This work is supported by grants from Canadian Institutes of Health Research (CIHR) and by Crohn's and Colitis Foundation of Canada (CCFC).

1. Murine model of DSS-induced acute colitis
<ol>
	<li>Add dextran sulphate sodium (DSS) to autoclaved drinking water to the desired final concentration (1-5%) (wt/vol) (i.e. To make a 5% DSS solution, add 25 g of DSS powder to 500 mL of autoclaved water). Stock solution can be left at room temperature for up to one week or at 4&deg;C until use.</li>
	<li>In a biosafety hood, pour stock DSS solution into 50 mL Falcon tubes (one needed per cage). Keep the stock solution to refill tubes when needed.</li>
	<li>Replace drinking water in each mouse cage with the DSS solution (in the 50 mL Falcon tubes) (The duration will depend on the DSS regimen that is used. For example, using 6-8 week old male C57BL/6 mice, we administer a 5% DSS solution for a total of five days). Mice should not have access to any other water source. Control mice are given autoclaved drinking water without DSS.</li>
	<li>Weigh mice daily and record the amount of DSS consumed per day. Top each bottle to 50 mL after recording DSS levels. This is to measure the approximate volume of DSS consumed per cage per mouse throughout the duration of the experiment. In our studies, we use a 5% DSS solution for 5 days with male C57BL/6 mice. Significant weight loss, altered stool consistency and signs of fecal blood are seen as early as day 3 using this particular DSS regimen. During the DSS administration, mice exhibit pronounced weight loss (around 5-10% of their initial weight on day 5) with weight loss greater than 20% of initial weight with dehydration and diarrhea to be the significant physiological indicator that an animal is at or near endpoint. If the animal is given access to normal water after the 5 days of 5% DSS, it will recover within 7 days. All experiments should be approved by the institution's animal ethics committee and be in accordance with the approved Animal Utilization Protocol (AUP).</li>
	<li>During the duration of the experiment, a disease activity index (DAI) score can be assessed to evaluate the clinical progression of colitis. The DAI is the combined score of weight loss compared to initial weight, stool consistency, and bleeding. Scores are defined as follows: weight loss: 0 (no loss), 1 (1-5%), 2 (5-10%), 3 (10-20%), and 4 (&gt;20%); stool consistency: 0 (normal), 2 (loose stool), and 4 (diarrhea); and bleeding: 0 (no blood), 1 (Hemoccult positive), 2 (Hemoccult positive and visual pellet bleeding), and 4 (gross bleeding, blood around anus). DAI can be scored daily during the duration of the DSS treatment.</li>
	<li>At the time point of choice, weigh and sacrifice mice. Mice can be euthanized by cervical dislocation following inhalation of isoflurane or by another method approved by the institution's animal facility.</li>
</ol>
2. Collect colonic tissue samples
<ol>
	<li>Expose the ventral side of the animal and wet the abdomen area with a 70% ethanol solution. At this point, refer to Table 1 and make note of any signs of rectal bleeding (blood present at the anal orifice) or rectal prolapse in each animal.</li>
	<li>Use standard dissecting scissors to incise the abdomen by making a ventral midline incision.</li>
	<li>Locate the colon and transect the colon as close to the colorectal margin as possible to free the distal colon.</li>
	<li>Carefully and slowly pull out the whole colon, detaching it from the surrounding mesentery.</li>
	<li>Transect the colon at the colonocecal margin to free the proximal end of the colon. Feces can be removed by rinsing colon with sterile PBS by using a gavage needle attached to a 3 or 5 mL syringe or by carefully squeezing it out using a pair of bent tweezers/forceps. Using the whole colon, assess for damage (See Section 3.1)</li>
	<li>Tissue sampling for histological analysis and other assays can be done by cutting 0.5 cm to 1.0 cm long colonic fragments making note of which area the sample is from (i.e. proximal, middle, or distal).</li>
