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 &mu;m 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 &ldquo;sausages&rdquo;. If histologic analysis only is desired than proceed to Part 4 &ldquo;Making Swiss Rolls for the Histological Analysis of Acute Colitis&rdquo;.</li>
</ol>
Part 3: Processing as &ldquo;Sausages&rdquo; 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 &ldquo;sausage&rdquo;. </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 &ldquo;Swiss Roll&rdquo;. 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.remotexs.ntu.edu.sg/files/ftp_upload/1652/1652fig1.jpg"><img src="/files/ftp_upload/1652/1652fig1tmb.jpg" alt="Figure 1" border="0" /></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 &mu;m 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.remotexs.ntu.edu.sg/files/ftp_upload/1652/1652fig2.jpg"><img src="/files/ftp_upload/1652/1652fig2tmb.jpg" alt="Figure 2" border="0" /></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.remotexs.ntu.edu.sg/files/ftp_upload/1652/1652fig3.jpg"><img src="/files/ftp_upload/1652/1652fig3tmb.jpg" alt="Figure 3" border="0" /></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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+in+the+induction+of+reliable+experimental+acute+and+chronic+ulcerative+colitis+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Mtgr1+sensitizes+the+colonic+epithelium+to+dextran+sodium+sulfate-induced+colitis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+"Swiss+roll":+a+simple+technique+for+histological+studies+of+the+rodent+intestine.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+susceptibility+of+inbred+mouse+strains+to+dextran+sulfate+sodium-induced+colitis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+therapeutic+effect+of+glatiramer+acetate+on+several+murine+models+of+inflammatory+bowel+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+transforming+growth+factor+beta+signalling+in+the+intestine+contributes+to+tissue+injury+in+inflammatory+bowel+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenergic+modulation+of+Escherichia+coli+O157:H7+adherence+to+the+colonic+mucosa.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+experimental+colitis+induced+by+dextran+sulphate+sodium+(DSS)+is+characterized+by+Th1+and+Th2+cytokines.">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:
Mice are placed on three five-day cycles of 3% DSS with sixteen days of recovery between each cycle. Mice are sacrificed after the final 16-day rest period.
There are several issues that researchers need to be aware of with this model:
<ol>
<li>Variability in site of injury: In our hands, we see a predominantly distal injury pattern, with relative sparing of the proximal colon and cecum. Others have reported more proximal predominance to the injury pattern.</li>
<li>Environmental variability. There is significant environmental variability. This is likely, in part, due to differences in intestinal microflora, diet and other environmental considerations.</li>
<li>This model is ineffective if high molecular weight DSS is used (i.e. 500,000 Da).</li>
<li>There is considerable strain variability with this model 4.</li>
</ol>
Biochemical analysis of the colon can be performed by taking samples from the proximal or distal margins. Depending on the severity of injury this may impact your ability to generate a histologic injury score. In vivo BrdU labeling to measure proliferation can be achieved by intraperitoneal injection of 16.7 &mu;g/kg BrdU 2 hrs prior to sacrifice followed by &alpha;-BrdU IHC processing.
As an alternative to the &ldquo;sausage&rdquo;, the colon can be fixed completely flat. Line the bottom of a Tupperware container with Whatman paper and soak the paper with 10% buffered formalin phosphate. Dissect the colon as described in &ldquo;Part 2: Necropsy and Harvesting of Colon&rdquo;. Again, keep the distal end of the colon closest to the operator. Using fine-tipped scissors, incise longitudinally from distal to proximal end of the colon. With fine tipped forceps grasp either edge of the incision and open laterally working, thus displaying the colon as a flat sheet. Transfer this flat sheet to the pre-soaked Whatman paper. Cover the flattened colon with another piece of Whatman and wet with 10% buffered formalin phosphate. The Whatman should be completely covered in formalin but not so much that it lifts off the colon. Seal the Tupperware and fix for 24 hours. Remove the colon from the Tupperware and place it in 10 ml 70% ethanol in a 15 ml conical tube. (Colons can be stored in 70% EtOH indefinitely at room temperature.)
There are several methods for quantifying colonic injury 5,6,7. One method, for example, uses a multi-parameter scale including: inflammation, extent involvement, regeneration, crypt damage, and percent involvement 8.

