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Role of TRPM8 and TRPA1 for cold allodynia in patients with cold injury
Pain 139 (2008) 63–72 www.elsevier.com/locate/pain
Role of TRPM8 and TRPA1 for cold allodynia in patients with cold injury Barbara Namer a,*, Inge Petter Kleggetveit b, Hermann Handwerker a, Martin Schmelz c, Ellen Jorum b a
Department of Physiology and Pathophysiology, University of Erlangen, Universita¨tsstr. 17, 91054 Erlangen, Germany b Department of Clinical Neurophysiology, Rikshospitalet, Oslo, Norway c Department of Anesthesiology, Mannheim, University Heidelberg, Germany Received 18 September 2007; received in revised form 6 February 2008; accepted 3 March 2008
Abstract Local cold injury often induces hypersensitivity to cold and cold allodynia. Sensitisation of TRPM8 or TRPA1 could be the underlying mechanisms. This was evaluated by psychophysics and axon-reflex-flare induction following topical menthol and cinnamaldehyde application in cold injury patients and healthy subjects. The patients had no signs of neuropathy except cold allodynia. We applied 20% cinnamaldehyde and 40% menthol solutions in the cold-allodynic area of the patients and in a corresponding area in healthy subjects and obtained sensory ratings during application. Thermotesting and Laser Doppler Imaging were performed before and after exposure to the compounds. Menthol did not induce axon-reflex-erythema in patients or in controls. After menthol cold pain threshold was decreased in healthy subjects; however, no further sensitisation was observed in the patients moreover in some patients an amelioration of their cold allodynia was observed. Cinnamaldehyde-induced pain sensation did not differ between patients and controls. Heat pain thresholds following cinnamaldehyde were lowered to a similar extent in patients and controls (43– 39.8 and 44–39 °C) and also the axon-reflex-flare responses were comparable. No evidence for sensitisation of responses to TRPM8 or TRPA1-stimulation was found in patients with cold injury-induced cold allodynia. The lack of TRPM8 induced axon-reflex indicates that also de-novo expression of TRPM8 on mechano-insensitive C-nociceptors does not underlie cold allodynia in these patients. We conclude from these data that the mechanisms for the induction of cold allodynia in the patients with cold injury are independent of TRPM8 or TRPA1 and differ therefore from neuropathic pain patients. Ó 2008 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Cold hypersensitivity; Cold allodynia; Cold injury; Menthol; Cinnamaldehyde; Temperature threshold; Axon-reflex-flare; Psychophysics; TRPM8; TRPA1
1. Introduction Cold allodynia is a frequent finding following nerve lesions, but also in cold injury patients [27]. In a military environment 95% of examined soldiers with cold injury were found to have hypersensitivity to cold [27]. The exact physiologic role of various cold transducing recep*
Corresponding author. Tel.: +49 9131 85 22796; fax: +49 9131 85 22497. E-mail address: [email protected] (B. Namer).
tors and the mechanisms of cold allodynia in the different patients groups are still unclear. In the last years the molecular basis of cold transduction in primary afferent neurons has been expanded by cloning and characterization of two cold sensitive receptors, TRPM8 and TRPA1. TRPM8 is activated by moderate cold between 25 and 28 °C and can also be activated and sensitized chemically by menthol [19,24]. In humans, application of menthol results in cold hyperalgesia, possibly by sensitisation of this receptor on peripheral C-fibers [13,20,35]. Thus, the topical applica-
0304-3959/$34.00 Ó 2008 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2008.03.007
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tion of menthol could be a model for cold allodynia evoked by peripheral sensitisation in humans. TRPA1 can be activated by pungent agents such as cinnamaldehyde or mustard oil and it is still unclear whether this channel can be activated under physiological conditions by noxious cold below 17 °C or only when expressed heterologously [14,32]. In humans topical cinnamaldehyde application provokes burning warmth sensation and heat hyperalgesia [20]. In animal models a contribution of TRPA1 to cold hyperalgesia in neuropathic pain states has been suggested [15,22]. Moreover, NGFdependant increase in expression of TRPA1, but not TRPM8 in hyperalgesia following peripheral inflammation and nerve lesion has been observed [22]. Similarly, knocking down TRPA1 can alleviate cold allodyniarelated behaviour in rats [15]. TRPM8 seems to play a different role in cold hyperalgesia: stimulation of TRPM8 with icilin evoked analgesia in different animal models of neuropathic and inflammation pain and TRPM8 antisense oligonucleotides prevents this analgesia in CCl nerve injury [25]. Proalgesic involvement of TRPM8 in neuropathy-induced cold allodynia has not been proven so far. In the present study, we have investigated possible peripheral sensitisation mechanisms of menthol and cinnamaldehyde in Norwegian soldiers who had developed cold hypersensitivity and cold allodynia following cold injury. The aim of the study was to test the patients for hypersensitivity to menthol (TRPM8 involvement) and cinnamaldehyde (TRPA1 involvement) application. Psychophysics of sensations evoked by topical application of the substances and on changes of thermal thresholds were performed in soldiers and an age-matched control group of healthy subjects. Sensitisation of TRPA1 or de-novo expression of TRPM8 on mechanoinsensitive C-nociceptors was investigated indirectly by objective assessment of the axon-reflex-flare responses to menthol and cinnamaldehyde by Laser Doppler Imaging.
chosen to take part in the study. The diagnosis of cold allodynia was established by the assessment of thermal thresholds with a thermotest device (Somedic Sweden). Cold pain detection thresholds above 20 °C were regarded as pathological. Healthy control subjects were recruited from university staff and students in Erlangen, Germany and underwent a short clinical neurological examination and a short interview regarding neurological and dermatological diseases. They were included only if there was no hint for a neurological, dermatological or severe chronic disease. 2.1. Experimental protocol In the healthy subjects and patients, psychophysical experiments were conducted in a double blinded and randomized cross-over design. On day 1 the three substances were applied in random order for 20 min to the back of one foot in three different sites. The application sites were marked before to ascertain that distances between the application sides were at least 2 cm (Fig. 1a). To make sure that the experimenter could not identify the applied substance by its characteristic smell, he was wearing a face mask with small amounts of cinnamaldehyde dropped into the mask tissue. Before and after the application of the substances the temperature thresholds were tested in the respective areas. On day 2 the two test substances was applied to the contralateral foot to test for axon-reflexflare by Laser Doppler Imager (Perimed AB, Stockholm, Periscan PIM II, Sweden) (Fig. 1b). For details of the time course of the protocol see Fig. 1.
2. Methods Ten healthy subjects and 12 patients with cold allodynia participated after giving their written informed consent. The study was approved by the Local Ethics Committee. The patients were recruited from the outpatients of the neurophysiological department of the Rikshospitalet University Hospital, Oslo, Norway. The patients had experienced cold injury of the type ‘‘non-freezing cold injuries” in the lower extremities during their service in the Norwegian army. Most soldiers had been exposed to temperatures slightly above freezing and wet conditions. At the time of the investigation they did not have any visible sequelae of the cold injury. However since these incidences they suffer from cold hypersensitivity in their lower extremities (pain upon exposure to cold) for more than 2 years. Patients with a combination of symptoms of cold hypersensitivity and the findings of cold allodynia were
Fig. 1. Schematic diagram illustrating the timing of the protocol and placement of stimuli on the foot. (a) On day 1 the psychophysical study was performed. At the dorsum of the foot the three rectangular places for application substances were marked. In the first randomly chosen place thermotesting was performed. During application of the first substance for 20 min the subject had to rate every minute cold, warm, itch and pain. After the removal of the substance thermotesting was performed again in the same place. Then in the second randomly chosen application place the protocol started again with thermotesting. After that in the third place the same was repeated. (b) On day 2 the axon-reflex-flare was scrutinized. At the dorsum of the other foot two rings were fixed with silicone. Two baseline scans with a Laser Doppler Imager were performed. Then menthol and cinnamaldehyde were applied each in one ring and airtight covered. During the application time of 20 min, 7 scans were performed. After removing the covers and substances one additional scan was performed.