	<li>Tissue samples to be used for assays can be individually placed in 1.5 mL eppendorf tubes and frozen in liquid nitrogen and stored until use at -70&deg;C.</li>
</ol>
3. Assessment of colitis severity
<ol>
	<li>Macroscopic or disease severity score is assessed terminally by an unbiased observer using a previously published scoring system (Table 1).9 Stool consistency can be assessed by using a pair of forceps and pressing down on the feces to determine consistency. To determine a score for blood in the feces, note the colour of the feces (i.e. black stool versus light brown stool) and further validate using a Hemoccult test. Using the scoring system, determine a score for each of the conditions. The final macroscopic score for each animal is the sum of each individual score.</li>
	<li>To evaluate histological damage of colitis severity, cut a small fragment (0.5 cm) of the colon, place in a tissue cassette and submerge in buffered 10% formalin solution. Prepare 5 &mu;m paraffin embedded cross sections and stain sections with hematoxylin/eoxin (H&amp;E) using the appropriate procedures. Colon fragments can be taken from the proximal, mid-colon, or distal section of the colon.</li>
	<li>H&amp;E stained colonic tissue sections are scored by a blinded observer using a previously published system for the following measures: crypt architecture (normal, 0 - severe crypt distortion with loss of entire crypts, 3), degree of inflammatory cell infiltration (normal, 0 - dense inflammatory infiltrate, 3), muscle thickening (base of crypt sits on the muscularis mucosae, 0 - marked muscle thickening present, 3), goblet cell depletion (absent, 0- present, 1) and crypt abscess (absent, 0- present, 1).7 The histological damage score is the sum of each individual score. It should be noted that unlike human UC, crypt abscesses are not characteristic of this model and are rarely seen; microscopic ulcerations are also rare. If multiple colon sections were stained, histological scores between similar sections should be used to determine the final score for each area (i.e. histological score in proximal colon versus histological score in distal colon).</li>
</ol>
4. Prepare stock solutions of reagents for assays
<ol>
	<li>Prepare a 50 mM solution of Potassium Phosphate buffer by adding solution B (K2HPO4, 8.7 g of dibasic potassium phosphate in 1 L of dH2O) to solution A (KH2PO4, 6.8 g of monobasic potassium phosphate in 1L of dH2O) until a pH of 6.0 is achieved. Remaining solutions can be stored in fridge (2-8&deg;C) until future use.</li>
	<li>Prepare hexadecyltrimethylammonium bromide (HTAB) buffer by adding 5 g HTAB into 1 L of Potassium Phosphate buffer (50 mM, pH=6.0). Gently heat to dissolve and store at 2-8&deg;C until use. When required, heat to re-dissolve.</li>
	<li>Prepare lysis buffer for tissue homogenization for protein analysis by adding 10 mL of 1M tris-hydrochloric acid (pH=8.0), 6 mL of 5M sodium chloride and 2 mL of Triton X-100 to 182 mL of sterilized distilled water. Triton X-100 is very viscous at room temperature and thus, should be gently warmed prior to use. The prepared lysis buffer can be stored at -20&deg;C until use.</li>
</ol>
5. Sample preparation for assays
<ol>
	<li>Sample preparation for MPO analysis.
	<ol>
		<li>Remove samples from -70&deg;C and place on ice. Record the weight of each sample after removing any visible feces or fat by using a bent forcep/tweezer and place into a 2mL Eppendorf Safe-Lock microcentrifuge tube (or any tube that can be used with a homogenizer). Samples should be kept on ice at all times. It is important to note that similar colon fragments should be used from each biological replicate (i.e. distal sections only or proximal sections only).</li>
		<li>Add homogenizer bead to each sample tube.</li>
		<li>Add the appropriate amount of HTAB buffer according to tissue weight. If tissue weight is less than 25 mg, add the buffer at a ratio of 12.5mg/mL; if tissue weight is between 25-50, add at a ratio of 25mg/mL. If tissue weight is greater than 50, add buffer at a ratio of 50mg/mL.</li>
