Diurnal Variation in Trigeminal Pain Sensitivity in Mice

Abstract

Management of time and circadian disruption is an extremely important factor in basic research on pain and analgesia. Although pain is known to vary throughout the day, the mechanism underlying this circadian variation remains largely unknown. In this study, we hypothesized that the process of pain transmission to the central nervous system (after receiving nociceptive stimuli from outside the body) would show day-night differences. Ten-week-old male mice were kept under a strict 12/12-h light/dark cycle for at least 10 days. Formalin was then injected into the second branch region of the trigeminal nerve and the duration of pain-related behaviors (PRBs) was assessed. Immunohistochemical staining was then performed, and the c-Fos-immunopositive cells in the trigeminal spinal tract subnucleus caudalis (Sp5C) were counted. The results showed that the duration of PRBs was longer and the number of c-Fos immunopositive cells in the Sp5C was higher at nighttime than during the day. In addition, the trigeminal ganglia (TG) were extracted from the mice and examined by quantitative real-time PCR to evaluate the daytime and nighttime expression of nociceptive receptors. The results showed that the mRNA expression of transient receptor potential ankyrin 1 in the TG was significantly higher at night than during the day. These results suggest that pain in the trigeminal nerve region is more intense at nighttime, when rodents are active, than during the daytime, partly due to differences in nociceptor expression.

Keywords: TRPA1; day-night difference; formalin test; pain; trigeminal nervous system.

FIGURE 1

Injection site of the solution and temporal differences in pain-related behaviors (PRBs) in mice. Injection site of the solution (A). Time courses of PRBs observed after the injection of either saline or formalin into the upper lip at daytime and nighttime. The mean number of seconds that mice spent rubbing is plotted for each 3-min bin over the 45-min post-injection observation period (B). Total duration of PRBs observed at 45 min after formalin injection. Statistical analysis was performed using two-way ANOVA followed by Scheffé’s F-test. ^∗P < 0.05 and ^∗∗∗P < 0.001, respectively, (C). Subtotal durations of PRB during phases I (0–3 min) and II (15–39 min) (Statistical analysis was performed using two-way ANOVA followed by Scheffé’s F-test. ^∗∗P < 0.01 and ^∗∗∗P < 0.001, respectively) (D). Sample size, n = 8 in each group. Each value presents the mean ± standard error of the mean.

FIGURE 2

Temporal differences in the c-Fos expression in the spinal tract subnucleus caudalis (Sp5C). The cytoarchitecture and lamina structure of the Sp5C (A–D). Calcitonin gene-related peptide (CGRP) immunoreactivity was observed selectively in laminae I/II, but not laminae III/IV, of the Sp5C (C,D). c-Fos immunoreactivity in laminae I/II of the Sp5C ipsilateral to the saline injection site [(E) daytime; (F) nighttime]or formalin site [(G) daytime; (H) nighttime)] injection. The representative distribution of c-Fos positive cells in laminae I/II of the Sp5C (J–M). The c-Fos-positive cells in each section were plotted onto a coronal plane under a microscope with a 20 × objective lens (SPlanApo20; numerical aperture = 0.7; Nikon Tokyo, Japan), while frequently changing the microscopic focus. Images of c-Fos positive cells at high magnification with a 40 × objective lens (UPlanSApo40; numerical aperture = 0.9; Olympus Tokyo, Japan) (I). The number of c-Fos immunoreactive cells in laminae I/II of the Sp5C [(N) mean ± standard error of the mean, ^∗P < 0.05, ^∗∗∗P < 0.001, Scheffé’s F-test). In the formalin group, the number of c-fos-expressing cells was higher at nighttime than at daytime. cu, cuneate fasciculus; MdD, medullary reticular nucleus, dorsal part; MdV, medullary reticular nucleus, ventral part; pyx, pyramidal decussation; sp5, spinal trigeminal tract; for the other abbreviations, see the text. The scale bar in panel (C) applies to panels (A,C), that in panel (D) applies to panels (B,D–H), and that in panel (M) applies to panels (J–M).

FIGURE 3

Temporal differences in the levels of transient receptor potential ankyrin 1 (TRPA1) mRNA expression in trigeminal ganglia. The relative value at nighttime when the daytime mRNA expression was set to 1 (mean ± standard error of the mean, ^∗∗∗P < 0.001, Student’s t-test).

FIGURE 4

Schematic diagram of the mechanism of diurnal variation in pain sensitivity. During the nighttime, trigeminal ganglia (TG) might express transient receptor potential ankyrin 1. (TRPA1) to a greater extent than that during the daytime and activate the pain-sensation pathway more intensively. This would cause more vigorous and long-lasting pain-related behaviors (PRBs) in mice at nighttime than at daytime. See text for more details.