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Epidermal expression of human TRPM8, but not of TRPA1 ion channels, is associated with sensory responses to local skin cooling

Weyer-Menkhoff, Irisa; Pinter, Andreasb; Schlierbach, Hannahc; Schänzer, Annec; Lötsch, Jörna,d,*

doi: 10.1097/j.pain.0000000000001660
Research Paper
Editor's Choice

Human cold perception and nociception play an important role in persisting pain. However, species differences in the target temperature of thermosensitive ion channels expressed in peripheral nerve endings have fueled discussions about the mechanism of cold nociception in humans. Most frequently implicated thermosensors are members of the transient receptor potential (TRP) ion channel family TRPM8 and TRPA1. Regularly observed, distinct cold pain phenotype groups suggested the existence of interindividually differing molecular bases. In 28 subjects displaying either high or medium sensitivity to local cooling of the skin, the density at epidermal nerve fibers of TRPM8, but not that of TRPA1 expression, correlated significantly with the cold pain threshold. Moreover, reproducible grouping of the subjects, based on high or medium sensitivity to cooling, was reflected in an analogous grouping based on high or low TRPM8 expression at epidermal nerve fibers. The distribution of TRPM8 expression in epidermal nerve fibers provided an explanation for the previously observed (bi)modal distribution of human cold pain thresholds which was reproduced in this study. In the light of current controversies on the role of human TRPA1 ion channels in cold pain perception, the present observations demonstrating a lack of association of TRPA1 channel expression with cold sensitivity–related measures reinforce doubts about involvement of this channel in cold pain in humans. Since TRP inhibitors targeting TRPM8 and TRPA1 are currently entering clinical phases of drug development, the existence of known species differences, in particular in the function of TRPA1, emphasizes the increasing importance of new methods to directly approach the roles of TRPs in humans.

The distribution of TRPM8 but not TRPA1 expression in epidermal nerve fibers provides an explanation of the (bi)modal distribution of human cold pain thresholds.

aInstitute of Clinical Pharmacology, Goethe-University, Frankfurt, Germany

bDepartment of Dermatology, Venerology, and Allergology, University Hospital Frankfurt, Frankfurt, Germany

cInstitute of Neuropathology, Justus Liebig University, Giessen, Germany

dFraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology TMP, Frankfurt, Germany

*Corresponding author. Address: Goethe-University, Theodor-Stern-Kai 7, Frankfurt 60590, Germany. Tel.: +49-69-6301-4589; fax: +49-69-6301-4354. E-mail address: (J. Lötsch).

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (

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1. Introduction

The major role of thermal stimuli in pain perception20,49,98,122 and clinical hyperalgesia22,27,37,39,86 continues to drive research on this area in pain.64,115 Thermal stimuli affect the nociceptive system through several different types of nociceptors.98 Particularly important in this context are members of the transient receptor potential (TRP) ion channel family49,64 sensing temperatures from noxious heat (TRPV1 ≥42°C,23,59,120,122 TRPM3 ≥40°C43,124,125), warmth (TRPM2 ≥37°C91,112,114), and cold (37°C ≥TRPC5 ≥25°C,139 TRPM8 ≤25°C81,101,123) to noxious cold (TRPA1 ≤17°C11,61).

Among TRP ion channels, TRPM8 and TRPA1 have received particular interest with respect to their role in human cold nociception10,64,100 and as analgesic drug targets.128 However, although a role of TRPM8 in human cold perception seems to be generally accepted,13,26,55,56 controversies persist regarding the contribution of TRPA1. The role of TRPA1 in human cold sensation and pain has been both supported50,51,85,94 and denied.13,55,138 Controversy has also arisen from human genetic assessments which implicate TRPA1 variants in both cold withdrawal53 and also in heat pain perception.53,59 In addition, some studies pointed to the involvement of TRPA1 in mechanotransduction,62,121,138 emphasizing the polymodal character of nociception.

