This work was supported by La Direction G&#233;n&#233;rale de L&#39;Armement, l&#8217;Agence de l&#8217;Innovation de D&#233;fense and &#201;cole Polytechnique. We thank our colleague Mr Yann Plantier from &#201;cole Polytechnique who provided insight and expertise that greatly assisted the production of the video file. The authors thank Mr Benoit Peuteman and Ms Charlotte Auriau from INSERM Lavoisier (SEIVIL) US 33, H&#244;pital Paul Brousse, Villejuif for their animal well-being and care expertise provided during the course of this project.

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
<ol>
	<li>Prepare a burning device for the procedure and set it to 80 &#176;C using the temperature controller (Figure 1A). Verify the temperature of the brass template (Figure 1B,C) using the infrared thermal imaging camera.</li>
	<li>Ensure that the digital manometer is operating correctly.</li>
	<li>Cover the stage with a surgical drape and adjust the height of the table (Figure 1A).</li>
</ol>
2. Pre-operative and intra-operative animal care
<ol>
	<li>Acquire BALB/c and SKH1-Hrhr mice, 6-8 weeks of age.</li>
	<li>Add paracetamol suspension at 3 mg/mL to drinking water and supply 12 hours before and up to 72 hours after the procedure.</li>
	<li>Using a 1 mL syringe and a 26 G needle, administer buprenorphine subcutaneously at 0.05 &#181;g/g 30 min before the procedure and every 6 hours for the first 72 hours after the procedure.</li>
	<li>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 &#181;g/g subcutaneously 15 min before the induction of the burn wound.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Apply lubrication gel on the eyes of the mouse to prevent dehydration of the cornea.</li>
	<li>Use the toe pinch withdrawal reflex to assess the depth of anesthesia.</li>
	<li>Using a 1 mL syringe and a 26 G needle administer 200 &#181;L of Lactated Ringer&#8217;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.</li>
</ol>
3. Full thickness burn wound induction
<ol>
	<li>Shave the anaesthetized mouse with the hair clippers.</li>
	<li>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.</li>
	<li>Place the mouse on the stage covered with a surgical drape and move the stage upward closer to the preheated brass template.</li>
	<li>Apply the circular brass template on the back of the mouse (80 &#176;C for 20 s) using constant pressure of 0.15 N (Figure 2).</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Harvesting of the donor graft
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Once prepared, keep the skin grafts in 0.9% saline solution at 4 &#176;C for up to 2 hours.</li>
</ol>
5. Surgical excision and skin grafting
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Apply inert paraffin gauze dressing and adhesive secondary dressing over the grafted wound.</li>
	<li>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.</li>
	<li>Provide toys and enrich the environment.</li>
</ol>
6. Digital imaging and post-mortem wound collection
<ol>
	<li>Photograph wounds with a digital camera by placing a ruler next to the wound (Figure 3).</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
</ol>
7. Skin histology, immunohistochemistry and collagen visualization
<ol>
	<li>Embed samples of skin into paraffin, cut to 4 &#181;m sections and place onto positively charged slides.</li>
	<li>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.</li>
	<li>Use histological sections (4 &#181;m thickness) prepared from formalin-fixed and paraffin-embedded tissue and subject them to immunohistochemistry.</li>
	<li>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.</li>
	<li>React sections with either: (i) HRP,3,3&#39;-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.</li>
	<li>To enable histological assessment of collagen deposition, perform trichrome staining on histological sections using a commercial kit.</li>
	<li>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).</li>
	<li>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.</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+outcomes+of+reconstruction+or+amputation+after+leg-threatening+injuries.">An analysis of outcomes of reconstruction or amputation after leg-threatening injuries.</a> New England Journal of Medicine. 347 (24), 1924-1931 (2002).</li><li>Braza, M. E., Fahrenkopf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> StatPearls. , (2019).</li><li>Duchesne, C., Banzet, S., Lataillade, J. 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The authors declare that they have no competing financial interests.

