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. 2013 Oct;154(10):2169-2177.
doi: 10.1016/j.pain.2013.06.043. Epub 2013 Jun 29.

TRPM8 is the principal mediator of menthol-induced analgesia of acute and inflammatory pain

Boyi Liu  1 , Lu Fan, Shrilatha Balakrishna, Aiwei Sui, John B Morris, Sven-Eric Jordt
Affiliations

TRPM8 is the principal mediator of menthol-induced analgesia of acute and inflammatory pain

Boyi Liu et al. Pain. 2013 Oct.

Abstract

Menthol, the cooling natural product of peppermint, is widely used in medicinal preparations for the relief of acute and inflammatory pain in sports injuries, arthritis, and other painful conditions. Menthol induces the sensation of cooling by activating TRPM8, an ion channel in cold-sensitive peripheral sensory neurons. Recent studies identified additional targets of menthol, including the irritant receptor, TRPA1, voltage-gated ion channels and neurotransmitter receptors. It remains unclear which of these targets contribute to menthol-induced analgesia, or to the irritating side effects associated with menthol therapy. Here, we use genetic and pharmacological approaches in mice to probe the role of TRPM8 in analgesia induced by L-menthol, the predominant analgesic menthol isomer in medicinal preparations. L-menthol effectively diminished pain behavior elicited by chemical stimuli (capsaicin, acrolein, acetic acid), noxious heat, and inflammation (complete Freund's adjuvant). Genetic deletion of TRPM8 completely abolished analgesia by L-menthol in all these models, although other analgesics (acetaminophen) remained effective. Loss of L-menthol-induced analgesia was recapitulated in mice treated with a selective TRPM8 inhibitor, AMG2850. Selective activation of TRPM8 with WS-12, a menthol derivative that we characterized as a specific TRPM8 agonist in cultured sensory neurons and in vivo, also induced TRPM8-dependent analgesia of acute and inflammatory pain. L-menthol- and WS-12-induced analgesia was blocked by naloxone, suggesting activation of endogenous opioid-dependent analgesic pathways. Our data show that TRPM8 is the principal mediator of menthol-induced analgesia of acute and inflammatory pain. In contrast to menthol, selective TRPM8 agonists may produce analgesia more effectively, with diminished side effects.

Keywords: Acetic acid; Acrolein; Analgesia; Capsaicin; Heat; Inflammatory pain; Menthol; TRPA1; TRPM8; TRPV1; Visceral pain.

