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
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	<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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		<col />
		<col />
		<col />
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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 ...

The nasal potential difference is a valuable method to characterize the phenotype of transgenic mice modeling cystic fibrosis. It allows investigation for the efficacy of new types of therapies for cystic fibrosis. The main advantages of the technique are that it provides reliable measurements in continuously breeding mice.It reduces related mortality and it allows repeating the test in the same animal. To prepare the double lumen nasal catheter, keep the middle of a 20-centimeter long polyethylene tube in the flame about 10 to 15 seconds until it is soft enough for pulling. Then, pull the two ends apart to obtain a very thin capillary tube of about 15 centimeters long and an outer diameter of 0.1 millimeters for the nasal probe.Afterward, clean and degrease the two capillaries with pure ethanol. Join them with tape and glue them together using cyanoacrylate glue. Cut away the excess length of the probes with a razor blade to obtain the double lumen catheter of about eight centimeters long.Then, inject water through both lumens to verify that they are permeable, and make a mark at a five millimeter distance from the tip. Subsequently, prepare a cream mix by mixing the electrolyte cream and saturated three molar potassium solution in a ratio of one to one, avoiding formation of tiny air bubbles. For the experimental setup, to prevent hypothermia during and after the test, use two heating pads, one for the measuring setup, and the other one for the box in which the animal will be placed for recovery at the end of the test.Turn on the computer, which is connected to the data memory high input impedance and high resolution voltmeter. Then open the software for data capture. Connect the electrodes to the voltmeter.Dip the tip of the electrodes together in the cream mix. Check the initial electrode offset value and reject the electrodes if the value is larger than plus or minus two millivolts. Then, switch on the peristaltic pump and insert the pump tube into the perfusing lumen of the nasal catheter.Fill one lumen of the nasal catheter with electrolyte cream mix through a 10 microliter pipette tip. Adapt the catheter to a silicone tube connector, and fill the connector with cream mix. Afterward, insert the positive electrode in the connector.Check the measuring electrode bridge offset value and reject the bridges if the value is larger than plus or minus two millivolts. Subsequently, connect the proximal outlet of the pump tube to the vial containing the selected solution for nasal perfusion. Then connect the distal outlet of the pump tube to the perfusion lumen of the nasal catheter.Next, fill the second lumen of the nasal catheter with 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 the length of the nasal catheter corresponding to 10 seconds of perfusion. Subsequently, fold a sheet of absorbing tissue to make a three centimeter wide pillow that will be used as a support for the mouse head. Cover the heating pad with the sheet of absorbing tissue.Then, lay the mouse on its back on the heating pad, and tape the limbs and the tail to the pad. Afterward, insert the reference intravenous catheter in the subcutaneous space of a hind limb. Remove the needle and adapt into the silicone tube connector.Fill the catheter and the connector with cream mix. Next, connect the positive measuring electrode to the nasal catheter, and the negative reference electrode to the catheter inserted in the subcutaneous space in the hind limb of the animal. Then, fix the catheter and the electrode with tape as required to prevent any displacement.After placing 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. Now, fix the mouse ears with tape on the heating pad in a free space between the two pieces of absorbing tissue.Fix the vibrancy without touching the eyes. To absorb fluid from the oral cavity, move the tongue sideways and insert a pointed wick of filter paper about one centimeter into the mouth. To absorb fluid running out of the perfused nostril, place a second piece of filter paper at the toi of the nose.Check the postive control value of the epithelial potential by placing the nasal catheter tip in the inside of the mouth. Holding it with fine forceps and under good direct illumination, delicately introduce the nasal catheter into one nostril, up to four to six millimeters from the nose tip. Five minutes after injecting the anesthetic drugs, gently tilt the heating pad by about 30 degrees with the animal head downwards.Monitor the maximal basal PD value. When it is stable over 30 seconds, start perfusing the nasal mucosa at 10 microliters per minute with the four buffered solutions in succession. Perfuse the first solution for 10 seconds, and each following solution for five minutes, or until a stable voltage has been reached.Stop perfusing Forskolin when the transient Forskolin response starts going down. After the assessment, release the mouse from the heating pad. Administer it with the anesthetic antagonist mix and transfer it to the recovery box with heating pad until full recovery.Here are the representative nasal PD tracings from a homozygous, normal mouse and a mouse homozygous for the F508 deletion CFTR mutation, together with the individual values obtained for the nasal PD parameters. Amiloride response is the difference between the values of nasal PD at the end and at the beginning of perfusion of the nasal mucosa with basal buffered salt solution containing amiloride. Chloride free response is the difference between the values of nasal PD at the end and at the beginning of perfusion of the nasal mucosa with chloride-free buffered salt solution, plus amiloride.Forskolin response is the difference between the values of nasal PD at the end and at the beginning of perfusion of the nasal mucosa with chloride free buffered salt solution, plus Forskolin and amiloride. Total chloride response is the sum of the last two parameters obtained under zero chloride perfusion. The project can be used to explore, provide the transport defectors in the mouse, but also in the brat, in the big, and in the ferret cystic fibrosis model.And sodium transport defectors in the eunuch mouse model. All the transporters can be studied by adopting the protocol. Working in the nasal cavity of rodents is extremely delicate because of the essential wall of sensory inputs from the nasal facial region.Adequate death of anesthesia is dosed particularly challenging when operating in the mouse nasal cavity.

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