Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice

Summary

Here, we present a protocol to measure nasal potential difference in mice. The test quantifies the function of transmembrane ion transporters such as the cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel. It is valuable to evaluate the efficacy of novel therapies for cystic fibrosis.

Abstract

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful in cases of attenuated, oligo- or mono-symptomatic forms of CF usually diagnosed later in life, and of CF-related disorders such as congenital bilateral absence of vas deferens, idiopathic chronic pancreatitis, allergic bronchopulmonary aspergillosis, and bronchiectasis. In both clinical and preclinical settings, the test has been used as a biomarker to quantify responses to targeted therapeutic strategies for CF. Adapting the test to a mouse is challenging and can entail an associated mortality. This paper describes the adequate depth of anesthesia required to maintain a nasal catheter in situ for continuous perfusion. It lists measures to avoid broncho-aspiration of solutions perfused in the nose. It also describes the animal care at the end of the test, including administration of a combination of antidotes of the anesthetic drugs, leading to rapidly reversing the anesthesia with full recovery of the animals. Representative data obtained from a CF and a wild-type mouse show that the test discriminates between CF and non-CF. Altogether, the protocol described here allows reliable measurements of the functional status of trans-epithelial chloride and sodium transporters in spontaneously breathing mice, as well as multiple tests in the same animal while reducing test-related mortality.

Introduction

For almost three decades, electrical potential difference (PD) measurements have been used to evaluate the functional status of transmembrane ion transporters expressed at the nasal mucosa, as representative of the distal airways¹. As a multistep dynamic test^2,3, nasal PD allows functional dissection of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and epithelial sodium channel (ENaC) activity, both localized at the apical membranes of epithelial cells and exerting critical roles in airway surface hydration. The major clinical application of the nasal PD test is to assist i....

Protocol

The studies and procedures were approved by the ethics committee for animal research of the UCL (2017/UCL/MD/015) and in agreement with the European Community regulations for animal use in research (CEE n° 86/609). The investigators are qualified for the animal experimentation following the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes.

1. Pre-test Assessment and Management

1. Prepare the double lumen nasal catheter.
   ​NOTE: A nasal catheter is made as a double lumen capillary tube, one lumen being used for the continuous perfusi....

Results

In order to illustrate the characteristic ion transport abnormalities in CF, nasal PD measurements were performed following the protocol described above in an F508del-CF mouse and in a wild-type control of the FVB/129 genetic background from the Brussels colony of Cftr^tm1Eur mice³⁰. This clinically relevant model, harboring the most common and one of the most severe F508del-CFTR mutation^23,24

Discussion

The purpose of this paper is to describe an adequate protocol for measuring nasal PD under continuous perfusion of solutions in spontaneously breathing mice for a length of time required for testing the integrity of ion transporters, mainly CFTR and ENaC. All steps of the protocol have been carefully optimized to ensure full animal recovery and good quality and reproducible data. In particular, critical steps are anesthesia assessment and management, and adequate animal position and care during and after the test.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Portex polyethylene tube	Smiths Medical, Hythe, Kent, England CT21 6JL	Portex 800/100/500;2.0mm ID, 3.0 mmOD	to prepare capillary tubes for nasal probe
Electrode cream	Parker, Fairfield, NJ, USA	Redux cream	to build electrode bridges
Ag/AgCl electrodes	Biomedical, Clinton Township, MI, USA	JNS BNT131-1,0	measuring and reference electrodes
amiloride hydrochloride	Sigma, St Louis, MI, USA	A7410	to prepare perfusion solutions
forskolin	Sigma, St Louis, MI, USA	F6886	to prepare perfusion solutions
Knick Portamess voltmeter	Elektronisch Meβgeräte, Berlin, Germany	Portavo 904 pH	to measure potential difference
Paraly SW 112 Software	Elektronisch Meβgeräte, Berlin, Germany	Paraly SW112 software	to capture potential difference data
midazolam	Mylan, Hoeilaart, Belgium	Dormicum 15mg/3ml	to serve as anaesthetic premedication
fentanyl	Janssen Cilag, Berchem, Belgium	Fentanyl-Janssen 0.05 mg/ml	to serve as anaesthetic medication
medetomidine	Orion Pharma, Espoo, Finland	Domitor 1 mg/ml	to serve as anaesthetic medication
droperidol	Janssen  Cilag, Berchem, Belgium	Dehydrobenzperidol 2.5 mg/ml	to serve as anaesthetic medication
clonidine	Boehringer Ingelheim Pharma KG, Ingelheim am Rhein, Germany	Catapressan 0.15 mg/ml,	to serve as anaesthetic medication
refernce IV catheter	Becton Dickinson, Sandy, UT, USA	24 GA x 0.75 IN, BD Insyte-W	to build electrode bridges
forceps	Fine science Tools, Heidelberg, Germany	Dumont #5, Fine science Tools	to place the nasal catheter
naloxone	Braun Medical, Brussels, Belgium	Narcan, 0.4 mg/ml	to serve as anaesthetic antagonist
atipamezole	Zoetis, Bloomberg, Belgium	Antisedan, 5 mg/ml	to serve as a medetomedine specific antidote
Heating pads	Harvard Apparatus, Holliston, MA, USA	18,8x37,5 cm; 15,5x15,5 cm	to avoid hypothermia during and after the test
Peristaltic pump P1	GE Life Sciences, Uppsala, Sweden	18111091	to perfuse solutions in the mouse nose
cyanoacrylate glue	Loctite, Henkel, Düsseldorf, Germany	super glue 3	to glue together two capillary tubes  for nasal probe
NaCl	Sigma, St Louis, MI, USA	RES0926S-A7	Pharma-Grade, USP
CaCl2.2H2O	Sigma, St Louis, MI, USA	M7304	Pharma-Grade, USP
MgCl2.6H2O	Sigma, St Louis, MI, USA	1551128	Pharma-Grade, USP
K2HPO4	Sigma, St Louis, MI, USA	1551139	Pharma-Grade, USP
Na gluconate	Sigma, St Louis, MI, USA	S2054	Pharma-Grade, USP
Ca gluconate	Sigma, St Louis, MI, USA	C8231	Pharma-Grade, USP
MgSO4.7H2O	Sigma, St Louis, MI, USA	RES0089M-A7	Pharma-Grade, USP
BD needle	Becton Dickinson, Franklin Lakes, USA	BD 26G (0.45x10 mm)	intraperitoneal injection