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Biology

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

Published: June 11th, 2021

DOI:

10.3791/62204

1Departamento de Fisiología, Biofísica y Neurociencias del Centro de Investigación y de Estudios Avanzados (CINVESTAV), 2Departamento de Biomedicina Molecular, del Centro de Investigación y de Estudios Avanzados (CINVESTAV), 3Unidad de Investigación Epidemiológica en Endocrinología, y Nutrición (UIEEN), Hospital Infantil de México Federico Gomez, 4Laboratorio de Investigación en Inmunología y Proteómica, Hospital Infantil de México Federico Gómez
* These authors contributed equally

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.

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.

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 a....

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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

  1. 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.
  2. For analyzing the passive paracellular permeability, prepare a working solution by di.......

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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.......

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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.......

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The research was partially supported by the SEP-Conacyt grant (No.179 to NV/PND) and supported by the sectorial funding for research and education via the grant for Basic Science from Conacyt (No. A1-S-20887 to PND). We want to extend our gratitude to Norma Trejo, M.V.Z. Raúl Castro Luna, M.C. Leonel Martínez, Felipe Cruz Martínez, Victor Manuel García Gómez and M.V.Z. Ricardo Gaxiola Centeno for their help and technical assistance.

....

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Name Company Catalog Number Comments
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

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