Mechanical stimulation by postnasal drip evokes cough

Cough affects all individuals at different times, and its economic burden is substantial. Despite these widespread adverse effects, cough research relies on animal models, which hampers our understanding of the fundamental cause of cough. Postnasal drip is speculated to be one of the most frequent causes of chronic cough; however, this is a matter of debate. Here we show that mechanical stimuli by postnasal drip cause chronic cough. We distinguished human cough from sneezes and expiration reflexes by airflow patterns. Cough and sneeze exhibited one-peak and two-peak patterns, respectively, in expiratory airflow, which were also confirmed by animal models of cough and sneeze. Transgenic mice with ciliary dyskinesia coughed substantially and showed postnasal drip in the pharynx; furthermore, their cough was completely inhibited by nasal airway blockade of postnasal drip. We successfully reproduced cough observed in these mice by injecting artificial postnasal drip in wild-type mice. These results demonstrated that mechanical stimulation by postnasal drip evoked cough. The findings of our study can therefore be used to develop new antitussive drugs that prevent the root cause of cough

Nasal mucociliary clearance was decreased in <i>Ttll1</i>^−/− mice.

<p>(A) Tosufloxacin (50 mg/kg) was administered to <i>Ttll1</i>^−/− mice with rhinosinusitis to inhibit cough for 7 days. Treatment with tosufloxacin did not decrease cough (bars indicate median values, n = 4 per group; NS = not significant). (B) Nasal sections (coronal) of the <i>Ttll1</i>^−/− mice treated with tosufloxacin. Neutrophils were decreased in the mucus. However, mucus accumulation persisted (*) (n = 4). (C) Representative computed tomography scans of the coronal nasal cavity. The area occupied by contrast material is bordered with a red line. (D) We calculated the percent changes in the area of contrast material [(area at 30 min − area at 150 min)/area at 30 min] to assess clearance of contrast material (mean ± SEM, n = 4, *P < 0.005 by two-tailed Student's t test). (E) Concentration of Evans blue in the nasal cavity and stomach 90 min after administration. In the <i>Ttll1</i>^−/− mice, a larger amount of Evans blue persisted in the nasal cavity and a lesser amount was swallowed compared with that in the wild-type (WT) mice (mean ± SEM, n = 5, *P < 0.005 by two-tailed Student's t test).</p

Respiratory reflexes in humans, guinea pigs, wild-type (WT) mice, and <i>Ttll1</i>^{<i>−/−</i>} mice.

<p>(A−D): Charts exhibiting airflow patterns of respiratory reflexes. Expiratory flow is indicated by the plus sign (upward) and inspiratory flow is indicated by the minus sign (downward). Airflow patterns of human reflexes (A). Airflow through the nose and mouth induced by cough, sneeze, and the expiration reflex was recorded using a spirometer (n = 3). Cough and the expiration reflex were evoked by inhaled capsaicin. Sneeze was evoked by mechanical stimuli applied by rubbing the nasal cavity with a tapered tissue paper. Airflow patterns of reflexes in guinea pigs (B) and WT mice (C; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141823#pone.0141823.s002" target="_blank">S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141823#pone.0141823.s005" target="_blank">S4</a> Videos). Airflows of cough and sneeze were analyzed using a whole body plethysmograph (WBP). Cough was evoked by inhaled citric acid in guinea pigs and capsaicin in mice. Sneeze was induced by intranasal instillation of ovalbumin in sensitized animals. In (A–C), cough and sneeze showed one-peak and two-peak expiration patterns with preceding inspiration, respectively. Airflow patterns of reflexes in <i>Ttll1</i>^−/− mice (D; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141823#pone.0141823.s006" target="_blank">S5</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141823#pone.0141823.s008" target="_blank">S7</a> Videos). These reflexes were analyzed by WBP and classified into three patterns: (i) one-peak expiration with preceding inspiration, (ii) two-peak expiration with preceding inspiration, and (iii) one-peak expiration without preceding inspiration. Patterns (i) and (ii) corresponded to cough and sneeze patterns, respectively. #: expiration during eupneic breathing, which was not accompanied by characteristic sound and motion. (E) Representative photos recorded by videofluoroscopy. The <i>Ttll1</i>^−/− mice were placed in a WBP device. Inspiration and expiration phases in cough and normal breathing are shown. Solid lines indicate the diaphragm of the <i>Ttll1</i>^−/− mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141823#pone.0141823.s009" target="_blank">S8 Video</a>). (F) The bar graph shows the calculated amplitude of the diaphragm while coughing [= b–a in (E)] and normal breathing [= d–c in (E)]. The maximal distance [two-headed arrow in (E)] between the dotted line connecting the costophrenic angles and the diaphragm [solid line in (E)] as measured during inspiration and expiration while coughing and normal breathing. Diaphragm motion in the <i>Ttll1</i>^−/− mice was larger during coughing than during normal breathing (n = 3; mean ± SEM; *P = 0.006 by two-tailed Student's t-test). (G) Number of respiratory reflexes of the <i>Ttll1</i>^−/− mice in ten minutes (mean ± SEM, n = 10).</p

