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Regulation of the NR2B-CREB-CRTC1 Signaling Pathway Contributes to Circadian Pain in Murine Model of Chronic Constriction Injury

Xia, Tianjiao PhD, MD; Cui, Yin MD; Qian, Yue MD; Chu, Shuaishuai MD; Song, Jia MD; Gu, Xiaoping PhD, MD; Ma, Zhengliang PhD, MD

doi: 10.1213/ANE.0000000000000991
Pain and Analgesic Mechanisms: Research Report

BACKGROUND: Numerous clinical investigations have revealed the circadian rhythm changes in the perception of chronic pain, and most clinical chronic pain types peak in the night. However, it is still undiscovered whether circadian rhythm of pain exists in rodents and the specific mechanism that may underlie it. Our study was conducted to investigate the rhythmic changes of hyperalgesia behavior in a chronic constrictive injury (CCI) model of rodents and to explore the role of the N-methyl-d-aspartate receptor 2B (NR2B)-cAMP response element binding protein (CREB)-CREB-regulated transcription coactivator 1 (CRTC1) signaling pathway in this pain rhythm.

METHODS: A CCI operation was performed to mimic clinical chronic pain. Paw mechanical withdrawal threshold and paw withdrawal thermal latency were used to test pain behavior in rats; a von Frey cilia test was used to test mechanical hyperalgesia in mice at Zeitgeber time (ZT) 4, ZT10, ZT16, and ZT22 for 14 contiguous days. The relative mRNA and protein expression of NR2B, CREB and CRTC1 in the suprachiasmatic nuclei and the dorsal horn were measured by real-time polymerase chain reaction and Western blot. CRTC1 and CREB interference adenovirus vectors were injected intrathecally at 2 time points, respectively (ZT12 and ZT0), to further explore the proper time point for pain treatment.

RESULTS: During the period of chronic pain state, the pain behavior of CCI rodents showed a circadian rhythm with the peak at ZT4 or ZT10 daily. The pain thresholds were significantly different between the activity period and the rest period. The expressions of NR2B, CRTC1, and CREB at the spinal level were consistent with the pain rhythm. The intrathecal treatment with CRTC1 or CREB interference adenovirus from day 7 to day 9 after CCI surgery markedly improved pain behaviors. Nevertheless, when given at ZT0, they were both more effective at relieving peak pain than drugs given at ZT12.

CONCLUSIONS: Pain behavior in the chronic pain of CCI displayed circadian rhythm and was associated with circadian secretion of pain-related receptors. The NR2B-CREB-CRTC1 signaling pathway may play a crucial role in this rhythm. Moreover, our results suggest that measures to relieve pain should be taken before pain reaches its peak.

Published ahead of print October 5, 2015

From the Department of Anesthesiology, Affiliated Drum Tower Hospital of Medical Department, Nanjing University, Nanjing, Jiangsu, China.

Accepted for publication July 20, 2015.

Published ahead of print October 5, 2015

Funding: This research was supported by National Natural Science Foundation of China (81371207, 81171047, 81070892, and 81171048), Natural Science Foundation of Jiangsu Province (BK2010105), and the Grant from the Department of Health of Jiangsu Province of China (XK201140, RC2011006).

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Zhengliang Ma, PhD, MD, Department of Anesthesiology, Affiliated Drum Tower Hospital of Medical Department of Nanjing University, 321 Zhong Shan Rd., Nanjing, Jiangsu 210008, P.R. China. Address e-mail to 13813996903@126.com; mazhengliang1964@nju.edu.cn.

Various physiologic functions of the organism usually establish regular cycles to adapt to the diurnal changes in the external environment. A variety of human physiologic activities have a clear circadian rhythm. Years of clinical investigation have found that different types and regions of chronic pain, such as spinal stenosis, carpal tunnel syndrome, and neuropathic pain caused by diabetes mellitus, are also affected by circadian rhythm.1–3 Therefore, a study on the rhythm changes and signal transduction mechanisms of chronic pain has significant meaning for exploring clinical pain treatment.

