causes a negative balance.^42,50 The resulting physiologic responses lead to an early onset of diabetes and massive obesity (up to 3 times normal body weight, about 80% of which is fat) at 3 to 4 weeks of age.⁵¹ The mutant mice have impaired sympathetic nervous system activity,⁵² a lower body temperature (35^˚C versus 37^˚C when housed at room temperature), and lower thyroid hormone activity—all of which contribute to their reduced energy expenditure.^42,53 Additionally, peripheral neuropathy through decreased numbers of epidermal nerves,⁵³ myocardial disease,^54–56 and impaired reproducibility,⁵⁷ are present.
Mice homozygous for LEPRdb show significant wound healing deficiency compared to the wild type mouse and makes this genotype interesting for wound healing studies.^{26,31,32,45–49}
Elevation in plasma insulin begins at 10 to 14 days, and blood sugar levels at 4 to 8 weeks.⁴⁰ Although most actions of leptin are thought to be centrally mediated, some studies show leptin influences in the periphery.^{43,44,58–60} Some examples of leptin action outside the central nervous system include: inhibition of insulin release in pancreatic beta cells,⁶⁰ stimulation of angiogenesis,⁶¹ proliferation of hematopoietic cells⁶² and effects, T-cell immunity,³⁶ and stimulation of re-epithelialization.⁴⁴ Dorsal excisional full-thickness skin wounds in the db/db mouse reached complete wound closure 7–10 days later than the wild-type mouse.²⁶ Slower wound closure in db/db mice has been attributed mainly to impaired wound contraction, regardless of the initial wound size.^26,32
The wild-type mouse has also been used to conduct wound repair studies. The strong tendency of wild-type mice to heal spontaneously, and the difficulties and limitations associated with this species for wound healing studies, have been reported in literature.^{26,27,29,32,33} Restrictions include: the high levels of contraction, re-epithelialization is almost impossible to capture over time, difficult to keep a dressing on the mouse during longer follow-up periods, and the fast re-growth of hair around the wound bed.³²
To address these limitations, wild-type mouse models have been customized, which include a splint model to inhibit contraction.^2,63 Researchers also developed more models by wounding different anatomical regions that are naturally unable to contract, such as the dorsum of the tail or the skull.^33,64
In the authors’ experience, the genetically diabetic mouse experimental model is one of the most suitable for use in wound healing studies. This model allows accurate quantification of the main aspects of a healing wound such as granulation tissue formation, collagen deposition, wound closure, re-epithelialization, and wound contraction. Given the epidemics of diabetes and its associated complications, the use of the diabetic mouse model will likely expand in the future, thus there is a need for a more accurate, reproducible development of this model.

Methods
Literature review. Three researchers independently conducted a literature review using PubMed to examine other diabetic murine models of wound healing. The keywords used to conduct this literature review were: db/db mouse, wound healing, wound closure, re-epithelialization, ob/ob, diabetic mouse model, wound contraction, wound re-epithelialization. The collected research was reviewed, and the methodologies and results were examined. All research used for comparison with the authors’ db/db model had similar methodology: hair removal, acute full-thickness skin excision on the mouse dorsum, application of semiocclusive dressings, and macroscopic and microscopic measurements of wound closure.
Wound model and study design. Excisional wounds can be created in different sizes and shapes, and can either be left open or covered with a semiocclusive dressing.^{26,30–32,35,37,43,46,49,65} The follow up period is either defined or adjusted to complete wound closure. Depending on the histological read out, tissue harvest can be performed at the end of the follow up period or at earlier time points.
In the authors’ studies, homozygous, genetically diabetic 8- to 12-week-old, db/db mice (strain C57BL/KsJ-LEPRdb) were used under an approved animal protocol in an AAALAC accredited facility. The day before surgery, hair was clipped and depilated (Nair^®, Church & Dwight Co., Princeton, NJ). On the day of the surgery, animals were weighed and anesthetized with 60-mg/kg pentobarbital. Dorsal skin was marked with a standardized template (1.0 cm² or 1.5 cm²) and skin plus panniculus carnosus was excised carefully. Tincture Benzodine Compound (Paddock Laboratories, Minneapolis, Minn) was applied at the surrounding skin and wounds were covered with semi-occlusive polyurethane dressings (Tegaderm^™, 3M, St. Paul, Minn). Tegaderm dressing was changed twice a week and wounds were photographed from a standard high using a tripod. When taking pictures, animals were anesthetized with Isoflurane inhalation (Isoflurane, USP, NovaPlus, Lake Forest, Ill). On days 9, 10, and 21, or when wounds were completely healed, animals were euthanized and entire wounds were harvested including a surrounding 0.5-cm skin area. The wound was cut through the middle, and 1 piece was fixed in 10% neutral-buffered formalin solution for 24 hours and stored in 70% alcohol at 4^˚C; the second piece was frozen in O.C.T. (Tissue-Tek O.C.T. Compound^®, Sakura Finetek, Torrance, Calif).
Before harvest, blood was draw by heart puncture through a 21-gauge needle and a drop of blood was applied onto a glucometer (CVS, Woonsocket, RI) for blood sugar determination.

