Name: ex vivo intestinal sacs to assess mucosal permeability in models of gastrointestinal disease
Uploaded: 2016-02-09T13:00:00.000+00:00
Duration: 6 min 4 s
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.

Alle tierischen Arbeit in diesem Protokoll wird bei strikter Einhaltung University of Newcastle Tierethikkommission genehmigten Verfahren durchgeführt. 1. Vorbereitung der Instrumente, Kultur, Medien und Gerichte <ol><li> Pre-warmen Medien 199 (TC199) oder Dulbeccos Modified Eagle Medium (DMEM) Medien bis 37 ° C. Pre-Oxygenat das Medium durch mit 95% O 2/5% CO 2 sprudeln. Überprüfen Sie, ob das Medium einen endgültigen pH-Wert von 7,3 hat. </li><li> Bereiten Faden durch Schneiden zwei 5 cm Abschnitte für jeden Sack. Schleife die Nähte in eine unverschlossene Knoten. </li></ol> 2. Dissection und Vorbereitung des Gastrointestinaltraktes <ol><li> Ziehen Sie feste Nahrung 12 Stunden vor der Euthanasie. Falls gewünscht, legen Tiere auf Nährgel Ergänzungen während dieser Zeit. </li><li> Euthanize Mäusen durch Natriumpentobarbiton Überdosierung ([200 mg / Kg], intraperitoneale Injektion), gefolgt durch zervikale Dislokation nach Ethik protoSpalten und 70% Ethanol auf den Bauch und Brustkorb sprühen. </li><li> Mit einer Schere, einen horizontalen Schnitt in der Mitte des Bauches machen und das Bauchfell aus. </li><li> Gehen Sie zu trennen und das Entfernen des Magen-Darm-Trakt, indem der obere Dünndarm aus dem Magen in die Magenpförtner Schneiden und den Dickdarm in den analen Rand schneiden. Verwenden einer Pinzette vorsichtig auf die Gekröse entfernen. Setzen Sie den Darmtrakt in vorgewärmten, sauerstoffreiches Medium. </li><li> Identifizieren Sie den Abschnitt des Darms, der für Durchlässigkeit (Abbildung 1) geprüft und schneiden Sie diesen Abschnitt frei von dem Rest des Verdauungstraktes. <ol><li> Um die Konsistenz zwischen den Tieren zu halten, messen Abschnitte von Duodenum und Jejunum in Bezug auf den Magen, und messen Abschnitte des Dickdarms und Ileum gegenüber dem Blinddarm. </li><li> Wenn Gewebesegmente auszuwählen, beachten Sie das Vorhandensein von Schleimhaut-assoziierten lymphatischen Gewebe wie Peyer-Plaques. Diese können als kleine Anspielung identifiziert werdenules auf der Serosa-Seite des Lumens. </li><li> Unter Verwendung einer 1 ml Spritze, spülen sanft die luminalen Inhalte des Darmsegments in eine Petrischale mit vorgewärmtem PBS (37 ° C). Diese fäkal Inhalt kann bei entsorgt oder gelagert werden -80 ° C für zukünftige Analyse wie gewünscht. </li></ol></li></ol> 3. Herstellung von Darm Sacs <ol><li> Bereiten einer 1 ml Spritze mit einer 300 ul Volumen der Testverbindung oder Molekül. Zur mukosalen Integrität, einer 1 mg / ml Lösung von FITC-Dextran M.Wt. 4400 verwendet werden. Sonden im Bereich von 4,400-70,000 Da in der Größe kann für eine erhöhte Empfindlichkeit verwendet werden. passen sicher ein kleines Tier Gefäßkatheter auf die Spritze. </li><li> Maßnahme 5 cm von der Öffnung des Darm Segment und das Segment binden fest an dieser Stelle mit einer Naht-Schleife geschlossen. Sanft legen Sie eine vorgebunden Naht-Schleife um die Öffnung des Darms und die abgestumpften Katheter einsetzen. Ziehen Sie die Schlinge geschlossen, um die Darmsegment zu sichernund Loslassen der 300 ul-Volumen aus der Spritze in den Darm, so dass die gesamte Lösung eingespritzt wird. </li><li> Sie vorsichtig den Katheter zu entfernen, während gleichzeitig die Naht Schlinge ziehen Schließung des Darmsack zu sichern. Schneiden Sie das Darmsack lose aus dem Darm und in einen 50 ml konischen Röhrchen gefüllt mit 20 ml sauerstoffreiches Medium, vorgewärmt auf 37 ° C. </li></ol> 4. Messung der Durchlässigkeit <ol><li> Platz konische Röhrchen, die die Darmsäcken in einem beheizten Wasserbad enthält, auf 37 ° C. Bei 0, 30, 60, 90 und 120 min Zeitpunkten, nehmen 100 ul Probe aus dem konischen Rohr und in ein 96-Well-Platte, das Volumen mit 100 ul frisches Medium in jedem Fall zu ersetzen. </li><li> Nachdem die letzte Probe entnommen wird, aufgeschnitten Säcke an der Stelle der Naht und über die gesamte Länge des Segments, die Schleimhautoberfläche freigelegt wird. </li><li> Messen Sie die Länge und Breite jedes Darmsegment. wenn Desirot, einfrieren Schnapp die Segmente und bei -80 ° C für Protein oder biochemischen Analyse oder alternativ Geschäft in RNA-Stabilisierung Lösung für molekulare Tests. </li><li> Eine Standardkurve von log-Verdünnungen für FITC-markierte Moleküle von 1 bis 1 x 10 -6 bis hin. </li><li> Messen Proben und Standards für FITC auf einem Fluoreszenzplattenlesegerät, FITC Anregungs- / Emissions: 495 nm / 519 nm. </li></ol> 5. Berechnung der Scheindurchlässigkeit für jeden einzelnen Darm-Sac <ol><li> Konvertieren Zeiteinheiten zu sek. </li><li> Für jeden Zeitpunkt berechnen die kumulative Konzentration, Q Q t = (C t * V r) + (Q t Summe * V s), wo: Q t = Kumulative Konzentration zum Zeitpunkt t C t = Konzentration zum Zeitpunkt t V r = Volumen auf der Empfängerseite Q t Summe = Summe aller bisherigen Q t Vs =Volumen abgetastet </li><li> Plot Q gegen die Zeit (T) und die Berechnung der Steigung: &amp; dgr; Q / &amp; dgr; t </li><li> Berechnen Sie die scheinbare Durchlässigkeit (P app) P app = (&amp; dgr; Q / &amp; dgr; t) / (A * Co), wo: A = Fläche des Gewebes C 0 = Anfangskonzentration </li></ol>

