Name: Murine Colitis Modeling using Dextran Sulfate Sodium (DSS) (Video) | JoVE Journal
Uploaded: 2010-01-19T17:00:00.000+00:00
Duration: 8 min 39 s
Description: Learn to induce acute colitis in mice using dextran sulfate sodium in drinking water, followed by tissue preparation for histological analysis. This standardized protocol enables reproducible inflammatory bowel disease research.

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5 minutes

Funding provided by NIH (1 K08 DK080221-01) and Vanderbilt institutional development funds.

Part 1: Injury-Repair Colitis Model
<ol>
<li>Baseline weight should be obtained for each mouse prior to beginning DSS treatment.</li>
<li>Make a 3% (w/v) Dextran Sulfate Sodium Salt solution in water and filter with a 0.45 μm cellulose acetate filter.</li>
<li>Replace drinking water in the mouse cage with the 3% DSS solution for four days. The mice should not have access to any other source of water (i.e. exclusion tips placed on automated watering systems).</li>
<li>On day four, replace the DSS solution with water for an additional three days, allowing some colonic epithelial recovery. The mice should be weighed on day four in order to quantify systemic consequences of colitis. Weight loss is common with severe injury. </li>
<li>On day 7, weigh and sacrifice the mice. Mice can be euthanized by inhalational overdose of isoflurane, or other institutionally, IACUC approved methods.</li>
</ol>
Part 2: Necropsy and Harvesting of Colon
<ol>
<li>Expose the ventral side of the mouse and secure legs to ensure unobstructed access to the abdomen. Wet the abdomen completely with 70% ethanol.</li>
<li>Grasp the midline of the abdomen with tissue forceps and extend upwards thus tenting the skin. Use fine-tipped scissors to incise the abdomen thus exposing the abdominal contents. Extend the incision to the tip of the xyphoid process at the midline and then extend along the inferior aspect of the costal margins bilaterally. </li>
<li>Identify the small intestine, cecum, and colon. Carefully dissect/tease the colon from the surrounding mesentery. Transect the colon deep in the pelvis to free the distal colon and rectum. Transect the colon at the colonocecal margin to free the proximal colon. Care must be taken during this process not to damage the colon as cleaning the colon will be problematic if perforation occurs.</li>
<li>Using a 20G feeding needle and 10 ml syringe, intubate and flush the colon with ice-cold PBS until the eluate is completely clear of stool.</li>
<li>At this point, if macroscopic analysis with a dissecting microscope is desired, the colons can be fixed as “sausages”. If histologic analysis only is desired than proceed to Part 4 “Making Swiss Rolls for the Histological Analysis of Acute Colitis”.</li>
</ol>
Part 3: Processing as “Sausages” for Macroscopic Analysis of the Entire Colon
<ol>
<li>It is important to maintain the correct orientation of the colon, therefore, keep the distal end of the colon closest to the operator. Cut two pieces of non-absorbable suture (SP116 1.5 metric braided silk), one approximately 1 inch in length, the other 2 inches.</li>
<li>Use the 2 inch piece of suture to tie off the distal end as close to the margin as possible while still maintaining a good seal.</li>
<li>Place a 20G feeding needle containing 5 ml of 10% buffered formalin phosphate into the proximal end of the colon. Loosely tie the remaining piece of suture immediately proximal to the bulb of the feeding needle. Grasp the colon firmly at the bulb of the feeding needle and infuse formalin until the colon is expanded. Tighten the knot in the suture as you withdraw the feeding needle, thus leaving the infused, expanded colon as a “sausage”. </li>
<li>Fill a 15 ml conical tube with 10% buffered formalin phosphate and place the colon in the tube. Fix for 24 hours.</li>
<li>Pour off formalin and replace with 70% EtOH for an additional 24hrs. (Colons can be stored in 70% EtOH indefinitely at room temperature.)</li>
