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.
Table 1.
Distribution of TRPM8 mRNA in the DRG after nerve injury.
Table 2.
Distribution of TRPA1 mRNA in the DRG after nerve injury.
Table 3.
Distribution of TRPA1 mRNA in the DRG after inflammation.
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.
Table 4.
Temperature threshold and amplitude of Ca2+ transients in DRG neurons after nerve injury.