The circadian rhythm is an endogenous clock that has an approximately 24 hour cycle. Clinical trials have demonstrated significant differences in nociceptive sensitivities depending on the circadian rhythm in various diseases, such as burning mouth syndrome [1] and symptomatic knee osteoarthritis [2]. In animal studies, the thermal nociceptive sensitivity of male mice shows daily variation [3]. These findings suggest that pain perception could be affected by circadian rhythm.

Accumulating data from many studies imply that a nuclear receptor, REV-ERBα, regulates circadian rhythm. REV-ERBα is an orphan nuclear receptor, which is encoded by the NR1D1 gene [4,5]. REV-ERBα not only regulates cyclic Bmal1 expression but also controls Clock and Cry1 mRNA expression [6]. BMAL1 and CLOCK are transcription factors, which play a central role in the circadian rhythm by forming a BMAL1/CLOCK heterodimer [7]. Moreover, abnormal phase-shifting of the circadian system was observed in KO mice [6]. However, the role of REV-ERBα in nociceptive sensitivity is still unknown. In this study, we examined acute and chronic pain sensitivities in KO mice.

To investigate the nociceptive sensitivity of KO mice, we measured acute and chronic pain sensitivities. In addition, we examined whether nociceptive sensitivity of KO mice is affected by circadian rhythm. Both male and female mice were used in each pain test. There are no significant differences between both groups (Data not shown). The acute pain test and chronic pain test were performed at CT 08-12 and CT 22-02 because KO mice had previously been shown to have abnormal mood and anxiety-like behavior at CT 08-12 but not CT 22-02 [10]. First, we examined acute nociceptive sensitivity by measuring their basal thermal perception. In the tail flick test, KO mice and WT mice did not show any significant difference in their latency to respond (Fig. 1A). However, in the hot plate test, the latency to respond to thermal stimuli was decreased in both KO mice and WT mice at CT 08-12 (Fig. 1B). There was no difference in acute nociceptive sensitivity depending on the genotype.

Next, we decided to examine whether KO mice showed any change in chronic pain depending on circadian rhythm. Baseline mechanical nociceptive sensitivity on Day 0 did not show any significant difference between both genotypes (Fig. 2A). However, paw withdrawal thresholds were significantly reduced on Day 3 and Day 7 in both genotypes compared with Day 0. One-way repeated measures ANOVA analysis suggests that mechanical nociceptive sensitivity was increased by injection of CFA and maintained until Day 7 in all groups (Fig. 2A). Interestingly, the withdrawal threshold in KO mice decreased on Day 3 (genotype p=0.0034) compared with WT mice, but not Day 7 (genotype p=0.7066) (Fig. 2B~D). Overall, all groups did not show any alteration of mechanical nociceptive sensitivity depending on circadian rhythm. Taken together, our results suggest that the KO of REV-ERBα partially increased mechanical nociceptive sensitivity but not thermal nociceptive sensitivity.

In the present study, we showed that KO mice exhibit increased mechanical nociceptive sensitivity in electronic von Frey test, and this phenomenon was not dependent on the circadian rhythm. Both WT and KO mice showed altered acute nociceptive sensitivity in accordance with circadian rhythm in the hot plate test. Therefore, our hot plate test results support a previous study showing that thermal nociceptive sensitivity of male mice undergoes daily variation [3]. On the other hand, in the tail flick test, both WT and KO mice did not show altered acute nociceptive sensitivity in relation to the circadian rhythm, and this discrepancy may stem from the differences between the methods.

Deletion of REV-ERBα increased mechanical nociceptive sensitivity but the increase was not maintained. This suggests that the deletion of REV-ERBα only transiently increases the mechanical nociceptive sensitivity. Although the transient nature of the increased nociception is yet to be explained, we propose several explanations to account for the enhanced mechanical nociceptive sensitivity in KO mice. A previous study has shown that KO mice have elevated dopamine levels in the midbrain and that this dopaminergic hyperactivity induces mood-related behavioral changes [10]. Accumulating evidence implies that pain perception is affected by both dopamine activity and mood differences. For example, it is well known that elevated dopamine activity produces anti-nociceptive effects [11,12,13]. In spite of elevated dopamine activity, KO mice showed increased mechanical nociceptive sensitivity. Dopamine is known to contribute to mood regulation [14,15]. A number of studies reported that mood alteration and pain are reciprocally associated [16,17,18]. Chung et al. reported that KO mice showed abnormal mood-related behavior observed in CT12 [10]. However, our results revealed that KO mice exhibited mechanical nociceptive sensitivity changes in both CT0 and CT12. Therefore, our results support the point that mechanical nociceptive sensitivity changes are not coincident with mood alteration in KO mice. Taken together, our results suggest that enhanced dopamine levels and mood alteration may not directly modulate mechanical nociceptive sensitivity in KO mice. The detailed mechanisms should be further investigated.

One possible explanation for the enhanced nociceptive sensitivity in KO mice is the increased level of pro-inflammatory cytokines [19]. Gibbs et al. reported that KO mice have elevated expression of pro-inflammatory cytokines, such as IL-6, CXCL1, CCL5 and CCL2, with a significant increase of IL-6 expression in particular [20]. Previous findings provide evidence that injection of IL-6 to rodent hind paws induces hyperalgesia in a dose-dependent manner [21]. In contrast, IL-6 deficiency in mice leads to lowered nociceptive responses to mechanical and thermal stimulation compared to WT controls [22]. Therefore, increased levels of IL-6 and pro-inflammatory cytokines may account for hyperalgesia observed in KO mice.

Another interpretation of the increased mechanical nociceptive sensitivity of KO mice may be that fatty acid binding proteins (FABP) are dysfunctional in pain perception. FABPs act as intracellular transporters and bind to endocannabinoids such as anandamide (AEA) [23] and N-acylethanolamines (NAEs) [24]. The endocannabinoid ligands activate cannabinoid receptor 1 (CB1) and produce anti-nociceptive effects [25,26]. Overexpression of FABPs enhances AEA uptake and, subsequently, hydrolysis of AEA, whereas inhibition of FABPs attenuates inactivation of AEA [23]. Therefore, inhibition of FABPs has caused analgesia in various pain models [27]. FABP5- and FABP7-deficient mice have a high level of AEA and NAEs in the brain and show analgesia in inflammatory pain models [28]. FABP7 mRNA expression is known to be regulated by REV-ERBα, and FABP7 is highly expressed in KO mice [29]. Therefore, the excessive expression of FABP7 may have contributed to causing the mechanical nociceptive sensitivity change in the KO mice in the present study.

In conclusion, we found that KO mice exhibit temporally increased sensitivity in mechanical nociception, and this transient change in mechanical nociceptive sensitivity was not dependent on circadian rhythm. Further studies are required to investigate the molecular mechanisms of nociceptive sensitivity changes in KO mice and examine the nociceptive sensitivity of KO mice in other pain models.