We are grateful to all the members of the Dunnwald Lab who have contributed to the optimization of this protocol over the years, and to Gina Schatteman whose persistence in promoting the use of serial sectioning for wound analysis made its creation possible. This work was supported by funding from NIH/NIAMS to Martine Dunnwald (AR067739).

All experiments were completed in accordance and compliance with federal regulations and University of Iowa policy and procedures have been approved by the University of Iowa IACUC.
1. Animals and husbandry
<ol>
	<li>Use adult mice of the desired mouse line at 8-10 weeks of age when the hair follicle stage is in telogen.</li>
	<li>On the day of surgery, separate mice into clean cages and individually house to minimize wound disruption.</li>
</ol>
2. Surgery
NOTE: It is unnecessary to maintain sterile surgical conditions. While care should be taken to maintain sterility between animals, the punch biopsy itself is done on a clean, but nonsterile surface. The surgery duration per animal is between 10 and 15 min.
<ol>
	<li>Anesthetization
	<ol>
		<li>Anesthetize the animal for 1-2 min in an induction chamber with the isoflurane vaporizer set to a 4-5% flow rate and the oxygen flow meter set at 1 liter per minute. See Discussion for alternative anesthesia options.</li>
		<li>Confirm proper anesthetization before beginning the procedure. The depth of anesthesia can be confirmed by a firm toe pinch.</li>
		<li>Transfer the mouse from the induction container to a nose cone and reduce the isoflurane flow rate to 1.5% and the oxygen flow meter to 0.5 L/min.</li>
		<li>Apply ophthalmic ointment to both eyes as the procedure exceeds 5 min.</li>
		<li>Maintain normal body temperature using a thermal pad.</li>
	</ol>
	</li>
	<li>Preparation of the wound site
	<ol>
		<li>Use an electric razor clipper in a caudal rostral motion to remove the fur on the back of the mouse at the shoulder level. Remove hair lower on the back as needed if performing more than two wounds.</li>
		<li>Remove the remaining hair by using a razor blade in a rostral caudal motion held at 20&#730; from the back of the mouse to closely shave the clipped area (Figure 1A).</li>
		<li>Clean the shaved area with a povidone-iodine swab.</li>
		<li>Wipe the skin with a sterile 70% isopropyl alcohol prep pad to reduce potential cutaneous irritation from the iodine swab.</li>
	</ol>
	</li>
	<li>Wounding
	<ol>
		<li>Pinch the skin between the shoulder blades along the dorsal midline and pull the sandwiched skinfold away from the body (Figure 1B).</li>
		<li>Position the mouse on its side with the skinfold on a flat surface draped with a clean paper-based towel or equivalent. Use a sheet of dental wax underneath the towel to protect the underlying surface from damage (Figure 1C).</li>
		<li>Place the biopsy punch of desired size as close to the body as possible and allow the skin to relax. Do not stretch the skin, or the wound size will be larger than the designated punch size (Figure 1D).</li>
		<li>Punch the skin by pressing down, a rocking motion may be used to ensure all layers of the skin on both sides have been penetrated (Figure 1E). Use a new biopsy punch for each animal.</li>
		<li>Remove the punch biopsies from the wounds (Figure 1F). If there are still sites of attachment use sterile scissors and tweezers to free the punch from the surrounding skin. Process the punch biopsy control tissue as required based on downstream plans for wound healing analyses (see Discussion for suggestions).</li>
		<li>Take macroscopic photographs from an equal distance to the wound sites or with a ruler in the frame in order to measure the initial wound area and eliminate outliers from analysis.</li>
		<li>Administer analgesia for a minimum of 24 h in accordance with an approved animal protocol. For example: Buprenorphine SR-LAB, injected as a single dose subcutaneously at 0.5-2 mg/kg for 48 h of pain relief (see Discussion for alternate suggestions and considerations).</li>
		<li>Monitor the mouse as it comes out of anesthesia until it maintains an upright posture and is walking normally around the cage.</li>
	</ol>
	</li>
</ol>
3. Post wound monitoring
<ol>
