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>Prepare the double lumen nasal catheter. 
	&#8203;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.
	<ol>
		<li>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).</li>
		<li>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).</li>
		<li>Clean and degrease two such capillaries with pure ethanol, join them with tape (Figure 1c) and glue them together using cyanoacrylate glue.</li>
		<li>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).</li>
		<li>Inject water through both lumens to verify that they are permeable.</li>
		<li>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.</li>
		<li>Store the double lumen catheter in a dry box. 
		NOTE: It can be used 6 - 10 times if appropriately cleaned immediately after the test.</li>
	</ol>
	</li>
	<li>Prepare the cream mix.
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57934/57934fig1.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57934/57934fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Solution Label</td>
			<td>Solution description</td>
		</tr>
		<tr>
			<td>A</td>
			<td>Basal buffered salt solution</td>
		</tr>
		<tr>
			<td>B</td>
			<td>Chloride-free buffered salt solution</td>
		</tr>
		<tr>
			<td>C</td>
			<td>10-2 M amiloride stock solution</td>
		</tr>
		<tr>
			<td>D</td>
			<td>10-3 M forskolin stock solution</td>
		</tr>
	</tbody>
</table>
Table 1: Stock solution for the nasal PD test in mice.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Salt</td>
			<td>mM</td>
			<td>Molecular weight</td>
			<td>g/L</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>135</td>
			<td>58.44</td>
			<td>7,889</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2.2H2O)</td>
			<td>2.25</td>
			<td>147</td>
			<td>0.331</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate (MgCl2.6H2O)</td>
			<td>1.2</td>
			<td>203.3</td>
			<td>0.244</td>
		</tr>
		<tr>
			<td>Dipotassium phosphate (K2HPO4)</td>
			<td>2.4</td>
			<td>174.2</td>
			<td>0.418</td>
		</tr>
		<tr>
			<td>Monopotassium phosphate (KH2PO4)</td>
			<td>0.4</td>
			<td>136.1</td>
			<td>0.054</td>
		</tr>
	</tbody>
</table>
Table 2: Composition of basal buffered salt solution (stock solution A).
<ol start="3">
	<li>Prepare the solutions.
	<ol>
		<li>Prepare the four stock solutions required for the experimental protocol (Table 1).&#160;
		<ol>
			<li>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 -&#160;7.6) using 1 N NaOH to raise pH, 1 N HCl to lower pH. Check osmolarity of the solution (275 mOsm/L).</li>
			<li>Store in labeled glass bottles at 4 &#176;C for up to 3 months or frozen in plastic bottles for up to 6 months.</li>
			<li>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 &#176;C. As amiloride is sensitive to light, keep solution C in a dark container. Label the container and store at 4 &#176;C for up to 3 months.</li>
			<li>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 &#176;C for up to 6 months.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare fresh solutions A1, B1 and B2 (Table 4).
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Salt</td>
			<td>mM</td>
			<td>Molecular weight</td>
			<td>g/L</td>
		</tr>
		<tr>
			<td>Sodium gluconate (monosodium salt)</td>
			<td>135</td>
			<td>218.1</td>
			<td>29,444</td>
		</tr>
		<tr>
			<td>Calcium gluconate (anhydrous powder)</td>
			<td>2.2</td>
			<td>430.4</td>
			<td>0.947</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate (MgCl2.6H2O)</td>
			<td>1.2</td>
			<td>246.5</td>
			<td>0.296</td>
		</tr>
		<tr>
			<td>Dipotassium phosphate (K2HPO4)</td>
			<td>2.4</td>
			<td>174.2</td>
			<td>0.418</td>
		</tr>
		<tr>
			<td>Monopotassium phosphate (KH2PO4)</td>
			<td>0.4</td>
			<td>136.1</td>
			<td>0.054</td>
		</tr>
	</tbody>
</table>
Table 3: Composition of chloride-free buffered solution (stock solution B).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57934/57934fig2.jpg" /> 
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). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57934/57934fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Assessment and Management During the Test