		<li>Homogenize with a tissue homogenizer for 4 min at 30 Hz. Repeat if tissue is not fully homogenized.</li>
		<li>Remove homogenizer bead and centrifuge solution for 6 min (13400 x g, 4&deg;C).</li>
		<li>Collect supernatant and discard the resulting pellet. Supernatent can be stored at -70&deg;C until use.</li>
	</ol>
	</li>
	<li>Sample preparation for cytokine analysis.
	<ol>
		<li>Repeat steps 5.1.1. to 5.1.2.</li>
		<li>Add 50 &mu;l of protease inhibitor cocktail (PIC) to 10 mL of prepared lysis buffer.</li>
		<li>Add 1 mL of the PIC and lysis buffer solution to each sample irrespective of weight.</li>
		<li>Homogenize for 5 min at 30 Hz. Repeat if tissue is not fully homogenized.</li>
		<li>Remove homogenizer bead and centrifuge solution for 5 min at 3300 x g.</li>
		<li>Collect supernatant and discard the resulting pellet. Supernatant can be stored at -70&deg;C until use.</li>
	</ol>
	</li>
</ol>
6. Quantification of inflammatory markers
<ol>
	<li>MPO activity assay
	<ol>
		<li>Prepare o-dianisidine dihydrochloride (o-dianisidine) solution by combining 16.7 mg of o-dianisidine dihydrochloride, 90 mL of dH2O, and 10 mL of potassium phosphate buffer. This solution should be prepared fresh for every assay.</li>
		<li>Add 7 &mu;L of tissue homogenate (prepared in section 5.1) in triplicate into a 96-well plate.</li>
		<li>Add 50 &mu;L of diluted H2O2 (4 &mu;L of 30% H2O2 diluted in 96 &mu;L of dH2O) to the o-dianisidine mixture.</li>
		<li>Use a multi-channel pipette to add 200 &mu;L of the o-dianisidine mixture containing H2O2 to each of the wells.</li>
		<li>Measure absorbance at 450 nm using a spectrophotometer. Take three readings at 30 second intervals.</li>
		<li>Calculate MPO activity. MPO activity is measured in units (U) of MPO/mg tissue, where one unit of MPO is defined as the amount needed to degrade 1 &mu;moL of H2O2 per minute at room temperature. Considering that one unit (U) of MPO= 1 &mu;moL of H2O2 split and that 1 &mu;moL of H2O2 gives a change of absorbance of 1.13 x 10-2 nm/min, units of MPO in each sample is determined as change in absorbance [&Delta;A(t2-t1)]/&Delta;min x (1.13 x 10-2). To get units per mg of tissue, use the tissue: buffer ratio. For example, if a tissue: buffer ratio of 50 mg/mL was used, in 7 &mu;L of homogenate, there is 0.35 mg of tissue. Therefore, to get units per mg tissue, divide the units of MPO by 0.35. A sample calculation using absorbance values (nm) is included below (assuming that the sample has been added in triplicate):</li>
	</ol>
	</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td>Sample</td>
			<td colspan="3">Time 0 sec</td>
			<td colspan="3">Time 30 sec</td>
			<td colspan="3">Time 60 sec</td>
		</tr>
		<tr>
			<td>A</td>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>1'</td>
			<td>2'</td>
			<td>3'</td>
			<td>1"</td>
			<td>2''</td>
			<td>3"</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>0.048</td>
			<td>0.048</td>
			<td>0.051</td>
			<td>0.061</td>
			<td>0.061</td>
			<td>0.065</td>
			<td>0.074</td>
			<td>0.073</td>
			<td>0.078</td>
		</tr>
	</tbody>
</table>
<ol class="lroman">
	<li>Average at Time 0 sec= (0.048 + 0.048 + 0.051)/3= 0.049 nm</li>
	<li>Average at Time 30 sec= 0.0623 nm</li>
	<li>Average at Time 60 sec= 0.075 nm</li>
	<li>Change in absorbance (&Delta;A) from 0 to 30 sec/ mg tissue (assuming 50 mg/mL of tissue: buffer ratio)= [(0.0623-0.049)/(1.13 x 10-2)]/0.35= 3.363</li>
	<li>Change in absorbance (&Delta;A) from 30 to 60 sec/ mg tissue= 3.211</li>
	<li>*MPO activity (U/mg tissue)= average of &Delta;A(0-30) and &Delta;A(30-60)= 3.287</li>
</ol>
<ol start="2">
	<li>Quantification of pro-inflammatory cytokines by ELISA
	<ol>
		<li>Cytokine (IL-1&beta;, IL-6 and TNF-&alpha;) levels are determined using commercially available enzyme-linked immunosorbent assay (ELISA) kit (Quantikine Murine; R&amp;D Systems).</li>
		<li>Absorbance values from each ELISA is normalized using a Bradford protein assay respective to each sample and is expressed in units of pg/mg of protein.</li>
