In these studies, a low concentration of menthol applied to the eye induced TRPM8-dependent tear secretions, as evidenced by the absence of menthol-evoked tears in TRPM8 knockout mice. At a high concentration, however, menthol produced tear secretions even in mice lacking TRPM8 channels. This higher concentration of menthol also evoked ocular nocifensive behaviors in the eye wipe test irrespective of the mouse genotype, while low concentrations of menthol had no effect on nocifensive behaviors. In parallel experiments, a low concentration of menthol increased the activity of corneal cool cells, whereas a high concentration caused these neurons to inactivate after a brief period of activity. Taken together, these results indicate that menthol activation of cool cells via TRPM8 channels can evoke tearing without eliciting nociceptive responses.
The TRPM8 channel is a nonselective cation channel found on lightly myelinated and unmyelinated A-delta and C primary afferent neurons.
18–20 Although results are not completely consistent between studies, they are expressed in trigeminal and dorsal root ganglion (DRG) neurons, including corneal afferents,
18 and the majority do not appear to express markers typical of the two main classes of unmyelinated nociceptive neurons; TRPM8 expressing neurons do not appear to include IB-4 positive neurons, and the majority of them are not peptidergic (do not express the peptides substance P or calcitonin gene-related peptide).
27 TRPM8 channels are activated by moderate cold (15–30°C) and by cooling compounds, such as menthol and icilin.
28–30 (reviewed by McCoy et al.
17 and Nilius and Voets
31 ). Menthol and icilin appear to modulate TRPM8 through different mechanisms that may have unique downstream consequences,
32–34 and various TRPM8 independent effects have been reported for menthol.
35–37 One study in particular found evidence for activation of nociceptive neurons following hind paw injection of 40 mM menthol in TRMP8 knockout mice.
37 This result is consistent with our finding that 50 mM menthol induced tearing and eye-wipe behaviors in TRPM8 knockout animals. The mechanism of action for the effect of high concentrations of menthol remain unknown, it appears unlikely to involve TRPA1, the receptor for wasabi and other related noxious chemicals.
37 However, other studies have shown that menthol can activate TRPA1 receptors.
38
Over the course of the three baseline tear measurements taken before adding solutions to the eye a small, yet significant, increase in tearing was noted, which may have been caused by mild irritation produced by the cotton thread used for measuring tears. In contrast to an earlier report on tearing in TRPM8
−/− mice,
14 we did not find a decrease in baseline tearing in these animals. In addition, we did not find evidence of corneal abrasions after fluorescein was applied to the ocular surface, or differences in the rate of spontaneous blinks. A simple methodologic difference may explain the discrepancy between the results found in basal tear levels. While our tear measurements were performed in awake animals, the previous study measured tears under ketamine/xylazine anesthesia. Measuring tears in awake animals may affect tearing by increasing stress, and it is well established that general anesthesia reduces tearing.
39,40 The absence of any difference in baseline tearing between TRPM8
+/+ and TRPM8
−/− mice was not entirely surprising, since cold-evoked activity likely involves additional cold-sensitive channels.
41 Corneal cool cells also are sensitive to hyperosmotic stimuli,
10,15 which may initiate tear secretions upon evaporation of the tear film even in the absence of a cooling response. Our data indicated that TRPM8 knockout mice retain the ability to respond to hyperosmotic tears.
The increase in tear secretions after the low concentration of menthol application likely is due to the activation of corneal cool cells. In this and in previous studies, menthol at relatively low concentrations (≤0.2 mM) has been demonstrated to increase the ongoing activity of cool cells innervating the cornea and depolarize cool sensitive corneal afferent nerve terminals.
10,14,15 While different species of animals were used in the recording and behavioral experiments, previous studies have found similar electrophysiologic properties in multiple species of animals, and consistent correlations between the activity of these neurons and sensory function in humans.
11 The results of our study are consistent with earlier findings that demonstrated increased tear production by cooling the cornea in TRPM8
+/+, but not TRPM8
−/− mice.
14 In addition, decreased corneal temperatures in human subjects led to an increase in tearing in human subjects.
14 However, it should be noted that an additional study found that non-noxious cooling of the cornea did not alter tearing, perhaps because of the relatively brief nature (3 seconds) of the stimulus.
7
Several additional lines of evidence indicate that corneal cool cell activity is involved in regulating tear secretions. Cool cells are the only known corneal primary afferent neuron with spontaneous activity at room temperature
10,12,42–44 and topical anesthesia of the ocular surface decreases tear secretions,
45 indicating that this spontaneous activity is responsible for basal tearing. In addition, diabetes and LASIK surgery, conditions that result in damage to corneal afferents, can cause dry eye conditions.
