The authors would like to thank Kevin Carlson of the Brown University Flow Cytometry and Sorting Facility for consultation and assistance with flow cytometry experiments. Images in Figure 1B and C were created with BioRender. Kayla Lee and Gregory Serpa are thanked for their photographic assistance. This work was supported by grants from the following: Defense Advanced Research Projects Agency (DARPA) YFAA15 D15AP00100, Dean&#8217;s Areas of Emerging New Science Award (Brown University), National Heart Lung and Blood Institute (NHLBI) 1R01HL126887-01A1, National Institute of Environmental Science (NIES) T32-ES7272 (Training in Environmental Pathology), and the Brown University Research Seed Award.

All animal studies described here were approved by the Brown University Institutional Animal Care and Use Committee and carried out in accordance with the Guide for the Care and Use of Animals of the National Institutes of Health.&#160; NOTE: in the video, the surgical drape has been omitted for demonstration purposes.&#160;
1. Subcutaneous implantation of PVA sponges
<ol>
	<li>Use scissors to cut sheets of PVA sponge into 8 mm x 8 mm x 4 mm pieces. Rehydrate the pieces of PVA sponge by submerging them in sterile 1x PBS in a beaker.</li>
	<li>Autoclave the sponges in 1x PBS to sterilize, and cool completely. Store autoclaved sponges in sterile 1x PBS at 4 &#176;C.</li>
	<li>Prior to performing surgical implantation, aliquot the necessary number of sponges into a sterile culture dish under sterile conditions in a laminar flow hood. Store the extra sponges in sterile 1x PBS at 4 &#176;C for future use. Ensure that the sponges swell to 1 cm x 1 cm x 0.5 cm after rehydration. An image of dehydrated PVA sponges is shown in Figure 1A. 
	NOTE: To maintain the sterility of the surgical site, the PVA sponge implantation procedure requires one individual to handle the mouse and prepare the surgical site, and a second person to perform the implantation.</li>
	<li>Anesthetize an 8&#8211;12 week-old male C57BL/6J mouse by intraperitoneal injection of 80 mg/kg ketamine.&#160;Administer 40 mg/kg of xylazine intraperitoneally for analgesia.&#160; Confirm the depth of anesthesia by the loss of a pedal reflex. Prepare the surgical site by removing the hair along the dorsum with clippers. Using sterile gauze, apply povidone-iodine solution to the shave area 2x, followed by 70% ethanol.</li>
	<li>Place the mouse on sterile surgical drapes. Place a heated pad below the surgical drapes to maintain the mouse&#8217;s core temperature.</li>
	<li>Wear sterile surgical gloves for performing the surgery. Pull the dorsal skin away from the underlying tissue with forceps, and using sterile surgical scissors, make a 2 cm incision along the dorsal midline approximately 2 cm anterior to the base of the tail. While holding the incision open with sterile forceps, use sterile curved, blunt-tipped surgical scissors to form a subcutaneous pocket along the dorsum in one of the positions indicated in Figure 1B.</li>
	<li>Continue to use forceps to lift the skin away from the underlying tissue. Using sterile surgical scissors, gently squeeze one PVA sponge in the culture dish to remove excess PBS. Pick up the PVA sponge by one corner using sterile surgical scissors and, leading with the corner held by the scissors, place the sponge into the subcutaneous pocket formed in step 1.4.</li>
	<li>Repeat this process 5x, placing a total of six sponges into subcutaneous pockets as shown in Figure 1B.</li>
	<li>Pinch the incised dorsal skin together with sterile forceps and close the incision with two stainless steel wound clips.</li>
	<li>Use a new set of sterile surgical instruments for each mouse. Alternatively, use a bead sterilizer to sterilize instruments between mice.</li>
</ol>
2. Isolation of fluids from PVA sponges
