Lung Histological Assessment in Mice

Twenty-four hours after the final challenge, to harvest the lungs, mice were euthanized by an intraperitoneal injection of a lethal dose of 10% chloral hydrate (0.3 mL/100 g, i.p). After fixing in formaldehyde for 24 hours, lungs were dissected and paraffin-embedded. The lung tissue was cut into thin slices four microns thick and then stained with hematoxylin and eosin (H&E; Sigma-Aldrich). At least five bronchi were selected from each mouse based on size (150–350 mm in diameter) for analysis. In order to reduce evaluator bias, the degree of airway inflammatory cell infiltration was scored in a single-blind fashion. Lung lesions were scored semi-quantitatively using a measurement tool as previously described^{62 (link)}. Images were captured under a Nikon Eclipse E200 microscope connected to a Nikon Coolpix 995 camera (Nikon, Tokyo, Japan). The severity of inflammation was evaluated by assigning a value of 0 point for normal; 1 point for few cells; 2 points for a ring of inflammatory cells 1 cell layer deep; 3 points for a ring of inflammatory cells 2 to 4 cells deep; 4 points for a ring of inflammatory cells of >4 cells deep.