<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)

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.

Watch this Scientific Journal Video about Murine Colitis Modeling using Dextran Sulfate Sodium DSS at JoVE.com

Murine Colitis Modeling using Dextran Sulfate Sodium DSS

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.

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 ...

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

Nanyang Technological University

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JoVE Journal

Biology

49.8K Views.  Vanderbilt University. 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.

Video: Murine Colitis Modeling using Dextran Sulfate Sodium DSS

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

Medicine

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of experimental models needs to be as rigorous as that applied for human samples. Indeed, drawing reliable and accurate conclusions is critically influenced by the quality of tissue section preparation. Here, we describe a protocol for histopathological analysis of murine tissues that implements several automated steps during the procedure, from the initial preparation to the paraffin embedding of the murine samples. The reduction of methodological variables through rigorous protocol standardization from automated procedures contributes to increased overall reliability of murine pathological analysis. Specifically, this protocol describes the utilization of automated processing and embedding robotic systems, routinely used for the tissue processing and paraffin embedding of human samples, to process murine specimens of intestinal inflammation. We conclude that the reliability of histopathological examination of murine tissues is significantly increased upon introduction of standardized and automated techniques.

Lack of standardization for murine tissue processing reduces the quality of murine histopathological analysis as compared to human specimens. Here, we present a protocol to perform histopathological examination of murine inflamed and uninflamed colonic tissues to show the feasibility of robotic systems routinely used for processing and embedding&#160;human samples.

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of ...

Immunology and Infection

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. ...

Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal cancer, inflammatory bowel disease, and Parkinson&#8217;s disease. To study the function of the appendix, a gut disease-associated murine appendectomy model has been established and its step-by-step protocol is described here. This report introduces a facile protocol for caecal patch removal in mice followed by the chemical induction of chronic colitis-associated colorectal cancer using a combination of dextran sulfate sodium (DSS) and azoxymethane (AOM). IgA specific cells and IgA concentration were significantly reduced upon removal of the caecal patch in male C57BL/6 mice&#160;compared to those in the sham group. Simultaneously administering 2% DSS and AOM resulted in nearly 80% mice survival in both sham and appendectomy groups without significant body weight loss. Histological results confirmed colonic inflammation and different degrees of adenocarcinoma. This model can be used for the study of the functional role of the appendix in maintaining gut microbiota homeostasis and pathogenesis of gut colitis and malignancies, as well as for the potential development of drug targeting therapies.

The presented protocol describes a facile surgical removal of the appendix (caecal patch) in a mouse followed by the induction of inflammatory bowel disease-associated colorectal cancer. This murine appendectomy model enables investigation of the biological role of the appendix in the pathogenesis of human gastrointestinal disease.

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal ...

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.

Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa.

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier ...

Hematoxylin and Eosin Staining to Assess Colon Damage in DSS-Induced Colitis

Source: Du, Y., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58668">Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model.</a> J. Vis. Exp. (2018)
This video demonstrates the hematoxylin and eosin (H&#38;E) staining method to evaluate colon damage in a murine model of DSS-induced colitis &#8212; an inflammatory bowel disease. Upon isolating the colon from a DSS-treated mouse and obtaining sections of the tissue, H&#38;E staining of the sections helps identify the inflammation-mediated tissue damage.

Source: Du, Y., et al. Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host-Pathogen Interaction

A Technique to Generate a Murine Model of Dextran Sulfate Sodium-Induced Colitis

Source: Du, Y., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58668">Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model.</a> J. Vis. Exp. (2018)
This video demonstrates a technique to generate a chemical-induced murine model of colitis &#8212; an inflammatory bowel disease. The mouse is allowed to consume a dextran sodium sulfate (DSS) solution for an adequate duration. Upon consumption, DSS damages the mucus and epithelial layer barrier in the colon, allowing gut microbes to enter the underlying tissue &#8212; where they are sensed by the resident immune cells to mediate an exaggerated immune response, resulting in inflammation.