All procedures involving animals were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. This study was approved by the Animal Care and Use Committee of Kagoshima University (permission #D17008, D19036).

Ten-week-old male C57BL/6J mice were purchased from Japan SLC, Inc. (Shizuoka, Japan) and housed in a temperature-controlled (24–25°C) quiet room under a 12/12-h light/dark cycle [i.e., light on was defined as Zeitgeber time (ZT) 0, light off was defined as ZT12] for at least 10 days, with food and water available ad libitum. Thirty-two mice were used for the formalin test and the immunohistochemistry experiments (saline-daytime; n = 8, saline-nighttime; n = 8, formalin-daytime; n = 8, formalin-nighttime, n = 8), and 18 mice were used for quantitative RT-PCR (qPCR) experiments (daytime; n = 9, nighttime; n = 9). Although the sample size was not pre-determined using statistical methods, the sample size for this study was set with reference to previous studies in which the formalin test was performed in the facial region of mice (Luccarini et al., 2006; Zhang et al., 2018).

Mice were placed individually in custom-made experimental cages (315 mm × 185 mm × 245 mm), with mirrors on three sides of a clear plastic box. Formalin was prepared from stock formalin (an aqueous solution of 37% formaldehyde; FUJIFILM Wako Chemicals, Osaka, Japan) and diluted to 5% in saline. After a 30-min habituation, either formalin (5%, 10 μL) or saline (10 μL) was injected subcutaneously into the right upper lip using a 26-gauge needle attached to a Hamilton (701, Hamilton Company, Reno, NV, United States) syringe. After the injection, the mouse was immediately returned to the experimental cage and recorded for 45 min using an infrared camera (HX-A1H; Panasonic, Tokyo, Japan). Because the camera was capable of capturing images in the dark field, we performed recordings under infrared light using C-Light (Sun Mechatronics, Tokyo, Japan), which did not affect the mice at night. Rubbing the injection site with the forepaw was defined as pain-related behavior (PRB), and the duration of time for which animals exhibited this behavior was measured cumulatively. The recording time of 45 min was divided into 15 blocks of 3 min each, and we distinguished two phases following the formalin injection [phase I (0–3 min) and phase II (15–39 min)], in accordance with a previous report (Luccarini et al., 2006). The duration of PRB was assessed by an experimenter who was blinded to the treatment condition. The experiments were conducted at two time points: at daytime (ZT6) and nighttime (ZT18).

After the formalin test (2 h after formalin injection, ZT8 or ZT20), the mice were anesthetized by an intraperitoneal injection of sodium pentobarbital (60 mg/kg) and perfused intracardially with 10 mL of 5 mM sodium phosphate (pH 7.4)-buffered 0.9% saline (PBS), followed by 50 mL of 4% formaldehyde, 75%-saturated picric acid, and 0.1 M Na₂HPO₄ (adjusted with NaOH to pH 7.0). The brains were removed and post-fixed for 3 h at 4°C with the same fixative. After cryoprotection with 30% sucrose in PBS, the caudal brainstems were cut into 40-μm-thick serial coronal sections on a freezing microtome, and the sections were collected serially in PBS. Every third section was processed for further immunohistochemical analysis and all subsequent incubations were performed at room temperature (24–26°C). The sections were incubated overnight with 1 μg/mL rabbit antibody to c-Fos (ABE457; Sigma-Aldrich, St Louis, MO, United States) or 1 μg/mL rabbit antibody to calcitonin gene-related peptide (CGRP) (C8198; Sigma) in PBS containing 0.3% Triton X-100 (PBS-X), 0.12% lambda-carrageenan, 0.02% sodium azide, and 1% donkey serum (AB237258; Jackson Immuno Research Laboratories, Inc., Bar Harbor, ME, United States) (PBS-XCD). After rinsing with PBS-X, the sections were incubated for 1 h with 10 μg/mL biotinylated anti-rabbit IgG donkey antibody (AP182P; Merck Millipore, Burlington, MA, United States) and then for 1 h with avidin-biotinylated peroxidase complex (ABC) (1:100; Elite variety, Vector, Burlingame, CA, United States) in PBS-X. After rinsing in PBS, the bound peroxidase was developed into a brown precipitate by reaction for 10–20 min with 0.02% diaminobenzidine-4HCl (DAB; D5637-1G, Sigma) and 0.003% H₂O₂ in 50-mM Tris-HCl (pH 7.6). All stained sections were serially mounted onto glass slides, dried, dehydrated in an ethanol series, cleared in xylene, and finally mounted with a coverslip. Sections adjacent to sections stained for c-Fos were counterstained for Nissl with 0.2% cresyl violet to identify the cytoarchitecture. The cytoarchitecture of the brainstem, including the Sp5C, was mainly determined according to the atlas of Paxinos and Franklin (2012). Laminae boundaries were determined with reference to previous studies using mice (Romero-Reyes et al., 2013) and in CGRP-immunostained sections with adequate reference to the study by Sugimoto et al. (1997), since CGRP-immunoreactivity is considered to be selectively located in laminae I/II of the Sp5C.

