Anti gdnf

SD Rats were anesthetized with 360 mg/kg chloral hydrate (Sigma, St. Louis, MO, USA) through intraperitoneal injection, and positioned in a stereotaxic apparatus (David Kopf Instrument, Tujunga, CA, USA). Two microliters of the viral vectors were injected into the right hippocampus of the SD rats (AP: −3.8 mm; ML: −2.4 mm; DV: 3.0 mm, relative to the bregma), according to the brain atlas [16 ]. Infusions of viral vectors were performed at a rate of 0.1 µl/min over 20 min using 30-gauge injection needles connected to a 10 µl Hamilton syringe. The needle was maintained in position for an additional 5 min to diffuse the materials before being slowly retracted.
To examine the contribution of neurotrophic signal pathways for neuroprotection, we used neutralizing antibodies (NA) [10 (link), 14 (link)]. Three weeks after virus injection, 200 ng of NA alone or in combination with 20 U of thrombin (Sigma) dissolved in 4 µl phosphate-buffered saline (PBS) were injected into the right hippocampal CA1 region as described above [13 (link), 14 (link)]. The following NA were used: anti-BDNF (Santa Cruz Biotechnology, Dallas, TX, USA), anti-GDNF (R&D Systems, Minneapolis, MN, USA), and anti-GFRα-1 (Sigma).

SH-SY5Y human neuroblastoma cells were plated into a poly-L-lysine-coated 6-well plate containing growth medium (MEM/F12 containing 10% fetal bovine serum and 1% P/S) at a density of 1×10⁵ cells/well and cultured for 24 h. Cells were washed twice and then incubated with conditioned medium (CM) of Mock-hNSPCs (Mock-CM) or GDNF-hNSPCs (GDNF-CM) for 24 h. To quantify neurite length, cells were observed under an Olympus IX71 microscope and analyzed with NeuronJ Software. For immunodepletion, GDNF-CM was pre-incubated with 5 μg/mL anti-GDNF (R&D Systems) or isotype-matched IgG antibody (R&D Systems) for 30 min at 37°C. After 24 h, neurite lengths were measured as described previously [31 (link)]. Each data point represents the average of the data of three independent replicates.