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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Functional Assessment of Intestinal Tight Junction Barrier and Ion Permeability in Native Tissue by Ussing Chamber Technique

Published: May 26th, 2021

DOI:

10.3791/62468

1Laboratory of Physiology, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka

Intestinal epithelium confers not only nutrient absorption but protection against noxious substances. The apical-most epithelial intercellular junction, i.e., the tight junction, regulates paracellular solute and ion permeability. Here, a protocol for the preparation of mucosal sheets and assessment of the ion selectivity of tight junctions using Ussing chamber technique is described.

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog skin. Since then, this technique has been applied to many different tissues to study the physiological parameters of transport across membranes. The Ussing chamber method is preferable to other methods because native tissue can be used, making it more applicable to what is happening in vivo. However, because native tissue is used, throughput is low, time is limited, and tissue preparation requires skill and training. These chambers have been used to study specific transporter proteins in various tissues, understand disease pathophysiology such as in Cystic Fibrosis, study drug transport and uptake, and especially contributed to the understanding of nutrient transport in the intestine. Given the whole epithelial transport process of a tissue, not only transepithelial pathways, but also paracellular pathways are important. Tight junctions are a key determinant of tissue specific paracellular permeability across the intestine. In this article, the Ussing chamber technique will be used to assess paracellular permselectivity of ions by measuring transepithelial conductance and dilution potentials.

The Ussing chamber method was first developed by the Danish scientist Hans Ussing. Ussing first used it to measure the short-circuit current of sodium transport across frog skin after it was observed that NaCl could be transported across the skin against a steep concentration gradient1. His system consisted of the frog skin mounted between two chambers with access to either side of the skin. Each chamber contained Ringer's solution which was circulated and aerated. Two narrow agar ringer bridges situated near the skin and connected to saturated KCl-calomel electrodes measured the potential difference as read by a potentiator. A second pair ....

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All animals used in these experiments were maintained in the animal care facility at the University of Shizuoka and the experiments were conducted according to the guidelines for animal research set out by the University of Shizuoka. All experiments were carried out with approval from the Animal Care and Use Committee at the University of Shizuoka (Permits #205272 and #656-2303).

1. Preparation of NaCl electrodes

NOTE: The electrodes used in these experiments consist .......

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The results shown in this paper are results that were part of larger project that has been completed (see ref.4,23,24).

Transepithelial electrical conductance of the small intestine is decreased in Cldn15-/- mice.
The baseline transmucosal conductance (under short circuit conditions) of the middle small intes.......

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In this experiment, Ussing chambers were used to measure the baseline electrical parameters and the dilution potential of NaCl in the small intestine of Cldn15-/- and WT mice. It is very important when doing Ussing chamber experiments to verify that the membrane preparation used in the experiments is viable. This is usually done by adding glucose or the adenylate cyclase activator forskolin and seeing whether there is an appropriate rise in Isc (100-300 µA/cm2 in mi.......

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This work is supported by 17K00860 (to HH) and 19K20152 (to NI). WH would like to acknowledge the Otsuka Toshimi Scholarship Foundation for their financial support from 2018-2021.

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Name Company Catalog Number Comments
#3 polyethyl tubing Hibiki outer diameter 1.0 mm; inner diameter 0.5 mm
#7 polyethyl tubing Hibiki outer diameter 2.3 mm; inner diameter 1.3 mm
10 mL locking syringe Terumo SS-10LZ Locking syringes are necessary to prevent the needle from dislodging during filling
19 g needle Terumo NN-1938R Please use caution when working with needles and dispose of in sharps container
23 g needle Terumo NN-2332R Please use caution when working with needles and dispose of in sharps container
5 mm punch NA NA Use to punch holes in filter paper and parafilm
acupuncture needles Seirin NS Used as dissection pins to pin tissue to dissection plate
Agar Fujifilm Wako 010-15815
Alligator clips NA NA Connects the electrode to the amplifier
CaCl2 Fujifilm Wako 038-00445
D(-)-Mannitol Fujifilm Wako 133-00845 This is used to correct for the osmolality difference in dilution HEPES buffer
D(+)-Glucose Fujifilm Wako 049-31165
Dissection kit You will need, scissors and curved forceps
Dissection plates We used 10 cm cell culture plates and covered with silicon rubber
DMSO Sigma 472301-500ML For making forskolin stock
Electrical recorder TOA Electronics PRR-5041 Other equivalent electrical recorders are available commercially
Epithelial voltage clamp amplifier Nihon Kohden CEZ9100 Other equivalent amplifiers are available commerically
filter paper, cut into squares NA NA Punched with a 5 mm punch, used to hold intestinal preparation
fine forceps Fast Gene FG-B50476 For blunt dissection of the muscle layer
Forskolin Alomone Labs F-500 Make 10 mM stock in DMSO, final concentration will be 10 µM
HEPES Sigma H4034-1KG
Indomethacin Sigma I7338-5G Make a 1 mM stock in 21 mM NaHCO3, final concentration is 10 µM
K2HPO4 Fujifilm Wako 164-04295
KCl Fujifilm Wako 163-03545
KCl/calomel electrode Asch Japan Co. SCE-100
KH2PO4 Kanto chemical 32379-00
L(+)-Glutamine Fujifilm Wako 074-00522
MgCl2 Fujifilm Wako 135-00165
Mixed Gas (95% O2/5% CO2) Shizuoka Oxygen Company Used for bubbling Ringer solution and chambers when using Ringer solution
NaCl Fujifilm Wako 191-01665
NaCl electrode NA NA Handmade electrodes which require concentrated NaCl and Silver wire
NaHCO3 Fujifilm Wako 191-01305
O2 Gas Shizuoka Oxygen Company Used for bubbling chambers when using HEPES buffer
parafilm Bemis PM-996 Used to help seal Ussing chambers
pH meter DKK-TOA Corp HM-305 HEPES buffer needs to be adjusted to pH 7.4 at 37 °C
pH meter electrode DKK-TOA Corp GST-5311C
silicone rubber Shinetsu Chemical KE-12 Used to fill dissection plates
silver wire Used for making NaCl electrodes
Small jars w/ plastic lids NA NA Use for NaCl electrodes
stereomicroscope Zeiss Stemi 305 A stereomicroscope allows you to see depth, so you can dissect the tissue more easily
Tris (Trizma base) Sigma T1503-1KG Make a 1M solution to adjust pH of HEPES buffers
Ussing chambers Sanki Kagaku Kougei These chambers are custom made continuous perfusion Ussing chambers with a window diameter of 5 mm
Water pump and heating system Tokyo Rikakikai Co. Ltd. NTT-110

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