	<li>Monitor mice daily for experimental endpoints as determined by the investigator and in accordance and compliance with institutional protocols. Examples include: infection, visible weight loss, or a hunched posture.</li>
	<li>Take daily macroscopic photographs in a controlled manner as was done after the initial surgery.</li>
</ol>
4. Harvesting wounds
<ol>
	<li>Euthanize mice at the desired time point post-wounding in accordance with an approved animal protocol.</li>
	<li>Take macroscopic photographs of the wound sites in a controlled manner consistent with previous photograph acquisition (Figure 2A,C).</li>
	<li>Cut a wide rectangle around the wound sites using a scalpel (Figure 2D,E).</li>
	<li>Free the rectangular piece of tissue using scissors and tweezers to peel back and cut the skin away from the underlying tissue (Figure 2F).&#160;&#160;Place in a Petri dish (Figure 2G).</li>
	<li>Harvest the wounds. Trim down to 2 mm of unwounded tissue surrounding all sides of the wound in a rectangular shape&#160;(Figure 2H,I). See Discussion for alternative options to harvest the wound.</li>
	<li>Process the wounds as required for subsequent studies. Reserve at least one wound per mouse for paraffin embedding and histological analysis.</li>
</ol>
5. Wound fixation and embedding
<ol>
	<li>Fix the wound
	<ol>
		<li>Fix the wound tissue in a freshly prepared 4% paraformaldehyde solution8 for 3 h at room temperature then transfer to 4 &#730;C overnight. Electron microscopy (EM) grade paraformaldehyde and solution filtration is not required.</li>
		<li>Wash the wounds twice for 30 min in 1x PBS.</li>
		<li>Replace PBS with 70% ETOH and store at 4 &#730;C until embedding. Process tissues within 24-48 h to avoid antigen loss or within 1-2 weeks if only evaluating histological characteristics.</li>
	</ol>
	</li>
	<li>Process and embed the wound
	<ol>
		<li>Transfer each wound to an embedding cassette. Label embedding cassettes in pencil as the process will remove inks.</li>
		<li>Process the tissue either manually or using an automated processor by dehydrating the tissue with increasing ethanol percentages, clearing with xylene, and then infiltrating the tissue with paraffin wax (Table 1).</li>
		<li>Embed the wound 90&#730; (&#8220;standing&#8221;) from the horizontal surface of the embedding mold (Figure 3A,B).</li>
	</ol>
	</li>
</ol>
6. Day 0 wound area analysis
<ol>
	<li>Download NIH-Image J or NIH-Fiji free software (https://imagej.net/Fiji/Downloads).</li>
	<li>Open a file with a photograph of day 0 wounds.</li>
	<li>Check the box for &#8220;Area&#8221; under Analyze | Set Measurements.</li>
	<li>Select &#8220;Set Scale&#8221; under Analyze. Enter the distance in pixels, the known corresponding distance and the unit of the distance (= unit of length) if macroscopic measurements are part of the study or skip this step if only relative measurements are required.</li>
	<li>Select &#8220;Freehand selections&#8221; on the Fiji toolbar.</li>
	<li>Outline the perimeter of the wound.</li>
	<li>Click Measure under Analyze.</li>
	<li>Create a spreadsheet to keep track of the measurements per animal per wound.</li>
	<li>Copy the measurement of the wound area in the spreadsheet.</li>
	<li>Calculate the mean area and the standard deviation of all wounds for a given experiment.</li>
	<li>Exclude any wounds outside two standard deviations of the mean from histological analysis.</li>
</ol>
7. Serial sectioning
<ol>
	<li>Chill the paraffin-embedded wound blocks at 4 &#730;C overnight.</li>
	<li>Insert the paraffin block on the block holder of the microtome and orient so the blade will cut straight across the block. Orient the block such that the tissue &#8220;stands&#8221; at 90&#730; allowing the simultaneous sectioning of the epidermis and dermis (Figure 3C,D).</li>
	<li>Make 2-4 ribbons of 20-30 paraffin sections of 7 &#181;m each.</li>
	<li>Use a dry paint brush and a dissection teasing needle to transfer each ribbon to a firm yet manipulatable surface such as a firm black plastic sheet.</li>
	<li>Detach the top section of each ribbon with a razor blade and place on a microscope slide.</li>
	<li>Observe the unstained sections under a brightfield microscope to determine which ones contain wounded tissue, which can be identified by absence of hair follicle, changes in the appearance of the connective tissue or the epidermis, and/or the presence of a scab (Figure 4A,B).</li>
