Semmes weinstein monofilaments

Perivaginal mechanical sensitivity was determined using Semmes-Weinstein monofilaments (Stoelting, Wood Dale, IL) according to Brennan et. al ^{10 (link)}. Rats were acclimated for 30 min in a Plexiglas box on a steel mesh floor and analyses performed using calibrated touch-test sensory evaluators (monofilaments). Pressure was applied for 3–5 sec to the injected vestibular region in an upward motion starting with the monofilament of lowest force. After 3–5 min test free period, the test was repeated with increasing evaluator force until the rat responded aversively (movement or vocalization). This was repeated three times and the lowest force from 3 tests producing withdrawal response was averaged. This was defined as the mechanical withdrawal threshold (MWT). Behavioral tests were performed between 8:00 and 10:00AM at 24h prior to and 1, 3 and 6d following vestibular injection.

Mice were acclimated to the testing room for two days prior to the assessment of perigenital mechanical threshold testing. On the day of testing, naïve and NMS mice were placed beneath individual clear plastic chambers (11×5×3.5cm) on a wire mesh screen elevated 55 cm above a table and allowed to acclimate for 1 hour prior to testing. The experimenter was blinded to the group status of the mice. The up-down method was performed to test mechanical sensitivity using a standard set of Semmes-Weinstein monofilaments (1.65, 2.36, 2.83, 3.22, 3.61, 4.08, 4.31, 4.74g; Stoelting, Wood Dale, IL) [18 ]. Beginning with the 3.22g monofilament, mice received a single application to the scrotum, avoiding the midline. A negative response was followed by the next larger filament and a positive response (considered a jerk or licking) was followed by the next smaller filament. The experimenter continued to move up or down the series, depending on the previously elicited response, for an additional four applications after the first positive response was observed for a minimum of five or a maximum of nine total monofilament applications. The value in log units of the final monofilament applied in the trial series was used to calculate a 50% g threshold for each mouse and group means were determined as previously described [9 (link)].

The presentation of neuropathic pain was determined by the development of hypersensitivity to mechanical and cold non-noxious stimuli. The mechanical hypersensitivity was assessed by the decrease in paw withdrawal mechanical thresholds using the von Frey test. Mice were placed in a clear plastic chamber with a wire mesh floor, which provided full access to the planter surface of the hind paws. Mice were allowed to habituate for at least 15 min before testing. The paws were touched with one of a series of 10 von Frey filaments (Semmes-Weinstein Monofilaments; Stoelting Co., Wood Dale, IL) with logarithmically incremental stiffness (0.005–3.630 g) starting with the filament of 0.407 g. The 50% mechanical withdrawal thresholds were determined using the up–down method.^{20 (link)} Cold hypersensitivity to non-noxious cold stimulus was assessed by counting the number of forepaw-shaking behavior of mice placed on a cold plate (LHP-1700CP; TECA, Chicago, IL) set at 15 °C (15 °C cold plate test) during a 2.5 min test period. The cold plate test was performed following the von Frey test. Behavioral testing was performed in a blinded manner with respect to all drug administration.

Testing was conducted blind with respect to group assignment. Rats received at least three 60-min habituations to the test environment before behavioral testing. The von Frey test (Chaplan et al., 1994 (link)) was performed at the distal region of the heel in the hindpaws, within the region of sciatic innervation as previously described (Chacur et al., 2001 (link)). Assessments were made before injection (baseline), and 2, 4, 6, and 24 h after the last injection. A logarithmic series of 10 calibrated Semmes–Weinstein monofilaments (von Frey hairs; Stoelting) was applied to the hindpaw to define the threshold stimulus intensity required to elicit a paw withdrawal response. Log stiffness of the hairs ranged from manufacturer-designated 3.61 (0.40 g) to 5.18 (15.14 g) filaments. The behavioral responses were used to calculate absolute threshold (50% probability of response) by fitting a Gaussian integral psychometric function using a maximum-likelihood fitting method (Harvey, 1986 ) as described (Milligan et al., 2000 (link)).