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Figures

Fig.1
Fig.1
Effects of L-menthol on thermal- and capsaicin-induced pain behavior in wild-type and Trpm8−/− mice. (A) Paw withdrawal latencies in the hot plate test (52 or 55°C) in wild-type (black) and Trpm8−/−mice (blue) treated with L-menthol (Ment, 10 mg/kg) or vehicle (PBS, Veh) through oral gavage (p.o.) 30 min prior to testing. n=7–10 mice per group. (B)Paw withdrawal latencies of wild-type mice in the hot plate test (52°C) following topical application of 30% L-menthol and ethanol (Veh). The two hind paws were immersed into vehicle or L-menthol solution for 5 seconds and allowed to dry for 1 min. n=7–9 mice per group. (C) Paw withdrawal latencies of Trpm8−/− mice in the hot plate test following topical application of 30% L-menthol and ethanol. Experiment as in (B). n=8 mice per group. (D) Tail flick latencies of wild-type and Trpm8−/− mice treated with vehicle or L-menthol (10 or 20 mg/kg, i.p.) 30 min before testing. n=6–7 mice per group. (E) Effects of L-menthol on capsaicin-induced nocifensive behavior (flinching, lifting and biting) in wild-type mice, Trpm8−/− or Trpa1−/− mice. Vehicle (PBS), L-menthol (50 nmol), capsaicin (Cap, 5 nmol) or capsaicin in combination with L-menthol (5nmol+50 nmol) were injected (in 50 μl volume) into the plantar surface of the hind paw and nocifensive behavior was recorded for 10 min. n=6–12 mice per group. (F) Effects of L-menthol on capsaicin-induced mechanical hyperalgesia in wild-type mice. Capsaicin (400 pmol) or capsaicin plus L-menthol (20 nmol) were injected into the plantar surface of the hind paw in 20 μl volume. Paw withdrawal thresholds were measured with von Frey hair filaments at time points indicated. n=6 mice per group. (G) Effects of L-menthol on capsaicin-induced mechanical hyperalgesia in Trpm8−/− mice. Experiment performed as in (E). n=6 mice per group. **p < 0.01, *p < 0.05, NS: not significant (p > 0.05).
Fig.2
Fig.2
Effects of L-menthol on acute acrolein-induced pain and CFA-induced mechanical hyperalgesia in wild-type, Trpm8−/−, or TRPM8 antagonist-treated mice (A) Nocifensive behavior in wild-type (black) or Trpm8−/− (blue) mice quantified over 10 min following hindpaw injection of vehicle (Veh, PBS), acrolein alone (Acrol, 25 nmol in 50 μl PBS) or acrolein plus L-menthol (50 nmol, co-injected). n=9–10 mice per group. **p < 0.01, NS: not significant (p > 0.05). (B) Effects of L-menthol on CFA-induced mechanical hyperalgesia in wild-type mice, measured by von Frey hair analysis. Baseline thresholds were measured before CFA injection (day 0). On days 1–3, mice were injected into the plantar surface of the paw with L-menthol (Ment, 60 nmol in 20 μl, green) or vehicle (PBS, red) 30 min prior to testing. Non CFA-treated mice are shown for comparison (black). n=7–8 mice per group. *p < 0.05 vs. CFA+Veh, ##p < 0.01 vs. Control, NS: not significant from CFA+Veh. (C) Effect of TRPM8 antagonists, AMG2850, on L-menthol inhibition of CFA-induced mechanical hyperalgesia in wild-type mice. Experiment performed as in (B). AMG2850 was administered (30 mg/kg, i.p.) 45 min before von Frey test. L-menthol was injected into hindpaw 15 min later. n=7–8 mice per group. (D) Effects of L-menthol on CFA-induced mechanical hyperalgesia in Trpm8−/− mice. Experiment performed as in (B) except that Trpm8−/− mice were used. n=7–8 mice per group.
Fig.3
Fig.3
Effects of L-menthol and eucalyptol on mouse visceral pain behavior in the acetic acid writhing test. (A) Quantification of writhing behavior in wild-type (black) or Trpm8−/− mice (blue) over 20 min following i.p. injection of 0.6% acetic acid (10 ml/kg). 30min prior to testing, mice were injected i.p. with L-menthol (10 mg/kg or 20 mg/kg, Ment) or eucalyptol (200 mg/kg, Eucaly) in 2.5 ml/kg volume in corn oil. Acetaminophen (Acet, 50 mg/kg) was used as a positive analgesic control.. n=5–8 mice per group. (B) Effects of acetic acid i.p. injection in Trpa1−/−, Trpv1−/− and Trpa1−/−/Trpv1−/− -mice. Treatment and analysis are as in (A). n=6–9 mice/ group. *p < 0.05, NS: not significant (p > 0.05)
Fig. 4
Fig. 4
Effects of cooling agent WS-12 on mouse DRG neurons, measured by ratiometric Ca2+-imaging. (A) Representative Ca2+-signals in wild-type DRG neurons elicited by WS-12 (1 μM), followed by mustard oil (MO, 70 μM), capsaicin (Cap, 1 μM) and KCl (40 mM). (B) Representative Ca2+-signals in Trpm8−/− DRG neurons. Experiments performed as in (A). (C) Percentage of wild-type, Trpm8−/− and Trpa1−/−/Trpm8−/−DRG neurons responding to Cap, MO, WS-12 (1 or 10 μM) and L-menthol (Ment, 250 μM or 1 mM). Each column shows average percentages from 5 to 8 separate tests and each test contains 40–60 neurons. Neurons were defined as responsive when the increase in Fura-2 emission ratio (340 nm/380 nm) in a given neuron exceeded 10% of the KCl response. **p <0.01, *p < 0.05, NS: no significance (p > 0.05). (D) Population analysis of wild-type DRG neurons responding to WS-12, MO and Cap. Agonists were applied as in (A). (n = 241 neurons, 6 fields).
Fig. 5
Fig. 5
Effects of WS-12 on acute thermal-, capsaicin- and acrolein-induced nocifensive behavior. (A) Nocifensive behavior of wild-type (black) or Trpm8−/− (blue) mice following local injection of vehicle (corn oil), WS-12 (6 nmol), capsaicin (2 nmol), and capsaicin (2 nmol) in combination with WS-12 (6 nmol), all administered in 20 μl injection volume. **p < 0.01, NS: not significant (p > 0.05). n=6–12 mice per group. (B) Nocifensive behavior in response to paw injections of vehicle (corn oil), acrolein (30 nmol/20 μl), and acrolein and WS-12 (6 nmol/20 μl) together, 20 μl injection volume each recorded as in (A). n=6–7 mice per group. *p < 0.05. (C) Paw withdrawal latencies in the hot plate test (52°C) in wild-type (black) or Trpm8−/− mice, injected i.p. with vehicle (corn oil) or WS-12 (10 mg/kg) in a volume of 5 ml/kg, 30 min prior to testing. n =6–8 mice per group. (D) Effects of naloxone on WS-12 induced analgesia in the hot plate test (52°C). WS-12 (10 mg/kg) was administered as in (C) 30 min before testing. 15 min later, naloxone (Nalox, 2 or 5 mg/kg, i.p.) or vehicle (Veh, PBS) in a volume of 5 ml/kg was injected. n=8–12 mice per group.
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