Measuring Lung Vascular Permeability

Lung vascular permeability was measured in each experiment. The Evans blue assay was used to test vascular permeability in lung tissue and was performed as described previously (Radu and Chernoff, 2013 (link); Pati et al., 2018 (link); Zhou et al., 2019 (link)). Briefly, the mice received an intravenous injection of 1% Evans blue dye solution (Sigma, St. Louis, MO, USA). After 1 h, the mice were sacrificed and perfused via the right ventricle with 4°C PBS for 10 min to introduce the dye intravascularly, and the lung tissues were collected. The lungs were placed in 1 ml of formamide (Avantor, Center Valley, PA, USA) at 60°C for 24 h to extract the Evans blue dye. The lung tissues were then centrifuged at 2,000 rpm for 10 min, and the supernatants were collected. The concentrations of Evans blue dye in the supernatant were measured at an absorbance of 620 nm. For lung water content, the left lung was harvested and weighed to determine the wet weight in each group. The lungs were then dried to a constant weight at 60°C for 48 h and were weighed to determine the dry weight. The lung water content was calculated as the ratio of wet weight to dry weight.