Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Summary

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.

Abstract

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.

Introduction

The gastrointestinal (GI) mucosa creates a physical barrier that separates the extracellular environment and the internal host milieu, and is involved in the absorption of nutrients, water and electrolytes. The intestinal barrier encompasses a mucus layer constituted of glycoproteins, a monolayer of epithelial cells, and the underlying lamina propria are immune and stromal cells reside. Intestinal epithelial cells forming the physical barrier are linked together by different protein complexes, which includes the adherens junction (AJ), the tight junction (TJ) and the desmosomes (DMs). Impairment in the epithelial barrier function augments intestinal permeability and allows the translocation of harmful substances and pathogens from the lumen to the interstitium¹. There is an increasing number of illnesses where the epithelial barrier is compromised, such as the inflammatory bowel diseases (IBD) like Crohn’s disease (CD), ulcerative colitis (UC) and indeterminate colitis (IC). The incidence of IBD is increasing worldwide, with a prevalence approaching 0.5% in the West. Although the causes of IBD are unclear, the excessive immune/inflammatory response triggered in the gut wall directly contributes to the epithelial barrier disruption by limiting the reestablishment of intestinal epithelial homeostasis^2,3,4. In addition, patients with long-standing colonic inflammation are at high risk of developing colorectal cancer (CRC)⁵. Other pathologies associated with intestinal epithelial barrier disruption are irritable bowel syndrome, obesity, celiac disease, non-celiac gluten sensitivity, and food allergies⁶. For these reasons, there is an urgent need for the development of experimental approaches that allow analysis of the integrity of the intestinal epithelial barrier in animal models mimicking the pathogenesis occurring in humans.

Here, we evaluated the gastrointestinal passive paracellular and the transcellular permeability associated to an inflammatory process in the colonic epithelium using a simple technique. To investigate the transmural flow of macromolecules, we measured the passive diffusion of FITC-dextran (4 kDa) and RITC-dextran (10 kDa) in colonic sacs ex vivo. Furthermore, by injecting a fluorescent 10 kDa lysine-fixable dextran into the lumen of the intestine sacs, we specifically identified the areas with high permeability in the inflamed mucosa. The use of apoptosis markers and antibodies against AJ proteins allowed us to demonstrate that high permeability areas in the inflamed mucosa correspond to specific regions where epithelial cells undergo apoptosis and cell-cell junctions are disrupted. This new technique can be used to evaluate the integrity of the epithelium in any model where the intestinal epithelial barrier is compromised.

Protocol

All procedures were reviewed and approved by the CINVESTAV Institutional Committee for Care and Use of Laboratory Animals (CICUAL). 1. Preparation of materials and reagents Pre-warm Hartmann's solution (130 mM NaCl, 28 mM lactate, 4 mM KCl, 1.5 mM CaCl2) to 37 °C while bubbling with 95% O2/5% CO2. Maintain physiological pH (7.4) for the solution. For analyzing the passive paracellular permeability, prepare a working solution by di…

Representative Results

In the inflamed mucosa, superficial erosions or microerosions compromise the integrity of the epithelial cell monolayer and represent sites of high permeability7,8. To assess such possibilities, we analyzed the passive permeability in the inflamed colonic mucosa in a dextran sodium sulfate colitis murine model. In brief, for 5 days, C57BL/6J mice received 2.5% DSS (w/v, 40-50 kDa) dissolved in drinking water. This model is characterized by inducing epithelial cel…

Discussion

Epithelial homeostasis resulting from balancing cell proliferation and epithelial apoptosis maintains a proper and functional intestinal barrier. Many clinical disorders, such as IBD, are accompanied or characterized by alterations in intestinal permeability, inflammation of the mucosa and disruption of the epithelial homeostasis¹. The interplay between those processes is still highly controversial. Therefore, the development of new research approaches to properly investigate those processes is an…

Materials

Active Caspase-3 antibody (1:1000)	Cell signaling	9664	Cleaved caspase-3 (Asp175)(5AE1) Rabbit mAb
Alexa Fluor 488  anti rabbit (1:1000)	Invitrogen	A21206
Alexa Fluor 594 anti rat (1:1000)	Invitrogen	A21209
Confocal microscope (Leica TCS SP8x)	Leica		HyD detectors  and White Light Laser
E-Cadherin antibody (1:750)	Sigma	MABT26	Rat monoclonal Delma-1 antibody
Ethanol 70%	Generic
Fixable-Dextran	Invitrogen	D22914	Dextran, Alexa Fluor, 10,000 MW, anionic, fixable
FITC Dextran	Sigma	46944	Fluorescein isothiocyanate–dextran M. Wt. 4 kDa
Hartmann's Solution	PiSA	HT PiSA
Incubator (AutoFlow NU-8500)	Nuaire
Microplate reader (Tecan Infinite 200 PRO)	Tecan
Nunc F96 MicroWell Black and White Polystyrene Plate	ThermoFisher Scientific
Paraformaldehyde	Sigma	P6148
Phalloidin (1:1000)	Invitrogen	A12380	Alexa Fluor 568 Phalloidin
RITC Dextran	Sigma	R8881-100MG	Rhodamine B Isothiocyanate-Dextran. M. Wt. 10 kDa
Secondary antibodies (1:10000)	Jackson ImmunoResearch Laboratories		HRP-conjugated secondary antibodies
Suture threads	Generic		Braided silk and braided polyester surgical sutures are prefered.
ZO-1 (1:1000)	Invitrogen	40-2200	Rb anti-ZO-1