Protocol to Create Chronic Wounds in Diabetic Mice

This mouse model can help answer questions in the medical field of wound chronicity. The major advantage of this model is that the chronic wounds in mice mimic many aspects of the chronic wounds in humans. To begin, place the mouse on a clean surface and hold it in place by pinching the base of its tail.

Then prepare the hair clippers and be ready to quickly pull them away if the mouse makes any sudden movements. Shave the whole back from the neck to the tail including all around the tail. Lightly run the blade against the direction of hair growth for the most efficient cut.

Do not press the clippers deep into the skin as this can bruise or cut. Next, prepare the skin for an application of depilatory lotion. Moisten the skin with a water soaked paper towel to prevent chemical burns.

Press lightly with enough pressure to wet the cut hair down against the skin. Then while the skin is wet, rub a small dollop of depilatory lotion into the shaved region for 15 to 20 seconds. Once applied, let the lotion react with skin for 20 to 45 seconds.

Before rinsing off the lotion, lightly wipe away some lotion at various locations to check the reaction. If the skin is pink and hairless, the reaction is complete. Then, grip the mouse to wash it off.

Using the nondominant hand with the thumb press down the base of its tail against the palm and close the other fingers. Then with mouse's nose up and away from the water, let water run over the animal's back. Then quickly, but gently, rub the back of the mouse with the other hand to wash away the lotion.

After removing all the depilatory cream, dry the skin with a paper towel. It is important to not apply lotion to the ears, tail, or anywhere near the face. If this should happen, immediately wipe away the lotion with a wet towel and then run water over the mouse to prevent damage to these areas.

If any dark patches are noticed after using the cream, reapply the cream to those areas. The dark patches of skin will grow hair faster throughout the duration of the experiment. So clip the hair short every three to five days when necessary.

A patch or two that are not over the wounding site is acceptable, but if the back of the mouse has more dark patches, do not use it for the chronic wound model. After removing the hair, house the mouse alone for at least one day before wounding because the skin will be too sensitive from the depilatory lotion. 30 minutes before the surgery, inject the mouse with 120 microliters of buprenorphine intraperitoneally in sterile PBS.

All the volumes in this procedure assume a 60 gram mouse. 20 minutes before the surgery, inject the mouse with 480 microliters of ATZ intraperitoneally in sterile PBS. Split the volume on either side of the abdomen.

After administering the ATZ, place the mouse in a small plastic container on top of a warm heating pad until the surgery begins. Cover the small container with a paper towel to help contain the heat. The mouse should calm down as it warms.

After confirming an anesthetized state, proceed with the wounding. To begin the wounding procedure, spray a chem wipe with 70%ethanol to wipe the back of the mouse to clean the wound site and the skin surface. Do not wipe excessively or there will be a risk of killing the bacteria present on the skin.

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 bacterial microbiome that resides on the skin is crucial for the subsequent initiation and development of chronic wounds upon treatment with inhibitors of antioxidant enzymes.

Now consider where the place the wound. Ideally, use the dorsal side of the mouse centered and away from dark patches of skin. Now perform the wounding to dressing procedure in 30 to 45 seconds.

First lightly press a seven millimeter skin biopsy punch onto the desired wound site and twist the punch around just deep enough to leave an impression of the punch. Then excise the outlined skin by pulling up the center of the punch with tweezers and cutting along the outline with surgical scissors. Once completed, cover the wound firmly with half a piece of transparent film dressing.

Within 10 minutes of wounding, apply a 60 microliter aliquot of MSA in sterile PBS and deposit MSA topically onto the wound. Later, six hours after the surgery, administer another does of buprenorphine. Extra doses may be given as needed.

Chronic wound initiation takes place in less than six hours and the wound margin is visibly altered from oxidative stress. A chronic wound is a wound that remains open, enlarged comparing to the initial wound and contains biofilm. After 20 days, the wound is at full chronicity.

Biofilm forming bacteria that dominate the wound site include Pseudomonas aeruginosa, Enterobacter cloacae, and various Staphylococcus and Corynebacterium species. These bacteria are also found in human chronic wounds. After watching this video you should have a good understanding of how to create chronic wounds in mice.

Once you have established these chronic wounds, you can then use them to investigate many of cellular molecular mechanisms involved in the initiation of wound chronicity.