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.

Tutti i lavori degli animali in questo protocollo viene eseguita con la stretta aderenza alle Università di Newcastle comitato etico degli animali procedure approvate. 1. Preparazione degli strumenti, Cultura Media e Piatti <ol><li> Pre-caldo media 199 (TC199) o Dulbecco Modified Eagle Medium (DMEM) supporti a 37 ° C. Pre-ossigenare il supporto facendo gorgogliare con il 95% O 2/5% di CO 2. Verificare che il mezzo ha un pH finale di 7,3. </li><li> Preparare sutura tagliando due sezioni 5 cm per ogni uscita. Loop i punti di sutura in un nodo non chiusa. </li></ol> 2. dissezione e preparazione del tratto gastrointestinale <ol><li> Ritirare solido cibo 12 ore prima di eutanasia. Se lo si desidera, inserire gli animali sugli integratori gel nutrienti durante questo periodo. </li><li> Euthanize topi per overdose pentobarbitone sodio ([200 mg / Kg], iniezione intraperitoneale) seguita da dislocazione cervicale in conformità con l&#39;etica proto istituzionaleCols e spruzzare il 70% di etanolo sul addome e torace. </li><li> Usando una forbice, fare una incisione orizzontale nel mezzo dell&#39;addome ed esporre il peritoneo. </li><li> Procedere per separare e rimuovere il tratto gastrointestinale tagliando il piccolo intestino superiore dallo stomaco al sfintere piloro e taglio crasso al margine anale. Utilizzare una pinza per rimuovere delicatamente il mesentere. Posizionare il tratto intestinale in pre-riscaldato, medio ossigenato. </li><li> Identificare la sezione dell&#39;intestino da valutare per permeabilità (Figura 1) e tagliare questa sezione libera dal resto del tratto intestinale. <ol><li> Al fine di mantenere la coerenza tra animali, misurare le sezioni di duodeno e nel digiuno rispetto allo stomaco, e misurare le sezioni di colon e dell&#39;ileo rispetto al cieco. </li><li> Quando si seleziona segmenti di tessuto, notare la presenza di tessuti linfoidi mucosa-associati, come le placche di Peyer. Questi possono essere identificati come piccolo cenno del capoORME sul lato sierose del lume. </li><li> Usando una siringa da 1 ml, lavare delicatamente il contenuto luminale del segmento intestinale in una capsula di Petri con PBS preriscaldato (37 ° C). Questi contenuti fecali possono essere scartati o conservati a -80 ° C per le analisi future, se lo desideri. </li></ol></li></ol> 3. Preparazione intestinale Sacs <ol><li> Preparare una siringa da 1 ml con un volume di 300 microlitri del composto di prova o molecola. Per l&#39;integrità della mucosa, un 1 mg / ml soluzione di FITC-destrano M.Wt. 4.400 possono essere utilizzati. Le sonde che vanno da 4,400-70,000 Da dimensioni possono essere utilizzati per una maggiore sensibilità. Trovare e montare un piccolo catetere vascolare animali sulla siringa. </li><li> Misura 5 cm dall&#39;apertura del segmento intestinale e legare il segmento completamente chiuse con una sutura-loop a questo punto. Posizionare delicatamente una sutura-loop pre-legata intorno all&#39;apertura dell&#39;intestino e inserire il catetere smussata. Tirare il cappio chiuso in modo da assicurare il segmento intestinalee rilasciare il volume 300 microlitri dalla siringa nell&#39;intestino, assicurando che tutta la soluzione viene iniettata. </li><li> Rimuovere delicatamente il catetere e contemporaneamente tirare il cappio di sutura per fissare la chiusura del sacco intestinale. Tagliare il sacco intestinale sciolto dall&#39;intestino e posto in un tubo da 50 ml riempito con 20 ml di terreno ossigenato, preriscaldata a 37 ° C. </li></ol> 4. la misura della permeabilità <ol><li> Luogo provette coniche contenenti le sacche intestinali in un bagno d&#39;acqua riscaldata impostati su 37 ° C. A 0, 30, 60, 90 e 120 punti min tempo, prelevare un campione di 100 microlitri dalla provetta conica e trasferimento ad una piastra da 96 pozzetti, sostituendo il volume con 100 ml di mezzi freschi in ciascun caso. </li><li> Dopo aver prelevato il campione finale, tagliare sacche aperte al punto di sutura e la lunghezza del segmento, esponendo la superficie mucosa. </li><li> Misurare la lunghezza e la larghezza di ogni segmento intestinale. Se Desirosso, scatto congelare i segmenti e conservare a -80 ° C per le proteine ​​o l&#39;analisi biochimica, o, in alternativa, conservare in soluzione di stabilizzazione RNA per saggi molecolari. </li><li> Costruire una curva standard di diluizioni di registro per le molecole FITC-etichettati da 1 a 1 x 10 -6. </li><li> Misure di campioni e standard per FITC su un lettore di piastre a fluorescenza, FITC di eccitazione / emissione: 495 nm / 519 nm. </li></ol> 5. Calcolo di permeabilità apparente per ogni intestinale individuale Sac <ol><li> Convertire unità di tempo di sec. </li><li> Per ogni punto di tempo, calcolare la concentrazione cumulativa, Q Q t = (C t * V r) + (Q t somma * V s), dove: Q t = concentrazione cumulativa al tempo t C t = concentrazione al tempo t V r = Volume sul lato ricevente Q t somma = Somma di tutte le precedenti Q t V s =volume campionato </li><li> Trama Q in funzione del tempo (T) e calcolare la pendenza: AQ / DT </li><li> Calcolare la permeabilità apparente (P app) P app = (AQ / T) / (A * Co), dove: A = area di tessuto C 0 = concentrazione iniziale </li></ol>