<li>Remove the colon from the conical tube and cut the strings on each end with a scalpel, being careful not to damage the colon. Remember that the distal end has the longest piece of string. Cut longitudinally from the distal to proximal end of the colon so that it forms a long sheet. At this point, the colon can be viewed under a dissecting microscope.</li>
</ol>
Part 4: Making Swiss Rolls for the Histological Analysis of Acute Colitis
<ol>
<li>It is important to maintain the correct orientation of the colon, therefore, keep the distal end of the colon closest to the operator.</li>
<li>Measure the colon length. This metric is another indicator of the severity of injury. Colitis increases edema and shortens the overall colon length.</li>
<li>Using fine-tipped scissors, incise longitudinally from distal to proximal end of the colon. Use fine tipped forceps to grasp either edge of the incision and open laterally working your way distally-&gt;proximally, thus displaying the colon as a flat sheet.</li>
<li>Rolling the colon requires a pair of forceps and a two handed technique. Grasp either edge of the distal margin with forceps. Proceed to sequentially roll the colon by rotating and releasing the forceps proceeding to the proximal margin. Thus generating a spiral with a third dimension or “Swiss Roll”. To maintain the rolled form, grasp the roll firmly with forceps and transect it with a 27G1/2 needle. Secure the needle by bending the needle as it exits the roll. </li>
<li>Place the roll in an appropriately labeled tissue cassette and place in a jar of 10% buffered formalin phosphate at room temperature for 24 hrs to ensure tissue fixation.</li>
<li>Pour off formalin and replace with 70% EtOH for an additional 24hrs. (Colons can be stored in 70% EtOH indefinitely at room temperature.)</li>
<li>Once the sample has been fixed, it can be paraffin-embedded, sectioned and mounted on slides for histologic analysis.</li>
</ol>
Part 5: Representative Results
The DSS model for acute colitis allows the researcher to obtain, fix, and analyze a colon that models acute colitis. When the Swiss roll is cut and mounted, it should form a representative slice of the entire colon if rolled properly (Figure 1). The mounted roll can be stained with H&amp;E in order to determine the extent of damage to the colon, from the distal (inside) end to the proximal (outside) end (Figure 2). Immunohistochemistry can also be performed on the Swiss roll to identify and quantify inflammatory infiltrates. If the sausage method has been performed correctly, the fixed colon will be dilated and the entire mucosal surface can be easily manipulated and viewed under the dissecting microscope (Figure 3).
<a href="https://www.jove.com/files/ftp_upload/1652/1652fig1.jpg"><img src="/files/ftp_upload/1652/1652fig1tmb.jpg" alt="Histology tissue sample preparation; microscopy image; paraffin embedding; section analysis." border="0" data-altupdatedat="1788235205000" data-alt="Figure 1"></a> Figure 1. The properly-executed "Swiss roll". (A) The colon is rolled from the distal to proximal end, transected with a needle and secured by bending the end of the needle. It is then placed in a tissue cassette for fixation. (B) H&amp;E stained 5 μm section of a Swiss roll made from the colon of a mouse treated with DSS (d= distal colon p= proximal colon).
<a href="https://www.jove.com/files/ftp_upload/1652/1652fig2.jpg"><img src="/files/ftp_upload/1652/1652fig2tmb.jpg" alt="Histology comparison, water vs DSS, colon tissue, 4x and 20x magnification, microscope images." border="0" data-altupdatedat="1788235205000" data-alt="Figure 2"></a> Figure 2. DSS treated colons show signs of acute colitis. Inflammation and crypt damage are apparent in the DSS-treated colon compared to a water treated control.
<a href="https://www.jove.com/files/ftp_upload/1652/1652fig3.jpg"><img src="/files/ftp_upload/1652/1652fig3tmb.jpg" alt="Tendon suture diagram; non-absorbable suture placement from proximal to distal ends for repair." border="0" data-altupdatedat="1788235205000" data-alt="Figure 3"></a> Figure 3. An example of a "sausage". The sausage infused with formalin and completely expanded. A slight angle will be present secondary to the natural curvature of the colon. The opened sausage should lie flat.

