Plantar test device

Mechanical allodynia was estimated by determining the hind paw withdrawal response to von Frey filament stimulation. Thus, mice were positioned in methacrylate cylinders (20 cm high, 9 cm diameter) with a wire grid bottom through which the von Frey filaments (North Coast Medical, Inc., San Jose, CA, USA) were applied according to the up–down paradigm [46 (link)]. A filament of 0.4 g was applied first, and one of 3.5 g as a cut-off. The strength of the next filament was reduced or augmented depending on the response. Withdrawal, shaking, or licking the paw were considered nociceptive-like reactions.
Thermal hyperalgesia was assessed according to previous methods [47 (link)]. Paw withdrawal latency in reply to a radiant heat was assessed using the plantar test device (Ugo Basile, Italy). Mice were placed in methacrylate cylinders, 20 cm high × 9 cm diameter, situated on a glass surface. The heat source was situated under the plantar surface of the hind paw and activated with a light beam. A cut-off time of 12 s was utilized to avoid tissue damage. The mean paw withdrawal latencies were calculated from the average of 2–3 separate trials, taken at 5 min intervals to prevent thermal sensitization.
In both tests, the animals were habituated to the environment for 1 h before the experiment. Both ipsilateral and contralateral paws were tested.

To assess hind paw heat sensitivity, Hargreaves’ test was conducted using a plantar test device (Ugo Basile, Italy). Animals were allowed to freely move within an open-topped transparent plastic box on a glass floor 20 min before the test. A mobile radiant heat source was then placed under the glass floor and focused onto the hind paw. Paw withdrawal latency was measured with a cutoff time of 15 s to prevent tissue damage. The heat stimulation was repeated three times with a 10 min interval to obtain the mean latency of paw withdrawal. Results are expressed as paw withdrawal latency.

Thermal thresholds were established through the Hargreaves’ method [40 (link)]. Mice were placed in individual plastic cages over an elevated glass surface and habituated for 45 min. Radiant heat was applied to the hind paw at a constant intensity using a Plantar test device (Ugo Basile, Italy), with a 30 s cut-off to prevent tissue damage. Two measurements were made and the mean latency of the paw withdrawal was considered as the thermal nociceptive threshold [41 (link)].