Wound Closure Analysis
Three independent observers who were blinded to the genotype, measured the initial wound size (I) on day 0, wound contraction (C), re-epithelialization (E), and the open wound (O) using planimetric methods (Image J, National Institutes of Health, Bethesda, Md). Wound kinetics were quantified by measuring contraction (C), re-epithelialization (E), and the open wound (O) as a percentage of the initial wound area (I). The sum of contracted, re-epithelialized, and open wound areas were equal to 100% of the original wound size (Figure 1).⁶⁶

Central wound cross-sections were embedded in paraffin, sectioned, and stained according to routine hematoxylin and eosin (H&E) protocols (Figure 2). Panoramic cross-sectional digital images of each wound were prepared using Adobe^® Photoshop^® CS software (Adobe Systems Inc, San Jose, Calif) to analyze granulation tissue area and thickness (Figure 2B). Two independent observers, blinded to the genotype, used digital planimetry (Image J, NIH, Bethesda, MD) to quantify the area and thickness of granulation tissue in the middle part of each section at 10-x magnification. Capillary density was evaluated using 3 fields per slide viewed at 40-x magnification. Mean values of the middle and 2 lateral areas of the granulation tissue were evaluated as average capillary density. The images were viewed with Adobe Photoshop CS software. Blood vessels in each high-powered field were marked and counted.

The epithelial gap (EG) and dermal gap (DG) were measured using a Nikon shadowgraph (Nikon Inc, Melville, NY). Epithelial gap was defined as the distance between the advancing edges of clear, multiple layer neoepidermis. An EG of zero represented a completely re-epithelialized wound.⁶⁷ The dermal gap was defined as the distance between uninjured dermis on both sides of the wound. Two serial sections were averaged to determine EG and DG (Figure 2B).
Immunohistochemistry. Paraffin-embedded sections were rehydrated and antigen retrieval for Ki-67 (LabVision, Freemont, Calif) was accomplished by microwaving the sections in 10 mM sodium citrate (pH 6.0) for 10 minutes. Frozen sections were fixed with acetone and stained for platelet endothelial cell adhesion molecule 1 (PECAM-1, Pharmingen, San Jose, Calif, [Figure 3]). PECAM-1 primary antibody was incubated at 4^˚C overnight, while the Ki-67 primary antibody was incubated for 1 hour at room temperature. PECAM-1 signal was intensified using the tyramide amplification system (Perkin Elmer, Boston, Mass).

Vessel density quantification. Digital color images of the wound sections were preprocessed before quantification to ensure uniform contrast of PECAM-1 positive areas relative to the background. A mask of positive staining was created using the color mask function of the program Corel Photo Paint v.10 by sampling 5 different chromogen color tones represented in positively stained areas. The masked vessel areas were converted to pure black while the background was made pure white. The black and white representations were used for area quantification in IP Lab software by applying the segmentation function. Tissue regions were defined by projecting the original H&E image over the processed image. Blood vessel density, quantified over the entire image, is expressed as the ratio of vessel area to total granulation tissue area (Figure 3). Between 4 and 7 microscopic fields (40 x) were used to evaluate vessel density for each wound.
Quantification of cell proliferation. Wounds were analyzed for cell proliferation using image analysis of Ki-67 stained sections in a manner similar to the method of vessel density quantification. High-power digital images of Ki-67 stained wound sections were used to measure the number of Ki-67 positive cells relative to the total number of nuclei (Figure 3). The degree of proliferation was quantified over the entire wound section using 4–6 fields at 20-x magnification and expressed as a ratio of proliferating nuclei (Ki-67 positive) to total nuclei.
Statistical analysis. Values are expressed as mean ± standard deviation (SD) in the text and figures. Unpaired student’s t-tests were used to determine the significance of differences between genotypes.
Genotyping. Tails were collected in dry ice and the polymerase chain reaction (PCR) product was amplified before RsaI digest. The positive control was obtained from Jackson Labs (Bar Harbor, Me).

