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...

<ol>
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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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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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. ...

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