Standardized Lung Tissue Preservation and Histological Analysis

After whole lung lavage, lungs were harvested, inflated with 1 ml 10% formalin, and hung under a pressure of 20 cmH₂O for 1 day while submerged in 10% formalin for optimal preservation of lung parenchymal architecture as previously described (Poole, et al. 2014 (link)). Fixed lung tissues were standardly processed and embedded in paraffin, and sections (4–5 μM) cut and stained with hematoxylin and eosin. Each slide was reviewed in entirety, using (2x, 4x, and 10x objectives; Nikon Eclipse Model E600 microscope, Nikon, Tokyo, Japan) and semi-quantitatively assessed for the degree and distribution of lung inflammation utilizing a previously published scoring system by a lung pathologist blinded to the treatment conditions (Poole, et al. 2009 (link)). The scoring system is a Likert scale (0–3) with higher values representing increased histopathologic inflammation.