Measurement of neurotrophic factors

The levels of neurotrophic factors were determined by commercial ELISA kits (Abfrontier, Korea) according to the manufacturer’s instructions. The samples were added onto a 96-well plate with coating of anti-rat NGF/BDNF/GDNF antibody and incubated at 37 °C for 90 min. Then, the samples were replaced by biotinylated anti-rat NGF/BDNF/GDNF antibody and incubated at 37 °C for 60 min. After washing four times with PBS, avidin-biotin-peroxidase complex solution was added and incubated at 37 °C for 90 min, which was replaced by tetra-methylbenzidine solution with another incubation of 30 min at 37 °C. At last, 1 M sulfuric acid was added to stop the reaction and absorbance of 450 nm was measured immediately. The absorbance of non-specific binding was taken consideration for sample analysis and each sample in duplicate was employed to minimize inter-assay variation⁴⁹.