Topical menthol mitigates pruritus and inhibits spinal microglial activation in mice

Mentha piperita L. (Lamiaceae), commonly known as peppermint, has a long history in traditional medicine. Its diverse essential oil composition, with menthol as a key bioactive component, supports a range of medicinal applications, including pruritus relief. This suggests a potential role in modulating itch via neuropharmacological mechanisms.

This study aimed to investigate the anti-pruritic mechanisms of menthol by assessing its effects on scratching behavior and spinal microglial activation in a mouse model of pruritus.

Male ICR mice were used for the compound 48/80 and GRP_18-27-induced scratching models (n = 5-17/group, N = 179 total). C57BL/6 wild-type (n = 5/group, N = 10 total) and Trpm8^-/- mice (n = 7/group, N = 14 total) were used to confirm TRPM8 involvement. Mice received a single topical application of 8 % menthol to the forepaws for 10 s. Pruritus was induced either by subcutaneous (s.c.) injection of compound 48/80 (5 mg/kg in ICR mice or 20 mg/kg in C57BL/6 and Trpm8^-/- mice) or by GRP_18-27 (3.5 mg/kg) into the nape of the neck. Scratching behavior was recorded for 30 min (compound 48/80) or 60 min (GRP_18-27). Minocycline, N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide (BCTC), U50488 and nor-binaltorphimine (nor-BNI) were administered intraperitoneally (i.p.) to elucidate underlying mechanisms. Spinal microglial activation was assessed by Iba1 immunohistochemistry and by Iba1/phospho-p38 co-labeling with confocal microscopy. Western blotting quantified CD11b in the spinal cord.

Scratching behavior was markedly elevated in mice treated with compound 48/80. Topical application of 8 % menthol significantly reduced scratching by approximately 63.02 % compared with compound 48/80 alone (p < 0.001). This antipruritic effect was blocked by BCTC (a TRPV1/TRPM8 antagonist, p < 0.001), and menthol had no effect in Trpm8^-/- mice, strongly suggesting a TRPM8-dependent mechanism. The decrease in scratching behavior by 8 % menthol was not significantly affected by nor-BNI, a κ-opioid receptor antagonist. Minocycline (a microglial inhibitor) pretreatment also reduced scratching (by approximately 24.71 %, p < 0.05). Histological analysis revealed that compound 48/80 induced marked microglial activation (characterized by enlarged cell bodies and thickened processes) attenuated by menthol. Confocal imaging showed increased phospho-p38/Iba1 co-expression after compound 48/80 treatment, which was also reduced by topical menthol application. Western blot showed the upregulation of CD11b in compound 48/80-treated mice, and this upregulation was decreased by topical menthol application. In addition, topical 8 % menthol also significantly reduced GRP_18-27-induced scratching (by approximately 60.94 %, p < 0.05).

Topical menthol application effectively reduces pruritus in a mouse model via a TRPM8-dependent mechanism that inhibits spinal microglial activation. These findings support the traditional use of peppermint for itch relief and highlight TRPM8 and microglia as therapeutic targets for pruritus management.