<ol><li>Okayasu, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+method+in+the+induction+of+reliable+experimental+acute+and+chronic+ulcerative+colitis+in+mice%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice.</a> Gastroenterology. 98 (3), 694-702 (1990).</li>
<li>Martinez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deletion+of+Mtgr1+sensitizes+the+colonic+epithelium+to+dextran+sodium+sulfate-induced+colitis%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Deletion of Mtgr1 sensitizes the colonic epithelium to dextran sodium sulfate-induced colitis.</a> Gastroenterology. 131 (2), 579-588 (2006).</li>
<li>Moolenbeek, C., Ruitenberg, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+%22Swiss+roll%22%3A+a+simple+technique+for+histological+studies+of+the+rodent+intestine%5BTitle%5D)+AND+%22Lab+Anim%22%5BJournal%5D)">The "Swiss roll": a simple technique for histological studies of the rodent intestine.</a> Lab Anim. 15 (1), 57-59 (1981).</li>
<li>Mahler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+susceptibility+of+inbred+mouse+strains+to+dextran+sulfate+sodium-induced+colitis%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis.</a> Am J Physiol. 274, G544-551 (1998).</li>
<li>Aharoni, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunomodulatory+therapeutic+effect+of+glatiramer+acetate+on+several+murine+models+of+inflammatory+bowel+disease%5BTitle%5D)+AND+%22J+Pharmacol+Exp+Ther%22%5BJournal%5D)">Immunomodulatory therapeutic effect of glatiramer acetate on several murine models of inflammatory bowel disease.</a> J Pharmacol Exp Ther. 318 (1), 68-78 (2006).</li>
<li>Hahm, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loss+of+transforming+growth+factor+beta+signalling+in+the+intestine+contributes+to+tissue+injury+in+inflammatory+bowel+disease%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">Loss of transforming growth factor beta signalling in the intestine contributes to tissue injury in inflammatory bowel disease.</a> Gut. 49 (2), 190-198 (2001).</li>
<li>Green, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adrenergic+modulation+of+Escherichia+coli+O157%3AH7+adherence+to+the+colonic+mucosa%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Adrenergic modulation of Escherichia coli O157:H7 adherence to the colonic mucosa.</a> Am J Physiol Gastrointest Liver Physiol. 287 (6), G1238-1246 (2004).</li>
<li>Dieleman, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+experimental+colitis+induced+by+dextran+sulphate+sodium+%28DSS%29+is+characterized+by+Th1+and+Th2+cytokines%5BTitle%5D)+AND+%22Clin+Exp+Immunol%22%5BJournal%5D)">Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines.</a> Clin Exp Immunol. 114 (3), 385-391 (1998).</li>
</ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by Vanderbilt IACUC.

This protocol can be modified to model acute injury, injury-repair, or chronic colonic injury processes.
Acute Injury Modification:
DSS treatment ad lib for 4-6 days followed by immediate sacrifice
Injury-Repair Modification:
Injury with 4-6 days of DSS treatment followed by recovery period of 3-4 days and sacrifice (as described in above protocol).
Chronic Colitis Modification:
<p ...

<table><tbody><tr><td>Dextran Sulfate Sodium Salt</td><td>USB Corp., Affymetrix</td><td>9011-18-1</td><td>mol wt 40,000-50,000</td></tr><tr><td>10% Buffered Formalin Phosphate</td><td>Fisher Scientific</td><td>SF100-4</td><td></td></tr><tr><td>Isoflurane, USP</td><td>Phoenix Pharmaceuticals, Inc.</td><td>NDC 57319-474-06</td><td></td></tr><tr><td>TISSUE PATH Macrosette Cassettes</td><td>Fisher Scientific</td><td>15182706</td><td></td></tr><tr><td>BD 10 ml Syringe</td><td>BD Biosciences</td><td>309604</td><td></td></tr><tr><td>Ethyl Alcohol</td><td>Pharmco-AAPER</td><td>E190</td><td>Dilute to 70% with distilled water</td></tr><tr><td>20G Straight Feeding Needle</td><td>VWR international</td><td>20068-612</td><td></td></tr><tr><td>27G1/2 PrecisionGlide Needle</td><td>BD Biosciences</td><td>305109</td><td></td></tr><tr><td>Dissecting Scissors Sharp/Blunt Tip</td><td>VWR international</td><td>82027-588</td><td></td></tr><tr><td>Waugh Forceps</td><td>VWR international</td><td>82027-428</td><td></td></tr><tr><td>Non-absorbable Suture </td><td>LOOK Surgical</td><td>SP116</td><td></td></tr><tr><td>Whatman Blotting Paper</td><td>VWR international</td><td>28298-020</td><td>Cut as needed</td></tr><tr><td>Nalgene Surfactant-Free Cellulose Acetate (SFCA) Filter</td><td>Cole-Parmer</td><td>EW-06731-2</td><td></td></tr><tr><td>Carbon fiber composites digital caliper</td><td>Fisher Scientific</td><td>15-007-958</td><td></td></tr></tbody></table>

murine colitis modeling using dextran sulfate sodium (dss)

