Plantar test apparatus

Thermal hyperalgesia was tested according to the Hargreaves procedure [30 (link)], slightly modified by us for mice [31 (link)], with a plantar test apparatus (Ugo Basile, Gemonio, Italy). Briefly, mice were placed in Plexiglass boxes (w 11 x h 11 cm) and a constant intensity radiant heat source (beam diameter 0.5 cm and intensity 20 I.R.) was aimed at the mid-plantar area of the hind paw until the animal removed it. Paw withdrawal latency (PWL) was expressed in seconds (sec). The test was conducted on both the OA paw (ipsilateral) and the healthy paw (contralateral).

The Basso Mouse Locomotor Scale (BMS) is used to evaluate locomotor function after thoracic spinal cord contusion or transection injury (Basso et al., 2006 (link)). It is scored from 0 (hind limb paralysis) to 9 (normal locomotion). BMS scores were recorded at days 4, 14, and 28 post-SCI for each hind limb, and averaged to give one value per mouse per test. Two independent examiners who were blinded to the experimental results tested the mice at days 1, 14, 21, and 28 post-SCI for mechanical allodynia and thermal sensitivity. Allodynia sensitivity was tested using a Dynamic Plantar Aesthesiometer (Ugo Basile, Comerio, Italy; Martucci et al., 2008 (link)). In this test, the withdrawal threshold (expressed in grams) is determined five times and the mean is reported. Thermal sensitivity at the plantar hindpaws was examined using a Plantar Test Apparatus (Ugo Basile; Hargreaves et al., 1988 (link)). In this test, the time between application of the thermal stimulus until hindpaw withdrawal (latency) is recorded (in seconds), as well as any other reaction to the stimulus (e.g., gazing at the affected paw, sniffing, licking, or attacking the stimulus). The latency was calculated using data from six tests after rejecting the longest and shortest latencies (Hoschouer et al., 2010 (link)).

Thermal antinociceptive activity in rats was assessed by measuring hind paw withdrawal latency in response to radiant heat using a plantar test apparatus (Ugo Basile, Comerio, Italy) according to the method used by Hargreaves et al. (42 (link)). Briefly, each rat was placed into a compartment enclosure on a glass surface. A mobile heat source was then positioned under the plantar surface of the hind paw and activated with a light beam. A digital timer automatically recorded the response latency for paw withdrawal to the nearest 0.1 s. The mean withdrawal latency (seconds) for the left hind paw was determined from the average of three trials separated by a 10-min interval to prevent thermal sensitization.
To confirm that JDTic (10 mg/kg) was producing its effect on self-administration by blocking kappa receptors, we examined whether U-69,593-induced antinociception could be blocked using the rats that underwent self-administration testing. Paw withdrawal latency was measured in rats that were pretreated with JDTic or vehicle after completion of the self-administration experiments. One week after the JDTic administration, rats received an injection of U-69,593 (0.3 mg/kg, s.c.) or vehicle and were tested for paw withdrawal latency 15 min post-U-69,593 administration.

All behavioral tests were performed by a blinded observer. Animals were tested on day 0 (before SNI), day 7 (7 days after SNI) and day 14 (14 days after SNI) to evaluate mechanical allodynia and thermal hyperalgesia. After that, all animals were sacrificed. Mechanical allodynia was assessed using the von Frey filament test (Ugo Basile) [34 (link),35 (link)]. Filaments were applied under the plantar surface of the right paw in the sural region, in either ascending or descending strength. The threshold was set as the lowest force that evoked a consistent, brisk withdrawal response. The time of response to a progressive force applied to the hind paw limb was assessed six times, with an interval of 5 min between stimulations. Nociceptive thresholds to radiant heat (infrared) were quantified using the plantar test apparatus (Ugo Basile) [36 (link)]. The heat source was positioned under the plantar surface of the right hind paw and activated for 20 s (cut-off) to prevent tissue damage, at a setting intensity of 7.0 to record the response latency for paw withdrawal. The injured hind limb was tested twice at each time point, with an interval of 5 min between stimulations.

Thermal paw withdrawal latency time was assessed 24 h post-surgery using the Ugo Basile Plantar Test apparatus (37370, Ugo Basile, Varese, Italy) following the Hargreaves radiant heat method [59 (link)]. Mice were placed in individual mouse runs at least 30 min prior to testing to acclimatize to the room conditions. An infrared radiant heat source (intensity set as 60) under a heat-acrylic plastic floor was focused on the surgical paw. The time to withdrawal was automatically measured, and the cut-off time was set as 20 s to avoid thermal injury. One biological replicate was averaged from three measurements separated by 5-min intervals.