Name: analyzing beneficial effects of nutritional supplements on intestinal epithelial barrier functions during experimental colitis
Uploaded: 2017-01-05T15:00:00
Description: Watch this Scientific Journal Video about Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis at JoVE.com

This work was supported by grants from the Mexican Council for Science and Technology (Conacyt, 207268 and 233395 to Michael Schnoor). KFCO is a recipient of a Conacyt stipend (396260) to obtain an MSc degree.

All animal experiments have been approved by the Institutional Animal Care and Use Committee of Cinvestav.
1. Preparation of DSS Drinking Water and the Induction of Colitis
<ol>
	<li>Prepare a 3.5% w/v dextran sulfate sodium (DSS) solution in autoclaved drinking water. DSS dissolves easily and it is not required to filter the final solution. 
	NOTE: 7 days of DSS treatment induces severe colitis. If inducing mild colitis, 3 - 4 days of treatment are recommended. As long as the DSS drinking water stays clear...

<ol>
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In order to use nutritional supplements in clinical studies, the benefits and safety of such supplements must be carefully evaluated in vivo in animal studies. In the case of colitis, several appropriate animal models that resemble the clinical signs of IBD have been established, including chemical models using DSS, TNBS, or acetic acid; knock-out (KO) models such as IL10-KO; and immune-cell-mediated colitis using adoptive T-cell transfer19-21. The DSS model of colitis is a rapid and reliable method o...

Colitis is an inflammatory condition of the colon that can cause diarrhea and abdominal pain. Colitis can be acute, in response to infection or to stress, or it can develop as a chronic disease, such as in ulcerative colitis (UC), which belongs to the group of inflammatory bowel diseases (IBD). Although the clinical signs of UC have been well described, the pathogenesis is still poorly understood1. It is accepted among experts that UC is a multifactorial disease, with genetic mutations and aberrant immune responses playing a major role2. However, environmental factors, such as style of living and nutrition, also contribute to disease development and progression3.
Unfortunately, IBD is not curable, but there are many treatment options that aim to alleviate clinical symptoms. Current treatments include anti-inflammatory drugs, such as sulfasalazine and corticosteroids; immunosuppressants, such as azathioprine; monoclonal antibodies that capture pro-inflammatory cytokines, such as tumor necrosis factor-&#945; (TNF-&#945;), or block adhesion molecules, such as integrins, to reduce excessive leukocyte recruitment; and inhibitors that target kinases that trigger pro-inflammatory pathways, such as Janus kinase (JAK)4. Not all patients respond to all treatments, so therapeutic strategies must be individualized5. Furthermore, most of these therapeutic drugs interfere with the metabolism and immune responses, often causing severe side effects. For this reason, alternative treatment options have been investigated.
Alternative treatments include probiotics and nutritional supplements, which have been applied in animal models and clinical trials with varying levels of success6,7. We recently found that the application of different single nutritional supplements, such as anti-oxidative vitamins or &#969;3-poly-unsaturated fatty acids (PUFAs), is inferior to the application of a combination of such supplements in alleviating colitis and cardiovascular disease symptoms8,9. These studies were performed in mice, so clinical studies must be performed in humans to determine whether these findings will also be applicable to humans. Before clinical studies are initiated, the effectiveness and safety of new treatment options must be evaluated in animal models.
For IBD, the dextran sulfate sodium (DSS) model has been widely used to study the mechanisms of disease development and the beneficial effects of drugs and nutritional supplements6,10. In most studies, only an acute disease over a time period of seven days is induced; nevertheless, the clinical signs observed in these animals closely resemble those observed in IBD patients (i.e., bloody diarrhea, weight loss, epithelial dysfunction, and immune cell infiltration)10. DSS induces erosions in the mucosa, resulting in barrier dysfunction and in increased intestinal epithelial permeability11. The exact mechanism remains unknown. However, a study suggests that DSS interacts with medium chain-length fatty acids to form nano-lipocomplexes that are able to enter epithelial cells and to induce inflammatory signaling pathways12. In this article, we describe in detail how colitis is induced and analyzed in mice, how nutritional supplements are applied by gavage to ensure a constant dosage in each animal, and how the effects of such supplements on various colitis symptoms are examined.

