Tissue samples from the right lung were either immediately shock frozen and stored at −70 °C until biochemical analyses were performed or used to assess the degree of lung oedema. Plasma samples of arterial blood were obtained by centrifugation (3000 rpm for 15 min, 4 °C). Levels of inflammatory and oxidation markers were determined in plasma and 10% (weight/volume) lung homogenate in 0.1 M phosphate buffer (PBS, pH 7.4). The concentration of IL-1β, TNFα, IL-6, and IL-8 was quantified using rabbit-specific ELISA kits (Cloud-Clone Corp., Houston, Texas, USA) and expressed in pg/mL. Oxidative modifications were determined using the following kits (Cell Biolabs Inc., San Diego, CA, USA): OxiSelectTM Nitrotyrosine ELISA Kit for protein oxidation expressed in 3-nitrotyrosine nanomolar concentration (nM 3NT) and OxiSelect™ TBARS Assay Kit for lipid oxidation expressed as malondialdehyde in micromolar concentration (μM MDA). All biochemical analyses were performed in duplicates according to the manufacturers’ instructions.
The extent of the lung oedema was expressed as a wet-to-dry (W/D) lung weight ratio. Strips of the right lung were weighed before and after drying in an oven at 60 °C for 48 h to calculate the W/D ratio. The total protein content in the BALF was determined by the Bradford colourimetric method, as described previously [24 (link)].
The extent of the lung oedema was expressed as a wet-to-dry (W/D) lung weight ratio. Strips of the right lung were weighed before and after drying in an oven at 60 °C for 48 h to calculate the W/D ratio. The total protein content in the BALF was determined by the Bradford colourimetric method, as described previously [24 (link)].