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 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.
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.
The authors have no conflicts of interest to declare.
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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