Name: Murine Model of Wound Healing (Video) | JoVE Journal
Uploaded: 2013-05-28T13:00:00.000+00:00
Duration: 5 min 39 s
Description: Learn a murine splinted wound healing model that prevents contraction to promote epithelialization, proliferation, and angiogenesis in mice. This method enables robust preclinical testing of therapeutic agents with each animal serving as its own control.

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5 minutes

The authors would like to acknowledge funding support from the National Health and Medical Research Council (NHMRC) of Australia (Project Grant ID: 632512). Louise Dunn was supported by an NHMRC Early Career Fellowship and Christina Bursill by a National Heart Foundation Career Development Fellowship.

1. Preparation of Splints and Occlusive Dressings
<ol>
 <li>Outline 10 mm circles on 0.5 mm thick silicone sheeting and use scissors or a biopsy punch to create silicone disks.</li>
 <li>Centre a 5 mm biopsy punch in the middle of the 10 mm circle and press firmly to create a hole to form a "donut"-like disc that will be used as a splint.</li>
 <li>Outline 10 mm circles on a transparent occlusive dressing such as Opsite and use scissors to create circular dressings.</li>
</ol>
2. Experimental Animals
<ol>
 <li>Obtain Animal Ethics Committee approval for all experiments that will be performed.</li>
 <li>Use 8-week old (22-26 grams) male C57BL/6J mice from a commercial breeder (e.g. The Jackson Laboratories).</li>
 <li>Keep mice in standard conditions of 21 &deg;C and a 12 hr light-dark cycle with free access to food and water.</li>
 <li>(Optional): Diabetes can be induced in 6 to 7 week old mice by a bolus intraperitoneal injection of 165 mg/kg streptozotocin (in citrate buffer, pH 4.5), with hyperglycemia confirmed by regular blood glucose monitoring (AccuChek glucometer). Diabetic mice may have polyuria and so their bedding may need to be changed more frequently to eliminate wetness and their weights should be monitored closely.</li>
</ol>
3. Anesthesia and Operative Preparation
<ol>
 <li>Induce general anesthesia using 5% isoflurane in 100% oxygen (flow rate 1 L/min) and maintain anesthesia using 1-3% isoflurane.</li>
 <li>Ensure the deep pedal reflexes of the mouse are suppressed and place the mouse in the prone position.</li>
 <li>Prepare the operative region by removing fur with clippers from the base of the neck to 3 cm further down the back and between the two shoulder blades.</li>
 <li>A light application of depilatory cream may be applied for no longer than 2 min. Wet gauze swabs can be used to ensure all cream and remaining fur is removed.</li>
 <li>Wipe the skin with an alcohol swab and two applications of 10% povidone-iodine (Betadine) and drape the mouse.</li>
</ol>
4. Excision and Splinting of Wound
<ol>
 <li>Diagrammatic representation of the murine wound-healing model is presented in Figure 1.</li>
 <li>Use a sterile 4 mm biopsy punch to outline two circular patterns for the wound on either side of the mouse's midline at the level of the shoulders (see Figures 2a-2b).</li>
 <li>Use serrated forceps to lift the skin in the middle of the outline and iris scissors to create a full-thickness wound that extends through the subcutaneous tissue (Figures 2c-2d), including the panniculus carnosus (Figure 2e), and excise the circular piece of tissue.</li>