<ol>
	<li>Prepare the experimental setup.
	<ol>
		<li>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.</li>
		<li>Switch on the computer loaded with the software for data capture connected to the data memory high input impedance (&#62;1012&#8486;) and high resolution (0.1 mV) voltmeter (Figure 2b).</li>
		<li>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.</li>
		<li>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 (&#177;) 2 mV (ideally 0 mV).</li>
		<li>Switch on the peristaltic pump and insert the pump tube into position, as illustrated in Figure 2.</li>
		<li>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).</li>
		<li>Fill one lumen of the nasal catheter with electrolyte cream mix through a 10 &#181;L 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 (&#177;) 2 mV (ideally 0 mV).</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Begin handling the mouse.
	<ol>
		<li>Record the mouse weight to calculate the exact dose of anesthetics to be injected by the intraperitoneal (ip) route17.</li>
		<li>Using a 26G (0.45 x 10 mm) needle, inject premedication intraperitoneally to promote the induction of anesthesia: a fixed dose of 50 &#181;L of 5 mg/mL midazolam. Wait for 5 min.</li>
		<li>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 &#181;g) 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.</li>
		<li>Fold a sheet of absorbing tissue to make a 3 cm wide &#39;pillow&#39; (Figure 2f) that will cover the upper part of the heating pad (Figure 2a) as a support for the mouse head.</li>
		<li>Cover the heating pad with a sheet of absorbing tissue (&#39;bed sheet&#39;; Figure 2g). Lay the mouse on its back on the heating pad. Tape out the limbs and the tail (Figure 2).</li>
		<li>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).</li>
		<li>Fill the catheter and the connector with cream mix and insert the negative electrode in the connector (Figure 1f).</li>
		<li>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 (&#177;) 2 mV).</li>
		<li>Fix the mouse ears with &#34;gentle&#34; tape on the heating pad in a free space between the two pieces of absorbing tissue (&#39;pillow&#39; (Figure 2f) and &#39;bed sheet&#39;; (Figure 2g)). Fix the vibrissae (the stiff hairs growing near the nostrils) without touching the eyes.</li>
		<li>To absorb fluid from the oral cavity, move the tongue sideways (Figure 3a) and insert a pointed wick of filter paper (the &#39;pipe&#39;; 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 &#39;handkerchief&#39;; Figure 3c) held at the tip of the nose.</li>
		<li>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.</li>
		<li>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 value17,20.</li>
		<li>5 min after injection of the anesthetic drugs, gently tilt the heating pad by about 30&#176; with the animal head downwards. Monitor the maximal basal PD value.</li>
		<li>When it is stable over a period of about 30 s, start perfusing the nasal mucosa, at a constant rate of 10 &#181;L/ 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.</li>
		<li>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.</li>
		<li>Stop data capture at the end of perfusion with solution B2. Save data as a spreadsheet file.</li>
		<li>Correct data by subtracting the final offset value.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57934/57934fig3.jpg" /> 
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). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57934/57934fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Post-test Assessment and Management
<ol>
	<li>Release the mouse from the heating pad.</li>
	<li>Wipe the mouse nose with the &#39;bed sheet&#39; (Figure 2g).</li>
	<li>Inject intraperitoneally the anesthetic antagonist mix composed of a fixed dose (4 &#181;g) of naloxone, a competitive morphine antagonist, and atipamezole, a medetomidine specific antidote (2 mg/kg body weight).</li>
	<li>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.</li>
</ol>