The important neurophysiologic basis of chronic pain is the pain sensitization, which is one of the manifestations of neuronal plasticity changes. N-methyl-D-aspartate receptors (NMDARs) are pain-related substances expressed in the postsynaptic membrane, and they are related to neuronal plasticity. NMDAR channels are heteromers composed of the key receptor subunit NMDAR1 and NMDAR2 subunits: NMDAR2A, NMDAR2B (NR2B), NMDAR2C, and NMDAR2D. The NMDAR2 subunit acts as the agonist binding site for glutamate (Glu) and is the predominant excitatory neurotransmitter receptor in the mammalian brain. The NR2B subunit is important in the formation and development of a variety of pain types. Nonselective NMDA receptor antagonists, or selective antagonists of the NR2B subunit, can relieve a variety of pain types, including neuropathic pain.4,5 In addition, the overexpression of NR2B or reduction of NR2B degradation can significantly prolong long-term potentiation.6 NR2B-mediated Ca2+ influx can stimulate adenylate cyclase to produce second messenger stimulation of adenylyl cyclase adenosine 3′, 5′-cyclic phosphate (3′, 5′-cyclic adenosine monophosphate [cAMP]), which can activate a family of kinases, such as PKA (protein kinase A), CaMKII (calcium/calmodulin-dependent protein kinase II), and CaMKIV (calcium/calmodulin-dependent protein kinase type IV), initiating downstream signal transduction.7 Among these, the transcription factor cAMP response element (CRE) binding protein (CREB) is closely linked to synaptic plasticity, and its expression is regulated by circadian rhythms.8

CREB is a nucleoprotein located in the nucleus that can highly selectively bind to CRE and is required for the transcription of many genes.9 At the same time, studies have found that the CREB/CRE transcriptional pathway is a possible molecular mechanism for adjusting the biological clock, and light affects the biological rhythms of the suprachiasmatic nuclei area.10 In addition to CREB itself, there are some coactivators that participate in the CREB signaling pathway; CRTCs (CREB-regulated transcription coactivator) is one of the CREB cofactor families.11 It has a highly selectivity for the CRE of genes and plays an important role in synaptic plasticity and pain modulation. When intracellular calcium or cAMP is increased, CRTCs are dephosphorylated via a calcineurin-dependent mechanism and facilitate CREB-mediated transcription.12 As a pain-related factor, CRTC1 is related to a significant increase in pain. In addition, in physiologic conditions, CRTC1 secretion had more rhythmic activity in daylight than in the evening in mice.13

On the basis of research, we propose the following hypothesis: pain perception could have a connection with circadian rhythm, and the NR2B-CREB-CRTC1 signaling pathway may play a role in it. This study observed the rhythmic changes of chronic pain symptoms, including allodynia and hyperalgesia, as well as the expression of NR2B, CREB, and CRTC1 in the central oscillator and the dorsal horn. The chronic constrictive injury (CCI) model was performed to mimic clinical chronic pain. CREB and CRTC1 interference adenoviral vectors were used as exogenous interventions to investigate the mechanism of the NR2B-CREB-CRTC1 signaling pathway in pain rhythm. Our study aimed to provide new ideas for the treatment of chronic pain.

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METHODS

Animals

Adult male Sprague-Dawley rats and 2-month-old male C57BL/6 mice were housed under a 12-hour light, 12-hour dark cycle (light, Zeitgeber time [ZT] 0–12; dark, ZT12–24), with food and water available ad libitum. All animal procedures were approved by the Institutional Animal Care and Use Committee and were performed by individuals with appropriate licenses. Every effort was made to minimize animal suffering and to use the minimal number of animals necessary to obtain valid results.

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Surgery

Mice were anesthetized by sodium pentobarbital anesthesia (50 mg/kg intraperitoneally). The CCI surgery was performed in accordance with the method described by Bennett and Xie.14 The plantar aspect of the right hind paw was prepared in a sterile manner with povidone-iodine solution, and the foot was placed under a sterile drape with a hole. A longitudinal 1-cm incision was made through the skin and fascia. After exposing the sciatic nerve, chromic catgut was used to perform 2 ligations, spaced approximately 1 mm, to not affect the blood supply to the outer membrane of the nerve. After hemostasis with gentle pressure, the skin was closed with two 5-0 nylon mattress sutures.

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Tissue Collection

Tissues were sampled after cervical dislocation, and fresh tissue samples from spinal cord dorsal horn were collected by laminectomy. Lumbar spinal segments were divided into the ipsilateral and contralateral side, and then the dorsal part separated. Coronal hypothalamic slices (500 μm) were prepared through the suprachiasmatic nuclei (SCN) using a rodent brain matrix (RBM-2000C; ASI Instruments, Warren, MI). The SCNs were taken using a flat-tripped 25-G needle (internal diameter approximately 0.5 mm) under the optical microscope. Tissues were immediately frozen at −80°C.

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Behavioral Testing

Hyperalgesia of CCI was tested in the murine right hind limb at 4 time points (ZT4, ZT10, ZT16, and ZT22) after modeling.