According to the thickness classification of burn injury23, full thickness burns are characterized by evident involvement of the whole thickness of the skin and certain portion of the subcutaneous tissue. This type of wound can heal only by contraction or with skin grafting2. An inherent limitation of the method described in this article is that only full thickness grafts, as opposed to the split thickness grafts, which are often used in the clinical setting, were harvested from the tail of a mouse. This was due to the technical difficulty, as the mouse skin is too thin to obtain split thickness grafts. It must be pointed out that full thickness grafts require a well vascularized wound bed, whereas split thickness skin grafts are able to survive at donor sites with less vascularity24. Previous studies showed that a burn wound induced on the back of the mouse was associated with a robust formation of new vasculature5. This suggests that a well vascularized area, such as the dorsum of the mouse, could be considered as the anatomical landmark for the induction of burn wounds.
Burn wound depth is an important factor to consider. The depth of the burn wound must be consistent between individual mice. The reproducibility of the burn wound depth depends on the temperature of the brass template, pressure and duration of the heat exposure. The burn wound depth must be verified histologically. It is important to keep in mind that excessive pressure or prolonged exposure of the skin to the preheated brass template may injure the underlying tissue. The tissue surrounding the vertebral column, including the components of the central and peripheral nervous system, are sensitive to heat, and if damaged may result in hind leg paralysis.
Although no postoperative mortality was directly associated with the surgical procedure, a small number of hairless SKH1-Hrhr mice, which are especially sensitive to cold, developed hypothermia and failed to recover after the general anaesthesia. Therefore, supplementary heat must be provided during all aesthetic events and constant surveillance is required while the mouse is anaesthetized.
The method described in this study was not associated with the surgical site infection. However, aseptic technique must be used to prevent the transfer of microorganisms into the surgical wound during the perioperative period. Inoculation of the wound with bioluminescent or fluorescent microorganisms may be incorporated into the procedure. This technique may be useful in studying infectious organisms and their pathogenesis25. For example, exogenous addition or injection of bioluminescent bacteria, may permit the monitoring of the microbial burden using the in vivo whole animal imaging25. Given that mouse hair is known to interfere with the in vivo whole animal fluorescence and bioluminescence imaging, hairless SKH1-Hrhr mice are ideal hosts for the studies involving fluorescent or bioluminescent reporters.
Wound tissue samples may be collected at different time points and processed for histological and immunohistochemical analysis. Protein and RNA may be isolated from the skin biopsy and molecular biology techniques may be used to assess the expression of key molecules involved in wound healing.
In the present study, we described an experimental model of burn wound healing and allogeneic skin engraftment. This procedure can be modified and serve as a model for preclinical studies.