The authors have nothing to disclose.

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.
Previous studies showed that plane 2 stage III of anesthetic exposure, which can be achieved by applying the cocktail mixture used here17, is associated with regular breathing and with absence of negative inotropic effect and of blink, pupillary and pedal withdrawal reflexes. At this level of anesthetic depth, the nasal catheter can be kept in situ for continuous perfusion of the nasal cavity and for use as a bridge of the measuring electrode with good tolerance. Also, insertion of the catheter in the subcutaneous space, serving as the bridge of the reference electrode, was not followed by any painful reaction or sign of harmful effect. In rodents, the essential role of sensory input from the nasofacial region in the control of behavior vis-a-vis an external situation, including a threat, makes adequate depth of anesthesia particularly challenging when operating in the nasal cavity. Nebulization instead of continuous nasal perfusion has been applied to perform nasal PD test in some mouse studies33,34. However, this method leads to non-reliable results, owing to repeated removals and reinsertions of the nasal probe. As a matter of fact, owing to non-homogenous distribution of cell types in the mouse nasal mucosa20, repositioning the tip of the probe at the same location in the nostril is critical. Moreover, responses to changes of solutions, in particular chloride-free solutions, rapidly vanish when perfusion is interrupted.
Broncho-aspiration of solutions leading to respiratory arrest is a critical limitation of the procedure and is the major cause of mortality of the test. Several essential measures aim at preventing it, including a dorsal decubitus position of the animal, lightly tilting it with its head downwards, and absorbing excess fluid from the oral and nasal cavities17. A rapid and reversible level of anesthesia with complete recovery of animals is ensured by applying antidotes of anesthetic drugs at the end of the test. The protocol presented here allows reliable measurements in spontaneously breathing mice and repeating the test in the same animal. It impacts on the number of mice required to get statistical significance35 and on complying to the 3R (replace, refine and reduce) rules for animal use in experimental procedures36. In humans as in mice, the lowest between-test variability has been found for total chloride response, suggesting that it as the most reliable nasal PD parameter to detect efficacy of new CF therapeutic strategies. In CF mice, the measurement error of the total chloride response was shown to be less than &#177;1.7 mV35. In other words, when evaluating the bioactivity of a CFTR-correcting drug in F508del-CF mice, a difference between total chloride response in the absence and in the presence of treatment larger than 2 mV indicates 95% chance of a drug-related improving effect.
Data interpretation of representative tracings illustrates the ability of the nasal PD test to discriminate between CF and wild-type mice18,19,20,21,35 and shows that the F508del-CF Erasmus mouse model30 mimics the human nasal mucosa with respect to the typical clinical ion transport abnormalities. However, in the animal model, a residual chloride conductance is detectable, resulting either from a residual F508del-CFTR function or from a contribution of alternative non-CFTR-dependent chloride channels. Translating results from CF research from preclinical into clinical settings implies dealing with several major differences between the two settings. The mouse CF phenotype displays an attenuated respiratory syndrome. The absence of multiple therapies together with the fact that the mouse model is housed in privileged conditions with hygienic barriers also contribute to the differences37. The protocol described here shows a very low variability35 and it has been adapted to the pig38,39 and the ferret models40. In a previous study, the experimental protocol was modified by including perfusion of the mouse nasal mucosa with an inhibitor of alternative chloride transporters, to explore the possible contribution of non-CFTR-dependent calcium-activated chloride channels18. The test has also been used to study sodium transport in the &#946;-ENaC overexpressing mouse model41, engineered to mimic CF-lung disease42. In the future, further applications of the test could be considered to study other transporters, such as the ATP12A, a CFTR-independent H+-pump protein expressed in human and in pig but absent in mouse airways43. Altogether, the protocol described here allows reliable measurements of the functional status of transepithelial chloride and sodium transporters in spontaneously breathing mice, reduced test-related mortality and multiple tests in the same animal.

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.

