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.

Alla djur arbete i detta protokoll utförs med strikt följsamhet till University of Newcastle Animal etikkommitté godkända förfaranden. 1. Framställning av instrument, odlingsmedia och rätter <ol><li> Pre-varm Media 199 (TC199) eller Dulbeccos Modified Eagle Medium (DMEM) media till 37 ° C. Pre-syresätta mediet genom bubbling med 95% O2 / 5% CO2. Kontrollera att mediet har ett slutligt pH av 7,3. </li><li> Förbered sutur genom att skära två 5 c...

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J., Bzik, V. A., Lewis, A. L., Illum, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CriticalSorb+promotes+permeation+of+flux+markers+across+isolated+rat+intestinal+mucosae+and+Caco-2+monolayers.">CriticalSorb promotes permeation of flux markers across isolated rat intestinal mucosae and Caco-2 monolayers.</a> Pharmaceutical research. 29, 2543-2554 (2012).</li><li>Hubbard, D., Ghandehari, H., Brayden, D. 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I. Studies in isolated rat and human colonic mucosae.</a> European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences. 38, 291-300 (2009).</li><li>Balda, M. 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Här har vi detaljerat isolering och beredning av tarmsäckar för att bedöma mukosal barriärfunktion ex vivo. Tarm sac preparat har huvudsakligen använts i läkemedelsforskning, undersöka absorptionen av läkemedelskandidater hela tarmen. Emellertid är denna analys lika väl lämpad för studier av tarmsjukdom. Intestinal permeabilitet kan variera kraftigt från region och platsspecifik bedömning av genomtränglighet tillåter en bättre förståelse av den regionala betydelsen av slemhinnans integritet ...

Tarm epitelial barriär hos mag-tarmkanalen är en mukosal yta uppskattas till 400 m 2 i vuxen människa. Följaktligen är det ständigt utsätts för ifrågasätta från mikrober, intagna läkemedel, näringsämnen och bakterietoxiner. Värden får inte bara skilja mellan tolerabla bakteriefloran och potentiella patogener, men måste förhindra att dessa arter och deras utsöndrade molekyler från att korsa epitelbarriären, medan samtidigt tillåter upptag av näringsämnen. Sålunda är rollen för tarmepitelet för att fungera som en selektiv barriär för de luminala innehållet 1. Detta uppnås, delvis genom det medfödda epiteliala försvarssystem vid slemhinnan, som verkar via ett responsiv biologiskt system som består av konstitutiva och inducerbara mekanismer 2. Förlust av epitelial barriär funktion är en patologi som är kännetecknande för ett antal gastrointestinala sjukdomar. In vivoundersökning av epitelial barriärfunktion kan bedömas genom oral sondmatning av en spårmolekyl och efterföljande serumanalys 3. Dock erbjuder denna teknik ingen uppgift om platsen för barriärfunktion. In vitro och ex vivo bedömning av transepitelial motstånd med hjälp av Transwell-system 3 och Ussing kamrar 4,5 respektive vanligen användes som surrogatmarkörer för epitelial barriärfunktion, men saknar den bidragande sjukdoms fysiologi djurmodeller 6. I detta protokoll beskriver vi en ex vivo vävnadspreparat modell som möjliggör direkt och lokal bedömning av intestinal integritet och som kan användas för att bedöma mukosal barriärfunktion vid ett antal nivåer. Viktigare, kan denna teknik appliceras på djurmodeller av sjukdom, eller kan vara farmakologiskt manipuleras för att möjliggöra på djupet förhör av slemhinnebarriären dysfunktion.

<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

Epitelbarriären är den första medfödda försvaret av mag-tarmkanalen och selektivt reglerar transport från lumen till den underliggande vävnaden fack, begränsa transport av mindre molekyler över epitel och nästan helt förbjuder epitel makromolekylära transport. Denna selektivitet bestämmes av slem gelskikt, vilket begränsar transporten av lipofila molekyler och både de apikala receptorer och snäva junctionala proteinkomplex av epitelet. Cellodling in vitro modeller av epitelet är bekvämt, men som modell, de saknar den komplexiteten hos samspelet mellan mikrobiota, slemhinnor-gel, epitel och immunsystemet. Å andra sidan, kan in vivo bedömning av intestinal absorption eller permeabilitet utföras, men dessa analyser mäter totalt gastrointestinal absorption, utan angivande av plats specificitet. Ex vivo permeabilitet analyser med hjälp av &quot;tarmsäckar&quot;; är en snabb och känslig metod för att mäta antingen total intestinal integritet eller jämförande transport av en specifik molekyl, med den extra fördelen av intestinal-ställespecificitet. Här beskriver vi framställningen av tarmsäckar för permeabilitet studier och beräkningen av den skenbara permeabiliteten (P app) av en molekyl genom tarmbarriären. Denna teknik kan användas som en metod för att bedöma läkemedelsabsorption, eller för att undersöka regional epitelial barriär dysfunktion i djurmodeller av gastrointestinal sjukdom.

Detta protokoll kan användas för att undersöka regionala förändringar i tarmbarriärfunktion i djurmodeller av mag-tarmsjukdomar. Genom att mäta flödet av en paracellulär sond tvärs slemhinneytan vid varierande områden av mag-tarmkanalen 7, kan bedömas integriteten för de epiteliala tight junctions. Dessutom, genom att variera naturen hos den para sonden genom storlek (Figur 2) eller hydrofobiciteten (figur 3), graden av epitelial ...

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 att bedöma Mukös permeabilitet i Modeller av gastrointestinal sjukdom

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

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

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