<ol>
	<li>To assess the acute wound cytokine milieu, isolate sponges between 1&#8211;14 days postimplantation.</li>
	<li>Prepare a fluid collection tube by nesting the barrel of a 5 mL syringe into a 16 mL culture tube.</li>
	<li>Euthanize a mouse with implanted PVA sponges by CO2 asphyxiation followed by cervical dislocation.</li>
	<li>Remove the surgical staples and open the incision with toothed forceps. Use scissors to extend the incision along the dorsal midline.</li>
	<li>Using forceps, extract one sponge from its subcutaneous pocket and transfer it to the prepared syringe barrel, being careful to minimize pressure to the sponge. Disassociate any connective tissue that remains adhered to the surface of the sponge. Use scissors to dissect the sponge from the surrounding tissue if necessary. Repeat the sponge removal process with the remaining sponges. Place the tube containing the sponges on ice.</li>
	<li>To isolate the wound fluid, centrifuge the culture tube containing the 5 mL syringe with the sponges at 700 x g for 10 min.</li>
	<li>After the centrifugation, discard the syringe and the sponges. The wound fluid will have collected in the 16 mL culture tube. A representative image of fluid collected from a day 7 wound is shown in Figure 1D. Transfer the wound fluid to a clean tube for long-term storage at -80 &#176;C.</li>
</ol>
3. Isolation of cells from PVA sponges
<ol>
	<li>To assess the acute wound healing cellular milieu, collect sponges between 1&#8211;14-days post-sponge implantation. Sponges obtained from a wound 7 days after implantation are shown in Figure 1E.</li>
	<li>Prepare collection medium containing 1x HBSS (+calcium/+magnesium/&#8211;phenol red) supplemented with 1% FBS, 1% HEPES, and 1% penicillin-streptomycin.</li>
	<li>Aliquot 5 mL of HBSS collection medium into a 15 mL conical tube.</li>
	<li>Extract the PVA sponges from euthanized mice as described in 2.2&#8211;2.4. Place the sponges into the conical tube containing 5 mL of HBSS collection medium.</li>
	<li>Transfer the sponges and the HBSS collection medium to an 80 mL blender bag by pouring. Hang the blender bag from the hatch of the paddle blender, positioned so that the paddles strike the sponges and media. Adjust the settings of the paddle blender to run on high for 60 s. Press start.</li>
	<li>Transfer the media from the blender bag back to the 15 mL conical tube with a pipette, leaving the sponges behind in the bag. Squeeze the sponges to thoroughly release the media. Adjust the settings of the paddle blender to run for 30 s on high. Add 5 mL of media to the blender bag and repeat the stomaching process 2x for a total of three sponge washes.</li>
	<li>Centrifuge the conical tube at 250 x g for 5 min. A red pellet of cells and red blood cells will be visible at the bottom of the conical tube after centrifugation.</li>
	<li>Discard the supernatant and perform a red blood cell lysis: add 900 &#956;L of dH2O to the cell pellet and pipette to mix. Neutralize with 100 &#956;L of 10x PBS. Add 4 mL of 1x PBS to the tube to fully neutralize the lysis, then centrifuge at 250 x g for 5 min. 
	NOTE: The lysis step should be brief; leaving the water in contact with the cells for more than 3&#8211;5 s could result in lysis of non-red blood cells.</li>
	<li>After centrifugation, a white cell pellet containing wound cells devoid of red blood cells will be present at the bottom of the conical tube. Discard the supernatant and resuspend the cell pellet in the desired medium for downstream analyses.</li>
</ol>
4. Optional flow cytometry analysis of innate leukocytes isolated from PVA sponge wounds
<ol>
	<li>Resuspend 0.5&#8211;1 x 106 wound cells in 25 &#956;L of a 2x solution of blocking antibody containing 10 &#956;g/mL anti-CD16/CD32 FcR&#947;II/III antibody diluted in 1x PBS + 1% BSA. 
