Effect of Menthol on Human Temperature Sensitivity - Springer Link

ISSN 0362-1197, Human Physiology, 2008, Vol. 34, No. 2, pp. 221–225. © Pleiades Publishing, Inc., 2008. Original Russian Text © T.V. Kozyreva, E.Ya. Tkachenko, 2008, published in Fiziologiya Cheloveka, 2008, Vol. 34, No. 2, pp. 99–103.

Effect of Menthol on Human Temperature Sensitivity T. V. Kozyreva and E. Ya. Tkachenko Institute of Physiology, Siberian Branch, Russian Academy of Medical Sciences, Novosibirsk, 630117 Russia Received March 28, 2007

Abstract—Application of 1% menthol, which, along with cold, activates specific thermosensitive ionic channels, changes the number of functioning cold receptors on the skin of the forearm similarly to the cold exposure test; however, it does not affect the number of heat receptors and does not significantly change the threshold of cold sensation. Group variants of responses to menthol that indicate individual differences in the sensitivity of skin receptors to the effects of menthol and cold have been found. The results obtained give grounds to suggest that, from the variant of response to menthol (a decrease, increase, or absence of changes in the number of functioning cold receptors 5 min after menthol application), it is possible to predict specific features of response to cold. DOI: 10.1134/S0362119708020138

INTRODUCTION Humans and animals constantly obtain information on the infinite diversity of changes in the external and internal environments. One of important factors that affect the body is ambient temperature. The mechanisms that form the basis of temperature sensitivity are not quite clear thus far. Temperature sensation is formed from the combination of functioning of thermoreceptors that ensure the perception of the temperature signal and its transduction into the codes of the nervous system, carrying of this signal to the structures of the CNS and to cortical cells, its analysis and synthesis by specific cortical areas, with the subsequent formation of sensation. Temperature sensations reflect the relations between the processes of heat exchange and thermoregulation on the whole. It is known that analyzer systems are capable of changing their activity due to changes in the pulse activity of receptors and/or the number of functioning receptors, depending on the environmental conditions and the functional state of the body. For instance, the number of functioning cold receptors changes upon a decrease in skin temperature [1] and continuous adaptation to cold, heat, and physical loads [2]. During recent years, numerous studies on the cellular and molecular mechanisms of cold sensitivity have been perfomed. It is assumed that thermosensitive TRP channels are detectors of temperature change and the main sensors in the peripheral nervous system [3, 4]. It has been shown that TRPM8 (a member of the family of TRP channels) is an ionic channel activated by cold and menthol [5, 6]. In addition, there are data that menthol increases the sensitivity of cold-sensitive peripheral vasoactive C nociceptors and activates cold-specific A-delta fibers [7, 8]. The involvement of mentholdependent thermoreceptors in the formation of the rec-

ognized temperature sensation in humans has been poorly studied. We studied the effect of menthol application on the temperature sensitivity and cold sensation thresholds in humans. EXPERIMENTAL Fifty apparently healthy volunteers, men and women aged 24.3 ± 1.15 years, whose height-to-weight ratio was 2.8 ± 0.06, were examined. The subjects were informed about the goal of the study and the planned exposures in the course of the experiment according to the principles of Helsinki Declaration. A lightly dressed patient was in a thermostated chamber at an air temperature of 22–23°ë, sitting in an armchair. During examination, the skin temperature, cold sensitivity initially and after local cooling, heat sensitivity, as well as the temperature threshold of cold sensation, were measured. Skin temperature on the inner surface of the left and right forearms throughout the experiment was constantly measured using TSD 2002B thermistors of the measuring device from BIOPAC. The estimation of temperature sensitivity was performed by the method of counting the number of cold and warm spots (an estimate of the number of functioning cold and heat receptors) in a 100-point matrix on the inner surface of the forearm. The distance between the tested points was 5 mm; the total area of the matrix was 25 cm2. For testing cold spots, a thermode filled with melting ice was used. The diameter of the tip of the thermode was 1 mm, the time of contact of the thermode with each marked point was 2 s. A similar method was used to count the number of warm spots (the temperature of the thermode was maintained at the level of 41°ë using a thermostat).

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45 40 35 30 25 20 15 10 45 40 35 30 25 20 15 10 34 33 32 31 30

Number of CSs

Number of warm spots

Skin temperature,°ë

Initial

5 min after application of menthol


Fig. 1. The effects of application of menthol on the temperature sensitivity of the forearm. *Significant difference from the initial parameters (p < 0.05).

