Seven days following the induction of SE with KA, all mice, including the vehicle-treated, were euthanized. Blood samples were collected by cardiac puncture into lithium heparin-coated tubes, centrifuged at 2,000 × g for 20 min at 4°C for the isolation of plasma, and stored at −80°C. Brains were removed, and the whole hippocampi and cerebral cortices including the tissue surrounding the electrodes were dissected, collected in cryovials, and snap-frozen in liquid nitrogen. Brain and plasma samples were stored at −80°C until required.

Hippocampi and cortical tissue samples were thawed on ice, dabbed with a sterile tissue paper to remove the water, and weighed before performing assays. Tris lysis buffer containing 1:50 protease inhibitor was added to each sample at a volume of 10 μl/mg of tissue. Samples were then homogenized using a TissueRuptor, sonicated, and centrifuged at 10,000 × g for 20 min at 4°C. The resulting supernatants were aliquoted, and protein concentrations were determined using a Bradford assay (Sigma Aldrich, UK). Bovine serum albumin (BSA) standards were prepared using a stock solution containing 4 g/ml BSA. BSA standards were diluted with distilled water to concentrations ranging from 100 to 1,400 μg/ml. A volume of 10 μl of each sample and BSA standards were added in duplicates to a 96-well plate. Bradford reagent (200 μl) was added to each well, and the 96-well plate was incubated at room temperature for 5 min. The maximum absorbance frequency for each sample was measured at 595 nm using a multimode plate reader (DTX 880; Beckman Coulter, USA), and the protein content of samples was determined by comparing to a standard curve generated using a serial dilution of BSA. Tissue supernatants were diluted to 1:50 with distilled water before protein assay. Plasma samples were thawed on ice, vortexed, and centrifuged at 13,000 × g for 10 min at 4°C. Hippocampi lysates were used for proteome analysis, and cortical lysates and plasma were used for MSD V-PLEX assay.

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