Nebulized hypertonic saline triggers nervous system-mediated active liquid secretion in cystic fibrosis swine trachea

Abstract

Inhaled hypertonic saline (HTS) treatment is used to improve lung health in patients with cystic fibrosis (CF). The current consensus is that the treatment generates an osmotic gradient that draws water into the airways and increases airway surface liquid (ASL) volume. However, there is evidence that HTS may also stimulate active secretion of ASL by airway epithelia through the activation of sensory neurons. We tested the contribution of the nervous system and airway epithelia on HTS-stimulated ASL height increase in CF and wild-type swine airway. We used synchrotron-based imaging to investigate whether airway neurons and epithelia are involved in HTS treatment-triggered ASL secretion in CFTR^-/- and wild-type swine. We showed that blocking parasympathetic and sensory neurons in airway resulted in ~50% reduction of the effect of HTS treatment on ASL volume in vivo. Incubating tracheal preparations with inhibitors of epithelial ion transport across airway decreased secretory responses to HTS treatment. CFTR^-/- swine ex-vivo tracheal preparations showed substantially decreased secretory response to HTS treatment after blockage of neuronal activity. Our results indicated that HTS-triggered ASL secretion is partially mediated by the stimulation of airway neurons and the subsequent activation of active epithelia secretion; osmosis accounts for only ~50% of the effect.

Figure 1

Experimental design and phase contrast imaging using synchrotron x-rays. (A) Schematic showing the set-up for ASL height measurement in the lumen of the trachea using phase contrast imaging. When x-rays pass through the preparation, the difference in refractive index between the ASL and the air results in a phase shift of x-rays that causes a distinct interference pattern detected as variations in x-ray intensities on the CCD (see for ex vivo diagram). (B) Synchrotron-based phase contrast imaging measurement of ASL height in an isolated swine trachea. (C) HTS or ITS aerosol were delivered at time 0 for 90 seconds, and images were acquired at time −3, 6, 12, and 18 minutes. Representative sample of the images acquired from an ex vivo preparation treated with (D) HTS and (E) ITS nebulization at −3, 6, 12, and 18 min.

Figure 2

HTS triggers ASL secretion in vivo and ex vivo preparations. (A) scatter plot of HTS and ITS treatment on ASL height in live swine and (B) change in ASL height (HTS, n = 6 beads from 4 swine; ITS, n = 6 beads from 5 swine). (C) scatter plot of HTS and ITS treatment on ASL volume in ex vivo trachea preparation and (D) change in ASL height (HTS, n = 45 beads from 15 tracheas; ITS, n = 49 beads from 14 tracheas; control, n = 12 beads from 5 tracheas). (E) Amiloride did not affect the HTS treatment result (HTS, n = 45 beads from 15 tracheas; ITS, n = 49 beads from 14 tracheas; HTS + Amil, n = 12 beads from 5 tracheas). Data are presented as mean ± SEM and values at 18 min were analyzed with ANOVA and Tukey’s multiple comparison test. Data sets labeled with different letters differ significantly, p < 0.05.

Figure 3

The nervous system contributes to HTS-triggered ASL secretion. (A) Atropine combined with lidocaine reduced HTS-triggered ASL secretion but had no effect in ITS-treated swine in vivo (HTS, n = 6 beads from 4 animals; ITS, n = 6 beads from 5 animals; HTS + Atro + Lido, n = 6 beads from 5 animals; ITS + Atro + Lido, n = 5 beads from 4 animals). (B) Stimulating C-fibers with capsaicin increased the secretion during ITS but not HTS treatment in live swine (HTS, n = 6 beads from 4 animals; ITS, n = 6 beads from 5 animals; HTS + Capsaicin, n = 6 beads from 4 animals; ITS + Capsaicin, n = 5 beads from 4 animals). Data are presented as mean ± SEM and values at 18 min were analyzed with ANOVA and Tukey’s multiple test. Data sets labeled with different letters differ significantly, p < 0.05.