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Thermal Hyperalgesia Test in Rats

Thermal hyperalgesia was determined according to the method adopted by Hargreaves et al.15 Rats were placed in clear plastic cages on an elevated glass plate. Before testing, they were allowed to acclimatize for 30 minutes. A radiant thermal stimulator (Dynamic Plantar Analgesiometer, Model 37370; Ugo Basile, Varese, Italy) was focused onto the plantar surface of the hind paw through the glass plate. The characteristic lifting or licking of the hind paw was used to evaluate the nociceptive end points in the radiant heat test, and the time to the end point was considered the paw withdrawal thermal latency (PWTL). Heat stimuli were delivered at 5-minute intervals for 5 trials per rat. A cutoff time of at least 30 seconds was used to avoid potential tissue damage.

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Mechanical Allodynia Test in Rats

Mechanical allodynia was assessed using a Dynamic Plantar Analgesiometer (Model 37370, Ugo Basile) on each hind paw. Metal wire was placed on the center of the plantar surface with increased pressure applied, and the pressure of the wire stopped increasing when the rat’s paws lifted. The instrument automatically recorded a value that was regarded as the paw withdrawal mechanical threshold (PWMT). Each stimulus was delivered at 5-minute intervals for 5 trials per rat. A cutoff time of at least 30 seconds was used to avoid potential tissue damage.

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Mechanical Allodynia Test in Mice

Mechanical allodynia in mice was assessed using calibrated von Frey filaments (0.008–300g bending force; Stoelting Co., Wood Dale, IL). Mice were placed in clear plastic cages on a transparent Plexiglass box placed at a distance of approximately 30-cm-high bench wire rack (grid: 0.5 cm × 0.5 cm) above the desk and allowed to acclimatize for 30 minutes before testing. After various exploratory activities disappeared, we used a series of standardized von Frey cilia (0.16, 0.4, 0.6, 1.0, 1.4, and 2.0 g) to vertically stab the right hind plantar skin of the mice and keep the cilia bent for 6 to 8 seconds. Lifted foot or paw-licking behaviors were deemed a positive reaction within the stimulation time or removal of von Frey cilia and vice versa for negative reactions. The PWMT was determined by sequentially increasing and decreasing the stimulus strength (the “up-and-down” method), as described previously.16 Each stimulus strength was tested 5 times at an interval of at least 30 seconds, and the minimal cilia strength that generated a positive reaction 3 times would be treated as PWMT.

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Western Blot

Tissue samples were homogenized in lysis buffer. The homogenate was centrifuged at 12,000 rpm for 20 minutes at 4°C and the supernatant removed. The protein concentration was determined using the bicinchoninine acid assay (BCA) method, a detergent-compatible protein assay with bovine serum albumin as a standard. Samples (50 μg) were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8%) and transferred onto a polyvinylidene fluoride (PVDF) membrane. The filter membranes were blocked with 5% bovine serum albumin for 2 hours at room temperature and incubated with the primary antibody (β-actin [1:1000 dilution; Abcam, Cambridge, UK]; anti-phosphor-Tyr1472 NR2B [1:1000 dilution; Abcam]; anti-phosphor-Ser133 CREB [1:1000 dilution; Abcam]; anti-CRTC1 [1:1000 dilution; Abcam]) for a night at 4°C. The membrane was washed with Tris-Buffered Saline with Tween 20 (TBST) buffer and incubated with the secondary antibody for 2 hours at room temperature and visualized using the Immobilon Western Chemiluminescent HRP Substrate (Thermo Fisher Scientific, Waltham, MA). The loading and blotting of equal levels of proteins were verified by reprobing the membrane with antibody against β-actin. Densitometry was performed using the Quantity One software (Bio-Rad, Hercules, CA) and expressed as the mean of the integrated volume after subtracting the background.

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Real-Time Polymerase Chain Reaction

Total RNA from the tissue was extracted using a PureLink RNA Mini Kit (12183020; Invitrogen, Waltham, MA) according to the manufacturer’s protocol. The RNA concentrations were determined using UV spectrophotometry (Biotek, Winooski, MA). Total RNA was reverse transcribed with a cDNA Reverse Transcriptase Kit (TaKaRa, RR036A; TaKaRa, Shiga, Japan). Real-time quantitative polymerase chain reaction (PCR) was performed using an ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, MA) and SYBR Premix Ex Taq (TaKaRa, RR420A), with the following gene-specific primers: β-actin forward primer 5′-CTGTCCCTGTATGCCTCTG-3′ and reverse primer 5′-ATGTCACGCACGATTTCC-3′; NR2B forward primer 5′-GGATCTACCAGTCTAACATG-3′ and reverse primer 5′-GATAGTTAGTGATCCCACTG-3′; CREB forward primer 5′-TTCTACAGTATGCACAGACCACTG-3′ and reverse primer 5′-GGTATGTTTGTACATCGCCTGA-3′; CRTC1 forward primer 5′-AGACAGACAAGACCCTTTCTAAGCA-3′ and reverse primer 5′-CAGGACTTGGGCCTGGAA-3′. β-actin was used as a housekeeping gene. To ensure specificity of the PCR amplification, a temperature-controlled melting curve analysis was performed as the last step of the PCR. As expected, each melting curve revealed a single peak corresponding to the desired specific amplification product.

