Citric Acid-Induced Cough Model in Conscious Guinea Pigs

The cough model was established by subjecting the guinea pigs to repeated inhalation of citric acid, as described previously (Xu et al., 2018). Awake animals were placed in a whole-body plethysmograph (EMKA, Bourré, France), where they were permitted to move freely, and then exposed to 0.4 M aerosolized citric acid for 3 min, twice daily to induce cough (Supplementary Figure S1A). The aerosol was generated via a jet nebulizer (ANP-1000; EMKA, Bourré, France) and delivered to the body plethysmograph at an output of 0.5 mL/min, with a median diameter of the particles between 0.5 and 2 μm. The cough module of the EMKA system, a respiratory flow analyzer (iox2.9 analysis system, EMKA, Bourré, France), was used to monitor the number of coughs during the inhalation of citric acid, by detecting the changes in the pressure waveform and sound waveform.

Control animals inhaled 0.9% sterile saline instead of citric acid. A previous study showed that repeated inhalation of 0.4 M citric acid for 25 days increased cough in a time-dependent manner and reached a peak on the 15th day, with a corresponding pattern in airway neurogenic inflammation, as indicated by similar changes in SP and calcitonin gene-related peptide in the airways over time (Xu et al., 2018). Taking this into account, we established a cough model for the guinea pigs that involved repeated inhalation of citric acid for 15 consecutive days.