The research leading to these results has received funding from the People Program (Marie Curie Actions) under REA grant agreement number 302170 of the European Union&#180;s Seventh Framework Programme (FP7/2007-2013); the Interdisciplinary Center for Clinical Research (IZKF) of the University Erlangen-Nuremberg; the Collaborative Research Center TRR241 and the Clinical Research Group KFO257 of the German Research Council (DFG); and the DFG.

The following protocol has been approved by the relevant local authorities in Erlangen (Regierung von Unterfranken, W&#252;rzburg, Germany). Mice were housed under specific pathogen-free conditions.
NOTE: Inhibition of GGTase-mediated prenylation within IECs causes a severe alteration of intestinal permeability in Pggt1bi&#916;IEC mice24. Therefore, this mouse model was used to demonstrate how the protocol can be useful to study intestinal barrier defects. However, this protocol could be used for the study of any other mouse line.
1. Surgical preparation and mouse intestinal mucosa staining
NOTE: The surgical preparation is based on previously described protocols25. Keep the anesthetized mouse under a red lamp during the surgical preparation, to avoid a drop in body temperature.
<ol>
	<li>Anesthetize mouse by intraperitoneal injection of ketamine/xylazin (96 mg/kg ketamine; and 12.8 mg/kg xylazin). Verify the anesthesia by checking for the lack of an eyelid reflex.</li>
	<li>Apply eye protection cream using a cotton bud.</li>
	<li>Make an incision (1 cm) on the left ventral area using standard forceps and straight fine scissors.</li>
	<li>Exteriorize a segment of the intestine (5-7 cm, approximately).</li>
	<li>Open the exteriorized intestinal segment longitudinally by electrocauterization at the antimesenteric side.</li>
	<li>Expose the mucosa and rinse shortly with saline solution to remove fecal content. 
	NOTE: Optionally, apply xylazin directly on the gut to avoid motion artifacts due to peristalsis.</li>
	<li>Stain the surface of the intestinal mucosa with acriflavine and rhodamine-B dextran (10 kDa).
	<ol>
		<li>Apply the 1 mg/mL acriflavine solution (100 &#181;L) by pipetting drop by drop on the mucosa and incubate for 3 min. Wash out the remaining solution with PBS.</li>
		<li>Apply the 2 mg/mL rhodamine-dextran solution (100 &#181;L) by pipetting drop by drop on the mucosa and incubate for 3 min. Wash out the remaining solution with PBS. 
		NOTE: Optionally, remove blood from the preparation using aseptic cotton.</li>
	</ol>
	</li>
	<li>Place the anesthetized mouse supine on a cover slide mounted in a chamber rinsed with pre-warmed saline solution (37 &#176;C). 
	NOTE: The opened intestinal segment is then placed luminal surface down, on the cover slide.</li>
	<li>Place the preparation (anesthetized mouse in the chamber) on the inverted microscope stage.</li>
	<li>Cover the animal with an isothermal pad (approx. 37 &#176;C).</li>
	<li>Proceed immediately to intravital microscopy. 
	NOTE: Keep the surgical preparation rinsed with pre-warmed saline solution to avoid dehydration of the tissue and cell death.</li>
</ol>
2. Intravital microscopy
NOTE: Perform step 2.1 before starting the surgical preparation, to avoid long waiting times between anesthesia, surgery and image acquisition. If necessary, additional doses of anesthetics can be given to surgically prepared animals to keep them under anesthesia for the imaging experiments.
<ol>
	<li>Set up the CLSM microscope.
	<ol>
		<li>Start the CLSM microscope by turning on the microscope base and the scanner box. Turn on the computer by pressing the Start button.</li>
		<li>Launch the image acquisition software (e.g., LAS X) by double clicking on the icon. Select the appropriate configuration (Configuration: machine; Microscope: DMI6000) and click OK.
		<ol>
			<li>Define the appropriate Resolution. Go to Configuration | Hardware | Resolution | Bit depth. Select 12.</li>
		</ol>
		</li>
		<li>Go to the Acquisition menu. Select xyzt for the image acquisition mode from the drop-down menu. Select the objective from the drop-off menu (20x or 40x).</li>
		<li>Design the sequential acquisition setting. Click on Seq. Add a second sequence, by clicking on the add button (+). Select Between frames.
		<ol>
			<li>Configure Sequence 1 (detection of acriflavine; 416 nm excitation; 514 nm emission). Turn on the visible laser box. Activate the PMT1 (ON). Define the emission wavelength window (490-550 nm).</li>
			<li>Configure Sequence 2 (detection of rhodamineB-dextran; 570 nm excitation; 590 nm, emission). Turn on the visible laser box. Activate the PMT2 (ON). Define the emission wavelength window (550-760).</li>
