This article describes a method for isolating wound beds in murine models following colonoscopic-guided biopsies. The isolated tissues are used for molecular and histological studies to investigate wound healing processes in gastrointestinal inflammatory diseases.
Colonoscopic-guided biopsies in murine models induce wound sites in the colon. These wound beds are isolated for molecular and histological studies to understand the process of wound healing in inflammatory diseases of the gastrointestinal, or GI tract.
To begin wound bed isolation, place a euthanized experimental mouse with a colonoscopic-induced wound in the supine position. Incise the abdominal skin and underlying muscles to expose the body cavity.
Place closed scissors under the colon and lift it. Cut the colon at its midpoint, and at the anus to separate it from the body. Flush the colon with an appropriate buffer to remove fecal content.
Place the colon on filter paper and cut it open longitudinally, with the mesenteric side facing down on the filter paper. Now, add methylene blue dye over the colon and observe under the microscope.
The dye stains the dead and granulated tissue in the wound bed, making it appear darker than the surrounding healthy tissue, allowing for its easy identification. Preserve the isolated wound bed for gene expression studies.
At the appropriate experimental endpoint, open the skin and abdominal muscle layers to expose the body cavity of the experimental animal, and place closed forceps under the colon. Gently lift the colon to release it from the underlying mesentery, and cut the tissue at its midpoint and at the anus to collect it from the mouse.
Use a 20-milliliter syringe filled with ice-cold PBS, and equipped with a rat gavage needle, to flush out the fecal contents; and place the cleared colon onto a piece of filter paper. Open the colon longitudinally, taking care that the mesenteric side is face down against the filter paper, and use a Pasteur pipette to cover the mucosa with 0.2% methylene blue.
After a few seconds, drain off the excess stain and view the colon under a dissecting microscope to locate the wound bed. Use 4-inch micro iris scissors to cut around the edge of the bed, being careful not to cut into the muscle layer, and use fine point tweezers to transfer the dissected tissue into a tube for snap freezing and/or storage.
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