Name: visualizing scar development using scad assay - an ex-situ skin scarring assay
Uploaded: 2022-04-28T13:40:00
Description: Watch this Scientific Journal Video about Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay at JoVE.com

We acknowledge all the co-authors of Jiang et al. 2020 for contributing to the development of SCAD methodology13. We thank Dr. Steffen Dietzel and the Bioimaging core facility at the Ludwig-Maximilans-Universit&#228;t for access to the Multiphoton system. Y.R. was supported by the Else-Kr&#246;ner-Fresenius-Stiftung (2016_A21), the European Research Council Consolidator Grant (ERC-CoG 819933), and the LEO Foundation (LF-OC-21-000835).

The model presented below provides a detailed step-by-step description of the generation of SCAD assay as briefly described in Jiang et al., 202013. SCAD sample preparations were performed after sacrificing the animals as per the international and the Government of Upper Bavaria guidelines. Animals were housed at the animal facility of the Helmholtz Centre Munich. The rooms were maintained with optimal humidity and constant temperature with a 12 h light cycle. Animals were supplied with food and w...

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Several models have already been developed to understand scar formation after injury. While a lot of advances have been rendered in this regard but, actual mechanisms are still not clear. In contrast to the previous technique, the SCAD model incorporates all cell types and cutaneous layers, thereby maintaining the complexity of native skin18,19. This methodology is capable of generating fundamental datasets that are important in understanding molecular mechanisms...

Wound healing is a process of restoration of breached wounds. Tissue injuries in invertebrates result in partial or complete regeneration. In contrast, mammals respond to deep injury by scarring, a process tailored to quickly seal wounds with dense plugs of matrix fibers that minimize the breached area and at the same time permanently deform the injured site1,2,3. Large skin burns or deep open wounds in mammals result in pathological phenotypes such as hypertrophic or keloid scars4,5. These exuberant scars cause a tremendous burden on clinical and global healthcare systems. In the US alone, scar management costs about $10 billion annually6,7. Therefore, the development of relevant methodologies are required to better understand the fundemental processes and mechanisms involved in scar formation.
In recent years, a wide range of studies in mice has revealed heterogeneous fibroblast populations with distinct functional potencies based on their origins in certain skin locations8,9,10. In back skin, Rinkevich et al., 2015, identified that a specific fibroblast population with an early embryonic expression of Engrailed-1 (En1), termed EPF (Engrailed positive fibroblast) contributes to cutaneous scarring upon wounding. Conversely,&#160; another fibroblast lineage with no history of engrailed expression, Engrailed negative fibroblast (ENF), does not contribute to scar formation8. Fate mapping of these En1 lineages using Cre-driven transgenic mouse lines crossed to fluorescence reporter mouse lines such as R26mTmG (En1Cre x R26mTmG) allows visualization of EPF and ENF populations.
Studying fibroblast migration in vivo over several days is limited by ethical and technical constraints. Furthermore, compound, viral and neutralizing antibody library screens to modulate pathways involved in scarring is technically challenging. Previously used in vitro or ex vivo models lack the ability to visualize fibroblast migration and scar formation in genuine skin microenvironments, uniformity in scar development, as well as tissue complexity that emulates in vivo skin environments11,12. To overcome the above limitations, we developed an ex vivo scarring assay termed SCAD (SCar-like tissue in A Dish)13,14. This simple assay can be performed by excising 2 mm full-thickness skin containing the epidermis, the dermis, and the subcutaneous fascia regions and culturing them in serum-supplemented DMSO media for up to 5 days. Scars generated from SCAD reliably replicate transcriptomic and proteomic hallmarks of in vivo scars. In addition, SCADs generated from relevant transgenic mouse lines (e.g., En1 mice) crossed with fluorescent reporter mouse lines allow the visualization of fibroblast migration dynamics and scar development at an unprecedented resolution. Furthermore, this model can be easily adapted for any high throughput applications (e.g., compound library, antibody library, or viral screening)13,14. In this article, we describe an optimized protocol to generate SCADs and subsequent downstream processing applications to study cellular and matrix dynamics in scar development.

