This method provides an ideal approach for studying colonic wound healing and the molecular and histologic changes that occur following injury. This method can also shed light on IBD pathogenesis. The main advantage of this technique is to precisely control the location of injury and the timing of the wound healing process.
Individuals performing this method for the first time may have difficulty inserting the colonoscope into the mouse's colon and creating consistent wounds. Repetitively practicing these techniques should overcome these issues. Given the multiple coordinated steps and nuance involved with this procedure, visual demonstration of this method is critical.
Before beginning the procedure, insert a 1.9 millimeter rigid bore endoscope into an endoscope sheath. And attach the assembled endoscope to the light source and video imaging device per the manufacturer's instructions. Use the provided tubing to attach the air pump to the gas valve on the left side of the sheath next to the working channel.
Ensure that the working channel is in the open position and insert three French biopsy forceps through the working channel. Advance the forceps to the end of the sheath without protruding out of the sheath. Next, place the anesthetized mouse onto an endoscopic staging platform on its ventral side and confirm the appropriate level of sedation by lack of response to pedal reflex.
After applying eye ointment, fill a three milliliter syringe with an attached rat gavage needle with room temperature PBS. And insert the needle approximately one centimeter into the mouse's anus. Gently infuse with PBS until the fecal material has been cleared.
Several fecal pellets should exit the mouse along with the PBS that was infused. Insert the assembled endoscope 0.5 centimeters into the anus and advance the biopsy forceps into the cleared lumen of the rectum until the full jaws of the forceps are beyond the end of the sheath. Turn the forceps 90 degrees so that the jaws open in an east-west orientation and open the tips and advance the forceps approximately one centimeter, closing and retracting the forceps in one smooth, quick motion to harvest the biopsy.
To avoid fully insufflating the colon while performing the biopsy, leave the right side of the gas valve open. Immediately after the biopsy, depress the foot pedal attached to the colonoscope recording device to initiate the video recording. And firmly press an index finger against the right side of the gas valve to completely cover the opening, forcing air into the endoscope and thus into the colon.
Retract the forceps back out of the sheath and into the rectal lumen while in the closed position. And place the forceps against the rectal wall immediately above the wound until the base of the jaws is aligned with the top edge of the viewing field. Continue fully insufflating the colon until a clear view of the wound can be observed.
To obtain the best measurements of the wound bed, be patient then place the colonoscope at a precise angle so that you get your best image of the wound bed. Open the video in an appropriate software program and advance the video to a frame showing a point in time at which the wound bed can be easily visualized. And at which the closed forceps are above the wound bed and against the rectal wall, and the wall is taut.
At each time point, obtain a snapshot of this frame and code the file name to ensure that the measurements of the wound beds are carried out in a blinded manner. To quantify the size of the wound bed, open the images in ImageJ and use the Free Hand selections tool to draw a perimeter around the wound. Under Analyze, select Measure.
And the value of that measurement will automatically populate in the Results window. When all of the measurements have been acquired, calculate the size of the wound on subsequent days relative to the size on day zero in a spreadsheet. 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 four-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. At the appropriate experimental endpoint, open the harvested colon longitudinally on a piece of filter paper, mesenteric side down.
Gently cover the tissue with your fixative of choice. Cover the tissue with parafilm in a sealed container for 4 to 6 hours before storage in 70%ethanol. On the day of the processing, remove the parafilm and add 0.2%methylene blue to the tissue, as demonstrated.
After draining off the excess stain, place the tissue under a dissecting microscope to locate the wound bed. Using a scalpel with a number 10 blade, cut directly through the center of the wound bed and continue cutting through the remainder of the colon in a straight line, such that the colon is cut in half lengthwise. For cryosectioning, embed the colon pieces such that the side that was cut by the scalpel is face down in a base mold half-filled with tissue freezing medium.
Secure the tissue in place with fine tweezers and place the base mold onto a metal plate or thick aluminum foil on top of dry ice to harden the freezing medium. Once the bottom portion of the medium is frozen, fill the remaining volume of the base mold with freezing medium at room temperature. And return the mold to the dry ice until the entire tissue block is frozen.
Then, transfer the mold to a 80 degrees Celsius freezer until sectioning. Here, representative images of acceptable views of the wound bed for an accurate quantification of the size of the wound bed and closure rate of the wound are shown. In this ex-vivo view of a wound bed, indicators of the perimeter of the wound bed and where to cut the tissue to enable visualization of the wound bed upon sectioning can be observed.
In this representative image, an H and E stain section of a wound bed can be clearly observed. It is important to acquire instant images of the wound bed at all time points to ensure an accurate assessment of the wound healing rate. Following the pinch biopsy, different agents of interest can be injected into the wound bed to test their effects on the wound healing rate.