<ol>
	<li>Goggins, B. J., Chaney, C., Radford-Smith, G. L., Horvat, J. C., Keely, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+Integrin-Mediated+Epithelial+Restitution+during+Mucosal+Inflammation.">Hypoxia and Integrin-Mediated Epithelial Restitution during Mucosal Inflammation.</a> Frontiers in immunology. 4, 272 (2013).</li><li>Otte, J. M., Kiehne, K., Herzig, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+in+innate+immunity+of+the+human+intestine.">Antimicrobial peptides in innate immunity of the human intestine.</a> Journal of gastroenterology. 38, 717-726 (2003).</li><li>Robinson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+protection+by+hypoxia-inducible+factor+prolyl+hydroxylase+inhibition.">Mucosal protection by hypoxia-inducible factor prolyl hydroxylase inhibition.</a> Gastroenterology. 134, 145-155 (2008).</li><li>Feighery, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+intestinal+permeability+in+rats+subjected+to+traumatic+frontal+lobe+percussion+brain+injury.">Increased intestinal permeability in rats subjected to traumatic frontal lobe percussion brain injury.</a> The Journal of trauma. 64, 131-137 (2008).</li><li>Keely, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride-led+disruption+of+the+intestinal+mucous+layer+impedes+Salmonella+invasion:+evidence+for+an+'enteric+tear'+mechanism.">Chloride-led disruption of the intestinal mucous layer impedes Salmonella invasion: evidence for an 'enteric tear' mechanism.</a> Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology. 28, 743-752 (2011).</li><li>Keely, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+epithelial+innate+immunity+to+systemic+protection+afforded+by+prolyl+hydroxylase+inhibition+in+murine+colitis.">Contribution of epithelial innate immunity to systemic protection afforded by prolyl hydroxylase inhibition in murine colitis.</a> Mucosal immunology. 7, 114-123 (2014).</li><li>Sourisseau, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+PCNA+and+cyclin+D1+expression+and+epithelial+morphogenesis+by+the+ZO-1-regulated+transcription+factor+ZONAB/DbpA.">Regulation of PCNA and cyclin D1 expression and epithelial morphogenesis by the ZO-1-regulated transcription factor ZONAB/DbpA.</a> Mol Cell Biol. 26, 2387-2398 (2006).</li><li>Ruehl-Fehlert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+guides+for+organ+sampling+and+trimming+in+rats+and+mice--part+1.">Revised guides for organ sampling and trimming in rats and mice--part 1.</a> Exp Toxicol Pathol. 55, 91-106 (2003).</li><li>Barthe, L., Woodley, J. F., Kenworthy, S., Houin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+everted+gut+sac+as+a+simple+and+accurate+technique+to+measure+paracellular+transport+across+the+small+intestine.">An improved everted gut sac as a simple and accurate technique to measure paracellular transport across the small intestine.</a> European journal of drug metabolism and pharmacokinetics. 23, 313-323 (1998).</li><li>Marks, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+Delivery+of+Prolyl+Hydroxylase+Inhibitor:+AKB-4924+Promotes+Localized+Mucosal+Healing+in+a+Mouse+Model+of+Colitis.">Oral Delivery of Prolyl Hydroxylase Inhibitor: AKB-4924 Promotes Localized Mucosal Healing in a Mouse Model of Colitis.</a> Inflammatory bowel diseases. 21, 267-275 (2015).</li><li>Keely, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor-dependent+regulation+of+platelet-activating+factor+receptor+as+a+route+for+gram-positive+bacterial+translocation+across+epithelia.">Hypoxia-inducible factor-dependent regulation of platelet-activating factor receptor as a route for gram-positive bacterial translocation across epithelia.</a> Mol Biol Cell. 21, 538-546 (2010).</li><li>Brayden, D. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial+transport+of+PAMAM+dendrimers+across+isolated+rat+jejunal+mucosae+in+ussing+chambers.">Transepithelial transport of PAMAM dendrimers across isolated rat jejunal mucosae in ussing chambers.