Based on the reproducible observation that human cold pain thresholds (CPTs) are multimodally distributed,74,77,130 the hypothesis was derived that individual cold sensitivity is modulated by a relative dominance on the skin of the expression of either TRPM8 or TRPA1 in 2 distinct phenotype groups. This was observed in the distribution of CPTs in cohorts of n = 180 subjects,77 n = 32974 and n = 148130 healthy subjects. Specifically, mean CPTs at 25 or 18°C observed in subjects displaying either high or medium sensitivity to cooling of the skin, respectively, were similar to the initially reported activation temperatures of 24 to 25°C81,122 and 17°C85,101 for TRPM8 and TRPA1, respectively. Supported by immunohistochemical evidence of the expression of TRPM8 and TRPA1 in peripheral nerves of the human skin,2,9 in this study, we assessed the local expression of these 2 types of ion channels in epidermal nerve fibers. Healthy volunteers were enrolled who displayed either high or medium sensitivity to skin cooling.

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2. Material and methods

2.1. Subjects and study design

The study followed the Declaration of Helsinki and was approved by the Ethics Committee of the Medical Faculty of the Goethe-University Frankfurt am Main, Germany (protocol number 110/16). Informed written consent was obtained from all subjects. Healthy volunteers with a Caucasian background by self-assignment (n = 28, 13 men), aged 21 to 32 years (mean ± SD: 25.5 ± 3.3 years), were enrolled based on CPTs acquired in a previous study.130 Exclusion criteria were drug intake during the previous week, except for oral contraceptives and vitamins, a current clinical condition involving pain, and current diseases according to questioning and medical examination.

Participants belonged to a cohort of 148 subjects in whom a multimodal distribution of CPTs had been described previously,130 using a Gaussian mixture model (GMM) with M = 3 modes. The first 2 modes, in decreasing order of sensitivity to cooling of the skin, comprised subjects with mean CPTs of 25 and 18°C, respectively, which are referred to phenotype groups with either high or medium sensitivity to cooling of the skin. The observations on these groups confirmed a previous finding74 based on which it had been proposed that a different relative expression of TRPM8 vs TRPA1 ion channels in epidermal nerves was an underlying mechanism. Based on the activation temperatures of TRPM8 and TRPA1 at 24 to 25°C81 and 17°C,11,61 respectively, relatively more pronounced expression of TRPM8 in the group with high sensitivity to cooling was proposed in comparison to the group with medium sensitivity. The reverse was expected for the expression of TRPA1. By contrast, no specific molecular hypothesis had been derived at the time for the third Gaussian mode, suggesting a contribution of other thermosensitive proteins. Therefore, subjects belonging to the formerly identified third Gaussian mode were not assessed in this hypothesis-driven study.

To test whether a differential expression pattern of TRPM8 and TRPA1 exists between subjects in groups with high and medium sensitivity to cooling, 14 subjects who had been assigned previously to either of these 2 phenotypes,130 who had CPTs close to the mean of the respective group, were invited to participate in this study. The study followed a parallel group, single-blind design. Group assignment of the subjects with respect to cold pain sensitivity was revised according to a reassessment of the CPT. Subsequently, skin biopsies were taken for morphometric analyses of intraepidermal nerve fiber density (IENFD) and the expression of TRPM8 and TRPA1 ion channels at epidermal nerve fibers.

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2.2. Assessment of cold pain thresholds

The interval between the previous130 and the actual measurements was 210 to 377 days (mean ± SD: 301.01 ± 50.55 days). Therefore, before the skin punch biopsy, CPTs were reassessed in the same manner as previously.130 That is, the standardized procedure of the quantitative sensory testing proposed by the German Research Network on Neuropathic Pain (DFNS)106,107 was used. It was preferred to alternative implementations of human experimental models involving cold pain as it produces standardized results that can be compared directly with both a reference data set76 and clinical pathological values acquired in independent cohorts.32,73 The latter, however, was not planned in this study.