Surgical debridement and skin grafting are common clinical practices used in the management of chronic wounds1, burn wounds2, and acute wounds such as traumatic wounds3. Skin grafting refers to the surgical procedure, which involves the removal of healthy skin from one part of the body and transferring it to another. Donor grafts replace the lost tissue and provide a structural scaffold for cellular migration and growth. Following integration into the recipient site, skin grafts replace the lost skin barrier by providing protection from microbial invasion, harmful effects of the external environment and excessive loss of moisture4. Successful skin graft integration depends on several factors. These include adequate immune responses in the presence of microbial infections and timely resolution of inflammation, robust angiogenesis at the wound site and establishment of vascular anastomoses between the recipient bed and the donor graft5. As the graft begins to degrade, resident dermal cells must be replaced by cells capable of producing new extracellular matrix. At the same time, the epidermal keratinocytes must crawl over the newly produced matrix to form the neo-epidermis and re-epithelialize the wound. It is, therefore, evident that efficient migration of cells from the recipient bed into the donor graft is another determining factor that influences successful graft incorporation. Given the vast number of factors involved in wound healing6, which may be impossible to control in the human trials due to ethical limitations, models of pre-clinical experimental skin grafting are necessary. Development of pre-clinical models of burn wound healing and associated skin grafting will be important for understanding of complex mechanisms involved in cutaneous tissue repair and essential for the testing of new therapeutic agents. The in vitro models of wound healing are unable to accurately mimic the complexity of the cutaneous tissue. The in vivo animal models are an indispensable investigative tool in understanding the mechanisms involved in tissue repair.
Several methods of skin grafting technique were developed in rodents to mimic surgical excision and burn wound reconstruction7,8,9. However, most of the previously described procedures failed to induce a thermal burn injury prior to skin grafting. Instead of the burn wound, a full thickness excisional wound was induced, which was then reconstructed with a full thickness skin allograft7. Various anatomical landmarks such as the ear, tail and back have been used for harvesting of the donor skin in rodents7,8. Different graft fixation and stabilization techniques were reported, including a &#8220;no suture technique&#8221;9, sutures7 and surgical glue10,11,12.
The purpose of this study was to develop a murine model of a full thickness burn wound that would recapitulate the current gold standard approach in burn treatment, which involves nonviable tissue excision and skin grafting. A thermal burn was induced on the dorsum surface of a mouse using a preheated brass template. Burn eschar was excised and replaced with a full thickness graft harvested from the tail of a donor mouse. There are three key advantages to this experimental model. First, more than one burn wound may be induced on the back of the recipient mouse, and four donor skin grafts may be harvested from a single tail of the donor mouse. This means that several experimental and control treatments may potentially be compared using the same recipient and donor animals. Depending on the desired route of administration, the control treatment may include local or systemic administration of the vehicle or placebo control (e.g., topical application of ointment, subcutaneous, intraperitoneal or intravenous injection of solution). Second, timing of the treatment and the endpoint of the experiment may be controlled. Third, this model depends on the reconstruction of wounds using full thickness grafts harvested from the tail, which are known to have a higher probability for successful incorporation into the donor site compared to the skin harvested from the back13. This may be due to the lower number of epidermal Langerhans cells, which play a key role in cutaneous immunobiology, and are associated with the skin graft rejection14.
The proposed model of wound healing and graft integration may well be applied to transgenic and knockout mice. The use of genetically modified mice will assist in elucidating the roles that certain genes may play during wound repair. Exogenous application of topical wound preparations or subcutaneous administration of therapeutic antibodies at the site of the injury may also be considered.
Due to technical difficulties, split thickness skin grafts consisting of the epidermis and part of the dermis are difficult to obtain in mice. Full thickness skin grafts consisting of the epidermis and full thickness dermis are known to require a well-vascularized wound bed for successful integration. The inability to harvest split thickness skin grafts in mice may be regarded as a limitation of this model. The fixation of the skin graft to the recipient wound bed was achieved via the application of the surgical adhesive glue, which is associated with less trauma and rapid degradation compared to other means of tissue fixation15. Previous studies have shown that suturing is associated with stronger tissue fixation than the surgical glue at 24 h after the surgical procedure15, which may be considered as a disadvantage of the procedure. However, at later timepoints, the biomechanical strength of wounds treated with a surgical adhesive becomes comparable to sutures15 and better than staple fixation16. Following tissue fixation with the surgical glue, wounds must be covered with a wound dressing. Although wounds on the dorsal surface of the mouse are difficult for the animal to reach, the wound dressing, on the other hand, is easy for the animal to manipulate and remove. Frequent wound dressing changes may be warranted.
Anesthesia-induced hypothermia in small rodents is a well-documented phenomenon17. Hypothermia is a side effect of this procedure, which causes complications, and potentially compromises both animal health and data quality. Therefore, this method warrants the implementation of temperature management strategies, especially if hairless SKH1-Hrhr are used.
The most significant limitation of using mice to mimic human wound closure is the difference between the skin anatomy and physiology. Mouse wounds heal mostly via contraction, whereas human wounds heal through granulation tissue formation and re-epithelialization18. To account for this discrepancy, the current model may be modified and used in combination with a splinting ring tightly adhered around the wound to prevent skin contraction19. Given some advantages and disadvantages of this in vivo protocol, this model could serve as a tool to study certain processes involved in wound healing that are impossible to study in vitro.

