Male, C57Bl/6 mice aged 8 weeks were given 20% ethanol (EtOH) in their drinking water along with ad lib access to standard mouse chow. Mice were acclimated to EtOH by increasing the EtOH concentration in 5% increments from 0% to the target 20% (w/v) over the course of two weeks then maintaining the 20% concentration for an additional ten weeks. This regimen replicates blood alcohol levels following chronic EtOH ingestion in human subjects (Jerrells et al., 2007 (link), Song et al., 2002 (link)). In preliminary studies, blood alcohol concentrations were measured with a rapid, high-performance plasma alcohol analyzer (Analox Instruments Ltd., London, UK) according to the manufacturer's protocol. These analyses confirmed that this ethanol regimen produced clinically relevant elevations in blood alcohol concentration (0.12% ± 0.03, n=24). During the final week of EtOH treatment, all mice were gavaged daily for 7 days with either rosiglitazone (10 mg/kg/day in 100 μl methylcellulose vehicle) or vehicle alone as previously reported (Hwang et al., 2007 (link), Nisbet et al., 2010 (link)). Selected mice were treated with Escherichia coli lipopolysaccharide (Sigma-Aldrich, St. Louis MO, 2 mg/kg IP at 6 and 3 hours prior to sacrifice) as an inflammatory stimulus to promote pulmonary dysfunction. The timing of these studies was based on recent reports showing significant LPS-mediated increases in lung leak in C57Bl/6 mice 6-hours after LPS administration (Rojas et al., 2005 (link)). After sacrifice, blood was collected via cardiac puncture, and bronchoalveolar lavage (BAL) performed via tracheotomy. Protein concentration in the BAL fluid was measured using the bicinchoninic acid (BCA) assay (Thermo Scientific, Rockford IL), and values were corrected for dilution based on the ratio of blood to BAL urea nitrogen, assayed with a commercially available kit (Pointe Scientific, Inc., Canton MI) as previously reported (Rennard et al., 1986 (link)). Lung tissue was collected for subsequent analyses as described below following perfusion of the pulmonary artery with sterile phosphate buffered saline (PBS, Cellgro, Manassas, VA). All animal studies were approved by the Atlanta Veteran's Affairs Medical Center Animal Care and Use Committee.