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

Summary

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.

Abstract

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.

Introduction

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 gut^1,2. Loss of epithelial integrity and increased tight junction permeability is well known to be associated with Inflammatory Bowel Disease (IBD)^3,4,5,....

Protocol

The following protocol has been approved by the relevant local authorities in Erlangen (Regierung von Unterfranken, Würzburg, 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 Pggt1b^iΔIEC mice²⁴. Therefore, this mouse model was used to demonstrate how the protocol can be useful to study intestinal barrier de.......

Discussion

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.......

Materials

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