von Frey Test

Mechanical hypersensitivity was assessed using paw withdrawal thresholds in response to the von Frey filaments (OptiHair, Marstock Nervtest, Schriesheim, Germany). After 30 min of habituation to the plastic enclosure and the mesh platform, a calibrated monofilament was applied perpendicularly to the hind paw plantar surface at the midsole (Figure 1C), pressed upward, and left for approximately 3 s with Dixon’s up-down method.¹⁰ Brisk paw withdrawals, licking, or shaking of the paw were scored as positive responses. The mechanical force required to elicit paw withdrawal in 50% (50% withdrawal threshold) of animals was determined based on Dixon’s statistical formula, as previously described.^11,12