 <li>Repeat the process for the wound on the other side of the midline (Figure 2f).</li>
 <li>Remove plastic protective coating from each side of the silicone splint.</li>
 <li>Apply cyanoacrylate adhesive (e.g. Super Glue or Krazy Glue) to one side of a silicone splint.</li>
 <li>Centre the splint over the wound (Figure 2g) and anchor the splint with interrupted 6-0 nylon sutures to ensure positioning (Figure 2h).</li>
 <li>Repeat the splinting process on the other wound.</li>
 <li>If required at this time-point, apply the therapeutic compound to be tested (up to 100 &mu;l) to one wound (Figure 2i), and the vehicle control to the other.</li>
 <li>Cover the wound with a transparent occlusive dressing (for example, OpSite) (Figure 2j-2k).</li>
 <li>A ruler is placed below the splints and a photomicrograph taken (Figure 2l).</li>
</ol>
6. Postoperative Management
<ol>
 <li>Carprofen (5 mg/kg) is administered once daily via sub-cutaneous injection for post-operative pain relief.</li>
 <li>After the surgery animals are individually caged and maintained on heat mats until fully recovered.</li>
 <li>Monitor animals twice daily for manifestations of pain and weight loss. We observed no gross behavioral displays of pain or weight loss.</li>
</ol>
7. Wound Measurement and Treatment
<ol>
 <li>The wound can be measured daily.</li>
 <li>Induce general anesthesia using 5% isoflurane gas (flow rate 1 L/min), and then ensure the deep sensory reflexes of the mouse are suppressed using 1-3% isoflurane.</li>
 <li>Gently peel back the occlusive dressing with forceps.</li>
 <li>Use surgical calipers to measure the wound diameter. We take the average of three measurements along the X, Y and Z axis (Figures 2m-2o) as well as a photomicrograph for future reference (Figure 2p).</li>
 <li>Optional: At this time the animal can be assessed for blood perfusion using a laser Doppler imager (Figure 2q).</li>
 <li>Re-application of the therapeutic compound and the vehicle control can be performed at this point.</li>
 <li>A clean transparent occlusive dressing is then re-applied and the animals kept warm until fully recovered.</li>
 <li>Note: if splints are not secured properly the wound will readily contract (Figure 2r).</li>
</ol>
8. Histological Analysis
<ol>
 <li>Euthanize the mice after 10 days with an overdose of anesthesia.</li>
 <li>Using forceps and a scalpel blade remove the sutures and carefully peel away the splint.</li>
 <li>Use iris scissors to create a wide, full excision around and under the wound area and incubate the tissue in 4% paraformaldehyde in phosphate buffered saline (PBS) at 4 &deg;C overnight.</li>
 <li>Transfer tissue to 17% sucrose in PBS solution for a further 24 hr at 4 &deg;C then remove excess solution by dabbing carefully on tissue and place in O.C.T compound and freeze at -80 &deg;C.</li>
 <li>Hematoxylin and eosin staining can be used to visualize the wound structure and epithelial gap. Neovascularisation can be assessed by immunohistochemical analyses to determine the number of capillaries (using Von Willebrand factor).</li>
</ol>