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of Crohn&rsquo;s Disease - Crohn&rsquo;s Colitis. Acute colitis may present with abdominal pain and distention, malabsorption, diarrhea, hematochezia and mucus in the stool. We are beginning to understand the complex interactions between the environment, genetics, and epithelial barrier dysfunction in Inflammatory Bowel Disease and animal models of colitis have been essential in advancing our understanding of this disease. One popular model involves supplementing the drinking water of mice with low-molecular weight Dextran Sodium Sulfate (DSS), resulting in epithelial damage and a robust inflammatory response in the colon lasting several days 1.Variations of this approach can be used to model acute injury, acute injury followed by repair, and repeated cycles of DSS interspersed with recovery modeling chronic inflammatory diseases 2. After a single four-day treatment of 3% DSS in drinking water, mice show signs of acute colitis including weight loss, bloody stools, and diarrhea. Mice are euthanized at the conclusion of the treatment course and at necropsy dissected colons are processed and can be 'Swiss rolled" 3 to allow microscopic analysis of the entire colon or infused with formalin as "sausages" to allow macroscopic analysis. Tissue is then embedded in paraffin, sectioned, and stained for histologic review.

Dextran sulfate sodium (DSS) administered in the drinking water is an established murine inflammatory injury model of acute colitis. This protocol outlines the method for DSS treatment and the preparation of tissues.

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of ...

