Hot plate meter

We assessed behavioral pain using the von Frey assay and the hotplate test. Mechano-sensitivity experiments were performed using von Frey filaments (Ugo Basile Biological Research Apparatus Company, Milan, Italy). For the thermal hyperalgesia test, mice were placed on the hot plate meter (Ugo Basile Biological Research Apparatus Company, Milan, Italy), setting PSU40. The mice were placed in a plexiglass cylinder and kept quiet for 1–2 h. The incubation period was recorded until licking and/or shaking or jumping of the hind paws, at which point the mice were immediately moved away from the hot plate. Each mouse was measured at 3 points at a time for 3 times, with an interval of 30 min for each time. Investigators were blinded to specific groups.

The hot plate test was applied to analyze the pain response in joints. The normal, vehicle, and BNTA (0.015, 0.15, and 1.5 mg kg⁻¹)-treated ACLT rats were placed on the hot plate meter (UGO BASILE srl, VA, ITALY) at 55 °C. The response time was recorded when the hindlimb responses, such as shaking, jumping, or licking, appeared. Rats were taken out if the hindlimb response time exceeded 30 s, avoiding scalds. Each rat was measured for three times. And the observers were blinded to the experiment.

The pain response in the joints of rats/mice in different experimental groups were evaluated by the hot plate test. Briefly, the animals were placed on a hot plate meter (Ugo Basile SRL, Italy) at 55 °C, and the time from the point when both hind limbs touched the hot plate until the appearance of hind limb responses, such as shaking, jumping, or licking, was measured. Each animal was tested three times with 15 min intervals, and the average of the three response times was considered the final pain threshold of the rat/mouse. The observers were blinded to the experimental treatments.