Cooling Relief of Acute and Chronic Itch Requires TRPM8 Channels and Neurons

Abstract

Cooling or the application of mentholated liniments to the skin has been used to treat itch for centuries, yet remarkably little is known about how counter-stimuli such as these induce itch relief. Indeed, there is no clear consensus in the scientific literature as to whether or not cooling does in fact block the transduction of itch signals or if it is simply a placebo effect. This gap in our understanding led us to hypothesize that cooling is antipruritic and, like cooling analgesia, requires function of the cold-gated ion channel TRPM8, a receptor for menthol expressed on peripheral afferent nerve endings. Using a combination of pharmacologic, genetic, and mouse behavioral assays, we find that cooling inhibits both histaminergic and non-histaminergic itch pathways, and that inhibition of itch by cooling requires TRPM8 channels or intact and functional TRPM8-expressing afferent neurons. The cold mimetic menthol is also effective in ameliorating itch in a TRPM8-dependent manner. Moreover, we find that chronic itch can be ameliorated by cooling, demonstrating that this counter-stimulus activates a specific neural circuit that leads to broad itch relief and a potential cellular mechanism for treatment of chronic itch.

Figure 1.. Hind paw pruritogen injections promote itch-like behaviors in mice.

(a) In wild-type mice, unilateral intradermal hind paw injections of both pruritogens and algogens induce robust lick and biting behaviors (P < 0.001 compared to vehicle, one-way ANOVA followed by Neuman-Keuls post-test) measured as duration of behaviors in a 30-minute period post-injection, with no differences between injections of 100 or 0.5 μg histamine (P > 0.05, one-way ANOVA, Neuman-Keuls post-test). Capsaicin, α-methyl 5-HT (αMe5-HT), and a high dose of histamine (100 μg, n = 8) produced flinching and hind paw lifts (P < 0.01 compared to vehicle, one-way ANOVA, Neuman-Keuls post-test), whereas chloroquine (Cqx; 200 μg, n = 8), 48/80 (0.5 μg, n = 6), and a low dose of histamine (0.5 μg, n = 6) did not induce these behaviors (P > 0.05 compared to vehicle, one-way ANOVA, Neuman-Keuls post-test). The duration of flinches/lifts between histamine doses was significantly different (***P < 0.001, one-way ANOVA, Neuman-Keuls post-test). All behaviors were combined (flinches/lifts/bites/licks) for comparisons. (b) The number of behavioral bouts produced similar results with no significant difference in the number of licks/bites between the different stimuli (^nsP < 0.05 compared to vehicle, one-way ANOVA, Neuman-Keuls post-test). The number of flinches/lifts between histamine doses of was significantly different (***p < 0.001, one-way ANOVA, Neuman-Keuls post-test). All behaviors were combined (flinches/lifts/bites/licks) for comparisons.

Figure 2.. Cooling inhibits acute itch.

The duration of licking and biting behaviors at different plate temperatures (30, 24, 20, 17, and 10°C) in mice injected with chloroquine (Cqx) (a, n = 8–10), 48/80 (b, n = 8), or histamine (c, n = 9). Neither Cqx (d) or 48/80 (e) induced flinches or lifts, whereas 100 μg histamine (f) induced robust pain-like behaviors that were inhibited at plate temperatures of 17°C and 10°C. (g–i) Duration of all behaviors showed cooling inhibition to all three substances tested. ***P < 0.001, **P < 0.01 and all data compared to 30°C via one-way ANOVA, Neuman-Keuls post-test.

Figure 3.. Antipruritic cooling requires constant stimulation.

