For the in vivo cytokine experiment, the hippocampus, prefrontal cortex, and amygdala of mice were obtained at day 5 postsurgery, or at 3 h after scopolamine or vehicle treatment after the neurobehavioral tests. Samples were stored at − 80 °C until use. To measure the levels of TNF-α, IL-1β, and IL-18 in the three different regions, brains were lysed using tissue protein extraction reagent (Tissue Protein Extraction Reagent, Thermo Scientific, Waltham, MA, USA) containing protease and phosphatase inhibitor cocktail (100 × Halt protease and phosphatase inhibitor cocktail, #1861281 Thermo Scientific). The tissues were then homogenized and centrifuged at 13,000 rpm for 10 min to obtain sample supernatants. Supernatant protein concentrations were measured with a BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer’s specifications. Levels of TNF-α, IL-1β, and IL-18 in the lysates were assayed using high-sensitivity ELISA kits (R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer’s specifications. Briefly, samples were added to the assay plates at a volume of 50 μL/well and incubated for 2 h at room temperature. After washing plates with the wash buffer from the kit, TNF-α, IL-1β, and IL-18 conjugates were added to each well and incubated for 2 h. The absorbance of each well was measured at 450 nm using a microplate reader. To measure the levels of NLR family pyrin domain-containing protein 3 (NLRP3), apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), and caspase-1 in lysates, ELISA kits from MyBioSource (San Diego, CA, USA) were used for this assay, and all procedures followed manufacture’s instruction.

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