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 in the diagnosis of CF, the most common fatal genetic disorder in Caucasian populations with an average incidence of 1 out of 2,500 live births in European countries. The test has long proved helpful in the diagnosis 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 bronchiectasis⁴. More recently, clinometric evaluation of the therapeutic modulation of the basic CFTR defect^{5,6,7,8,9,10,11,12,13,14,15,16} has made use of the nasal PD in clinical trials of new CF therapies. In the preclinical setting, the test has been adapted to the mouse¹⁷ to allow investigation of the bioactivity of new CF target therapies^18,19,20,21. In mice, the technique is delicate, based on species-related anatomical differences in size of the nasal region between rodents and humans, and mainly on the essential role of sensory inputs from the nasofacial region in rodents. It requires trained and skilled operators, dedicated equipment and supplies.

CF is a multi-systemic disorder of exocrine glands, in which chronic respiratory disease dominates the clinical picture. The disease is caused by mutations in the gene encoding the cyclic adenosine monophosphate (cAMP)-regulated CFTR chloride channel²². To date, more than 2,000 CFTR mutations have been identified²³. The most common mutation^24,25, found in almost 90% of CF alleles, corresponds to a deletion of the phenylalanine in position 508 of the polypeptide chain of the protein (F508del-CFTR). The CFTR protein is a purely ohmic small conductance chloride channel. There is also considerable evidence that CFTR regulates other transport mechanisms, in particular, ENaC^26,27. Defective electrolyte transport, including reduced CFTR-dependent chloride conductance and increased ENaC-dependent sodium conductance, is a hallmark of CF epithelia. The former defect is reflected by a reduced or abolished repolarization in response to both an electrochemical gradient favoring chloride efflux and addition of isoprenaline (a β-adrenergic agonist that increases intracellular cAMP) or forskolin (an adenylate cyclase agonist, not approved for clinical use). The latter defect is reflected by a basal hyperpolarization of the nasal mucosa (a more negative PD) and an increased response to amiloride, a diuretic drug that blocks ENaC²⁸.

CF mouse models have been frequently used in CF research and have been invaluable in dissecting CF pathology. Nowadays, at least fifteen models have been described²⁹, three of which are homozygous for the most clinically relevant F508del mutation^30,31,32. One of these three strains³⁰, developed at Erasmus University in Rotterdam, has been used for nearly 20 years in the Université catholique de Louvain (UCL) laboratory. The Cftr^tm1Eur model³⁰ has proved to be very useful to study the multiorgan pathophysiology of CF disease and to test the efficacy of new therapeutic strategies^18,19,20,21. Numerous problems may occur during or early after (<24 h) the nasal PD test in mice. In this paper, the adequate depth of anesthesia required for keeping a nasal catheter in situ for continuous perfusion, and measures to avoid broncho-aspiration of solutions perfused in the nose are described. The animal care at the end of the test is also described, including administration of a combination of antidotes of anesthetic drugs, leading to rapidly reversing the anesthesia with complete recovery of the animals. Altogether, these procedures allow reliable measurements in spontaneously breathing mice, reduced test-related mortality and repeating the test in the same animal. Representative data obtained from the nasal PD test in a CF and in a wild-type mouse are shown and discussed.

The murine nasal PD test protocol is reported in three sessions: assessment and management before, during, and after the test. In the pre-test assessment and management, the protocol of preparation of the double lumen nasal catheter and of solutions used for continuous nasal perfusion is described in detail. During the assessment and management portions of the test, the experimental setup and the handling of the mouse is minutely dissected. Finally, management of the animal at the end of the test is described to improve full animal recovery.