	<li>Discard unwounded sections up to 20 sections before the beginning of the wound.</li>
	<li>Section through the wound by repeating steps 7.3 and 7.4 until no wound is detected in unstained sections.</li>
</ol>
8. Mounting of paraffin sections
<ol>
	<li>Separate paraffin sections every 5 sections with a razor blade, starting with the first ribbon (Figure 3E).</li>
	<li>Label microscope slides with both the slide number and all the section numbers.</li>
	<li>Grab the group of 5 sections with a wet paint brush and float them on the surface of the water of a warm water bath (40-45 &#730;C) to flatten them out.</li>
	<li>Pick the group of 5 sections out of the water bath using one of the labelled microscope slides (Figure 3F) and place on a slide warmer set at 37 &#730;C for up to 24 h.</li>
	<li>Store the slides upright in a slide box.</li>
</ol>
9. Histological staining
<ol>
	<li>Transfer every 8th microscope slide (equivalent to every 40th paraffin section) to a staining rack and stain with hematoxylin and eosin.</li>
</ol>
10. Microscopic imaging
<ol>
	<li>Acquire images using a bright field microscope equipped with a 4x objective and digital acquisition capabilities. Record the scale at which the image is taken.</li>
	<li>Image the entire wound of the top section of each stained slide and make sure to include some unwounded tissue on either side. Take multiple overlapping pictures if the wound is larger than the frame of a single picture.</li>
	<li>Save the file including the section number for morphometric analysis. Use the section number followed by a, b, c, etc. for overlapping pictures of the same wound.</li>
</ol>
11. Morphometric analysis
NOTE: When the wound spans multiple pictures, sum the measurements taken from the individual pictures to obtain one value per metric per wound section to record in the spreadsheet.
<ol>
	<li>In Image J, open a digital file of a stained wound picture. Do not use stitched pictures for analysis. Perform measurements on zoomed-in overlapping pictures by finding landmarks to leave off and pick up measurements from picture to picture.</li>
	<li>Set the scale and measurement preferences.
	<ol>
		<li>Select &#8220;Set Scale&#8221; under Analyze. Enter the distance in pixels, the known corresponding distance and the unit of the distance (= unit of length). The scale should appear in the window and should correspond to the scale the image was acquired at.</li>
		<li>Check the box &#8220;Global&#8221; to keep the scale the same for each open image.</li>
		<li>Repeat steps 11.2.1 to 11.2.2 every time Image J is closed and reopened.</li>
		<li>Check the box for &#8220;Area&#8221; under Analyze | Set Measurements.</li>
	</ol>
	</li>
	<li>Measure the wound length
	<ol>
		<li>Select &#8220;Freehand selections&#8221; on the Fiji toolbar.</li>
		<li>Measure starting from the last hair follicle of the uninjured tissue on one side of the wound to the first hair follicle of the uninjured tissue on the other side of the wound (Figure 4A,B).</li>
		<li>Trace along the dermo-epidermal junction to reach these two landmarks. If the epidermis does not cover the entire wound, follow the dermo-epidermal junction on one side of the wound and where the migrating tongue ends continue following the superior aspect of the granulation tissue or the junction between the granulation tissue and the scab until you reach the migrating tongue and then finally the first hair follicle of the uninjured tissue on the other side (Figure 4E,F).</li>
		<li>Under Analyze, click Measure. The length of the measurement will appear in the same units as set in the scale.</li>
		<li>Create a spreadsheet to keep track of the measurements (Supplementary Table 1).</li>
		<li>Copy the wound length into the spreadsheet.</li>
	</ol>
	</li>
	<li>Measure the epidermal length
	<ol>
		<li>If the epidermis covers the entire wound, the epidermal length is the same as the wound length.
		<ol>
			<li>Copy the &#8220;wound length&#8221; measurement into the &#8220;epidermal length&#8221; column in the Excel spreadsheet and skip to step 11.5.</li>
		</ol>
		</li>
		<li>If the epidermis does not cover the entire wound, select the &#8220;Freehand selections&#8221; and measure the distance between each epidermal leading edge following the superior aspect of the granulation tissue or the junction between the granulation tissue and the scab to the first hair follicle (Figure 4C,D).
