Th 115

At the designated interval, mice were anesthetized with 2.5% isoflurane and transcardially perfused with ice-cold phosphate-buffered saline (PBS) for 5 min. Following perfusion, the brains were rapidly removed, and the ipsilateral dorsal hippocampus was dissected and snap frozen in a 2-mL screw-top tube in liquid nitrogen. All dissected hippocampi were stored at − 80 °C for subsequent biochemical evaluation. Hippocampi were processed for protein extraction using a high shear homogenizer (Omni TH115) using lysis buffer at a 1:10 weight to volume ratio. Tissue lysis buffer consisted of PBS containing 1 mM PMSF and 1 mM EDTA. Hippocampal homogenate was centrifuged at 12,000×g for 20 min at 4 °C in a Heraeus Megafuge 16R. Supernatants were collected for measurement of cytokines and chemokines using MesoScale Discovery (MSD) custom multiplex high-sensitivity (V-Plex) ELISA kits, as we have previously described [13 (link)].

The protein levels of a panel of inflammatory cytokines were measured in the neocortex by Meso Scale Discovery (MSD) multiplex immunoassay (sector imager 2400, Meso Scale Discovery; Gaithersburg, MD) as previously described [38 (link)]. Brain cortex was homogenized using high shear homogenizer (Omni TH115), in a 1:10 (w/v) of ice-cold lysis buffer consisting of PBS containing 1 μg/ml Leupeptin, 1 mM PMSF, and 1 mM EDTA. The cortical homogenate was centrifuged at 14,000 × g for 20 min at 4 °C in a microcentrifuge. Fifty microliters of the resulting supernatant was loaded per well of the custom MSD plate, and cortical cytokine levels were determined by MSD assay (Mouse Proinflammatory 7-Plex Ultra-Sensitive (K15012C)). Cytokine levels in the cortex were normalized to the total amount of protein in the sample loaded as determined by BCA Protein Assay (Pierce).

To measure the amount of FITC-dextran that had leaked into the lung, lung tissue was thawed and homogenized using a radioimmunoprecipitation assay (RIPA) buffer and tissue homogenizer (TH-115, Omni International, Kennesaw, GA, USA). A 100 µL sample of this solution was measured for fluorescence using a spectrophotometer (spectramax M2, molecular devices, San Jose, CA, USA).