<table><tbody><tr><td>Portex polyethylene tube&amp;nbsp;</td><td>Smiths Medical, Hythe, Kent, England CT21 6JL</td><td>Portex 800/100/500;2.0mm ID, 3.0 mmOD</td><td>to prepare capillary tubes for nasal probe</td></tr><tr><td>Electrode cream</td><td>Parker, Fairfield, NJ, USA</td><td>Redux cream</td><td>to build electrode bridges</td></tr><tr><td>Ag/AgCl electrodes</td><td>Biomedical, Clinton Township, MI, USA</td><td>JNS BNT131-1,0</td><td>measuring and reference electrodes</td></tr><tr><td>amiloride hydrochloride</td><td>Sigma, St Louis, MI, USA</td><td>A7410</td><td>to prepare perfusion solutions</td></tr><tr><td>forskolin</td><td>Sigma, St Louis, MI, USA</td><td>F6886</td><td>to prepare perfusion solutions</td></tr><tr><td>Knick Portamess voltmeter</td><td>Elektronisch Me&amp;beta;ger&amp;auml;te, Berlin, Germany</td><td>Portavo 904 pH</td><td>to measure potential difference</td></tr><tr><td>Paraly SW 112 Software&amp;nbsp;</td><td>Elektronisch Me&amp;beta;ger&amp;auml;te, Berlin, Germany</td><td>Paraly SW112 software</td><td>to capture potential difference data</td></tr><tr><td>midazolam&amp;nbsp;</td><td>Mylan, Hoeilaart, Belgium</td><td>Dormicum 15mg/3ml</td><td>to serve as anaesthetic premedication</td></tr><tr><td>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><td>medetomidine</td><td>Orion Pharma, Espoo, Finland</td><td>Domitor 1 mg/ml</td><td>to serve as anaesthetic medication</td></tr><tr><td>droperidol&amp;nbsp;</td><td>Janssen&amp;nbsp; Cilag, Berchem, Belgium</td><td>Dehydrobenzperidol 2.5 mg/ml</td><td>to serve as anaesthetic medication</td></tr><tr><td>clonidine&amp;nbsp;</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><td>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><td>forceps&amp;nbsp;</td><td>Fine science Tools, Heidelberg, Germany</td><td>Dumont #5, Fine science Tools</td><td>to place the nasal catheter</td></tr><tr><td>naloxone&amp;nbsp;</td><td>Braun Medical, Brussels, Belgium</td><td>Narcan, 0.4 mg/ml</td><td>to serve as anaesthetic antagonist</td></tr><tr><td>atipamezole&amp;nbsp;</td><td>Zoetis, Bloomberg, Belgium</td><td>Antisedan, 5 mg/ml</td><td>to serve as a medetomedine specific antidote&amp;nbsp;</td></tr><tr><td>Heating pads&amp;nbsp;</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><td>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><td>cyanoacrylate glue</td><td>Loctite, Henkel, D&amp;uuml;sseldorf, Germany&amp;nbsp;</td><td>super glue 3</td><td>to glue together two capillary tubes&amp;nbsp; for nasal probe</td></tr><tr><td>NaCl</td><td>Sigma, St Louis, MI, USA</td><td>RES0926S-A7</td><td>Pharma-Grade, USP</td></tr><tr><td>CaCl2.2H2O</td><td>Sigma, St Louis, MI, USA</td><td>M7304</td><td>Pharma-Grade, USP</td></tr><tr><td>MgCl2.6H2O</td><td>Sigma, St Louis, MI, USA</td><td>1551128</td><td>Pharma-Grade, USP</td></tr><tr><td>K2HPO4</td><td>Sigma, St Louis, MI, USA</td><td>1551139</td><td>Pharma-Grade, USP</td></tr><tr><td>Na gluconate</td><td>Sigma, St Louis, MI, USA</td><td>S2054</td><td>Pharma-Grade, USP</td></tr><tr><td>Ca gluconate</td><td>Sigma, St Louis, MI, USA</td><td>C8231</td><td>Pharma-Grade, USP</td></tr><tr><td>MgSO4.7H2O</td><td>Sigma, St Louis, MI, USA</td><td>RES0089M-A7</td><td>Pharma-Grade, USP</td></tr><tr><td>BD needle&amp;nbsp;</td><td>Becton Dickinson, Franklin Lakes, USA</td><td>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 mutation23,24,25, is the best currently available CF mouse model30,31,32.
Representative nasal PD tracings, obtained in a 4-month old female mouse homozygous for the F508del-CF mutation and in an age- and sex-matched wild-type littermate, are shown in Figure 4. During the first two phases of the test, the functional status of ENaC was studied by perfusing solutions A and A1, the latter containing amiloride. The functional status of CFTR (and of alternative chloride transporters in the absence of forskolin) was assessed during the last two phases of the test, when the contribution of ENaC remained blocked by amiloride.