Results
Wild type versus diabetic wound healing kinetics. Delayed wound closure in db/db mice with a 1.5-cm² full-thickness wound on the dorsum was observed after 4–6 weeks, compared to their nondiabetic counterparts, who healed in 10–16 days.⁶⁵ After 2–3 weeks, db/db mice showed almost no signs of healing with wound contraction of 10%–20%, while wild type animals at the same time point were almost healed. Similarly, in another study using db/db mice,³² 4-mm to 6.0-mm db/db wounds on the dorsum of each animal healed in 27.75 days ± 1.49.^32,49
In previous studies that corroborate these results, different sizes of full-thickness wounds in the db/db showed significant delay compared to their wild-type counterparts.²⁶
However, in the authors’ experience, using a 1.5 cm² excisional wound in db/db mice showed slightly faster wound closure, reaching 34% after only 9 days. On the same day, wild type animals showed 84% wound closure (Figure 4).^26,65

Wound closure kinetics of the db/db mouse. Considering the 2 main mechanisms of wound healing—contraction and re-epithelialization—db/db mice were found to heal mainly by re-epithelialization (Figure 5, Table 1) during the first 10 days of the follow-up, and contraction in the last 2 weeks. Although it has been reported that contraction is the main wound healing deficiency of this animal model,^65,68 the authors of the present study found that it plays a main role to facilitate complete wound closure that starts at the second week of the follow-up period. On day 10, wounds displayed a peak in new epithelial formation from the wound edges.^30,31,69

One cm² full-thickness wounds on the back of the diabetic mice reached 50% wound closure in 13.7 ± 2.9 days.^30,31 Diabetic mice with 0.6-, 1.0-, and 1.5-cm² full-thickness wounds, regardless of initial wound size, demonstrated similar delays in wound healing.²⁶ Ninety percent closure was reached in 19.9 ± 8.6 days post wounding by 1.0 cm² wounds in the diabetic mice and consistently reached 80% wound closure by day 21 (Figure 5).^30,31

Histological Analysis
Wound tissues for histological assessment were collected in the period when 50% closure was reached (8–13 days post operation).²⁶
The level of contraction of a wound can be measured by the dermal gap. The dermal gap of 1.0 cm² reduced in size of 8.3 mm ± 1 mm on day 9. Microscopic quantification of the epithelial gap (the open portion of the wound) on the same day resulted in 5.8 mm ± 2 mm (Figure 2A, 2B).^30,31
Microscopic analysis of wounds mid-healing demonstrated less inflammatory infiltrates in diabetic wounds than wild-type wounds.²⁶ The granulation tissue formed by the db/db mouse in the middle of the lesion of a 1.0 cm² full-thickness wound was 78% less when compared to wild type animals. Similar results were seen when granulation tissue thickness was measured—79% reduction (Figure 2A, 2C).^26,30,31 The number of blood vessels on the same day was 5.7 ± 5.8 per high power field at
40-x magnification.
Blood vessel density in the granulation tissue reached 4 ± 3% and cell proliferation was found up to 20 ± 7% on day 9, as assessed by immunohistochemistry.
Diabetic mice and hyperglycemia. Db/db mice showed a diabetic-hyperglycemic phenotype with significantly greater blood sugar levels (590 mg/dL ± 6 mg/dL) compared to their corresponding wild-type counterparts (285 mg/dL ± 7 mg/dL).²⁶
Genotype analysis. Genotype analysis revealed a band at 135 bp in wild type mice (Figure 6A). Mice heterozygous (db/+) for the point mutation, showed a 135 bp and 108 bp bands (Figure 6B). Mice homozygous for leptin receptor mutation (db/db) showed only the 108 bp band (Figure 6C).