<ol><li>Sen, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+skin+wounds%3A+a+major+and+snowballing+threat+to+public+health+and+the+economy%5BTitle%5D)+AND+%22Wound+Repair%22%5BJournal%5D)">Human skin wounds: a major and snowballing threat to public health and the economy.</a> Wound Repair. 17, 763-771 (2009).</li>
<li>Sen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wound+healing+essentials%3A+let+there+be+oxygen%5BTitle%5D)+AND+%22Wound+Repair+Regen%22%5BJournal%5D)">Wound healing essentials: let there be oxygen.</a> Wound Repair Regen. 17, 1-18 (2009).</li>
<li>Gurtner, G. C., Werner, S., Barrandon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wound+repair+and+regeneration%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Wound repair and regeneration.</a> Nature. 453, 314-321 (2008).</li>
<li>Lindblad, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Considerations+for+selecting+the+correct+animal+model+for+dermal+wound-healing+studies%5BTitle%5D)+AND+%22J.+Biomater.+Sci.+Polym.+Ed%22%5BJournal%5D)">Considerations for selecting the correct animal model for dermal wound-healing studies.</a> J. Biomater. Sci. Polym. Ed. 19, 1087-1096 (2008).</li>
<li>Grose, R., Werner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wound-healing+studies+in+transgenic+and+knockout+mice%5BTitle%5D)+AND+%22Mol.+Biotechnol%22%5BJournal%5D)">Wound-healing studies in transgenic and knockout mice.</a> Mol. Biotechnol. 28, 147-166 (2004).</li>
<li>Reid, R. R., Said, H. K., Mogford, J. E., Mustoe, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+future+of+wound+healing%3A+pursuing+surgical+models+in+transgenic+and+knockout+mice%5BTitle%5D)+AND+%22J.+Am.+Coll.+Surg%22%5BJournal%5D)">The future of wound healing: pursuing surgical models in transgenic and knockout mice.</a> J. Am. Coll. Surg. 199, 578-585 (2004).</li>
<li>Fang, R. C., Mustoe, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+wound+healing%3A+utility+in+transgenic+mice%5BTitle%5D)+AND+%22J.+Biomater.+Sci.+Polym.+Ed%22%5BJournal%5D)">Animal models of wound healing: utility in transgenic mice.</a> J. Biomater. Sci. Polym. Ed. 19, 989-1005 (2008).</li>
<li>Wong, V. W., Sorkin, M., Glotzbach, J. P., Longaker, M. T., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surgical+approaches+to+create+murine+models+of+human+wound+healing%5BTitle%5D)+AND+%22J.+Biomed.+Biotechnol%22%5BJournal%5D)">Surgical approaches to create murine models of human wound healing.</a> J. Biomed. Biotechnol. 2011, 969618(2011).</li>
<li>Galiano, R. D., Michaels, J. t, Dobryansky, M., Levine, J. P., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+and+reproducible+murine+model+of+excisional+wound+healing%5BTitle%5D)+AND+%22Wound+Repair%22%5BJournal%5D)">Quantitative and reproducible murine model of excisional wound healing.</a> Wound Repair. 12, 485-492 (2004).</li>
<li>Galiano, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Topical+vascular+endothelial+growth+factor+accelerates+diabetic+wound+healing+through+increased+angiogenesis+and+by+mobilizing+and+recruiting+bone+marrow-derived+cells%5BTitle%5D)+AND+%22The+American+Journal+of+Pathology%22%5BJournal%5D)">Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells.</a> The American Journal of Pathology. 164, 1935-1947 (2004).</li>
<li>Thangarajah, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+basis+for+impaired+hypoxia-induced+VEGF+expression+in+diabetic+tissues%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 13505-13510 (2009).</li>
<li>Raza, A., Bayles, C., Biebel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Investigation+of+Bacterial+Growth+and+Moisture+Handling+Properties+of+Transparent+Dressings%3A+3M+Tegaderm+Transparent+Dressing%2C+3M+Tegaderm+HP+Transparent+Dressing%2C+and+Opsite+IV3000+Transparent+Dressing%5BTitle%5D)+AND+%22Smith+and+Nephew+Report%22%5BJournal%5D)">Investigation of Bacterial Growth and Moisture Handling Properties of Transparent Dressings: 3M Tegaderm Transparent Dressing, 3M Tegaderm HP Transparent Dressing, and Opsite IV3000 Transparent Dressing.</a> Smith and Nephew Report. , (1998).</li>
<li>Chung, T. Y., Peplow, P. V., Baxter, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Testing+photobiomodulatory+effects+of+laser+irradiation+on+wound+healing%3A+development+of+an+improved+model+for+dressing+wounds+in+mice%5BTitle%5D)+AND+%22Photomed.+Laser+Surg%22%5BJournal%5D)">Testing photobiomodulatory effects of laser irradiation on wound healing: development of an improved model for dressing wounds in mice.</a> Photomed. Laser Surg. 28, 589-596 (2010).</li>
</ol>