Cold stimuli were applied using a 3 × 3-cm2 thermode placed on the skin of the volar forearm at the site where the biopsies were planned to be taken. The temperature was lowered at a constant rate of −1°C/second from a baseline temperature of 32°C. The technical cutoff temperature was 0°C. Subjects were instructed to press a button at the first sensation of pain, ie, the moment when the sensation of cold changed to an additional impression of a “burning,” “stinging,” “drilling,” or “aching” sensation. Pressing the button ended the cooling of the thermode. Measurements were repeated 5 times at an interval of 30 seconds. The CPT was defined as the median of these measurements. Before the actual measurements, subjects were acquainted with the procedures during a separate training session that usually took place the day before. Standardization and reference data served to control the quality of the newly acquired data.76 A comparison with the reference data indicated that the actual data did not cross the upper limit of CPT temperature of at 29°C, as recorded in the reference cohort from women and of 27°C from men.76 This confirms that all trained volunteers responded to the first perception of cold pain rather than to the detection of cold. A possible trend across single measurements was analyzed using a Jonckheere-Terpstra test,48,113 which produced a nonsignificant result (P > 0.2).

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2.3. Quantification of intraepidermal nerve fiber density and TRPM8 and TRPA1 expression

Biopsies of normal skin were taken using a 6-mm circular punch from the middle third of the volar forearm at the same site where the thermosensitivity was tested. The procedure was performed under sterile conditions after local anesthesia with 1% mepivacaine hydrochloride (Mecain; Puren Pharma, Munich, Germany). Tissue was immediately fixed for 30 minutes in 4% phosphate-buffered paraformaldehyde (pH 7.4) at room temperature, rinsed 3 × 10 minutes in phosphate-buffer, cryoprotected in 10% sucrose, and stored at −80°C pending further processing.33 From each biopsy, 50-µm-thick frozen sections were cut vertically in relation to the epidermis. Sections from the beginning and the end of the specimen were discarded. For immunofluorescence staining, the sections were incubated with primary antibody (Table 1) overnight at room temperature in a phosphate-buffered saline solution, containing 0.1% Triton X-100, 0.1% Tween 20, and 0.1% bovine serum albumin.

Table 1

Table 1

The sections were rinsed and incubated with the secondary antibody for 3 hours at room temperature. The following primary antibodies were used: rabbit antiprotein gene product 9.5-antibody (PGP 9.5, 516-3341; Zytomed, Berlin, Germany; 1:1000), mouse antiprotein gene product 9.5-antibody (PGP 9.5, Clone 31A3, NB600-1160; Novus Biological, United Kingdom; 1:500), sheep anti-TRP M8 antibody (TRPM8, OST00028W; Thermo Fisher, Waltham, MA; 1:1000), and rabbit anti-TRP A1 antibody (TRPA1, NB110-40763; Novus Biological, Abingdon, United Kingdom; 1:500). As secondary antibodies, the following were used: Alexa Fluor 488-conjugated goat anti-rabbit IgG (A11008; Thermo Fisher; 1:1000), Alexa Fluor 488-conjugated goat anti-mouse (A11029; Thermo Fisher; 1:1000), and Alexa Fluor 568-conjugated donkey anti-sheep IgG (A21099; Thermo Fisher; 1:1000), or Alexa Fluor 568-conjugated goat anti-rabbit IgG (A11011; Thermo Fisher; 1:1000). These antibodies have been used previously, both in the present laboratory and in several independent studies.19,40,93,95 For antibody validation of TRPM8-control immunofluorescence, staining was performed with hTRPM8 transfected HEK cells (Supplementary Figure 1, available at When double staining was performed, the antibodies were incubated as a cocktail (Table 1). Nuclei were stained with DAPI fluoroshield mounting medium (abcamI), to assess cell morphology, especially to distinguish between the dermis and epidermis. Negative controls included omission of the primary antibodies to evaluate nonspecific staining of the secondary antibodies. The stratum corneum and collagen fibers in the dermis were stained. Samples were examined using a Leica DM 2000 fluorescence microscope equipped with a Leica DFC450C camera and Leica Application-Suite Version 4.7.1 (Leica Microsystems, Wetzlar, Germany).

