Western blotting

Brain tissue samples were harvested and stored at −80 °C. Total protein samples for Western blot analysis were extracted from the CA1 and DG of the hippocampus or superficial prefrontal cortex of the beagle, and the detailed preparation protocol was as follows. Frozen brain tissues were lysed in a solution containing 40% SDS, 60% RIPA, and 1% protease inhibitor. The homogenate was then centrifuged at 16 700 × g at 4 °C for 30 min, and the supernatants (containing cytosolic and membrane fractions) were collected. The concentration of proteins was detected spectrophotometrically using a BCA kit (Universal Microplate Spectrometer; Bio-Tek Instruments, Winooski, VT, USA). Protein samples were fractionated by 10% SDS-PAGE gels and then transferred onto a nitrocellulose membrane. Anti-BDNF (1:1000, ab108319, Abcam, MA, USA) and anti-HIF-1α (1:1000, 12-2180, Assay Biotechnology, CA) were used as primary antibodies. β-Actin (1:1000, AT-09, ZSGB-BIO, China) was selected as an internal control. Blots were detected using an Odyssey Infrared Imaging System (Licor, Lincoln, NE, USA) and were quantified with Odyssey v1.2 software by measuring the protein intensity (area × optical density) in each group. The final results were expressed as fold changes compared with the control values.