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2.2. Application of substances L-Menthol was applied as a 40% w/v solution in ethanol (99.8%). Trans-cinnamaldehyde (99%) was applied as a 20% v/v solution in ethanol (99.8%). The control solution was ethanol (98%). Two hundred microliters of respective substances were applied on a 2.5 5 cm cellulose pad, which was placed under a 2.5 5 cm silicon chamber on the skin to avoid evaporation and leakage. Sensory ratings were obtained every minute during the 20 min drug application on a numerical rating scale (minimum 0 maximum 10) for pain, temperature (warm or cold) and itch separately. An interval of 20 min was kept between the applications.
2.3. Thermal thresholds Thermal detection thresholds and pain thresholds were determined directly before and after drug application at the area of substance application using a Peltier driven thermotest device (slope 1 °C/s) by the method of limits. Thresholds were determined as average of five successive stimuli for thermal detection and three successive stimuli for pain detection (10 s inter-stimulus intervals for pain for detection; SENSElab, Somedic, Horby, Sweden). The probe had a contact area of 2.5 5 cm (SENSElab, Somedic, Horby, Sweden). 2.4. Mechanical testing Hyperalgesia to pin prick was assessed using a 256 mN von Frey bristle with a flat tip. The von Frey bristle was applied at 1 Hz frequency and steps of 0.5 cm towards the application site starting from a distance of 5 cm or more when possible. The subject was instructed to verbally indicate, when the slight stinging changed to a more painful prolonged stinging. Dynamic mechanical allodynia was tested similarly by applying gentle strokes with a brush (brush 05, SENSElab, Somedic, Horby, Sweden). 2.5. Laser Doppler Imaging A Laser Doppler Imager (Perimed AB, Stockholm, Periscan PIM II, Sweden) was used to measure the area of axonreflex-erythema. For this part of the experiment 100 ll of the substances was applied to a cellulose pad that was placed inside a plastic ring (inner chamber: 1.5 cm diameter and 3 mm depth). This ring was carefully attached to the skin of the foot with silicone to prevent leakage. Scans were made at intervals of 3 min with two baseline scans before application and one scan thereafter. The flare reaction around the application site was analyzed offline with commercial software (LDPIwin; Perimed AB, Stockholm, Sweden). The area was calculated from 8 radii using a model for ellipses. All patients had undergone the testing with Menthol, but only 9 patients were tested with cinnamaldehyde due to the technical failure or time constrains of the patients. 2.6. Statistical analyses For comparison of the magnitude of sensations to the stimulation with the substances the area under the curve (AUC) was calculated for 20 ratings over 20 min.
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Statistical analysis was performed using the statistical software package (Statistica, TULSA). The Wilcoxon matched pairs test was used for statistical evaluation of temperature thresholds. For the comparison of the ratings an ANOVA with post hoc LSD testing was used. A p values < 0.05 were considered to be significant. All values are given as means ± SEM.
3. Results 3.1. Patients and healthy subjects Twelve young patients (mean age 28.5 years; range from 21.9 to 37.8 years) presented symptoms of cold hypersensitivity (localised pain upon exposure to cold) in their feet after suffering a cold injury. When thermotesting was performed in the affected area at their feet clear cold allodynia was found (mean cold pain threshold: 24.2 °C in comparison to healthy subjects 14.8 °C). However, in contrast to typical cold allodynia neuropathic pain patients, who generally report on rapidly increasing intolerable pain upon cooling up to pain threshold, our patients reported only minor pain intensity at threshold level that gradually increased upon further cooling. In comparison to an age-matched control group of the laboratory at Rikshospitalet the warm and cold detection thresholds were within the normal range (patients: mean warm detection threshold: 37.7 °C; mean cold detection threshold: 28.8 °C). Cold allodynia was the only abnormal finding in the patient group. There were no clinical signs of large fiber neuropathy (such as impaired reflexes or touch sensitivity) or mechanical hyperalgesia or allodynia. Nerve conduction studies including conduction velocity, motor amplitude and distal latency in posterior tibial and peroneal nerve as well as sensory amplitude and conduction velocity in the sural nerve were in the normal range. As a control group 10 healthy subjects (mean age 29.7 years min/max 19.3–38.9 years) participated in the study. Their temperature detection thresholds were normal (healthy subjects: warm detection threshold: 37.6 °C; cold detection threshold: 27.8 °C), when compared to the control values of the clinical control sample of the Rikshospitalet in Oslo. 3.2. Flare In all patients a weak local erythema at the menthol application site was observed by Laser Doppler scanning after the menthol was removed from the skin. However, none of the patients developed an axonreflex-flare during or after application of menthol. Cinnamaldehyde provoked an axon-reflex-flare in 7 of the 9 patients tested. The maximal mean flare area in these 7 patients was 10.2 ± 2.3 cm2. A specimen of the axon-reflex-flare after cinnamaldehyde application
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in contrast to the lacking axon-reflex-flare after menthol application is shown in 1 patient in Fig. 2. In 2 patients no widespread flare was observed, but only a small erythema exceeding the application site by 2 mm (mean value of 4 trajectories) was detectable. These 2 patients did not have any special common prop-
Fig. 2. Specimen of the axon-reflex-flare in a patient after stimulation with menthol (left ring) and Cinnamaldehyde (right ring) as depicted with a Laser Doppler Imager. Increased blood flow (vasodilation) is depicted in yellow and red colours. Menthol produced mild local vasodilation at the application site inside the ring, but no axon-reflexflare, whereas cinnamaldehyde evoked both local vasodilation and a widespread axon-reflex-erythema.
erties regarding clinical symptoms or experimental sensory ratings to the substances or results of thermotesting. 3.3. Psychophysics Time course of sensory ratings to cinnamaldehyde, menthol and ethanol control are shown in Fig. 3 for patients and controls. Menthol-induced pain and cold sensations in the patients did not exceed the ratings in the controls. Cinnamaldehyde initially provoked higher pain and warmth sensations in the control group. However, the area under the curve for all 20 ratings (AUC) did not differ significantly for pain (Fig. 4). Both the patients and the healthy subjects group had significantly higher ratings for the test substances cinnamaldehyde and menthol than for the control substance ethanol (ANOVA p < 0.001; post hoc LSD ethanol versus menthol and cinnamaldehyde: menthol p = 0.045 and cinnamaldehyde p = 0.015). Cinnamaldehyde or menthol application did not evoke a significant mechanical hyperalgesia or allodynia in either group. 3.3.1. Menthol Menthol produced a cold sensation in 5 of 10 healthy subjects (mean maximal rating of these 5 healthy sub-
Fig. 3. Time-course of sensory ratings to topical menthol stimulation in (a) and cinnamaldehyde stimulation in (b) and ethanol control in (c). Values are given as mean ratings ± SE including those persons who rated ‘‘0”.