In the F508del-CF mouse, a hyperpolarized baseline value (a more negative PDmax compared to the wild-type mouse value) together with an increased amiloride response were observed; both findings reflect the CFTR-associated ENaC overactivity. More consistently, a drastically reduced repolarization in response to both an electrochemical gradient favorable to chloride efflux and addition of forskolin, named here as total chloride response, was observed. Even though the magnitude of the forskolin response in wild-type mice is small (-3 mV), in CF, the response is usually blunted, consistent with the CFTR loss-of-function.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57934/57934fig4.jpg" /> 
Figure 4&#160;- Representative nasal PD tracings. Representative nasal PD tracings from a homozygous normal mouse (A) and a mouse homozygous for the F508del-CFTR mutation (B), together with the individual values obtained for the nasal PD parameters (C and D). PDmax: maximal baseline stable value. Amiloride response: 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 (solution A1). Chloride-free response: 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 (solution B1). Forskolin response: 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 (solution B2). Total chloride response: sum of the last two parameters obtained under zero-chloride perfusion. Arrows indicate changes of solutions perfused into the nostril. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57934/57934fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Antidotes of the anesthetics were applied at the end of the tests, thus reducing the duration of the anesthesia, which can be up to 45 min beyond test completion. As recovery of the animals occurred without after-effects, they were tested again after an interval of seven days, when the same protocol was applied. The same nostril was explored during both tests. Examples of the second test and paired differences of each individual nasal PD parameter between the two tests are shown in Figure 5. As previously reported35, the between-test differences were close to nil, in particular for total chloride response, reflecting the functional status of CFTR-dependent chloride transport, defective in CF.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57934/57934fig5.jpg" /> 
Figure 5 - Individual values of nasal PD parameters. Values were obtained in a second test (t2) performed in a homozygous normal mouse (A) and a mouse homozygous of the F508del-CFTR mutation (B), together with the paired differences between the second and the first test (t1) for each corresponding parameter. PDmax: maximal baseline stable value. Amiloride response: difference between the values of nasal PD at the end and at the beginning of perfusion of the nasal mucosa with the basal buffered salt solution containing amiloride (solution A1). Chloride-free response: difference between the values of nasal PD at the end and at the beginning of perfusion of the nasal mucosa with the chloride-free buffered salt solution plus amiloride (solution B1). Forskolin response: difference of the values of nasal PD at the end and at the beginning of perfusion of the nasal mucosa with the chloride-free buffered salt solution plus forskolin and amiloride (solution B2). Total chloride response: sum of the last two parameters obtained under zero-chloride perfusion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57934/57934fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Solution Label</td>
			<td>Solution description</td>
		</tr>
		<tr>
			<td>A1</td>
			<td>Basal buffered salt solution (A) plus 10-4&#160;M amiloride</td>
		</tr>
		<tr>
			<td>B1</td>
			<td>Chloride-free buffered salt solution (B) plus 10-4&#160;M amiloride</td>
		</tr>
		<tr>
			<td>B2</td>
			<td>Chloride-free buffered salt solution (B) plus&#160; 10-4&#160;M amiloride plus 10-5 M forskolin</td>
		</tr>
	</tbody>
</table>
Table 4: List of the fresh solutions for the nasal PD test in mice.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

