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 ...

Research

JoVE Journal

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