46–55 Furthermore, dry eye prevalence increases with age, which is correlated with a loss of corneal sensation.
56,57
The utility of targeting cool cells as a means to increasing tear production depends on the selectivity of the response to cool cell activation. In addition to regulating tear secretions, corneal afferents can elicit nocifensive responses, such as blinking, irritation, and pain. This constellation of responses is initiated by the activation of polymodal and mechanoreceptive neurons to protect the eye from potentially damaging stimuli. In contrast, mild cooling of the ocular surface in humans (<2.0°C decrease), which selectively activates cool cells, does not appear to elicit irritation or pain.
12,13
While increasing cool cell activity may increase tearing without producing irritation or pain, the effectiveness of TRMP8 agonists as potential therapies would depend on its ability to activate cool cells selectively. Subtypes of corneal polymodal neurons, some of which respond to cold stimuli, have been reported. In one study of corneal afferents in cats, 6/26 polymodal neurons responded to cold stimuli with a low frequency of activity evoked only at temperatures lower than that needed to evoke activity in cool cells.
12 These corneal temperatures also produced irritation of pain in human subjects.
12 This cold-evoked activity in polymodal neurons would be consistent with the finding in cultured DRG neurons of cells sensitive to menthol and capsaicin.
58,59 Those neurons responsive to menthol and capsaicin were less responsive to menthol than those that were sensitive only to menthol.
58,59 These neurons may represent polymodal nociceptive neurons. Alternatively, it has been suggested that TRPV1 and TRPM8 co-expression in DRG neurons may explain the “paradoxical” heat responses in cool cells.
60
The effect of menthol in corneal polymodal nociceptive neurons remains to be described in detail. However, one study reported no changes in thermal or chemical responsiveness after application of menthol to seven polymodal corneal units.
12 We used the eye wipe test to gain insight into potential nociceptive effects of menthol. This test has been used previously to examine nociceptive responses produced by application of capsaicin and hyperosmotic solutions to the ocular surface.
22–24,61 The amount of time spent wiping at the eye correlates with the concentration of these irritants and is reduced by analgesic drugs. The absence of eye wipe behaviors after low concentrations of menthol suggests that TRPM8 activation may increase tearing selectively without producing irritation or pain. However, it still is possible that the behavioral measure was not sensitive enough to detect the possible activation of a small subpopulation of menthol-sensitive corneal nociceptors. In a previous report, a similar concentration of menthol (0.2 mM) produced a feeling of cooling and in some cases slight discomfort in human volunteers.
12
Unlike the absence of nociceptive responses in response to low concentrations of menthol, the highest concentration evoked an increase in eye wipe behaviors in TRPM8
+/+ and TRPM8
−/− mice. Similarly, the same concentration of menthol induced tearing that was unaffected in the TRPM8
−/− mice. These responses are unlikely to be the result of cool cell activity. While low and high concentrations of menthol increased cool cell activity over the first 10 seconds, only the low concentration of menthol produced a prolonged increase in cool cell activity. The high concentration of menthol tended to inactivate cool cells after a brief period of activity. These results indicated that this higher concentration of menthol activates nociceptive neurons in a TRPM8-independent fashion. A previous study also has demonstrated that a relatively high concentrations of menthol (6.4–64 mM) enhanced cold-evoked activity in trigeminal subnucleus caudalis nociceptive neurons with intraoral receptive fields.
62 Similarly, intraoral menthol (19 mM) enhanced lingual cold pain in human subjects,
63 and even higher concentrations of menthol have been used to model cold pain in humans.
64–66 The results of studies using these high concentrations of menthol should be interpreted with caution, since such high concentrations produce effects that are not mediated by TRPM8 channels.
In summary, these results indicated that TRPM8 and potentially other channels expressed on corneal cool cells represent potential targets for increasing tearing without eliciting nociceptive responses. In this way, new therapies for treating DES could include TRPM8-mediated activation of corneal cold cells to drive the basal tearing reflex loop. Furthermore, our study raised an additional consideration regarding a common current therapy for DES: not only is repeated application of artificial tears often inadequate, it also may be detrimental to the overall disease progression. Repeated application of artificial tears would reduce the afferent drive for endogenous tear production.
3 This decrease in afferent drive would reduce the release of trophic factors from the parasympathetic neurons that innervate the lacrimal gland, which may result in atrophy of the gland. Such a consequence would be consistent with previous studies that have reported atrophy of the salivary glands following denervation.
67–69 Thus, increasing the activity of neurons involved in basal tearing would be beneficial to the overall health of the tear-producing glands.