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Fig. 4. Cumulative sensory ratings (area under the curve = AUC) are given for application of menthol in (a) and cinnamaldehyde in (b) and the control substance ethanol in (c). Values are given as means ± SE for all patients and subjects including those, who rated ‘‘0”. Significant differences between patients and subjects were found only for warmth sensation induced by cinnamaldehyde (bold line) (b). In the patient group warm sensation induced by menthol was significantly lower than cold and pain sensation (a), whereas cinnamaldehyde induced higher pain ratings as compared to warmth and cold (b). In healthy subjects menthol-induced more cold than pain (a) and cinnamaldehyde induced more warmth and pain as compared to cold sensation (b). Stars indicate a significant difference (ANOVA p < 0.05 post hoc LSD).
jects: 5.3) and a warm sensation in 7 of 10 healthy subjects (maximal rating of these 7 healthy subjects: 3.8). Three of these healthy subjects reported at the same time warm and cold sensations. In 5 of 10 healthy subjects menthol produced some moderate pain (maximal rating in these healthy subjects: 3.2). In patients menthol evoked a cold sensation in 9 of 12 patients (maximal rating of the 9 patients: 2.6). Two of these patients reported at the same time warm and cold sensations (mean warm maximal rating: 2; mean cold maximal rating: 2.5). Three patients did not report any temperature sensation. In 8 of 12 patients menthol produced some moderate pain (maximal rating of 8 patients: 2.5) (details for maximal ratings in Fig. 5).
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Fig. 5. Maximal ratings of healthy subjects and patients to topical menthol (a) and cinnamaldehyde (b) application and control substance ethanol (c). Values are given as means ± SE for those patients and subjects, whose rating was >0. The number of persons reporting the respective quality of sensation (cold, warmth panel, pain, itch) is depicted under the columns (n).
There were no significant differences in cold, warmth and pain ratings to menthol between healthy subjects and patients, neither in the maximal ratings (Fig. 5) of all healthy subjects and patients nor in the area under the curve (AUC) (Fig. 4) of all healthy subjects and patients. However the profile of sensations which menthol evoked was different between healthy subjects and patients (Figs. 4 and 5). Healthy subjects felt mostly cold to menthol followed by warmth and then by pain (ANOVA post hoc LSD p = 0.01 for cold against pain, AUC). In contrast, patients felt similar amounts of cold and pain whereas they hardly felt any warmth (ANOVA post hoc LSD p = 0.006 for cold against warmth and p = 0.02 for warmth against pain, AUC) (Fig. 4). 3.3.2. Cinnamaldehyde When stimulated with cinnamaldehyde, healthy subjects reported mainly burning heat pain (9/10 healthy subjects; mean AUC of 10 healthy subjects: heat: 33.3 SE 10.2; pain: 35.7 SE 6.6; cold: 25 SE 7) (post hoc
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LSD AUC p < 0.001 for heat and pain against cold). Six healthy subjects reported a pure warmth sensation (maximal rating of these: 4.4) and three subjects reported simultaneous warmth and cold sensations (maximal rating of these: cold: 4), but none reported a pure cold sensation (details for AUC in Fig. 4 and for maximal ratings in Fig. 5). The predominant sensation of the patients with cold allodynia to cinnamaldehyde was pain (8 of 12 patients; mean maximal rating of these 8 patients: 3.6; mean AUC of all 12 patients: 26 SE 6.7) (AUC rating of pain against heat and cold, post hoc LSD both p < 0.01). Patients felt significantly less warmth than healthy subjects to cinnamaldehyde (AUC rating of heat sensation, post hoc LSD p = 0.02) (details for AUC in Fig. 4 and for maximal ratings in Fig. 5). In contrast to the healthy subjects, patients reported simultaneous warmth and cold sensations of very low intensity to cinnamaldehyde (warmth: 3 of 12 patients; mean AUC of 12 patients: 10.9 SE 6.9; cold: 4 of 12 patients; mean AUC of 12 patients: 11.2 SE 5.2). Two patients reported pure warmth sensation (maximal rating: 4.5) and 3 patients a pure cold sensation (maximal rating: 2.3). Two of these had pure cold sensation without pain. One patient experienced simultaneous warm (4/10) and cold (4/10) sensations. 3.3.3. Ethanol Ethanol produced weak sensations of cold in 6 of 12 patients, warmth in 5 of 12 patients and mild pain in 2 healthy subjects (mean maximal rating in all: 1) (see Figs. 4 and 5). 3.3.4. Itch Itch was not the predominant sensation to any of the test substances or ethanol. Most itch was evoked by cinnamaldehyde (8 of 10 healthy subjects and 3 of 12 patients; mean maximal rating healthy subjects 2.9: SE 0.9; mean AUC: 6.9 SE 3.7; mean maximal rating patients: 0.6 SE 0.3; mean AUC 4.6: SE 2.5). Mentholinduced weak itch (3 of 10 healthy subjects and 2 of 12 patients) and only spurious itch was evoked by ethanol (0 of 10 healthy subjects and 1 of 12 patients) (see Figs. 4 and 5). 3.4. Thermal thresholds 3.4.1. Healthy subjects In all healthy subjects menthol produced a cold allodynia. The mean cold pain thresholds were sensitized from 14.4 to 21.1 °C (p = 0.01 Wilcoxon matched pairs). Warm and cold detection and heat pain detection thresholds did not change significantly following menthol. Cinnamaldehyde did not change the cold pain threshold, but affected temperature detection thresh-
olds and heat pain threshold. As expected from previous experiments, cinnamaldehyde induced a marked heat hyperalgesia. Heat pain threshold dropped from 44.1 to 39.2 °C (p = 0.005 Wilcoxon). The cold detection threshold was desensitized (26.7–24.4 °C; p = 0.02 Wilcoxon), whereas warmth detection threshold was sensitized (38.8–36.3 °C; p = 0.005 Wilcoxon) (Fig. 6). Ethanol did not significantly change any temperature thresholds (Fig. 6). 3.4.2. Patients In contrast to healthy subjects, menthol did not sensitize cold pain detection, i.e. the pre-existing cold allodynia was not enhanced (cold pain threshold before 24 after 25.6 °C). In 3 patients, the cold pain threshold was higher after menthol (less cold allodynia than before). In the other 8 patients the cold pain threshold decreased (4 patients) or did not change (4 patients). The 3 patients with an alleviation of cold allodynia did not differ in the cold detection thresholds compared to the other patients. The heat pain threshold was slightly but significantly sensitized from 43.4 to 42.6 °C, but cold and warmth detections were virtually unchanged. Cinnamaldehyde application induced a heat hyperalgesia (heat pain threshold from 43 to 39.8 °C) (p < 0.05). Similarly, warmth detection was sensitized. In contrast, cold detection and the cold pain detection thresholds were significantly desensitized (p < 0.05) (Fig. 6). 4. Discussion 4.1. TRPM8 and menthol In previous studies in healthy humans it was shown that topical menthol produced a marked cold allodynia in the forearm [13,20,35]. It was suggested that menthol sensitizes peripheral nociceptive C-fibers via the TRPM8 receptor and induces cold hyperalgesia by this peripheral receptor sensitisation. In our study, we could reproduce this result by application of menthol at the feet. In contrast, the pre-existing cold allodynia in the patients was not worsened by topical menthol. 4.1.1. Sensitisation One possible explanation for this unexpected finding could be a pre-existing peripheral sensitisation of TRPM8 receptors in the patients, induced by the cold injury. In this case, we would expect menthol to induce increased cold and pain ratings by the patients as they report following exposure to cold temperatures. Alternatively, the TRPM8 receptor could be sensitized specifically for physical cold without facilitating the menthol transduction. However, as cold detection thresholds were normal in the patients, this option is less probable.
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Fig. 6. Mean temperature thresholds ± SD before and after topical application of menthol, cinnamaldehyde and control (ethanol). * indicates significance p < 0.5; ** indicates significance p < 0.01. (a) Baseline warm detection thresholds were similar in healthy subjects and patients. Cinnamaldehyde induced in both healthy subjects and patients lowered warm detection thresholds. (b) Baseline cold detection thresholds were similar in healthy subjects and patients. Cinnamaldehyde induced in both healthy subjects and patients lowered cold detection thresholds. (c) Baseline heat pain detection thresholds were similar in healthy subjects and patients. Cinnamaldehyde evoked heat hyperalgesia in both subjects and patients. (d) Patients had a significant higher baseline cold pain threshold than healthy subjects (cold allodynia). Menthol sensitized cold pain in the subjects, resulting in cold hyperalgesia, whereas in patients no significant increase in (c).
