Generating an Excisional Skin Wound in a Mouse Model via Punch Biopsy

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Mouse Source and Housing

1. Derive Lysozyme M- enhanced green fluorescent protein (LysM-eGFP) mice on a C57BL/6J genetic background as described previously. Derive LysM-EGFP× myeloid differentiation primary response 88 (MyD88)-/- mice by crossing LysM-EGFP mice with MyD88-/- mice on a C57BL/6J background.
2. House mice in a vivarium. For these studies, animals were housed at the University of California, Davis in groups prior to surgery and singly housed following surgery. Use mice between the ages of 10-16 weeks.

2. Excisional Skin Wounding

1. Administer 100 µL of 0.03 mg/mL buprenorphine hydrochloride (~0.2 mg/kg) to each mouse via intraperitoneal injection.
2. Twenty minutes post-injection, place 2-4 mice in a chamber with 2-3 liters per minute (LPM) oxygen with 2-4% isoflurane. Once mice are fully anesthetized, transfer the mice one at a time to a nose cone connected to 2-3 LPM oxygen with 2-4% isoflurane. Verify mice are fully anesthetized by firmly pinching each rear paw between a thumb and forefinger. Proceed to the next step if the animal does not respond to the pinch.
3. Shave a 1-inch by 2-inch section on the back of the mouse with electric clippers and clear the area of fur clippings using a clean wipe or gauze. Avoid using depilatory lotion because it may cause excess inflammation.
4. Clean the back of the mouse first with 10% povidone-iodine-soaked gauze and then with 70% ethanol-soaked gauze. Clean the area in a spiral pattern, moving outward from the center of the surgical area. Wait approximately 1 minute for the surgical area to dry prior to surgery.
5. Hold the shaved back of the mouse loosely between two fingers and firmly press a sterile 6 mm punch biopsy at the center of the prepared surgical area. Do not pull the skin taut.
   1. Twist the punch biopsy to create a circular outline on the skin that fully cuts through the skin in at least one section of the outline. Be careful not to cut into the underlying fascia or tissue.
   2. Use sterile scissors and forceps to cut through the epidermis and dermis following the circular pattern imprinted by the punch biopsy.