Name: scratch migration assay and dorsal skinfold chamber for in vitro and in vivo analysis of wound healing
Uploaded: 2019-09-26T13:00:00
Description: Watch this Scientific Journal Video about Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing at JoVE.com

We thank Dr. Petra Weissgerber and the Transgene Unit of the SPF animal facility (project P2 of SFB 894) of the Medical Faculty and the animal facility at the Institute of Clinical and Experimental Surgery at the Medical Faculty of Saarland University, Homburg. We thank Dr. Andreas Beck for critical reading of the manuscript. This study was funded by the Deutsche Forschungsgemeinschaft (DFG) Sonderforschungsbereich (SFB) 894, project A3 to A.B. and V.F.).

All experimental procedures were approved and performed in accordance with the ethics regulations and the animal welfare committees of Saarland and Saarland University.
1 Primary cell culture and siRNA transfection
NOTE: In the described method, primary fibroblasts are used. These cells play a crucial role in wound healing and tissue remodeling11. In this experiment, the Cacnb3 gene, encoding the Cav&#946;3 subunit of high voltage-gate...

<ol>
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In this manuscript, we describe an in vitro and in vivo wound healing assay and correlate the results obtained. For the in vitro assay, we used primary mouse fibroblasts4,14,15 which play an important role in wound healing and tissue remodeling11. Other adherent cell types growing as monolayers (e.g., epithelial cells, endothelial cells, keratinocytes) can be used as well. Plating the same number of viabl...

Skin wound healing starts immediately after the skin injury in order to restore the skin&#39;s integrity and to protect the organism from infections. The wound healing process goes through four overlapping phases; coagulation, inflammation, new tissue formation, and tissue remodeling1. Cell migration is crucial during these phases. Inflammatory cells, immune cells, keratinocytes, endothelial cells, and fibroblasts are activated at different time points and invade the wound area2. Methods to investigate wound healing in vitro and in vivo are of great interest not only to understand the underlying mechanisms but also to test new drugs and to develop new strategies aiming to ameliorate and accelerate skin wound healing.
To monitor and analyze cell migration, the scratch migration assay can be used. It is often referred to as in vitro wound healing assay. This method requires a cell culture facility3. It is a simple procedure, there is no need of high-end equipment and the assay can be performed in most cell biology laboratories. In this assay, a cell-free area is created by the mechanical disruption of a confluent cell monolayer, preferably epithelial- or endothelial-like cells or fibroblasts. Cells on the edge of the scratch will migrate in order to repopulate the created gap. Quantification of the decreasing cell-free area over time resembles the migration rate and indicates the time, which the cells need to close the gap. For this purpose, investigators can use either acutely isolated cells from WT mice or mice lacking a gene of interest4, or immortalized cells available from reliable cell repositories. The scratch assay allows studying the influence of pharmacologically active compounds or the effect of transfected cDNAs or siRNAs on cell migration.
In vivo, wound healing is a complex physiological process, requiring different cell types including keratinocytes, inflammatory cells, fibroblasts, immune cells and endothelial cells in order to restore the skin&#8217;s physical integrity as fast as possible1. Different methods to study in vivo wound healing have been developed and used in the past5,6,7,8. The dorsal skinfold chamber described in this article was previously used for wound healing assays9. It is used as a modified dorsal skinfold chamber preparation for mice. The modified skinfold chamber model has several advantages. 1) It minimizes skin contraction, which prevents observing the wound healing process and might influence wound repair in mice. 2) This chamber makes use of covering the wound with a glass coverslip, reducing tissue infections and desiccation, which could delay the healing process. 3) Blood flow and vascularization can be directly monitored. 4) It allows repetitive topical application of pharmacologically active compounds and reagents in order to treat the wound and accelerate healing9,10.
We identified the &#946;3 subunit of high voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence skin wound healing using two independent protocols, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. For the in vitro assay, we used primary fibroblasts, these cells do express the Cacnb3 gene encoding the Cav&#946;3 protein but lack depolarization-induced Ca2+ influx or voltage-dependent Ca2+ currents. We described a novel function of Cav&#946;3 in these fibroblasts: Cav&#946;3 binds to the inositol 1,4,5-trisphosphate receptor (IP3R) and constraints calcium release from the endoplasmic reticulum. Deletion of the Cacnb3 gene in mice leads to increased sensitivity of the IP3R for IP3, enhanced cell migration and increased skin wound repair4.

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	<tbody>
		<tr>
			<td>0.9 % NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringes</td>
			<td>BD Plastipak</td>
			<td>303172</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy punch</td>
			<td>Kai Industries</td>
			<td>48201</td>
			<td>2 mm</td>
		</tr>
		<tr>
			<td>Cacnb3 Mouse siRNA Oligo Duplex (Locus ID 12297)</td>
			<td>Origene</td>
			<td>SR415626</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilation cream</td>
			<td></td>
			<td></td>
			<td>any depilation cream</td>
		</tr>
		<tr>
			<td>Dexpanthenol 5% (BEPANTHEN)</td>
			<td>Bayer</td>
			<td>3400935940179.00</td>
			<td>(BEPANTHEN)</td>
		</tr>
		<tr>
			<td>Dihydroxylidinothiazine hydrochloride (Xylazine)</td>
			<td>Bayer Health Care</td>
			<td></td>
			<td>Rompun 2%</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco by life technologies</td>
			<td>41966-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco by life technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexagon full nut</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>Zoetis</td>
			<td></td>
			<td>KETASET</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Keyence, Osaka, Japan</td>
			<td>BZ-8000</td>
			<td>Similar microscopes might be used</td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX Transfection Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>13778075</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse restrainer</td>
			<td>Home-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Nikon</td>
			<td>plan apo 10x/0.45</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco by life technologies</td>
			<td>51985-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene sutures</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Screwdriver</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Skin disinfectant (octeniderm)</td>
			<td>Sch&#252;lke &#38; Mayr GmbH</td>
			<td>118212</td>
			<td></td>
		</tr>
		<tr>
			<td>Slotted cheese head screw</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Snap ring</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Snap ring plier</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope with camera</td>
			<td>Leica</td>
			<td>Leica M651</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium frames for the skinfold chamber</td>
			<td>IROLA</td>
			<td>160001</td>
			<td>Halteblech M</td>
		</tr>
		<tr>
			<td>Wire piler</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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scratch migration assay and dorsal skinfold chamber for in vitro and in vivo analysis of wound healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

The scratch assay was performed on a confluent cell monolayer of wild-type and &#946;3-deficient MEFs (Figure 1c). After performing the &#34;scratch&#34;&#160;using a 200 &#956;L pipette tip, cells from both genotypes migrate into the scratch area and close the gap. Images were taken after 6, 10 and 30 h (Figure 1a). Cell migration was quantified as the percentage (%) of scratch area repopulated by migrating cells 6 hours after ...

Watch this Scientific Journal Video about Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing at JoVE.com

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

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