Von frey filament

After IDD, locomotor performance was assessed using the von Frey, horizontal ladder, and tail suspension tests. The von Frey test was used to evaluate behavior in response to pain once a week postoperatively. The rats were maintained to adapt to the testing environment for 15 min before measurement. The latency of the paw withdrawal response was measured by applying mechanical stimulation to the center of both hind paws using the von Frey filament (Ugo Basile, Varese, Italy). A positive avoidance response was indicated by lifting, whipping, licking, or rubbing the paw during stimulation, and the average value of three or more measurements was used. In the horizontal ladder test, the rats were made to walk on the metal runway (2.5 cm between grids) from left to right three times, and their movements were captured with a digital camcorder. The ladder score was calculated as follows:
The tail suspension test was performed as previously described [17 (link)]. The suspension box was made of acrylic (35 depth × 35 width × 80 cm height) with a ceiling steel hook to suspend the rats in the center. The rats were suspended 50 cm above the floor using adhesive tape attached approximately 3 cm from the tail tip. Immobility (extending without moving) and mobility (attempting to reach a floor or wall) were recorded for 6 min and analyzed by an observer blinded to the experimental conditions.

The pain-related behavior of OA rats after intra-articular injection of various therapeutic agents was evaluated by weight-bearing index (WBI) by a 3D gait analysis system (Kinama Tracer, Japan) and paw-withdrawal threshold (PWT) by the von Frey filament (Ugo Basile, Varese, Italy). Briefly, for WBI detection, rats walked along the runway equipped with mechanical sensors, which can record the ground reaction force for each foot, reacting to the weight supported by the corresponding limb. The values were represented by the following formula: WBI = ipsilateral weight/(ipsilateral weight + contralateral weight) × 100%. PWT was conducted to evaluate the efficacy on pain threshold as described previously (Micheli et al., 2019 (link)). In addition, PWT, reflecting the mechanical allodynia (hypersensitivity), was investigated according to the force exerted by the von Frey filament ranging from 0 to 40 g with a .2-g accuracy. The paw sensitivity threshold was recorded as the minimum force for leading to a strong and immediate withdrawal reflex of the paw.

Changes in the mechanical paw withdrawal threshold were measured at 1, 4, 7, and 14 days after nerve or sham surgery. Rats were habituated for 10 min to the testing cages, which consisted of metal mesh floors under plastic domes. Mechanical allodynia was measured by assessing thresholds for hind paw withdrawal upon stimulation with an electrically controlled von Frey filament (Ugo Basile, Varese, Italy). Licking or rapid withdrawal of the hind paw was considered a positive response. Mechanical forces were recorded for each withdrawal. The responses were measured seven times, and the means were calculated after the maximum and minimum values were excluded. Nociceptive tests were performed after MCS in awake rats for 10 days. Electrodes were connected to a stimulator (A385, WPI, Sarasota, FL, USA) for repetitive MCS. For MCS, the M1 of awake animals were stimulated for 30 min (continuously at 50 μA, 50 Hz, and 300 μs pulses). Sham MCS group underwent the same procedure without any electrical stimulation. After the last behavioral test, the animals were anesthetized with urethane (1.25 mg/kg, i.p.), and tissue was collected for western blot and immunohistochemistry assays.

Sciatic cuff group: In a subset of animals, von Frey testing was performed 7 d after surgery as previously described (Torres-Rodriguez et al., 2023 (link)). Male mice were habituated in the von Frey apparatus in a red-lit room for 2 h prior to testing. The smallest filament that elicited a paw withdrawal response in at least three of five trials was taken as the mechanical threshold for each hindpaw. Tactile sensitivity was determined for both hindpaws by an experimenter blind to pain treatment.
CCI-ION group: We confirmed the presence of CCI-induced, mechanical allodynia in subset of mice (5 sham and 5 CCI-ION, male ∼10 weeks old) using a 1.4 g von Frey filament (Ugo Basile). Mice were gently held in the hand of the experimenter, and the filament was applied to the dorsal edge of the vibrissae pad ipsilateral to the ligated nerve. The number of nocifensive responses to five applications of the filament was recorded. Nocifensive responses were defined as the animal actively withdrawing its head or aggressively biting or swiping at the filament. We tested for differences in the fraction of nocifensive responses between groups at each time point (pre-surgery/baseline and again 1 and/or 2 weeks after surgery) using a mixed-effects model with Sidak's post hoc multiple-comparisons test.

Mechanical allodynia was evaluated through mechanical touch sensitivity using a blunt probe (Von Frey filament, 0.5 mm diameter, ranging up to 10 g in 10 s) on central plantar surface of hind-paw by dynamic plantar aesthesiometer (UgoBasile, Gemonio, Italy). Responses to mechanical stimuli (Paw Withdrawal Thresholds, PWT) were expresses in grams (g) [29 (link)]. Three different measurements for each paw were recorded and the mean calculated.

We used three tests to assess functional recovery after inducing stenosis. The Basso, Beattie, and Bresnahan (BBB) scale was performed as previously described [26 (link)]. Two independent observers analyzed the hindlimb motion in an open field for 4 min. The average value was used for analysis. The ladder walking test was used to test the ability of rats to maintain balance. Rats walked on a metal runway (2.5 cm between grids) from left to right three times, and their movements were recorded with a digital camera. The score was calculated as follows: ladder score (%) = erroneous steps of the hind limb/total steps of the hind limb × 100 [26 (link)]. Locomotor functions were examined in each group every 7 days until sacrifice. The Von Frey test was used to measure the response of the rats to pain. We measured the latencies of paw withdrawals in response to mechanical stimulation applied to the center of both hind paws using a Von Frey filament (Ugo Basile, Varese, Italy). The average value from three or more measurements was used. All locomotor tests were recorded using a digital camera and were assessed by two observers who were blinded to the treatment conditions.