2.1. Nociceptive Behavioral Tests

Six-week-old male C57BL/6J mice were used. Time engaged in hind paw licking and flicking behavior was quantitated for ~10 min as previously described [9,10,11] (Supplementary Figure S8A). For eliciting TRPV1-activation-induced behaviors, 100 ng capsaicin-containing 10 μL saline was intraplantarily injected; for eliciting TRPA1-activation-induced behaviors, 100 ng AITC in 10 μL saline or 26.4 μg cinnamaldehyde in 20 μL saline was injected. For eliciting depolarization-induced behaviors mediated by voltage-gated channel activation, 140 mM KCl in 10 μL water was injected. For conventional TRPV4-mediated flinch assays, we used intraplantar injection of 10 μL deionized water for hypotonic stimulation 30 min after prostaglandin E2 (PGE2) priming (intraplantar pretreatment with 10 μL saline containing 100 ng PGE2) [11]. TRPV4-mediated nociceptive behaviors were observed by counting the number of hind paw flinching events for 10 min immediately after the hypotonic stimulus. For observing formalin-induced acute and tonic pain, 5% or 0.5% formalin in saline (20 μL) was intraplantarily injected and the cumulative time that a mouse spent licking and flinching the injected paw was measured every 5 min for 50 min.

For CFA-induced inflammation, 10 μL CFA was injected into a hind paw (Supplementary Figure S8D). For determining changes in mechanical or thermal behaviors by 24 and 48 h CFA inflammation, mice were acclimated to the test environment for 30 min before performing the following assays. Hargreaves assay (using a Plantar Analgesia meter; IITC, Woodland Hills, CA, USA) for acute heat avoidance or thermal hyperalgesia, von Frey assay by up-and-down paradigm (using von Frey filaments; Stoelting, Dale Wood, IL, USA) for mechanical allodynia, and Randall–Selitto assay (using Analgesy-meter, UGO Basile, Italy) for flexion reflex to noxious pressures, were carried out as described previously [12,13,14,15,16] (Supplementary Figure S8C).

For neuropathic pain development, the CCI model was established as described previously [17] (Supplementary Figure S8D). Briefly, the mice were anesthetized with inhalation of 3% isoflurane in a mixture of N₂O/O₂ gas. The full circumference of the sciatic nerve of the left hind limb was exposed and loosely tied three times with two silk sutures (7-0; Ailee, Busan, Korea). Sham surgery was conducted by exposing the sciatic nerve in the same manner but without tying the nerve. Hargreaves and von Frey assays were performed with the injured mice 14 days after the surgery.

For a postoperative pain model, an incision pain model was modified according to a previous study [18] (Supplementary Figure S8D). Briefly, mice were anesthetized with 2% isoflurane and hind paws was sterilized with 10% povidone–iodine solution. With a no. 10 scalpel blade, a 5 mm longitudinal incision was made in the glabrous skin and fascia of a hind paw plantar. The underlying muscle was elevated with a sterile curved forcep, with the muscle origin and insertion intact. The incised skin was apposed with two nylon sutures (8-0; Ailee, Korea). Sham mice were not incised or sutured but anesthetized for the same duration.