The overall goal of this procedure is to utilize a polyvinyl alcohol sponge to model wound healing in mice. This is accomplished by first hydrating and loading the sponge for implantation. Next, the sponge is inserted into a pocket between the abdominal muscle and the skin of the mouse.
After implantation, each sponge can then be injected with a treatment. The final step is to remove the sponges from the mouse. Ultimately, sectioning staining and quantitative assays can be performed to observe changes and variations in the wound healing process.
Though this method can provide insight into wound healing, it can also be applied to other areas of study such as tumor angiogenesis, drug delivery, and implanted cell survival. And engraftment Begin by hydrating the sponges with overnight stirring in a 0.9%aqueous sodium chloride solution. Then autoclave 75 milliliters of the hydrated sponge suspension for 25 minutes at 121 degrees Celsius.
To sterilize the sponges, place the autoclave sponge solution in a sterile hood, and then use sterile tweezers to pick up one sponge, squeeze the sponge with the tweezers, and use a vacuum tip to remove as much solution as possible from the sponge. Now, place the rung out sponge in a tissue culture plate, being careful to leave space between sponges, pipette the treatment solution directly onto the center of a sponge. Then press down gently on the sponge with the pipette tip to help the sponge absorb the solution.
Once all the sponges are loaded, store the plate on ice until the sponges are implanted into the mouse. After confirming sedation with the pinch reflex test, begin by placing the mouse in a nose cone in the supine position. Then using an electric shaver, remove a one by one inch area of hair on the lower ventral side of the mouse below the rib cage.
Wiping the area clean of cut hair after. Sterilize the shaved area with Betadine, followed by 70%alcohol. Next, holding the skin taut.
Use the scalpel to make a vertical incision, one and a half to two times the diameter of the sponges to be inserted. Being careful not to cut through the body cavity. Use the tweezers to grasp the edge of the skin.
Then use Metzenbaum scissors to slowly separate the skin from the muscle on both sides of the incision. Extend the pocket across the width of the mouse and from the rib cage to the hind legs. Now, use the tweezers to pick up the first sponge to be placed in the animal by pinching it from the sides.
Then transfer the sponge to a pair of straight tweezers so that it is held from the top and bottom. Next, grasp and lift the edge of the skin with another pair of tweezers, and slowly insert the sponge, avoiding touching the skin or the muscle underneath. Once all the sponges have been inserted, use a pair of tweezers to hold both sides of the incision together and then thread the suture through both layers of skin.
Tighten the first throw just enough to close the skin edges and finish the square knot with one more throw from the other direction. This time pulling the knot tight. Then tie two more square knots to finish the suture.
Place additional sutures to close the entire incision. Finally, remove the animal from the anesthesia system and monitor it until consciousness is resumed. The mouse should quickly begin ambulating and return to preoperative mobility.
Begin by filling a syringe with the desired solution and attach a 28 gauge needle to the syringe. Then after confirming sedation by toe pinch, push the needle into the animal at a 45 degree angle through the skin at the center of the sponges to make sure the needle is in the sponge. Slowly lift the needle vertically.
If the needle is in the sponge, the sponge will move up with the needle. Then slowly inject the substance into the sponge and remove the needle. After the animal has been sacrificed, hold the skin with a pair of tweezers and make an incision near the desired sponge.
Finally, carefully separate the sponges from the surrounding capsule, avoiding harvesting extra tissue surrounding the sponge removed. Sponges can be stored under different conditions depending on the type of analysis that will be performed. For sectioning, the retrieve sponges can be embedded in a freezing medium, such as optimal cutting temperature compound, or placed in 10%formalin for embedding and sectioning in paraffin blocks, as was done for these h and e stained sponges.
And in these sections of trireme stained sponges, the best results for sectioning are obtained when the sponge is cut in half across the diameter as shown here, and then is embedded cut surface down. As demonstrated here, sections of the sponge should be taken so that the entire width of the sponge is present in this figure. The sponge on the left was embedded or sectioned incorrectly as evidenced by the disrupted tissue border along the right side of the section, whereas the intact sponge on the right was processed correctly.
After watching this video, you should have a good understanding of how to prepare, implant, inject, and remove polyvinyl alcohol sponges to study wound healing in mice.