	</ol>
	</li>
</ol>
7. Representative Results
Administration of the appropriate DSS regimen will induce acute colitis in mice. During the duration of the DSS treatment, DAI can be used to assess and evaluate the clinical progression of disease. Animals treated with DSS will show significant weight loss compared to their initial weights, loose stools and fecal bleeding (Figure 1). Upon sacrifice and examination of the colon, the severity of colitis is macroscopically scored based on shortening of colon length, colonic bleeding, fecal bleeding, loosening of stool consistency, and signs of rectal bleeding compared to controls treated with water only (Figure 2 &amp; Table 1). Cross sections of colonic tissue samples stained with H&amp;E will have higher histological scores for DSS-treated colons versus water-treated controls (Figure 3). To further characterize the extent of inflammation in DSS-treated mice, MPO activity can be assessed from homogenized colonic tissue samples. DSS-treated colons will have higher MPO activity compared to controls (Figure 4). In addition, this is associated with increased levels of pro-inflammatory cytokines (IL-1&beta;, IL-6, TNF-&alpha;) (Figure 5).
<img alt="Figure 1." src="/files/ftp_upload/3678/3678fig1.jpg" /> 
Figure 1. Male, C57BL/6 mice were given 5% DSS in drinking water for 5 days. DAI scores were assessed daily for each animal and were averaged per day for each group (mean &plusmn; SEM, n=4 mice/group).
<img alt="Figure 2." src="/files/ftp_upload/3678/3678fig2.jpg" /> 
Figure 2. C57BL/6 mice were given 5% DSS in drinking water for 5 days. Control mice received water without DSS. Macroscopic damage score/disease severity scores were blindly assessed on day 5 post DSS-induced colitis. Colons isolated from mice that received DSS have higher macroscopic damage scores (rectal bleeding, rectal prolapse, diarrhea, colonic bleeding) indicating greater disease severity (mean &plusmn; SEM, n=4 mice/group).
<img src="/files/ftp_upload/3678/3678fig3.jpg" alt="Figure 3" /> 
Figure 3. C57BL/6 mice were given 5% DSS solution in drinking water in order to induce colitis. Control mice received water without DSS. (A) Histological scores were blindly scored using H&amp;E stained colonic tissue sections collected on day 5 post-DSS administration. (B) DSS-treated samples show more histological damage (more cellular infiltration, more goblet cell depletion, greater distortion/damage to crypt architecture) compared to (C) controls (mean &plusmn; SEM, n=4 mice/group). In (B) and (C), asterisk (*) indicates area of goblet cell depletion and distortion of crypt architecture; number sign (#) indicates cellular infiltration. 
<img alt="Figure 4." src="/files/ftp_upload/3678/3678fig4.jpg" /> 
Figure 4. All mice were sacrificed on day 5 post administration of DSS and colonic tissue samples were collected to assess MPO activity. Severity of DSS induced colitis is associated with higher levels of MPO activity compared to controls (mean &plusmn; SEM, n=4 mice/group).
<img alt="Figure 5." src="/files/ftp_upload/3678/3678fig5.jpg" /> 
Figure 5. In addition to higher MPO levels, severity of DSS-induced colitis is also associated with an increased level of pro-inflammation cytokines such as IL-1&beta;, IL-6, TNF-&alpha; (mean &plusmn; SEM, n=4 mice/group).
<table border="1">
	<tbody>
		<tr>
			<td>Score</td>
			<td>Rectal bleeding</td>
			<td>Rectal Prolapse</td>
			<td>Stool Consistency</td>
			<td>Blood</td>
		</tr>
		<tr>
			<td>0</td>
			<td>None</td>
			<td>None</td>
			<td>Normal</td>
			<td>Normal</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Red</td>
			<td>Signs of prolapse</td>
			<td>Soft</td>
			<td>Red</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Dark Red</td>
			<td>Clear prolapse</td>
			<td>Very Soft</td>
			<td>Dark red</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Gross Bleeding</td>
			<td>Extensive prolapse</td>
			<td>Diarrhea</td>
			<td>Black</td>
		</tr>
	</tbody>
</table>
Table 1. Macroscopic/Disease Severity Score

