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Spinal CCK1 Receptors Contribute to Somatic Pain Hypersensitivity Induced by Malocclusion via a Reciprocal Neuron-Glial Signaling Cascade

  • Ting Xiang
    Affiliations
    Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, Research Center of Stomatology, Xi'an Jiaotong University College of Stomatology, Xi'an, Shaanxi, China

    Department of Orthodontics, Xi'an Jiaotong University College of Stomatology, Xi'an, Shaanxi, China
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  • Jia-Heng Li
    Affiliations
    Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, Research Center of Stomatology, Xi'an Jiaotong University College of Stomatology, Xi'an, Shaanxi, China
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  • Han-Yu Su
    Affiliations
    Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, Research Center of Stomatology, Xi'an Jiaotong University College of Stomatology, Xi'an, Shaanxi, China
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  • Kun-Hong Bai
    Affiliations
    Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, Research Center of Stomatology, Xi'an Jiaotong University College of Stomatology, Xi'an, Shaanxi, China
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  • Shuang Wang
    Affiliations
    Department of Orthodontics, Xi'an Jiaotong University College of Stomatology, Xi'an, Shaanxi, China
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  • Richard J. Traub
    Correspondence
    Address reprint requests to Dong-Yuan Cao, PhD, Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, Xi'an Jiaotong University College of Stomatology, 98 West 5th Road, Xi'an, Shaanxi 710004, China.
    Affiliations
    Department of Neural and Pain Sciences, School of Dentistry; Center to Advance Chronic Pain Research, University of Maryland Baltimore, Baltimore, Maryland
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  • Dong-Yuan Cao
    Correspondence
    Address reprint requests to Richard J. Traub, PhD, Department of Neural and Pain Sciences, School of Dentistry, University of Maryland Baltimore, 650 W Baltimore St, Baltimore, MD 21201.
    Affiliations
    Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, Research Center of Stomatology, Xi'an Jiaotong University College of Stomatology, Xi'an, Shaanxi, China
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      Highlights

      • Malocclusion induces widespread somatic pain hypersensitivity, a typical characteristic of fibromyalgia syndrome.
      • Cholecystokinin -dependent descending facilitation contributes to the development of temporomandibular disorders and fibromyalgia syndrome comorbidity.
      • IL-18 in the spinal cord is important for the development of somatic pain hypersensitivity.
      • Cholecystokinin /IL-18-mediated neuronal and glial cascades are involved in the somatic pain hypersensitivity.

      Abstract

      Recent studies have shown that the incidence of chronic primary pain including temporomandibular disorders (TMD) and fibromyalgia syndrome (FMS) often exhibit comorbidities. We recently reported that central sensitization and descending facilitation system contributed to the development of somatic pain hypersensitivity induced by orofacial inflammation combined with stress. The purpose of this study was to explore whether TMD caused by unilateral anterior crossbite (UAC) can induce somatic pain hypersensitivity, and whether the cholecystokinin (CCK) receptor-mediated descending facilitation system promotes hypersensitivity through neuron-glia cell signaling cascade. UAC evoked thermal and mechanical pain hypersensitivity of the hind paws from day 5 to 70 that peaked at week 4 post UAC. The expression levels of CCK1 receptors, interleukin-18 (IL-18) and IL-18 receptors (IL-18R) were significantly up-regulated in the L4 to L5 spinal dorsal horn at 4 weeks post UAC. Intrathecal injection of CCK1 and IL-18 receptor antagonists blocked somatic pain hypersensitivity. IL-18 mainly co-localized with microglia, while IL-18R mainly co-localized with astrocytes and to a lesser extent with neurons. These findings indicate that the signaling transduction between neurons and glia at the spinal cord level contributes to the descending pain facilitation through CCK1 receptors during the development of the comorbidity of TMD and FMS.

