The authors thank Dr. Sven Flemming of the University of Wuerzburg for his contributions to the establishment of the proximal colon loop model, Sean Watson for the management of the mouse colonies and Chithra K. Muraleedharan for helping with the acquisition of the pictures of the iLoop model. This work was supported by the German Research Foundation/DFG (BO 5776/2-1) to KB, R01DK079392, R01DK072564, and R01DK061379 to C.A.P.

The authors have nothing to disclose.

The mechanisms responsible for dysregulation of intestinal barrier function and immune cell recruitment under pathologic conditions such as IBD are incompletely understood. Here, we detail a robust in vivo murine model that employs a well-vascularized exteriorized intestinal segment of either ileum or proximal colon and allows for assessment of intestinal permeability, neutrophil migration studies as well as other applications.
The iLoop is a non-recovery surgery that is performed on live anim...

The intestinal mucosa encompasses a single layer of columnar intestinal epithelial cells (IECs), underlying lamina propria immune cells and the muscularis mucosae. Besides its role in the absorption of nutrients, the intestinal epithelium is a physical barrier that protects the body interior from luminal commensal bacteria, pathogens, and dietary antigens. In addition, IECs and lamina propria immune cells coordinate the immune response inducing either tolerance or response depending on the context and stimuli. It has been reported that the disruption of the epithelial barrier can precede the onset of pathologic mucosal inflammation and contribute to inflammatory bowel disease (IBD) encompassing both ulcerative colitis and Crohn&#39;s disease1,2,3,4,5,6,7. Individuals with ulcerative colitis present excessive transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) forming crypt abscesses, a finding that has been associated with severity of disease8,9. Although compromised epithelial barrier function and excessive immune responses are hallmarks of IBD, there is a lack of experimental in vivo assays to perform quantitative assessments of intestinal permeability and immune cell recruitment into the intestinal mucosa.
The most common methods used to study intestinal epithelial permeability and PMN TEpM employ ex vivo chamber-based approaches using IEC monolayers cultured on semi-permeable porous membrane inserts10,11,12. The epithelial barrier integrity is monitored either by measurements of transepithelial electrical resistance (TEER) or the paracellular flux of the Fluorescein isothiocyanate (FITC)-labeled dextran from apical to basal compartment13,14,15. Similarly, PMN TEpM is typically studied in response to a chemoattractant that is added in the lower chamber16. PMN are placed in the upper chamber and after an incubation period, PMN that have migrated into the basal compartment are collected and quantified. While these methods are useful, easy to perform and very reproducible, they are obviously reductionist approaches and do not necessarily represent an accurate reflection of in vivo conditions.
In mice, a common assay to study intestinal paracellular permeability is by oral gavage of FITC-dextran and subsequent measurement of FITC-dextran appearance in the blood serum13,17. The disadvantage of this assay is that it represents an assessment of overall barrier integrity of the gastrointestinal tract rather than that of regional intestinal contributions. In addition, Evans blue is commonly used to evaluate vascular leakage in vivo18 and has also been employed to evaluate intestinal mucosal permeability in mouse and rat19,20,21. The quantification of Evans blue in the intestinal mucosa requires extraction from tissue employing incubation in formamide overnight. Therefore, the same tissue cannot be used to study intestinal epithelial permeability and neutrophil infiltration.
Here we highlight a simple protocol that reduces the number of animals needed to collect reproducible data on colonic mucosal permeability and leukocyte transepithelial migration in vivo. We, therefore, recommend the use of FITC-dextrans that are easily detectable in blood serum without compromising the integrity of intestinal loops which can be harvested for further analysis. Of note, the intestinal ligated loops have been used in various species (including mouse, rat, rabbit, calf) to study bacterial infection (such as Salmonella, Listeria monocytogenes and Escherichia coli)22,23,24,25 as well as intestinal permeability26; however, to the best of our knowledge there are no studies investigating mechanisms of PMN TEpM in specific regions in the intestine such as ileum or colon that are commonly involved in IBD.
Here we describe the mouse intestinal loop (iLoop) model that is a robust and reliable microsurgical in vivo method that employs a well-vascularized and exteriorized intestinal segment of either the ileum or proximal colon. The iLoop model is physiologically relevant and allows the assessment of intestinal barrier integrity and PMN TEpM on living mice under anesthesia. We demonstrate two applications: 1) quantification of serum levels of 4 kDa FITC-dextran after intraluminal administration in the iLoop 2) quantification of transmigrated PMN in the iLoop lumen after intraluminal injection of&#160;the potent chemottractant Leukotriene B4 (LTB4)27. Moreover, utilizing the iLoop model with Jam-a-null mice or mice harboring selective loss of JAM-A on IECs (Villin-cre;Jam-a fl/fl) compared to control mice, we are able to corroborate previous studies that have reported a major contribution for tight junction-associated protein JAM-A to intestinal permeability and neutrophil transmigration15,28,29,30,31.
The iLoop model is a highly functional and physiological method that can be used to corroborate in vitro assays. Furthermore, this is a versatile experimental model that allows the study of various reagents that can be injected into the loop lumen, including chemokines, cytokines, bacterial pathogens, toxins, antibodies and therapeutics.

