After evaluation of cognitive performance, the mice in each group were divided into three sets and were euthanized by cervical dislocation under light anesthesia. Subsequently, the brains were rapidly dissected and washed with ice-cold saline. In the first set (n = 3 per group), brains were fixed in 10% (v/v) formalin for 24 h to perform histopathological staining with hematoxylin and eosin (HE), Congo red, and Nissl stain. In the other sets, the hippocampal tissues were excised from each brain on an ice-cold glass plate. In the second set (n = 6 per group), the hippocampi were homogenized in ice-cold physiological saline (10% w/v), then the hippocampal homogenates were used for determination of the levels of acetylcholine (Ach), amyloid β1-42, oxidative stress parameters (malondialdehyde, MDA; nuclear factor erythroid 2-related factor 2, Nrf-2; heme oxygenase-1, HO-1; and glutathione, GSH), inflammatory markers (nuclear factor kappa beta, NF-κB; NLRP3; interleukin 1β, IL-1β; and tumor necrosis factor-alpha, TNF-α), caspase-3, and insulin-degrading enzyme (IDE). In the third set (n = 3 per group), hippocampi were used for the assessment of protein expression of phosphorylated tau, glycogen synthase kinase 3β (GSK-3β), and mammalian target of rapamycin (mTOR), as well as the determination of extracellular signal-regulated protein kinase (ERK1/2) and p38 mitogen-activated protein kinase (p38-MAPK) gene expression.

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