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 perfusion of solutions, and the other one as a measuring channel.
   1. Heat up the central section of a piece, about 20 cm long, of polyethylene tube (2.0 mm inner diameter; 3.0 mm outer diameter; Figure 1a) in the flame of a gas burner until it is soft enough for pulling (10 - 15 s).
   2. Pull the two ends apart to obtain a very thin capillary tube of appropriate length (~15 cm) and outer diameter (~0.1 mm) for the nasal probe (Figure 1b).
   3. Clean and degrease two such capillaries with pure ethanol, join them with tape (Figure 1c) and glue them together using cyanoacrylate glue.
   4. Cut away the excess length of the probes with a razor blade or a scalpel to obtain an optimal length of the double lumen catheter of about 8 cm (Figure 1d).
   5. Inject water through both lumens to verify that they are permeable.
   6. Apply a mark at a 5 mm distance from the tip.
      NOTE: The probe is inserted up to the mark into the nostril, so that all measurements are made at the same site inside the nasal cavity.
   7. Store the double lumen catheter in a dry box.
      NOTE: It can be used 6 - 10 times if appropriately cleaned immediately after the test.
2. Prepare the cream mix.
   1. Gently mix electrolyte cream and saturated 3 M KCl solution in a 1:1 ratio (volume/volume), avoiding formation of tiny air bubbles.
      NOTE: The cream mix is used to build the electrode bridges for the measuring and the reference Ag/AgCl electrodes, as illustrated in Figure 1.

Figure 1: Probes with electrodes and bridges. The figure illustrates the polyethylene tube before (a) and after heating and pulling (b), the tape joining the two capillary parts (c) of the double lumen catheter (d), the silicone tube connectors (e) and the electrodes (f). For picture clarity, the connectors have not been filled with cream mix. Please click here to view a larger version of this figure.

Solution Label	Solution description
A	Basal buffered salt solution
B	Chloride-free buffered salt solution
C	10-2 M amiloride stock solution
D	10-3 M forskolin stock solution

Table 1: Stock solution for the nasal PD test in mice.

Salt	mM	Molecular weight	g/L
Sodium chloride (NaCl)	135	58.44	7,889
Calcium chloride dihydrate (CaCl2.2H2O)	2.25	147	0.331
Magnesium chloride hexahydrate (MgCl2.6H2O)	1.2	203.3	0.244
Dipotassium phosphate (K2HPO4)	2.4	174.2	0.418
Monopotassium phosphate (KH2PO4)	0.4	136.1	0.054

Table 2: Composition of basal buffered salt solution (stock solution A).

3. Prepare the solutions.
   1. Prepare the four stock solutions required for the experimental protocol (Table 1). 
      1. Prepare the basal buffered solution (stock solution A; Table 2) and the chloride-free buffered solution (stock solution B; Table 3) by mixing the salts in pure water at room temperature. Adjust pH to 7.4 (range 7.0 - 7.6) using 1 N NaOH to raise pH, 1 N HCl to lower pH. Check osmolarity of the solution (275 mOsm/L).
      2. Store in labeled glass bottles at 4 °C for up to 3 months or frozen in plastic bottles for up to 6 months.
      3. Prepare the 10^-2 M amiloride solution (stock solution C) by adding 26.6 mg of amiloride hydrochloride to 10 mL of pure water. Heat the mixture for 5 to 10 minutes at 70 °C. As amiloride is sensitive to light, keep solution C in a dark container. Label the container and store at 4 °C for up to 3 months.
      4. Prepare the 10^-3 M forskolin solution (stock solution D) by adding 10 mg of forskolin to 24.36 mL of pure water. Label 0.1 mL aliquots of the forskolin stock solution and store at -20 °C for up to 6 months.
4. Prepare fresh solutions A1, B1 and B2 (Table 4).
   1. Prepare fresh solution A1 (basal buffered salt solution plus 10^-4 M amiloride) by obtaining 10 mL of basal salt solution (stock solution A) and adding 0.1 mL of 10^-2 M amiloride solution (stock solution C). Label the container and use within 24 h of preparation.
   2. Prepare fresh solution B1 (chloride buffered solution plus 10^-4 M amiloride) by obtaining 10 mL of chloride-free buffered salt solution (stock solution B) and adding 0.1 mL of 10^-2 M amiloride solution (stock solution C). Label the container and use within 24 hours of preparation.
   3. Prepare fresh solution B2 (chloride-free buffered salt solution plus 10^-4 M amiloride and 10^-5 M forskolin) by obtaining 10 mL of chloride-free buffered salt solution (stock solution B) and adding 0.1 mL of 10^-2 M amiloride solution (stock solution C) and 0.1 mL of 10^-3 M forskolin solution (stock solution D). Label the container and use within 2 hours of preparation.