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			<td height="21" style="height:21px;width:439px;">0.3% of gelatin&#160;</td>
			<td style="width:184px;">Bioxon</td>
			<td style="width:139px;">158</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">10% formaldehyde</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">2106-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">96-well plate with flat bottom</td>
			<td style="width:184px;">Corning</td>
			<td style="width:139px;">3368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Absolute ethanol&#160;</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">9000-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Absolute xylene</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">9490-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">ABTS</td>
			<td style="width:184px;">Sigma Aldrich</td>
			<td style="width:139px;">H5882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Bovine Serum Albumin</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">9048-46-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">ColoScreen</td>
			<td style="width:184px;">Helena Laboratories</td>
			<td style="width:139px;">5072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Corabion (Kindly provided by Merck, Naucalpan, Mexico)</td>
			<td style="width:184px;">Merck</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Dextran Sulfate Sodium Salt</td>
			<td style="width:184px;">Affymetrix</td>
			<td style="width:139px;">9011-18-1</td>
			<td style="width:191px;">(M.W. 40,000-50,000)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Dihydroethidium</td>
			<td style="width:184px;">Life Technology</td>
			<td style="width:139px;">D11347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Eosin-Y&#160;</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">L087-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Evans blue</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">314-13-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Feeding&#160; needle</td>
			<td style="width:184px;">Cadence Science Inc.</td>
			<td style="width:139px;">9921</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Glass slide rack with handle</td>
			<td style="width:184px;">Electron Microscopy</td>
			<td style="width:139px;">70312-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Glass Slides</td>
			<td style="width:184px;">Corning</td>
			<td style="width:139px;">2947</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Harris hematoxylin&#160;</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">H3136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Hexadecyltrimethyammonium (HTAB)</td>
			<td style="width:184px;">Sigma Aldrich</td>
			<td style="width:139px;">H6269</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Histosette (Embedding cassette)</td>
			<td style="width:184px;">Simport</td>
			<td style="width:139px;">M498.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Hydrochloric acid 1 M</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">9535-62</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Hydrogen peroxide</td>
			<td style="width:184px;">Sigma Aldrich</td>
			<td style="width:139px;">H3410</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Ketamine</td>
			<td style="width:184px;">PiSA Agropecuaria</td>
			<td style="width:139px;">Q-7833-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Liquid paraffin&#160;</td>
			<td style="width:184px;">Paraplast</td>
			<td style="width:139px;">39501-006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Lithium carbonate&#160;</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">2362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">N,N-dimethylformamide</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">68-12-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">N-acetylcysteine</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">A9165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Plastic cubes</td>
			<td style="width:184px;">Electron Microscopy</td>
			<td style="width:139px;">70181</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Poly-L-Lysine Solution</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">25988-63-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Prism 5 statistical software</td>
			<td style="width:184px;">GraphPad Software</td>
			<td style="width:139px;">Prism 5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Saline Solution 0.9% NaCl</td>
			<td style="width:184px;">CS PiSA</td>
			<td style="width:139px;">Q-7833-009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Sodium citrate</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">W302600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Synthetic resin&#160;</td>
			<td style="width:184px;">Poly Mont</td>
			<td style="width:139px;">7987</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Taq DNA polymerase</td>
			<td style="width:184px;">Invitrogen</td>
			<td style="width:139px;">11615-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Tissue-tek. O.C.T Compound</td>
			<td style="width:184px;">Sakura Finetek</td>
			<td style="width:139px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Tuberculine Syringe</td>
			<td style="width:184px;">BD Plastipak</td>
			<td style="width:139px;">305945</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Tween 20</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">9005-64-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">VECTASHIELD Antifade Mounting Medium with DAPI</td>
			<td style="width:184px;">Vector Laboratories</td>
			<td style="width:139px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Xylazine</td>
			<td style="width:184px;">PiSA Agropecuaria</td>
			<td style="width:139px;">Q-7833-099</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing beneficial effects of nutritional supplements on intestinal epithelial barrier functions during experimental colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Diet Supplements Can Protect from DSS Colitis
Anti-inflammatory and anti-oxidative diet supplements, such as vitamin C, vitamin E, the nitric oxide (NO)-source L-arginine, and &#969;3-polyunsaturated fatty acids (&#969;3-PUFAs), have been used with varying success in animal models to alleviate signs of inflammatory diseases3,6,15. Malnutrition, oxidative stress, and the production of pro-inflammatory cytokines can trigge...

Watch this Scientific Journal Video about Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis at JoVE.com

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

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

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.

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