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Cell Culture of Primary Murine Spinal Cord Neurons

The mice that were pregnant for 12 to 14 days were killed by cervical dislocation and soaked in 75% alcohol soak for 5 to 10 minutes. The pregnant mice were dissected under sterile conditions; we then carefully removed the sac and placed them in ice-cold sterile phosphate-buffered saline (PBS) solution. The spinal cord was stripped of the meninges and nerve roots under a dissecting microscope and was transferred to a new dish with cold sterile PBS. We washed the spinal cord with PBS 3 to 4 times before and after adding 4 mL of 0.125% trypsin at 37°C incubator for approximately 6 minutes. After cleaning, the spinal cord was tipped by pipetting softly for approximately 10 times and cells were collected in a new centrifuge tube. This step was repeated 3 times to collect all cells. Newly collected cells were filtered and centrifuged at 1200 rpm for 5 minutes. After centrifugation, the supernatant was added to the cell complete medium, counted, plated at a density of 7–8 × 105/mL, and the medium was changed every day until the seventh day for subsequent tests.

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Treatment of CREB and CRTC1 Gene Adenoviral Vectors in Neurons and CCI Mice

On the seventh day, we treated murine spinal cord neurons with Glu stimulation (100 mmol/L) for 10 to 15 minutes at 37°C to induce neuron activation. Both CREB and CRTC1 gene interference adenovirus (CREB-miR and CRTC1-miR) were identified spinal cord neurons.

In CCI mice, adenoviral vectors and CREB-miR and CRTC1-miR were injected intrathecally at 2 time points, respectively (ZT0 and ZT12), from day 7 to day 9 after modeling to further explore the changes of pain behavior and to clarify the link in the mechanism.

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Statistical Analyses

Data were expressed as mean ± SD. The data of pain behaviors were analyzed using repeated measures analysis of variance across testing time points. Data from Western blot and RT-PCR experiments were analyzed by 1-way analysis of variance to detect the differences among the experimental groups. Bonferroni post hoc tests were performed to determine the sources of the differences when significant main effects were observed. The Dunnett T3 post hoc tests were used when equal variances were not assumed. Data were managed with SPSS 16.0 software (IBM Corporation, Armonk, NY). The statistical significance criterion was P < 0.05.

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RESULTS

Circadian Rhythm of Pain Behavior After CCI in Rodents

Figure 1

Figure 1

Figure 2

Figure 2

Pain behaviors were tested at 4 time points (ZT4, ZT10, ZT16, and ZT22) in CCI rodents. Significant allodynia and hyperalgesia were shown on day 3 in CCI rats. From day 7 after CCI surgery, the pain behavior tested by the PWTL showed a peak at ZT4, while the relative pain relief at ZT16 (ZT4 versus ZT16: 11.85 ± 0.77 vs 20.38 ± 4.80; P < 0.001). This pain rhythm lasted at least up to day 14 (ZT4 versus ZT16: 11.64 ± 1.34 vs 19.73 ± 1.63; P < 0.001) after CCI surgery in rats (Fig. 1A). Moreover, in the test of thermal hyperalgesia, the PWTL of CCI rats showed more sensitivity at the rest period than the activity period on day 7 (ZT4 versus ZT16: 9.65 ± 1.49 vs 14.13 ± 1.39; P < 0.001). This circadian rhythm also lasted at least up to day 14 (ZT4 versus ZT16: 10.14 ± 1.94 vs 15.03 ± 1.32; P < 0.001; Fig. 1B). Similar results were also found in mice after CCI surgery. In the mechanical allodynia test, from day 7 (ZT4 versus ZT16: 0.70 ± 0.16 vs 1.29 ± 0.34; P = 0.001) to day 14 (ZT4 versus ZT16: 0.48 ± 0.10 vs 0.98 ± 0.25; P < 0.001) after surgery, the pain behavior showed regularity with the peak at ZT4, while the relative pain relief at ZT16 (Fig. 2).