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No conflicts of interest declared.

DSS colitis is a widely used chemically induced model of intestinal inflammation. In this model, mice are given drinking water supplemented with DSS, which is thought to be toxic to gut epithelial cells and disrupt the integrity of the mucosal barrier.10 Administration of DSS induces an acute colitis that is characterized by loose stool, fecal bleeding, and infiltration with granulocytes.10 During DSS administration, colitis is usually associated with significant weight loss and presence of blood in...

<table border="1">
<tbody>
 <tr>
 <td>Name of reagent/equipment:</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Eppendorf Safe-Lock Microcentrifuge Tube (2mL)</td>
 <td>eppendorf</td>
 <td>0030 120.094</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Biotek EL808 Absorbance plate reader </td>
 <td>Biotek </td>
 <td>EL808 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dextran sulphate sodium salt reagent grade (molecular weight 36,000-50,000 Da)</td>
 <td>MP Biomedicals</td>
 <td>160110</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Gen5 (software)</td>
 <td>Biotek</td>
 <td>Version 1.10.8</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hexadecyltrimethylammonium bromide (HTAB)</td>
 <td>Sigma-Aldrich</td>
 <td>H5882-100G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hydrogen peroxide, 30 wt.% solution in water</td>
 <td>Sigma-Aldrich</td>
 <td>216763</td>
 <td>Store at 2-8&deg;C</td>
 </tr>
 <tr>
 <td>Microtest plate 96-well flat bottom</td>
 <td>Sarstedt</td>
 <td>82.1581</td>
 <td>For single use only</td>
 </tr>
 <tr>
 <td>o-Dianisidine</td>
 <td>Sigma-Aldrich</td>
 <td>D-3252</td>
 <td>Light sensitive. Store at 2-8&deg;C</td>
 </tr>
 <tr>
 <td>Potassium phosphate, dibasic</td>
 <td>Caledon</td>
 <td>6620-1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium phosphate, monobasic</td>
 <td>EMD Chemicals</td>
 <td>PX1565-1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Protease inhibitor cocktail</td>
 <td>Sigma-Aldrich </td>
 <td>P8340</td>
 <td>Store at -20&deg;C</td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>Sigma-Aldrich</td>
 <td>T8787</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tungsten Carbide beads for Tissue Lyser II</td>
 <td>QUIAGEN</td>
 <td>69997</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Investigating Intestinal Inflammation in DSS-induced Model of IBD at JoVE.com

Investigating Intestinal Inflammation in DSS-induced Model of IBD

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 ...

investigating-intestinal-inflammation-in-dss-induced-model-of-ibd

Research

JoVE Journal

Biology

66.8K Views.  McMaster University. Modelli sperimentali di malattia infiammatoria intestinale, ci hanno permesso di esaminare il complesso di risposte innate e adattative immunitario associate con patogenesi. Utilizzando il punteggio istologico, la quantificazione di citochine pro-infiammatorie e di attività mieloperossidasi, si può cominciare a valutare queste risposte visto nella malattia infiammatoria intestinale.

Video: Investigating Intestinal Inflammation in DSS-induced Model of IBD

Catheterization of Intestinal Loops in Ruminants

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the outside world. As such, the intestine plays a pivotal role in immunosurveillance and protection from enteric pathogens. Investigating intestinal physiology and immunology commonly employs 'intestinal loops' as an experimental model. The majority of these loop models are non-recovery surgical procedures that study short-term (&lt;24 hr) changes in the intestine (1-3). We previously created a recovery intestinal loop model to specifically measure long-term (&lt;6 mo) immunological changes in the intestine of sheep following exposure to vaccines, adjuvants, and viruses (4). This procedure localized treatments to a specific 'loop', allowing us to sample this area of the intestine. A significant drawback of this method is the single window of opportunity to administer treatments (i.e. at the time of surgery). Furthermore, samples of both the intestinal mucosa and luminal contents can only be taken at the termination of the project. Other salient limitations of the above model are that the surgical manipulation and requisite post-operative measures (e.g. administration of antibiotics and analgesics) can directly affect the treatment itself and/or alter immune function, thereby confounding results. Therefore, we modified our intestinal loop model by inserting long-term catheters into the loops. Sheep recover fully from the procedure, and are unaffected by the exteriorized catheters. Notably, the establishment of catheters in loops allows us to introduce multiple treatments over an extended interval, following recovery from surgery and clearance of drugs administered during surgery and the post-operative period.

We describe a novel surgical method for catheterizing 'intestinal loops' within the ileum of sheep. Once animals have recovered from surgery and have cleared antibiotics and analgesics, multiple treatments can be deposited directly in loops via the catheters.

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the ...