		</ol>
		</li>
		<li>Activate the corresponding lasers (488 and 552). Go to Configuration | Lasers. Activate 488 and 552 nm lasers (turn ON).</li>
	</ol>
	</li>
	<li>Adjust the setting to the specific features of the current experiment.
	<ol>
		<li>Turn ON the light source (press power). Place the preparation (anesthetized mouse in the chamber) on the microscope stage and change the xy position until the illumination axis is focused on the tissue preparation.</li>
		<li>Select the field of interest using the standard light source and the eyepieces.
		<ol>
			<li>Select the filter cube (I3). Open the shutter. Focus on the surface of the intestinal mucosa by using the macro- and micro-wheel. Search for an area where several villi can be visualized within the field of view by changing the XY position.</li>
		</ol>
		</li>
		<li>Verify that the rhodamine-dextran staining is also visible in that area. Change the filter cube (N2.1). Check the image through the eyepieces.</li>
		<li>Start the CLSM image acquisition from the software. Optimize settings for the two sequences.
		<ol>
			<li>Select Sequence 1. Adjust laser power, gain and offset for Sequence 1.</li>
			<li>Select Sequence 2. Adjust laser power, gain and offset for Sequence 2.</li>
		</ol>
		</li>
		<li>Define the z stack range.
		<ol>
			<li>Open the Z-stack drop-off menu. Focus on the surface of the mucosa using the z-axis control. Press Begin.</li>
			<li>Focus on the bottom limit where the signal is still detectable. Press End.</li>
			<li>Define the numbers of z stacks (10). Avoid time lapses longer than 2 min for two consecutive time points.</li>
		</ol>
		</li>
		<li>Define the time settings. Go to the Time menu. Click Minimize. Select Acquire until stopped.</li>
		<li>Define the line average. Select Seq 1. Select Line Average 2. Select Seq 2. Select Line Average 2.</li>
	</ol>
	</li>
	<li>Acquire corresponding images.
	<ol>
		<li>Select Format (1024 x 1024) and Speed (400). Press Start.</li>
		<li>Press Stop after the desired image acquisition time.</li>
	</ol>
	</li>
	<li>Save the file. Go to Project. Right click on the corresponding file. Press Save as.
	<ol>
		<li>Name the file appropriately. Select the adequate folder. Press Save.</li>
	</ol>
	</li>
	<li>Euthanize the animal (cervical dislocation) and collect tissues for ulterior analysis, if needed.</li>
</ol>
3. Image analysis: Determine cell shedding rate and the intestinal epithelium
<ol>
	<li>Cell shedding rate
	<ol>
		<li>Launch the image acquisition software.</li>
		<li>Go to Open projects. Select the appropriate file.</li>
		<li>Define the total time of image acquisition.
		<ol>
			<li>Select the last complete z stack. Select the last Z position from the previously selected time point. Read the time on the right bottom corner of the screen (total time of image acquisition).</li>
		</ol>
		</li>
		<li>Select a villus.
		<ol>
			<li>Select the appropriate Z stack position (surface of the villus, but deep enough to avoid the interference with already shed cells and other components present at the lumen).</li>
			<li>Measure the length of basal membrane. Select the ruler tool. Divide the villus in several lines to cover or draw the whole villus perimeter. Add the different values (total length of the basal membrane). Delete the segments of the Ruler tool.</li>
		</ol>
		</li>
		<li>Count the number of events occurring during the whole duration of the image acquisition.
		<ol>
			<li>Divide the villus into segments with an adequate size. Analyze the sequence of events/segment by sliding the bar through the different time points. Annotate the identified cell shedding events.</li>
		</ol>
		</li>
		<li>Calculate the number of shedding events/time/length of basal membrane, which indicates the cell shedding rate. Count up to 5 villi/video, and at least two videos/mouse. Calculate the mean of these 10 measurements.</li>
	</ol>
	</li>
	<li>Leakage
	<ol>
		<li>Select a villus.</li>
		<li>Select the appropriate Z stack position (surface of the villus, but deep enough to avoid the interference with already shed cells and other components present at the lumen). Count total number of epithelial cells/villus.</li>
		<li>Count the number of leakage points (Para-cellular presence of rhodamine dextran. This event is transitory, which can be confirmed by checking previous and ulterior acquired pictures).</li>
		<li>Calculate the number of leakage/total number of epithelial cells. Analyze 10 different villus/sample/video and calculate the average number of leakage and permeable cells/villus.</li>
	</ol>
	</li>
</ol>