Salt	mM	Molecular weight	g/L
Sodium gluconate (monosodium salt)	135	218.1	29,444
Calcium gluconate (anhydrous powder)	2.2	430.4	0.947
Magnesium sulfate heptahydrate (MgCl2.6H2O)	1.2	246.5	0.296
Dipotassium phosphate (K2HPO4)	2.4	174.2	0.418
Monopotassium phosphate (KH2PO4)	0.4	136.1	0.054

Table 3: Composition of chloride-free buffered solution (stock solution B).

Figure 2: Position of the mouse during perfusion of the nasal mucosa. The figure illustrates the heating pad (a), the voltmeter (b), the reference electrode inserted in the subcutaneous space in a hind limb (c), the proximal (d) and the distal (e) outlets of the peristaltic pump, the pillow (f), and the bed sheet (g). Please click here to view a larger version of this figure.

2. Assessment and Management During the Test

1. Prepare the experimental setup.
   1. To prevent hypothermia during and after the test, use two heating pads maintained at physiological temperatures, one for the measuring setup (18.8 x 37.5 cm; Figure 2a), and the other one (15.5 x 15.5 cm) for the box in which the animal will be placed for recovery at the end of the test.
   2. Switch on the computer loaded with the software for data capture connected to the data memory high input impedance (>10¹²Ω) and high resolution (0.1 mV) voltmeter (Figure 2b).
   3. Connect the electrodes to the voltmeter. Connect the positive measuring electrode to the nasal catheter and the negative reference electrode to the catheter inserted in the subcutaneous space in a hind limb, as shown in Figure 2c.
   4. Dip the tips of the electrodes together in the cream mix. Check the initial electrode offset value. Reject the electrodes if the value is larger than (±) 2 mV (ideally 0 mV).
   5. Switch on the peristaltic pump and insert the pump tube into position, as illustrated in Figure 2.
   6. Connect the proximal outlet of the pump tube to the vial containing the selected solution for nasal perfusion (Figure 2d). Connect the distal outlet of the pump tube to the perfusion lumen of the nasal catheter (Figure 2e).
   7. Fill one lumen of the nasal catheter with electrolyte cream mix through a 10 µL pipette tip. Adapt the catheter to a silicone tube connector (Figure 1e), fill the connector with cream mix and insert the positive electrode in the connector (Figure 1f). Check the measuring electrode bridge offset value. Reject the bridges if the value is larger than (±) 2 mV (ideally 0 mV).
   8. Fill the second lumen of the nasal catheter with the fresh solution A1. Dip the tip of the nasal catheter in the vial containing solution A. Reverse the direction of the peristaltic pump to fill a length of the nasal catheter corresponding to 10 s of perfusion.
      NOTE: This allows recording basal PD values for 10 s before applying amiloride. Dip the tip of the nasal catheter in the cream mix inside the connector of the reference electrode (Figure 1).
2. Begin handling the mouse.
   1. Record the mouse weight to calculate the exact dose of anesthetics to be injected by the intraperitoneal (ip) route¹⁷.
   2. Using a 26G (0.45 x 10 mm) needle, inject premedication intraperitoneally to promote the induction of anesthesia: a fixed dose of 50 µL of 5 mg/mL midazolam. Wait for 5 min.
   3. Prepare the anesthetic mix (fentanyl, medetomidine, droperidol at final concentrations of 0.05, 0.40, and 20 mg/kg body weight respectively, and clonidine at the fixed dose of 0.375 µg) allowing optimal and stable depth of anesthesia (stage III, plane 2)¹⁷. Inject the volume of anesthetic mix intraperitoneally, and then inject the volume of clonidine.
      NOTE: After 15 min, the mouse should be asleep and the anesthesia can last for at least 30 min.
   4. Fold a sheet of absorbing tissue to make a 3 cm wide 'pillow' (Figure 2f) that will cover the upper part of the heating pad (Figure 2a) as a support for the mouse head.
