Name: an intravital microscopy-based approach to assess intestinal permeability and epithelial cell shedding performance
Uploaded: 2020-12-03T13:00:00
Description: Watch this Scientific Journal Video about An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance at JoVE.com

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 de...

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+In+vivo+Approaches+to+Determine+Intestinal+Epithelial+Cell+Permeability.">In vitro and In vivo Approaches to Determine Intestinal Epithelial Cell Permeability.</a> Journal of Visualized Experiments. (140), (2018).</li><li>Marchiando, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epithelial+barrier+is+maintained+by+in+vivo+tight+junction+expansion+during+pathologic+intestinal+epithelial+shedding.">The epithelial barrier is maintained by in vivo tight junction expansion during pathologic intestinal epithelial shedding.</a> Gastroenterology. 140 (4), 1208-1218 (2011).</li><li>Hoytema van Konijnenburg, D. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+barrier+function+in+vivo+is+sustained+despite+gaps+in+epithelial+layers.">Epithelial barrier function in vivo is sustained despite gaps in epithelial layers.</a> Gastroenterology. 129 (3), 902-912 (2005).</li><li>Haep, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon+Gamma+Counteracts+the+Angiogenic+Switch+and+Induces+Vascular+Permeability+in+Dextran+Sulfate+Sodium+Colitis+in+Mice.">Interferon Gamma Counteracts the Angiogenic Switch and Induces Vascular Permeability in Dextran Sulfate Sodium Colitis in Mice.</a> Inflammatory Bowel Diseases. 21 (10), 2360-2371 (2015).</li><li>Lopez-Posadas, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibiting+PGGT1B+Disrupts+Function+of+RHOA,+Resulting+in+T-cell+Expression+of+Integrin+alpha4beta7+and+Development+of+Colitis+in+Mice.">Inhibiting PGGT1B Disrupts Function of RHOA, Resulting in T-cell Expression of Integrin alpha4beta7 and Development of Colitis in Mice.</a> Gastroenterology. , (2019).</li><li>Fischer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+alpha4beta7+and+GPR15+on+homing+of+effector+and+regulatory+T+cells+from+patients+with+UC+to+the+inflamed+gut+in+vivo.">Differential effects of alpha4beta7 and GPR15 on homing of effector and regulatory T cells from patients with UC to the inflamed gut in vivo.</a> Gut. 65 (10), 1642-1664 (2016).</li></ol>

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.

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	<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...

Watch this Scientific Journal Video about An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance at JoVE.com

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. ...

Using intravital microscopy, this protocol enables the real-time visualization of intestinal tissue up to a single-cell resolution and the study of highly dynamic processes such as cell shedding. In combination with the standard tracer experiments, this protocol permits the identification of both intestinal permeability disturbances in vivo and the distinction between para and transcellular permeability. This protocol could be used as the basis for the development of additional intravital microscopy approaches for visualizing other highly dynamic cellular processes of interests within various tissues.Visual demonstration of this method is essential to understanding the surgical preparation steps and to position the living animal in a way that facilitates image acquisition on the intestinal mucosal surface. Before beginning the procedure, turn on the base and scanner box of a confocal laser scanning microscope and press start to turn on the computer. Launch the image acquisition software and select the appropriate configuration.To set the appropriate image resolution, select configuration, hardware, resolution, bit depth, and 12. Under the acquisition menu, select XYZT for the image acquisition mode and set the objective to 20 or 40X. To set up the sequential acquisition setting, click seek and add and select between frames.To configure sequence one, turn on the visible laser box. Set photomultiplier tube one to on and define the emission wavelength. To configure sequence two, turn on the visible laser box.Set photomultiplier tube two to on and define the emission wavelength. Then activate the appropriate lasers. After confirming a lack of response to toe pinch, use a cotton bud to apply ointment to the eyes of the anesthetized mouse and make a one centimeter incision in the left ventricle region of the abdomen.Use forceps to exteriorize a three to five centimeter segment of the intestine. Make two small incisions at the top and bottom of the gut segment and introduce a glass micropipette into the intestinal lumen. Then use electrocauterization to open the exteriorized intestinal segment longitudinally along the anti-mesenteric side and expose the mucosa.Rinse the tissue briefly with saline solution to remove the fecal content. For staining of the intestinal mucosa surface, apply 100 microliters of a one milligram per milliliter of acriflavine solution in droplets onto the exposed mucosal surface and allow the stain to develop for three minutes before washing out the remaining solution with PBS. Next, apply 100 microliters of a two milligram per milliliter rhodamine dextran solution in droplets to the mucosa for three-minute incubation before washing with PBS.Then place the anesthetized mouse supine on a cover slide mounted on the chamber rinsed with 37 degrees Celsius saline, adjusting the position if necessary, and place the preparation onto the inverted microscope stage. Immediately after placing the mouse onto the stage, turn on the light source and adjust the XY position until the illumination axis is focused on the tissue preparation. In order to select the field of vision, select the appropriate filter cube.Open the shutter and use the macro and micro wheels to focus on the surface of the intestinal mucosa. Adjust the XY position to locate an area in which several villi can be visualized within the field of view and change the filter cube to verify that the rhodamine dextran staining is also visible in that area. When an area of interest has been identified, start the image acquisition and select sequence one to adjust the laser power, gain, and offset for sequence one.Then select sequence two and adjust the laser power, gain, and offset for sequence two. To define the Z-stack range, open the Z-stack drop-off menu. Use the Z-access control to focus on the surface of the mucosa and click begin.Focus on the bottom limit of where the signal is still detectable and click end. Define the numbers of Z-stacks, avoiding time-lapses longer than two minutes for two consecutive time points and open the time menu to select minimize and acquire until stop. To define the line average, select seek one, line average two, seek two, and line average two.Then to acquire images, set the format to 1024 x 1024 pixels and the speed to 400 and click start. Intestinal epithelial cell-specific GGTase deficient conditional mice developed severe intestinal pathology as demonstrated by an increase in the histological damage score of the small intestine and increased intestinal epithelial permeability can also be detected via tracer in in-vivo experiments using orally administered FITC-dextran. Cell shedding events could be identified as cells moving out of the epithelial monolayer into the lumen, leading to temporary gaps in the ceiling of the epithelium that are finally closed by the contact between neighboring cells, a so-called zipper effect.These gaps are observed in both control and GGTase deficient mice, although the frequency of these phenomenon is higher in the latter. Interestingly, other cells also appear to uptake dextran. These so-called permeable cell events also occur mainly in conditional knockout mice.The immunostaining of cytoskeleton-related and tight junction proteins of interest can be performed to identify specific targets that contribute to potential epithelial permeability disruptions determined via intravital microscopy. This method can be used for the analysis of other phenomena on the surface of the intestinal mucosa, such as endothelial leakage or immune epithelial communication in the context of intestinal infection.

Research

JoVE Journal

Immunology and Infection

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

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 ...

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...