	NOTE: All antibodies should be titrated to determine optimal concentrations for flow cytometry analysis.</li>
	<li>Incubate the cells with blocking antibody for 10 min on ice.</li>
	<li>Make a 2x master mix of antibodies containing 2.5 mg/mL of PerCP-eFluor710-Ly6G, 5 mg/mL of FITC-Ly6C, APC-Fire750-CD45.2, APC-R700-Siglec-F, and 10 mg/mL of eFluor660-F4-80 diluted in 1x PBS + 1% BSA. Directly add 25 &#956;L of the antibody cocktail to the cells incubating in blocking antibody.</li>
	<li>Incubate the cells for 20 min on ice, protected from light.</li>
	<li>Wash the cells 2x with 1x PBS.</li>
	<li>Make a master mix of amine-reactive fixable viability dye-eFluor506 diluted 1:1,000 in 1x PBS. Resuspend the cell pellet in 50 &#956;L of the viability dye solution. 
	NOTE: Serum must be excluded from the fixable viability step, as it will prevent binding of the dye to endogenous proteins. Incubate the cells for 20 min on ice, protected from light.</li>
	<li>Wash the cells 2x with 1x PBS. Resuspend the cell pellet in 50 &#956;L of 1% paraformaldehyde.</li>
	<li>Incubate the cells with 1% paraformaldehyde for 15 min on ice, protected from light.</li>
	<li>Wash the cells 1x with 1x PBS and resuspend the pellet in 1x PBS for flow cytometric analysis.</li>
</ol>
5. Tail skin excision
<ol>
	<li>Anesthetize an 8&#8211;12 week-old male C57BL/6J mouse by intraperitoneal injection of 80 mg/kg of ketamine.&#160;Administer 40 mg/kg of xylazine intraperitoneally for analgesia.&#160;Confirm the depth of anesthesia by the loss of a pedal reflex.</li>
	<li>Prepare the surgical site on the tail of the anesthetized mouse by using sterile gauze to apply povidone-iodine solution 2x, followed by one application of 70% ethanol.</li>
	<li>Define a 10 mm x 3 mm section on the dorsal surface of the tail, 10 mm from the base of the tail (Figure 1C) using a permanent marker to trace from a premade template.</li>
	<li>Place the anesthetized mouse on sterile surgical drapes.</li>
	<li>Wear sterile surgical gloves to perform the surgical procedures. With a sterile scalpel blade, make a full thickness incision along the right, bottom, and left edges of the wound area.</li>
	<li>Using sterile forceps, peel the excised skin away from the tail, and use sterile surgical scissors to cut away the top edge of the wound area.</li>
	<li>Apply pressure with sterile gauze to stop bleeding.</li>
	<li>Apply a spray barrier film to the wound bed.</li>
	<li>Photograph the wounds from a fixed distance at regular time intervals. Analyze photographs by planimetric analysis to determine wound area measurements. Alternatively, use calipers to obtain wound length and width measurements.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MIP-1alpha+as+a+critical+macrophage+chemoattractant+in+murine+wound+repair.">MIP-1alpha as a critical macrophage chemoattractant in murine wound repair.</a> Journal of Clinical Investigation. 101 (8), 1693-1698 (1998).</li><li>Wetzler, C., K&#228;mpfer, H., Stallmeyer, B., Pfeilschifter, J., Frank, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+and+Sustained+Induction+of+Chemokines+during+Impaired+Wound+Healing+in+the+Genetically+Diabetic+Mouse:+Prolonged+Persistence+of+Neutrophils+and+Macrophages+during+the+Late+Phase+of+Repair.">Large and Sustained Induction of Chemokines during Impaired Wound Healing in the Genetically Diabetic Mouse: Prolonged Persistence of Neutrophils and Macrophages during the Late Phase of Repair.</a> Journal of Investigative Dermatology. 115 (2), 245-253 (2000).</li><li>Kim, D. J., Mustoe, T., Clark, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+wound+healing+in+aging+small+mammals:+a+systematic+review.">Cutaneous wound healing in aging small mammals: a systematic review.</a> Wound Repair and Regeneration. 23 (3), 318-339 (2015).</li></ol>