Effects of antitussive drugs on cough in <i>Ttll1</i>^−/− mice.

<p>(A, B, D, F, H) Graphs displaying the pre- and post-treatment number of coughs. (C) Graph displaying the dose–response relationship of the increased number of coughs in <i>Ttll1</i>^−/− mice. (E, G) Graph displaying the number of coughs evoked by inhaled capsaicin in wild-type (WT) mice treated with control and capsazepine (CPZ) or HC-030031. (A) Codeine phosphate (10 mg/kg) or saline was administered by gavage. Codeine phosphate significantly decreased cough in the <i>Ttll1</i>^−/− mice. (B) The <i>Ttll1</i>^−/− mice were nebulized with salbutamol (5 mg/ml) or phosphate-buffered saline (PBS). Salbutamol did not decrease cough in the <i>Ttll1</i>^−/− mice. (C) Moguisteine (3, 10, and 30 mg/kg) or control [0.5% dimethylsulfoxide (DMSO)] was intraperitoneally administered. Administration of 10 and 30 mg/kg moguisteine significantly inhibited cough in the <i>Ttll1</i>^−/− mice. (D) The <i>Ttll1</i>^−/− mice were nebulized with CPZ (300 μM) or control (10% DMSO). CPZ did not decrease cough in the <i>Ttll1</i>^−/− mice. (E) After treatment with nebulized CPZ (300 μM) or control (10% DMSO), the WT mice were nebulized with capsaicin to evoke cough. Nebulization with 300 μM CPZ was sufficient to inhibit cough evoked by capsaicin in the WT mice. (F) Vehicle (0.5% methyl cellulose in sterile saline) or HC-030031 (300 mg/kg) was administered intraperitoneally to the <i>Ttll1</i>^−/− mice. HC-030031 did not decrease cough in the <i>Ttll1</i>^−/− mice. (G) After administration of HC-030031 (300 mg/kg) or vehicle, the WT mice were nebulized with acrolein (10 mM) to evoke cough. Administration of HC-030031 was sufficient to inhibit cough evoked by acrolein in the WT mice. (H) Lidocaine (4%) or saline was administered to each nostril. Lidocaine decreased cough in the <i>Ttll1</i>^−/− mice [bars indicate median values; n = 5–10 mice in each group; *P < 0.05, number of coughs compared between pre- and post-treatment (in A−D, F, H) or between control and treated groups (in E and G) using Wilcoxon signed-rank test; †P < 0.05, increased number (post-treatment–pretreatment) compared with that in the control group using Mann–Whitney U-test; NS = not significant].</p

Laryngeal stimuli by postnasal drip-evoked cough in mice.

