Nebulized methacholine

Lung function was measured in two ways. In the first, it was measured as the change in airway resistance to increasing concentrations of nebulized methacholine (0.5–8.0 mg/ml) (Sigma-Aldrich). Mice were anesthetized, tracheostomized, and mechanically ventilated at a fixed breathing rate of 140 breaths/min using the FinePointe Series RC Sites (Buxco Research Systems, Wilmington, NC), and airway resistance and specific dynamic compliance were recorded. Results are expressed as a percentage of respective basal values in response to PBS.
The second used the Pulmonary Function Test Plethysmograph system (Buxco Research Systems) as recommended by the manufacturer (https://www.datasci.com/products/buxco-respiratory-products/pulmonary-function-testing). Mice were anesthetized with a mixture consisting of ketamine/medetomidine/saline. Sedation was ensured to be deep enough before the trachea was cannulatcd and connected to a built-in ventilator using a tracheal cannula. The data of breathing, airflow obstruction, and lung volumes were acquired using FinePointe software by measuring, and the parameters of total lung capacity (TLC) and forced expiratory volume were determined according to the manufacturer's recommendations. The results shown are TLC and forced expiratory volume at 100 ms (FEV100).

Briefly, 48h after the last PM exposure, mice were anesthetized by i.p. administration of ketamine/xylazine and tracheotomized before insertion of an 18-gauge cannula into the trachea. Mice were paralyzed with suxamethonium chloride (3 mg/kg), intubated and respirated at a rate of 120 breaths per minute with a constant tidal volume (0.2 ml). After a stable baseline was achieved, mice were exposed to 30 mg/ml nebulized methacholine (Sigma). After 10 seconds, dynamic airway pressure (cm H₂0×s) was recorded for 3 min. Following airway reactivity measurements, BAL fluid, and lungs were collected, and processed as previously described (Lajoie et al. 2010 (link); Lewkowich et al. 2008 (link)).