Sample collection and biochemical detection

Blood of all experimental mice were collected from a caudal vein subsequent to the behavioral tests. All mice were euthanized by an intraperitoneal injection of 200 mg/kg pentobarbital. Following this, the brains and hypothalamus were immediately collected. The cerebral cortex, separated from the brains (n=20/group) and the hypothalamus (n=20/group) were homogenized in ice-cold phosphate buffered saline (PBS).

Subsequent to detecting the protein concentration using a BCA assay kit (Merck KGaA), the levels of acetyl-choline (Ach; cat. no. CK-E20536), acetylcholinesterase (AchE; cat. no. CK-E93899), choline acetyltransferase (ChAT; cat. no. CK-E94456), super oxidase dismutase (SOD; cat. no. CK-E20348), glutathione peroxidase (GSH-Px; cat. no. CK-E92669) and ROS (cat. no. CK-E91516) in serum, the cerebral cortex and the hypothalamus, and Aβ1-42 (cat. no. CK-E94157) in the serum and cerebral cortex were detected using enzyme-linked immunosorbent assay kits according to the manufacturers protocols (Shanghai Yuanye Biotechnology Co., Ltd.).