<table>
	<tbody>
		<tr>
			<td>1 ml syringue</td>
			<td>Terumo</td>
			<td>SS + 01T1</td>
			<td></td>
		</tr>
		<tr>
			<td>26 G needle</td>
			<td>Terumo Agani</td>
			<td>NN-2613R</td>
			<td>1/2&#39;&#39; - 0,45 X 12mm</td>
		</tr>
		<tr>
			<td>96X21 mm Petri Dish</td>
			<td>Dutscher</td>
			<td>193199</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal Weighing scale</td>
			<td>Kern</td>
			<td>EMB 5.2K5</td>
			<td></td>
		</tr>
		<tr>
			<td>BALB/c mouse</td>
			<td>Janvier labs</td>
			<td>BALB/cAnNRj</td>
			<td>6-weeks old</td>
		</tr>
		<tr>
			<td>Biopsy foam pads 30.2X25.4X2mm</td>
			<td>Simport</td>
			<td>M476-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Bond polymer Refine Red</td>
			<td>Leica Biosystems</td>
			<td>DS9390</td>
			<td></td>
		</tr>
		<tr>
			<td>Brass block</td>
			<td>BVG</td>
			<td>custom-designed</td>
			<td>Circular 10 mm in diameter</td>
		</tr>
		<tr>
			<td>Buprenorphine (BUPRECARE)</td>
			<td>Axience</td>
			<td>FR/V/6328396 3/2011</td>
			<td>administered subcutaneously at a dose of 0.05 &#956;g/ g</td>
		</tr>
		<tr>
			<td>Burning apparatus Kausistar 400</td>
			<td>Tra&#231;aMatrix</td>
			<td>34010</td>
			<td></td>
		</tr>
		<tr>
			<td>CaseViewer</td>
			<td>3DHISTECH Ltd.</td>
			<td></td>
			<td>3Dhistech, Budapest, Hungary</td>
		</tr>
		<tr>
			<td>Collagen I antibody</td>
			<td>Abcam</td>
			<td>ab34710</td>
			<td>Recommanded concentration 1:50; 1:200</td>
		</tr>
		<tr>
			<td>D-(+)- glucose (Dextrose)</td>
			<td>Sigma Aldrich</td>
			<td>G-8769-100 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>DAB</td>
			<td>Leica Biosystems</td>
			<td>AR9432</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>NIKON</td>
			<td>D3400</td>
			<td>objective: SIGMA 18-250mm F3.5-6.3 DC MACRO C45</td>
		</tr>
		<tr>
			<td>Depilating cream</td>
			<td>Veet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable scalpels</td>
			<td>Swann Morton</td>
			<td>6601</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>PAN biotech</td>
			<td>P04-36300</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>VWR chemicals</td>
			<td>20821.310</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin antibody</td>
			<td>Abcam</td>
			<td>ab23750</td>
			<td>Recommanded dilution 1:1000</td>
		</tr>
		<tr>
			<td>Filter 0.22um</td>
			<td>Sartorius</td>
			<td>16532</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>F.S.T.</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont</td>
			<td>F.S.T.</td>
			<td>11295-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair clippers</td>
			<td>AESCULAP</td>
			<td>B00VAQ4KUY (ISIS)</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Petelevage</td>
			<td>120070</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorane</td>
			<td>Piramal healthcare</td>
			<td>FR/V/03248850/2011</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Imalgene</td>
			<td>FR/V/0167433 4/1992</td>
			<td>surgical anesthetic, administered intraperitoneally at a dose of 100mg/kg</td>
		</tr>
		<tr>
			<td>Lactated Ringers solution</td>
			<td>Flee-Flex</td>
			<td>1506443</td>
			<td></td>
		</tr>
		<tr>
			<td>Lamina multilabel slide scanner</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LAS software</td>
			<td>Leica</td>
			<td></td>
			<td>version 2.7.3</td>
		</tr>
		<tr>
			<td>Leica Bond III</td>
			<td>Leica Biosystem</td>
			<td>1757</td>
			<td></td>
		</tr>
		<tr>
			<td>Leukosilk dressing</td>
			<td>BSN medical</td>
			<td>72669-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td>Aguettant</td>
			<td>N01BB02</td>
			<td>local analgesic, administered subcutaneously at a dose of 0.05 &#956;g/ g</td>
		</tr>
		<tr>
			<td>Manometer</td>
			<td>Kern</td>
			<td>HDB-5K5</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson Trichrome Staining kit</td>
			<td>Sigma-Aldrich</td>
			<td>HT15-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromesh Biopsy cassettes</td>
			<td>Simport</td>
			<td>M507</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiphoton inverted stand Leica SP5 microscope</td>
			<td>Leica microsystems</td>
			<td>DM500</td>
			<td>Scanner 8000Hz NDD PMT detectors</td>
		</tr>
		<tr>
			<td>Non adhering dressing Adaptic</td>
			<td>Systagenix</td>
			<td>A6222</td>
			<td>12.7cm X 22.9 cm</td>
		</tr>
		<tr>
			<td>Ocrygel</td>
			<td>Tvm France</td>
			<td>###</td>
			<td></td>
		</tr>
		<tr>
			<td>Paracetamol 300mg</td>
			<td>Dolliprane</td>
			<td>Liquiz</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldheyde 4%</td>
			<td>VWR chemicals</td>
			<td>1169945</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>MEDA pharma</td>
			<td>D08AG02</td>
			<td>diluted to 1:2</td>
		</tr>
		<tr>
			<td>SKH1-Hrhr mouse</td>
			<td>Charles river</td>
			<td>686SKH1-HR</td>
			<td>6-weeks old</td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>Thermoscientific</td>
			<td>AGAA000080</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical adhesive</td>
			<td>BSN medical</td>
			<td>9927</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Gauze</td>
			<td>Hartmann</td>
			<td>418545/9</td>
			<td>10 X 10 cm</td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>Versylene Fresenius</td>
			<td>B230521</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical drape</td>
			<td>Hartmann</td>
			<td>2775161</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti:Sapphire ChameleonUltra</td>
			<td>Coherent</td>
			<td>DS 16-02-16 F</td>
			<td>690-1040 nm</td>
		</tr>
		<tr>
			<td>Thermal imaging Camera</td>
			<td>Testo</td>
			<td>Testo 868</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine (Rompum 2%)</td>
			<td>Bayer</td>
			<td>FR/V/ 8146715 2/1980</td>
			<td>surgical anesthetic, administered intraperitoneally at a dose of 10 mg/kg</td>
		</tr>
	</tbody>
</table>