   5. Cover the heating pad with a sheet of absorbing tissue ('bed sheet'; Figure 2g). Lay the mouse on its back on the heating pad. Tape out the limbs and the tail (Figure 2).
   6. Insert the reference intravenous catheter (Figure 1g) in the subcutaneous space of a hind limb. Remove the needle and adapt the silicone tube connector (Figure 1e, 2c).
   7. Fill the catheter and the connector with cream mix and insert the negative electrode in the connector (Figure 1f).
   8. Fix the catheter and the electrode with tape as required to prevent any displacement. After the placement of the reference electrode and bridges, verify the measuring electrode bridge offset value again by dipping the tip of the nasal catheter in the electrode cream mix inside the connector of the IV catheter. Record the stable final offset value (ideally less than (±) 2 mV).
   9. Fix the mouse ears with "gentle" tape on the heating pad in a free space between the two pieces of absorbing tissue ('pillow' (Figure 2f) and 'bed sheet'; (Figure 2g)). Fix the vibrissae (the stiff hairs growing near the nostrils) without touching the eyes.
   10. To absorb fluid from the oral cavity, move the tongue sideways (Figure 3a) and insert a pointed wick of filter paper (the 'pipe'; Figure 3b) about 1 cm into the mouth. To absorb fluid running out of the perfused nostril, place a second piece of filter paper (the 'handkerchief'; Figure 3c) held at the tip of the nose.
   11. Check a positive control value of the epithelial potential by placing the tip of the nasal catheter in contact with the inside of the mouth.
       NOTE: The reading should be stable and range between -10 mV and -20 mV.
   12. Holding it with fine forceps and under good direct illumination, delicately introduce the nasal catheter into one nostril up to 4 - 6 mm from the nose tip.
       NOTE: The nasal PD catheter is located in the middle nasal concha at the position that gives the maximal stable baseline PD value^17,20.
   13. 5 min after injection of the anesthetic drugs, gently tilt the heating pad by about 30° with the animal head downwards. Monitor the maximal basal PD value.
   14. When it is stable over a period of about 30 s, start perfusing the nasal mucosa, at a constant rate of 10 µL/ min, with the 4 buffered solutions (A, A1, B1, B2) in succession. Perfuse the first solution (step 1.3.1.1) for 10 s and each following solution (step 1.4) for 5 min or until a stable value has been reached. Stop perfusing forskolin (B2) when the transient forskolin response starts going down.
   15. Select a 1 s interval for data recording. Start recording data at the time of starting perfusion. Display data as a function of time on the computer screen.
   16. Stop data capture at the end of perfusion with solution B2. Save data as a spreadsheet file.
   17. Correct data by subtracting the final offset value.

Figure 3: Position of the mouse on the heating pad with the nasal catheter and the filter papers in place. The figure illustrates the tongue put sideways (a), the pipe (b), and the handkerchief (c). Please click here to view a larger version of this figure.

3. Post-test Assessment and Management

1. Release the mouse from the heating pad.
2. Wipe the mouse nose with the 'bed sheet' (Figure 2g).
3. Inject intraperitoneally the anesthetic antagonist mix composed of a fixed dose (4 µg) of naloxone, a competitive morphine antagonist, and atipamezole, a medetomidine specific antidote (2 mg/kg body weight).
4. Lay the mouse on the smaller heating pad in the recovery box until full recovery, that is usually observed after 1 to 2 h.
   NOTE: In the UCL laboratory, about 10% mortality, mainly related to bronchoaspiration of solutions perfused in the nasal cavity during the test, is observed irrespective of the gender or genotype.

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