Menthol

After allowing the neurites to freely extend for 48 hours, the DRG neurons were pretreated with capsazepine (a TRPV1 antagonist, 10 μM) (Sigma-Aldrich) for 30 minutes. Thereafter, the neurons were treated and cultured for 24 hours as follows: (1) formaldehyde group (n = 5), DRG neurons were exposed to formaldehyde (Sigma-Aldrich) 10 μM; (2) menthol group (n = 5), DRG neurons were exposed to menthol (Sigma-Aldrich) 300 μM; (3) PD98059 + formaldehyde group (n = 5), PD98059 (an ERK1/2 inhibitor, 10 μM) (Cell Signaling Technology, Danvers, MA, USA) was added 30 minutes before formaldehyde (10 μM) exposure; (4) PD98059 + menthol group (n = 5), PD98059 (10 μM) was added 30 minutes prior to menthol (300 μM) exposure; (5) control group (n = 5), DRG neurons treated only with the TRPV1 receptor antagonist capsazepine (10 μM). Because TRPA1 and TRPV1 channels interact in neurons (Ruparel et al., 2011), capsazepine, a TRPV1 antagonist, was used to block the effects of TRPV1.