von Frey test of paw withdrawal threshold

In all cases, behavioural tests were performed immediately prior to anaesthesia for electrophysiological recordings to quantify the development of tactile hypersensitivity characteristic of CIBP. Rats were placed in a transparent Plexiglas box containing 0.5 cm diameter holes spaced 1.5 cm apart on the floor to allow full access to the paws.^22–24 Animals were allowed to habituate to the box until cage exploration and major grooming activities had ceased.

von Frey filaments (Stoelting Co., Wood Dale, IL) were applied to the plantar surface of the ipsilateral hind paw to determine mechanical withdrawal thresholds using the up-down method of Dixon.²⁵ A von Frey filament was applied five times for 3–4 s each at 3-second intervals to a different spot on the plantar surface of the ipsilateral hind paw. Filaments were applied in ascending order of force until a clear withdrawal response was observed. When this occurred, the next lightest filament was reapplied, and the process continued until a 50% withdrawal response threshold was derived.²⁶ Brisk foot withdrawal in response to the mechanical stimulus was interpreted as indicating mechanical hypersensitivity.