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Although technically challenging, intravital microscopy-based methodology represents a unique experimental approach to visualize highly dynamic cellular process in real time, such as cell shedding performance. Thus far, there is no alternative experimental approach to visualize cell extrusion in vivo. We believe that this protocol can contribute to the description of diverse cellular processes playing a role in the maintenance of intestinal homeostasis.
Taking advantage of intravital microscop...

The intestine is a highly specialized organ with a tightly regulated function enabling conflicting processes, namely nutrition and protection against harmful luminal substances. Lining between the human body and the environment, the intestinal epithelium acts as a physical and immunological barrier and contributes to the maintenance of mucosal homeostasis in the gut1,2. Loss of epithelial integrity and increased tight junction permeability is well known to be associated with Inflammatory Bowel Disease (IBD)3,4,5,6. Epithelial alterations are then considered as causes and secondary amplifiers for chronic intestinal inflammation in IBD. Thus, an improved understanding of early epithelial alterations in the gut of IBD patients would be of immense value for the development of new strategies to restore epithelial integrity for reliable prediction and subsequent prevention of IBD relapses.
Intestinal epithelium follows a complex and tightly regulated turnover process. From the crypt bottom, terminally differentiated intestinal epithelial cells (IECs) derived from pluripotent stem cells migrate upwards to the villus tip, where aged/damaged cells are shed into the lumen7. The equilibrium between division and cell extrusion enables the maintenance of intestinal epithelial cell numbers, avoiding the formation of gaps and leakage, as well as the accumulation of epithelial cells potentially leading to cell masses and tumorigenesis8,9,10. Despite the key role of epithelial cell shedding in the physiological renewal of the gut epithelium, the knowledge about the molecular mechanisms driving the extrusion of cells at the villus tip is limited. Thus, there is a need for basic research providing a precise description of the sequence of molecular events involved in epithelial cell shedding.
Complex interactions between different cell types within the intestinal mucosa are key to understand the molecular mechanisms regulating epithelial turnover and intestinal homeostasis. Thus, in vivo studies offer high advantages over in vitro and ex vivo approaches in this context. Moreover, real-time imaging techniques permit the description of the sequence of events controlling specific phenomena. In this context, the study of highly dynamic processes demands the use of optimized high resolution techniques for the direct observation of the tissue. Intravital imaging techniques appear as unique suitable tools for the study of epithelial cell shedding in the gut.
The term &#34;intravital microscopy&#34; refers to experimental approaches taking advantage of high-resolution imaging techniques (multiphoton or confocal microscopy) to directly visualize cells and tissues in their native environs within a living animal11. It enables real time acquisition of in vivo information up to single-cell resolution, and entails clear advantages over static or low resolution methods. Intravital microscopy provides complementary information and overcome some limitations from classical and/or high-end techniques, such as artifacts due to tissue processing. In contrast, the main limitation of intravital microscopy is that the tissue should be directly exposed to the microscope, which in most cases requires surgery. Although sophisticated approaches preserve the vitality and minimize the impact of the imaged tissue (skinfold chambers and imaging windows)12,13, in most cases a simple skin incision is performed for the externalization of the tissue (skin flaps)14. In the last decade, these approaches have contributed key evidence about highly dynamic processes, which were previously inscrutable. Translationally, real-time imaging provided new biological insights on stem cell and leukocytes homing15, as well as cancer dissemination and metastasis formation13,16. In the clinical context, endomicroscopy is currently exploited as a diagnostic tool of cancer17 and gastrointestinal diseases, such as IBD18,19; while confocal mosaicking microscopy became a rapid pathology tool during surgery20. Together, intravital microscopy has lately emerged as a valuable and versatile tool for biomedical research and future application in the clinic.
Intravital microscopy is here implemented for real-time visualization of intestinal epithelial leakage and observation of epithelial cell shedding events. Leakage of intestinal permeability can be identified by other in vivo noninvasive techniques, such as quantification of orally administration of fluorescent tracers in serum21. However, this technique does not allow the direct observation of shedding performance nor the segregation between para- and trans-cellular permeability. The combination of standard tracer experiments and intravital microscopy represents a suitable approach to: i) identify disturbances in intestinal permeability, and ii) segregate between para- and trans-cellular epithelial permeability. Besides cell shedding, intravital microscopy in combination with in vivo fluorescence labelling enables the study of other cellular and molecular mechanisms (e.g., tight junction redistribution during cell shedding using fluorescent reporter mice22 or interactions between IECs and other cells within the intestinal mucosa23).
The method presented here represents an adaptation of intravital microscopy to enable real-time observation of intestinal mucosa, using confocal laser scanning microscopy (CLSM). Therefore, we use conditional knock-out mice of GGTase (Geranylgeranyltransferase) in intestinal epithelial cells (IECs) in (Pggt1bi&#916;IEC mice), since they suffer from a severe intestinal disease and increased epithelial permeability24. Surgical preparation of the mouse and staining of the intestinal mucosa, as well as appropriate settings used for imaging acquisition and post-acquisition analysis are described. This protocol could enable future studies contributing to the current knowledge about dynamics and kinetics of intestinal epithelial cell shedding. Moreover, the protocol could serve as a basis for various adaptations to study other phenomena occurring at the surface of the intestinal mucosa, and even at other tissues.