		<ol>
			<li>Under Analyze, click Measure.</li>
			<li>Subtract this measurement from the wound length and record the number under &#8220;epidermal length&#8221; in the Excel spreadsheet.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Measure the wound area
	<ol>
		<li>Select &#8220;Freehand selections&#8221; on the Fiji toolbar.</li>
		<li>Measure starting from the last hair follicle of the uninjured tissue on one side of the wound to the first hair follicle of the uninjured tissue on the other side of the wound (Figure 4A,B,E,F).</li>
		<li>Trace along the superior aspect of the epidermis (do not include the scab) or the superior aspect of the granulation tissue if the wound is not fully covered by the epidermis.</li>
		<li>Continue to trace vertically along the hair follicle into the granulation tissue once the opposite hair follicle is reached and until adipose tissue or muscle is reached. Follow the inferior border of the granulation tissue to the opposite side of the wound and join the starting point along the hair follicle to close the area (Figure 4E,F).</li>
		<li>Under Analyze, click Measure.</li>
		<li>Copy the wound area into the spreadsheet under &#8220;wound area measured.&#8221;</li>
	</ol>
	</li>
	<li>Measure the epidermal area
	<ol>
		<li>Select &#8220;Freehand selections&#8221; on the Fiji toolbar.</li>
		<li>If the wound is fully epithelialized:
		<ol>
			<li>Trace along the superior aspect of the epidermis until the opposite hair follicle is reached and complete the area by &#8220;returning&#8221; to the starting point following the dermo-epidermal junction between the epidermis and dermis (Figure 4D).</li>
			<li>Under Analyze, click Measure.</li>
			<li>Copy the epidermal area into the Excel spreadsheet under &#8220;epidermal area measured&#8221; and skip to step 11.7.</li>
		</ol>
		</li>
		<li>If the wound is not fully epithelialized:
		<ol>
			<li>Trace along the superior aspect of the epidermis until the leading edge and return to the starting point following the dermo-epidermal junction (Figure 4C).</li>
			<li>Under Analyze, click Measure.</li>
			<li>Repeat step 11.6.3.1 and 11.6.3.2 on the opposite side of the wound.</li>
			<li>Under Analyze, click Measure.</li>
			<li>Sum the two numbers obtained in steps 11.6.3.2 and 11.6.3.4 and enter the result under &#8220;epidermal area measured&#8221; in the spreadsheet.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Repeat steps 11.3 to 11.6 on every 40th section&#160;(every 8th slide).</li>
	<li>Calculate the epidermal area of the entire wound.
	<ol>
		<li>Create a new column in the spreadsheet &#8220;epidermal area calculated&#8221; next to &#8220;epidermal length.&#8221;</li>
		<li>Multiply the number for &#8220;epidermal length&#8221; by 280 for each section except the last one (7 &#181;m thick section x 40 sections).</li>
		<li>Multiply the number for &#8220;epidermal length&#8221; by 7 for the last section (thickness of the section).</li>
		<li>Sum the value of the &#8220;epidermal area calculated&#8221; for each section to obtain the epidermal area of the entire wound.</li>
	</ol>
	</li>
	<li>Calculate the wound area of the entire wound.
	<ol>
		<li>Repeat steps 11.8.1 to 11.8.4 using the wound length measurements.</li>
	</ol>
	</li>
	<li>Calculate the epidermal volume of the entire wound.
	<ol>
		<li>Repeat steps 11.8.1 to 11.8.4 using the &#8220;epidermal area measured&#8221; measurements.</li>
	</ol>
	</li>
	<li>Calculate the wound volume of the entire wound.
	<ol>
		<li>Repeat steps 11.8.1 to 11.8.4 using the &#8220;wound area measured&#8221; measurements.</li>
	</ol>
	</li>
	<li>Calculate the percentage of epidermal volume in the wound.
	<ol>
		<li>Divide the total epidermal volume by the total wound volume and multiply by 100 to obtain the percentage (do not do this ratio for each section).</li>
	</ol>
	</li>
	<li>Calculate the percentage of epidermal area among the wound area.
	<ol>
		<li>Divide the total epidermal area by the total wound area and multiply by 100 to obtain the percentage. If a wound is fully epithelialized, this number should be 100.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

