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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of Mice and DSS
<ol>
	<li>Keep the knockout (&#945;-gustducin-/-) mice and age-, gender-, and body weight-matched wild-type control (&#945;-gustducin+/+) C57BL/6 mice individually in clean cages. 
	NOTE: The knockout mice have been backcrossed with C57BL/6 mice for over 20 generations and have a nearly 100% C57BL/6 genetic background.</li>
	<li>Dissolve 30 g of dextran sulfate sodium (DSS) powder in 1 L of autoclaved water. Mix until the solution becomes clear to ensure that the final working concentration is 3% (w/v). 
	NOTE: The DSS solution can be stored at room temperature for up to 1 week or at 4 &#176;C until use.</li>
</ol>
2. Induction and Evaluation of DSS Colitis in Mice
<ol>
	<li>Weigh and record each mouse&#39;s initial body weight. Place the mice individually into standard plastic cages and label the cages.</li>
	<li>Replace regular drinking water with 3% DSS solution for a total of 7 days to which both groups of mice have access ad libitum.</li>
	<li>Measure the mouse&#39;s body weight, record DSS solution consumption, and collect and examine the stool of each mouse daily during the DSS administration. Observe the severity of diarrhea and rectal bleeding and convert this to the DSS-induced disease index.</li>
	<li>During the experiment, the percentage of weight loss compared to initial weight and the disease index are calculated to evaluate the symptoms of colitis. 
	NOTE: The disease index is scored by combining observations of diarrhea and rectal bleeding and is defined as follows: 0 (normal stool, no blood), 1 (soft stool, no blood), 2 (soft stool, little blood), 3 (very soft stool, modest bleeding), and 4 (watery stool, significant bleeding). The disease index is analyzed daily for each mouse.</li>
	<li>By the end of the 7-day DSS treatment, sacrifice the mice by cervical dislocation and proceed with the remaining experiments.</li>
</ol>
3. Preparation of Tissue Samples
<ol>
	<li>Place the mouse in the supine position and clean the skin of the abdomen with 70% ethanol. Make a 3 cm-long midline incision in the abdomen with a pair of small scissors to expose the abdominal cavity.</li>
	<li>Use a pair of forceps to carefully separate the spleen from other tissues, then remove the spleen and measure its size.</li>
	<li>Identify and lift the colon with forceps and separate it from the surrounding mesentery. Pull out the whole colon until the cecum and rectum are visible.</li>
	<li>Isolate the colon by transecting it at the colonocecal margin and deep in the pelvis to free the proximal and distal colon, respectively. Then, measure and record the length of the isolated colon. Be careful not to damage the colonic tissue during the dissecting procedure.</li>
	<li>Flush the colon with 10 mL of ice-cold phosphate-buffered saline (PBS) with a 10 mL syringe equipped with a gavage needle to remove the feces and blood until the eluate is completely clear.</li>
	<li>For histological identification, divide the tissue samples equally into three parts: proximal, middle, and distal. Then, fix the tissue with 4% paraformaldehyde (PFA) overnight at 4 &#176;C.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran Sulfate Sodium Salt (DSS)</td>
			<td>MP Biomedicals</td>
			<td>2160110</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Sangon Biotech</td>
			<td>B548117</td>
		</tr>
		<tr>
			<td>BD 10 ml Syringe</td>
			<td>BD Biosciences</td>
			<td>309604</td>
		</tr>
		<tr>
			<td>Instruments and equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Balance</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a technique to generate a murine model of dextran sulfate sodium-induced colitis

Source: Du, Y., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/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.

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

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

To induce colitis &#8212; an inflammatory bowel disease &#8212; in a murine model, take a mouse housed in a cage.
Replace the drinking water with a solution of dextran sulfate sodium, DSS, a colitis-inducing polysaccharide. Allow the mouse to consume the solution to develop colitis.
Upon consumption, DSS enters the lumen of the mouse&#39;s colon. The colon contains a thick mucus layer, providing a physical barrier between the commensal microbes and the underlying epithelial cell monolayer.
DSS penetrates the mucus layer, entering the epithelial cells. Inside the cells, DSS alters the expression of tight-junction proteins and induces apoptosis, resulting in the disruption of the epithelial layer and mucosal barrier. This allows the entry of luminal microbes into the lamina propria &#8212; the underlying tissue with resident dendritic cells and macrophages.
The immune cells recognize the microbes, activating downstream signaling to secrete pro-inflammatory cytokines. Cytokines cause the infiltration of a large number of T cells which mount an exaggerated immune response, resulting in inflammation.
Monitor the treated mouse for gastrointestinal disorders and weight loss, indicating the onset of colitis.
Euthanize the mouse; surgically isolate the entire colon. Divide the colon into proximal, middle, and distal sections and add a fixative for tissue preservation.
The tissue is ready for downstream assays to evaluate colitis.

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191 Views. Source: Du, Y., et al. Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model. J. Vis. Exp. (2018) This video demonstrates a technique to generate a chemical-induced murine model of colitis — 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 t...

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

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. ...

JoVE Journal

Immunology and Infection

A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting approximately 20 per 1,000,000 in Europe and USA. CD is characterized by transmural inflammation, intestinal fibrosis, and luminal stenosis. Although anti-inflammatory therapies may help control inflammation, they have no efficacy on fibrosis and stenosis in CD. The pathogenesis of CD is not well understood. Current studies focus mainly on delineating dysregulated gut immune response mechanisms. While CD-associated transmural inflammation, intestinal fibrosis, and luminal stenosis all represent mechanical stress to the gut wall, the role of mechanical stress in CD is not well defined. To determine if mechanical stress plays an independent pathogenic role in CD, a protocol of TNBS-induced CD-like colitis model in rodents has been developed. This TNBS-induced transmural inflammation and fibrosis model resembles pathological hallmarks of CD in the colon. It is induced by intracolonic instillation of TNBS into the distal colon of adult Sprague-Dawley rats. In this model, transmural inflammation leads to stenosis at the TNBS instillation site (Site I). Mechanical distention is observed in the portion proximal to the instillation site (Site P), representing mechanical stress but not visible inflammation. Colonic portion distal to inflammation (Site D) presents neither inflammation nor mechanical stress. Distinctive changes of gene expression, immune response, fibrosis, and smooth muscle growth at different sites (P, I, and D) were observed, highlighting a profound impact of mechanical stress. Therefore, this model of CD-like colitis will help us better understand CD&#39;s pathogenic mechanisms, particularly the role of mechanical stress and mechanical stress-induced gene expression in immune dysregulation, intestinal fibrosis, and tissue remodeling in CD.

The present protocol describes the development of a Crohn's-like colitis model in rodents. Transmural inflammation leads to stenosis at the TNBS instillation site, and mechanical enlargement is observed in the segment proximal to the stenosis. These changes allow studying mechanical stress in colitis.

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

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

Transcript

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

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

A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4⁺ T Cells to Immunodeficient Mice