A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.
Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.
Impaired wound healing is responsible for significant morbidity and mortality worldwide; this is particularly true for sufferers of diabetes mellitus1,2. In humans, wound healing is a continuum of processes, in which there is significant overlapping3. Immediately following wounding, inflammatory processes are initiated. Inflammatory cells release factors that encourage the processes of cell proliferation, migration and angiogenesis. After re-epithelialization and new tissue formation there is a phase of remodeling that entails both apoptosis and re-organization of matrix proteins such as collagen.
The complexity of wound healing cannot currently be replicated in vitro and this necessitates the use of animal models. To date, wound-healing studies have been limited to large animal models, such as swine, to ensure that the healing processes are equivalent and comparable to humans. However, using large animals for such studies can be difficult to house and are not always practical4. The laboratory mouse represents an economical animal model that can be easily genetically manipulated for mechanistic investigation5-7. However, murine wounds heal differently to human’s, primarily due to the process of contraction8. This is in part, due to an extensive subcutaneous striated muscle layer called the panniculus carnosus that is largely absent in humans. In mice, this muscle layer allows the skin to move independently of the deeper muscles and is responsible for the rapid contraction of skin following wounding.
To overcome this limitation, murine wound healing can be adapted to replicate human wound healing by use of a splint (Figure 1)8,9. In this video we demonstrate the splinted murine wound model that eliminates wound contraction and more closely approximates the human processes of re-epithelialization and new tissue formation. In this model two full-thickness excisions that include the panniculus carnosus are created on the dorsum, one on each side of the midline of the mouse. A silicone splint is placed around the wound with the assistance of adhesive and the splint then secured with interrupted sutures. Each mouse acts as its own control, with one wound receiving treatment and the other vehicle control, thereby reducing animal numbers. Following topical applications, a transparent occlusive dressing is applied. The dressing can be removed when required for further topical applications and/or measurement of the wound area10,11. At the completion of experiments, wound closure, morphological architecture and degree of neovascularization can be assessed by immunohistochemistry. This economical and easy to perform model can also be utilized to assess wound healing in the context of diabetes mellitus or other pathophysiologies.
1. Preparation of Splints and Occlusive Dressings
2. Experimental Animals
3. Anesthesia and Operative Preparation
4. Excision and Splinting of Wound
6. Postoperative Management
7. Wound Measurement and Treatment
8. Histological Analysis
A wound closure curve is determined by calculating the average diameter of the wound and expressing the results as a percentage, i.e. 100 – (Day 0 diameter/Day X diameter). In this experiment a therapeutic compound (or vehicle control) was applied daily to the wound. The therapeutic compound greatly accelerated wound closure (Figure 3). It is important to note that the splints must be maintained for the duration of the experiment, as removal of splints will lead to rapid wound contraction (Figure 2r) and diverge from the pattern of wound healing observed in humans.
Figure 1. Schematic representation of the murine wound healing model. In this model two full thickness wounds are created on either side of the midline allowing each mouse to serve as their own control. Silicone splints are adhered and sutured to the wound perimeter to prevent wound contraction, providing a model replicable to that of humans.
Figure 2. Wound healing surgery and post-surgical measurements. Following hair removal and preparation of the skin with iodine and alcohol (a-b) a biopsy punch is gently used to outline two circles on the dorsum, either side of the midline. (c) A small incision is then created and (d) a circular piece of skin is removed, (e) including the panniculosus carnosus, (f) to create two full-thickness wounds. (g) Adhesive is then applied to the silicone splints and the splints adhered to the wound perimeter. (h) Splints are then secured with sutures. (i) Treatments are topically applied and (j-k) an occlusive, transparent dressing is placed over the wound and adhered to the splints (adhesive can be used if required). (l) Photomicrographs are taken daily and wound area is calculated from the average of three diameter measurements on the (m) y-axis, (n) x-axis and (o) z-axis. (p) A representative photo of the wounds at day 10, noting the smaller wound on the right that was treated with a therapeutic compound. (q) Representative laser Doppler image of blood perfusion of the wound at day 6. (r) Example of rapid wound contraction following removal of the silicone splints.
