Fig 1.
Schematic of laboratory set up for potential difference (PD) measurements in ferrets.
Cartoon representing hardware and electrical connections representing PD apparatus modified from the human instrument recommended by the standard operating procedure of the CF Therapeutics Development Network and the European Clinical Trial Network [19]. Ground Lead: Connects electrical apparatus to ground for reference from which voltage is measured. Headstage: Connects electrode inputs to bioamplifier and safely isolates from wall AC current. Bioamplifier: Serves as voltmeter to measure PD. Analog-Digital Converter: Converts analog signals to digital form. K/Cl Calomel Electrodes: Measure PD. Agar Bridge: Connects positively charged (+) calomel electrodes to ferret nasal epithelium or lower airway epithelium through Airway Catheter and negatively charged (-) calomel electrodes to subcutaneous tissue through Butterfly Needle. Syringe Pumps: Deliver test solutions perfused sequentially from pump 1 to 5 with a flow rate of 4ml/hr (see S1 Table). The PD changes are observed and recorded on the computer using LabChart 7.0 software. Following the procedure, the nasal cannula and subcutaneous needle were removed, and anesthesia reversed with atipamezole (5 mg/kg body weight, IM) delivered at an equal volume as dexmedetomidine. All ferrets recovered within 10–15 min, and remained on a warming pad until aroused and moving. Supplemental oxygen was not required during the NPD procedures in our experience, however supplemental oxygen was delivered during recovery from anesthesia.
Fig 2.
Measurement of nasal potential difference (NPD) in wild type ferrets.
A: Representative NPD tracing in ferrets using the apparatus described in Fig 1. Under deep sedation, PD changes following perfusion with Ringer’s solution, Ringer’s solution with amiloride (100 μM), chloride-free solution, chloride-free with forskolin (20 μM), and chloride-free with CFTR-specific inhibitor GlyH101 (10 μM). B: Summary tracing of mean PD measurements (mean ± SEM) in ferrets following infusion of these 5 sequential reagents, N = 7. Ion channel activity of CFTR is quantified by PD changes in response to chloride-free + forskolin or by GlyH101 inhibitor. Similarly, ENaC activity is attributed to PD changes following amiloride treatment. C: Change in NPD (ΔPD) measurements from the same cohort of male and female adult, wild type ferrets plotted as individual measurements. D: Summary tracing of PD measurements (mean ±SEM) in male and female, adult, wild type ferrets acquired at two different times to validate reproducibility, N = 7. No discernable differences in ENaC or CFTR activity were found over 7 weeks. E: PD measurements of each animal subject plotted individually for each perfusate at two time points conducted 7 weeks apart.
Table 1.
Potential difference measurements across nasal epithelium.
Table 2.
Differences in potential difference at separate measurements.
Table 3.
Sample size calculations to detect significant change in nasal potential difference if using the ferret model.
Fig 3.
Reproducibility of nasal potential difference (NPD) assays in ferrets.
A: Representative NPD tracing in ferrets one hour after acute intranasal administration with either vehicle (DMSO 0.001% in saline) or CFTR-specific inhibitor GlyH101 (10μM). B: Summary of CFTR-dependent PD differences in vehicle and GlyH101-treated ferrets. N = 4, * P<0.05.
Fig 4.
Measurement of lower airway potential difference (LAPD) in wild type ferrets.
A. Representative LAPD tracing in a sedated intubated ferret indicating PD changes following perfusion with Ringer’s solution, amiloride (100 μM), chloride-free, chloride-free + forskolin (20 μM), and CFTR-specific inhibitor GlyH101 (10 μM); N = 5. B: Summary tracing of PD changes in ferrets following infusion of the 5 sequential reagents, N = 5. C: Change in LAPD measurements from 5 (male and female) adult, wild type ferrets.
Table 4.
Potential difference measurements across lower airway epithelium.
Fig 5.
Measurement of short-circuit current (Isc) in cultured ferret bronchial epithelial cells and in explants of ferret trachea.
A: Representative hematoxylin and eosin stained image of a well-differentiated ferret bronchial epithelial cells grown at air-liquid interface. B: Representative Isc tracings of primary ferret bronchial epithelial cells sequentially exposed to forskolin (10 μM), followed by GlyH101 (20 μM) in the setting of amiloride (100 μM) and a chloride secretory gradient. C: Summary data of Isc measurements from different ALI cultures indicating stimulated Isc following acute addition of forskolin or GlyH101. N = 4/condition. D: Representative Isc tracing of an ex vivo ferret trachea sequentially exposed to Ringer’s, chloride-free, forskolin (20 μM), followed by GlyH101 (20 μM) in the setting of amiloride (100 μM). E: Summary data of Isc measurements from different trachea showing stimulated Isc following acute addition of amiloride, forskolin, or GlyH101. N = 7/condition.