Animals were anesthetized and perfused transcardially with PBS, followed by zinc formalin. Lungs were fixed in zinc formalin. For routine histology, tissue sections (~4 μm each) were stained with hematoxylin and eosin. The following criteria were used for scoring edema, hyaline membrane formation and necrotic cellular debris: 0- none; 1-uncommon detection in <5% lung fields (200x); 2- detectable in up to 33% of lung fields; 3- detectable in up to 33-66% of lung fields; 4- detectable in >66% of lung fields. For scoring neutrophil infiltration: 0- within normal limits; 1-scattered PMNs sequestered in septa; 2- #1 plus solitary PMNs extravasated in airspaces; 3-#2 plus small aggregates in vessel and airspaces. For scoring mononuclear infiltrates, thrombosis and hemorrhage: 0-none; 1- uncommon detection in <5% lung fields (200x); 2- detectable in up to 33% of lung fields; 3- detectable in up to 33-66% of lung fields; 4- detectable in >66% of lung fields.
For SARS-CoV-2 antigen detection, slides were incubated with blocking reagent (10% normal goat serum x 30 minutes) followed by rabbit monoclonal antibody against SARS-CoV2 N protein (1:20,000 dilution x 60 minutes, #40143-R019, Sino Biological US Inc., Wayne, PA, USA), then incubated with Rabbit Envision (Dako) and diaminobenzidine (Dako) as chromogen. Tissues were examined and scored in a post-examination method of masking by a boarded experimental pathologist32 . Ordinal scores for lesion parameters were assigned using the following tiers: 0 = within expected limits; 1 - uncommon, <5%; 2 - detectable in 5-33%; 3 - detectable in 34-66% and 4 - detectable in >66% of lung fields (200x objective magnification).