Nc12775 99

For this experiment, decursin was first dissolved in DMSO, and this solution was diluted in Dulbecco’s phosphate-buffered saline by 10-fold. Each mouse was intrathecally injected with 5 μL of diluted decursin three times at 2-day intervals, for 6 days, to examine whether decursin could improve mechanical allodynia in the mouse model of paclitaxel-induced neuropathic pain. Intrathecal injections into the L5-L6 intervertebral space of unanesthetized mice were made using a 10 µL Hamilton syringe connected to a 30-gauge needle [9 (link)]. The effect of this compound was measured using the 50% paw withdrawal threshold, based on the von Frey method [10 (link)]. The mice were habituated to an elevated platform with a mesh floor for 30 min. The plantar side of the hind paw was stimulated with calibrated von Frey filaments (NC12775-99, North Coast Medical, Morgan Hill, CA, USA). The 50% paw withdrawal thresholds were calculated using the up-down method [10 (link)]. The mechanical allodynia test for monitoring the analgesic activity of this compound was conducted every 2 days at an appropriate time after the injection of decursin, for a total of three times over 6 days.

At baseline (before surgery) and either 21 or 28 dpo (see below), a subset of mice was transferred to acrylic chambers in a behavioral testing room for the von Frey test of mechanical sensitivity. This assay was not performed 1 day after surgery since SCI mice had BMS scores of 0 or 1, indicating very little hindpaw placement. All mice were acclimated to the behavioral suite and testing apparatuses for at least 3 days prior to surgery. On testing days, following a 30-min acclimation period, individual animals were assayed for mechanical sensitivity (paw withdrawal responses) according to the established up-down method (Chaplan et al., 1994 (link)). Calibrated von Frey hairs (NC12775-99, North Coast Medical, Inc., Morgan Hill, CA, USA) starting with filament evaluator size 3.22 (target force 0.16 g) were administered from below a metal mesh platform to test each animal’s sensitivity to mechanical stimulation of the hindpaw. Right and left paw withdrawal thresholds were averaged to determine overall mechanical sensitivity. A reduction in von Frey withdrawal threshold values from their baseline levels corresponded to increased mechanical sensitivity.

At baseline (before surgery) and weekly (7, 14, 21 and 28 dpo), mice underwent the von Frey test of mechanical sensitivity. All mice were acclimated to the behavioral suite and testing apparatuses for at least 3 days prior to surgery. On the day of testing, they were acclimatized to the testing apparatus for 20 min. Mechanical assessment was undertaken according to the established up-down method (Chaplan et al., 1994 (link)). Briefly, calibrated von Frey hairs (NC12775-99, North Coast Medical, Inc., Morgan Hill, CA, USA) starting with filament evaluator size 3.22 (target force 0.16 g) were administered from below a metal mesh platform to test each animal’s sensitivity to mechanical stimulation of the hind-paw. Right and left paw withdrawal thresholds were averaged to determine overall mechanical sensitivity. A reduction in von Frey withdrawal threshold values from their baseline levels indicated enhanced mechanical hypersensitivity.