Von frey filament

On day 8 (study 4), the mechanical pain sensitivity of the abdominal and plantar region was evaluated with von Frey filaments (Bioseb, Vitrolles, France) by a modification of the method of Laird et al. (2001 (link)). The mice were habituated in small compartments on a perforated grid (Dynamic Plantar Aesthesiometer, Ugo Basile, Comerio, Italy) for 1 h before the test. Subsequently von Frey filaments were applied to the abdomen (between diaphragm and genitals) and the plantar side of the right and the left hind paw. The test was performed by a trained blinded observer. The individual filaments were tested in an ascending order covering 0.008, 0.02, 0.04, 0.07, 0.16, 0.4, and 0.6 g forces. Each force was applied 10 times to the abdominal surface, and 5 times to the right and the left hind paw. The maximal duration of each force application was 2 s, and the inter-stimulus interval was 2–3 min. Following each challenge, the withdrawal response was quantified either as 1 (withdrawal of abdominal wall or paw, licking or retraction of animal) or 0 (no response). All counts in response to an individual filament were averaged. Withdrawal responses to low forces reflect high mechanical pain sensitivity.

To evaluate the mechanical allodynia in the ipsilateral paw, we used a set of eight calibrated von Frey filaments (BioSeb, Vitrolles, France) as previously described (Blom et al., 2020 (link)). Animals were individually placed in small Plexiglas cubicles on a metal grid surface and allowed to acclimatize. The filaments were applied perpendicular to the plantar surface of the ipsilateral paw until the fibers bowed and were held for 2 s. The up-down staircase method was used to determine the threshold force necessary for eliciting withdrawal (Dixon, 1991 (link)). The grams of force that evoked a positive response more than 50% of the time was determined as described by Chaplan et al. (1994) (link).

Tactile allodynia was assessed using von Frey filaments (Bioseb, France). Mice were placed in a plastic cage with a wire mesh floor which allowed access to the paws. The plastic cage was covered with an opacity cup to avoid visual stimulation. The area tested was the mid-plantar left hind paw. Mechanical threshold was tested using a modification of the simplified up-down method (Bonin et al., 2014 (link)). A test round started with filament #6 (0.40 g) and progressed to higher or lower filament value depending on the animal’s response. Each animal went through three test rounds for each paw at each experimental condition. Mechanical threshold is expressed as percentage according to baseline (%).

Mechanical allodynia was determined using the von Frey filament test. Each rat was placed in a plastic compartment and behavioral settlement was permitted for at least 25 min; until cage adaptation and major grooming activities stopped. The plastic cages possess wire mesh bottom allowing for sufficient access to the rats’ paws. To evaluate the withdrawal threshold, the “up-down” approach was used by applying the von Frey filaments (2–15 g, with logarithmically incremental stiffness; Bioseb, Vitrolles, France) to the mid-plantar surface of each rat’s left hindpaw. The von Frey hair was held for about 6–8 s perpendicularly to the plantar surface of the left hind paw [24 (link),25 (link)]. Data were presented in grams as paw withdrawal thresholds (PWT).

Mice were placed in plastic chambers on a wire mesh grid and stimulated with von Frey filaments (Bioseb) using the up-down method19 (link) starting with 1 g and ending with 2.0 g filament as cutoff value. Baseline measures of untreated or DT-treated WT or GINIP-DTR mice were performed on separate lots.

The von Frey filament test was used to measure sensitivity to a punctuate pressure stimulus. Rats were placed in a plastic cage with a wire mesh bottom which allowed full access to the paws. Behavioral accommodation was allowed for at least 25min until cage exploration, and major grooming activities ceased. Subsequently, von Frey filaments (2-15g, with logarithmically incremental stiffness; Bioseb, Vitrolles, France) were applied to the mid-plantar aspect of the left hind paw using the “up-down” method to determine the withdrawal threshold; the von Frey hair was presented perpendicular to the plantar surface and held for approximately 6-8sec [23 (link), 24 ]. Data are expressed as means ± SEM of paw withdrawal threshold (PWT) in grams.

Hind paw mechanical threshold was assessed using von Frey filaments (Bioseb, France) as described^{20 (link)}. For facial testing, mice were subjected to a von Frey stimulus applied to the forehead surface, repeated three times (at minimum 30 s interval). The head withdrawal tactile sensory threshold was the lowest force to elicit withdrawal in 2 of 3 trials. Data points were normalized to the control sensory threshold values determined just before minipump implantation (i.e. at day 0) or before SNP injection (i.e. at day 21, H0).