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high concentrations in specific and tertiary granules of neutrophils. This protocol details how MUB40&#160;conjugated directly to a fluorophore can be used to stain neutrophils in fixed/permeabilized tissues as well as how this can be used in live-cell imaging to assay for neutrophil activation and de-granulation. Neutrophil detection methods are limited to species-specific monoclonal antibodies, which are not always suitable for certain applications. MUB40 does not penetrate the cell membrane and is thus excluded from lactoferrin stored in non-activated/non-permeabilized neutrophils. MUB40 has the added benefit of recognizing lactoferrin from a broad host range, making it especially useful for comparing results in studies involving multiple research models, reducing the number of duplicate reagents, and simplifying protocols through single-step staining.

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we present a protocol to detect the presence of neutrophils in fixed/permeabilized histology sections and assess the activation state of live purified neutrophils. In particular, the MUB40&#160;peptide binds lactoferrin present in neutrophil-specific and tertiary granules. Exposure of the granule contents through either permeabilization or neutrophil activation allows for the marking of neutrophils.

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the active proliferation of intestinal stem cells located at the bottom of intestinal crypts. However, during events of acute intestinal injury, these active intestinal stem cells undergo cell death. Gamma irradiation is a widely used colorectal cancer treatment, which, while therapeutically efficacious, has the side effect of depleting the active stem cell pool. Indeed, patients frequently experience gastrointestinal radiation syndrome while undergoing radiotherapy, in part due to active stem cell depletion. The loss of active intestinal stem cells in intestinal crypts activates a pool of typically quiescent reserve intestinal stem cells and induces dedifferentiation of secretory and enterocyte precursor cells. If not for these cells, the intestinal epithelium would lack the ability to recover from radiotherapy and other such major tissue insults. New advances in lineage-tracing technologies allow tracking of the activation, differentiation, and migration of cells during regeneration and have been successfully employed for studying this in the gut. This study aims to depict a method for the analysis of cells within the mouse intestinal epithelium following radiation injury.

The gastrointestinal tract is one of the most sensitive organs to injury upon radiotherapeutic cancer treatments. It is simultaneously an organ system with one of the highest regenerative capacities following such insults. The presented protocol describes an efficient method to study the regenerative capacity of the intestinal epithelium.

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the ...

Epithelial Effects of Intestinal Inflammation on Established Murine Colonoids

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

Author Spotlight: Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids  

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...

Construction of a 3D Intestinal Mucosa Model with Paraffin Embedding

Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the pharmacological screening of potentially beneficial substances, and for toxicity studies on potentially harmful food components. Of relevance, there is a current demand for the development of cell-based in vitro models to substitute animal models. Here, a protocol for a basic, &#8220;healthy tissue&#8221; three-dimensional (3D) intestinal equivalent model using cell lines is presented with the dual benefit of providing both experimental simplicity (standardized and easily repeatable system) and physiological complexity (Caco-2 enterocytes with a supporting immune component of U937 monocytes and L929 fibroblasts). The protocol also includes paraffin embedding for light microscopic evaluation of fixed intestinal equivalents, thereby providing the advantage of analyzing multiple visual parameters from a single experiment. Hematoxylin and eosin (H&#38;E) stained sections showing the Caco-2 columnar cells forming a tight and regular monolayer in control treatments are used to verify the efficacy of the model as an experimental system. Using gluten as a pro-inflammatory food component, parameters analyzed from sections include reduced monolayer thickness, as well as disruption and detachment from the underlying matrix (H&#38;E), decreased tight junction protein expression as shown from occludin staining (quantifiable statistically), and immune-activation of migrating U937 cells as evidenced from the cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. As shown by using lipopolysaccharide to simulate intestinal inflammation, additional parameters that can be measured are increased mucus staining and cytokine expression (such as midkine) that can be extracted from the medium prior to fixation. The basic three-dimensional (3D) intestinal mucosa model and fixed sections can be recommended for inflammatory status and barrier integrity studies with the possibility of analyzing multiple visual quantifiable parameters.

Author Spotlight: Investigating the Effects of Compounds on Intestinal Tissue Using 3D Human Cell Line Models 

Here we describe constructing a basic three-dimensional (3D) intestinal cell line model system and a paraffin embedding protocol for light microscopic evaluation of fixed intestinal equivalents. Staining of selected proteins permits the analysis of multiple visual parameters from a single experiment for potential use in preclinical drug screening studies.

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the ...