Sections were automatically captured in a large color image through the normal mode of a TOCO digital slide scanner (CLARO, Aomori, Japan) with a 10 × objective lens (Zeiss, Oberkochen, Germany). In the normal mode, the scanner obtained 11 images with differently focused images at each site, selected the best-focused image at each site, and fused the partially overlapping images into a large image (Ohno et al., 2012). For each image, the average number of c-Fos positive cells in the superficial layer (laminae I/II) of Sp5C (8.12–8.24 mm posterior to bregma) ipsilateral to the injection side was counted manually using CANVAS X software (ACD Systems International Inc., Victoria, BC, Canada). The c-Fos positive cells in each section were checked under a microscope equipped with a 20 × objective lens (SPlanApo20; numerical aperture = 0.7; Nikon Tokyo, Japan), while frequently changing the microscopic focus. At least three sections per animal were analyzed, and the average numbers of c-Fos-positive cells during daytime and nighttime with and without formalin were compared.

The mice of this group were housed under the same conditions as those used in the formalin test. After euthanizing the mice by cervical dislocation, the TG on both sides was immediately removed at ZT8 (daytime) and ZT20 (nighttime). Total RNA was extracted using ISOGEN (Nippon Gene, Toyama, Japan), as recommended by the manufacturer. One microgram of total RNA was reverse-transcribed to cDNA by using oligo-dT adapter primers and ReverTra Ace (TOYOBO, Osaka, Japan). A cDNA equivalent of 2 ng of total RNA was used for qPCR. The qPCR reactions were performed with an ABI 7300 real-time PCR system (Applied Biosystems, Foster City, CA, United States) using THUNDERBIRD^® qPCR Mix (TOYOBO, Osaka, Japan). β-actin was used as the loading control. cDNA was amplified as follows: one cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 10 s, 55°C for 10 s, and 72°C for 30 s. Each experiment was performed in triplicate. The primer sequences used in this experiment are provided below (Sasaguri et al., 2018; Tomita et al., 2019).

All results are presented as means ± standard errors of the means (SEM), and differences were considered statistically significant at P < 0.05. The two-way analysis of variance (ANOVA), post hoc Scheffé’s F tests, and unpaired Student’s t-tests were used for comparative analyses.

First, we reproduced a model of acute, persistent pain by injecting formalin into the right upper lip of mice (Figure 1A) and examined whether PRBs showed day-night differences at the behavioral level. Figure 1B shows the time course of the PRBs in the daytime and nighttime observed after the injection of saline or formalin into the right upper lip (saline-daytime; n = 8, saline-nighttime; n = 8, formalin-daytime; n = 8, formalin-nighttime, n = 8). The mean duration of the PRBs was plotted for each 3-min bin over the 45-min post-injection observation period. The PRB duration in the formalin group appeared to be longer in the nighttime than in the daytime, but there was no statistically significant difference in any 3-min bin. At nighttime, the formalin group showed a characteristic bimodal pattern with two peaks. The first peak emerged immediately after the administration of formalin, and the second one appeared at approximately 18–21 min.

Figure 1C shows the total duration of PRBs. First, for the two-way ANOVA, we defined the factor as the time of the experiment (daytime or nighttime) and the condition as the type of solution injected (saline or formalin). Next, we examined whether these two affect each other, and found that there was the interaction (F₁,₂₈ = 5.152, P = 0.031; two-way ANOVA). Accordingly, we conducted a multiple comparison test for all groups. The saline group did not show a significant difference in the duration of PRBs between the daytime and nighttime. Next, on comparing injections of 5% formalin and saline, there was no significant increase in the duration of PRBs in the daytime (saline-daytime; 69.4 ± 8.0 s, formalin-daytime; 128.6 ± 24.0 s), but there was a significant increase in the duration of PRBs in the nighttime (saline-nighttime; 69.1 ± 8.9 s, formalin-nighttime; 203.6 ± 19.4 s, P < 0.001, Scheffé’s F test). Interestingly, in the formalin group, the duration of PRBs was found to be significantly longer in the nighttime than in the daytime (P = 0.031, Scheffé’s F test). These results indicate that the pain sensitivity was higher at nighttime than at daytime when assessed at the animal behavioral level.