The bilateral excisional wound model is a highly customizable procedure which can be used to study many different aspects of wound healing. Before beginning a wound healing project, investigators should perform a power analysis to determine the number of wounds needed to detect a defect of a particular effect size. Inconsistencies exist within the literature on whether individual mice or wounds should be used as biological replicates, however, a recent study showed that there is no significant correlation between two wou...

Cutaneous wound healing is a complex biological process with sequentially overlapping phases. It requires the coordination of cellular and molecular processes that are temporally and spatially regulated in order to restore the barrier function of the damaged epithelium. In the first phase, inflammation, neutrophils and macrophages migrate into the wound, mobilizing local and systemic defenses1. Following and overlapping the inflammatory phase is the proliferation stage. Fibroblasts begin rapidly proliferating and migrating into the granulation tissue. Keratinocytes away from the leading edge directionally proliferate towards the wound as differentiated keratinocytes in the leading edge migrate to re-epithelialize the wound2. Finally, the remodeling and maturation phase begins, during which fibroblasts in the granulation tissue start to synthesize and deposit collagen. The remodeling and organization of the new matrix can last up to 1 year following injury3. Due to the complexity of overlapping events involving cross-talk between multiple cell types, and despite years of research, many of the cellular and molecular mechanisms underlying wound healing remain poorly understood.
The mouse model is the predominant mammalian model for investigating mechanisms of wound healing due to their ease of use, relatively low cost and genetic manipulability1,4,5. Although different types of wounds have been described in the murine model, the most common is an excisional wound (either bilateral punch or direct punch biopsy), followed by incisional wound models4. The excisional wound model has a distinct advantage over the incisional model as it inherently generates control tissue that has not undergone the healing process. The punch biopsy tissue that is excised as part of the surgical protocol can be processed in the same manner as the wounded tissue and used to establish the homeostatic conditions for a desired criterion. Excised control tissue may also be useful if assessing the effects of a skin pretreatment or confirming successful gene alteration at the time of injury4.
Healing parameters can be assessed by many different techniques, including planimetry or histology. However, planimetry can only evaluate visible characteristics of the wound, and due to the presence of a scab, often does not correlate to measurements of healing that are visualized by histology, thereby making histology the &#8220;gold standard&#8221; of analysis4. Despite histological analysis being the gold standard, it is most often performed on an arbitrary subset of the wound6,7. For instance, cutting the wound in &#8220;half&#8221; prior to embedding and sectioning the wound is currently common practice to reduce the time and resources spent on sectioning materials and data analysis. The method of morphometric analysis described in this protocol was developed to encompass the entire wound tissue, to accurately reflect the morphological characteristics of the wound, and to increase the likelihood of detecting wound healing defects with a small effect size. In this protocol, we detail a surgical method for generating the most commonly studied murine wound, the bilateral full-thickness excisional wound, as well as a detailed and rigorous method for histological analysis such is rarely used in the field.