Table 2

Table 2

Skin biopsies were taken and processed for the analysis of IENFD with antibody against PGP9.5 and colocalization with antibodies against TRPM8 or TRPA1 as shown in (Table 1). Colocalization of TRPM8 and TRPA1 was not assessed. All specimens were blinded, and 6 sections for each staining were analyzed by the same observer. Intraepidermal nerve fibers were (1) stained with the panaxonal marker PGP9.5 for all 3 staining sessions (“A,” “B,” and “C”; Table 1), counted at magnification 400 and assessed as density to the total length of the epidermis (nerve fibers/mm) according to international criteria from the European Federation of Neurological Science (EFNS guideline65,66). That is, small fibers originating in the dermis and crossing the dermal–epidermal junction to the epidermis were counted, whereas secondary branching was excluded from quantification. Nucleus staining provided guidance in the determination of IENF since other structural components of the skin were also found to be innervated by nerve fibers.9 The results of IENFD obtained with different staining sessions were assessed to verify the internal consistency of the staining. This served to allow comparability of the results with other studies.25,97 For staining sessions “B” and “C” (2), the coexpression of TRPM8 with PGP9.5 and TRPA1 with PGP9.5, respectively, in IENF was analyzed at magnification 1000. For this purpose, IENF, identified according to the description in (1), that were colocalized with immunolabelled expression of either TRPM89 or TRPA1,95 respectively, were counted as coexpressing nerves fibers and assessed as density relative to the total length of the epidermis (fibers/millimeter).

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2.4. Data analysis

Data were analyzed using the R software package (version 3.4.3 for Windows; Reassessment of the grouping of the subjects with respect to CPT was performed by analyzing the probability density function (PDF) of the CPTs using the Pareto density estimation (PDE), which is a kernel density estimator particularly suitable for the discovery of groups in data. A GMM was fit to the PDE, expressed as , where N (x|mi, si) denotes Gaussian probability densities (components) with mean values mi and SDs si. The wi denotes the mixture weights indicating the relative contribution of each M Gaussian component to the overall distribution. The parameters of GMM were adapted by applying the expectation maximization algorithm29 using our R package “AdaptGauss” ( A bimodal distribution of the CPTs was statistically assessed by comparing the goodness-of-fit obtained using M = 2 modes with that obtained using only M = 1 mode, using a likelihood ratio test. In addition, the difference between observed data and fit was assessed by means of a Pearson χ2 goodness-of-fit test.100 Furthermore, the suitability of the fit to describe the data was assessed using a quantile-quantile (QQ) plot, which is a probability plot comparing the model-based data distribution with the observed data distribution. Group assignment was performed based on the Bayesian decision border14 between Gaussian modes. The agreement between present and previous130 cold pain sensitivity phenotype group assignments was assessed by means of Cohen's κ and Fisher exact tests.35 The actual group assignment was used in all subsequent analyses.

Associations of nerve fiber density and TRPM8/TRPA1 expression with the cold pain sensitivity phenotype group structure were explored by means of analysis of variance (ANOVA). As visual inspection of the data of IENFD and TRPM8 and TRPA1 coexpression nerve fibers suggested partially multimodal distributions, a preprocessing step involving aligned rank transformation137 was applied, to enable the use of parametric ANOVA procedures with nonparametric data. Subsequently, ANOVA for repeated measures was performed, with “fiber” (3 levels) as within-subject factor and “Gaussian mode” (2 levels) and “sex” (2 levels) as between subject factors. These calculations were performed using the R package “ARTool” (,139 Mann–Whitney U tests78,133 were applied for post hoc pairwise comparisons. The α level was set at 0.05 and corrected for multiple testing according to Bonferroni.18 Fiber density measures that passed this analytical step were subsequently analyzed with respect to the hypothesized group structure. This was obtained by means of GMM analyses performed as described above. The association of fiber density groups with cold pain phenotype groups was assessed by means of a Fisher exact test.35 Finally, nonparametric correlations between fiber density measures and CPTs were analyzed by calculating Spearman ρ.110

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3. Results

Cold pain thresholds and skin biopsies were acquired completely in all 28 subjects. However, skin biopsies could be analyzed only in 26 of the subjects due to poor immunostaining of nerve fibers or the basement membrane in 2 subjects.

