Intrathecal injection of TNF-α

Intrathecal (i.t.) administration was performed in conscious mice according to the method of Hylden.^35,36 Before the experiment, we performed methylene blue dye tests to check the distribution consistency of the injected drug. The i.t. injection of 4 µl of 1% methylene blue was localized at L4–L5. The dye was distributed both rostrally and caudally within a short distance (about 1.0 to 1.5 cm); no dye was observed in the brain. We divided the CCI mice into four groups: CCI and GJG-treated CCI groups that were administered i.t. PBS, and CCI and GJG-treated CCI groups that were administered i.t. TNF-α (n = 4 per group). For these mice, 10 pg recombinant TNF-α (R&B, Calabasas, CA, USA) diluted with 4 µl PBS or PBS alone was i.t. injected at post-operative day 1. Behavioral tests were used to evaluate analgesic effects as described above. The areas under the time-course curves (AUC) in the cold-plate test were calculated using the trapezoidal rule.³⁷