Figure 3. Representative wound closure graph. Wound area is calculated from the average of three daily diameter measurements along the x, y and z-axes. Wound closure is expressed as a percentage of initial wound area at day 0.
This is an experimental murine model of cutaneous wound healing. A significant feature of this model is the use of silicone splints to prevent wound contraction so that re-epithelialization and new tissue formation may occur, making it similar to the process that occurs in humans. This model is versatile and can be used to assess wound healing in both physiological and pathophysiological (e.g. diabetes mellitus) settings. The model may also be used to assess potential wound healing or angiogenesis therapeutics in an economical setting. With each mouse acting as its own control, animal numbers are minimized. The surgical techniques required for this model are not highly sophisticated, thus this model can be widely used by those with relatively little surgical experience.
To ensure reproducibility and accurate quantification it is imperative that the splint is adequately adhered and anchored to the skin with sutures, and that there is minimal delay between creating the two wounds. The propensity of murine wounds to rapidly contract following loosening of the splint or partial removal due to scratching by the mouse requires daily monitoring of the splints. Careful application of adhesive is also required to minimize irritation of healthy skin around the splint that may promote scratching. It is also very important to follow aseptic techniques and thoroughly disinfect equipment, particularly the calipers, between mice. The application of the occlusive dressing must also be considered, especially if wounds are not going to be treated or dressed daily. Opsite and Tegaderm (3M) dressings are comparable 12, and it has been shown that Tegaderm dressings may only remain adhered for 1-2 days. Should longer-term wound dressings be required an alternative approach has been described by Chung and colleagues13.
Potential weaknesses of this model may include inflammation due to the anchoring of sutures, the diffusion of the treatment or vehicle between the wounds and the entry of the treatment into the systemic circulation. In regards to the sutures inducing local inflammation, the sutures are placed relatively far from the wound and as each wound is created the same, therefore any inflammation that may occur should be similar between wounds. Similarly, the distance between the wounds and a lack of edema between the wounds would minimize diffusion between the two beds. There is some evidence that a treatment may enter the systemic circulation, which may accelerate healing of the control wound 9,10. To determine the extent of a treatment entering the systemic circulation, littermate mice could be utilized, in which the two wounds are only treated with the vehicle. The differences in rates of wound closure between mice treated with vehicle only, and the mice receiving both vehicle and treatment could then be compared.
In conclusion, we have demonstrated a relatively simple murine model of wound healing that exhibits many of the features observed in human wound healing.
The authors have nothing to disclose.
The authors would like to acknowledge funding support from the National Health and Medical Research Council (NHMRC) of Australia (Project Grant ID: 632512). Louise Dunn was supported by an NHMRC Early Career Fellowship and Christina Bursill by a National Heart Foundation Career Development Fellowship.
Name of Reagent/Material | Company | Catalog Number | Comments |
Press-to-seal silicone sheeting 0.5 mm thick | Invitrogen | P18178 | Cut into “donuts” with external diameter of 1cm external, 0.5 cm internal diameter |
Biopsy punch 5 mm | Steifel | BC-B1-0500 | To outline wound area to be excised |
Vannas scissors 8.5 cm curved | World Precision Instruments | 501232 | For wound incision and excision |
Dumonte #7b forceps, 11 cm | World Precision Instruments | 501302 | To grip skin when creating incision and excising skin |
Graefe forceps, serrated 10cm | World Precision Instruments | 14142 | To help attach silicone splint to skin |
Needle holder, smooth jaws, curved, 12.5 cm | World Precision Instruments | 14132 | |
Malis forceps, smooth, straight, 12 cm | Codman and Shurtleff, Inc (J&J) | 80-1500 | To suture the silicon rings to the skin |
Ruler, 0.5 mm gradation | n/a | ||
Calipers 0.25 mm gradation | Duckworth and Kent | 9-653 | To measure wound area |
Opsite FlexiFix transparent adhesive film. 10 cm x 1 m | Smith & Nephew | 66030570 | |
Rimadyl (Carprofen) | Pfizer | 462986 |