Inhaled Nitric Oxide Therapy Fails to Improve Outcome in Experimental Severe Influenza

Darwish, Ilyse; Miller, Chris; Kain, Kevin C.; Liles, W. Conrad

Lungs were harvested and frozen at -80°C. Lungs were thawed, weighed, and homogenized in 1 ml PBS for 30 sec using a Tissue Miser homogenizer (Fisher Scientific, ON, CA). Lung homogenates were spun at 10,000xg for 10 min, aliquoted, and stored at -80°C for viral yield titration. Influenza WSN/33 viral yield in lung homogenates was quantified by plaque assay in MDCK canine kidney epithelial cells (ATCC, VA, USA). Cells were maintained in Eagle's MEM (ATCC, VA, USA) supplemented with 10% fetal bovine serum and antibiotics. MDCK cells were cultured at 37°C with 5% CO2. Cells were plated at a concentration of 8x10 6 cells/plate in 6 well culture plates. 12-24 hours later, medium was removed and MDCK cells were washed twice with PBS. 10-fold dilutions of lung homogenates were added to MDCK cells in 500 μL Eagle's MEM, in duplicate, and incubated at 37°C with 5% CO2 for 1 hour with plates rocked every 15 min. After incubation, 1 mL of serum-free 2X Eagle's MEM supplemented with 8 μl/ml trypsin, 60 μl/ml of 7.5% sodium bicarbonate and 20 μl/ml antibiotics, combined with 1 ml of 1.2% agarose, was added to each well. Once the agarose set, plates were incubated at 37°C for 42-72 hours until syncitia were observed. Plates were fixed with Carnoy's fixative (3:1, methanol:glacial acetic acid) for 30 min then stained with 0.1% crystal violet in 20% ethanol to visualize plaques. Viral load is expressed as plaque forming units per gram of lung tissue (PFU/g).