Increased expression of TRPM8 has been shown in animal models of chronic constriction injury [11,25]. However, the interpretation of these findings is controversial. In one case increased expression of TRPM8 was thought to result in cold hyperalgesia [11]. Another study showed that the activation of TRPM8, which was overexpressed in a model of nerve injury, resulted in analgesia [25]. In human material from patients with traumatic nerve injury and diabetic neu-
ropathy [9], increased expression has been found, too. In different models of neuropathic pain in rodents, it has been found that stimulation of TRPM8 with icilin was effective in evoking analgesic effects [25]. The authors show evidence for increased expression of TRPM8 and a central mechanism in which glutamate is released from TRPM8-expressing cells, which inhibits nociceptive inputs [25]. Thus, stimulation of TRPM8 by menthol could trigger a central analgesic
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mechanism, by which the cold allodynia evoked during thermotesting could be suppressed by increasing the innocuous cold input by menthol. The analgesic effect was not seen in all patients. This could depend on the dose of applied menthol. Proudfoot et al. used a low dose of menthol (4–16 mM) to evoke analgesic effects. With higher doses they induced also proalgesic effects. Previously it has been shown that the blocking of innocuous cold input via thinly myelinated (A-deltafibers) could unmask cold allodynia mediated by Cfibers [12,23,34]. This notion has been supported by the clinical findings that in some patients cold allodynia coexists with cold hypesthesia [23]. However our patients did not have cold hyposensitivity as in previous reports [23]. 4.1.2. Expression of TRPM8 on other C-fiber classes New pathological expression of TRPM8 on neuron classes, which are usually involved in pain processing and sensitisation processes, could also contribute to cold hyperalgesia. In rodents, TRPM8 is expressed on small diameter trigeminal and DRG cells [16,21]. There is evidence for expression on nociceptive and non-nociceptive neurons [2]. In humans, based on the results from selective nerve blocking experiments, it was suggested that TRPM8 is expressed on C-fibers [35]. In microneurography experiments noxious and innocuous cold activated neurons in humans have been observed, which have a low conduction velocity suggesting that these are C-fibers [4,5]. Generally in humans, afferent C-nociceptors can be divided into different functional different classes: the classical mechano-sensitive polymodal nociceptors are thought to be responsible for the acute, well-localized burning pain. In contrast, mechano-insensitive-fibers play a special role in tonic pain, inflammation pain, central sensitisation and are responsible for the widespread axon-reflex-flare [29,36]. A significant new expression of TRPM8 on mechano-insensitive C-fibers could lead to cold allodynia via central sensitisation. However, menthol failed to provoke an axon-reflex-erythema in the patients. It could be argued that TRPM8 receptors on mechano-insensitive nociceptors are expressed to cause pain, but insufficient release of CGRP to provoke a measurable flare response. However it was shown that even very low frequency stimulation at 0.125 Hz provokes a strong flare response [29]. Thus, our results clearly speak against any role of TRPM8 expression on mechano-insensitive nociceptors as a mechanism for cold allodynia in patients with cold injury. Taken together, our results do not support peripheral TRPM8 sensitisation, over-expression or new expression on a different nociceptor class as mechanisms for cold allodynia in our patients.
4.2. TRPA1 and cinnamaldehyde In animal models TRPA1 had a proalgesic role in neuropathic pain. Increased expression of TRPA1 in hyperalgesia following peripheral inflammation and nerve lesion has been observed [22] and knocking down of TRPA1 can alleviate cold allodynia-related behaviour in rats [15]. We did not observe increased pain ratings to cinnamaldehyde or changed heat hyperalgesia in patients compared to healthy subjects. The effect of cinnamaldehyde on symptomatic areas in the patients was similar to the sensations and threshold changes produced in healthy subjects. TRPA1 is highly coexpressed with TRPV1 in small diameter peptidergic nociceptors [16]. As discussed above, the axon-reflex-flare reaction in humans depends crucially on mechano-insensitive C-fibers, that also vigorously respond to Capsaicin as TRPV1 agonist [29,30]. The area of axon-reflex-erythema has previously been used to test for thin fiber neuropathy [3,17]. The normal area of cinnamaldehyde induced flare response in the patients therefore speaks against any small fiber neuropathy, as also shown by their normal warmth and cold detection thresholds. In 3 of 12 patients the axon-reflex-flare was not tested due to technical reasons. However, the missing values are not expected to change the result as these patients did not differ in clinical picture or thermal thresholds from the other patients. The predominant sensation of healthy subjects to cinnamaldehyde was warmth and burning pain. The burning pain sensation did not differ significantly between patients and controls even though the time course was faster in healthy subjects. Interestingly, chemically induced warmth sensation was reduced in the patients following cinnamaldehyde and there was also a tendency of less warmth sensation to menthol stimulation. However, warmth detection- and heat pain thresholds were in the normal range. The exact role of TRPA1 in innocuous warmth sensation is unclear, but our results would suggest that warm sensations induced by temperature and chemical stimulation might be transduced differentially. 4.3. Mechanisms of cold allodynia evoked by cold injury Patients suffering from cold allodynia are heterogeneous: even though cold pain thresholds might be similar between neuropathic pain patients and cold injury, the neuropathic pain patients use to report intense pain at threshold level and will not tolerate further decrease of the temperature, whereas the cold injury patients have a pain sensation at threshold level which linearly increase upon further cooling. For neuropathic pain patients, even a slight cold breeze could evoke intense pain whereas the cold injury patients report of pain, but not intense pain in their limbs when exposed to colder temperatures.
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Patients, who suffer from sequelae of cold injury not only have pain, but also their autonomic and neurovascular response patterns are changed [1,10,26,31]. Among other changes they have a decreased cold-induced reflex vasodilation (Lewis’ ‘‘hunting” reaction) [1]. It has been shown previously that there is a correlation between skin vasoconstriction and magnitude of pain [18]. Thus, local changes in reflex vasodilation or other neurovascular or autonomic processes could lead to the unpleasantness of cold, without an involvement of specific changes of TRPM8 or TRPA1. Other cold specific receptors or channels such as background potassium channels [33] might contribute. Pronounced cold-induced slowing of conduction velocity might explain part of the symptoms as they can desynchronize the afferent signals in damaged nerves [8]. The sensory components induced by a cold challenge are complex [6,7] and cold pain threshold therefore might also be sensitized in patients suffering from cold intolerance [28] rather than from a specific sensitisation in the cold pain pathway. In our patients suffering from cold allodynia following cold injury, we found no evidence for sensitisation of TRM8 or TRPA1 or pathologic expression of TRPM8 on silent C-nociceptors. In patients with cold injury, cold allodynia might be evoked by local changes of neurovascular mechanisms in the skin and deeper tissue rather than from specific changes of TRPM8 or TRPA1. This pattern is expected to be different in patients with cold allodynia following traumatic nerve injury; further studies comparing different groups of cold allodynia patients are therefore warranted, especially to differentiate cold allodynia from cold intolerance. Conflict of interest There is no conflict of interest.
Acknowledgment The work was supported by the DFG Grant HA 183 14/2.
