4.5. Hot Plate Test

The hot plate test was done based on our laboratory protocols [23]. The effects of treatment on the thermal nociceptive threshold were measured. Mice were placed on a 54.5 °C hot plate (Socrel model DS37; Ugo Basile, Gemonio, Italy), and the response latency to either a hind paw lick or jump was recorded. The animal was removed from the hot plate at 60 s to prevent serious tissue injury in the absence of a response, then a latency response of 60 s was assigned.

Morphine (20 mg/kg) dissolved in 300 µL of normal saline (M) or an equal volume of saline alone (NS) was administered intraperitoneally twice a day for five consecutive days. Each group was then orally administered either PBA (0.25, 0.5, or 1 g/kg dissolved in 300 µL of NS), TUDCA (0.25, 0.5, or 1 g/kg dissolved in 300 µL of NS), or saline (300 µL of NS) twice a day for 5 consecutive days. The hot plate test was performed after the first and tenth drug administrations on days 1 and 5, respectively for the experiments in Figure 1, Figure 2 and Figure 3. For the experiment in Figure 4, morphine (20 mg/kg) dissolved in 300 µL of normal saline was administered intraperitoneally twice a day for 5 consecutive days. Intraperitoneal morphine (20 mg/kg) and oral saline were then administered twice in the NS + morphine group, while intraperitoneal morphine and oral PBA (1 g/kg) were administered twice in the PBA + morphine group on day 6. Hot plate tests were performed after the tenth drug administration on day 5 and after intraperitoneal morphine injection on day 7.

Hot plate latency was measured at 5, 15, 30, 45, and 60 min after drug injection. Before drug administration, hot plate latency was measured three times, and the average was used as the pre-drug response latency at time 0 min. To analyze the effects of the drugs in the hot plate test, we calculated the %MPE as ([post-drug maximum response latency − pre-drug response latency]/[60-s cut-off time − pre-drug response latency]) × 100. The post-drug maximum response latency was defined as the single longest response latency.