Name: ex vivo intestinal sacs to assess mucosal permeability in models of gastrointestinal disease
Uploaded: 2016-02-09T13:00:00
Description: Watch this Scientific Journal Video about Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease at JoVE.com

This work was funded by National Health and Medical Research Project Grant APP1021582 and a Hunter Medical Research Institute grant sponsored by Sparke Helmore/NBN Triathlon and the Estate of the late Leslie Kenneth McFarlane.

All animal work in this protocol is performed with strict adherence to University of Newcastle Animal Ethics Committee approved procedures.
1. Preparation of Instruments, Culture Media and Dishes
<ol>
	<li>Pre-warm Media 199 (TC199) or Dulbecco&#39;s Modified Eagle Medium (DMEM) media to 37 &#176;C. Pre-oxygenate the medium by bubbling with 95% O2/5% CO2. Check that the medium has a final pH of 7.3.</li>
	<li>Prepare suture by cutting two 5 cm sections for ea...

<ol>
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Here, we have detailed the isolation and preparation of intestinal sacs to assess mucosal barrier function ex vivo. Intestinal sac preparations have primarily been utilized in pharmaceutical research, examining the absorption of candidate drugs across the intestine. However, this assay is equally well suited for the study of intestinal disease. Intestinal permeability may vary greatly by region and site specific assessment of permeability allows a better understanding of the regional importance of mucosal integr...

The intestinal epithelial barrier of the gastrointestinal tract is a mucosal surface area estimated at 400 m2 in the human adult. Consequently, it is constantly exposed to challenge from microbes, ingested drugs, nutrients and bacterial toxins. The host must not only distinguish between tolerable commensal bacteria and potential pathogens, but must prevent these species and their secreted molecules from crossing the epithelial barrier, while at the same time allowing absorption of nutrients. Thus, the role of the intestinal epithelium is to act as a selective barrier to the luminal contents 1. This is achieved, in part, by the innate epithelial defense system at the mucosa, which acts through a responsive biological system consisting of constitutive and inducible mechanisms 2.
Loss of epithelial barrier function is a pathology that is characteristic of a number of gastrointestinal diseases. In vivo examination of epithelial barrier function may be assessed through oral gavage of a tracer molecule and subsequent serum analysis 3. However, this technique offers no indication as to the site of barrier dysfunction. In vitro and ex vivo assessment of transepithelial resistance using Transwell systems 3 and Ussing chambers 4,5 respectively, are commonly employed as surrogate markers of epithelial barrier function, but lack the contributing disease physiology of animal models 6. In this protocol we describe an ex vivo tissue preparation model that allows direct and localized assessment of intestinal integrity and which may be used to assess mucosal barrier function at a number of levels. Importantly, this technique may be applied to animal models of disease, or may be pharmacologically manipulated to allow in depth interrogation of mucosal barrier dysfunction.

<table border="0" cellpadding="0" cellspacing="0" width="904">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Dekantel&#160; Non-absorbable Silk suture</td>
			<td style="width:227px;">Braintree Scientific</td>
			<td style="width:227px;">SUT-S 116</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Media 199 (TC199)&#160;</td>
			<td style="width:227px;">Life Technologies</td>
			<td style="width:227px;">11043-023</td>
			<td style="width:227px;">No phenol red as this interferes with fluorescence</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:227px;">Life Technologies</td>
			<td style="width:227px;">21063-045</td>
			<td style="width:227px;">No phenol red as this interferes with fluorescence</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">N-acetylcysteine</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;"></td>
			<td style="width:227px;">Use at 10mM in media</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Small animal vascular cathether: Physiocath</td>
			<td style="width:227px;">Data Sciences International</td>
			<td style="width:227px;">277-1-002</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">FITC-Dextran 4400 MW</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;">FD-4</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">FITC-Dextran 20,000 MW</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;">FD-20</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">FITC-Dextran 70,000 MW</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;">FD-70</td>
			<td style="width:227px;"></td>
		</tr>
	</tbody>
</table>

ex vivo intestinal sacs to assess mucosal permeability in models of gastrointestinal disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

This protocol may be used to examine regional changes in intestinal barrier function in animal models of gastrointestinal disease. By measuring the flux of a paracellular probe across the mucosal surface at varying areas of the gastrointestinal tract7, the integrity of the epithelial tight junctions can be assessed. In addition, by varying the nature of the paracellular probe by size (Figure 2) or hydrophobicity (Figure 3), the degree of epithe...

Watch this Scientific Journal Video about Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease at JoVE.com

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

The overall goal of this procedure is to assess the localized intestinal integrity in animal models of gastrointestinal disease.This method can help answer key quesitons about gastrointestinal disease, such as whether musosal integrity is lost or if there is an increase in uptake of a specific molecule or bacteria during disease.The main advantage of this technique is that it can be used to assess specific areas within the gastrointestinal tract.To prepare the gastrointestinal tract, begin by spraying the abdomen and thorax of the mouse with 70 percent ethanol and use scissors to make a horizontal incision in the middle of the abdomen, to expose the peritoneum.Next, cut the upper small intestine from the stomach at the pyloric sphincter and the large intestine at the anal verge.Then use forceps to gently remove the mesentery and place the intestinal tract in prewarmed, oxyginated medium.Identify the area to be assessed for permeability, measuring the sections of the duodenum and jejunum relative to the stomach in the sections of the colon and ilium relative to the cecum to maintain consistency between the experimental animals.Note the presence of mucosa associated lymphoid tissues such as Peyer's patches, which can be identified as small nodules on the sarosal side of the lumen.Then, cut the selected section and use a 1 ml syringe to gently flush the lumenal contents of the segment into a petri dish containing 37

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