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			<td>10% Tween 20, Nonionic Detergent</td>
			<td>Biorad Laboratories</td>
			<td>1610781</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin, Cold ethanol fract</td>
			<td>Sigma</td>
			<td>A4503-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, HEPES, no phenol red-500 mL</td>
			<td>LIFE Technologies</td>
			<td>11039021</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Epredia Cryostar NX70 Cryostat</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Epredia SuperFrost Plus Adhesion slides</td>
			<td>Fisher scientific</td>
			<td>J1800AMNZ</td>
			<td>Adhesion slides</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated, E.U.-approved, South America Origin-500 mL</td>
			<td>LIFE Technologies</td>
			<td>&#160;10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G with DAPI</td>
			<td>Life Technologies</td>
			<td>00 4959 52</td>
			<td>Mounting medium with DAPI</td>
		</tr>
		<tr>
			<td>Forceps curved with fine points with guidepinstainless steel(tweezers)125 mm length</td>
			<td>Fisher Scientific</td>
			<td>12381369</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>Sigma</td>
			<td>G2500-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement-100 mL</td>
			<td>LIFE Technologies</td>
			<td>35050038</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, calcium, magnesium, no phenol red-500 mL</td>
			<td>LIFE Technologies</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibidi Gas incubation system for CO2 and O2</td>
			<td>Ibidi</td>
			<td>11922</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibidi Heating system</td>
			<td>Ibidi</td>
			<td>10915</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8 upright microscope - Multiphoton excitation 680&#8211;1300 nm</td>
			<td>Leica</td>
			<td></td>
			<td>Equipped with a 25x water-dipping objective (HC IRAPO L 25x/1.00&#8201;W) in combination with a tunable laser (Spectra-Physics, InSight DS&#8201;+&#8201;Single)</td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>LIFE Technologies</td>
			<td>11140035</td>
			<td></td>
		</tr>
		<tr>
			<td>NuSieve GTG Agarose ,25 g</td>
			<td>Biozym /Lonza</td>
			<td>859081</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Embedding Matrix</td>
			<td>Carlroth</td>
			<td>6478.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 16% W/V AQ. 10 x10 mL</td>
			<td>VWR International</td>
			<td>43368.9M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-Strep</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Stiefel Biopsy-Punch 2 mm</td>
			<td>Stiefel</td>
			<td>270130</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Sharp/Sharp Dissecting Scissors 11.4 cm</td>
			<td>Fisher Scientific</td>
			<td>15654444</td>
			<td></td>
		</tr>
		<tr>
			<td>Thimerosal Bioxtra, 97%&#8211;101%</td>
			<td>Sigma-Aldrich</td>
			<td>T8784-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axioimager M2 upright microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing scar development using scad assay - an ex-situ skin scarring assay

The mammalian global response to sealing deep tissue wounds is through scar formation and tissue contraction, mediated by specialized fascia fibroblasts. Despite the clinical significance of scar formation and impaired wound healing, our understanding of fascia fibroblast dynamics in wound healing is cursory due to the lack of relevant assays that enable direct visualization of fibroblast choreography and dynamics in complex environments such as in skin wounds. This paper presents a protocol to generate ex- situ skin scars using SCAD or &#34;SCar-like tissue in A Dish&#34; that emulate the complex environment of skin wounds. In this assay, 2 mm full-thickness skin is excised and cultured upside down in media for 5 days, during which scars and skin contractures develop uniformly. This methodology, coupled with fibroblast-lineage specific transgenic mouse models, enables visualization of individual fibroblast lineages across the entire wound repair process. Overall, this protocol aids&#160;researchers in understanding fundamental processes and mechanisms of wound repair,&#160;directly exploring the effects of modulators on wound healing outcomes.

Generation of SCADs can be separated into three essential steps: Harvesting&#160;back skin from P0-P1 mice, generating of full-thickness biopsy punches, and subsequent culture of individual scads up to 5 days in 96-well plates. As a readout, this assay can further be applied to analyze the spatial and temporal aspects of scarring. The spatial analysis utilizes 2D and 3D immune-labeling of tissues to study spatial localization of cellular and matrix components within developing scar tissue. Spatiotemporal studies allow vi...

Watch this Scientific Journal Video about Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay at JoVE.com

Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay

This protocol describes the generation of a skin-fascia explant termed "SCar like tissue in A Dish" or SCAD. This model allows unprecedented visualization of single fibroblasts during scar formation.

Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay

The mammalian global response to sealing deep tissue wounds is through scar formation and tissue contraction, mediated by specialized fascia fibroblasts. ...

