Brain derived neurotrophic factor (bdnf)

The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the serum and the levels of brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) in the spinal cord tissues were determined using commercially available ELISA kits according to the manufacturer’s protocols (IL-1β and TNF-α, Invitrogen, Carlsbad, California, USA; BDNF, MyBioSource, SanDiego, California, USA; GDNF, Abnova, Taipei city, Taiwan). The OD value was determined by an ELISA reader at a wavelength of 450 nm and calculated in the linear part of the curve.

The hippocampi and prefrontal cortex were dissected and flash-frozen in liquid nitrogen and stored at − 80 °C. Before Western blot analysis, all samples’ homogenization was done in the suitable lysis buffer, [19 (link)] and total protein extract was prepared by centrifuging in 15,000 rpm for 5 min. Bradford’s method [20 (link)] was applied for measuring the protein concentration in the supernatants. After loading standardized lysates equivalent to 60 µg of protein on SDS-12.5% poly acrylamide gel electrophoresis and transferring to PVDF membrane (Chemicon Millipore Co. Temecula, USA), blots were blocked in 2% Electrochemiluminescence (ECL) advanced kit blocking reagent (Amersham Bioscience Co. Piscataway, USA) and probed with Klotho-α, FGF23, BDNF and c-fos (1/1000, MyBioSource Inc. CA, USA) antibodies overnight. Then, the membranes' incubation was done with rabbit IgG-horseradish peroxidase-conjugated secondary antibody (1/3000, Cell Signaling Technology Co. New York, USA) that was directly noticeable by chemiluminescence kit reagent (Amersham Bioscience Co. Piscataway, USA). For detecting β-actin as an internal control, stripping buffer (pH 6.7) was used to strip the blots and then probing was done with anti β-actin antibody (1/1000, Cell Signaling Technology Co. New York, USA) [19 (link)].

Following transcardiac perfusion with ice-cold PBS, spinal cords of mice were harvested and homogenized using a lysis buffer containing protease inhibitor cocktail and phosphatase inhibitor. Levels of proinflammatory cytokines including interleukin-1β (IL-1β; RayBiotech, USA), interleukin-6 (IL-6; RayBiotech, USA) and tumor necrosis factor-α (TNF-α; RayBiotech, USA), and growth factors including brain-derived neurotrophic factor (BDNF; MyBioSource, USA), glial cell line-derived neurotrophic factor (GDNF; MyBioSource, USA) and vascular endothelial growth factor (VEGF; MyBioSource, USA) in the homogenates were determined using mouse ELISA kits according to the manufacturer’s instructions.