Histological Assessment of Murine Lung Pathology

Lungs were dissected from mice euthanized by intraperitoneal sodium pentobarbital injection as described above. The left lung lobe of each animal was removed at necropsy, instilled intratracheally with 4% paraformaldehyde (PFA), and post-fixed for 24 h by immersion in 4% PFA, prior to transfer into 70% ethanol and storage at 4 °C. Lung samples were then routinely processed to paraffin blocks and 5-µm thick serial sections prepared by cutting through the whole paraffin block and mounting sections 1:10 onto glass slides. These slides were stained with hematoxylin and eosin according to standard procedure and examined microscopically by an experienced veterinary pathologist. Microscopic lung findings were scored semi-quantitatively following accepted principles^{74 (link)}. Briefly, all serial sections per animal were first evaluated blinded to treatment at low magnification (×40 to ×100) to select the lung level(s) with the most severe and extensive lesions for each animal. The extent of the lesions across this section was then estimated (and scored as 0 = < 5%; 1 = 5–33%; 2 = 33–66%; 3 = >66%) and this parameter taken as a weighing factor to multiply with the sum of all the individually scored lesions, calculating an overall combined lung pathology score per animal. The individual lesions were scored at high magnification (×200–400) and these included alveolar interstitial inflammatory cells (neutrophils, macrophages, and/or lymphocytes/plasma cells), perivascular mixed inflammatory cell infiltrate and edema, necrosis, intra-alveolar neutrophils, macrophages, and/or hemorrhage (each scored as 0 = none; 1 = mild, 2 = moderate, 3 = severe). Alveolar septal thickening (scored as 0 = none, 1 = 2-fold increase, 2 = 2–4-fold increase, 3 = more than 4-fold increase compared with unaffected septa), hyaline membranes, and intra-alveolar proteinaceous fluid (the latter two scored as 0 = none, 1 = 1, 2 = more than 1 per alveolus) were scored as well.