Demonstration of the Rat Ischemic Skin Wound Model

The overall goal of this procedure is to create a reproducible ischemic wound model. This is accomplished by first utilizing a sterile punch biopsy tool to create two excisional ischemic wounds inside the designated flap out line on the dorsum of the rat. Next, a bi pedicled flap is created by making two incisions along the pre-drawn lines lateral to the two ischemic wounds.

Then the bi pedicled flap is raised and a sterile silicone sheet is placed in between the fascia and muscles to support the wounds and prevent regrowth of blood vessels. Finally, both incisions are closed and two excisional non-ischemic control wounds are created lateral to the suture lines. Ultimately, digital photography is used to show changes in wound size over time.

Laser doppler is used to show changes in blood flow, and finally, wound tissues are harvested for histology and molecular analysis. The main advantage of this technique over existing methods like the McFarland flap, is that it is a reproducible model of persistent ischemia with open wounds that can have added topical agents and can be studied with both histology and molecular analysis. Visual demonstration of this method is critical.

The wound excision and suture placement steps are somewhat difficult to learn. It is easy to make the wounds too deep and the flat must be sutured without distorting the wounds. To begin precut strips of non reinforced 0.01 thickness medical grade silicone cheating and autoclave to sterilize, clean and sterilize suitable surgical instruments including scissors, forceps, and drapes or towels to create a sterile field during surgery.

After inducing general anesthesia on an adult male or female rat, weighing 250 to 300 grams according to the text protocol. Perform a toe pinch to check the depth of sedation and apply a nose cone supplying one to 2%isof fluorine with oxygen To maintain anesthesia, place the rat in the prone position and use clippers to shave the dorsum from the base of the neck down approximately 11 centimeters with permanent marker stencil. The outline for the 3.0 centimeter by 10.5 centimeter flap.

Move the rat to a clean designated surgical area equipped with an approved heating pad and sterile surgical drapes or towels. Inject five milligrams per kilogram of ketoprofen subcutaneously prior to the first surgical incisions. For pain management.

Use 70%isopropyl alcohol to swab the skin. Then 0.2%chlorhexidine, and then apply sterile drapes to create a sterile field using a sterile disposable six millimeter biopsy punch tool. Create two circular ischemic wounds in the center of the designated flap area that reaches the underlying fascia of the panus carno muscle.

Using forceps, lift the skin in the middle of the wound outline and then use iris scissors with curved tips to excise the circular piece of tissue, including the panus carno muscle. The result will be a full thickness wound with the fascia as the base of the wound. Next, with a sterile scalpel, create a bi pedicled flap by making incisions along the tron lines 10.5 centimeters in length and 3.0 centimeters apart, and to the depth of the paraspinous muscles on each side of the ischemic wounds.

Then using iris scissors, separate the panus carno fascia from the paraspinous muscles, being careful to keep the fascia intact as the base of the six millimeter punches. Place a sterile precut silicone sheet in between the panus carno fascia and the paraspinous muscles, ensuring that the sheet does not buckle or fold using black non-absorbable 4.0 sutures. Close both incisions by anchoring the silicone sheet to the skin with at least eight interrupted stitches on each side along the length of the flap.

Then with the sterile disposable biopsy punch tool, create two internal control non-ischemic wounds, one centimeter lateral to the ischemic wounds on either side of the flap. Place a ruler below the wounds and take digital photos for wound measurement purposes. Use laser doppler to monitor blood flow or perform topical drug treatments as desired.

Apply an approved liquid adhesive, both cranial and coddle to the wounds and a transparent film dressing to keep the wound environment moist and clean. Place an additional dressing at the coddle end of the flap to prevent the animal from removing the most coddle sutures. After surgery, place the animal by itself in a cage equipped with a shallow feeder so as to prevent the surgical site from rubbing against the feeder.

Place a heating pad under half of the cage for up to two days during recovery. When the animal has regained sufficient consciousness to maintain sternal recumbent and exhibits purposeful movement, return it to the company of other animals to manage pain postoperatively the morning after surgery, administer five milligrams per kilogram ketoprofen subcutaneously and follow up one time per day for up to 48 hours post-surgery. The animal should also be monitored daily for prolonged signs of pain, weight loss, or site infections to measure ischemic and non-ischemic wounds.

After anesthetizing the animal as described earlier in the video, gently remove the dressing so as not to pull the adhesive from the skin. Take digital photos for wound measurements and perform laser doppler imaging. If desired, then reapply topical treatments including adhesive and clean dressing, and allow the animal to recover as before to harvest ischemic and non-ischemic wounds.

After anesthetizing the animal, use a scalpel to make a square shaped incision around the wound to include some healthy tissue, excise the wound and healthy tissue, place the excision into a 1.5 milliliter snap cap tube and snap freeze in liquid nitrogen for future molecular analysis or transfer to 10%buffered formin and incubate overnight at room temperature for histological processing. Since the development of this model, laser doppler imaging has increasingly been used to measure blood perfusion, and this technique will give adequate information about the ischemic state of the bi pedicled flap. This figure shows a Doppler image for a dorsal bi pedicled flap showing both a perfused area and the non perfused area.

For the purposes of this demonstration, wound closure progression is represented as percent of initial wound area over a time course of 28 days. As shown here in Image J, a digital picture was opened and the scale was set to 10 millimeters. On the ruler, a line drawn 10 millimeters in length equates to pixel count, which can be converted to a unit of choice in this case millimeters.

Then the circumference of the wound is traced, and by giving the measure command, the area is presented in millimeters as seen. In this graph, the data can be presented as a percentage of initial wound area on the Y axis and days on the x axis. Once mastered, this technique can be performed in about 20 minutes if properly.Done.

While attempting this procedure, it's important to once again remember to suture the flap without stretching the wounds. If the wounds are distorted to an oval, they will heal more quickly than round wounds leading to variability in the healing results.