Plantar Heat Withdrawal Latency Assay

Paw withdrawal latencies (PWLs) were measured with the IITC Plantar Analgesia Meter (IITC Life Science Inc., Woodland Hills, CA, United States) in a double-blinded manner as described in our previous studies (Liu et al., 2011 (link), 2018 (link); Zhang H. et al., 2017 (link); Zhang S. et al., 2017 (link)). Mice were placed in transparent acrylic enclosures (10 × 10 × 20 cm) on a glass plate in a temperature-controlled and noise-free room. The mice were allowed to habituate for 1 h before the behavioral test. A heat-producing radiant light source was used to stimulate the plantar surface of the left hind paw. Time from the “light on” to a typical withdrawal or licking of the tested hind paw was recorded as paw withdrawal latency. The basal PWLs were set to 9–15 s by adjusting the radiant light intensity. To prevent tissue damage, the radiant heat illumination was automatically cut off at 25 s. The PWLs were measured for five times/time points/animal with the last three used for analysis.