Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

The overall goal of this protocol is to evaluate the effects of supplemental nutrients in a murine model of colitis induced by dextran sulfate sodium. This method can help answer key questions in the intestinal immunology field about the development of clinical science throughout the direction of the colitis model experiment. The main advantages of this technique are that it is low cost, does not require sophisticated equipment, and provides a clear analysis of the morphology of the entire colon.

This method can be used to evaluate oxidative stress in the colon as a consequence of excessive neutrophil recruitment. For nutritional supplement administration by gavage, first manually restrain the mouse by the scruff, placing the tail between the third finger and the base of the thumb. Next, holding the mouse in the upright position, insert the gavage needle into the left side of the animal's mouth and carefully follow the roof of the mouth to locate the esophagus.

If no resistance is encountered, advance the needle towards the stomach. Slowly deliver the entire volume of supplement, followed by careful removal of the syringe and measure the animal's body weight. Assign a score of zero to four according to the weight loss percentage, then collect a fresh stool sample and use the provided applicators to apply a thin smear of sample onto a slide from a guaiac fecal occult blood test kit.

Close the cover on the front of the kit and open the back window to apply two drops of the developer solution to the back of the sample location. Read the results after 30 seconds, assigning a score of zero to four depending upon the signal strength. Then observe a second fresh stool sample to assess the stool consistency, assigning a score of zero to four as appropriate.

To determine the intestinal epithelial permeability in vivo, place an anesthetized animal in the supine position and make a midline incision to expose the intestines. Locate the cecum and make a small incision in the proximal segment of the colon ascendants. Then insert a 22 gauge feeding needle and secure the needle with a common silk thread ligature.

Carefully flush an abundant volume of PBS through the needle to rinse all of the feces from the colon, followed by the installation of Evans blue solution until the dye reaches the anus. After 15 minutes, flush the tube with an excess volume of PBS until the perianal washout is clear. Then excise the colon and rinse the tissue with more PBS, followed by one milliliter of six millimolar N-acetylcysteine to eliminate any dye sticking to the colonic mucus.

Now cut the colon longitudinally and rinse the tissue one more time with PBS and N-acetylcysteine, then record the weight and length of the colon and place the tissue into two milliliters of N, N-dimethylformamide at room temperature with gentle agitation to extract the remaining dye, measuring the dye concentrations spectrophotometrically at 610 nanometers the following morning. After seven days of dextran sulfate sodium, or DSS-induced colitis, dissect the entire colon and record its length. For swiss rolls, open the colon longitudinally, carefully remove any fecal matter, and use a thin, round, wooden stick or fine-tipped forceps to roll the tissue with the mucosa facing inwards.

Then carefully place the tissue inside an embedding cassette, followed by fixation with five milliliters of 10%formaldehyde at room temperature. It is critical to roll up the colon tightly without folding to avoid problems during the tissue sectioning, which could result in a clinical tissue damage and a false interpretation as colitis-associated damage. After 48 hours, wash the sample with tap water for 18 hours, followed by dehydration by an ethanol series.

After the last absolute ethanol immersion, treat the tissue with four one-hour incubations in xylene. After the last xylene immersion, place the sample in 50 milliliters of liquid paraffin for one 17-hour and one three-hour incubation. At the end of the three hour incubation, submerge the sample in a 22 cubic millimeter square plastic mold in eight to 10 milliliters of fresh liquid paraffin for 24 hours.

When the paraffin has solidified, use a microtome to acquire five micron thick tissue sections, then briefly immerse the cross-sections in a one liter water bath containing 0.3%gelatin and immediately mount the samples on glass slides treated with polylysine for H&E staining. For oxidative stress analysis, first create a barrier around the tissue using a hydrophobic pen. Next, incubate eight micron tissue cryo cross-sections in five micromolar DHE dissolved in water for 30 minutes at 37 degrees celsius in the dark.

Incubation is followed by three 15-minute washes in PBS with gentle agitation at room temperature. Air dry the samples and add a drop of mounting medium to protect the fluorescence. Then observe the oxidized ethidium fluorescence on a laser scanning confocal microscope at a 40x magnification.

DSS treatment leads to a continuously increasing disease activity index as expected, reaching a maximum score on day seven post-induction. Anti-inflammatory diet supplementation delays the early onset of colitis, while the protective effects are only marginal at later time points. Dietary intervention may also prevent DSS-induced increases in intestinal epithelial permeability, as well as colon length shortening.

Hematoxylin and eosin staining of paraffin-embedded distal colon tissues reveals that control samples demonstrate a normal morphology, whereas animals treated with DSS exhibit the typical signs of colitis. Of note, parallel treatment with diet supplementation clearly alleviates colitis-induced morphological changes with an overall morphology similar to that of the control group. Immunofluorescence staining of the distal colon tissue also indicates that the tight inadherens junction molecules are internalized during DSS-induced colitis and that co-localization of these proteins is partly lost at crypt epithelial cell contacts.

Importantly, this internalization is reduced when DSS-treated mice receive the anti-inflammatory and anti-oxidative diet supplement. Further, robust neutrophil recruitment to the colon in response to DSS-induced colitis is counteracted by diet supplementation, resulting in the concomitant amelioration of neutrophil-induced oxidative stress and a slight reduction in inflammatory cytokine production. Following this procedure, other methods like oozing chamber measurements can be performed to analyze the electrical resistance of the epithelial barrier in the colon.

After its development, this technique paved the way for researchers in the field of colitis research and exploring the molecular mechanism of ulcerative colitis pathogenesis in a reproducible mouse model. After watching this video, you should have a good understanding of how to analyze the development of colitis and the beneficial effects of nutritional supplements on intestinal epithelial barrier functions. Don't forget that working with live animals requires the adherence to high ethical standards to avoid unnecessary suffering.