a murine model of a burn wound reconstructed with an allogeneic skin graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The results demonstrate that the protocol developed is a straightforward method, which permits the induction of a full thickness burn wound in mice. Burns are induced using a preheated brass template (Figure 1A-C). The burned area appears as a circular wound with a white eschar and a hyperemic zone. The size of the burn wound is slightly larger at 24 hours after the burn injury as a result of the well-described phenomenon known as the burn injury progression, which is possibly due to acute inflammation22. After excision, burn wounds are reconstructed using allogeneic skin graft (Figure 3). On day 7 after the burn injury, wounds become vascularized5, which is an indication of successful engraftment. Epidermal keratinocytes migrate from the adjacent recipient skin in the effort to close the wound and bridge the gap between the two edges of the wounds. Microscopic analysis of H and E stained section of wounds revealed that the length of the neo-epidermis becomes significantly longer on day 7 after burn injury compared to day 3 after burn injury (Figure 4B). Prior to performing a large study, it is highly recommended that researchers complete a pilot study, which enables the exploration of a novel intervention, assessment of feasibility, identification of modifications to the method to ensure reproducibility. Statistically significant effects are difficult to detect in smaller samples, whereas increasing the sample size is one way to boost the statistical power of a test. For example, to detect a statistically significant difference (p &#60; 0.05) in the rate of wound re-epithelialization (Figure 4) between groups, the sample size should be between six and eight mice per group. All experiments should be repeated at least twice. As matrix producing cells, such as fibroblasts, migrate from the recipient tissue into the graft, key components of the extracellular matrix, including collagen I and fibronectin become highly expressed in the newly formed matrix (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61339/61339fig01.jpg" /> 
Figure 1: Burning device setup.&#160;(A) Placement and set-up of the burning device. The burning device is connected to the temperature controller and is attached to the digital monometer to enable the monitoring of pressure. The burning device is suspended above an adjustable stage &#8211; flat surface onto which mice are placed for the induction of burn. (B-C) A close up image of the brass template used to induce wound burns. (C) The diameter of the brass template is 1 cm. <a href="https://www.jove.com/files/ftp_upload/61339/61339fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61339/61339fig02.jpg" /> 
Figure 2: Schematic illustration of the different steps required to reproduce the experimental model described in this article.&#160;There are three main steps to the procedure: (i) induction of the burn wound using a preheated brass template; (ii) surgical excision of the non-viable necrotic tissue at 24 hours after the burn injury; (iii) surgical wound reconstruction using a full thickness allogeneic skin graft harvested from a donor mouse. <a href="https://www.jove.com/files/ftp_upload/61339/61339fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61339/61339fig03.jpg" /> 
Figure 3: The macroscopic view of reconstructed mouse burn wounds.&#160;Representative digital images of burn wounds reconstructed with allogeneic skin grafts on days 0, 1, 3 and 7 after burn injury. The ruler on images is in millimeters. <a href="https://www.jove.com/files/ftp_upload/61339/61339fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61339/61339fig04.jpg" /> 
Figure 4: Microscopic appearance of wounds at 3 and 7 days after burn injury.&#160;H&#38;E-stained sections of wounds 3 and 7 days after burn injury. The length of neo-epidermis (dotted line) is significantly increased in (A) day 3 wounds compared to (B) day 7 wounds. In (A) and (B), the scale bar is 100 &#181;m. (C) Graphical representation of the percentage of wound re-epithelialization. This was evaluated by measuring the length of neo-epidermis at day 3 and 7 post-burn injury and expressed as a percentage of the whole wound length. Results represent mean &#177; S.E.M. (n = 6 mice in day 3 group; n = 6 mice in day 7 group, *p &#60; 0.05; Student&#8217;s t-test). <a href="https://www.jove.com/files/ftp_upload/61339/61339fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61339/61339fig05.jpg" /> 
Figure 5: Assessment of the extracellular matrix and collagen I visualization.&#160;Representative images of immunohistochemistry analysis on day 7 mouse wounds stained for (A) collagen and (B) fibronectin. Note intense red staining in elongated spindle-shaped collagen I-positive cells. Note brown staining in fibronectin-positive cells in the dermis of day 7 wounds. Scale bar = 50 &#181;m in all images. In (A) and (B), e denotes epidermis and d denotes dermis. (C) Visualization of collagen fibers and histological assessment of collagen deposition. (D) Representative TPEF/SHG collagen image of day 7 mouse wounds. Simultaneous TPEF/SHG acquisition using circular polarization and SHG signals were selectively processed to obtain a binary distribution of SHG after applying a threshold. TPEF images (green) and SHG images (white) were pseudo-colored and overlaid. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61339/61339fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft at JoVE.com

