The contribution of TRPM8 and TRPA1 channels to cold allodynia and neuropathic pain

Abstract

Cold allodynia is a common feature of neuropathic pain however the underlying mechanisms of this enhanced sensitivity to cold are not known. Recently the transient receptor potential (TRP) channels TRPM8 and TRPA1 have been identified and proposed to be molecular sensors for cold. Here we have investigated the expression of TRPM8 and TRPA1 mRNA in the dorsal root ganglia (DRG) and examined the cold sensitivity of peripheral sensory neurons in the chronic construction injury (CCI) model of neuropathic pain in mice.In behavioral experiments, chronic constriction injury (CCI) of the sciatic nerve induced a hypersensitivity to both cold and the TRPM8 agonist menthol that developed 2 days post injury and remained stable for at least 2 weeks. Using quantitative RT-PCR and in situ hybridization we examined the expression of TRPM8 and TRPA1 in DRG. Both channels displayed significantly reduced expression levels after injury with no change in their distribution pattern in identified neuronal subpopulations. Furthermore, in calcium imaging experiments, we detected no alterations in the number of cold or menthol responsive neurons in the DRG, or in the functional properties of cold transduction following injury. Intriguingly however, responses to the TRPA1 agonist mustard oil were strongly reduced.Our results indicate that injured sensory neurons do not develop abnormal cold sensitivity after chronic constriction injury and that alterations in the expression of TRPM8 and TRPA1 are unlikely to contribute directly to the pathogenesis of cold allodynia in this neuropathic pain model.

Figure 1. Time course of cold and (-)-menthol sensitivity following sciatic nerve ligation.

(a) Cold sensitivity assessed by acetone response score where 0 = no response, 0.5 = licking, 1 = flinching and brushing of the paw, 2 = strong flinching, 3 = strong flinching and licking. Circles, CCI operated animals (n = 6) and triangles, control animals (n = 6). *P<0.05 CCI against control, two-way repeated measures ANOVA followed by Student-Newman-Keuls test. (b), Menthol evoked paw licking duration. Filled circles, CCI operated mice treated with (-)-menthol (250 mM). Open circles, CCI mice with vehicle (90%DMSO, 10% PBS). Filled triangles, control mice (-)-menthol. Open triangles, control mice vehicle. All values are mean±SEM. *P<0.05 CCI (-)-menthol against control (-)-menthol, two-way repeated measures ANOVA followed by Student-Newman-Keuls test.

Figure 2. Quantitative reverse-transcription PCR.

mRNA levels for (a), TRPM8 (b), TRPA1 (c), galanin, and (d) TREK-1 in the mouse. Levels are expressed relative to GAPDH in control animals and at 2, 7 and 14 days after surgery. I indicates ipsilateral to the injury, C, contralateral. (e) qRT-PCR for TRPM8 and (f) TRPA1 in the rat at 14 days post injury. L4 and L5 indicate respective ganglia. *P<0.05 ipsilateral versus contralateral, paired T-test. All values are mean±SEM (n = 6 animals for each group).

Figure 3. Distribution of TRPM8 and TRPA1 in identified neuronal subpopulations in DRG.

Combined in situ hybridization of TRPM8 (red) with (a), immunohistochemistry (green) for CGRP, (b), IB4 and (c), NF200. TRPA1 mRNA expression (red) with (d), CGRP, (e), IB4 and (f), NF200 (green). Scale bar 40 µm.

Figure 4. Representative recordings of Ca²⁺ transients in DRG neurons from control mice.

Responses to (a), cooling, (b), mustard oil and (c), KCl (note different Y-axis scale for KCl).

Figure 5. Proportions of cold, (-)-menthol and mustard oil responsive DRG neurons in control animals, at 7 and 14 days post-CCI and at 48 hours post CFA.

(a), Cell-size histogram in control mice and in mice at 7 and 14 days post CCI injury. (b), Percentage of cold-responsive neurons in the DRG (n = 6–15 mice). (c), Percentage of neurons responsive to 100 µM menthol (n = 6–15 mice). (d), Concentration-response profile for menthol. (e), Percentage of mustard oil-responsive neurons (n = 3 mice in each group). (f), Percentage of neurons responding to mustard oil in DRG ipsilateral and contralateral to CFA injection (n = 3 mice). (g), Maximum amplitude of Ca²⁺ transients in mustard oil sensitive neurons following CFA injection. (h), Percentage of neurons in the DRG responsive to both (-)-menthol and cold (n = 6–11). (i), Percentage of neurons in the DRG insensitive to (-)-menthol but responsive to cold (n = 6–11). I indicates ipsilateral to the injury, C contralateral. *P<0.05 ipsilateral versus contralateral, paired T-test. All values are mean±SEM.