Mice are given 3%DSS at libido for four days. While weight and presence of diarrhea and fecal blood is recorded daily TSS is replaced by autoclave water after day four. After three days on water, mice are euthanized and their colons are removed.Colons are measured and weighed. The colons are then either formed into Swiss rolls or sausages and examined under a microscope. This video article will demonstrate the harvesting and processing of colons in a murn model of colitis.This procedure is used to study colitis in the laboratory of Dr.Christopher Williams of the Department of Cancer Biology at Vanderbilt University prior to beginning dextrin sulfate sodium treatment. Baseline weights are obtained throughout the course of the experiment. Daily weights are taken after the mice have been weighed, make a 3%weight per volume, dextrin sulfate sodium salt solution, or DSS in water.The DSS must be of a low molecular weight for this model to be effective, make enough DSS solution to fill a water bottle for each cage of mice and filter the solution with a 0.45 micrometer cellulose acetate filter. Replace the drinking water bottle in the mouse cage with a bottle containing 3%DSS solution. If your institution uses an automated watering system, place exclusion tips on watering spouts to ensure that the mice do not have access to any other source of water.After four days, the mice are weighed again to quantify systemic consequences of colitis. Weight loss is common with severe injury, however, may not be apparent for several more days on the same day. Day four, replace the DSS solution with water for an additional three days to allow some colonic epithelial recovery.Three days later, day seven, the mice are weighed again. The colon can then be harvested from the euthanized mice as shown in the next section. To harvest the colon, expose the ventral side of the euthanized mouse to ensure unobstructed access to the abdomen.Wet the abdomen completely with 70%ethanol. Grasp the midline of the abdomen with tissue forceps and elevate. Thus tenting the skin using fine tipped scissors.Incise the abdomen to expose the abdominal contents. Extend the incision to the tip of the xiphoid process at the midline, and then extend along the inferior aspect of the costal margins bilaterally. Identify the small intestine cecum and colon.Carefully dissect slash tease the colon from the surrounding mesentery. After that, transect the colon deepen the pelvis to free. The distal colon from the rectum.Care should be taken so that no arteries or veins are cut. Also, be aware that the colon can tear easily. So gently remove the colon from the mesentery.Transect the colon at the Colona sequel margin to free the proximal colon. Remove the colon and total using a 20 gauge feeding needle and 10 milliliter syringe intubate and flush the colon with ice cold PBS until the EIT is completely clear of stool. It is important to maintain the correct orientation of the colon by keeping the distal end of the colon closest to you.Start by measuring the colon length using a digital caliper. Colon length is another indicator of the severity of injury. Colitis increases edema and shortens the overall colon length.At this point, the harvested colons can either be processed as sausages for macroscopic analysis or made into Swiss roll for histological analysis of acute colitis. We will demonstrate these techniques in the following sections to process the colon as a sausage For macroscopic analysis of the entire colon, it is important to maintain the correct orientation of the colon. Therefore, keep the distal end of the colon closest to you.Cut two pieces of non-absorbable suture, one approximately one inch in length and the other two inches in length. Use the two inch piece of suture to tie off the distal end of the colon as close to the margin as possible while still maintaining a good seal. Then place a 20 gauge feeding needle containing five milliliters of 10%buffered formalin phosphate into the proximal end of the colon.Loosely tie the remaining piece of suture immediately proximal to the bulb of the feeding needle. Now grasp the colon firmly at the bulb of the feeding needle and infuse sufficient amount of formalin ensuring the colon is expanded. Tighten the knot in the suture as you withdraw the feeding needle, thus leaving the infused expanded colon.As a sausage, fill a 15 milliliter conical tube with 10%buffered, formal and phosphate, and label the tube. Place the colon in the tube and fix for 24 hours. After 24 hours, pour off the formin into an appropriate waste container and replace it with 70%ethanol for an additional 24 hours.Colons can be stored in 70%ethanol indefinitely at room temperature. When you are ready to analyze the colon, remove it from the conical tube. Cut the strings on each end with a scalpel being careful not to damage the colon.Remember that the distal end has the longest piece of string. Cut the colon longitudinally from the distal to proximal end so that it forms a long sheet. The colon can now be viewed under a dissecting microscope.Cut the colon longitudinally and lay out the colon as a flat sheet. Rolling the colon requires two pairs of forceps and a two handed technique. Grasp both lateral edges of the distal colon margin with forceps proceed to sequentially roll the colon by rotating and releasing the forceps proceeding to the proximal margin, thus generating a spiral with a third dimension or Swiss roll.If the colon adheres to the watman paper, wet the paper and colon with a small amount of PBS to maintain the form. Grasp the roll firmly with forceps and transect with a 27 and a half gauge needle. Secure the roll by bending the needle as it exits the roll.Now, place the roll in a labeled tissue cassette and place the cassette in a jar of 10%buffered formalin phosphate. Leave it room temperature for 24 hours to fix the tissue. After 24 hours, pour off the formalin and replace with 70%ethanol for an additional to four hours.Colons can be stored in 70%ethanol indefinitely at room temperature. This model allows the researcher to obtain, fix and analyze colons, thus allowing for analysis of severity of colitis. For macroscopic analysis of the entire colon, the colon can be processed as a sausage that is infused with formalin and completely expanded.The open sausage should lie flat. If the sausage method has been performed correctly, the fixed colon will be dilated and the entire mucosal surface can be easily manipulated and viewed under the dissecting microscope. For histological analysis, the colon can be processed as a Swiss roll.The roll is secured with a needle and placed in a tissue cassette for fixation. When the roll is cut and mounted, it should form a representative slice of the entire colon if rolled properly, sections of the Swiss roll can be stained with hematin and ASIN in order to determine the extent of colonic injury. Remembering the distal end of the colon is in the center of the roll on the slide.In this example, inflammation and crypt damage are apparent in the DSS treated colon compared to a water treated control. We have just shown you how to harvest and process colons from a murine model of acute colitis. When performing this procedure, it's important that you are well organized prior to sacrificing.The mice, for example, have all tubes and cassettes labeled for each mouse and all of the tools and reagents readily available. In addition, maintain detailed records of mouse weight and colon length. Finally, make sure that you have allotted enough time for processing.It usually takes 15 minutes per mouse for a novice and six to eight minutes for someone more experienced in this technique.

murine-colitis-modeling-using-dextran-sulfate-sodium-dss

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Learn to induce acute colitis in mice using dextran sulfate sodium in drinking water, followed by tissue preparation for histological analysis. This standardized protocol enables reproducible inflammatory bowel disease research.

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

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