A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

All experiments were approved by the French Department of Higher Education and Research (Study Number: 122162017111616517670v2 and DAP180012). All mice were single-housed upon arrival in plastic cages and were allowed a 7-day acclimatization period prior to the study. The animal room was maintained at a 12/12 h light/dark cycle (lights on at 07:00). Food and tap water were provided ad libitum. BALB/c and SKH1-Hrhr mice were fed traditional wheat/soy-based diet. Sawdust bedding was provided along with nesting material. 1. Equipment preparation Prepare a burning device for the procedure and set it to 80 °C using the temperature controller (Figure 1A). Verify the temperature of the brass template (Figure 1B,C) using the infrared thermal imaging camera. Ensure that the digital manometer is operating correctly. Cover the stage with a surgical drape and adjust the height of the table (Figure 1A). 2. Pre-operative and intra-operative animal care Acquire BALB/c and SKH1-Hrhr mice, 6-8 weeks of age. Add paracetamol suspension at 3 mg/mL to drinking water and supply 12 hours before and up to 72 hours after the procedure. Using a 1 mL syringe and a 26 G needle, administer buprenorphine subcutaneously at 0.05 µg/g 30 min before the procedure and every 6 hours for the first 72 hours after the procedure. Using a 1 mL syringe and a 26 G needle, administer lidocaine to the dorsum of the mouse and 2-3 mm distal to the area of the burn wound. Inject lidocaine at 0.05 µg/g subcutaneously 15 min before the induction of the burn wound. Anesthetize mice using an intraperitoneal injection of xylazine at 10 mg/kg and ketamine 100 mg/kg. Use a 1 mL syringe and a 26 G needle to administer the injection. Critical step: Place the anaesthetized mouse on a heated pad and keep the mouse warm to prevent hypothermia for the first 30 minutes after the induction of anesthesia and for at least 15 minutes after the recovery from anesthesia. In addition to the heated pad, other modalities including heat lamps, circulating warm liquids or air, and pre-warmed heat reservoirs may be used to regulate the body temperature. Apply lubrication gel on the eyes of the mouse to prevent dehydration of the cornea. Use the toe pinch withdrawal reflex to assess the depth of anesthesia. Using a 1 mL syringe and a 26 G needle administer 200 µL of Lactated Ringer’s Solution supplemented with 5% dextrose. Administer fluid replacement subcutaneously immediately after the induction of anesthesia and 6 hours after the procedure to prevent dehydration. 3. Full thickness burn wound induction Shave the anaesthetized mouse with the hair clippers. Apply depilating cream on the dorsum surface of the mouse for 1 minute. Wipe off the cream using some sterile gauze and clean the area with a piece of damp gauze. Blot the skin with some gauze until dry. Place the mouse on the stage covered with a surgical drape and move the stage upward closer to the preheated brass template. Apply the circular brass template on the back of the mouse (80 °C for 20 s) using constant pressure of 0.15 N (Figure 2). Critical step: Immediately after the burn induction, place the anaesthetized animal on the heated pad to prevent hypothermia and keep the mouse warm during and after the procedure. Once recovered from anesthesia, return the mouse back to the cage. Critical step: Provide a mashed diet on the cage floor for the first 72 hours after the surgical procedure. Mice are sometimes reluctant to reach up to a sipper tube to drink water after a burn wound injury. 4. Harvesting of the donor graft Make a longitudinal incision with a scalpel in the upper part of the tail of the donor mouse and gently remove the skin using surgical forceps. Place the tail skin into a sterile Petri dish filled with 10 mL of sterile 0.9% saline solution. Use a ruler to measure out individual grafts and cut the tail skin into pieces, each measuring 15 mm, using a scalpel. Once prepared, keep the skin grafts in 0.9% saline solution at 4 °C for up to 2 hours. 5. Surgical excision and skin grafting Twenty-four hours after the burn induction, prepare the mouse for anesthesia by inhalation of isoflurane. Place the mouse into the induction chamber and induce anesthesia using 5% isoflurane in 100% oxygen at a flow rate of 4 L/min. To maintain anesthesia during surgery, use 2% isoflurane at 2 L/min. Place a surgical drape on the mouse and cut out a window to expose the surgical field. Using the aseptic technique, swab the wound first with povidone-iodine and then with 70% alcohol. Gently pick up the burned tissue with a pair of surgical tweezers and excise all necrotic and nonviable tissue with sterile surgical scissors and tweezers. Remove the panniculus carnosus layer of the hypodermis to create a stable recipient bed. Place the skin graft on the freshly prepared wound bed. Gently pull the surrounding skin toward the skin graft using surgical tweezers. Apply some surgical adhesive to attach the graft to the wound bed and gently press to align the skin edges. Critical step: The size of the wound bed must be slight larger than the size of the skin graft to ensure successful engraftment. Allow the mouse to recover from anesthesia. Critical step: Place the mouse on a heated pad. Keep the mouse warm during and after the procedure to prevent hypothermia. Apply inert paraffin gauze dressing and adhesive secondary dressing over the grafted wound. Place the mouse into an individual cage. Critical step: Provide some mashed diet on the cage floor for the first 72 hours after the surgical procedure and monitor daily. Provide toys and enrich the environment. 6. Digital imaging and post-mortem wound collection Photograph wounds with a digital camera by placing a ruler next to the wound (Figure 3). At the end point of the experiment, euthanize animals by exposure to carbon dioxide and cervical dislocation. Critical step: Excessive pulling action of the skin during the cervical dislocation may damage the graft. At post-mortem, surgically excise the dorsal burn wounds to the fascia using scissors. Bisect wounds. Fix half in 10% buffered formalin and process for histology and immunohistochemistry. Fast freeze the other half in liquid nitrogen for RNA extraction and protein quantitation and keep at -80 °C. 7. Skin histology, immunohistochemistry and collagen visualization Embed samples of skin into paraffin, cut to 4 µm sections and place onto positively charged slides. Use slides stained with hematoxylin and eosin to evaluate the rate of re-epithelialization (% of the original wound). The area of the wound that is covered with neo-epidermis may be expressed as a percentage of the entire wound (Figure 4). Use a digital microscope application and ImageJ software to perform the microscopic analysis of the sections. Use histological sections (4 µm thickness) prepared from formalin-fixed and paraffin-embedded tissue and subject them to immunohistochemistry. To assess collagen I and fibronectin expression in wounds, apply primary antibodies and incubate for 1 h. Detection may be performed by species-specific horseradish peroxidase (HRP) or alkaline phosphatase (AP)-conjugated secondary antibodies. React sections with either: (i) HRP,3,3'-diaminobenzidine (DAB) or (ii) AP, Bond Polymer Refine Red (Table of Materials), which yields a bright red color (Figure 5). Scan the sections using an instrument and analyze with the digital microscope application and ImageJ. To enable histological assessment of collagen deposition, perform trichrome staining on histological sections using a commercial kit. For collagen fiber visualization, use multiphoton microscopy and second harmonic generation technique (Figure 5). Use a multiphoton microscope for tissue imaging as previously described20. Use a Ti:Sapphire laser with a center wavelength at 810 nm as the laser source for generating second harmonic and two-photon excited fluorescence signals (TPEF). Use a laser beam equipped with a 25x/0.95 W objective to collect and excite second harmonic generation (SHG) and TPEF. Detect signals as described previously21 by NDD PMT detectors. Use software for laser scanning control and image acquisition.