Primary cell culture

The E18 Sprague–Dawley rats were killed, and the spinal cords were dissociated. Then, spinal cord tissues were digested in papain (Invitrogen, Carlsbad, CA, USA) after being minced. Cells were seeded at 1 × 10⁴ cells/well in 96‐well plates for cell viability assays and at 3 × 10⁴ cells/well in 100‐mm culture dishes for western blot analysis. The cells were then coated with poly‐d‐lysine (Sigma‐Aldrich, St Louis, MO, USA) in a mixture of minimum essential medium, 10% fetal bovine serum, and 0.6% glucose supplemented by antibiotics (penicillin/streptomycin). The medium was discarded a day after neuroplating, and the cells were cultured in neurobasal media including B27, GlutaMAX, and penicillin/streptomycin (Invitrogen, Carlsbad) in a humidified 5 % CO₂ incubator at 37°C. The cells were used in the subsequent experiment after being cultured for 8 days to 10 days.