Von Frey testing

Animals were tested in acrylic test chambers (12 cm W × 16 cm L × 15 cm H) placed on a 6 × 6 mm grid wire mesh flooring. Mechanical allodynia was assessed by measuring the 50% mechanical paw withdrawal threshold (PWT), using nylon von Frey filaments (Semmes Weinstein Monofilament Kit, Stoelting, Woodale, IL) applied to the rat hind paw for a maximum of 10s. Beginning with a stimulus intensity of 2 g, the force of the filament used was either increased following a lack of response or decreased after a positive paw withdrawal response. The sequence ended once five responses were recorded following the first positive response, or after three changes in the direction of the pattern of responses. Minimum and maximum intensities were 0.25 g and 15 g, respectively. Based on the response pattern, the PWT was calculated using the following formula: 50% g threshold = (10^{[xf + kδ]})/10,000, where x_f = strength (in log units) of von Frey filament applied last; k = value for the pattern of positive/negative responses; δ = mean difference (in log units) between stimuli.²⁷