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

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

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9.8K Views.  Sorbonne Université, CNRS. The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

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

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed (1,2,3) and validated (4,5) for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients. The EpiDerm SIT utilizes the 3D in vitro reconstructed human epidermal (RHE) model EpiDerm. The procedure described in this protocol allows for discrimination between irritants of GHS category 2 and non-irritants (6). The test is performed over the course of a 4 day time period, consisting of pre-incubation, 60 minute exposure, 42 hour post-incubation and MTT viability assay. After tissue receipt and overnight pre-incubation (Day 0), tissues are topically exposed to the test chemicals (Day 1), which can be liquid, semi-solids, solid or wax. Three tissues are used for each test chemical, as well as for the positive control (5% aq. SDS solution), and a negative control (DPBS). Chemical exposure lasts for 60 minutes, 35 min of which the tissues are kept in an incubator at 37&deg;C. The test substances are then removed from the tissue surface by an extensive washing procedure. The tissue inserts are blotted and transferred to fresh medium. After a 24 hr incubation period (Day 2), the medium is exchanged. The medium can be saved for further analysis of cytokines or other endpoints of interest. After the medium exchange, tissues are incubated for an additional 18 hours. At the end of the entire 42h post-incubation (day 3), the tissues are transferred into yellow MTT solution and incubated for 3 hours. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, is extracted for 2 hours using isopropanol. The optical density of the extracted formazan is determined using a spectrophotometer. A chemical is classified as an irritant if the tissue viability relative to the negative control treated tissues is reduced below 50%. This procedure can be used as full replacement of the in vivo rabbit skin irritation test for hazard identification and labeling of chemicals in line with EU regulations (7).

An In Vitro Skin Irritation Test SIT using the EpiDerm Reconstructed Human Epidermal RHE Model

In this video, we demonstrate the EpiDerm Skin Irritation test (EpiDerm SIT) developed and validated for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients.