Discussion
Considering the increased prevalence of diabetes type 2 in the general population, the development of a reliable animal model, and the standardization of measurement techniques and study designs, are crucial aspects to facilitate improvements in therapies that seek to provide an efficient clinical translation. Among all animal models used, the db/db mouse model is the one that is most commonly used in wound care.
Different mechanisms contribute to wound closure kinetics, such as wound contraction and re-epithelialization. In the db/db mouse, the possibility to capture the different wound closure mechanisms makes it possible to quantify early effects of treatments, whereas vigorous wound contraction in wild type mice easily plaster effects. In the authors’ experience with the db/db mouse it is crucial to study the relative contribution to healing from re-epithelialization and contraction in order to better understand the effects of a therapy. For example, while for some wounds, such as a chronic foot ulcer, the most important goal is to achieve faster wound closure regardless from its mechanism, others would benefit mainly from inhibition of contraction (eg, joint wounds, wounds in cosmetic relevant positions, or burn wounds covering a larger surface).
In the authors’ experience with the db/db mouse model, 80% closure was consistently achieved by day 21.³⁰ This differs significantly from the original observations of Greenhalgh et al⁶⁵ who reported only a 40% wound closure on the same day. In this study, a glass slide was traced onto the wound edges to evaluate wound closure—planimetric analysis was used to determine wound area. This method might change closure data compared to the authors’ method, taking a picture from a standard height using a tripod without touching the wound. Likewise, in a study performed by Sullivan et al³² using db/db mice, multiple 6-mm wounds on the dorsum of each animal healed in 27.75 days ± 1.49. In this study, digital pictures were taken of the wound area with a metric ruler in the frame. The raw, open area of the wound was measured in millimeters squared. These photographs, however, were taken with the dressing over the wound, thus, reducing the accuracy of the wound measurements due to photographic glare and blurred edges. Deformation of the wound shape through the dressing also influenced the measurements.³² When animals lost the dressing, faster wound closure was seen compared to wounds that continued with the dressing, suggesting that the dressing was not consistently maintained over the wound for the entire follow-up.³²
The frequency of dressing change noted in different studies might influence wound healing kinetics mainly due to infection rate and wound fluid preservation. Although direct comparison between different studies is difficult due to discrepancies in methodology, the authors believe that differences in wound closure time using the same animal model are significant. It may be possible that differences in wound dressing and rate of infection play an important role. It has been reported that the dressing itself acts as a splint on the wound and that the accuracy in maintaining the dressing through the follow up is crucial to prevent wound contamination. The meticulous application and maintenance of the dressing, as well as stringent infection control, are important aspects in model standardization.^26,30–32 Differently defined exclusion criteria with regard to dressing maintenance might explain the variability among wound closure data in the db/db mouse model.
Other details, such as the quality of digital wound photos and reliability of software to analyze the results, can enhance a study’s sensitivity.
To achieve reliable data in wound closure kinetics, macroscopic and histologic analysis has to be well defined and easily repeatable. The re-epithelialized area in macroscopic pictures of the wound is widely measured but the correlation between macroscopically and microscopically re-epithelialization is not always well verified.^{30,31,49,65,70} In the authors’ studies, macroscopically revealed wound closure showed a similar trend when compared to the epithelial gap measured in histological specimens among treated and nontreated wounds.³¹ Other studies comparing these two parameters found differences between wounds that maintained the dressing and wounds that did not maintain the dressing.³² More detailed and reliable analysis in clinically-relevant read outs is needed to ensure treatment effects.
Discrepancies in the results can be partially explained also with the variability in healing ability of the db/db strain. It is clear from the authors’ experience that although these animals consistently develop a diabetic phenotype between 8 to 12 weeks of age, they express different levels of wound healing impairment as they progress in the pathology course. To narrow these differences, it may be important to consistently use animals of the same age (ie, 10 weeks), and strictly monitor their glucose concentration and weight. A number of animals per group (15–30), as well as the consistent use of control and case animals from the same order, may reduce the genetic variability. It has been suggested that the use of multiple wounds on one animal may correct for these changes,³² although they cannot be used if a systemic effect is hypothesized. In the authors’ experience, all the animals used for the studies should be phenotyped, as some heterozygous have been found among batches of db/db directly ordered from a company (data not shown).

Conclusion
Currently, a variety of clinicians evaluate wounds in humans and their evaluation is a subjective, macroscopic assessment of wound healing. Better knowledge of the early events in wound healing, such as vasculature changes and cell proliferation, might provide clinicians with substantial information regarding the stages of wound healing. In the genetically diabetic mouse model, the maximal effect of a treatment can be seen at approximately day 10 when wounds reach nearly 50% wound closure. Quantification of vasculature and proliferation at about 50% wound closure can be used as predictive parameters for eventual wound outcome. The introduction of a staging system in wound healing would help physicians assess the most appropriate therapy and the efficacy of the treatment during the follow-up period.
The excisional db/db mouse model is suitable for wound healing research, especially so considering the urgent need for more effective diabetic ulcer treatments. Consistency in methodologies and definitions are needed to generate more comparable and reproducible results.