Mechanical allodynia was assayed using nylon von Frey filaments according to the ‘up-down’ algorithm described by Chaplan et al. [19] as previously described [14,20]. In these experiments, mice were placed on wire mesh platforms in clear cylindrical plastic cylinders. After 15 min of acclimation, fibers of sequentially increasing stiffness were applied to the plantar surface of one hind paw, pressed upward to cause a slight bend in the fiber and left in place for 5 seconds. Withdrawal of the hind paw from the fiber was scored as a response. When no response was obtained the next stiffest fiber in the series was applied to the same paw; if a response was obtained a less stiff fiber was applied. Testing proceeded in this manner until four fibers had been applied after the first one causing a withdrawal response allowing the estimation of the mechanical withdrawal threshold [21]. This data-fitting algorithm allowed the use of parametric statistics for analysis. This assay is sufficiently sensitive to detect mechanical thresholds as low as 0.02 g [14].