<table><tbody><tr><td>100% ethanol</td><td></td><td></td><td></td></tr><tr><td>70% ethanol</td><td></td><td></td><td></td></tr><tr><td>80% ethanol</td><td></td><td></td><td></td></tr><tr><td>95% ethanol</td><td></td><td></td><td></td></tr><tr><td>Alcohol Prep</td><td>NOVAPLUS</td><td>V9100</td><td>70% Isopropyl alcohol, sterile</td></tr><tr><td>Ammonium hydroxide</td><td></td><td></td><td></td></tr><tr><td>Biopsy pads</td><td>Cellpath</td><td>22-222-012</td><td></td></tr><tr><td>Black plastic sheet</td><td></td><td></td><td>Something firm yet manipulatable about the size of a sheet of paper</td></tr><tr><td>Brightfield microscope</td><td></td><td></td><td>With digital acquisition capabilities and a 4X objective</td></tr><tr><td>Cotton tipped applicators</td><td></td><td></td><td></td></tr><tr><td>Coverslips</td><td></td><td></td><td>22 x 60 #1</td></tr><tr><td>Dental wax sheets</td><td></td><td></td><td></td></tr><tr><td>Digital camera</td><td></td><td></td><td>Include a ruler for scale, if applicable</td></tr><tr><td>Dissection teasing needle (straight)</td><td></td><td></td><td></td></tr><tr><td>Embedding molds</td><td></td><td></td><td>22 x 22 x 12</td></tr><tr><td>Embedding rings</td><td>Simport Scientific Inc.</td><td>M460</td><td></td></tr><tr><td>Eosin Y</td><td></td><td></td><td></td></tr><tr><td>Glacial acetic acid</td><td></td><td></td><td></td></tr><tr><td>Hair clipper</td><td></td><td></td><td></td></tr><tr><td>Heating pad</td><td>Conair</td><td></td><td>Moist dry Heating Pad</td></tr><tr><td>Hematoxylin</td><td></td><td></td><td></td></tr><tr><td>Microtome</td><td></td><td></td><td></td></tr><tr><td>Microtome blades</td><td></td><td></td><td></td></tr><tr><td>Paint brushes</td><td></td><td></td><td></td></tr><tr><td>Paraffin Type 6</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td></td><td></td><td></td></tr><tr><td>Permount</td><td></td><td></td><td></td></tr><tr><td>Phosphate buffer solution (PBS)</td><td></td><td></td><td></td></tr><tr><td>Povidone-iodine</td><td>Aplicare</td><td>82-255</td><td></td></tr><tr><td>Processing cassette</td><td>Simport Scientific Inc.</td><td>M490-2</td><td></td></tr><tr><td>Razor blades</td><td>ASR</td><td></td><td>.009 Regular Duty</td></tr><tr><td>Scalpel blades #10</td><td></td><td></td><td></td></tr><tr><td>Scalpel handle</td><td></td><td></td><td></td></tr><tr><td>Sharp surgical scissors</td><td></td><td></td><td>sterile for surgery</td></tr><tr><td>Skin biopsy punches</td><td></td><td></td><td>Size as determined by researcher</td></tr><tr><td>Slide boxes</td><td></td><td></td><td></td></tr><tr><td>Slide warmers</td><td></td><td></td><td></td></tr><tr><td>Superfrosted microscope slides</td><td>Fisher Scientific</td><td>22 037 246</td><td></td></tr><tr><td>Temperature control water bath</td><td></td><td></td><td></td></tr><tr><td>Tissue embedding station</td><td></td><td></td><td>Minimum of a paraffin dispenser and a cold plate</td></tr><tr><td>Tissue processor</td><td></td><td></td><td>Minimum of a oven with a vacuum pump</td></tr><tr><td>Triple antibiotic opthalmic ointment</td><td></td><td></td><td></td></tr><tr><td>tweezers, curved tip</td><td></td><td></td><td>sterile for surgery</td></tr><tr><td>tweezers, tapered tip</td><td></td><td></td><td>sterile for surgery</td></tr><tr><td>WypAll X60</td><td>Kimberly-Clark</td><td>34865</td><td></td></tr></tbody></table>