Figure 4

HTS stimulates ex vivo ASL secretion via activation of sensory neurons and release of acetylcholine in wild-type and CF airways. (A) Treatment with lidocaine (Lido, 80 mg/ml aerosol) plus tetrodotoxin (TTX, 1 µM) (HTS, n = 45 beads from 15 tracheas; ITS, n = 49 beads from 14 tracheas; HTS + Lido + TTX, n = 18 beads from 4 tracheas; ITS + Lido + TTX, n = 9 beads from 4 tracheas). (B) Incubation with CFTRinh172 (172, 100 µM) (HTS, n = 45 beads from 15 tracheas; ITS, n = 49 beads from 14 tracheas; HTS + 172, n = 59 beads from 12 tracheas; ITS + 172, n = 29 beads from 8 tracheas). (C) Incubation with lidocaine plus TTX on CFTRinh172-treated airway (HTS + 172, n = 59 beads from 12 tracheas; HTS + 172 + Lido + TTX, n = 9 beads from 4 tracheas; ITS + 172, n = 29 beads from 8 tracheas; ITS + 172 + Lido + TTX, n = 21 beads from 6 tracheas). (D) Effect of L-703606 (NK-1 blocker, 1 µM) (HTS, n = 45 beads from 15 tracheas; ITS, n = 49 beads from 14 tracheas; HTS + NK-1 blocker, n = 36 beads from 12 tracheas; ITS + NK-1 blocker, n = 16 beads from 5 tracheas). (E) Effect of L-703606 on CFTRinh172-treated preparations (HTS + 172, n = 59 beads from 12 tracheas; HTS + 172 + NK-1 blocker, n = 10 beads from 4 tracheas; ITS + 172, n = 29 beads from 8 tracheas; ITS + 172 + NK-1 blocker, n = 17 beads from 6 tracheas). (F) Effect of atropine (1 µM) (HTS, n = 45 beads from 15 tracheas; ITS, n = 49 beads from 14 tracheas; HTS + Atropine, n = 22 beads from 5 tracheas; ITS + Atropine, n = 11 beads from 4 tracheas). (G) Effect of atropine (1 µM) on CFTRinh172-treated preparations (HTS + 172, n = 59 beads from 12 tracheas; HTS + 172 + Atropine, n = 18 beads from 5 tracheas; ITS + 172, n = 29 beads from 8 tracheas; ITS + 172 + Atropine, n = 19 beads from 5 tracheas). (H) Effect of lidocaine, TTX, and atropine on CFTR^−/− swine trachea (HTS, n = 6 beads from 2 tracheas; HTS + Lido + TTX + Atro, n = 4 beads from 2 tracheas; ITS, n = 4 beads from 2 tracheas). Data are presented as mean ± SEM and values at 18 min were analyzed with ANOVA and Tukey’s multiple test. Data sets labeled with different letters differ significantly, p < 0.05.

Figure 5

HTS treatments stimulate active ASL production by airway epithelia. (A) Treatment with the CFTR blocker CFTRinh172 (100 µM) reduced HTS-triggered ASL height increase, and treatment with CFTRinh172, bumetanide (100 µM), and niflumic acid (100 µM) in HCO₃⁻-free saline solution bath reduced HTS-triggered ASL height increase even further (HTS, n = 45 beads from 15 tracheas; HTS + 172, n = 59 beads from 12 tracheas; HTS + Bumet + NA + 172 + HCO₃⁻-free, n = 24 beads from 7 tracheas). (B) In ITS-treated preparations incubation with the ion transport blocker cocktail (CFTRinh172, bumetanide, and niflumic acid in HCO₃⁻-free bath) had a similar effect as CFTRinh172 alone (ITS, n = 49 beads from 14 tracheas; ITS + 172, n = 29 beads from 8 tracheas; ITS + Bumet + NA + 172 + HCO₃⁻-free, n = 18 beads from 7 tracheas). (C) Approximately 50% of the ASL produced by HTS in airways without CFTR function is the result of the osmotic effect. After blocking all ion transport with CFTRinh172, bumetanide, and niflumic acid in HCO₃⁻-free saline, HTS produced ~50% less ASL secretion than that produced by preparations incubated with CFTRinh172 alone (HTS + 172, n = 59 beads from 12 tracheas; HTS + Bumet + NA + 172 + HCO₃⁻-free, n = 24 beads from 7 tracheas; ITS + Bumet + NA + 172 + HCO₃⁻-free, n = 18 beads from 7 tracheas). Data are presented as mean ± SEM and values at 18 min were analyzed with ANOVA and Tukey’s multiple comparison test. Data sets labeled with different letters differ significantly, p < 0.05.