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Die Autoren erklären, dass sie keine finanziellen Interessen haben.

Hier haben wir die Isolierung und Herstellung von Darmsäcken detailliert Schleimhautbarrierefunktion ex vivo zu beurteilen. Darmsack Vorbereitungen sind in der pharmazeutischen Forschung genutzt, die Prüfung der Aufnahme von Wirkstoffkandidaten über den Darm in erster Linie ist. Allerdings ist dieser Test gleichermaßen gut für die Untersuchung von Darmerkrankungen geeignet. Darmdurchlässigkeit kann stark von Region und standortspezifische Bewertung der Durchlässigkeit ermöglicht ein besseres Verständni...

Die intestinale epitheliale Barriere des Gastrointestinaltraktes ist Bereich einer Schleimhautoberfläche bei 400 m 2 in dem erwachsenen Menschen geschätzt. Folglich wird es ständig ausgesetzt von Mikroben, eingenommene Drogen, Nährstoffe und bakterielle Toxine in Frage zu stellen. Der Host muss nicht nur zwischen tolerierbaren kommensalen Bakterien und potentielle Pathogene unterscheiden, aber müssen diese Arten und ihre sezernierten Moleküle vor der Kreuzung die Epithelbarriere, während zur gleichen Zeit ermöglicht die Aufnahme von Nährstoffen zu vermeiden. Somit ist die Rolle des Darmepithels als selektive Barriere zu der luminalen Inhalte 1 zu wirken. Dies wird dadurch erreicht, teilweise durch das angeborene epithelialen Abwehrsystem an der Schleimhaut, die eine reaktions biologischen System wirkt über 2 konstitutiver und induzierbarer Mechanismen aus. Verlust von epithelialen Barrierefunktion ist eine Pathologie, die charakteristisch für eine Reihe von Magen-Darm-Erkrankungen ist. In vivoPrüfung der epithelialen Barrierefunktion kann durch orale Gabe von Tracer-Molekül und anschließende Serumanalyse 3 bewertet werden. Allerdings bietet diese Technik keinen Hinweis auf den Ort der Schrankenstörung. In-vitro- und Ex-vivo-Bewertung der transepithelialen Widerstand mit Transwell-Systeme 3 und Ussing-Kammern 4,5 bzw. werden als Surrogatmarker der epithelialen Barrierefunktion häufig verwendet, aber nicht über die beitragenden Krankheit Physiologie von Tiermodellen 6. In diesem Protokoll beschreiben wir eine ex vivo Gewebepräparationsmodell, das direkte und lokale Bewertung der Darm-Integrität ermöglicht und die verwendet werden können Schleimhautbarrierefunktion in mehreren Ebenen zu beurteilen. Wichtig ist, kann diese Technik zu Tiermodellen von Krankheiten angewendet werden, oder pharmakologisch manipuliert werden kann, in der Tiefe Abfrage von Schleimhaut-Barriere Dysfunktion zu ermöglichen.

