Name: nasal potential difference to quantify trans-epithelial ion transport in mice
Uploaded: 2018-07-04T14:30:00
Description: Watch this Scientific Journal Video about Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice at JoVE.com

The authors thank Prof. J. Lebacq for critically editing the manuscript. Cftrtm1Eur (homozygous F508del-CFTR (FVB/129) mice were developed by the Erasmus MC, Rotterdam, The Netherlands, with the support of European Economic Community European Coordination Action for Research in Cystic Fibrosis EU FP6 LHHM-CT-2005-018932.

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&#176; 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
<ol>
	<li>Prep...

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.
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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 test2,3, 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 defect5,6,7,8,9,10,11,12,13,14,15,16 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 therapies18,19,20,21. 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 mutation24,25, 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, ENaC26,27. 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 &#946;-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 mutation30,31,32. One of these three strains30, developed at Erasmus University in Rotterdam, has been used for nearly 20 years in the Universit&#233; catholique de Louvain (UCL) laboratory. The Cftrtm1Eur model30 has proved to be very useful to study the multiorgan pathophysiology of CF disease and to test the efficacy of new therapeutic strategies18,19,20,21. Numerous problems may occur during or early after (&#60;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.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Portex polyethylene tube&#160;</td>
			<td style="width:403px;">Smiths Medical, Hythe, Kent, England CT21 6JL</td>
			<td style="width:225px;">Portex 800/100/500;2.0mm ID, 3.0 mmOD</td>
			<td style="width:167px;">to prepare capillary tubes for nasal probe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Electrode cream</td>
			<td style="width:403px;">Parker, Fairfield, NJ, USA</td>
			<td style="width:225px;">Redux cream</td>
			<td>to build electrode bridges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Ag/AgCl electrodes</td>
			<td style="width:403px;">Biomedical, Clinton Township, MI, USA</td>
			<td style="width:225px;">JNS BNT131-1,0</td>
			<td>measuring and reference electrodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">amiloride hydrochloride</td>
			<td style="width:403px;">Sigma, St Louis, MI, USA</td>
			<td style="width:225px;">A7410</td>
			<td>to prepare perfusion solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">forskolin</td>
			<td style="width:403px;">Sigma, St Louis, MI, USA</td>
			<td style="width:225px;">F6886</td>
			<td>to prepare perfusion solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Knick Portamess voltmeter</td>
			<td>Elektronisch Me&#946;ger&#228;te, Berlin, Germany</td>
			<td style="width:225px;">Portavo 904 pH</td>
			<td>to measure potential difference</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraly SW 112 Software&#160;</td>
			<td>Elektronisch Me&#946;ger&#228;te, Berlin, Germany</td>
			<td style="width:225px;">Paraly SW112 software</td>
			<td>to capture potential difference data</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">midazolam&#160;</td>
			<td style="width:403px;">Mylan, Hoeilaart, Belgium</td>
			<td style="width:225px;">Dormicum 15mg/3ml</td>
			<td>to serve as anaesthetic premedication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">fentanyl</td>
			<td>Janssen Cilag, Berchem, Belgium</td>
			<td>Fentanyl-Janssen 0.05 mg/ml</td>
			<td>to serve as anaesthetic medication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">medetomidine</td>
			<td>Orion Pharma, Espoo, Finland</td>
			<td>Domitor 1 mg/ml</td>
			<td>to serve as anaesthetic medication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">droperidol&#160;</td>
			<td>Janssen&#160; Cilag, Berchem, Belgium</td>
			<td>Dehydrobenzperidol 2.5 mg/ml</td>
			<td>to serve as anaesthetic medication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">clonidine&#160;</td>
			<td>Boehringer Ingelheim Pharma KG, Ingelheim am Rhein, Germany</td>
			<td>Catapressan 0.15 mg/ml,</td>
			<td>to serve as anaesthetic medication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">refernce IV catheter</td>
			<td>Becton Dickinson, Sandy, UT, USA</td>
			<td>24 GA x 0.75 IN, BD Insyte-W</td>
			<td>to build electrode bridges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">forceps&#160;</td>
			<td>Fine science Tools, Heidelberg, Germany</td>
			<td>Dumont #5, Fine science Tools</td>
			<td>to place the nasal catheter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">naloxone&#160;</td>
			<td>Braun Medical, Brussels, Belgium</td>
			<td>Narcan, 0.4 mg/ml</td>
			<td>to serve as anaesthetic antagonist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">atipamezole&#160;</td>
			<td>Zoetis, Bloomberg, Belgium</td>
			<td>Antisedan, 5 mg/ml</td>
			<td>to serve as a medetomedine specific antidote&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Heating pads&#160;</td>
			<td>Harvard Apparatus, Holliston, MA, USA</td>
			<td>18,8x37,5 cm; 15,5x15,5 cm</td>
			<td>to avoid hypothermia during and after the test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Peristaltic pump P1</td>
			<td>GE Life Sciences, Uppsala, Sweden</td>
			<td>18111091</td>
			<td>to perfuse solutions in the mouse nose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">cyanoacrylate glue</td>
			<td style="width:403px;">Loctite, Henkel, D&#252;sseldorf, Germany&#160;</td>
			<td style="width:225px;">super glue 3</td>
			<td>to glue together two capillary tubes&#160; for nasal probe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">NaCl</td>
			<td>Sigma, St Louis, MI, USA</td>
			<td style="width:225px;">RES0926S-A7</td>
			<td>Pharma-Grade, USP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">CaCl2.2H2O</td>
			<td>Sigma, St Louis, MI, USA</td>
			<td style="width:225px;">M7304</td>
			<td>Pharma-Grade, USP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">MgCl2.6H2O</td>
			<td>Sigma, St Louis, MI, USA</td>
			<td style="width:225px;">1551128</td>
			<td>Pharma-Grade, USP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">K2HPO4</td>
			<td>Sigma, St Louis, MI, USA</td>
			<td style="width:225px;">1551139</td>
			<td>Pharma-Grade, USP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Na gluconate</td>
			<td>Sigma, St Louis, MI, USA</td>
			<td style="width:225px;">S2054</td>
			<td>Pharma-Grade, USP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Ca gluconate</td>
			<td>Sigma, St Louis, MI, USA</td>
			<td style="width:225px;">C8231</td>
			<td>Pharma-Grade, USP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">MgSO4.7H2O</td>
			<td>Sigma, St Louis, MI, USA</td>
			<td style="width:225px;">RES0089M-A7</td>
			<td>Pharma-Grade, USP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">BD needle&#160;</td>
			<td>Becton Dickinson, Franklin Lakes, USA</td>
			<td style="width:225px;">BD 26G (0.45x10 mm)</td>
			<td>intraperitoneal injection</td>
		</tr>
	</tbody>
</table>

nasal potential difference to quantify trans-epithelial ion transport in mice

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.

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 Cftrtm1Eur mice30. This clinically relevant model, harboring the most common and one of the most severe F508del-CFTR mutation23,24<sup...

Watch this Scientific Journal Video about Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice at JoVE.com

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

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.

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 ...

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