Name: a technique to generate a murine model of dextran sulfate sodium-induced colitis
Uploaded: 2023-08-31T09:44:30.000+00:00
Duration: 1 min 52 s
Description: Source: Du, Y., et al. Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model. J. Vis. Exp. ...

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

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

To induce dextran sulfate sodium, or DSS colitis, replace the regular drinking water with 3% DSS solution ad libitum for seven days, measuring each mouse&#39;s body weight, DSS solution consumption, and stool production daily. At the end of the seven-day treatment, place the mouse in the supine position, and use small scissors to make a three-centimeter-long midline incision in the abdomen.
Then, use forceps to lift the colon to allow it to be separated from the surrounding mesentery, and extract the whole colon until the cecum and rectum are visible. Transect the tissue at the colonocecal margin and deep within the pelvis, to free the proximal and distal colon respectively.
Measure and record the length of the isolated colon, and use a 10-milliliter syringe equipped with a gavage needle to flush the colon with 10 milliliters of ice-cold PBS to remove the feces and blood until the eluate runs clear. For histological identification, divide the tissue samples into equally-sized proximal, middle, and distal sections, and fix the tissues in 4% paraformaldehyde overnight at four degrees Celsius.

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Nanyang Technological University

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JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host Defense Mechanism

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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>

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.

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

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