Reassessment of the cold pain sensitivity group structure verified the bimodal distribution underlying subject enrolment. This was supported (1) by a statistically significant better fit of the PDF obtained using M = 2 Gaussian modes (Fig. 1, top) rather than a unimodal distribution (likelihood ratio test: Δlog likelihood: −4.426, P = 0.031), (2) a nonsignificant χ2 test (χ2 = 8.29, P = 0.34), and (3) the vicinity of the observed quantiles to the expected quantiles in the QQ plot (Fig. 1, left). The cold pain sensitivity phenotype group assignment based on GMM agreed with the previous assignment on which subject enrolment had been based (Cohen's κ of 0.79, Fisher exact test: P = 0.0001077). However, in 3 subjects, cold pain sensitivity had increased, with CPTs passing the Bayesian decision border between the Gaussian modes. This resulted in group sizes of 17 subjects and 11 subjects for the phenotypes of high and medium sensitivity to cooling, respectively.

Figure 1.

Figure 1.

Skin biopsies were analyzed from 16 subjects assigned to the phenotype of high sensitivity to cooling of the skin and from 10 subjects assigned to the phenotype of medium sensitivity to cooling of the skin. The forearm skin, in particular the epidermis, was found to be richly innervated, whereas TRPM8 and TRPA1 staining in the epidermis was observed as a dot-like expression pattern (Fig. 2), similar to that in other reports investigating TRP coexpression in the dermal afferents of skin from healthy volunteers.9,95 The IENFDs of single-stained controls and double-stained sections were significantly correlated (P = 0.027), indicating internal consistency of the methods. Median IENFD in the control samples was 15.73 fiber/mm and agreed with reference values for fiber densities in forearm skin.25,97 Median fiber densities of TRPM8- or TRPA1-coexpressing nerve fibers were 0.62 fibers/mm (12.8% of IENFD) and 0.71 fibers/mm (11.4% of IENFD), respectively (Fig. 3).

Figure 2.

Figure 2.

Figure 3.

Figure 3.

Statistically, the fiber densities differed significantly, as indicated by the effect of “fiber” in the ANOVA (Table 2). The differences were also dependent on the pain phenotype group, as indicated by a significant ANOVA interaction of “fiber” by “Gaussian mode.” In addition, a significant effect of the factor sex on the fiber densities was observed. Post hoc tests specified that the density of TRPM8-coexpressing nerve fibers, but not of other fibers, differed significantly between the 2 phenotype groups (Mann–Whitney U: P = 0.00673), particularly in women (Mann–Whitney U for Gaussian modes #1 vs #2 in women: P = 0.0420). It is important to bear in mind that subjects were selected according to their mean CPT of 25 and 18°C from a previous study;thus, the present cohort does not represent a random sample.

The density of TRPM8-coexpressing nerve fibers was significantly correlated with the CPTs (Spearman ρ = 0.47, P = 0.015; Fig. 4). Moreover, the density of TRPM8-coexpressing nerve fibers was bimodally distributed as indicated by a significantly better GMM fit with M = 2 modes than with M = 1 (Δlog likelihood: −9.2813, P = 0.00037). A satisfactory fit with M = 2 modes was supported by a nonsignificant χ2 test (χ2 = 4.74, P = 0.73) and the closeness of observed quantiles to the expected quantiles in the QQ plot. A group association based on the TRPM8 fiber density was in agreement with the group association based on cold pain sensitivity (Fisher exact test: P = 0.0039). Specifically, subjects belonging to the group with higher densities of TRPM8-coexpressing nerve fibers also belonged to the high-sensitivity phenotype group.

Figure 4.

Figure 4.

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4. Discussion

The results of this study indicate that the bimodal distribution of CPTs in humans, reported from previous assessments, eg, in cohorts with n = 329,74 n = 148130 or in n = 180 healthy volunteers and in n = 1236 patients with neuropathic pain (Fig. 2 in 77), is reflected in the distribution of TRPM8-coexpressing epidermal nerve fibers. This was concluded first from the statistically significant correlation of the TRPM8 density with the CPTs. Higher TRPM8 density was associated with higher sensitivity to cooling. Second, the conclusion was based on the bimodal distribution of the density of TRPM8-expressing nerve fibers, which allowed a group of subjects with comparatively high TRPM8 expression to be distinguished. This high expression proved to be significantly overrepresented among subjects belonging to a phenotype of high sensitivity to cooling of the skin.

