Histological Analysis of Lung Injury

For histological analyses, the right lung of each mouse was perfused with PBS, inflated with 10% buffered formalin (ThermoFisher Scientific, Waltham, MA, USA), and stored at 4 °C until processing. Slices from each lobe of the right lung were trimmed and sent to the UAB Comparative Pathology Laboratory to be processed, paraffin embedded, sectioned onto slides and stained with hematoxylin and eosin (H and E). Semiquantitative grading of histopathological changes of all lung sections was performed by a board-certified surgical pathologist (L.N.). Severity of lung tissue injury included alveolar, peribronchial inflammation, perivascular neutrophil infiltration, pleuritis and tissue necrosis. Severity of tissue damage from all lung lobes was graded. Grade 0 showed no neutrophil infiltrate in lung tissue; grade 1 showed rare neutrophils in the alveolar, peribronchial or perivascular tissue; grade 2 showed dense neutrophil infiltrate within the alveolar spaces with no injury to alveolar tissue; grade 3 showed dense neutrophil infiltrate within the alveolar space and necrosis of involved alveolar tissue. H-score is a cumulative score, determined by the percentage of tissue area assigned each grade (0–3). Maximum grade was assigned to the most severely damaged lobe, representing the maximum infection per animal. Images of lung sections were taken on a Lionheart FX Automated Imaging microscope (BioTek, Winooski, VT, USA) using 10× objective. Scale bar represents 200 µm.