The overall goal of this procedure is to create a wound, which is splinted, to prevent contraction and promote epithelialization sole proliferation and angiogenesis. This is accomplished by first preparing the dorsum and creating two full thickness excisional wounds either side of the midline. Next, a splint is placed over the wound and sutured in place.
The wound is then measured and treatments are applied. Lastly, an occlusive dressing is placed over the wound and the mouse is allowed to recover. Ultimately, the cutaneous excisional wound healing model is used to assess wound, epithelialization and associated processes, including proliferation and angiogenesis.
The main advantage of this technique over other methods is that wound contraction is prevented. This encourages epithelization proliferation and angiogenesis, which more closely approximates the wound healing process. In humans, This method is particularly useful for assessing the efficacy of therapeutic compounds, and as each mouse serves as its own control, it minimizes the number of animals needed for a study.
Begin by preparing the splints and occlusive dressings to make a splint. First outline a 10 millimeter circle on a sheet of silicone, and then use scissors or a biopsy punch to create silicone discs. Next, center, a five millimeter biopsy punch in the middle of the 10 millimeter disc, and press firmly to create a donut like ring that will be used as a splint.
Create circular dressings of the same size using transparent occlusive dressing such as ops site. To begin, confirm the proper depth of anesthesia in an anesthetized mouse. After removing all of the hair from the surgical area, clean the skin with repeated applications of Betadine and alcohol.
When ready, use a sterile four millimeter biopsy punch to make an outline of two circular patterns on either side of the mouse's midline at shoulder level. Next, use serrated forceps to lift the skin in the middle of the outline and iris scissors to create the wound. Be sure to cut through the subcutaneous tissue, including the panus osis.
Excise the tissue from both sides. To reveal two circular wounds. Remove the plastic protective coating from each side of the silicone splint and apply cyanoacrylate adhesive to one side.
Center the splint over the wound and suture it in place with interrupted six oh nylon sutures. Repeat the splinting process on the other wound. At this point, a therapeutic compound can be applied to one wound while using the other.
For a vehicle control, place a ruler below the splints and take a photo micrograph. Cover the wound with one of the circular pieces of dressing prepared earlier. After administering an analgesic, allow the animal to recover in a heated cage.
The animals are monitored twice daily for manifestations of pain and weight loss. To measure the wound, anesthetize the animal as shown earlier and gently peel back the occlusive dressing with forceps. Use a surgical caliper to measure the wound diameter at multiple points to get an average and take a photo micrograph for future reference.
At this time, the animal can be assessed for blood perfusion. Using a laser doppler imager, apply a clean, transparent occlusive dressing to the wound and keep the animal warm until fully recovered. It is important to note that if the splints are not secured properly, the wound will readily contract after euthanasia by anesthetic overdose, use forceps and a scalpel blade to remove the sutures and carefully peel away the splint from the wounds.
Using iris scissors create a wide, full excision around and under the wound area. Once removed, fix the tissue following standard procedures. Hematin and eosin staining can be used to visualize the wound structure and epithelial gap.
In addition, neovascularization can be assessed by immunohistochemical analyses to determine the number of capillaries. A wound closure curve is determined by calculating the average diameter of the wound and expressing the results as a percentage. In this example, the therapeutic compound greatly accelerated wound closure when compared to controls.
Once mastered, this technique can be done in 30 minutes if performed properly. While attempting this procedure, it is important to attach the splint securely with equally placed sutures to maintain consistency between experiments.
