Measurement of lung mechanics and FVC
Measurement of lung mechanics and FVC was performed with a computer-controlled small-animal ventilator connected to a negative pressure reservoir (FlexiVent; SCIREQ, Montreal, Canada), as previously described (K. Tanaka, Azuma, Miyazaki, Sato, & Mizushima, 2012). Mice were anaesthetised with three types of mixed anesthetic agents (0.75 mg/kg medetomidine, 4.0 mg/kg midazolam, and 5.0 mg/kg butorphanol), a tracheotomy was performed, and an 8 mm-long section of metallic tube (outer and inner diameters of 1.27 mm and 0.84 mm, respectively) was inserted into the trachea. Mice were mechanically ventilated at a rate of 150 breaths/min, using a tidal volume of 8.7 ml/kg and a positive end-expiratory pressure of 2–3 cmH2O.
Total respiratory system elastance and tissue elastance were measured by snap shot and forced oscillation techniques, respectively. For determination of FVC, lungs were inflated to 30 cmH2O over one second and held at this pressure. After 0.2 sec, the pinch valve (connected to the ventilator) was closed, and after 0.3 sec, the shutter valve (connected to the negative pressure reservoir) was opened, exposing the lung to the negative pressure, which was held for 1.5 sec to ensure complete expiration. All data were analysed using FlexiVent software (version 5.3; SCIREQ, Montreal, Canada).