(a) Chloroquine (Cqx; 200 μg) induced licking/biting behaviors resolved within 25 minutes post injection when the test plated was held at 24°C (n = 7). A plate temperature of 20°C showed inhibition with robust behaviors produced when the plate was warmed to 24°C 15 minutes after Cqx injections, responses not observed in vehicle-injected mice. (b) Summary of the data in (a) shows inhibition in the first 15-minute period post-injection when the plate is at 20°C (***P < 0.001 compared to 24°C, one-way ANOVA, Neuman-Keuls post-test), with behaviors uncovered in the second 15–45 minute period when the plate was warmed to 24°C (*P < 0.05 compared to when the plate was held constant at 24°C, one-way ANOVA, Neuman-Keuls post-test). Summary of the total behaviors showed no difference between the two stimulation protocols (P>0.05, one-way ANOVA, Neuman-Keuls post-test). (c) In a test of the duration in which cooling inhibits itch, α-methyl 5-HT (aMe5-HT; 30 μg) behaviors were blocked by cooling to 17°C, but were observed when the plate was warmed to 24°C after 30 minutes. (d) Summary of the αMe5-HT responses in mice on a 17°C plate for 30 minutes followed by warming to 24°C compared to mice placed at 24°C only. *P < 0.05, ^nsP> 0.05, n = 6–8, one-way ANOVA, Neuman-Keuls post-test.

Figure 4.. Antipruritic cooling requires TRPM8 channels and neurons.

(a) Chloroquine (Cqx)-induced licking/biting behaviors were inhibited in wild-type mice (n = 8–10) at 20°C and 17°C (***p < 0.001 compared to 24°C, one-way ANOVA, Neuman-Keuls post-test). (b) In mice lacking functional TRPM8 channels (Trpm8^−/−, n = 8–9), 20°C was ineffective in blocking these behaviors (^nsP > 0.05, one-way ANOVA, Neuman-Keuls post-test), while a colder temperature of 17°C did inhibit (**P < 0.01 compare to both 24° and 20°C, one-way ANOVA, Neuman-Keuls post-test). (c) In comparison, mice in which TRPM8-expressing afferent neurons are ablated, cooling was unable to inhibit Cqx-induce behaviors, even at 10°C (^nsP > 0.05 compared to 24°C for all temperatures tested, one-way ANOVA, Neuman-Keuls post-test, n = 8).

Figure 5.. Menthol inhibits Cqx-induced itch in a TRPM8-dependent manner.

(a) Topical menthol (8%) produced robust cheek wiping behaviors in wild-type mice (***P < 0.001 compared to vehicle, one-way ANOVA, Neuman-Keuls post-test, n = 8) that were significantly reduced in Trpm8^−/− animals (***P < 0.001 compared to vehicle, one-way ANOVA, Neuman-Keuls post-test, n = 6–8). Menthol did evoke significant wiping compared to vehicle (*P < 0.05, one-way ANOVA, Neuman-Keuls post-test, n = 6). (b) Topical menthol (8%) significantly inhibited Cqx-induced cheek scratches in wild-type (*P < 0.05, one-way ANOVA, Neuman-Keuls post-test, n = 8), an effect that was not observed in Trpm8^−/− animals (^nsP > 0.05, one-way ANOVA, Neuman-Keuls post-test, n = 6–8). Cqx, chloroquine; PBS, phosphate buffered saline.

Figure 6.. Cooling inhibits chronic itch.

(a) A chronic itch model of dry skin (acetone, ether, and water [AEW]) was induced by twice daily treatments with an acetone/ether/water treatment protocol for 15 days. At 30°C, spontaneous hind-paw behaviors observed in the treated hind paw (ipsilateral) were significantly different than those in the untreated paw (contralateral, ***P < 0.001, one-way ANOVA, Neuman-Keuls post-test, n = 10), and inhibited when the plate was cooled to 20°C (***P < 0.001, one-way ANOVA, Neuman-Keuls post-test, n = 10). At 20°C, there was no significant difference between the ipsilateral and contralateral paws (^nsP > 0.05, one-way ANOVA, Neuman-Keuls post-test, n = 10). (b) Control, water alone–treated hind paws displayed no significant difference in spontaneous behaviors at either temperature (^nsP > 0.05, one-way ANOVA, Neuman-Keuls post-test, n = 6).