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

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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 .......

Discussion

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 I_sc (100-300 µA/cm² in mi.......

Materials

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