BDNF and NGF Levels in the Hippocampus

After the behavioral assessments, mice were sacrificed, and their brains were rapidly removed and chilled in an ice-cold saline solution. The hippocampi were dissected on a cold plate, frozen, and stored at −80 °C until assay. On the day of experiments, tissues were thawed on ice and homogenized (1:9 w/v) in phosphate-buffered saline (4 °C) and protease inhibitor cocktail was added. The 10% homogenates were prepared and homogenized for 30 s with TissueRuptor homogenizer. The homogenized tissues were centrifuged (2500×g at 4 °C for 20 min), and the supernatants were collected for further assays.

Protein concentrations of BDNF and NGF in homogenates from hippocampi were determined using the enzyme-linked immunosorbent assay (ELISA) kits (BDNF: DZE201020014, SunRed Biotechnology Company; NGF: MBS825100, MyBioSource) according to the manufacturer’s instructions. Serial dilutions of the standards were performed to make the standard curve within the range of this assay (BDNF 0.1–10 ng/mL; NGF 31.2–2000 pg/mL). The samples were analyzed in duplicates, and the mean concentrations were calculated. BDNF and NGF antibodies are high selectivity and thus did not cross-react with any other cytokines. The reaction was terminated after the stop solution was added. The intensity of the color was read at 450 nm. Absorbance was measured in a multifunction plate reader (POLARstar Omega, BMG Labtech, Germany). The concentration of the samples was interpolated from the standard curve using GraphPad Prism Version 6.00 software.