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The Expression of NR2B, CREB, and CRTC1 in the Dorsal Horn and the SCN at 4 Time Points (ZT4, ZT10, ZT16, and ZT22) on Day 10 After CCI in Mice

Figure 3

Figure 3

To test our initial hypothesis that NR2B-CREB-CRTC1 signaling pathway may play a role in the circadian nociceptive transmission, the expression of NR2B, CREB, and CRTC1 was examined by RT-PCR and Western blot in both the dorsal horn and SCN on day 10 in CCI mice. As expected, the mRNA expression level of NR2B, CREB, and CRTC1 genes in the dorsal horn and SCN showed robust circadian expression (Fig. 3, A and C). The relative mRNA expression of NR2B increased at ZT4 and decreased at ZT16 in both SCN (0.54 ± 0.08, P < 0.001 compared with ZT4) and the dorsal horn (0.53 ± 0.09, P < 0.001 compared with ZT4). In both tissues, the relative mRNA expression of CREB peaked at ZT22 (SCN: 1.23 ± 0.10, P = 0.002 compared with ZT4; the dorsal horn: 1.31 ± 0.12, P = 0.003 compared with ZT4). CRTC1 mRNA expression in SCN did not show any significant differences among 4 time points, while the expression peaked at ZT4 in the dorsal horn. The protein expressions of pNR2B, pCREB, and CRTC1 in SCN and the dorsal horn also presented a circadian rhythm (Fig. 3, B and D). Similar to mRNA level, the expression of pNR2B turned peaked at ZT4 and lowest at ZT16 in both SCN (ZT4 versus ZT16: 1.00 ± 0.07 vs 0.42 ± 0.04; P < 0.001) and the dorsal horn (ZT4 versus ZT16: 1.00 ± 0.07 vs 0.21 ± 0.02; P < 0.001). Consistent with pNR2B expression, the expression of pCREB and CRTC1 also peaked at ZT4 in both tissues.

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The Effects of CREB and CRTC1 Gene Interference Adenovirus in Cultured Spinal Cord Neurons

Figure 4

Figure 4

With the help of cell culture and adenovirus vector technology, the relative mRNA expression of CREB and CRTC1 was detected after stimulation by CREB and CRTC1 gene interference adenovirus. Compared with control group, no significant differences in CREB (P = 0.182) and CTRC (P = 1.000) mRNA expression were found after Glu stimulation, while the interference adenoviral vector of CRTC1 altered the expression of CRTC1 (0.27 ± 0.07; P < 0.001). In the meantime, the treatment of CREB gene interference adenovirus decreased the relative mRNA expression of both CRTC1 (0.47 ± 0.07; P < 0.001) and CREB (0.47 ± 0.05; P < 0.001). These data suggest that CRTC1 is a downstream molecule of CREB and both may participate in circadian pain transmission (Fig. 4).

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The Effects of Intrathecal Injection of CRTC1 and CREB Gene Interference Adenovirus (CREB-miR and CRTC1-miR) on the Pain Rhythm in CCI Mice

From day 7 to day 9 after CCI surgery, mice were continuously treated with CRTC1 or CREB gene interference adenovirus at ZT0 or ZT12, respectively. We found that adenovirus vector had no effect on the PWMT of CCI mice when treated either at ZT0 or at ZT12 (Fig. 5).

Figure 5

Figure 5

CREB-miR or CRTC1-miR was intrathecally injected, and the pain behavior was significantly improved. Four hours after the first injection at ZT12 on day 6 after CCI surgery (ZT16 on day 7), compared with control group (1.29 ± 0.34), the PWMT markedly increased in the CREB-miR group (1.73 ± 0.28; P = 0.032) and the CRTC1-miR group (1.79 ± 0.10; P = 0.013). Similar analgesic effects were observed at ZT16 on day 8 (CREB-miR versus control: 1.73 ± 0.17 vs 1.08 ± 0.13, P < 0.001; CRTC1-miR versus control: 1.40 ± 0.40 vs 1.08 ± 0.13; P < 0.001) and day 9 (CREB-miR versus control: 1.90 ± 0.11 vs 1.07 ± 0.13; P < 0.001; CRTC1-miR versus control: 1.72 ± 0.17 vs 1.07 ± 0.13; P < 0.001). The pain behavior of CCI mice gradually recovered to control level after we stopped drug administration (Fig. 6, A and B). In addition, 4 hours after the first injection at ZT0 on day 7 after CCI surgery (ZT4 on day 7), compared with control group (0.70 ± 0.16), the PWMT in the CREB-miR group were 1.36 ± 0.25 (P < 0.001) and the PWMT in the CRTC1-miR group were 1.60 ± 0.20 (P < 0.001). These effects were also observed at ZT4 on day 8 (CREB-miR versus control: 1.47 ± 0.26 vs 0.70 ± 0.13, P < 0.001; CRTC1-miR versus control: 1.85 ± 0.05 vs 0.70 ± 0.13, P < 0.001) and day 9 (CREB-miR versus control: 1.74 ± 0.09 vs 0.63 ± 0.09, P < 0.001; CRTC1-miR versus control: 1.75 ± 0.18 vs 0.63 ± 0.09, P < 0.001). The analgesic effect diminished to control level when CREB-miR and CRTC1-miR were stopped treated (Fig. 6, A and B).

