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.
