Brain derived neurotrophic factor (bdnf)

Animals were sacrificed after 2 weeks of treatment. Rats were anesthetized with 10% chloral hydrate and decapitated. The rat brains were fixed in 4% paraformaldehyde in PBS for 24 h at 4^°C, and then dehydrated in a graded series of alcohol, placed in xylene until transparent and embedded in paraffin via treatment at 60^°C oven. Brain tissues were cut to 4 mm-thick sections using a microtome. The sections were blocked in 3% H₂O₂ for 10 min at room temperature, and 3% normal goat serum (OriGene Technologies, Inc.), and then incubated with rabbit polyclonal antibodies against phospho cAMP response element-binding protein (pCREB; 1:800; cat. no. 9198; Cell Signaling Technology, Inc.), CREB (1:3,000; cat. no. 9197; Cell Signaling Technology, Inc.), BDNF (1:200; cat. no. ab6201; Abcam) at 4˚C overnight. HRP-labeled goat anti-rabbit secondary antibody (1:1,000; cat. no. TA140003; OriGene Technologies, Inc.) for 30 min at 37˚C and diaminobenzidine from the Streptavidin-Peroxidase kit (OriGene Technologies, Inc.) were used to visualize the signals. Hematoxylin was used for counterstaining for 1-2 min at room temperature. The controls included incubation without any primary or secondary antibodies at 37^°C for 30 min. Finally, the sections were observed under a NIKON fluorescence microscope (magnification, x40, x200 and x400; Nikon Corporation).

Total protein was extracted using RIPA lysate. Protein concentration was measured with BCA protein assay kit (Pierce, Bonn, Germany) according to the manufacturer's instructions. Total cellular proteins were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). After blocking in 5% skim milk for 1 h, the membranes were incubated with primary antibodies (β-actin, A1978, 1:2,000; Sigma-Aldrich, St. Louis, MO, USA) (ERK, cat. no. 9102, 1:2,000; p-ERK, cat. no. 9101, 1:2,000; CREB, cat. no. 9197, 1:150; P-CREB, cat. no. 9191, 1:150; all from Cell Signaling Technology, Inc., Danvers, MA, USA) (BDNF, cat. no. ab108383, 1:1,200; Abcam, Cambridge, MA, USA) overnight at 4°C, followed by the incubation with goat anti-rabbit secondary antibodies (ERK, 1:2,000; CREB 1:150; BDNF 1:1,200) for 2 h at room temperature. Protein bands were visualized on X-ray film using enhanced chemiluminescence ECL substrate (Pierce).
The levels of BDNF were detected with enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instruction.