Figure 6

Figure 6

Interestingly, analgesic effects varied when CREB-miR and CRTC1-miR were treated at different time points. As mentioned earlier, pain behavior peaked at ZT4 on day 7 after CCI surgery. At this time point, compared with the treatment of CREB-miR at ZT12, better pain relief was found when the administration was at ZT0 (P = 0.025). This effect was also observed on day 8 (P = 0.001) and day 9 (P < 0.001) after surgery. Meanwhile, we found similar results with CRTC1-miR. Stronger analgesic effects were observed when drugs were injected at ZT0 compared with ZT12 on day 8 (P < 0.001) and day 9 (P < 0.001).

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The Protein Expression of pNR2B, pCREB, and CRTC1 in the Dorsal Horn and SCN at 4 Time Points (ZT4, ZT10, ZT16, and ZT22) on Day 10 After CCI in Mice After Intrathecal Injection of CREB-miR and CRTC1-miR

Figure 7

Figure 7

Figure 8

Figure 8

Figure 9

Figure 9

Figure 10

Figure 10

The protein expression of pNR2B, pCREB, and CRTC1 showed significant differences, depending on the time point of intrathecal CREB-miR and CRTC1-miR treatments. When CREB-miR were treated at ZT12, the expression of all protein at ZT16 was significantly decreased compared with ZT4 in the dorsal horn (pNR2B: 0.67 ± 0.06 vs 1.00 ± 0.08, P < 0.001; pCREB: 0.55 ± 0.05 vs 1.00 ± 0.09, P < 0.001; CRTC1: 0.74 ± 0.06 vs 1.00 ± 0.05, P < 0.001; Fig. 7). Meanwhile, when treated at ZT0, the expression of proteins was markedly depressed at ZT4 compared with ZT16 in both the dorsal horn (pNR2B: 1.00 ± 0.09 vs 1.64 ± 0.08, P < 0.001; pCREB: 1.00 ± 0.11 vs 2.40 ± 0.10, P < 0.001; CRTC1: 1.00 ± 0.12 vs 1.66 ± 0.13, P < 0.001) and SCN (pNR2B:1.00 ± 0.08 vs 1.91 ± 0.09, P < 0.001; pCREB: 1.00 ± 0.14 vs 2.90 ± 0.09, P < 0.001; CRTC1: 1.00 ± 0.08 vs 1.24 ± 0.11, P = 0.004; Fig. 8). Similar results were found with CRTC1-miR. After CRTC1-miR were intrathecally injected at ZT12, the expression of CRTC1 and pCREB protein at ZT16 was significantly decreased compared with ZT4 in the dorsal horn (pCREB: 0.46 ± 0.03 vs 1.00 ± 0.03, P < 0.001; CRTC1: 0.64 ± 0.06 vs 1.00 ± 0.06, P < 0.001; Fig. 9). However, when treated at ZT0, the expression of proteins were markedly depressed at ZT4 compared with ZT16 in both the dorsal horn (pCREB: 1.00 ± 0.07 vs 1.56 ± 0.05, P < 0.001; CRTC1: 1.00 ± 0.10 vs 2.21 ± 0.07, P < 0.001) and SCN (pCREB: 1.00 ± 0.04 vs 2.16 ± 0.10, P < 0.001; CRTC1:1.00 ± 0.07 vs 1.36 ± 0.07, P < 0.001; Fig. 10).

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DISCUSSION

Clinical surveys have found that many types of chronic pain in different regions generally displayed a characteristic change with circadian rhythm. For example, patients with caries experience the most pain in the morning and the least in the late afternoon,17 while cancer pain shows an evening peak.18 In our study, a circadian rhythm for chronic pain was established experimentally in rodents. We tested the pain states of rodents after CCI operation and found that the pain threshold was significantly different between the activity period (nighttime for rodents) and the rest period (daytime for rodents). The pain induced by CCI was worse at night than during the day in both mice and rats. At the same time, the expressions of target genes in the spinal cord were different between day and night, which is consistent with the behavior. We also observed the hyperalgesia and gene expressions in normal mice and found that mechanical allodynia was at its lowest point at ZT10 (rest period) but that the other 3 time points showed no statistical differences (data not shown). Besides, there are no statistically differences in the gene expressions at the spinal level (data not shown). The persistent pain signals induced by CCI may act as a trigger for the significant changes in pain rhythm. Our data support the notion that chronic pain is another physiologic process with a circadian rhythm, closely associated with the secretion of pain-related proteins.