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[31] Shafer JC, Thompson AW. Local cold injury; a report of sequelae. AMA Arch Dermatol 1955;72:335–47. [32] Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, et al. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 2003;112:819–29. [33] Viana F, de la Pena E, Belmonte C. Specificity of cold thermotransduction is determined by differential ionic channel expression. Nat Neurosci 2002;5:254–60. [34] Wahren LK, Torebjork E, Jorum E. Central suppression of coldinduced C fibre pain by myelinated fibre input. Pain 1989;38:313–9. [35] Wasner G, Schattschneider J, Binder A, Baron R. Topical menthol – a human model for cold pain by activation and sensitization of C nociceptors. Brain 2004;127:1159–71. [36] Weidner C, Schmelz M, Schmidt R, Hansson B, Handwerker HO, Torebjork HE. Functional attributes discriminating mechanoinsensitive and mechano-responsive C nociceptors in human skin. J Neurosci 1999;19:10184–90.
Role of TRPM8 and TRPA1 for cold allodynia in patients with cold injury Barbara Namer a,*, Inge Petter Kleggetveit b, Hermann Handwerker a, Martin Schmelz c, Ellen Jorum b a
Department of Physiology and Pathophysiology, University of Erlangen, Universita¨tsstr. 17, 91054 Erlangen, Germany b Department of Clinical Neurophysiology, Rikshospitalet, Oslo, Norway c Department of Anesthesiology, Mannheim, University Heidelberg, Germany Received 18 September 2007; received in revised form 6 February 2008; accepted 3 March 2008
Abstract Local cold injury often induces hypersensitivity to cold and cold allodynia. Sensitisation of TRPM8 or TRPA1 could be the underlying mechanisms. This was evaluated by psychophysics and axon-reflex-flare induction following topical menthol and cinnamaldehyde application in cold injury patients and healthy subjects. The patients had no signs of neuropathy except cold allodynia. We applied 20% cinnamaldehyde and 40% menthol solutions in the cold-allodynic area of the patients and in a corresponding area in healthy subjects and obtained sensory ratings during application. Thermotesting and Laser Doppler Imaging were performed before and after exposure to the compounds. Menthol did not induce axon-reflex-erythema in patients or in controls. After menthol cold pain threshold was decreased in healthy subjects; however, no further sensitisation was observed in the patients moreover in some patients an amelioration of their cold allodynia was observed. Cinnamaldehyde-induced pain sensation did not differ between patients and controls. Heat pain thresholds following cinnamaldehyde were lowered to a similar extent in patients and controls (43– 39.8 and 44–39 °C) and also the axon-reflex-flare responses were comparable. No evidence for sensitisation of responses to TRPM8 or TRPA1-stimulation was found in patients with cold injury-induced cold allodynia. The lack of TRPM8 induced axon-reflex indicates that also de-novo expression of TRPM8 on mechano-insensitive C-nociceptors does not underlie cold allodynia in these patients. We conclude from these data that the mechanisms for the induction of cold allodynia in the patients with cold injury are independent of TRPM8 or TRPA1 and differ therefore from neuropathic pain patients. Ó 2008 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Cold hypersensitivity; Cold allodynia; Cold injury; Menthol; Cinnamaldehyde; Temperature threshold; Axon-reflex-flare; Psychophysics; TRPM8; TRPA1
1. Introduction Cold allodynia is a frequent finding following nerve lesions, but also in cold injury patients [27]. In a military environment 95% of examined soldiers with cold injury were found to have hypersensitivity to cold [27]. The exact physiologic role of various cold transducing recep*
Corresponding author. Tel.: +49 9131 85 22796; fax: +49 9131 85 22497. E-mail address: [email protected] (B. Namer).
tors and the mechanisms of cold allodynia in the different patients groups are still unclear. In the last years the molecular basis of cold transduction in primary afferent neurons has been expanded by cloning and characterization of two cold sensitive receptors, TRPM8 and TRPA1. TRPM8 is activated by moderate cold between 25 and 28 °C and can also be activated and sensitized chemically by menthol [19,24]. In humans, application of menthol results in cold hyperalgesia, possibly by sensitisation of this receptor on peripheral C-fibers [13,20,35]. Thus, the topical applica-
0304-3959/$34.00 Ó 2008 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2008.03.007
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tion of menthol could be a model for cold allodynia evoked by peripheral sensitisation in humans. TRPA1 can be activated by pungent agents such as cinnamaldehyde or mustard oil and it is still unclear whether this channel can be activated under physiological conditions by noxious cold below 17 °C or only when expressed heterologously [14,32]. In humans topical cinnamaldehyde application provokes burning warmth sensation and heat hyperalgesia [20]. In animal models a contribution of TRPA1 to cold hyperalgesia in neuropathic pain states has been suggested [15,22]. Moreover, NGFdependant increase in expression of TRPA1, but not TRPM8 in hyperalgesia following peripheral inflammation and nerve lesion has been observed [22]. Similarly, knocking down TRPA1 can alleviate cold allodyniarelated behaviour in rats [15]. TRPM8 seems to play a different role in cold hyperalgesia: stimulation of TRPM8 with icilin evoked analgesia in different animal models of neuropathic and inflammation pain and TRPM8 antisense oligonucleotides prevents this analgesia in CCl nerve injury [25]. Proalgesic involvement of TRPM8 in neuropathy-induced cold allodynia has not been proven so far. In the present study, we have investigated possible peripheral sensitisation mechanisms of menthol and cinnamaldehyde in Norwegian soldiers who had developed cold hypersensitivity and cold allodynia following cold injury. The aim of the study was to test the patients for hypersensitivity to menthol (TRPM8 involvement) and cinnamaldehyde (TRPA1 involvement) application. Psychophysics of sensations evoked by topical application of the substances and on changes of thermal thresholds were performed in soldiers and an age-matched control group of healthy subjects. Sensitisation of TRPA1 or de-novo expression of TRPM8 on mechanoinsensitive C-nociceptors was investigated indirectly by objective assessment of the axon-reflex-flare responses to menthol and cinnamaldehyde by Laser Doppler Imaging.
chosen to take part in the study. The diagnosis of cold allodynia was established by the assessment of thermal thresholds with a thermotest device (Somedic Sweden). Cold pain detection thresholds above 20 °C were regarded as pathological. Healthy control subjects were recruited from university staff and students in Erlangen, Germany and underwent a short clinical neurological examination and a short interview regarding neurological and dermatological diseases. They were included only if there was no hint for a neurological, dermatological or severe chronic disease. 2.1. Experimental protocol In the healthy subjects and patients, psychophysical experiments were conducted in a double blinded and randomized cross-over design. On day 1 the three substances were applied in random order for 20 min to the back of one foot in three different sites. The application sites were marked before to ascertain that distances between the application sides were at least 2 cm (Fig. 1a). To make sure that the experimenter could not identify the applied substance by its characteristic smell, he was wearing a face mask with small amounts of cinnamaldehyde dropped into the mask tissue. Before and after the application of the substances the temperature thresholds were tested in the respective areas. On day 2 the two test substances was applied to the contralateral foot to test for axon-reflexflare by Laser Doppler Imager (Perimed AB, Stockholm, Periscan PIM II, Sweden) (Fig. 1b). For details of the time course of the protocol see Fig. 1.
2. Methods Ten healthy subjects and 12 patients with cold allodynia participated after giving their written informed consent. The study was approved by the Local Ethics Committee. The patients were recruited from the outpatients of the neurophysiological department of the Rikshospitalet University Hospital, Oslo, Norway. The patients had experienced cold injury of the type ‘‘non-freezing cold injuries” in the lower extremities during their service in the Norwegian army. Most soldiers had been exposed to temperatures slightly above freezing and wet conditions. At the time of the investigation they did not have any visible sequelae of the cold injury. However since these incidences they suffer from cold hypersensitivity in their lower extremities (pain upon exposure to cold) for more than 2 years. Patients with a combination of symptoms of cold hypersensitivity and the findings of cold allodynia were
Fig. 1. Schematic diagram illustrating the timing of the protocol and placement of stimuli on the foot. (a) On day 1 the psychophysical study was performed. At the dorsum of the foot the three rectangular places for application substances were marked. In the first randomly chosen place thermotesting was performed. During application of the first substance for 20 min the subject had to rate every minute cold, warm, itch and pain. After the removal of the substance thermotesting was performed again in the same place. Then in the second randomly chosen application place the protocol started again with thermotesting. After that in the third place the same was repeated. (b) On day 2 the axon-reflex-flare was scrutinized. At the dorsum of the other foot two rings were fixed with silicone. Two baseline scans with a Laser Doppler Imager were performed. Then menthol and cinnamaldehyde were applied each in one ring and airtight covered. During the application time of 20 min, 7 scans were performed. After removing the covers and substances one additional scan was performed.