<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

Die epitheliale Barriere ist der erste angeborenen Abwehr des Gastrointestinaltraktes und regelt selektiv Verkehr von dem Lumen zu dem darunter liegenden Gewebe Fächer, den Transport von kleineren Molekülen über das Epithel zu beschränken und nahezu vollständig zu verbieten epithelialen makromolekulare Transport. Diese Selektivität wird durch die Schleimhaut Gelschicht bestimmt, die den Transport von lipophilen Molekülen und sowohl den apikalen Rezeptoren und enge Verbindungsprotein-Komplexe des Epithels begrenzt. In-vitro-Zellkulturmodellen des Epithels sind bequem, aber als ein Modell, es fehlt ihnen die Komplexität der Wechselwirkungen zwischen der Mikroflora, Schleim-Gel, Epithel und Immunsystem. Auf der anderen Seite, in vivo Beurteilung der intestinale Absorption oder Durchlässigkeit kann durchgeführt werden, aber diese Tests insgesamt Magen-Darm-Absorption zu messen, ohne Angabe von Ortsspezifität. Ex-vivo-Durchlässigkeit Tests &quot;Darmsäcken&quot; können; sind eine schnelle und empfindliche Verfahren entweder Gesamtdarm Integrität oder Vergleichs Transport eines spezifischen Moleküls, mit dem zusätzlichen Vorteil der intestinalen Ortsspezifität messen. Hier haben wir die Herstellung von Darmsäcken für Durchlässigkeit Studien und die Berechnung des scheinbaren Durchlässigkeit (P app) beschreiben eines Moleküls durch die Darmbarriere. Diese Technik kann als ein Verfahren zur Beurteilung der Arzneimittelabsorption verwendet werden, oder regional Epithelbarriere Dysfunktion in Tiermodellen von gastrointestinalen Erkrankungen zu untersuchen.

Dieses Protokoll kann verwendet werden, um regionale Veränderungen in Darmbarrierefunktion in Tiermodellen von gastrointestinalen Erkrankungen untersuchen. Durch Messung des Flusses eines parazellulären Sonde über der Schleimhautoberfläche in unterschiedlichen Bereichen des Gastrointestinaltraktes 7, die Integrität der epithelialen tight junctions beurteilt werden. Außerdem kann durch die Art der parazellulären Sonde von unterschiedlicher Größe (Abbildung 2)</...

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-Darm-Sacs zu beurteilen Durchlässigkeit der Schleimhaut in Modelle von Magen-Darm-Krankheit

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

21.0K Aufrufe.  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.

Serie: 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.

Echtzeitmessung der epitheliale Barriere Permeabilität im menschlichen Darm Organellen

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

Forschung

Entwicklungsbiologie

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.

Analyse wohltuende Wirkung von Nahrungsergänzungsmitteln auf Darmepithel Barrierefunktionen Während Experimental ulcerosa

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

Medizin

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.

Ein intravitaler, auf Mikroskopie basierender Ansatz zur Bewertung der Darmdurchlässigkeit und der Epithelzellvergießen-Performance

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

Immunologie und Infektion

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.

Funktionelle Beurteilung der Intestinalpermeabilität und der neutrophilen transepithelialen Migration bei Mäusen mit einem standardisierten Darmschleifenmodell

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.

Bestimmung der Darmpermeabilität mit Lucifer Yellow in einem apikalen enteroiden Modell

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.

Untersuchung des Darmbarriereabbaus bei lebenden Organoiden

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 und In Vivo Ansätze Darm Epithelzelle Durchlässigkeit zu bestimmen

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

Biologie

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.

Beurteilung der Integrität der Darmbarriere bei Mäusen unter Verwendung von Fluorescein-Isothiocyanat-markiertem 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 ...

Konstruktion eines 3D-Darmschleimhautmodells mit Paraffineinbettung

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.

Autor im Rampenlicht: Untersuchung der Auswirkungen von Verbindungen auf das Darmgewebe mit Hilfe von 3D-Modellen menschlicher Zelllinien 

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.

Grundlegendes dreidimensionales (3D) Darmmodellsystem mit einer Immunkomponente

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 ...