Involvement of the melanocortin-1 receptor in acute pain and pain of inflammatory but not neuropathic origin

Abstract

Background: Response to painful stimuli is susceptible to genetic variation. Numerous loci have been identified which contribute to this variation, one of which, MC1R, is better known as a gene involved in mammalian hair colour. MC1R is a G protein-coupled receptor expressed in melanocytes and elsewhere and mice lacking MC1R have yellow hair, whilst humans with variant MC1R protein have red hair. Previous work has found differences in acute pain perception, and response to analgesia in mice and humans with mutations or variants in MC1R.

Methodology and principal findings: We have tested responses to noxious and non-noxious stimuli in mutant mice which lack MC1R, or which overexpress an endogenous antagonist of the receptor, as well as controls. We have also examined the response of these mice to inflammatory pain, assessing the hyperalgesia and allodynia associated with persistent inflammation, and their response to neuropathic pain. Finally we tested by a paired preference paradigm their aversion to oral administration of capsaicin, which activates the noxious heat receptor TRPV1. Female mice lacking MC1R showed increased tolerance to noxious heat and no alteration in their response to non-noxious mechanical stimuli. MC1R mutant females, and females overexpressing the endogenous MC1R antagonist, agouti signalling protein, had a reduced formalin-induced inflammatory pain response, and a delayed development of inflammation-induced hyperalgesia and allodynia. In addition they had a decreased aversion to capsaicin at moderate concentrations. Male mutant mice showed no difference from their respective controls. Mice of either sex did not show any effect of mutant genotype on neuropathic pain.

Conclusions: We demonstrate a sex-specific role for MC1R in acute noxious thermal responses and pain of inflammatory origin.

Figure 1. Behavioural responses to acute mechanical or noxious thermal stimuli in naïve animals.

Adult male and female Mc1r^e and their littermate controls Tg-Mc1r^e, or A^y-Jkn1 and their littermate control non-A^y-Jkn1 mice were assessed for responses to acute noxious thermal or non-noxious mechanical stimuli. The paw withdrawal latency (PWL; n = 8–11 per genotype) [A], paw withdrawal threshold (PWT; n = 8–11 per genotype) [B] as measures of thermal hyperalgesia and mechanical allodynia respectively of both hind-paws (right and left hind-paw responses combined) in naïve animals are shown as mean responses ± SEM. Significant differences between Mc1r^e and A^y-Jkn1 and their respective controls were only seen in female Mc1r^e mice in [A] paw withdrawal latency (***P<0.01).

Figure 2. Effect of MC1R functionality on the Formalin Response.

Data represents the number of paw flinch or flick responses per min ± SEM following the intraplantar injection of formalin in [A] male and [B] female adult Mc1r^e, Tg-Mc1r^e, A^y-Jkn1and control non-A^y-Jkn1 mice (n = 5–9 per genotype). There was no significant difference in the formalin response occurring over time (0–60 min post-injection) for male Mc1r^e or A^y-Jkn1 when compared to their control groups Tg-Mc1r^e and control non-A^y-Jkn1 respectively. Significant differences for female Mc1r^e or A^y-Jkn1 when compared to their respective control groups Tg-Mc1r^e and control non-A^y-Jkn1 are shown *P<0.05; **P<0.01 (Mc1r^e compared to Tg-Mc1r^e) and †P<0.05; ††P<0.01; †††P<0.001 (A^y-Jkn1 compared to control non-A^y-Jkn1).

Figure 3. Effect of MC1R functionality on CFA-induced thermal hyperalgesia and mechanical allodynia.

Data represents the paw withdrawal latency [A], paw withdrawal threshold [B] as measures of thermal hyperalgesia and mechanical allodynia prior to and post-injection of CFA (n = 4–5 per genotype), shown as mean responses ± SEM. Differences in behaviours prior to injection were observed in female Mc1r^e mice only, when compared to their control littermates, Tg-Mc1r^e in [A] paw withdrawal latency (††P<0.01). CFA-induced increased ipsilateral sensitivity (IPSI; injured hindlimb) compared to contralateral (CON; un-injured hindlimb) are shown (*P<0.05). Female Mc1r^e mice were found to have a significantly higher ipsilateral thresholds when compared to their control littermates, Tg-Mc1r^e ipsilateral thresholds in [A] paw withdrawal latency and [B] paw withdrawal threshold at 0.5–2 hours post-injection (¶P<0.05).

Figure 4. Behavioural analysis of CCI-induced thermal hyperalgesia and mechanical allodynia.

Data represent the paw withdrawal latency [A], paw withdrawal threshold [B] as measures of thermal hyperalgesia and mechanical allodynia respectively in male and female Mc1r^e, Tg-Mc1r^e, A^y-Jkn1 and control non-A^y-Jkn1 adult mice, prior to- and post-surgery shown as the area under the curve (AUC; which measures the total change over the time course (baseline to post-surgery day 21) of CCI-induced thermal hyperalgesia and mechanical allodynia; n = 5–6 per genotype). AUC's were calculated from raw data by the standard trapezoidal rule. No significant differences in either male or female mice were observed.

Figure 5. Concentration-dependent aversion to capsaicin consumption.

Concentration related aversion to capsaicin in adult [A] male and [B] female Mc1r^e and their control littermates Tg-Mc1r^e (n = 6–7 per genotype). Data is expressed as an aversion index ranging from −1 to 1, which measures the degree to which the solution was avoided during the 10 day exposure at each capsaicin concentration tested ± SEM and was calculated by subtracting capsaicin consumed from water consumed, differences in Mc1r^e consumption of capsaicin are shown (**P<0.01) compared to control Tg-Mc1r^e group. [C] Shows the concentration threshold for capsaicin (at 0.83 µM) in the first trial of male and female mice, followed by a repeat trial in naïve animals (not previously assessed for oral aversion) data is expressed as the volume capsaicin containing solution consumed as a percentage of body weight ± SEM during the 10 day exposure, differences in Mc1r^e consumption of capsaicin at 0.83 µM are shown (††P<0.01) compared to control Tg-Mc1r^e group.