The systematic scoring analysis is a technique for evaluating mucosal injury, facilitating histological interpretation that can be performed by trained researchers. The main advantages of this method is that it's free and easy to use. It allows for quantitative analysis of histological data obtained from the intestines of mice with colitis.
This protocol provides a rapid and reproducible way to interpret disease severity of the entire colon, which is necessary for comparing disease from animals of different genetic backgrounds. For tissue preparation, it's important to organize all reagents and materials. Begin by placing a euthanized mouse on a dissecting pad in a supine position and immobilize the mouse extremities using 20 gauge 1.5 inch needles.
Using forceps and scissors, make a small incision on the abdominal skin and pull it to the side to expose the peritoneum, then open the abdominal cavity with a midline incision in the peritoneum from the pubic bone to the sides of the abdomen. Carefully remove tissues and organs until the large intestine is visualized. Cut the pelvic bone on both sides of the colon to fully visualize the organ, extending from the anus towards the cecum.
Gently remove fat, small veins, and arteries attached to the colon while carefully dissecting the organ, cutting just proximal to the anus and distal to the cecum. Carefully flush the colon with PBS to remove fecal contents using a flexible plastic gavage needle inserted through the anus. Position the colon in a straight line and open longitudinally along the mesenteric artery.
Bisect the colon longitudinally from the distal to the proximal end. Use half of the tissue for histological analysis and the other half for Western blot, PCR, or rolled into a second Swiss roll for fresh frozen immunofluorescence microscopy. Trim extra tissue from the proximal colon with a razor blade until approximately the same width along the length of the whole colon is obtained.
Align the colon to expose the lumen and flatten the tissue completely using a flexible gavage needle. Add more PBS if needed to keep the tissue moist throughout the procedure. Remove the excess PBS using a paper wipe.
With a syringe and gavage needle, add 10%neutral buffered formalin solution for two to three minutes to fix and flatten the tissue, then use straight forceps to grab the end of the distal colon and twist the colon into concentric circles from the distal to proximal end. Insert a 27 gauge needle to pin the colon in the middle and hold its Swiss roll shape, then place the Swiss roll into an embedding cassette inside a histological specimen container orienting the tissue in parallel with respect to the cassette. Fix the tissue in 10%neutral buffered formalin solution overnight at four degrees Celsius.
After overnight fixation, wash the tissue three times with PBS. Add 70%ethanol prior to the paraffin embedding process and remove the needle from the Swiss roll before proceeding. The tissue can be stored in ethanol at room temperature until paraffin embedment.
After opening the scanned images in the image processing software, verify that the entire colon is visible and that there are no missing areas of the sample. Activate the label imager and scale bar tools to properly identify scanned slides by clicking label imager. Open the annotations tool by clicking annotations and create three different layers by clicking new layer to quantify the total length of the Swiss roll, inflammation or injury, and erosion or ulceration.
Choose a different color for each layer by clicking layer color. Measure the length of each layer or category by clicking the pen tool to draw a line following the muscularis mucosa, moving the pointer as needed to visualize the adjacent area for analysis. View the image at 400 micrometer zoom to facilitate adequate visualization of the muscularis mucosa.
Each time that the pen is stopped, a small new layer region will be generated. It can be visualized and edited with the layer regions tab. Review the total length, inflammation or injury, and erosion or ulceration measured using the muscularis mucosa as a reference.
Once all layers are defined, export the data using the export grid to text file button inside the layer regions options. Open the text files and copy the data to a spreadsheet software. Total all the segments from each region and calculate the percentage of injury and ulceration with respect to total length.
Consider three main characteristics to calculate the histological colitis score and to evaluate the severity of disease. Check for healthy intestinal mucosa, which is characterized by organized epithelial cells in the crypt luminal axis, lamina propria with few immune cells and subjacent muscularis mucosa. Next, check for inflammation or injury, which is characterized by epithelial crypts that are attenuated or partially missing epithelial cells and mucosal inflammation with neutrophil infiltration into crypts.
Finally, check for the presence of erosion or ulceration, which is characterized by areas devoid of surface epithelium or areas completely lacking epithelium crypts with or without associated leukocytes. To illustrate the reliability of this histological colitis score analysis in the context of mucosal damage, 2.5%DSS was administered in the drinking water of eight C57BL6 wild-type mice for five days, followed by a recovery period with regular water for five days. Body weight remained constant during the acute administration of DSS, but dramatically decreased when mice started drinking regular tap water.
Blood and soft stools appeared after three days of DSS administration and continued until day eight of the experiment. These observations suggested that the most detrimental effects of exposure to DSS were observed during the recovery period between days five and eight. Measurement of colon length on day 10 confirmed a significant shortening at the end of the experiment.
Colons were harvested at day zero, two, five, seven, eight, and 10 to make paraffin Swiss rolls. The tissue was stained with H&E and high definition scans were analyzed to calculate the histological colitis score and percentage of injury and ulceration. Normal crypt architecture was observed in untreated mice.
After two days of DSS administration, immune cell recruitment was observed. On day five, epithelial cells appear damaged and there was an infiltration of neutrophils across the epithelium associated with epithelial injury. From day five to eight, areas of epithelial loss with inflammation and ulceration were observed.
On day 10, epithelial cells began to regenerate and repopulate ulcerated areas, slowly restoring colonic mucosa. When attempting this protocol, it is important to create a properly oriented and well-preserved Swiss roll, as well as to correctly identify mucosal inflammation or injury and ulceration or erosion. This method can be used to quantitatively evaluate injury along the entire length of the colonic mucosa in other models of colitis, such as TMS or T-cell transfer model.