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed (1,2,3) and validated (4,5) for in vitro skin irritation testing of chemicals, including ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

A Murine Model of Vertical Sleeve Gastrectomy

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. Bariatric surgery is currently the most effective long-term treatment for obesity. In addition to weight loss, bariatric surgery produces additional health benefits such as remission of type 2 diabetes, remission of hypertension, and decreased risk of developing certain types of cancer. The mechanisms beyond weight loss for these benefits remain incompletely defined. Therefore, animal models of bariatric surgery are being developed and validated to identify the mechanisms leading to these benefits, with the goal of improving understanding of gastrointestinal physiology and identifying new therapeutic targets. VSG has become the most commonly performed bariatric procedure in the clinic in the United States because it is highly effective at producing weight loss and metabolic improvement, and is simpler to perform than other bariatric procedures. Therefore, we have developed and validated a murine model of VSG. This murine VSG model recapitulates many of the effects of VSG seen in humans, including improved glucose and blood pressure regulation. The method is based on isolation of the stomach, ligation of gastric vessels, and removal of 70% of the stomach by transecting along the greater curvature of the stomach. We have successfully applied this surgical protocol to various genetically modified mouse lines to define the mechanistic contributors to the benefits of VSG. Furthermore, this murine VSG model has been combined with other surgical techniques, to achieve deeper mechanistic insight. Therefore, this is a simple and versatile model for studying gastrointestinal physiology and the health benefits of bariatric surgery.

The following describes the performance of vertical sleeve gastrectomy in mice. This is a type of weight-loss surgery that involves removal of approximately 70% of the stomach.

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. ...

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling structure that forms rapidly post-amputation and is required for limb regeneration. However, methods to study its precise function at the molecular level over the last decades have been limited due to a paucity of precise functional techniques and genomic information available in salamander model systems. Excitingly, the recent plethora of sequencing technologies coupled with the release of various salamander genomes and the advent of functional genetic testing methods, including CRISPR, makes it possible to re-visit these foundational experiments at unprecedented molecular resolution. Here, I describe how to perform the classically developed full skin flap (FSF) surgery in adult axolotls in order to inhibit wound epidermis formation immediately following amputation. The wound epidermis normally forms via distal migration of epithelial cells in the skin proximal to the amputation plane to seal off the wound from the outside environment. The surgery entails immediately suturing full thickness skin (which includes both epidermal and dermal layers) over the amputation plane to hinder epithelial cell migration and contact with the underlying damaged mesenchymal tissues. Successful surgeries result in the inhibition of blastema formation and limb regeneration. By combining this surgery method with contemporary downstream molecular and functional analyses, researchers can begin to uncover the molecular underpinnings of wound epidermis function and biology during limb regeneration.

This article describes how to perform a surgical method to inhibit wound epidermis formation during axolotl limb regeneration by immediately suturing full thickness skin over the amputation plane. This method allows researchers to investigate the functional roles of the wound epidermis during the early stages of limb regeneration.

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling ...

Alkali Burn-Induced Corneal Neovascularization in Mice

A Mouse Model for Corneal Neovascularization by Alkali Burn

Corneal neovascularization (CoNV), a pathological form of angiogenesis, involves the growth of blood and lymph vessels into the avascular cornea from the limbus&#160;and adversely&#160;affects transparency and vision. Alkali burn is one of the most common forms of ocular trauma that leads to CoNV. In this protocol, CoNV is experimentally induced using sodium hydroxide solution in a controlled manner to ensure reproducibility. The alkali burn model is useful for understanding the pathology of CoNV and can be extended to study angiogenesis in general because of the avascularity, transparency, and accessibility of the cornea. In this work, CoNV was analyzed by direct examination under a dissecting microscope and by immunostaining flat-mount corneas using anti-CD31 mAb. Lymphangiogenesis was detected on flat-mount corneas by immunostaining using anti-LYVE-1 mAb. Corneal edema was visualized and quantified using optical coherence tomography (OCT). In summary, this model will help to advance existing neovascularization assays and discover new treatment strategies for pathologic ocular and extraocular angiogenesis.

Author Spotlight: Decoding Corneal Neovascularization with Alkali Burn Model for Future Therapeutic Strategies 

This protocol focuses on alkali burn-induced corneal neovascularization in mice. The method generates a reproducible and controllable corneal disease model to study pathological angiogenesis and the associated molecular mechanisms and to test new pharmacological agents to prevent corneal neovascularization.

Corneal neovascularization (CoNV), a pathological form of angiogenesis, involves the growth of blood and lymph vessels into the avascular cornea from the ...