Hippocampal Cytokine Profiling in Mice

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)].