The authors have nothing to disclose.

This is an experimental murine model of cutaneous wound healing. A significant feature of this model is the use of silicone splints to prevent wound contraction so that re-epithelialization and new tissue formation may occur, making it similar to the process that occurs in humans. This model is versatile and can be used to assess wound healing in both physiological and pathophysiological (e.g. diabetes mellitus) settings. The model may also be used to assess potential wound healing or angiogenesis therapeutics i...

Impaired wound healing is responsible for significant morbidity and mortality worldwide; this is particularly true for sufferers of diabetes mellitus1,2. In humans, wound healing is a continuum of processes, in which there is significant overlapping3. Immediately following wounding, inflammatory processes are initiated. Inflammatory cells release factors that encourage the processes of cell proliferation, migration and angiogenesis. After re-epithelialization and new tissue formation there is a phase of remodeling that entails both apoptosis and re-organization of matrix proteins such as collagen.
The complexity of wound healing cannot currently be replicated in vitro and this necessitates the use of animal models. To date, wound-healing studies have been limited to large animal models, such as swine, to ensure that the healing processes are equivalent and comparable to humans. However, using large animals for such studies can be difficult to house and are not always practical4. The laboratory mouse represents an economical animal model that can be easily genetically manipulated for mechanistic investigation5-7. However, murine wounds heal differently to human&#x2019;s, primarily due to the process of contraction8. This is in part, due to an extensive subcutaneous striated muscle layer called the panniculus carnosus that is largely absent in humans. In mice, this muscle layer allows the skin to move independently of the deeper muscles and is responsible for the rapid contraction of skin following wounding.
To overcome this limitation, murine wound healing can be adapted to replicate human wound healing by use of a splint (Figure 1)8,9. In this video we demonstrate the splinted murine wound model that eliminates wound contraction and more closely approximates the human processes of re-epithelialization and new tissue formation. In this model two full-thickness excisions that include the panniculus carnosus are created on the dorsum, one on each side of the midline of the mouse. A silicone splint is placed around the wound with the assistance of adhesive and the splint then secured with interrupted sutures. Each mouse acts as its own control, with one wound receiving treatment and the other vehicle control, thereby reducing animal numbers. Following topical applications, a transparent occlusive dressing is applied. The dressing can be removed when required for further topical applications and/or measurement of the wound area10,11. At the completion of experiments, wound closure, morphological architecture and degree of neovascularization can be assessed by immunohistochemistry. This economical and easy to perform model can also be utilized to assess wound healing in the context of diabetes mellitus or other pathophysiologies.

<table><tbody><tr><td>Press-to-seal silicone sheeting 0.5 mm thick</td><td>Invitrogen</td><td>P18178</td><td>Cut into &quot;donuts&quot; with external diameter of 1cm external, 0.5 cm internal diameter</td></tr><tr><td>Biopsy punch 5 mm</td><td>Steifel</td><td>BC-B1-0500</td><td>To outline wound area to be excised</td></tr><tr><td>Vannas scissors 8.5 cm curved</td><td>World Precision Instruments </td><td>501232</td><td>For wound incision and excision</td></tr><tr><td>Dumonte #7b forceps, 11 cm</td><td>World Precision Instruments</td><td>501302</td><td>To grip skin when creating incision and excising skin </td></tr><tr><td>Graefe forceps, serrated 10cm</td><td>World Precision Instruments</td><td>14142</td><td>To help attach silicone splint to skin</td></tr><tr><td>Needle holder, smooth jaws, curved, 12.5 cm</td><td>World Precision Instruments</td><td>14132</td><td></td></tr><tr><td>Malis forceps, smooth, straight, 12 cm</td><td>Codman and Shurtleff, Inc (J&amp;amp;J)</td><td>80-1500</td><td>To suture the silicon rings to the skin</td></tr><tr><td>Ruler, 0.5 mm gradation</td><td>n/a</td><td></td><td></td></tr><tr><td>Calipers 0.25 mm gradation</td><td>Duckworth and Kent</td><td>9-653</td><td>To measure wound area</td></tr><tr><td>Opsite FlexiFix transparent adhesive film. 10 cm x 1 m</td><td>Smith &amp;amp; Nephew</td><td>66030570</td><td></td></tr><tr><td>Rimadyl (Carprofen)</td><td>Pfizer</td><td>462986</td><td></td></tr></tbody></table>

murine model of wound healing

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 wound closure curve is determined by calculating the average diameter of the wound and expressing the results as a percentage, i.e. 100 - (Day 0 diameter/Day X diameter). In this experiment a therapeutic compound (or vehicle control) was applied daily to the wound. The therapeutic compound greatly accelerated wound closure (Figure 3). It is important to note that the splints must be maintained for the duration of the experiment, as removal of splints will lead to rapid wound contraction (<stro...

