Spectrophotometry

Lung wet weight and extravascular lung water (EVLW) were quantified in the same experiment. Body weight was recorded in anesthetized mice at the time of lung excision in untreated mice or at the time of IAV instillation in coinfected mice. We used our established methods (27 (link)) to exsanguinate anesthetized mice by cardiac puncture, then excised and weighed the lungs. Blood-free EVLW content was quantified by the method of Selinger and colleagues (80 (link)), which we have used previously (27 (link)). Lungs were cut with scissors, then processed using a handheld tissue homogenizer. Hemoglobin content was determined by spectrophotometry (Molecular Devices) using hemoglobin standards and a solution of Drabkin’s reagent and Brij L23 (MilliporeSigma). Homogenate, supernatant, and blood samples were dried for 24 hours in a vacuum oven at 57°C and –5 mmHg. Total EVLW content was normalized to body weight to account for increases of lung dry weight due to extravasated protein (81 (link)).

The BBB permeability was assessed by the extravasation of Evans blue dye (EB) as previously described [38 (link)]. The 2% Evans blue (4 mL/kg, Sigma Aldrich) in sterile saline was injected intravenously and circulated in the mice for 2 h before the mice were sacrificed. The mice were transcardially perfused with PBS, and their brains were dissected and weighed. The samples were then homogenized in n, n-dimethylformamide (Sigma Aldrich), and incubated at 60 °C for 72 h. After centrifugation, the absorption of the supernatant was determined by spectrophotometry (Molecular Devices, Sunnyvale, CA) at optical density (OD) 620 nm. The sample collection and ELISA analysis were performed by two researchers in a blinded manner.

The Indocyanine green (ICG, Sigma-Aldrich) assay measures the liver depuration capacity (34 (link)). Mice received a single intravenous injection of ICG (20mg/kg); 20 min later, blood samples were collected, and the serum was obtained following centrifugation 1500x g 10 minutes. ICG concentration was determined by spectrophotometry (Molecular Devices) at 800nm.