N2513

Glass coverslips were coated with 50 μl of poly-d-lysine in hydrobromide solution (P6407; Sigma-Aldrich) and incubated at 37°C for 30 min. After incubation, coverslips were washed twice with sterile water and again incubated with 100 μl of collagen solution (in 20 mM acetic acid) at 37°C for 45 min. Next coverslips were washed twice with phosphate-buffered saline (PBS; 10010-023; Life Technologies). PC12 cells were cultured on coated coverslips using DMEM containing 4.5 g/l d-glucose (31053-028; Life Technologies), 10% fetal bovine serum (FBS: L11-044; Biochrom AG), 5% horse serum (B15-023; PAA, GE Lifesciences, Fairfield, CT), 1% Glutamax (35050-061; Life Technologies) and 1% penicillin/streptomycin (10.00 U/ml; 12212; Biochrom AG) and incubated at 37°C and 5% CO₂. Transfection was accomplished 1 d after plating with Lipofectamine 2000 (11668-027; Life Technologies) following manufacturer’s protocol. At 24 h after transfection, culture medium was exchanged with differentiation medium containing DMEM with 4.5 g/l d-glucose (31053-028Life Technologies), 0.67% FBS (Biochrom AG, L11-044), 0.33% horse serum (B15-023; PAA), 1% GlutaMax, 1% penicillin/streptomycin (10,00 U/ml; 12212; Biochrom AG), and 1 μl of rat nerve growth factor-β (N2513; Sigma-Aldrich). The differentiating PC12 cells were imaged 5 d after plating at 10-s intervals.

In rats in the Lido group, 10% lidocaine hydrochloride (20 µl; 0219011125; MP Biomedicals, LLC, Santa Ana, CA, USA) was intrathecally administered, one day following the intrathecal catheter insertion. Rats in the NGF group were intrathecally administered with NGF (20 µg/20 µl; N2513 Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) 1 h prior to intrathecal administration of 10% lidocaine hydrochloride (20 µl). Rats in the Sham group were sham operated and intrathecally administered with normal saline (20 µl) without intrathecal injection of lidocaine or NGF.