The neurophysiologic basis for chronic pain was pain sensitization, which was one of the manifestations of neuronal plasticity. Overexpression of NR2B/CREB, or reduction in NR2B/CREB degradation, can significantly prolong long-term potentiation. NR2B subunits are mainly distributed in the sites of transmission and regulation nociceptive signals, such as the brain and shallow region of the spinal cord dorsal horn, which is involved in the delivery and regulation of nociceptive signals. Although some experiments with virus-mediated gene transfection demonstrated that overexpressing CRTC1 could strengthen long synaptic transmission and plasticity in the hippocampus, interference in the interaction between CRTC1 and CREB could block the long-term plasticity of synaptic transmission. These were indications that NR2B, CREB, and CRTC1 play important roles in synaptic plasticity and pain modulation.

The suprachiasmatic nucleus is the body’s master biological clock responsible for the biorhythm area, which is located in a ventral nucleus of the thalamus.19 NR2B and CREB/CRE have been experimentally demonstrated to play important roles in the regulation of the suprachiasmatic circadian clock.8,10,20 Recent research on the association between CRTC1 and the hypothalamic suprachiasmatic nucleus circadian clock revealed its importance in regulating circadian rhythms.21 In this study, we also confirmed that these receptors in chronic pain mice have a higher secretion in the rest period than in the activity period.

Based on this, we performed interference injection in chronic pain mice with CREB/CRTC1 adenovirus at 2 time points of day and night alternating (ZT12 and ZT0). The allodynia behavior of the mice was significantly alleviated after inhibiting the expression of CREB and CRTC1, which was matched with the effect of opioid analgesics in clinical. More interestingly, when drugs were given at ZT0 (from the activity period to the rest period), the original pain threshold of mice was increased significantly, and lasted longer, whereas the analgesic effect with CREB/CRTC1 adenovirus treatment was not so obvious when drugs were given at ZT12 (from the rest period to the activity period).

Combined with the abovementioned conclusion—that chronic pain had features that were worse at night and better in the day—such results hint at possible therapeutic strategies for using clinical painkillers because it seemed that medication at night is much more effective than during the day. Although clinical analgesic therapies for neuropathic pain were more likely to give intervention at the peak time of pain, a more appropriate treatment time point associated with pain rhythm should attract considerable attention.

In our previous study, we found that the protein and mRNA expression of pain-related protein NR2B, CRTC1, and CREB had circadian oscillation in spinal cord at stable period of sciatic nerve ligation mice and was highly consistent with pain circadian rhythm of CCI mice. In addition, we injected the inside of nerve cells with interference adenovirus vector for CREB and CRTC1 to explore whether there were correlated signaling pathways between CREB and CTRC1. Interestingly, CRTC1 interference adenovirus only affected the expression of CRTC1 and not CREB mRNA. In contrast, the expression of CREB and CRTC1 were significantly changed with interference for CREB. We observed that the accumulation and activation of NR2B and CREB were induced by Glu. The inhibition of CREB could only reduce the expression CREB and CRTC1 but did not affect NR2B phosphorylation. The interference of CRTC1 can only unilaterally reduce expression of oneself activity state, suggesting that the interaction between CREB and CRTC1 is a common key in pain signal conditioning. This indicates that there might be a CREB-CRTC1-CREB signaling pathway that participates in regulating the chronic pain rhythm.

In summary, we found that pain behavior in mice with chronic pain had a circadian rhythm that was associated with the circadian secretion of pain-related receptors. Circadian rhythm was affected by multiple regulation of various environmental and internal factors, and its association with pain rhythm may be particularly difficult to explore. Inhibition of the relevant receptors in mice could improve pain behavior and help identify an appropriate schedule of use for clinical painkillers. Our observation that drugs given at ZT0 was more effective at relieving peak pain than those given at ZT12 is meaningful. It suggests that measures to relieve pain should be taken before the time when pain reaches its peak. Furthermore, there was a huge effect of the modulation of NR2B-CRTC1-CREB signaling pathway in the process. Understanding the correlation between circadian rhythm and pain could allow us to find new interventions and provide ideas for further research into the pain rhythms.

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DISCLOSURES

Name: Tianjiao Xia, PhD, MD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Tianjiao Xia has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Yin Cui, MD.

Contribution: This author helped conduct the study, analyze the data, and write the manuscript.

Attestation: Yin Cui has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Yue Qian, MD.

Contribution: This author helped write the manuscript.

Attestation: Yue Qian approved the final manuscript.

Name: Shuaishuai Chu, MD.

Contribution: This author helped conduct the study and write the manuscript.

Attestation: Shuaishuai Chu approved the final manuscript.

Name: Jia Song, MD.

Contribution: This author helped conduct the study and write the manuscript.

Attestation: Jia Song approved the final manuscript.

Name: Xiaoping Gu, PhD, MD.

Contribution: This author helped design the study and write the manuscript.

Attestation: Xiaoping Gu approved the final manuscript.