<p>(A) Contrast material in the nasal airway and upper airway was scanned by computed tomography (CT). CT shows optimal nasal airway obstruction with contrast material (arrows). (B) Physical blockade of the nasal airway to dam postnasal drip. Cyanoacrylate glue was placed in the nasal airway of <i>Ttll1</i>^−/− mice, as illustrated. Sagittal section of the nasal airway stained with hematoxylin and eosin showing obstruction of the nasal airway with cyanoacrylate glue (#). (C) Graph displaying the pre- and post-treatment number of coughs in the <i>Ttll1</i>^−/− mice. Cough in the <i>Ttll1</i>^−/− mice was completely inhibited by nasal airway blockade with cyanoacrylate glue (bars: median values, n = 7, *P = 0.02 by Wilcoxon signed-rank test). (D−F) Artificial postnasal drip in the wild-type (WT) mice (n = 5). A blue-colored polyvinyl alcohol (PVAL) solution was intranasally administered to the WT mice to mimic postnasal drip. The photos show the lower jaw (D) and lung (F), and the photomicrographs show sagittal sections of the larynx (E) after administration of the PVAL solution. The PVAL solution (blue) was found in the larynx (white and black arrowheads). Bar, 1 mm. There was no finding of aspiration of the PVAL solution in the trachea and lungs (E and F). (G) Graph showing the pre- and post-treatment number of coughs in the WT mice. A PVAL solution (artificial postnasal drip) was intranasally administered to the WT mice. Cough was evoked by an artificial postnasal drip in the WT mice (n = 4).</p

Increased cough sensitivity in <i>Ttll1</i>^−/− mice.

<p>Wild-type (WT) and <i>Ttll1</i>^−/− mice were nebulized with saline and capsaicin (10 and 50 μM). A graph displaying the increased number of coughs (post-treatment–pretreatment). Even low doses (10 μM) of capsaicin increased the number of coughs in the <i>Ttll1</i>^−/− mice but not in the WT mice. High doses (50 μM) of capsaicin increased the number of coughs in the WT and <i>Ttll1</i>^−/− mice. (n = 5 mice per group; mean ± SEM; *P < 0.05, the increased numbers of coughs were compared between the WT and <i>Ttll1</i>^−/− mice; †P < 0.05 and ‡P < 0.01, the increased numbers of coughs in comparison with those induced by saline; Mann–Whitney U-test).</p

Increased upper airway resistance without airway remodeling in <i>Ttll1</i>^−/− mice.

<p>(A) Lung sections stained with periodic acid-Schiff (PAS) and elastic van Gieson's (EVG) stains. There were no apparent differences between the wild-type (WT) and <i>Ttll1</i>^−/− mice (n = 4 per group). Scale bar, 200 μm. (B) Measurement of smooth muscle area. To adjust airway size, the area of the smooth muscle layer was divided by the length of the basement membrane (bars indicate mean values, n = 4 per group, two-tailed Student's t test; NS = not significant). (C) Measurements of hydroxyproline in lung homogenates. Right lower lung homogenates used for the hydroxyproline assay (bars indicate mean values, n = 4 per group, two-tailed Student's t test) (D). The baseline enhanced pause (Penh) indices of mice (bars indicate mean values, n = 10 mice per group, * P < 0.0001 by two-tailed Student's t-test). (E and F) The flexiVent system was used to measure upper (E) and lower (F) airway resistance in the anesthetized and intubated mice. The upper airway resistance was increased, but lower airway resistance was not increased in the <i>Ttll1</i>^−/− mice compared with that in the WT mice (mean ± SEM, n = 4 mice per group, † P < 0.05 by two-tailed Student's t-test). (G) Assessment of airway hyperresponsiveness (AHP). The mice were exposed to nebulized saline and methacholine (2.5, 5, 10, 20 mg/ml), and airway resistance was measured in each nebulization. There was no difference in AHP to methacholine (mean ± SEM, n = 4 mice per group, two-tailed Student's t-test; NS = not significant).</p