</a> Biomacromolecules. 15, 2889-2895 (2014).</li><li>Keely, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+ex+vivo+intestinal+tissue+models+to+measure+mucoadhesion+of+poly+(methacrylate)+and+N-trimethylated+chitosan+polymers.">In vitro and ex vivo intestinal tissue models to measure mucoadhesion of poly (methacrylate) and N-trimethylated chitosan polymers.</a> Pharmaceutical research. 22, 38-49 (2005).</li><li>Maher, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+intestinal+absorption+enhancement+and+local+mucosal+toxicity+of+two+promoters.+I.+Studies+in+isolated+rat+and+human+colonic+mucosae.">Evaluation of intestinal absorption enhancement and local mucosal toxicity of two promoters. 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. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+dissociation+of+paracellular+permeability+and+transepithelial+electrical+resistance+and+disruption+of+the+apical-basolateral+intramembrane+diffusion+barrier+by+expression+of+a+mutant+tight+junction+membrane+protein.">Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein.</a> The Journal of cell biology. 134, 1031-1049 (1996).</li><li>Behrens, I., Stenberg, P., Artursson, P., Kissel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+lipophilic+drug+molecules+in+a+new+mucus-secreting+cell+culture+model+based+on+HT29-MTX+cells.">Transport of lipophilic drug molecules in a new mucus-secreting cell culture model based on HT29-MTX cells.</a> Pharmaceutical research. 18, 1138-1145 (2001).</li><li>Stefka, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commensal+bacteria+protect+against+food+allergen+sensitization.">Commensal bacteria protect against food allergen sensitization.</a> Proc Natl Acad Sci U S A. 111, 13145-13150 (2014).</li><li>Keely, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+fluid+transport+regulates+bacterial-epithelial+interactions+and+significantly+shifts+the+murine+colonic+microbiome.">Activated fluid transport regulates bacterial-epithelial interactions and significantly shifts the murine colonic microbiome.</a> Gut microbes. 3, 250-260 (2012).</li><li>Barrett, K. E., Keely, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride+secretion+by+the+intestinal+epithelium:+molecular+basis+and+regulatory+aspects.">Chloride secretion by the intestinal epithelium: molecular basis and regulatory aspects.</a> Annual review of physiology. 62, 535-572 (2000).</li><li>Soni, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat,+ovine+and+bovine+Peyer's+patches+mounted+in+horizontal+diffusion+chambers+display+sampling+function.">Rat, ovine and bovine Peyer's patches mounted in horizontal diffusion chambers display sampling function.</a> Journal of controlled release : official journal of the Controlled Release Society. 115, 68-77 (2006).</li><li>Justino, P. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+role+of+Lactobacillus+acidophilus+on+inflammation+and+gastric+dysmotility+in+intestinal+mucositis+induced+by+5-fluorouracil+in+mice.">Regulatory role of Lactobacillus acidophilus on inflammation and gastric dysmotility in intestinal mucositis induced by 5-fluorouracil in mice.</a> Cancer chemotherapy and pharmacology. , (2015).</li><li>Tran, C. D., Sundar, S., Howarth, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+zinc+supplementation+and+methotrexate-induced+small+intestinal+mucositis+in+metallothionein-knockout+and+wild-type+mice.">Dietary zinc supplementation and methotrexate-induced small intestinal mucositis in metallothionein-knockout and wild-type mice.</a> Cancer biology &amp; therapy. 8, 1662-1667 (2009).</li><li>Musch, M. W., Wang, Y., Claud, E. C., Chang, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lubiprostone+decreases+mouse+colonic+inner+mucus+layer+thickness+and+alters+intestinal+microbiota.">Lubiprostone decreases mouse colonic inner mucus layer thickness and alters intestinal microbiota.</a> Digestive diseases and sciences. 58, 668-677 (2013).</li></ol>