murine excisional wound healing model and histological morphometric wound analysis

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, proliferation and remodeling. Murine wounds have a histologically well-defined and easily recognizable wound bed over which these different phases of the healing process are measurable. Within the field, it is common to use an arbitrarily defined &#8220;middle&#8221; of the wound for histological analyses. However, wounds are a three-dimensional entity and often not histologically symmetrical, supporting the need for a well-defined and robust method of quantification to detect morphometric defects with a small effect size. In this protocol, we describe the procedure for creating bilateral, full-thickness excisional wounds in mice as well as a detailed instruction on how to measure morphometric parameters using an image processing program on select serial sections. The two-dimension measurements of wound length, epidermal length, epidermal area, and wound area are used in combination with the known distance between sections to extrapolate the three-dimension epidermal area covering the wound, overall wound area, epidermal volume and wound volume. Although this detailed histological analysis is more time and resource consuming than conventional analyses, its rigor increases the likelihood of detecting novel phenotypes in an inherently complex wound healing process.

Figure 5 depicts the range in measured and calculated values obtained by performing morphometric analysis on wild-type wounds generated in different mouse strains by multiple surgeons and analyzed by different individuals. Wild-type mice from different strains can display statistical differences as described both in our studies and in the literature9,10. Based on these representative results, we recommend that, within one study, mice...

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Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

This protocol describes how to generate bilateral, full-thickness excisional wounds in mice and how to subsequently monitor, harvest, and prepare the wounds for morphometric analysis. Included is an in-depth description of how to use serial histological sections to define, precisely quantify and detect morphometric defects.

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, ...

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Research

JoVE Journal

Developmental Biology

11.8K Views.  University of Iowa. This protocol describes how to generate bilateral, full-thickness excisional wounds in mice and how to subsequently monitor, harvest, and prepare the wounds for morphometric analysis. Included is an in-depth description of how to use serial histological sections to define, precisely quantify and detect morphometric defects.

Video: Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Medicine

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Immunology and Infection

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

Digital Planimetry for Assessing Wound Closure Kinetics in a Mouse Model

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, thereby reducing the risk of complications and alleviating the economic burden on healthcare systems. Although numerous experimental studies have investigated wound healing, most rely on qualitative observations or quantitative direct measurements. The objective of this study was to standardize an indirect wound measurement method using digital planimetry, incorporating digital scaling and segmentation. This approach addresses the lack of detailed, step-by-step methodologies for accurate wound assessment. A photodocumentation booth was designed and constructed, and computer-assisted digital planimetry tools were employed to minimize variability in measurements of the wound area, perimeter, and the distance from the wound center to its edges. A circular traumatic wound (5 mm in diameter) was created on the dorsal midline at the shoulder blade level of male CD1 mice (n = 4, 10 weeks old, 30-35 g). Wound evolution was photodocumented for 14 days using the custom-designed photo booth, which controlled lighting conditions, focal distance, and subject positioning. Scaling and wound measurements were performed using segmentation in ImageJ software, and statistical analysis was conducted using statistical analysis software. The kinetics of wound closure showed a slight increase in wound size and perimeter between day 0 and day 2, followed by a gradual decrease until complete closure by day 14. The photodocumentation booth and computer-assisted digital planimetry enabled quantitative measurements with minimal variability. In conclusion, these tools provide a reliable and reproducible method for evaluating wound closure kinetics in pre-clinical models.