<table><tbody><tr><td>&lt;strong&gt;Equipment and Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BD Alcohol Swabs</td><td>BD</td><td>326895</td><td></td></tr><tr><td>BD PrecisionGlide Needle, 25G X 5/8&quot;</td><td>BD</td><td>305122</td><td></td></tr><tr><td>BD PrecisionGlide Needle, 30G X 1/2&quot;</td><td>BD</td><td>305106</td><td></td></tr><tr><td>BD 1ml Tuberculin Syringe Without Needle</td><td>BD</td><td>309659</td><td></td></tr><tr><td>15ml Centrifuge Tube</td><td>Corning</td><td>14-959-53A</td><td></td></tr><tr><td>Corning 96-Well Solid Black Polystyrene Microplate</td><td>FisherScientific</td><td>07-200-592</td><td></td></tr><tr><td>Corning Non-treated Culture Dish, 10cm</td><td>MilliporeSigma</td><td>CLS430588</td><td></td></tr><tr><td>Cotton Tip Applicator (cotton swab), 6&quot;, sterile</td><td>FisherScientific</td><td>25806 2WC</td><td></td></tr><tr><td>Dynarex Cotton Filled Gauze Sponges, Non-Sterile, 2&quot; x 2&quot;</td><td>Medex</td><td>3249-1</td><td></td></tr><tr><td>EZ-7000 anesthesia vaporizer (Classic System, including heating units)</td><td>E-Z Systems</td><td>EZ-7000</td><td></td></tr><tr><td>Falcon Centrifuge Tube 50ml&amp;nbsp;</td><td>VWR</td><td>21008-940</td><td></td></tr><tr><td>Fisherbrand Colored Labeling Tape</td><td>FisherScientific</td><td>15-901-10R</td><td></td></tr><tr><td>Halsey Needle Holder (needle holder)&amp;nbsp;</td><td>FST</td><td>12001-13</td><td></td></tr><tr><td>Kimwipes, small (tissue wipe)</td><td>FisherScientific</td><td>06-666</td><td></td></tr><tr><td>1.7ml Microcentrifuge Tubes&amp;nbsp;</td><td>Thomas Scientific&amp;nbsp;</td><td>c2170</td><td></td></tr><tr><td>Micro Tube 1.3ml Z (serum clot activator tube)</td><td>Sarstedt&amp;nbsp;</td><td>41.1501.105</td><td></td></tr><tr><td>Moria Fine Scissors</td><td>FST</td><td>14370-22</td><td></td></tr><tr><td>5ml Polystyrene Round-Bottom Tube with Cell-Strainer Cap (35 &amp;micro;m nylon mesh)</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>Puralube Vet Ointment, Sterile Ocular Lubricant</td><td>Dechra</td><td>12920060</td><td></td></tr><tr><td>Ring Forceps (blunt tissue forceps)</td><td>FST</td><td>11103-09</td><td></td></tr><tr><td>Roboz Surgical 4-0 Silk Black Braided, 100 YD</td><td>FisherScientific</td><td>NC9452680</td><td></td></tr><tr><td>Semken Forceps (anatomical forceps)</td><td>FST</td><td>1108-13</td><td></td></tr><tr><td>Sofsilk Nonabsorbable Coated Black Suture Braided Silk Size 3-0, 18&quot;, Needle 19mm length 3/8 circle reverse cutting&amp;nbsp;</td><td>HenrySchein</td><td>SS694</td><td></td></tr><tr><td>Student Fine Forceps, Angled</td><td>FST</td><td>91110-10</td><td></td></tr><tr><td>10ml Syringe PP/PE without needle</td><td>Millipore Sigma&amp;nbsp;</td><td>Z248029</td><td></td></tr><tr><td>96 Well Cell Culture Plate</td><td>Corning</td><td>3799</td><td></td></tr><tr><td>Yellow Feeding Tubes for Rodents 20G x 30 mm</td><td>Instech</td><td>FTP-20-30</td><td></td></tr><tr><td>&lt;strong&gt;Solutions and Buffers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Accugene 0.5M EDTA</td><td>Lonza</td><td>51201</td><td></td></tr><tr><td>Ammonium-Chloride-Potassium (ACK) Lysing Buffer</td><td>BioWhittaker</td><td>10-548E</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution</td><td>Corning</td><td>21-023-CV</td><td></td></tr><tr><td>Phosphate-Buffered Saline without Calcium and Magnesium</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 647 Anti-Mouse Ly-6G Antibody (1A8)</td><td>BioLegend</td><td>127610</td><td></td></tr><tr><td>CD11b Monoclonal Antibody, PE, eBioscience (M1/70)</td><td>ThermoFisher</td><td>12-0112-81</td><td></td></tr><tr><td>CountBright Absolute Counting Beads</td><td>Invitrogen</td><td>C36950</td><td></td></tr><tr><td>Dithiotreitol</td><td>FisherScientific</td><td>BP172-5</td><td></td></tr><tr><td>Fetal Bovine Serum, heat inactivated</td><td>R&amp;amp;D Systems</td><td>511550</td><td></td></tr><tr><td>Fluorescein Isothiocyanate-Dextran, average molecular weight 4.000</td><td>Sigma</td><td>60842-46-8</td><td></td></tr><tr><td>Isoflurane</td><td>Halocarbon</td><td>12164-002-25</td><td></td></tr><tr><td>Leukotriene B&lt;sub&gt;4&lt;/sub&gt;</td><td>Millipore Sigma</td><td>71160-24-2</td><td></td></tr><tr><td>PerCP Rat Anti-Mouse CD45 (30-F11)</td><td>BD Pharmingen</td><td>557235</td><td></td></tr><tr><td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD FC Block)</td><td>BD Bioscience</td><td>553142</td><td></td></tr><tr><td>Recombinant Murine IFN-&amp;gamma;</td><td>Peprotech</td><td>315-05</td><td></td></tr><tr><td>Recombinant Murine TNF-&amp;alpha;</td><td>Peprotech</td><td>315-01A</td><td></td></tr></tbody></table>