Gli autori dichiarano di non avere interessi finanziari concorrenti.

Qui, abbiamo descritto l&#39;isolamento e la preparazione di sacche intestinali per valutare la funzione barriera della mucosa ex vivo. preparazioni sac intestinali sono principalmente utilizzati nella ricerca farmaceutica, esaminando l&#39;assorbimento di farmaci candidati attraverso l&#39;intestino. Tuttavia, questo test è ugualmente adatta per lo studio della malattia intestinale. La permeabilità intestinale può variare notevolmente da regione a regione e la valutazione specifica del sito di permeabilità...

La barriera epiteliale intestinale del tratto gastrointestinale è una superficie mucosa stimato a 400 m 2 nel umano adulto. Di conseguenza, si è costantemente esposto a sfidare da microbi, farmaci ingeriti, nutrienti e tossine batteriche. L&#39;host non solo deve distinguere tra batteri commensali tollerabili e potenziali patogeni, ma deve impedire queste specie e loro molecole secrete di attraversare la barriera epiteliale, mentre allo stesso tempo permette l&#39;assorbimento dei nutrienti. Pertanto, il ruolo dell&#39;epitelio intestinale è di agire come barriera selettiva al contenuto luminale 1. Questo risultato è ottenuto, in parte, dal sistema di difesa epiteliale innato alla mucosa, che agisce attraverso un sistema biologico reattivo costituito da costitutive e inducibili meccanismi 2. La perdita della funzione barriera epiteliale è una patologia che è caratteristica di un certo numero di malattie gastrointestinali. In vivoesame della funzione di barriera epiteliale può essere valutata attraverso sonda gastrica di una molecola tracciante e la successiva analisi del siero 3. Tuttavia, questa tecnica non offre alcuna indicazione sul sito di disfunzione barriera. In vitro e ex vivo valutazione della resistenza transepiteliale utilizzando sistemi Transwell 3 e ussing rispettivamente camere di 4,5, sono comunemente impiegati come indicatori surrogati di funzione di barriera epiteliale, ma manca la contribuendo malattia fisiologia dei modelli animali 6. In questo protocollo si descrive un modello di preparazione del tessuto ex vivo che permette una valutazione diretta e localizzata dell&#39;integrità intestinale e che può essere utilizzato per valutare la funzione barriera della mucosa a diversi livelli. È importante sottolineare che questa tecnica può essere applicata a modelli animali di malattia, o può essere farmacologicamente manipolato per permettere a fondo interrogatori di disfunzione barriera mucosa.