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. ...

The overall goal of this procedure is to create a wound, which is splinted, to prevent contraction and promote epithelialization sole proliferation and angiogenesis. This is accomplished by first preparing the dorsum and creating two full thickness excisional wounds either side of the midline. Next, a splint is placed over the wound and sutured in place.The wound is then measured and treatments are applied. Lastly, an occlusive dressing is placed over the wound and the mouse is allowed to recover. Ultimately, the cutaneous excisional wound healing model is used to assess wound, epithelialization and associated processes, including proliferation and angiogenesis.The main advantage of this technique over other methods is that wound contraction is prevented. This encourages epithelization proliferation and angiogenesis, which more closely approximates the wound healing process. In humans, This method is particularly useful for assessing the efficacy of therapeutic compounds, and as each mouse serves as its own control, it minimizes the number of animals needed for a study.Begin by preparing the splints and occlusive dressings to make a splint. First outline a 10 millimeter circle on a sheet of silicone, and then use scissors or a biopsy punch to create silicone discs. Next, center, a five millimeter biopsy punch in the middle of the 10 millimeter disc, and press firmly to create a donut like ring that will be used as a splint.Create circular dressings of the same size using transparent occlusive dressing such as ops site. To begin, confirm the proper depth of anesthesia in an anesthetized mouse. After removing all of the hair from the surgical area, clean the skin with repeated applications of Betadine and alcohol.When ready, use a sterile four millimeter biopsy punch to make an outline of two circular patterns on either side of the mouse's midline at shoulder level. Next, use serrated forceps to lift the skin in the middle of the outline and iris scissors to create the wound. Be sure to cut through the subcutaneous tissue, including the panus osis.Excise the tissue from both sides. To reveal two circular wounds. Remove the plastic protective coating from each side of the silicone splint and apply cyanoacrylate adhesive to one side.Center the splint over the wound and suture it in place with interrupted six oh nylon sutures. Repeat the splinting process on the other wound. At this point, a therapeutic compound can be applied to one wound while using the other.For a vehicle control, place a ruler below the splints and take a photo micrograph. Cover the wound with one of the circular pieces of dressing prepared earlier. After administering an analgesic, allow the animal to recover in a heated cage.The animals are monitored twice daily for manifestations of pain and weight loss. To measure the wound, anesthetize the animal as shown earlier and gently peel back the occlusive dressing with forceps. Use a surgical caliper to measure the wound diameter at multiple points to get an average and take a photo micrograph for future reference.At this time, the animal can be assessed for blood perfusion. Using a laser doppler imager, apply a clean, transparent occlusive dressing to the wound and keep the animal warm until fully recovered. It is important to note that if the splints are not secured properly, the wound will readily contract after euthanasia by anesthetic overdose, use forceps and a scalpel blade to remove the sutures and carefully peel away the splint from the wounds.Using iris scissors create a wide, full excision around and under the wound area. Once removed, fix the tissue following standard procedures. Hematin and eosin staining can be used to visualize the wound structure and epithelial gap.In addition, neovascularization can be assessed by immunohistochemical analyses to determine the number of capillaries. A wound closure curve is determined by calculating the average diameter of the wound and expressing the results as a percentage. In this example, the therapeutic compound greatly accelerated wound closure when compared to controls.Once mastered, this technique can be done in 30 minutes if performed properly. While attempting this procedure, it is important to attach the splint securely with equally placed sutures to maintain consistency between experiments.

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Learn a murine splinted wound healing model that prevents contraction to promote epithelialization, proliferation, and angiogenesis in mice. This method enables robust preclinical testing of therapeutic agents with each animal serving as its own control.

Murine Model of Wound Healing

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