The distribution of TRPM8-expressing nerve fibers provided an explanation for the previously observed and presently reproduced (bi)modal distribution of human CPTs. A role of TRPM8 ion channels in human cold pain perception is in agreement with their known molecular functions. Specifically, TRPM8 is activated by temperatures of 24 to 25°C,81,101 L-menthol, and several other compounds such as icilin and eucalyptol.11,81 Peripheral- and central-mediated TRPM8 activation leads to distinct sensations of cold including a feeling of innocuous and noxious cold,13,26,55 cold allodynia26,56 but also cooling analgesia,103 which qualifies both TRPM8 agonists and antagonists as analgesics,72,135 as in the context of cold allodynia.21

The study hypothesis had additionally included TRPA1 ion channels, proposing a more complex scenario in which different ratios between TRPM8 and TRPA1 would underlie the bimodal distribution of CPT. This had been derived from the observation of the location of the second Gaussian mode at a temperature of 13.3 and 18.4°C, respectively,74,130 fitting well with the initially reported working temperature of TRPA1 at 17°C.85,111 However, the results of this study indicate that TRPA1 provided no significant contribution to the modal distribution of human CPTs in healthy subjects. This agrees with evidence supporting TRPM8, but not TRPA1, as the principal transducer of cool and cold stimuli in humans.13,26,31,55,80,102

Although TRPA1 has been proposed to be involved in cold sensitivity in several studies,1,3,4,6,50,58,85,88,89,96 its contribution to thermal sensation, especially to cold sensation in humans, remains a topic of ongoing debate.13,55,59,84,138 A lack of association of TRPA1 expression with cold pain sensitivity is in line with results obtained with topical application of TRPA1 agonists, which sensitized the skin without producing clear effects on cold pain.3,4,88,89,96 Occasionally, TRPA1 agonists failed to induce sensations of cold while evoking spontaneous pain, inflammatory symptoms, and even warmth.3,4,8,88,89,96 Moreover, TRPA1 activation induced by topical cinnamaldehyde produced hyperalgesia to heat pain, to an extent comparable with that obtained with topical capsaicin.129 Indeed, TRPA1 has even been classified as a heat sensor in humans.38,53,59

It remains possible that TRPA1 plays some role as a human cold sensor.128,129 TRPA1 stimulation using mustard oil or cinnamaldehyde was followed by both cold hyperalgesia and hypoalgesia.3,8,88,89,96 Both outcomes seem biologically plausible since cold hypersensitization would be expected when considering observations that human TRPA1 channels are gated by cold stimuli.6,50,62,79,111 Cold hyperalgesia could also result through the excitation of TRPA1 expressed exclusively at nociceptive C but not at Aδ afferents, which would lead to a relative dominance of nociceptive C-fiber input over inhibitory Aδ input.57,126 On the other hand, cold hypoalgesia after application of mustard oil or cinnamaldehyde could result as a diffuse noxious inhibitory control effect,68,69 ie, through the induction of inflammation12,46 and itch44,134 that were alleviated by the cold stimulation.5

Although the present results point to a major role for TRPM8 in human cold pain perception, it is well known that mechanisms of cold perception and cold nociception are more complex than just relying on a single ion channel.28 Other TRP ion channels, such as TRPC5 and TRPC4, contribute to complex cold pain perception.139 In addition to TRPM8, cold thresholds in cultured trigeminal neurons of mice have been found to correlate with the ratio of the expression of TRPM8 and Kv1 potassium channels.75 This may explain why a few subjects assigned to the high sensitivity group in our study in fact had low TRPM8 expression. Indeed, the group correlation between CPTs and TRPM8 expression was not complete (Fig. 4). In addition to TRPM8, general factors known to modulate individual sensitivity to pain stimuli, such as aversive hedonic properties of the stimuli,132 arousal,105 expectations,52 attention bias,116 reward,90 or the subject's genetic background of variants in pain-relevant genes63,82 or epigenetic factors,71,108 probably also contributed to the present observations.