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2.2. Application of substances L-Menthol was applied as a 40% w/v solution in ethanol (99.8%). Trans-cinnamaldehyde (99%) was applied as a 20% v/v solution in ethanol (99.8%). The control solution was ethanol (98%). Two hundred microliters of respective substances were applied on a 2.5 5 cm cellulose pad, which was placed under a 2.5 5 cm silicon chamber on the skin to avoid evaporation and leakage. Sensory ratings were obtained every minute during the 20 min drug application on a numerical rating scale (minimum 0 maximum 10) for pain, temperature (warm or cold) and itch separately. An interval of 20 min was kept between the applications.
2.3. Thermal thresholds Thermal detection thresholds and pain thresholds were determined directly before and after drug application at the area of substance application using a Peltier driven thermotest device (slope 1 °C/s) by the method of limits. Thresholds were determined as average of five successive stimuli for thermal detection and three successive stimuli for pain detection (10 s inter-stimulus intervals for pain for detection; SENSElab, Somedic, Horby, Sweden). The probe had a contact area of 2.5 5 cm (SENSElab, Somedic, Horby, Sweden). 2.4. Mechanical testing Hyperalgesia to pin prick was assessed using a 256 mN von Frey bristle with a flat tip. The von Frey bristle was applied at 1 Hz frequency and steps of 0.5 cm towards the application site starting from a distance of 5 cm or more when possible. The subject was instructed to verbally indicate, when the slight stinging changed to a more painful prolonged stinging. Dynamic mechanical allodynia was tested similarly by applying gentle strokes with a brush (brush 05, SENSElab, Somedic, Horby, Sweden). 2.5. Laser Doppler Imaging A Laser Doppler Imager (Perimed AB, Stockholm, Periscan PIM II, Sweden) was used to measure the area of axonreflex-erythema. For this part of the experiment 100 ll of the substances was applied to a cellulose pad that was placed inside a plastic ring (inner chamber: 1.5 cm diameter and 3 mm depth). This ring was carefully attached to the skin of the foot with silicone to prevent leakage. Scans were made at intervals of 3 min with two baseline scans before application and one scan thereafter. The flare reaction around the application site was analyzed offline with commercial software (LDPIwin; Perimed AB, Stockholm, Sweden). The area was calculated from 8 radii using a model for ellipses. All patients had undergone the testing with Menthol, but only 9 patients were tested with cinnamaldehyde due to the technical failure or time constrains of the patients. 2.6. Statistical analyses For comparison of the magnitude of sensations to the stimulation with the substances the area under the curve (AUC) was calculated for 20 ratings over 20 min.
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Statistical analysis was performed using the statistical software package (Statistica, TULSA). The Wilcoxon matched pairs test was used for statistical evaluation of temperature thresholds. For the comparison of the ratings an ANOVA with post hoc LSD testing was used. A p values < 0.05 were considered to be significant. All values are given as means ± SEM.
3. Results 3.1. Patients and healthy subjects Twelve young patients (mean age 28.5 years; range from 21.9 to 37.8 years) presented symptoms of cold hypersensitivity (localised pain upon exposure to cold) in their feet after suffering a cold injury. When thermotesting was performed in the affected area at their feet clear cold allodynia was found (mean cold pain threshold: 24.2 °C in comparison to healthy subjects 14.8 °C). However, in contrast to typical cold allodynia neuropathic pain patients, who generally report on rapidly increasing intolerable pain upon cooling up to pain threshold, our patients reported only minor pain intensity at threshold level that gradually increased upon further cooling. In comparison to an age-matched control group of the laboratory at Rikshospitalet the warm and cold detection thresholds were within the normal range (patients: mean warm detection threshold: 37.7 °C; mean cold detection threshold: 28.8 °C). Cold allodynia was the only abnormal finding in the patient group. There were no clinical signs of large fiber neuropathy (such as impaired reflexes or touch sensitivity) or mechanical hyperalgesia or allodynia. Nerve conduction studies including conduction velocity, motor amplitude and distal latency in posterior tibial and peroneal nerve as well as sensory amplitude and conduction velocity in the sural nerve were in the normal range. As a control group 10 healthy subjects (mean age 29.7 years min/max 19.3–38.9 years) participated in the study. Their temperature detection thresholds were normal (healthy subjects: warm detection threshold: 37.6 °C; cold detection threshold: 27.8 °C), when compared to the control values of the clinical control sample of the Rikshospitalet in Oslo. 3.2. Flare In all patients a weak local erythema at the menthol application site was observed by Laser Doppler scanning after the menthol was removed from the skin. However, none of the patients developed an axonreflex-flare during or after application of menthol. Cinnamaldehyde provoked an axon-reflex-flare in 7 of the 9 patients tested. The maximal mean flare area in these 7 patients was 10.2 ± 2.3 cm2. A specimen of the axon-reflex-flare after cinnamaldehyde application
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in contrast to the lacking axon-reflex-flare after menthol application is shown in 1 patient in Fig. 2. In 2 patients no widespread flare was observed, but only a small erythema exceeding the application site by 2 mm (mean value of 4 trajectories) was detectable. These 2 patients did not have any special common prop-
Fig. 2. Specimen of the axon-reflex-flare in a patient after stimulation with menthol (left ring) and Cinnamaldehyde (right ring) as depicted with a Laser Doppler Imager. Increased blood flow (vasodilation) is depicted in yellow and red colours. Menthol produced mild local vasodilation at the application site inside the ring, but no axon-reflexflare, whereas cinnamaldehyde evoked both local vasodilation and a widespread axon-reflex-erythema.
erties regarding clinical symptoms or experimental sensory ratings to the substances or results of thermotesting. 3.3. Psychophysics Time course of sensory ratings to cinnamaldehyde, menthol and ethanol control are shown in Fig. 3 for patients and controls. Menthol-induced pain and cold sensations in the patients did not exceed the ratings in the controls. Cinnamaldehyde initially provoked higher pain and warmth sensations in the control group. However, the area under the curve for all 20 ratings (AUC) did not differ significantly for pain (Fig. 4). Both the patients and the healthy subjects group had significantly higher ratings for the test substances cinnamaldehyde and menthol than for the control substance ethanol (ANOVA p < 0.001; post hoc LSD ethanol versus menthol and cinnamaldehyde: menthol p = 0.045 and cinnamaldehyde p = 0.015). Cinnamaldehyde or menthol application did not evoke a significant mechanical hyperalgesia or allodynia in either group. 3.3.1. Menthol Menthol produced a cold sensation in 5 of 10 healthy subjects (mean maximal rating of these 5 healthy sub-
Fig. 3. Time-course of sensory ratings to topical menthol stimulation in (a) and cinnamaldehyde stimulation in (b) and ethanol control in (c). Values are given as mean ratings ± SE including those persons who rated ‘‘0”.
