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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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Juglone+ameliorates+skin+wound+healing+by+promoting+skin+cell+migration+through+Rac1/Cdc42/PAK+pathway.">Juglone ameliorates skin wound healing by promoting skin cell migration through Rac1/Cdc42/PAK pathway.</a> Wound Repair and Regeneration. 24, 786-794 (2016).</li></ol>

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>
	</tbody>
</table>

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 ...

The insurgent need for treatment of skin wounds. To identify your target molecules for drug treatments, in vitro and in vivo test systems are required. Appropriate systems are the in vitro scratch assay, and the in vivo dorsal skin fold chamber.Both are straight forward procedures for monitoring cell migration and skin wound healing, in the absence and presence of pharmacologically active compounds. Although both techniques are straight forward, investigators should practice the scratch application. And the dorsal skin fold chamber implantation, and wounding to obtain reliable and reproducible results.Demonstration of the procedures will be done by Dr.Matthias Laschke, a surgeon and professor from the institute of clinical and experimental surgery. Before beginning the procedure, use an ultra fine permanent marker to mark one six well plate per cell type, with a horizontal line at the bottom of each well to delineate the scratch region for each culture. Next plate primary fiber blasts isolated from wild type and beta 3 deficient mice, in a six well plate at a 5 times 10 to the fifth fiber blasts per well concentration.In 2mL of DMEM supplemented with 10%fetal bovine serum. Label the plates with the cell type, genotype and date. And place the plates in the cell culture incubator for 24 hours.The next morning add 9 micro liters of each lipid based transfection reagent of interest, to individual micro centrifuge tubes containing 150 micro liters of reduced serum medium per tube. Add 1.5 micro liters of siRNA to a second micro centrifuge tube per transfection reagent containing 150 micro liters of reduced serum medium per tube. And mix each siRNA solution with a single tube of transfection reagent solution.After briefly vortexing place the mixtures at 21 degrees Celsius for 5 minutes. During the incubation carefully replace the supernate in each well. With 2.25mL of fresh culture medium down the side of the wells.At the end of the incubation add 250 micro liters of the appropriate siRNA transfection reagent mixture, drop wise into each corresponding well of cells. And return the plates to the cell culture incubator for 72 hours. When the cells have reached 100%confluency, discard the culture supernatant from each well.And use a 200 micro liter pipette tip to create scratch in the confluent cell monolayer. Vertical to the marked horizontal line at the bottom of each well. Then carefully rinse each wounded well 2 times with 2mL of PPS per wash.To remove any released factors from the damaged cells, loose cells or debris from the scratched area. After the second wash add 2mL of fresh cell culture medium supplemented with 1 or 10 percent serum to each well. And capture images of the wounds in each plate on the stage of a light microscope.Immediately after scratching at a 10X magnification. Then return the plate to the cell culture incubator. Continuing to capture images at the appropriate experimental time points for the next 30 hours.At the end of the experiment quantify the initial cell free area and remaining area after 6, 10 and 30 hours of culture. To determine the percentage of the scratch area repopulated by the migrating cells relative to the initial scratch area. After confirming a lack of response to toe pinch, in the wild type or beta three knockout C57 black six mouse, carefully shave the dorsum.And apply ointment to the animal's eyes. Apply depilatory cream to eliminate any remaining hair. Removing the cream after 10 minutes.To prepare the titanium chamber, fix one part of the symmetrical titanium chamber frame, with the connecting screws with nuts. To maintain 400 to 500 micrometers between the two symmetrical parts of the chamber. To avoid compression of the blood vesicles in the skin.Disinfect the exposed skin with 70%ethanol. And grasp a fold of skin in front of a light source. Position the middle line of the double layer of skin to where the titanium chamber will be implanted.And cranially and caudally fix the skin fold with a polypropylene suture. Tighten the other side of the suture on a metal rack to lift the folded skin. And adjust the height of the rack to allow the mouse to sit comfortably.Suture the first frame of the titanium chamber by polypropylene sutures on it superior edge to the back of the dorsal skin fold. After reconfirming sedation use fine scissors to make small incisions in the skin. And pass the two connecting screws attached to the first half of the titanium chamber through the base of the skin fold.Remove the mouse from the rack, and place the animal in a lateral position. Place the second complimentary half of the titanium chamber on top of the 3 connecting screws. And manually apply slight pressure to pass these screws through the second half of the titanium frame.The folded skin layer is now sandwiched between the two symmetrical halves of the titanium frame. Then fix both symmetrical parts with stainless steel nuts. Using a standardized 2mL diameter biopsy punch, mark the wound area in the center of the skin, within the observation window of the skin fold chamber.And use fine forceps and scissors to remove the entire epidermis and dermis within the mark. To create a circular wound. Clean the 3 and a half to 4 and a half milometer squared wound with 0.5mL of steril saline.And cover the wound with a glass coverslip. Use the snap ring plier on the titanium chamber to fix the coverslip in place. And immediately transfer the mouse to the imaging stage of a stereomicroscope.Then image the wound at a 40X magnification. Before placing the mouse into a cage with monitoring until full recovery. After creating a scratch using a 200 micro liter pipette tip as demonstrated, in this representative experiment, cells from both genotypes migrated into the scratch area and closed the gap.The cell migration was then quantified as the percentage of scratch area repopulated by migrating cells 6 hours after performing the scratch. For example in this experiment migrating Cav beta 3 deficient fiber blasts closed the scratch area significantly earlier than fiber blasts from wild type mice. To confirm the Cav beta 3 dependent effect observed in Cav beta 3 deficient fiber blasts, wild type fiber blasts were transfected with siRNA to down regulate the Cav beta 3 protein.Fiber blasts treated with the Cav beta 3 specific siRNAs behaved like beta 3 deficient fiber blasts. To compare skin wound healing between both genotypes, the wound area in the skin fold chamber was photographed directly after wounding. And the wound was imaged over the next 2 weeks.The sizes of the wounds were measured on the images and the wound area, on a given day was expressed as, the percentage of the initial wound area. As expected the rate of wound closure was increased in Cav beta 3 deficient mice compared to wild type controls. Be sure to apply equal pressure to the pipette tip, for each scratch and to match the scratches to the marked lines on the plate bottom as closely as possible.The observation window of the dorsal skin fold chamber can be opened at anytime during the healing process. Allowing for the topical application of different pharmacologically active compounds. It is also possible to excise the wounded area at different stages of the healing process.For molecular protein biochemical or histological analysis.

scratch-migration-assay-dorsal-skinfold-chamber-for-vitro-vivo

Research

JoVE Journal

Medicine

12.7K vistas.  Saarland University. La necesidad insurgente de tratamiento de heridas cutáneas. Para identificar las moléculas diana para tratamientos farmacológicos, se requieren sistemas de prueba in vitro e in vivo. Los sistemas apropiados son el ensayo de arañazo in vitro, y la cámara de plegado de la piel dorsal in vivo.Ambos son procedimientos directos para monitorear la migración celular y la cicatrización de heridas en la piel, en ausencia y presencia de compuestos farmacológicamente activos. Aunque ambas...