Name: Zhengliang Ma, PhD, MD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Zhengliang Ma reviewed the analysis of the data and approved the final manuscript.

This manuscript was handled by: Jianren Mao, MD, PhD.

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REFERENCES

1. Auvil-Novak SE, Novak RD, Smolensky MH, Kavanagh JJ, Kwan JW, Wharton JT. Twenty-four hour variation in self-administration of morphine sulfate (MS) and hydromorphone (H) by post-surgical gynecologic cancer patients. Ann Rev Chronopharmacol. 1988;5:343–6
2. Goldman SM. Nocturnal neuropathic pain in diabetic patients may be caused by spinal stenosis. Diabet Med. 2005;22:1763–5
3. Forssell H, Teerijoki-Oksa T, Kotiranta U, Kantola R, Bäck M, Vuorjoki-Ranta TR, Siponen M, Leino A, Puukka P, Estlander AM. Pain and pain behavior in burning mouth syndrome: a pain diary study. J Orofac Pain. 2012;26:117–25
4. Rondón LJ, Privat AM, Daulhac L, Davin N, Mazur A, Fialip J, Eschalier A, Courteix C. Magnesium attenuates chronic hypersensitivity and spinal cord NMDA receptor phosphorylation in a rat model of diabetic neuropathic pain. J Physiol. 2010;588:4205–15
5. Bhatt JM, Prakash A, Suryavanshi PS, Dravid SM. Effect of ifenprodil on GluN1/GluN2B N-methyl-D-aspartate receptor gating. Mol Pharmacol. 2013;83:9–21
6. Slutsky I, Abumaria N, Wu LJ, Huang C, Zhang L, Li B, Zhao X, Govindarajan A, Zhao MG, Zhuo M, Tonegawa S, Liu G. Enhancement of learning and memory by elevating brain magnesium. Neuron. 2010;65:165–77
7. Lisman J, Yasuda R, Raghavachari S. Mechanisms of CaMKII action in long-term potentiation. Nat Rev Neurosci. 2012;13:169–82
8. Bendová Z, Sládek M, Svobodová I. The expression of NR2B subunit of NMDA receptor in the suprachiasmatic nucleus of Wistar rats and its role in glutamate-induced CREB and ERK1/2 phosphorylation. Neurochem Int. 2012;61:43–7
9. Schulman H, Hanson PI. Multifunctional Ca2+/calmodulin-dependent protein kinase. Neurochem Res. 1993;18:65–77
10. Luo J, Phan TX, Yang Y, Garelick MG, Storm DR. Increases in cAMP, MAPK activity, and CREB phosphorylation during REM sleep: implications for REM sleep and memory consolidation. J Neurosci. 2013;33:6460–8
11. Altarejos JY, Montminy M. CREB and the CRTC co-activators: sensors for hormonal and metabolic signals. Nat Rev Mol Cell Biol. 2011;12:141–51
12. Conkright MD, Canettieri G, Screaton R, Guzman E, Miraglia L, Hogenesch JB, Montminy M. TORCs: transducers of regulated CREB activity. Mol Cell. 2003;12:413–23
13. Sakamoto K, Norona FE, Alzate-Correa D, Scarberry D, Hoyt KR, Obrietan K. Clock and light regulation of the CREB coactivator CRTC1 in the suprachiasmatic circadian clock. J Neurosci. 2013;33:9021–7
14. Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain. 1988;33:87–107
15. Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain. 1988;32:77–88
16. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods. 1994;53:55–63
17. Proffit WR, Frazier-Bowers SA. Mechanism and control of tooth eruption: overview and clinical implications. Orthod Craniofac Res. 2009;12:59–66
18. Bruera E, Macmillan K, Kuehn N, Miller MJ. Circadian distribution of extra doses of narcotic analgesics in patients with cancer pain: a preliminary report. Pain. 1992;49:311–4
19. Welsh DK, Takahashi JS, Kay SA. Suprachiasmatic nucleus: cell autonomy and network properties. Annu Rev Physiol. 2010;72:551–77
20. Lindsay JH, Glass JD, Amicarelli M, Prosser RA. The mammalian circadian clock in the suprachiasmatic nucleus exhibits rapid tolerance to ethanol in vivo and in vitro. Alcohol Clin Exp Res. 2014;38:760–9
21. Jagannath A, Butler R, Godinho SI, Couch Y, Brown LA, Vasudevan SR, Flanagan KC, Anthony D, Churchill GC, Wood MJ, Steiner G, Ebeling M, Hossbach M, Wettstein JG, Duffield GE, Gatti S, Hankins MW, Foster RG, Peirson SN. The CRTC1-SIK1 pathway regulates entrainment of the circadian clock. Cell. 2013;154:1100–11
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