Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice
July 4th, 2018
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 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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Overview
This article presents a protocol for measuring nasal potential difference in mice, which quantifies the function of transmembrane ion transporters. The method is particularly useful for evaluating novel therapies for cystic fibrosis.
Key Study Components
Area of Science
- Neuroscience
- Biology
- Transgenic Models
Background
- Nasal potential difference is a method to characterize transgenic mice.
- It helps in assessing the efficacy of new cystic fibrosis therapies.
- The technique is advantageous for continuously breeding mice.
- It minimizes related mortality and allows repeated testing in the same animal.
Purpose of Study
- To measure nasal potential difference in mice.
- To evaluate the function of ion transporters like CFTR and ENaC.
- To assess the effectiveness of novel cystic fibrosis treatments.
Methods Used
- Preparation of a double lumen nasal catheter.
- Measurement of nasal potential difference in transgenic mice.
- Assessment of ion transporter function.
- Evaluation of therapy efficacy.
Main Results
- Reliable measurements obtained from continuously breeding mice.
- Reduction in mortality related to testing procedures.
- Ability to repeat tests in the same animal.
- Insights into the efficacy of new cystic fibrosis therapies.
Conclusions
- Nasal potential difference is a valuable method for cystic fibrosis research.
- The protocol enhances the reliability of measurements in mouse models.
- This technique supports the development of new therapeutic strategies.
Frequently Asked Questions
Executive Industry Relevance
Nasal potential difference (NPD) measurement in mice provides a functional readout of trans-epithelial ion transport, enabling preclinical evaluation of cystic fibrosis (CF) therapeutics. By quantifying CFTR and ENaC activity in vivo, the method supports target validation and mechanistic de-risking in early discovery. Repeated testing in the same animal reduces variability and improves predictive confidence for go/no-go decisions in CF therapy development.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypotheses by measuring functional chloride and sodium transport in CF mouse models.
- Operational Value: Enables biological de-risking of CFTR modulators through direct ion transport phenotyping.
- Scientific Value: Supports predictive confidence by discriminating CF from wild-type phenotypes via amiloride, chloride-free, and forskolin responses.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems for assay standardization using double lumen nasal catheters and controlled perfusion.
- Operational Value: Ensures reproducibility through stable electrode offset verification and consistent solution perfusion protocols.
- Scientific Value: Enables reliable compound evaluation by measuring ion transporter responses to amiloride, chloride-free, and forskolin solutions.
Translational & Preclinical Research
- Scientific Value: Maintains disease relevance by quantifying functional defects in CFTR and ENaC transporters in spontaneously breathing mice.
- Operational Value: Supports translational continuity by allowing multiple tests per animal, reducing inter-animal variability.
- Scientific Value: Informs risk-adjusted advancement decisions by providing quantitative PD tracings that correlate with genotype.
Pipeline & Workflow Integration
The NPD method integrates into the discovery continuum from target validation through preclinical evaluation, providing functional ion transport data that informs therapeutic efficacy assessments.
- Discovery Biology: Supports hypothesis testing by measuring basal and stimulated nasal potential differences in transgenic mice.
- Screening: Delivers assay readiness through standardized perfusion of buffered solutions and stable voltage readouts.
- Analytics: Generates quantitative dependent variable measurements (amiloride response, chloride-free response, forskolin response, total chloride response) for condition comparison.
- Translational Research: Connects to preclinical continuity by enabling repeated testing in the same animal to monitor longitudinal treatment effects.
- Enterprise Reuse: Functions as a reusable capability for CF model characterization across multiple therapeutic iterations.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in target validation through quantitative ion transport phenotyping.
- Operational Value: Standardization and reproducibility via catheter preparation, electrode calibration, and perfusion controls.
- Strategic Value: Better go/no-go decisions by reducing mechanistic ambiguity in CFTR and ENaC modulator efficacy.
- Portfolio Impact: Risk-adjusted prioritization based on functional transport defect correction in preclinical models.
Implementation Considerations
- Expertise in rodent anesthesia management and nasal catheterization techniques.
- Instrumentation including high-impedance voltmeter, peristaltic pump, and temperature-controlled heating pads.
- Cross-team standardization of solution preparation, electrode offset verification, and perfusion timing.
- Adaptation considerations for different rodent strains and CFTR mutation models.
- Practical limitations include surgical delicacy of nasal cavity access and anesthesia depth optimization to prevent broncho-aspiration.