The authors have nothing to disclose.

This article describes two tractable murine wound models that allow for the assessment of the acute wound healing response. The first method involves the surgical implantation of PVA sponges in the dorsal subcutaneous space. This approach offers a distinct advantage over biopsy-based wound models for studying the cellular wound healing response due to the large number of cells and quantity of wound fluids obtained from the isolated sponges. For the successful execution of this procedure, maintaining a sterile surgical fi...

There are many murine model systems available to examine wound healing processes, each possessing specific advantages and limitations1,2. The following methods present two murine wound models, each of which addresses a particular aspect of the wound healing response, and which can be used to identify the cause and effect of perturbations in the response to injury. The process of wound healing occurs in distinct phases. The first phase is inflammatory, characterized by the rapid influx of platelets, neutrophils, and monocytes/macrophages, as well as the production of proinflammatory cytokines and chemokines. Following resolution of inflammation, the environment transitions to a more reparative state with the induction of profibrotic and proangiogenic cytokines and growth factors. Granulation tissue is deposited and neovessels form with the migration of myofibroblasts, fibroblasts, epithelial cells, and endothelial cells. In the final stages, the provisional extracellular matrix is remodeled, and scar formation and wound closure proceeds2,3,4,5,6,7,8.
No single murine model provides a system to study all stages of wound healing2. Here, two surgical wound models are described: one elucidates acute cellular and cytokine wound healing responses, and the other allows for the assessment of wound closure as well as histological analyses. These two methods may be employed in a complementary fashion to assess the effects of a perturbation or comorbidity on different aspects of the wound healing response. The dorsal subcutaneous implantation of polyvinyl alcohol (PVA) sponges is a system that has been used in rodent models for decades to elucidate numerous aspects of cellular and granulation tissue responses9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24. This approach allows for the retrieval of cytokine-rich wound fluids and cellular infiltrates. In this model, 1 cm x 1 cm x 0.5 cm pieces of PVA sponge are placed into subcutaneous pockets through a 2 cm incision made at the posterior dorsal midline. The incision is closed with surgical clips, and the sponges can be retrieved at later time points for cell and fluid isolation. The cellular and cytokine milieu of isolated sponges reflects the normal stages of acute wound healing up to about 14 days postimplantation. At later time points the model is more advantageous for studying granulation tissue formation and the foreign body response1. With this system, it is possible to isolate &#62;106 cells, which offers a distinct advantage for phenotypic and functional assays and RNA isolation, over isolating cells from other biopsy-based methods1,22,23,25,26.
The rate of wound closure is determined using the tail skin excision model. In this model, as initially described by Falanga et al. and reported by others27,28,29,30, a 1 cm x 0.3 cm full thickness section of tail skin is removed near the base of the tail. The wound area is easily visualized and can be measured over time. Alternatively, tail tissue can be isolated for histological analysis. This approach can be used as an alternative to or in conjunction with the well-established dorsal punch biopsy method. The primary distinctions between these two models are the rate of wound closure, the presence or absence of fur, and the skin structure2,31,32. Tail skin wounds offer a longer timeframe in which to assess wound closure, as it takes approximately 21 days for full closure to occur. This is opposed to unsplinted dorsal punch biopsies, which heal much faster (~7&#8211;10 days), primarily by contraction due to the action of the panniculus carnosus. Splinted dorsal punch biopsies heal more slowly and diminish the effects of contractile healing, but rely on the presence of a foreign body to restrict contractile-based mechanisms1,2,27,30,31,33.
The described wound models are informative for understanding normal wound healing processes in the absence of perturbation. While the healing of rodent skin differs in very significant ways from human skin, including loose structure, reliance on contractile healing, and other anatomical differences, the murine system offers certain advantages for mechanistic and screening studies. Foremost among these is the availability of inbred strains and genetic mutants, genetic tractability, and lower cost. Mechanistic insight gained from murine studies can be translated to complex animal models that more closely mimic human skin healing, such as the porcine system2,31.
In addition to examining wound healing responses in the steady state, these models can be combined with comorbid conditions to understand the basis of wound healing defects at the cellular, cytokine, and gross tissue level. It is in this particular setting that the two models can be used in concert to assess the effects of a particular comorbid condition, such as postoperative pneumonia, on both the acute cellular wound healing response and the rate of wound closure30.