The temperature threshold of cold sensitivity was determined on the inner surface of the forearm upon a decrease in the thermode temperature from 34°ë at a rate of 1°C/min until the appearance of cold sensation, about which the subject reported by pressing a signal button. The value of the decrease in skin temperature under the thermode until the appearance of cold sensation was taken to be the cold threshold. Test cooling of the forearm in the region of the matrix for counting cold spots was performed using a thermode with an area of 25 cm2. The thermode temperature was 8−10°ë, the duration of cold exposure was 5 min. The application of 1% menthol solution in 50% ethyl alcohol was performed during 5 min. For this purpose, 1 ml of the solution of (–)Menthol (Sigma) was evenly spread over filter paper of the corresponding size (25 cm2) and applied to the skin of the right forearm at the place of the matrix for counting sensitive cold spots. According to published data, the solvent (ethanol) itself exerts no effect on the temperature sensitivity in humans in the used testing periods after application [7, 8]. Temperature was measured in the initial state on the skin of the inner surface of both forearms, and the number of cold spots was counted, as well as the number of warm spots on the right forearm; the cold threshold was also determined in the region of the matrix location on both forearms.

Test cooling was performed in the initial state prior to menthol application on the left forearm, and, after menthol application, on the right forearm, which permitted exclusion of a repeated cold effect on the same skin site. Immediately after test cooling, the number of cold spots was determined. After menthol application, 5 and 30 min later, the number of cold spots was repeatedly counted on the right forearm. Warm spots were determined 10 to 15 min after menthol application. The determination of the cold threshold was performed repeatedly on the right forearm 20 min after the menthol application. At the end of the experiment (40 min after the menthol application), test cooling of the right forearm was performed. Immediately after cooling, the number of cold spots was counted. RESULTS AND DISCUSSION In the initial state, the skin temperature (ST) was 32.2 ± 0.11°ë (from 30.5 to 33.7°ë) on the left forearm and 32.3 ± 0.09°ë (from 30.5 to 33.6°ë) on the right forearm, which indicates the absence of temperature asymmetry. As is known, the number of cold spots (CSs) on the skin considerably exceeds the number of warm spots (WSs), which agrees with our data (41.1 ± 2.33 versus 18.7 ± 2.9 per 100-point matrix). The thresholds of cold sensation did not differ on two forearms, being, on average, 0.6 ± 0.07°ë on the left and 0.5 ± 0.06°ë on the right. A considerable scatter of individual threshold values was observed. After menthol application, ST of the right forearm remained on the same level, while the number of CSs decreased 5 and 30 min after the menthol treatment (Fig. 1). Menthol did not change the number of WSs (18.7 ± 2.9 and 17.4 ± 2.0). The threshold of cold sensation upon the application of menthol did not change either (0.5 ± 0.07 and 0.5 ± 0.07°ë). When the ST decreased by 4.0 ± 0.12°ë as a result of test cooling, the number of CSs decreased, on average, by 35%. After the application of menthol, the test cooling of the same intensity (by 4.0 ± 0.13°ë) against the background of the amount of CSs already decreased under the effect of menthol caused an additional decrease in CS by 46% (Fig. 2). At the present time, there is no consensus on the mechanism of the action of cold or menthol. Some authors point to the heterogeneity of channels activated by cold and menthol [9, 10]. In addition, a suggestion has been made that cold and menthol have a common mechanism of the effect via activation of Ca2+ channels [11, 12]. A decrease in the number of CSs as a result of cooling or the action of menthol is possibly related to the activation of menthol- and cold-sensitive receptors TRPM8. The flow of Ca2+ ions through the membrane of receptor cells that arises in response to these factors may lead to inactivation of receptors and the loss of the HUMAN PHYSIOLOGY

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II

45

Number of CSs

40 35 30 25 20 15

Skin temperature, °ë

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30

25

Prior to cooling

After cooling

Prior to cooling

After cooling

Fig. 2. The effects of cooling (I) prior to and (II) after application of menthol on the number of cold spots. * Significant difference from the parameters prior to cooling (p < 0.05).

capacity for dynamic response to a short-term test cold exposure while CSs are being counted. The decrease in the amount of CS during cooling against the background of menthol action is apparently related to the activation of other types of cold receptors, insensitive or weakly sensitive to menthol [13]. Detailed analysis of individual specific features of the response to menthol application permitted revealing group variants of responses determined by differences in individual sensitivity to menthol. In the main group (70% of the examined subjects), changes typical of the total sample but more distinct were traced. For instance, 5 min after the application of menthol, the number of CSs in the region treated with menthol considerably decreased (by 45%). Subsequently, 30 min after, the effect of menthol in this group was retained; in some cases, it continued to increase, i.e., a subsequent decrease in the number of CSs was observed (Fig. 3a). Cooling of the forearm without the application of menthol was accompanied in this group by a decrease in the number of CSs (by 34.7%). Cooling of the same intensity and duration 40 min after the application of menthol caused an additional decrease in the number of sensitive CSs (by 43.9%) (table, group A). HUMAN PHYSIOLOGY