<table><tbody><tr><td>Dekantel&amp;nbsp; Non-absorbable Silk suture</td><td>Braintree Scientific</td><td>SUT-S 116</td><td></td></tr><tr><td>Media 199 (TC199)&amp;nbsp;</td><td>Life Technologies</td><td>11043-023</td><td>No phenol red as this interferes with fluorescence</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>21063-045</td><td>No phenol red as this interferes with fluorescence</td></tr><tr><td>N-acetylcysteine</td><td>Sigma Aldrich</td><td></td><td>Use at 10 mM in media</td></tr><tr><td>Small animal vascular cathether: Physiocath</td><td>Data Sciences International</td><td>277-1-002</td><td></td></tr><tr><td>FITC-Dextran 4,400 MW</td><td>Sigma Aldrich</td><td>FD-4</td><td></td></tr><tr><td>FITC-Dextran 20,000 MW</td><td>Sigma Aldrich</td><td>FD-20</td><td></td></tr><tr><td>FITC-Dextran 70,000 MW</td><td>Sigma Aldrich</td><td>FD-70</td><td></td></tr></tbody></table>

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

La barriera epiteliale è la prima difesa innata del tratto gastrointestinale e regola selettivamente trasporto dal lume ai compartimenti sottostanti, limitare il trasporto di molecole più piccole attraverso l&#39;epitelio e quasi completamente vieta epiteliale trasporto macromolecolare. Questa selettività è determinata dallo strato di gel mucoso, che limita il trasporto di molecole lipofile ed entrambi i recettori apicali e stretti complessi proteici giunzionali dell&#39;epitelio. Modelli in vitro su colture di cellule dell&#39;epitelio sono convenienti, ma come un modello, non hanno la complessità delle interazioni tra microbiota, muco-gel, epitelio e il sistema immunitario. D&#39;altra parte, la valutazione in vivo dell&#39;assorbimento intestinale o permeabilità può essere eseguita, ma questi test misurare il grado di assorbimento gastrointestinale, senza alcuna indicazione di specificità del sito. Ex vivo saggi permeabilità utilizzando &quot;sacche intestinali&quot;; sono un metodo rapido e sensibile per misurare sia l&#39;integrità intestinale globale o il trasporto comparativa di una molecola specifica, con l&#39;ulteriore vantaggio di specificità del sito intestinale. Qui si descrive la preparazione di sacche intestinali per gli studi di permeabilità e il calcolo della permeabilità apparente (P app) di una molecola attraverso la barriera intestinale. Questa tecnica può essere utilizzata come un metodo di valutazione assorbimento del farmaco, o per esaminare disfunzione barriera epiteliale regionale in modelli animali di malattia gastrointestinale.

Questo protocollo può essere utilizzato per esaminare i cambiamenti regionali in funzione di barriera intestinale in modelli animali di malattie gastrointestinali. Misurando il flusso di una sonda paracellular sulla superficie mucosa in diverse aree del tratto gastrointestinale 7, l&#39;integrità delle giunzioni strette epiteliali può essere valutata. Inoltre, variando la natura della sonda paracellular per dimensione (Figura 2) o idrofobicità (Fig...

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 intestinale Sacs per valutare mucosa Permeabilità in modelli di malattia gastrointestinale

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

ex-vivo-intestinal-sacs-to-assess-mucosal-permeability-models

Research

JoVE Journal

Medicine

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

20.9K Views.  University of Newcastle. 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.

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

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Misura in tempo reale di permeabilità della barriera epiteliale in Organoids intestinale umana

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Ricerca

Biologia dello sviluppo

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

Analizzando Effetti benefici dei supplementi nutrizionali sulle funzioni intestinali epiteliali barriera durante Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

Medicina

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

Un approccio intravitale basato sulla microscopia per valutare la permeabilità intestinale e le prestazioni di spargimento di cellule epiteliali

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

Immunologia e infezioni

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

Valutazione funzionale della permeabilità intestinale e della migrazione transepiteliale neutrofila nei topi utilizzando un modello standardizzato di loop intestinale