It is possible that the present grouping of CPTs reflects different groups of subjects according to their responses to cold stimuli rather than a local difference in cold sensor expression. This can be concluded from the differences in observed early and late behavior of preferred temperature zones among wild type, TRPM8−/−, TRPA1−/−, and DKO mice,136 or from differences in the words chosen in the McGill Pain Questionnaire to describe the same cold stimulus.15,42,83 Moreover, pain intensity ratings correlated with cold withdrawal times in a cold immersion test.24,54 However, this study focused on sensory-discriminative rather than on affective-motivational components of pain,7 which may be sustained by different neural structures.60 The standardized quantitative sensory testing protocol used for the present assessments did not include a cold pressor test.106,107 However, although tolerance to tonic cold was proposed as a grouping criterion for subjects with respect to a cold pain phenotype,24,54 the lack of such cold-tolerant subjects in the present cohort does not weaken the evidence for a TRPM8-related molecular basis for the presently observed phenotypes. Thus, had the present grouping occurred with cold-tolerant subjects rather than with average subjects, the fiber density difference would still exist.

A possible study limitation is the exclusive enrolment of healthy subjects. Therefore, the present findings might differ slightly from those obtained in clinical settings and in chronic pain conditions. However, as with the present results, in patients suffering from cold injury followed by cold allodynia, no association of TRPA1 activation and hypersensitivity to cold was observed.88 Moreover, in subjects with hereditary episodic pain syndrome (FEPS) caused by the TRPA1 N855S gain-of-function mutation, CPTs were similar to those measured in unaffected relatives.58 A further limitation lies in the fact that correlation of the sensitivity to cooling with mechanical thresholds was not assessed in this study. Such a correlation seems possible when considering mechanosensory functions of TRPM8 and TRPA1,41 along with the evidence for 2 different populations of sensory neurons expressing TRPM8 or TRPA1, either mechanically sensitive or insensitive.47,62 However, given the multimodality and complexity of pain, correlations between the nociceptive intensity of different physical stimuli have both been shown92 and denied,67 making a forecast from the present cohort difficult.106,107

Finally, to investigate the molecular background of the modal distribution of CPTs, the forearm was preferred to the leg, which is normally the clinical standard for diagnosing small fiber neuropathies.30,66 Nevertheless, IENFD obtained from healthy skin of the forearm has been shown to correlate with that obtained from the leg.25,97 In general, variation in nerve fiber density in different body areas might emerge from different surface areas of the dermatomes. The relative quantity of nerve fibers per dermatome, however, remains constant. Nerve fiber density decreases from the trunk to the extremities,17 while the trunk with 12 dermatomes has approximately the same surface area as both lower distal parts of the body with 7 dermatomes.70 Within the superficial dermis, where small nerve endings attached to Schwann cells branch out, complex interactions mediated through nerve growth factor87 and involving Schwann cells,117,127 keratinocytes,45 and other cell lines16,34,36 contribute to the individual innervation and neuronal signaling.

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5. Conclusions

A reproducible multimodal distribution of human CPTs was successfully related to an underlying pattern of multimodal expression of human TRPM8 ion channels in epidermal nerve fibers. In view of current controversies over the role of human TRPA1 ion channels in cold pain perception, the present lack of association between TRPA1 channel expression and pain-related measures fuels previously expressed doubts about its involvement in human cold pain. Present results are of potential importance for the clinical testing of TRP inhibitors, targeting TRPM8 and TRPA1, which are currently entering clinical phases of drug development.128

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The authors have no conflicts of interest to declare.

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Appendix A. Supplemental digital content

Supplemental digital content associated with this article can be found online at

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The authors thank Prof Mike Parnham for proofreading the manuscript.

This work has been funded by the Else Kröner-Fresenius Foundation (EKFS), Research Training Group Translational Research Innovation—Pharma (TRIP, J.L.) and by the Landesoffensive zur Entwicklung Wissenschaftlich—ökonomischer Exzellenz (LOEWE), LOEWE-Zentrum für Translationale Medizin und Pharmakologie (J.L.). The funders had no role in method design, data selection and analysis, decision to publish, or preparation of the manuscript.

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        Cold pain thresholds; Human; Healthy volunteers; Gaussian mixture model; Probability density function; Phenotype; TRP ion channels; TRPA1; TRPM8; Immunofluorescence; Nerve fibers; Immunostaining

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