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.

A schematic representation of the ileal loop and pcLoop models is depicted in Figure 1 and Figure 2, respectively. The anatomical pictures display the critical steps of the procedure including exteriorization of the intestinal segment (Figure 1B and Figure 2B), identification of an appropriate location for ligations that allows minimal disturbance of blood supply (<strong class=...

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

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.

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

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Research

JoVE Journal

Immunology and Infection

6.8K Views.  University of Michigan, Ann Arbor. Het darmmodel is een breed aanpasbare en fysiologische manier om te meten hoe lek de darm is en hoe leukocyten migreren in de darm bij muizen, in vivo. Deze methode helpt om mechanismen die ten grondslag liggen aan verhoogde darmpermeabiliteit en pathologische darmontsteking beter te begrijpen. De techniek kan worden gebruikt om te helpen bij het begrijpen van mechanismen die ten grondslag liggen aan de darmbarrièrefunctie en pathologische ontsteking, zoals te zien is bij colitis ulcerosa en...

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

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

Medicine

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

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

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.

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

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.

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

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.

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

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.

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.

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.

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

Biology

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.

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

Mouse Ileal Loop Model: A Surgical Model to Study Intestinal Permeability in the Mouse

Source: Boerner, K., et al. <a href="https://www.jove.com/v/62093">Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model.</a> J. Vis. Exp. (2021)
This video demonstrates the generation of an ileal loop model to study intestinal permeability in a mouse model. This method provides a live model to understand the epithelial barrier function and consequent immune response.

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

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