<table><tbody><tr><td>Acriflavine hydrochloride</td><td>Sigma Aldrich</td><td>A8251</td><td>1 mg/mL solution in PBS</td></tr><tr><td>Deltaphase isothermal pad</td><td>BrainTree</td><td>B-DP-PAD</td><td>-</td></tr><tr><td>Gemini Cautery System</td><td>BrainTree</td><td>B-GEM-5917</td><td>-</td></tr><tr><td>Ketamin</td><td>WDT</td><td>9089.01.00</td><td></td></tr><tr><td>LAS X</td><td>Leica</td><td>-</td><td>-</td></tr><tr><td>LSM microscope SP8</td><td>Leica</td><td>-</td><td>-</td></tr><tr><td>PBS</td><td>Biochrom</td><td>L182</td><td></td></tr><tr><td>Rhodamine B dextran</td><td>Invitrogen</td><td>D1824</td><td>10,000 kDa MW; 2 mg/mL solution</td></tr><tr><td>Standard forceps (Dumont SS)</td><td>Fine Science Tools</td><td>11203-23</td><td>-</td></tr><tr><td>Straight fine scissors</td><td>Fine Science Tools</td><td>14060-10</td><td>-</td></tr><tr><td>Tamoxifen</td><td>Sigma Aldrich</td><td>T5648</td><td>50 mg/mL in ethanol</td></tr><tr><td>Xylazin</td><td>Bayer</td><td>1320422</td><td></td></tr></tbody></table>

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.

The protocol presented here describes an intravital microscopy-based approach to visualize intestinal epithelial leakage and observe cell shedding performance in the gut in real-time. Briefly, mice are anesthetized and submitted to surgical preparation in order to expose the surface of the small intestine mucosa. IECs are then stained via topical application of acriflavine; while luminal rhodamine B-dextran is used as tracers to detect transmucosal passage from the lumen to the sub-epithe...

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

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. ...

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Research

JoVE Journal

Immunology and Infection

5.7K Views.  University Hospital of Erlangen. Met behulp van intravitale microscopie maakt dit protocol de real-time visualisatie van darmweefsel mogelijk tot een eencellige resolutie en de studie van zeer dynamische processen zoals celafstoten. In combinatie met de standaard tracer experimenten, dit protocol maakt het mogelijk de identificatie van zowel darmdoorlaatbaarheid verstoringen in vivo en het onderscheid tussen para en transcellulaire doorlaatbaarheid. Dit protocol kan worden gebruikt als basis voor de ontwikkeling van aanvulle...

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

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.

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

Developmental Biology

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 ...

Medicine

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 ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

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.

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.

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 ...

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

This protocol details the establishment of enteroids, a three-dimensional intestinal model, from fetal intestinal tissue. Immunofluorescent imaging of epithelial biomarkers was used for model characterization. Apical exposure of lipopolysaccharides, a bacterial endotoxin, using microinjection technique induced epithelial permeability in a dose-dependent manner measured by the leakage of fluorescent dextran.

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit ...

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

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

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

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