Topical application of L-menthol induces heat analgesia, mechanical allodynia, and a biphasic effect on cold sensitivity in rats

Abstract

Menthol is used in analgesic balms and also in foods and oral hygiene products for its fresh cooling sensation. Menthol enhances cooling by interacting with the cold-sensitive thermoTRP channel TRPM8, but its effect on pain is less well understood. We presently used behavioral methods to investigate effects of topical menthol on thermal (hot and cold) pain and innocuous cold and mechanical sensitivity in rats. Menthol dose-dependently increased the latency for noxious heat-evoked withdrawal of the treated hindpaw with a weak mirror-image effect, indicating antinociception. Menthol at the highest concentration (40%) reduced mechanical withdrawal thresholds, with no effect at lower concentrations. Menthol had a biphasic effect on cold avoidance. At high concentrations (10% and 40%) menthol reduced avoidance of colder temperatures (15 degrees C and 20 degrees C) compared to 30 degrees C, while at lower concentrations (0.01-1%) menthol enhanced cold avoidance. In a -5 degrees C cold plate test, 40% menthol significantly increased the nocifensive response latency (cold hypoalgesia) while lower concentrations were not different from vehicle controls. These results are generally consistent with neurophysiological and human psychophysical data and support TRPM8 as a potential peripheral target of pain modulation.

Fig. 1

Concentration-dependent antinociceptive effect of menthol on thermal hindpaw withdrawal latency. A: Thermal paw withdrawal latency (Hargreaves) test: ipsilateral hindpaw. The hindpaw receiving topical menthol exhibited a concentration-dependent increase in withdrawal latency (analgesia). Groups of animals tested at concentrations of 40%, 10% and 1% menthol were significantly different from vehicles. Forty percent menthol was different from all other concentrations (*: p<0.01, repeated-measures ANOVA), while 10% menthol was not different from 1% menthol (p=0.07). Data for 0.01% menthol are similar to 0.1% menthol group and omitted for clarity. Error bars: SEM; n=8/group. The stimulus was cut off at 20 sec if there was no withdrawal. B: Contralateral hindpaw. There was a weak mirror-image effect. The 40% menthol group was significantly different from all other concentrations (*: p<0.01), which were not different from vehicle (0.01% menthol omitted). C: Vehicle controls: ipsilateral hindpaw. There was a significant difference between groups (*: p<0.01). D: Vehicle controls: contralateral hindpaw. There was no significant difference between ethanol concentrations, both of which were ineffective.

Fig. 2

Mechanical paw withdrawal latencies and the lack of concentration-dependent antinociceptive effect of menthol. A: von Frey paw withdrawal threshold: ipsilateral hindpaw. The 0.1–10% menthol groups were not significantly different from vehicle (10% ethanol). Only the 40% menthol group was significantly different from all other groups (*: p<0.05, repeated measures ANOVA) indicating allodynia. Data for 0.01% menthol are similar to 0.1% menthol treated groups and omitted for clarity. N=8/group. B: Contralateral hindpaw. None of the menthol concentration groups were significantly different from the vehicles (0.01% menthol omitted). C: Vehicle controls: ipsilateral hindpaw. There was no significant difference between 10% + 1% Tween-80 and 50% ethanol + 5% Tween-80 vehicle groups. D: Vehicle controls: contralateral hindpaw. No significant difference between ethanol concentrations.

Fig. 3

Two-temperature preference test. Rats were placed on one of two adjacent thermoelectric plates whose temperatures could be set independently (range −5 to >50°C). One plate was set at 30 °C and the other at a warmer or colder temperature in 5 °C increments in a counterbalanced design. The rat was free to move from one surface to the other through an opening in a vertical barrier between the two plates. A. The graph plots the mean percentage of time naive rats spent on the warmer or colder plate relative to the thermoneutral (30°C) plate over a 20 min period. Rats significantly avoided temperatures <30°C and >35°C. B. As in A for mean number of crossings between the thermoneutral (30°C) and warmer/ cooler plate over a 20 min period. Error bars: SEM. Naive animals, *, p<0.05, paired t-test; n=16/group for 5°C–45°C; n=8/group for 0°C and 50°C).

Fig. 4

Biphasic effects of menthol on thermal preference. A: Graph plots % time rat stood on 30 vs. 15°C plate. Horizontal dashed line indicates that naive and vehicle-treated rats avoided the colder plate ∼80% of the time as a reference. At high (40, 10%; open and vertically-striped bars) menthol concentrations rats spent significantly less time on the 30°C plate compared to vehicle controls (diagonally-hatched and dark gray bars)(p<0.05), indicating cold hyposensitivity. At lower concentrations (0.01–1%; horizontally-striped, light gray and stippled bars), rats spent significantly more time on the 30°C plate (p<0.05), indicating cold hypersensitivity. *: significantly different vs. vehicle (p<0.05, n=16/group). B: As in A for 20 vs 30°C preference test. Note significant cold hyposensitivity with 40% menthol and significant hypersensitivity at the lowest menthol concentration of 0.01%. *: significantly different vs. vehicle (p<0.05, n=16/group).

Fig. 5

Antinociceptive effect of 40% menthol in −5°C cold plate test. A: Cold plate −5°C: menthol. Graph plots change in cold plate latency (% of pre-menthol baseline) vs time after topical menthol application at indicated concentrations. Mean baseline latency was 26.4 sec; animals were removed at 150 sec (cutoff) if they did not respond. Rats were tested 3 min after menthol or vehicle, and again at 15-min intervals out to 60 min and once more at 120 min. The 40% menthol group was significantly different from all other concentrations (*: p<0.001, repeated-measures ANOVA) which did not differ from each other or vehicle. This indicates a cold antinociceptive effect of 40% menthol. Error bars: SEM, n=8/group. B: Cold plate −5°C: vehicle. There was a significant difference between 10% and 50% ethanol (*:p<0.001, ANOVA). The ∼20% reduction in cold plate latency seen at 3 min post- 10% ethanol was comparable to the latency reductions in the 0.1–10% menthol groups at the same 3-min time point (A). C: Cold plate 0°C: menthol. Mean baseline latency was 105.9 sec. There was no significant difference among menthol concentration groups. D: Cold plate 0°C: vehicle. There was no significant difference between ethanol concentrations.