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

Riepilogo

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.

Introduzione

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.

Protocollo

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 dissolving 1 mg/mL of FITC-Dextran (4 kDa) and 1 mg/mL of RITC-Dextran (10 kDa) in pre-warmed Hartmann´s solution.
3. Prepare a 4 µg/mL solution of Alexa Fluor647 Fixable-Dextran (10 kDa) in Hartmann's solution. Store working solutions in a 15 mL conical tube and protect from the light until use.
   NOTE: 300 µL of working solution per colon will be necessary.
4. Prepare a surgical suture by cutting two 5 cm sections for each large intestine. Loop the sutures into an unclosed knot.

2. Dissection and preparation of the gastrointestinal trac

1. Withhold solid food for 6 hours before euthanizing the mice. Provide ad libitum drinking water.
   NOTE: If possible, place the mice on nutrient gel supplements (purified Water, Molasses, Pumpkin, Corn Syrup, Sunflower Seeds, Wheat Protein, Vegetable Oil, Food Acid, Hydrocolloids, Electrolytes, Corn Fiber, NIH-31M Mineral Mix, NIH-31M Vitamin Mix).
2. Euthanize mice in a CO₂ chamber followed by cervical dislocation in accordance with institutional ethics protocols.
3. Sterilize the abdomen and thorax with 70% ethanol.
4. Using a pair of scissors, make an incision in the middle of the abdomen and expose the peritoneal cavity.
5. For orientation purposes, separate and dissect the large intestine by cutting at the end of the small intestine (end portion of the ileum) right before the cecum and then at the anal verge. Use surgical forceps to gently remove the mesentery and place the colon in Hartmann's solution.
6. Importantly, in order to maintain consistency between animals, identify similar sections and use to evaluate permeability. Using regions close to the cecum is highly recommendable.
7. Use an insulin syringe equipped with a blunted plastic cannula to gently flush the luminal content present in the colon. If the stool is firm, carefully push with the help of blunt forceps. After the feces have been removed, wash 3 times with 400 µL of Hartmann's solution.
8. Tie the proximal region (closest one to the cecum) and place a pre-tied suture-loop in the distal region of the colon. With the help of a syringe equipped with a blunted plastic cannula fill the intestinal sac with the solution containing the desire probe. Carefully remove the plastic cannula and tie the loop in the distal region.
9. Place the intestinal sac in a 15 mL conical tube with 6 mL of Hartmann's solution and incubate for 1 h to evaluate the passive paracellular flow of FITC/RITC-Dextran or 30 min to analyze the flow of the Alexa Fluor Fixable-Dextran.
   1. Maintain the conical tubes containing the intestinal sacs at 37 °C with 5% CO₂ and protect from light.
10. To measure the passive permeability using FITC/RITC-Dextran. At 0 and 60 min, take a 100 µL sample from the conical tube and transfer to a 96 well plate. Add back 100 µL of fresh media to replace the volume lost.
11. Measure samples and standards for FITC/RITC on a fluorescent plate reader (FITC excitation/emission: 495 nm/519 nm; RITC excitation/emission: 570/595 nm).
12. To measure the passive permeability using Alexa Fluor 647 Fixable-Dextran, remove the intestines, cut close to the surgical tie knot, and cut the intestine to expose the lumen to remove the solution with the probe. Wash the lumen of the intestine 2 times with cold Hartmann's solution.
13. Place the tissue in a tissue-mold previously filled with Optimal Cutting Temperature compound (O.C.T.). Orient the tissue vertically or horizontally according to the side to be sectioned. Store the samples at -80 °C.

3. Immunofluorescent staining

1. Fix frozen sections of 20 micrometers with 3.7% paraformaldehyde (PFA) for 20 min at room temperature and then wash 3 times with cold phosphate buffered saline (PBS; 37 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂ PO₄).
   NOTE: Vertical sections from intestines tend to come off if the washes are very strong.
2. Permeabilize with 0.2% TX-100/PBS for 12 min at room temperature and then wash 3 times with cold PBS.
3. Block with 0.2% BSA/PBS for 1 hour at room temperature.
4. Dilute the primary antibody in blocking solution and incubate for 1 h at room temperature. Wash 3 times with cold PBS.
5. Incubate for 1 h with secondary antibodies in blocking solution. Wash 3 times with cold PBS.
6. Apply mounting medium to the cuts and seal with a coverslip. The slides can be stored for up to 3 months at -20 °C.

Risultati

In the inflamed mucosa, superficial erosions or microerosions compromise the integrity of the epithelial cell monolayer and represent sites of high permeability^7,8. 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...

Discussione

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...

Materiali

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