Previously used in vitro or ex vivo models lack dermal cell components and complexity of skin tissue. This ex situ SCAD assay overcomes a set limitation and enables studying the development of scars outside of wounded animal. The SCAD assay allows visualization of fibroblast migration and scar formation in skin microenvironments.It enables screening libraries of activators or inhibitors to understand the mechanistic basis of scar formation. High-throughput screening assays of flat libraries using the SCAD model in understanding fundamental process and the molecule course involved in wound repair. The SCAD assay is easy to perform using the skin explants.For consistent scar, it is recommended to select post-natal day zero or day one skin. After sacrificing post-natal day zero or day one newborn pup, use a sterile surgical scalpel to carefully excise 1.5-by-1.5-centimeter full thickness dorsal back skin till the skeletal muscle layer. Peel the skin using sterile curved forceps, ensuring that the superficial fascia is intact with the underlying panniculus carnosus muscle.Wash the excised tissue with 50 to 100 milliliters of cold DMEM F-12 medium to remove contaminating blood. Then, wash the tissue with Hanks balanced salt solution to maintain tissue and cell viability. Place the skin upside down with superficial fascia on top in a 10-centimeter Petri dish containing DMEM F-12 medium.Then, using a disposable two-millimeter biopsy punch, excise full-thickness round skin pieces to generate scabbed tissues, ensuring that superficial fascia is intact with underlying panniculus carnosus muscle until the epidermis. Add 200 microliters of fresh DMEM F-12 complete medium without phenol red into each well of a 96-well plate. Using sterile forceps, transfer and fully submerge individual scabbed tissue upside down to the wells of a 96-well plate.Transfer the plate to a cell culture incubator maintained under standard conditions. On days two and four of culture, remove the medium leaving 10 microliters in the well and add fresh pre-warmed DMEM F-12 complete medium along with the treatment compounds to maintain continued cell and tissue viability conditions. For live imaging of SCADs, prepare 30 milliliters of 2 to 3%low-melting agarose solution in PBS in a glass bottle by heating in a microwave.After boiling, immediately transfer the bottle and cool the liquid agarose solution in a 40-degrees-Celsius water bath. Next, transfer the SCAD tissue with fascia or scar facing upward onto the center of a 35-millimeter dish. Then embed the SCAD at room temperature by slowly pouring 40-degrees-Celsius liquid agarose onto the tissue using a 1000-microliter pipette tip.Agarose polymerizes within two minutes. After polymerization, add two milliliters of pre-warmed DMEM F-12 complete medium. Then using a confocal or a multiphoton microscope equipped with suitable incubation systems explained in the manuscript, acquire time-lapse images of day zero to day one.For tissue harvesting, wash the tissues at relevant time points by replacing the media with sterile PBS. Using sterile forceps, transfer each SCAD to a 1.5-milliliter microcentrifuge tube containing 500 microliters of 2%paraformaldehyde to fix the tissues overnight at four degrees Celsius. The next day, wash the tissues three times with PBS before proceeding to two-or three-dimensional immunofluorescence staining as described in the text manuscript.The representative whole-mount bright-field images of SCADs on days zero and five are shown here. The Masson's trichrome staining of vertical tissue sections reveals signatures of tissue scarring, tissue contraction, and accumulation of extracellular matrix at the scar core at days zero and five. The immunofluorescence staining of SCADs at days zero and five are shown.From early and late stage scar, the ingrained positive fibroblast is shown in green and the ingrained negative fibroblast in red. For three-dimensional imaging, tissues were embedded in agarose solution and topped with PBSGT. The representative images demonstrate exclusive localization of N-cadherin protein at the scar site of a day-five SCAD.The three-dimensional time lapse imaging setup equipped with an incubation chamber displayed early events of progression of the fibroblast swarms over the first 12 hours or early stages of scar development. The graphical representation of single-cell tracked cellular trajectories of individual fibroblasts over scar development is presented here. It's crucial to place the SCAD tissue upside down inside the well plate so the fascia faces upwards.Failure to do this would result in unavoidable variations in migration patterns. This procedure allows to examine dermal layers for treatments or culture using chemical modulators, neutralizing antibodies, or viral methods. This helps in the assessment of pathological fibrotic responses across diverse medical settings.We showed the expression of connexin-43 and N-cadherin as key molecules involved in fascia mobilization upon wounding. The SCAD methodology allows the identification of novel therapeutic strategies to curtail scarring and fibrosis.

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