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Fig. 4. Cumulative sensory ratings (area under the curve = AUC) are given for application of menthol in (a) and cinnamaldehyde in (b) and the control substance ethanol in (c). Values are given as means ± SE for all patients and subjects including those, who rated ‘‘0”. Significant differences between patients and subjects were found only for warmth sensation induced by cinnamaldehyde (bold line) (b). In the patient group warm sensation induced by menthol was significantly lower than cold and pain sensation (a), whereas cinnamaldehyde induced higher pain ratings as compared to warmth and cold (b). In healthy subjects menthol-induced more cold than pain (a) and cinnamaldehyde induced more warmth and pain as compared to cold sensation (b). Stars indicate a significant difference (ANOVA p < 0.05 post hoc LSD).
jects: 5.3) and a warm sensation in 7 of 10 healthy subjects (maximal rating of these 7 healthy subjects: 3.8). Three of these healthy subjects reported at the same time warm and cold sensations. In 5 of 10 healthy subjects menthol produced some moderate pain (maximal rating in these healthy subjects: 3.2). In patients menthol evoked a cold sensation in 9 of 12 patients (maximal rating of the 9 patients: 2.6). Two of these patients reported at the same time warm and cold sensations (mean warm maximal rating: 2; mean cold maximal rating: 2.5). Three patients did not report any temperature sensation. In 8 of 12 patients menthol produced some moderate pain (maximal rating of 8 patients: 2.5) (details for maximal ratings in Fig. 5).
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Fig. 5. Maximal ratings of healthy subjects and patients to topical menthol (a) and cinnamaldehyde (b) application and control substance ethanol (c). Values are given as means ± SE for those patients and subjects, whose rating was >0. The number of persons reporting the respective quality of sensation (cold, warmth panel, pain, itch) is depicted under the columns (n).
There were no significant differences in cold, warmth and pain ratings to menthol between healthy subjects and patients, neither in the maximal ratings (Fig. 5) of all healthy subjects and patients nor in the area under the curve (AUC) (Fig. 4) of all healthy subjects and patients. However the profile of sensations which menthol evoked was different between healthy subjects and patients (Figs. 4 and 5). Healthy subjects felt mostly cold to menthol followed by warmth and then by pain (ANOVA post hoc LSD p = 0.01 for cold against pain, AUC). In contrast, patients felt similar amounts of cold and pain whereas they hardly felt any warmth (ANOVA post hoc LSD p = 0.006 for cold against warmth and p = 0.02 for warmth against pain, AUC) (Fig. 4). 3.3.2. Cinnamaldehyde When stimulated with cinnamaldehyde, healthy subjects reported mainly burning heat pain (9/10 healthy subjects; mean AUC of 10 healthy subjects: heat: 33.3 SE 10.2; pain: 35.7 SE 6.6; cold: 25 SE 7) (post hoc
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LSD AUC p < 0.001 for heat and pain against cold). Six healthy subjects reported a pure warmth sensation (maximal rating of these: 4.4) and three subjects reported simultaneous warmth and cold sensations (maximal rating of these: cold: 4), but none reported a pure cold sensation (details for AUC in Fig. 4 and for maximal ratings in Fig. 5). The predominant sensation of the patients with cold allodynia to cinnamaldehyde was pain (8 of 12 patients; mean maximal rating of these 8 patients: 3.6; mean AUC of all 12 patients: 26 SE 6.7) (AUC rating of pain against heat and cold, post hoc LSD both p < 0.01). Patients felt significantly less warmth than healthy subjects to cinnamaldehyde (AUC rating of heat sensation, post hoc LSD p = 0.02) (details for AUC in Fig. 4 and for maximal ratings in Fig. 5). In contrast to the healthy subjects, patients reported simultaneous warmth and cold sensations of very low intensity to cinnamaldehyde (warmth: 3 of 12 patients; mean AUC of 12 patients: 10.9 SE 6.9; cold: 4 of 12 patients; mean AUC of 12 patients: 11.2 SE 5.2). Two patients reported pure warmth sensation (maximal rating: 4.5) and 3 patients a pure cold sensation (maximal rating: 2.3). Two of these had pure cold sensation without pain. One patient experienced simultaneous warm (4/10) and cold (4/10) sensations. 3.3.3. Ethanol Ethanol produced weak sensations of cold in 6 of 12 patients, warmth in 5 of 12 patients and mild pain in 2 healthy subjects (mean maximal rating in all: 1) (see Figs. 4 and 5). 3.3.4. Itch Itch was not the predominant sensation to any of the test substances or ethanol. Most itch was evoked by cinnamaldehyde (8 of 10 healthy subjects and 3 of 12 patients; mean maximal rating healthy subjects 2.9: SE 0.9; mean AUC: 6.9 SE 3.7; mean maximal rating patients: 0.6 SE 0.3; mean AUC 4.6: SE 2.5). Mentholinduced weak itch (3 of 10 healthy subjects and 2 of 12 patients) and only spurious itch was evoked by ethanol (0 of 10 healthy subjects and 1 of 12 patients) (see Figs. 4 and 5). 3.4. Thermal thresholds 3.4.1. Healthy subjects In all healthy subjects menthol produced a cold allodynia. The mean cold pain thresholds were sensitized from 14.4 to 21.1 °C (p = 0.01 Wilcoxon matched pairs). Warm and cold detection and heat pain detection thresholds did not change significantly following menthol. Cinnamaldehyde did not change the cold pain threshold, but affected temperature detection thresh-
olds and heat pain threshold. As expected from previous experiments, cinnamaldehyde induced a marked heat hyperalgesia. Heat pain threshold dropped from 44.1 to 39.2 °C (p = 0.005 Wilcoxon). The cold detection threshold was desensitized (26.7–24.4 °C; p = 0.02 Wilcoxon), whereas warmth detection threshold was sensitized (38.8–36.3 °C; p = 0.005 Wilcoxon) (Fig. 6). Ethanol did not significantly change any temperature thresholds (Fig. 6). 3.4.2. Patients In contrast to healthy subjects, menthol did not sensitize cold pain detection, i.e. the pre-existing cold allodynia was not enhanced (cold pain threshold before 24 after 25.6 °C). In 3 patients, the cold pain threshold was higher after menthol (less cold allodynia than before). In the other 8 patients the cold pain threshold decreased (4 patients) or did not change (4 patients). The 3 patients with an alleviation of cold allodynia did not differ in the cold detection thresholds compared to the other patients. The heat pain threshold was slightly but significantly sensitized from 43.4 to 42.6 °C, but cold and warmth detections were virtually unchanged. Cinnamaldehyde application induced a heat hyperalgesia (heat pain threshold from 43 to 39.8 °C) (p < 0.05). Similarly, warmth detection was sensitized. In contrast, cold detection and the cold pain detection thresholds were significantly desensitized (p < 0.05) (Fig. 6). 4. Discussion 4.1. TRPM8 and menthol In previous studies in healthy humans it was shown that topical menthol produced a marked cold allodynia in the forearm [13,20,35]. It was suggested that menthol sensitizes peripheral nociceptive C-fibers via the TRPM8 receptor and induces cold hyperalgesia by this peripheral receptor sensitisation. In our study, we could reproduce this result by application of menthol at the feet. In contrast, the pre-existing cold allodynia in the patients was not worsened by topical menthol. 4.1.1. Sensitisation One possible explanation for this unexpected finding could be a pre-existing peripheral sensitisation of TRPM8 receptors in the patients, induced by the cold injury. In this case, we would expect menthol to induce increased cold and pain ratings by the patients as they report following exposure to cold temperatures. Alternatively, the TRPM8 receptor could be sensitized specifically for physical cold without facilitating the menthol transduction. However, as cold detection thresholds were normal in the patients, this option is less probable.
