Mechanisms and time course of menthol-induced cutaneous vasodilation

This great paper by Craighead, McCartney,  Tumlinson and Alexander describes the vasodilation dose-response of topical menthol. In the post below, we’ve listed some important info from the study, but to read the full study, please see the link at the bottom of the article…

Microvasc Res. 2017 Mar;110:43-47. doi: 10.1016/j.mvr.2016.11.008. Epub 2016 Nov 27

Craighead DH, McCartney NB, Tumlinson JH, Alexander LM

This paper describes the vasodilation dose-response of topical menthol.

Microvasc Res. 2017 Mar;110:43-47. doi: 10.1016/j.mvr.2016.11.008. Epub 2016 Nov 27

Craighead DH, McCartney NB, Tumlinson JH, Alexander LM

Menthol is a vasoactive compound that is widely used in topical analgesic agents. Menthol induces cutaneous vasodilation, however the underlying mechanisms are unknown. Determining the rates of appearance and clearance of menthol in the skin is important for optimizing topical treatment formulation and dosing. The purpose of this study was to determine the mechanisms contributing to menthol-mediated cutaneous vasodilation and to establish a time course for menthol appearance/clearance in the skin. Ten young (23±1years, 5 males 5 females) subjects participated in two protocols. In study 1, four intradermal microdialysis fibers were perfused with increasing doses of menthol (0.1-500mM) and inhibitors for nitric oxide (NO), endothelium derived hyperpolarizing factors (EDHFs), and sensory nerves. Skin blood flow was measured with laser Doppler flowmetry and normalized to %CVCmax.

In study 2, two intradermal microdialysis fibers were perfused with lactated Ringer’s solution. 0.017mL·cm-2 of a 4% menthol gel was placed over each fiber. 5μL samples of dialysate from the microdialysis fibers were collected every 30min and analyzed for the presence of menthol with high performance gas chromatography/mass spectrometry. Skin blood flow (laser speckle contrast imaging) and subjective ratings of menthol sensation were simultaneously obtained with dialysate samples. In study 1, menthol induced cutaneous vasodilation at all doses ≥100mM (all p<0.05). However, inhibition of either NO, EDHFs, or sensory nerves fully inhibited menthol-mediated vasodilation (all p>0.05). In study 2, significant menthol was detected in dialysate 30min post menthol application (0.89ng, p=0.0002). Relative to baseline, cutaneous vasodilation was elevated from minutes 15-45 and ratings of menthol sensation were elevated from minute 5-60 post menthol application (all p<0.05). Menthol induces cutaneous vasodilation in the skin through multiple vasodilator pathways, including NO, EDHF, and sensory nerves. Topical menthol is detectable in the skin within 30min and is cleared by 60min. Skin blood flow and perceptual measures follow a similar time course as menthol appearance/clearance.

Introduction

Menthol is the active ingredient in many topical analgesic agents that elicits a cold sensation by acting on transient receptor potential melastatin 8 (TRPM8) channels (Peier et al., 2002). Because of its wide ranging use in topical products, it is important to understand how menthol is penetrating the skin and affecting blood flow and sensation. Utilizing laser speckle contrast imaging, we recently demonstrated the topically applied menthol gel induces vasodilation of the cutaneous microvasculature (Craighead and Alexander, 2016).

TRPM8 channels are expressed in vascular endothelium (Johnson et al., 2009) and smooth muscle (Yang et al., 2006). The literature on the vasoactive effects of menthol is varied, with most (Cheang et al., 2013; Craighead and Alexander, 2016; Johnson et al., 2009; Sun et al., 2014) but not all (Olive et al., 2010; Topp et al., 2013; Topp et al., 2011) studies finding that menthol possess vasorelaxent properties. However, there is not a consensus on the mechanism(s) through which menthol mediates vasodilation. There is evidence that menthol acts through nitric oxide (NO)(Johnson et al., 2009), RhoA/Rho kinase (Sun et al., 2014), and by altering smooth muscle calcium concentration (Cheang et al., 2013; Ito et al., 2008). The disparate findings may be due to the use of different animal models and vascular beds. Because of menthol’s presence in many topical agents, the human cutaneous circulation is a clinically relevant vascular bed in which to examine the vasoactive effects of menthol.

Utilizing reactive hyperemia and local heating to assess endothelium derived hyperpolarizing factor (EDHF)/sensory nerve and NO-dependent vasodilation respectively, we recently found that menthol likely caused vasodilation through EDHFs and sensory nerves (Craighead and Alexander, 2016). However, a ceiling effect of cutaneous vasodilation with local heating may have masked any contribution from NO to menthol-mediated vasodilation. Furthermore, while reactive hyperemia and local heating in the skin are in large part pathway specific, there is a certain degree of cross talk from multiple vasodilator pathways (Brunt and Minson, 2012; Engelke et al., 1996; Medow et al., 2007). Consequently, the mechanism(s) contributing to menthol-mediated vasodilation need to be more fully elucidated.

From a therapeutic standpoint, topical menthol containing products are used in clinical populations (i.e. arthritis, muscle strain, back pain) for their effects of sensation and pain relief. Along with elucidating menthol’s mechanism(s) of action, determining the amount of menthol delivered to the cutaneous tissue and the rate of menthol appearance/clearance is important for optimizing formulation of topical menthol containing products for improving its analgesic effects.

The goals of this study were to use intradermal microdialysis, to (1) pharmacodissect the mechanism(s) through which menthol induces cutaneous vasodilation, and (2) determine a time course for menthol appearance and clearance in the cutaneous tissue following topical menthol application. We hypothesized that NO, EDHFs, and sensory nerves would all contribute to menthol-mediated vasodilation. We also hypothesized that menthol would be detectable in dialysate samples from the cutaneous tissue within 30 minutes on topical exposure and remain present throughout the duration of our protocol.

Figure 1: Menthol dose response

Menthol elicited increased vasodilation at all concentrations ≥100 mM. Antagonism with any of L-NAME, TEA, or lidocaine prevented vasodilation. The control site exhibited significantly more vasodilation than the TEA site at all menthol doses ≥50mM and more than the L-NAME and lidocaine sites at all menthol doses ≥100mM. * p<0.05 between control and L-NAME, † p<0.05 between control and TEA, ‡ p<0.05 between control and lidocaine.

Figure 2: Perceptual and physiological responses to topical menthol

(A) Self-reported sensation from topical menthol was significant at every time point between minute 5 and minute 60 post menthol application. (B) Topical menthol significantly increased vasodilation by 15 minutes post-application. Vasodilation remained elevated relative to baseline through minute 45 post-application. (C) Menthol collected from dialysate (ng). Significant menthol was detected at 30 minutes post menthol application. Menthol detection was statistically insignificant at every following time point. * p<0.05 compared to baseline.

Conclusion

By using specific pharmacological inhibitors, we confirmed our previous finding that menthol induces vasodilation through EDHFs and sensory nerves. We also found that menthol induces NO-dependent vasodilation at thermoneutral skin temperatures. Additionally, we characterized the time course of perception, vasodilation, and appearance/clearance for topical menthol. Menthol peaks in the skin after 30 minutes, the vasoactive effects are apparent after 15 minutes and disappear after 45 minutes, while the sensation of menthol begins rapidly and persists for an hour post application.

For more info on this study, please see the link below…

https://www.ncbi.nlm.nih.gov/pubmed/27899298


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