Published: June 11th, 2021
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....
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
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.......
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.......
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.....
|Active Caspase-3 antibody (1:1000)
|Cleaved caspase-3 (Asp175)(5AE1) Rabbit mAb
|Alexa Fluor 488 anti rabbit (1:1000)
|Alexa Fluor 594 anti rat (1:1000)
|Confocal microscope (Leica TCS SP8x)
|HyD detectors and White Light Laser
|E-Cadherin antibody (1:750)
|Rat monoclonal Delma-1 antibody
|Dextran, Alexa Fluor, 10,000 MW, anionic, fixable
|Fluorescein isothiocyanate–dextran M. Wt. 4 kDa
|Incubator (AutoFlow NU-8500)
|Microplate reader (Tecan Infinite 200 PRO)
|Nunc F96 MicroWell Black and White Polystyrene Plate
|Alexa Fluor 568 Phalloidin
|Rhodamine B Isothiocyanate-Dextran. M. Wt. 10 kDa
|Secondary antibodies (1:10000)
|Jackson ImmunoResearch Laboratories
|HRP-conjugated secondary antibodies
|Braided silk and braided polyester surgical sutures are prefered.
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