Wounds represent a global health challenge. This study developed a standardized photo booth utilizing digital planimetry to minimize wound measurement variability. Monitoring wounds in mice over 14 days revealed an initial increase in wound area and perimeter, followed by gradual closure. This methodology may aid in evaluating wound closure kinetics in pre-clinical models.

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, ...

Bioengineering

An Epithelial Abrasion Model for Studying Corneal Wound Healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...

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 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 ...

Biology

Optimized Protocol for Mouse Wound Healing Model with Stem Cell-Derived Exosomes

Mouse Wound Models and Preparation of Single-Cell Suspensions 

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving diverse cell populations and the intricate wound microenvironment, complicates the assessment of treatment effects and their interactions using traditional experimental approaches. Single-cell transcriptome sequencing technology has revolutionized the understanding of cellular functions and mechanisms during wound healing. However, the variability in methodologies for creating mouse wound models introduces confounding factors that can impact experimental results. Furthermore, the process of dissociating mouse skin tissues presents significant technical hurdles, highlighting the critical need for high-quality single-cell suspensions for accurate transcriptome sequencing. Therefore, this study aims to optimize the construction of mouse wound models to accurately assess changes in wound closure while eliminating variables that could influence the subsequent sequencing result. Additionally, the preparation of single-cell suspensions was performed using both enzyme preparation protocols and test kit protocols to optimize cost and effectiveness.

Mouse Wound Models and Preparation of Single-Cell Suspensions

This study presents two crucial experimental techniques: the construction of a murine wound model for assessing wound closure and the preparation of single-cell suspensions from murine skin to preserve cellular viability.

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving ...

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

All animal organs, from the skin to eyes to intestines, are covered with sheets of epithelial cells that allow them to maintain homeostasis while protecting them from infection. Therefore, it is not surprising that the ability to repair epithelial wounds is critical to all metazoans. Epithelial wound healing in vertebrates involves overlapping processes, including inflammatory responses, vascularization, and re-epithelialization. Regulation of these processes involves complex interactions between epithelial cells, neighboring cells, and the extracellular matrix (ECM); the ECM contains structural proteins, regulatory proteins, and active small molecules. This complexity, together with the fact that most animals have opaque tissues and inaccessible ECMs, makes wound healing difficult to study in live animals. Much work on epithelial wound healing is therefore performed in tissue culture systems, with a single epithelial cell-type plated as a monolayer on an artificial matrix. Clytia hemisphaerica (Clytia) provides a unique and exciting complement to these studies, allowing epithelial wound healing to be studied in an intact animal with an authentic ECM. The ectodermal epithelium of Clytia is a single layer of large squamous epithelial cells, allowing high-resolution imaging using differential interfering contrast (DIC) microscopy in living animals. The absence of migratory fibroblasts, vasculature, or inflammatory responses makes it possible to dissect the critical events in re-epithelialization in vivo. The healing of various types of wounds can be analyzed, including single-cell microwounds, small and large epithelial wounds, and wounds that damage the basement membrane. Lamellipodia formation, purse string contraction, cell stretching, and collective cell migration can all be observed in this system. Furthermore, pharmacological agents can be introduced via the ECM to modify cell:ECM interactions and cellular processes in vivo. This work shows methods for creating wounds in live Clytia, capturing movies of healing, and probing healing mechanisms by microinjecting reagents into the ECM.

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

This paper describes a method to create wounds in the epithelium of a live Clytia hemisphaerica medusa and image wound healing at a high resolution in vivo. Additionally, a technique to introduce dyes and drugs to perturb signaling processes in the epithelial cells and extracellular matrix during wound healing is presented.

Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

Summary

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Chapters in this video

Murine Model of Wound Healing

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Digital Planimetry for Assessing Wound Closure Kinetics in a Mouse Model

An Epithelial Abrasion Model for Studying Corneal Wound Healing

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

Mouse Wound Models and Preparation of Single-Cell Suspensions

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica