Name: assessment of acute wound healing using the dorsal subcutaneous polyvinyl alcohol sponge implantation and excisional tail skin wound models.
Uploaded: 2020-03-25T12:30:00
Description: 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

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
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	<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 subm...

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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>
			<td style="width:104px;">BP3991</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">15 mL centrifuge tubes, Olympus</td>
			<td style="width:121px;">Genesee</td>
			<td style="width:104px;">28-103</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">1x HBSS (+Calcium, +Magnesium, &#8211;Phenol Red)</td>
			<td style="width:121px;">ThermoFisher Scientific</td>
			<td style="width:104px;">14025076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">5ml Syringe</td>
			<td style="width:121px;">BD</td>
			<td style="width:104px;">309646</td>
			<td></td>
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			<td height="21" style="height:21px;width:317px;">Anti-mouse CD45.2-APC Fire750</td>
			<td style="width:121px;">BioLegend</td>
			<td style="width:104px;">109852</td>
			<td>Clone 104</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Anti-mouse F4/80-eFluor660</td>
			<td style="width:121px;">ThermoFisher Scientific</td>
			<td style="width:104px;">50-4801-82</td>
			<td>Clone BM8</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Anti-mouse Ly6C-FITC</td>
			<td style="width:121px;">BD Biosciences</td>
			<td style="width:104px;">553104</td>
			<td>Clone AL-21</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Anti-mouse Ly6G-PerCP-eFluor710</td>
			<td style="width:121px;">ThermoFisher Scientific</td>
			<td style="width:104px;">46-9668-82</td>
			<td>Clone 1A8-Ly6g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Anti-mouse Siglec-F-APC-R700</td>
			<td style="width:121px;">BD Biosciences</td>
			<td style="width:104px;">565183</td>
			<td>Clone E50-2440</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Autoclip Stainless Steel Wound Clip Applier</td>
			<td style="width:121px;">Braintree Scientific</td>
			<td style="width:104px;">NC9021392</td>
			<td></td>
		</tr>
		<tr height="42">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Blender Bag, 80mL</td>
			<td style="width:121px;">Fisher Scientific</td>
			<td style="width:104px;">14258201</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Culture Tube, 16mL, 17x100</td>
			<td style="width:121px;">Genesee Scientific</td>
			<td style="width:104px;">21-130</td>
			<td></td>
		</tr>
		<tr height="42">
			<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>
			<td></td>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">ImageJ Software</td>
			<td style="width:121px;">NIH</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Povidone-iodine solution, 10%</td>
			<td style="width:121px;">Fisher Scientific</td>
			<td>3955-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

