Metal plate

Pain perception in male and female was assessed at the age of 13, 15, 17, and 20 months, using the hot plate test as described (Dere et al., 2014 (link)). Briefly, mice were placed on a preheated (55°C) metal plate (Ugo Basile Srl, Comerio, Italy) and the latency (s) to retract by jumping or licking of the hind paws was recorded. Mice were exposed for a maximum of 40 s, used as cut-off time, in case they did not show an aversive response to the heated plate.

Thermal nociception (hot plate test) was assessed with a commercially available apparatus consisting of a metal plate 25 × 25 cm (Ugo Basile, Italy) heated to a constant temperature of 55.0 ± 0.1 °C, on which a plastic cylinder (20 cm diameter, 18 cm high) was placed. The time of latency (s) was recorded from the moment the animal was placed in the cylinder on the hot plate until it licked its paws or jumped; the cut-off time was 60 s. The baseline was calculated as mean of three readings recorded before testing at intervals of 15 min. The time course of latency was then determined at 15, 30, 45, 60, 90 and 120 min after compound treatment. Data were elaborated as time-course curve of the percentage of maximum effect (%MPE) = (post drug latency – baseline latency)/(cut-off time – baseline latency) × 100. In these experiments, compounds were administered by intracerebroventricular (i.c.v.) or intravenous (i.v.) injections. For i.c.v. injections, mice were lightly anesthetized with isoflurane, and an incision was made in the scalp. Injections were performed using a 10 μL Hamilton microsyringe at a point 2-mm caudal and 2-mm lateral from the bregma at a depth of 3 mm in a volume of 10 μL as previously described^{64 (link)}.

The hot plate test was used as a measure of pain perception. Mice were placed on a metal plate (Ugo Basile Srl, Comerio, Italy) that was preheated up to 55°C. The latency to show hind paw licking or jumping was recorded. Immediately after showing the response, the mice were removed from the platform. A cut-off time of 40 s ensured that the mice were not injured. Animals were tested at the age of 27–28 weeks.