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			<td height="21" style="height:21px;width:317px;">10x Phosphate Buffered Saline</td>
			<td style="width:121px;">Fisher Scientific</td>
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			<td height="21" style="height:21px;width:317px;">15 mL centrifuge tubes, Olympus</td>
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			<td style="width:104px;">28-103</td>
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			<td height="42" style="height:42px;width:317px;">1x HBSS (+Calcium, +Magnesium, &#8211;Phenol Red)</td>
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			<td height="21" style="height:21px;width:317px;">5ml Syringe</td>
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			<td height="21" style="height:21px;width:317px;">Anti-mouse CD45.2-APC Fire750</td>
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			<td>Clone 104</td>
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			<td height="42" style="height:42px;width:317px;">Anti-mouse F4/80-eFluor660</td>
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			<td>Clone BM8</td>
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			<td height="21" style="height:21px;width:317px;">Anti-mouse Ly6C-FITC</td>
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			<td style="width:104px;">553104</td>
			<td>Clone AL-21</td>
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			<td height="42" style="height:42px;width:317px;">Anti-mouse Ly6G-PerCP-eFluor710</td>
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			<td style="width:104px;">46-9668-82</td>
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			<td height="21" style="height:21px;width:317px;">Anti-mouse Siglec-F-APC-R700</td>
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			<td>Clone E50-2440</td>
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			<td height="42" style="height:42px;width:317px;">Autoclip Stainless Steel Wound Clip Applier</td>
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			<td style="width:104px;">NC9021392</td>
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			<td height="42" style="height:42px;width:317px;">Autoclip Stainless Steel Wound Clips, 9mm</td>
			<td style="width:121px;">Braintree Scientific</td>
			<td style="width:104px;">NC9334081</td>
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			<td height="21" style="height:21px;width:317px;">Blender Bag, 80mL</td>
			<td style="width:121px;">Fisher Scientific</td>
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			<td height="42" style="height:42px;width:317px;">Fetal Bovine Serum - Standard</td>
			<td style="width:121px;">ThermoFisher Scientific</td>
			<td style="width:104px;">10437028</td>
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			<td height="42" style="height:42px;width:317px;">Fixable Viability Dye eFluor506</td>
			<td style="width:121px;">ThermoFisher Scientific</td>
			<td style="width:104px;">65-0866-14</td>
			<td></td>
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			<td height="42" style="height:42px;width:317px;">Hepes Solution, 1M</td>
			<td style="width:121px;">Genesee Scientific</td>
			<td style="width:104px;">25-534</td>
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			<td height="21" style="height:21px;width:317px;">ImageJ Software</td>
			<td style="width:121px;">NIH</td>
			<td style="width:104px;"></td>
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			<td height="42" style="height:42px;width:317px;">Penicillin-Streptomycin (5000 U/mL)</td>
			<td style="width:121px;">ThermoFisher Scientific</td>
			<td style="width:104px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Polyvinyl alcohol sponge - large pore size</td>
			<td style="width:121px;">Ivalon/PVA Unlimited</td>
			<td style="width:104px;"></td>
			<td>www.sponge-pva.com</td>
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			<td height="21" style="height:21px;width:317px;">Povidone-iodine solution, 10%</td>
			<td style="width:121px;">Fisher Scientific</td>
			<td>3955-16</td>
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			<td height="21" style="height:21px;width:317px;">Spray barrier film, Cavilon</td>
			<td style="width:121px;">3M</td>
			<td style="width:104px;">3346E</td>
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			<td height="21" style="height:21px;width:317px;">Stomacher 80 Biomaster, 110V</td>
			<td style="width:121px;">Seward</td>
			<td style="width:104px;">0080/000/AJ</td>
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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.