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out Enteroid Model

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown in the basolateral-out (BO) conformation, where the apical surface of the epithelial cell faces the inner lumen. In this model, access to the luminal surface of enteroids for treatment and experimentation is challenging, which limits the ability to study host-pathogen interactions. To circumvent this, a neonatal apical-out (AO) model for necrotizing enterocolitis was created. Since intestinal epithelial cell permeability changes are pathognomonic for NEC, this protocol outlines using lucifer yellow (LY) as a marker of paracellular permeability. LY traverses the intestinal epithelial barrier via all three major paracellular pathways: pore, leak, and unrestricted. Using LY in an AO model allows for a broader study of permeability in NEC. Following IRB approval and parental consent, surgical samples of intestinal tissue were collected from human preterm neonates. Intestinal stem cells were harvested via crypt isolation and used to grow enteroids. Enteroids were grown to maturity and then transformed AO or left in BO conformation. These were either not treated (control) or were treated with lipopolysaccharide (LPS) and subjected to hypoxic conditions for the induction of in vitro NEC. LY was used to assess for permeability. Immunofluorescent staining of the apical protein zonula occludens-1 and basolateral protein &#946;-catenin confirmed AO conformation. Both AO and BO enteroids treated with LPS and hypoxia demonstrated significantly increased paracellular permeability compared to controls. Both AO and BO enteroids showed increased uptake of LY into the lumen of the treated enteroids compared to controls. The utilization of LY in an AO enteroid model allows for the investigation of all three major pathways of paracellular permeability. It additionally allows for the investigation of host-pathogen interactions and how this may affect permeability compared to the BO enteroid model.

The present protocol outlines a method that utilizes lucifer yellow in an apical-out enteroid model to determine intestinal permeability. This method can be used to determine paracellular permeability in enteroids that model inflammatory bowel diseases such as necrotizing enterocolitis.

Determinazione della permeabilità intestinale utilizzando Lucifer Yellow in un modello enteroide apical-out

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

Indagare la rottura della barriera intestinale negli organiidi viventi

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

In Vitro e In Vivo approcci per determinare la permeabilità intestinale delle cellule epiteliali

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

Biologia

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.

Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa.

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier ...

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of plasma inflammatory markers and bacterial translocation to the spleen and lymph nodes, the gold standard directly quantifies the ability of selected molecules to traverse the gut mucosal layer toward systemic circulation. This article uses a non-invasive, cost-effective, and low-burden technique to quantify and follow in real time the intestinal permeability in mice using fluorescein-isothiocyanate-labeled dextran (FITC-dextran). Prior to oral supplementation with FITC-dextran, the mice are fasted. They are then gavaged with FITC-dextran diluted in phosphate-buffered saline (PBS). One hour after the gavage, the mice are subjected to general anesthesia using isoflurane, and the in vivo fluorescence is visualized in an imaging chamber. This technique aims to assess residual fluorescence in the abdominal cavity and the hepatic uptake, which is suggestive of portal migration of the fluorescent probe. Blood and stool samples are collected 4 h after oral gavage, and the mice are sacrificed. Plasma and fecal samples diluted in PBS are then plated, and the fluorescence is recorded. The concentration of FITC-dextran is then calculated using a standard curve. In previous research, in vivo imaging has shown that fluorescence rapidly spreads to the liver in mice with a weaker gut barrier induced by a low-fiber diet, while in mice supplemented with fiber to strengthen the gut barrier, the fluorescent signal is retained mostly in the gastrointestinal tract. In addition, in this study, control mice had elevated plasma fluorescence and reduced fluorescence in the stool, while inversely, inulin-supplemented mice had higher levels of fluorescence signals in the gut and low levels in the plasma. In summary, this protocol provides qualitative and quantitative measurements of intestinal permeability as a marker for gut health.

In the present study, fluorescein-isothiocyanate-labeled (FITC) dextran is administered to mice via oral gavage to evaluate intestinal permeability both in vivo and in plasma and fecal samples. As gut barrier function is affected in many disease processes, this direct and quantitative assay can be used in diverse research areas.

Valutazione dell'integrità della barriera intestinale nei topi utilizzando destrano marcato con fluoresceina-isotiocianato

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of ...

Costruzione di un modello 3D della mucosa intestinale con inclusione di paraffina

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.

Autore in primo piano: Indagine sugli effetti dei composti sul tessuto intestinale utilizzando modelli 3D di linee cellulari umane 

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.

Sistema modello intestinale tridimensionale (3D) di base con una componente immunitaria

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 ...