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In the second group differentiated by analysis (14% of the examined subjects), in contrast to the first group, the number of CSs 5 min after the application of menthol increased by 49%, returning to the initial level 30 min later (Fig. 3b). The subjects of this group did not exhibit significant changes in the number of CSs upon cooling both prior to the application of menthol and against the background of the aftereffect of menthol (table, group B), which may be evidence for a smaller effect of cold on the number of functioning cold receptors in subjects of this group. Finally, in some examined subjects (16%), the application of menthol was not accompanied by a change in the number of CSs either 5 or 30 min after the exposure (Fig. 3c). Note that, in subjects of this group, the initial number of CSs was significantly greater than in group A (51.4 ± 8.45 versus 36.3 ± 2.37;  < 0.05). In this group, a significant decrease in CS number during test cooling was also initially absent (table, group C). This suggests that a pronounced response to cold as such is provided by receptors highly sensitive to menthol, which, in the given group, have a decreased activity. Noteworthy is the fact that, in this group, the effect of menthol led to a distinct decrease in the number of CSs in response to test cooling: in contrast to the lack of

224 60 50 40 30 20 10 60 50 40 30 20 10 60 50 40 30 20 10

KOZYREVA, TKACHENKO Number of CSs Group Ä

Group B

Group C

Initial



Fig. 3. Group specific features of the effects of menthol on cold sensitivity. *Significant difference from the initial parameters (p < 0.05).

such response prior to the application of menthol, after its action, a highly significant decrease in the CS number by 45% was observed (table, group C). A possible explanation of this fact is the suggestion about the subliminal depolarization under the effect of menthol of the cellular membrane of thermoreceptors that are weakly sensitive to menthol. A partial depolarization of the membrane could facilitate the formation of the action potential, i.e., the activation of receptors during the subsequent cooling. The intergroup differences in responses to cold and menthol may be related to the involvement of different groups of A-delta and C fibers, as well as of different ionic channels of thermoreceptors. For instance, recent studies have shown that, besides menthol-dependent Group-specific features of response to cooling before and after application of menthol

Groups

Group A Group B Group C

Decrease in the number of CS under exposure to cooling (% of the data prior to cooling) before the application of menthol

after the application of menthol

34.7 ± 4.48* 19.2 ± 6.83 20.5 ± 7.05

43.9 ± 5.36* 22.5 ± 14.53 44.6 ± 6.44*

* Significant decrease in the number of CSs during cooling (p < 0.01).

TRPM8, other ionic channels activated by cold rather than menthol exist [14]. Note that no significant intergroup differences between the thresholds of cold sensation were found. The lack of the effect of menthol on cold and heat thresholds was also observed in the studies of other authors [7], who relate the effects of menthol to the activation of cold-specific A-delta fibers. The following suggestion can be made. It is known that the flow of sensory information (Ä) that comes from peripheral thermoreceptors to the corresponding central structures is the function (F) of the amount of functionally active receptors (N) and the level of their impulse activity (f): A = F(N, f). We judge about the number of functionally active cold receptors from the number of CSs that decreases under the effect of menthol. At the same time, the data obtained on neurons of the dorsal ganglion of the spinal cord, as well as on preparations of coldreception fibers, testify to an increase in the impulse activity of thermoreceptors under the effect of menthol and an enhanced response to cooling [14–17]. It is likely that menthol, considerably increasing the activity of receptors that are highly sensitive to it, excludes them from the subsequent process of cold perception. Receptors that are weekly sensitive to menthol, being depolarized under its effect, turn out capable of a more pronounced response to cold exposure. A decrease in the number of functioning thermoreceptors upon an increase in their impulse activity may slightly change the information flow from sensors to the central structures. It may be suggested that the formation of the general cold sensation and the change in its threshold are largely related to the flow of information on the environmental temperature. This suggestion is confirmed by a rather weak negative correlation between the number of CSs and the value of the cold sensation threshold that was observed in our study (r = –0.22; p < 0.05). CONCLUSIONS Thus, the results obtained showed that application of menthol, which, along with cold, activates specific thermosensitive ionic channels, changes the number of functioning cold receptors on the forearm skin, similarly to the cold exposure test; however, it does not affect the number of heat receptors. The found group variants of responses to menthol indicate individual differences in the sensitivity of skin receptors to menthol and cold. The obtained results suggest that it is possible to predict specific features of response to cold from the variant of response to menthol (a decrease, increase, or absence of changes in the number of functioning cold receptors 5 min after the application of menthol). REFERENCES 1. Kozyreva, T.V. and Yakimenko, M.A., On Human Temperature Sensitivity to Cold, Fiziol. Zh. SSSR, 1978, vol. 64, no. 2, p. 220. HUMAN PHYSIOLOGY

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