Since previous studies have suggested that phase I primarily reflects the pain response due to the direct stimulation of nerve endings by formalin and phase II reflects a secondary inflammatory pain response (Shibata et al., 1989), the duration of PRBs was divided into phase I (0–3 min) and phase II (15–39 min) (Figure 1D). In this figure, phase I and phase II represent a total PRB duration of 3 min and 24 min, respectively. In phase I, two-way ANOVA was performed for two factors, namely, the solution injected (saline or formalin) and the time of the experiment (daytime or nighttime); there was no significant difference for time of the experiment (F₁,₂₈ = 1.299, P = 0.264), but there was a significant difference for the solution injected (F₁,₂₈ = 7.109, P = 0.013), with no interaction (F₁,₂₈ = 3.436, P = 0.074). Thus, in phase I, there was a significant variation depending on the solution injected. In phase II, as in phase I, we first conducted a two-way ANOVA and found that there was the interaction between the solution injected and the time of the experiment (F₁,₂₈ = 5.365, P = 0.028). Next, a multiple comparison test showed that there was no significant difference in the saline group (daytime; 24.6 ± 4.4 s, nighttime; 32.3 ± 7.2 s). In the formalin group, PRB duration was of longer at nighttime than at daytime (daytime; 43.5 ± 5.3 s, nighttime; 91.5 ± 14.3 s, Scheffé’s F test, P = 0.006). These results indicate that the inflammatory pain response was greater during the nighttime than during the daytime.

We subsequently examined how nociceptive stimuli received by peripheral nerves were transmitted to secondary neurons in the central nervous system. We used c-Fos as a marker of nociception in the present study because c-Fos has previously been shown to be an excellent marker for examining neurons activated by various nociceptive stimuli (Hunt et al., 1987; Bullitt, 1991; Nomura et al., 2002). In previous studies in rats, c-Fos expression was overwhelmingly observed on the ipsilateral side in comparison with the contralateral side (Veres et al., 2017), and it was observed in the rostrocaudal direction throughout the Sp5C (Zhou et al., 1999). Furthermore, it has been reported in studies using mice that the expression of c-Fos-positive cells is mainly found in laminae I and II, which receive nociceptive-specific input, as well as in the spinal dorsal horn (Romero-Reyes et al., 2013). Therefore, in this study, we counted the number of c-Fos positive cells located in laminae I/II (Figures 2A–D) of the Sp5C on the ipsilateral side of the formalin injection and found a difference, as shown in Figures 2E–L. The results for c-Fos expression were obtained from eight animals in each group that underwent the formalin test without any deficiency. The numbers of c-Fos-positive cells in laminae I/II of the Sp5C per section in the daytime and nighttime were 10.3 ± 2.5 and 14.5 ± 2.9 after the injection of saline, and 63.0 ± 11.1 and 114.5 ± 19.1 after the injection of formalin, respectively, (Figure 2N). We also conducted a two-way ANOVA and found that there was the interaction between the solution injected and the time of the experiment (F₁,₂₈ = 4.435, P = 0.044). The formalin groups showed statistically significantly higher numbers of c-Fos-positive cells compared to those in the saline groups, both during the daytime and during the nighttime (daytime, P = 0.023 and nighttime, P < 0.001, Scheffé’s F test). Furthermore, the number of c-Fos-positive cells did not significantly differ between daytime and nighttime in the saline groups, but was significantly higher during the nighttime than during the daytime in the formalin groups (P = 0.028, Scheffé’s F test).

We searched for the causes of the day-night differences in nociception in the Sp5C. Because the stimuli applied to the upper lip were the same in both the daytime and the nighttime, we hypothesized that there would be day-night differences in the expression of transducers that convert nociceptive stimuli into electrical activity. Therefore, we performed qPCR to investigate gene expression changes in the TG, where the cell bodies of nociceptive neurons are located. We targeted TRPA1, which is reported to be receptive to a variety of chemical stimuli, including formalin. Figure 3 shows that the mRNA expression levels of TRPA1 in the TG were significantly higher at nighttime than during the daytime (P < 0.001, unpaired Student’s t-test).