These models can be implemented to assess various aspects of the wound healing response, including cellular and cytokine kinetics and wound closure. The PVA sponge model allows the recovery of millions of wound leukocytes for phenotypic and functional analyzes, while the tail skin excision model allows the easy visualization of slow-healing wounds. The PVA sponge and excisional tail wound models can be used in conjunction with comorbid conditions, such as diabetes or pneumonia, to understand their impact on wound healing.Demonstrating the procedures with Meredith Crane will be Bill Henry, a research assistant from my lab. Before beginning the experiment, use scissors to cut sheets of PVA sponge into eight-by-eight-by-four-millimeter pieces. Rehydrate the sponge pieces in sterile PBS in a beaker for 10 minutes before autoclave sterilizing the sponges in the PBS.When the sponges have cooled, store the sponges submerged in PBS at four degrees Celsius. On the day of the procedure, place six sponges per experimental animal into a sterile culture dish in a sterile laminar flow hood, and confirm a lack of response to toe pinch in an anesthetized, eight-to 12-week-old, male, C57-Black/6 mouse. Have an assistant use clippers to remove the hair along the dorsum and sterile gauze to apply povidone-iodine solution to the shaved area two times, followed by a single 70%ethanol application.The assistant should then transfer the mouse onto sterile surgical drapes placed over a heated pad. Wearing sterile gloves, use forceps to pull the dorsal skin away from the underlying tissue, and use sterile surgical scissors to make a two-centimeter incision along the dorsal midline approximately two centimeters anterior to the base of the tail. 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 as indicated in the figure.Once the scissors have been inserted, open and close the tips two times to form a pocket big enough to hold an inserted sponge. When the pocket has been created, use sterile surgical scissors to gently squeeze one PVA sponge in the culture dish to remove excess PBS. Next, lift the sponge by one corner, and, leading with the corner held by the scissors, place the sponge into the subcutaneous pocket.When all six sponges have been placed as demonstrated, use sterile forceps to pinch the incised dorsal skin together, and close the incision with two stainless steel wound clips. For PVA sponge fluid isolation, one to 14 days after implantation, remove the surgical staples, and open the incision with toothed forceps. Use scissors to extend the incision along the dorsal midline, and use forceps to extract one sponge from its subcutaneous pocket.Place the sponge into the barrel of a five-milliliter syringe nested in a 16-milliliter culture tube on ice, and use scissors to disassociate any connective tissue that remains adherent to the surface of the sponge, as necessary. When all of the sponges have been extracted and transferred as demonstrated, centrifuge the culture tube to collect the wound fluid. To isolate the cells from the PVA sponges, after collecting the sponges as just demonstrated, place them into a 15-milliliter conical tube containing five milliliters of HBSS collection medium.When all of the sponges have been collected, decant the sponge suspension into an 80-milliliter blender bag, and hang the bag from the hatch of the paddle blender so that the paddles will strike the sponges and medium. Set the paddle blender to run on high for 60 seconds, and press start. When the blender has stopped, squeeze the sponges in the bag to completely release the medium, and use a pipette to transfer the medium from the blender bag back into the 15-milliliter tube.Adjust the settings of the paddle blender to run for 30 seconds on high, and add five milliliters of medium to the blender bag. Repeat the stomaching process two more times before centrifuging the tube of collected medium. A red pellet of cells and red blood cells should be visible at the bottom of the conical tube.Mix 900 microliters of distilled water with the cells for three to five seconds before neutralizing the lysis with 100 microliters of 10x PBS and four milliliters of 1x PBS. Collect the cells by centrifugation, and resuspend the white cell pellet in the appropriate medium for downstream analysis. To create a tail skin wound, after confirming a lack of response to toe pinch in the anesthetized, eight-to 12-week-old, male, C57-Black/6 mouse, use sterile gauze to apply povidone-iodine solution two times to the surgical site, followed by one application of 70%ethanol.Using a permanent marker and a premade template, trace a 10-by-three-millimeter section on the dorsal surface of the tail, 10 millimeters from the tail base, and place the mouse on sterile surgical drapes. Wearing sterile surgical gloves, use a sterile scalpel blade to make a full-thickness incision along the right, bottom, and left edges of the wound area. 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.Use sterile gauze to apply pressure to the wound to stop bleeding, and apply a spray barrier film to the wound bed. Then, photograph the wounds from a fixed distance at regular time intervals, and analyze the photographs by planimetric analysis to determine the wound area measurements. PVA sponge implantation surgery generates a systemic inflammatory response, as demonstrated by the induction of IL-6 in the plasma one day after wounding.The number of cells that can be recovered from PVA sponge wounds increases over time, with neutrophils, monocytes, and macrophages comprising the primary cellular infiltrating populations within the sponges. Neutrophils are identified as Ly6G-positive, Siglec-F-negative cells. Siglec-F-positive cells are primarily eosinophils.Gating on the Ly6G-negative, Siglec-F-negative cells allows the identification of F4/80-positive monocytes and macrophages. F4/80-positive cells can be further differentiated by their Ly6C expression to distinguish Ly6C high inflammatory monocytes and Ly6C low monocyte-derived macrophages. The excisional tail skin model provides an alternative to the dorsal skin punch biopsy method to study wound closure in firm skin lacking dense fur.Wound closure can be quantified by measuring the area of the wound bed over time. The tail skin wound can also be observed in cross section by histological analysis via H and E and Masson's trichrome staining. In these images, the lateral margins of the excised skin are indicated by the arrowheads on the dorsal surface of the tail.To understand steady-state wound healing, therapeutic compounds or interventions can also be tested through their injection directly into the PVA sponge or through delivery to the tail wound bed.

Research

JoVE Journal

Immunology and Infection

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