7.7K Views.  Université Catholique de Louvain. Hier presenteren we een protocol voor het meten van de nasale potentiaalverschil in muizen. De test kwantificeert de functie van transmembraan ion vervoerders zoals de cystic fibrosis transmembrane huidgeleiding regelgever en de epitheliale natrium-kanaal. Het is waardevol om te evalueren van de werkzaamheid van nieuwe therapieën voor cystic fibrosis.

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

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Bioengineering

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Neuroscience

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) function are monitored by the change in voltage across the nasal epithelium after the superfusion of solutions that modify ion channel activity. This is enabled by the measurement of the potential difference between the subcutaneous compartment and the airway epithelium in the nostril, utilizing a catheter in contact with the inferior nasal turbinate.
The test allows the measurement of the stable baseline voltage and the successive net voltage changes after perfusion of 100 &#181;M amiloride, an inhibitor of Na+ reabsorption in Ringer&#39;s solution; a chloride-free solution containing amiloride to drive chloride secretion and 10 &#181;M isoproterenol in a chloride-free solution with amiloride to stimulate the cyclic adenosine monophosphate (cAMP)-dependent chloride conductance related to CFTR.
This technique has the advantage of demonstrating the electrophysiological properties of two key components establishing the hydration of the airway surface liquid of the respiratory epithelium, ENaC, and CFTR. Therefore, it is a useful research tool for phase 2 and proof of concept trials of agents that target CFTR and ENaC activity for the treatment of cystic fibrosis (CF) lung disease. It is also a key follow-up procedure to establish CFTR dysfunction when genetic testing and sweat testing are equivocal. Unlike sweat chloride, the test is relatively more time consuming and costly. It also requires operator training and expertise to conduct the test effectively. Inter- and intra-subject variability has been reported in this technique especially in young or uncooperative subjects. To assist with this concern, interpretation has been improved through a recently validated algorithm.

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD

Here, we present a standardized protocol to measure the nasal potential difference (NPD). Cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) function are evaluated by the change in the voltage across the nasal epithelium after superfusion of solutions that modify ion channel activity, providing an outcome measure.

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) ...

Medicine

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Immunology and Infection

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...

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

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.

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.

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog ...

Primary Human Nasal Epithelial Cells: Biobanking in the Context of Precision Medicine

Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and differentiated into a pseudo-stratified epithelium in air-liquid interface conditions to quantify cyclic AMP-mediated Chloride (Cl-) transport as an index of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function. If critical steps such as quality of nasal brushing and cell density upon cryopreservation are performed efficiently, HNE cells can be successfully biobanked. Moreover, short-circuit current studies demonstrate that freeze-thawing does not significantly modify HNE cells&#39; electrophysiological properties and response to CFTR modulators. In the culture conditions used in this study, when less than 2 x 106 cells are frozen per cryovial, the failure rate is very high. We recommend freezing at least 3 x 106 cells per cryovial. We show that dual therapies combining a CFTR corrector with a CFTR potentiator have a comparable correction efficacy for CFTR activity in F508del-homozygous HNE cells. Triple therapy VX-445 + VX-661 + VX-770 significantly increased correction of CFTR activity compared to dual therapy VX-809 + VX-770. The measure of CFTR activity in HNE cells is a promising pre-clinical biomarker useful to guide CFTR modulator therapy.

Here we describe the isolation, amplification, and differentiation of primary human nasal epithelial (HNE) cells at the air-liquid interface and a biobanking protocol allowing to successfully freeze and then thaw amplified HNE. The protocol analyzes electrophysiological properties of differentiated HNE cells and CFTR-related chloride secretion correction upon different modulator treatments.

Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and ...

A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery campaigns targeting recombinant ion channel proteins overexpressed in cells such as HEK-293 cells. However, there is increasing emphasis on the use of tissue-relevant cell lines for studying the effects of small molecule interventions. The following protocol describes the adaptation of a fluorescence-based membrane potential assay for the study of ion channels endogenously expressed in epithelial cell lines. The membrane potential assay details a high-throughput assay for chloride channel activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in two commonly studied epithelial cell lines, Caco-2 and Calu-3. In addition, this paper describes a novel application of this system to measure the activity of the Epithelial Sodium Channel (ENaC) in a high-throughput format in the same epithelial cell lines. Together, these fluorescence-based assays provide a robust and flexible platform for studying small molecule modulators, targeting two epithelial channels in a relevant cellular context.

This protocol describes a method for the study of electrogenic membrane proteins by measuring changes in membrane potential. This assay provides a platform for the functional readout of multiple ion channels endogenously expressed in epithelial cell lines.

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery ...

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

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