Systemic inflammatory response following PVA sponge implantation 
The PVA sponge implantation surgery generated a systemic inflammatory response, as demonstrated by the induction of IL-6 in the plasma 1 day after wounding (Figure 2A). Other proinflammatory cytokines including TNF-&#945; and IL-1&#946;, as well as an array of chemokines including CCL2 and CXCL1 were induced systemically in the first 7 days post-PVA sponge implantation, and have been described elsewhere<s...

Watch this Scientific Journal Video about Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models. at JoVE.com

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

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 ...

assessment-acute-wound-healing-using-dorsal-subcutaneous-polyvinyl

Research

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Immunology and Infection

8.2K Views.  Brown University. 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.

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

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

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 ...

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical research on infectious diseases. Described here are three models of humanized immunodeficient mice for studying the dynamics of HIV infection. The first is based on the intrahepatic injection of CD34+ hematopoietic stem cells in newborn mice, which allows for the reconstitution of several blood and lymphoid tissue-confined cells, followed by infection with a reference HIV strain. This model allows monitoring for up to 36 weeks post-infection and is hence called the chronic model. The second and third models are referred to as the acute and reactivation models, in which peripheral blood mononuclear cells are intraperitoneally injected in adult mice. In the acute model, cells from a healthy donor are engrafted through the intraperitoneal route, followed by infection with a reference HIV strain. Finally, in the reactivation model, cells from an HIV-infected donor under antiretroviral therapy are engrafted via the intraperitoneal route. In this case, a drug-free environment in the mouse allows for virus reactivation and an increase in viral load. The protocols provided here describe the conventional experimental approach for humanized, immunodeficient mouse models of HIV infection.

Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation.

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage where they execute crucial antimicrobial functions. Their trafficking to these loci is orchestrated by several inflammatory chemoattractants, including chemokines. At the molecular level, chemoattractant signaling is regulated by the intracellular trafficking of the corresponding receptors. However, it remains unclear how subcellular changes in chemokine receptors affect leukocyte migration dynamics at the cell and tissue level. Here we describe a methodology for live imaging and quantitative analysis of chemokine receptor dynamics in neutrophils during inflammatory responses to tissue damage. These tools have revealed that differential chemokine receptor trafficking in zebrafish neutrophils coordinates neutrophil clustering and dispersal at sites of tissue damage. This has implications for our understanding of how inflammatory responses are self-resolved. The described tools could be used to understand neutrophil migration patterns in a variety of physiological and pathological settings and the methodology could be expanded to other signaling receptors.

Here we describe protocols to perform live imaging and quantitative analysis of chemoattractant receptor dynamics in zebrafish neutrophils

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage ...

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery research less attractive. Antimicrobial drug discovery and subsequent commercialization can be made more lucrative if a fail-fast-and-fail-cheap approach can be implemented within the lead optimization stages where researchers have greater control over drug design and formulation. In this article, the setup of an ex vivo ovine wounded skin model infected with Staphylococcus aureus&#160;is described, which is simple, cost-effective, high throughput, and reproducible. The bacterial physiology in the model mimics that during infection as bacterial proliferation is dependent on the pathogen&#39;s ability to damage the tissue. The establishment of wound infection is verified by an increase in viable bacterial counts compared to the inoculum. This model can be used as a platform to test the efficacy of emerging antimicrobials in the lead optimization stage. It can be contended that the availability of this model will provide researchers developing antimicrobials with a fail-fast-and-fail-cheap model, which will help increase success rates in subsequent animal trials. The model will also facilitate the reduction and refinement of animal use for research and ultimately enable faster and more cost-effective translation of novel antimicrobials for skin and soft tissue infections to the clinic.

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

The protocol describes a step-by-step method to set up an ex vivo ovine wounded skin model infected with Staphylococcus aureus. This high-throughput model better simulates infections in vivo compared with conventional microbiology techniques and presents researchers with a physiologically relevant platform to test the efficacy of emerging antimicrobials.

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery ...