Cigarette smoke exposure & ebselen treatment
Mice were placed into an 18-litre Perspex chamber (The Plastic Man, Huntingdale, Victoria, Australia) in a standard fume cabinet (Aircare Extraction Systems LTD, Clayton, Victoria, Australia) and exposed to cigarette smoke generated from 9 Winfield Red Cigarettes (total particulate matter of 419 mg m-3, 16 mg or less of tar, 1.2 mg or less of nicotine and 15 mg or less of CO, Philip Morris, Moorabbin, Australia) for 5 days a week for 8 weeks. Mice were exposed to CS delivered 3 times per day with 3 cigarettes delivered at 9 AM, 12 noon and 3 PM, over a 1h time-period. CS was generated in 60 mL tidal volumes over 10 s, via a timed draw-back to mimic normal smoking inhalation and burn rates. We have previously shown that this CS exposure protocol in Balb/C mice replicates key clinical traits of early stage COPD in humans, including lung inflammation and pathology (mucous hypersecretion and impaired lung function), increased lung and systemic oxidative stress and comorbidities including skeletal muscle wasting (Austin, Crack, Bozinovski, Miller & Vlahos, 2016; Chan et al., 2019; Vlahos & Bozinovski, 2014). Thus, this model provides a robust clinically relevant platform to test therapies for COPD and its comorbidities. Sham-exposed mice are placed into an identical 18-liter Perspex chamber, but do not receive cigarette smoke. Mice were weighed every second day (prior to initial CS exposure) up to and including the day of cull. For the ebselen treatment studies, mice were administered 10 mg kg-1 of ebselen (Sapphire Bioscience, Australia) prepared in 5% w/v CM-cellulose in water (Sigma-Aldrich, USA) or vehicle treated with 5% CM-cellulose in water alone. Treatments were administered via oral gavage once daily, 1h prior to the initial CS exposure.