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Fig. 6. Mean temperature thresholds ± SD before and after topical application of menthol, cinnamaldehyde and control (ethanol). * indicates significance p < 0.5; ** indicates significance p < 0.01. (a) Baseline warm detection thresholds were similar in healthy subjects and patients. Cinnamaldehyde induced in both healthy subjects and patients lowered warm detection thresholds. (b) Baseline cold detection thresholds were similar in healthy subjects and patients. Cinnamaldehyde induced in both healthy subjects and patients lowered cold detection thresholds. (c) Baseline heat pain detection thresholds were similar in healthy subjects and patients. Cinnamaldehyde evoked heat hyperalgesia in both subjects and patients. (d) Patients had a significant higher baseline cold pain threshold than healthy subjects (cold allodynia). Menthol sensitized cold pain in the subjects, resulting in cold hyperalgesia, whereas in patients no significant increase in (c).
Increased expression of TRPM8 has been shown in animal models of chronic constriction injury [11,25]. However, the interpretation of these findings is controversial. In one case increased expression of TRPM8 was thought to result in cold hyperalgesia [11]. Another study showed that the activation of TRPM8, which was overexpressed in a model of nerve injury, resulted in analgesia [25]. In human material from patients with traumatic nerve injury and diabetic neu-
ropathy [9], increased expression has been found, too. In different models of neuropathic pain in rodents, it has been found that stimulation of TRPM8 with icilin was effective in evoking analgesic effects [25]. The authors show evidence for increased expression of TRPM8 and a central mechanism in which glutamate is released from TRPM8-expressing cells, which inhibits nociceptive inputs [25]. Thus, stimulation of TRPM8 by menthol could trigger a central analgesic
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mechanism, by which the cold allodynia evoked during thermotesting could be suppressed by increasing the innocuous cold input by menthol. The analgesic effect was not seen in all patients. This could depend on the dose of applied menthol. Proudfoot et al. used a low dose of menthol (4–16 mM) to evoke analgesic effects. With higher doses they induced also proalgesic effects. Previously it has been shown that the blocking of innocuous cold input via thinly myelinated (A-deltafibers) could unmask cold allodynia mediated by Cfibers [12,23,34]. This notion has been supported by the clinical findings that in some patients cold allodynia coexists with cold hypesthesia [23]. However our patients did not have cold hyposensitivity as in previous reports [23]. 4.1.2. Expression of TRPM8 on other C-fiber classes New pathological expression of TRPM8 on neuron classes, which are usually involved in pain processing and sensitisation processes, could also contribute to cold hyperalgesia. In rodents, TRPM8 is expressed on small diameter trigeminal and DRG cells [16,21]. There is evidence for expression on nociceptive and non-nociceptive neurons [2]. In humans, based on the results from selective nerve blocking experiments, it was suggested that TRPM8 is expressed on C-fibers [35]. In microneurography experiments noxious and innocuous cold activated neurons in humans have been observed, which have a low conduction velocity suggesting that these are C-fibers [4,5]. Generally in humans, afferent C-nociceptors can be divided into different functional different classes: the classical mechano-sensitive polymodal nociceptors are thought to be responsible for the acute, well-localized burning pain. In contrast, mechano-insensitive-fibers play a special role in tonic pain, inflammation pain, central sensitisation and are responsible for the widespread axon-reflex-flare [29,36]. A significant new expression of TRPM8 on mechano-insensitive C-fibers could lead to cold allodynia via central sensitisation. However, menthol failed to provoke an axon-reflex-erythema in the patients. It could be argued that TRPM8 receptors on mechano-insensitive nociceptors are expressed to cause pain, but insufficient release of CGRP to provoke a measurable flare response. However it was shown that even very low frequency stimulation at 0.125 Hz provokes a strong flare response [29]. Thus, our results clearly speak against any role of TRPM8 expression on mechano-insensitive nociceptors as a mechanism for cold allodynia in patients with cold injury. Taken together, our results do not support peripheral TRPM8 sensitisation, over-expression or new expression on a different nociceptor class as mechanisms for cold allodynia in our patients.
4.2. TRPA1 and cinnamaldehyde In animal models TRPA1 had a proalgesic role in neuropathic pain. Increased expression of TRPA1 in hyperalgesia following peripheral inflammation and nerve lesion has been observed [22] and knocking down of TRPA1 can alleviate cold allodynia-related behaviour in rats [15]. We did not observe increased pain ratings to cinnamaldehyde or changed heat hyperalgesia in patients compared to healthy subjects. The effect of cinnamaldehyde on symptomatic areas in the patients was similar to the sensations and threshold changes produced in healthy subjects. TRPA1 is highly coexpressed with TRPV1 in small diameter peptidergic nociceptors [16]. As discussed above, the axon-reflex-flare reaction in humans depends crucially on mechano-insensitive C-fibers, that also vigorously respond to Capsaicin as TRPV1 agonist [29,30]. The area of axon-reflex-erythema has previously been used to test for thin fiber neuropathy [3,17]. The normal area of cinnamaldehyde induced flare response in the patients therefore speaks against any small fiber neuropathy, as also shown by their normal warmth and cold detection thresholds. In 3 of 12 patients the axon-reflex-flare was not tested due to technical reasons. However, the missing values are not expected to change the result as these patients did not differ in clinical picture or thermal thresholds from the other patients. The predominant sensation of healthy subjects to cinnamaldehyde was warmth and burning pain. The burning pain sensation did not differ significantly between patients and controls even though the time course was faster in healthy subjects. Interestingly, chemically induced warmth sensation was reduced in the patients following cinnamaldehyde and there was also a tendency of less warmth sensation to menthol stimulation. However, warmth detection- and heat pain thresholds were in the normal range. The exact role of TRPA1 in innocuous warmth sensation is unclear, but our results would suggest that warm sensations induced by temperature and chemical stimulation might be transduced differentially. 4.3. Mechanisms of cold allodynia evoked by cold injury Patients suffering from cold allodynia are heterogeneous: even though cold pain thresholds might be similar between neuropathic pain patients and cold injury, the neuropathic pain patients use to report intense pain at threshold level and will not tolerate further decrease of the temperature, whereas the cold injury patients have a pain sensation at threshold level which linearly increase upon further cooling. For neuropathic pain patients, even a slight cold breeze could evoke intense pain whereas the cold injury patients report of pain, but not intense pain in their limbs when exposed to colder temperatures.
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Patients, who suffer from sequelae of cold injury not only have pain, but also their autonomic and neurovascular response patterns are changed [1,10,26,31]. Among other changes they have a decreased cold-induced reflex vasodilation (Lewis’ ‘‘hunting” reaction) [1]. It has been shown previously that there is a correlation between skin vasoconstriction and magnitude of pain [18]. Thus, local changes in reflex vasodilation or other neurovascular or autonomic processes could lead to the unpleasantness of cold, without an involvement of specific changes of TRPM8 or TRPA1. Other cold specific receptors or channels such as background potassium channels [33] might contribute. Pronounced cold-induced slowing of conduction velocity might explain part of the symptoms as they can desynchronize the afferent signals in damaged nerves [8]. The sensory components induced by a cold challenge are complex [6,7] and cold pain threshold therefore might also be sensitized in patients suffering from cold intolerance [28] rather than from a specific sensitisation in the cold pain pathway. In our patients suffering from cold allodynia following cold injury, we found no evidence for sensitisation of TRM8 or TRPA1 or pathologic expression of TRPM8 on silent C-nociceptors. In patients with cold injury, cold allodynia might be evoked by local changes of neurovascular mechanisms in the skin and deeper tissue rather than from specific changes of TRPM8 or TRPA1. This pattern is expected to be different in patients with cold allodynia following traumatic nerve injury; further studies comparing different groups of cold allodynia patients are therefore warranted, especially to differentiate cold allodynia from cold intolerance. Conflict of interest There is no conflict of interest.
Acknowledgment The work was supported by the DFG Grant HA 183 14/2.
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