      Perspective

      CCK1 receptor-dependent descending facilitation may mediate central mechanisms underlying the development of widespread somatic pain via a reciprocal neuron-glial signaling cascade, providing novel therapeutic targets for the clinical treatment of TMD and FMS comorbidities.

      Key words

      Introduction

      Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage or similar experiences.

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      Therefore, we chose a unilateral anterior crossbite (UAC) rat model causing TMJ-OA to explore whether TMD alone can induce the characteristic symptoms of FMS.
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      It has been shown that IL-1β upregulates genes which are involved in activation of nuclear factor-кB (NF-кB) cascade associated with CCK receptor signaling.
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      However, there is no report clarifying the relationship between CCK receptors and IL-18 in the development of pain, and there is no evidence that CCK receptors, IL-18, and glial cell signaling cascade in the development of comorbidities of TMD and FMS.
      Therefore, the purpose of this study was to establish a TMJ-OA animal model to explore whether it can induce widespread somatic pain hypersensitivity in rats, which is a typical symptom of FMS, and further explore the role of CCK receptors and IL-18-mediated neuron-glia cell signaling cascade in the spinal cord in the TMD-induced somatic pain hypersensitivity.

      Materials and Methods

      Animals

      Female Sprague-Dawley rats weighing 180 to 200 g (about 8 weeks of age) were obtained from Xi'an Jiaotong University Laboratory Animal Center (Xi'an, Shaanxi, China) and housed in a climate-controlled room on a 12-hour light/dark cycle (lights on at 7:00 AM). Food and water were available ad libitum. All experiments were approved by the Institutional Animal Care and Use Committees of Xi'an Jiaotong University and the Biomedical Ethics Committee of Xi'an Jiaotong University Health Science Center (approved No. 2019-950), China. The experiments were also adhered to guidelines for experimental pain in animals published by the International Association for the Study of Pain.

      UAC Rat Model

      Every rat has 2 pairs of incisors, and the upper incisor occludes the labial side of the lower incisor ordinarily. UAC surgery was performed on 8-week-old rats according to previous studies.
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      • Li JL
      • Wang MQ.
      Malocclusion generates anxiety-like behavior through a putative lateral habenula-mesencephalic trigeminal nucleus pathway.
      Briefly, after each rat was anesthetized with 2 to 3% isoflurane, a section of metal tube (length, 2.5 mm; inside diameter, 3 mm) was pasted to the left maxillary incisor and a curved section of metal tube (length, 4.5 mm; inside diameter, 3.5 mm) was pasted to the left mandibular incisor. The end of the latter tube was bent to create a 135°-angle leaning toward the labial side to create a cross-bite relationship between the top and bottom incisors (Supplemental Fig 1). Each operation was completed within 3 minutes and all efforts were made to minimize suffering. The tubes were carefully glued with light-curing resin and inspected every day. During the experiment, the metal tubes were not found to drop off. In the sham group, the rats experienced the same procedures without fixing the metal tubes so that the tubes dropped off in a few hours. All animals were fed cylindrical compressed food pellets.

      Histological Staining and Micro– Computed Tomography (Micro-CT) Scanning

      UAC and sham operated rats were anesthetized with isoflurane (5%) and sacrificed at 2- and 4-weeks post-UAC. Based on the previous studies, there were no differences in the degenerative changes between the left and right TMJs of UAC rats.
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      Cartilage degradation in temporomandibular joint induced by unilateral anterior crossbite prosthesis.
      Consequently, the left TMJ tissues in each group were used for histological analysis which was hematoxylin and eosin (H&E) staining (n = 4), and the right TMJ tissues were fixed in 4% paraformaldehyde for Micro-CT scanning (n = 4).
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      Death and proliferation of chondrocytes in the degraded mandibular condylar cartilage of rats induced by experimentally created disordered occlusion.
      the surface of condylar cartilage was divided equally into the anterior, middle, and posterior portions. The thickness of hypertrophic layer in each section was measured.
      Micro-CT was performed on condyles by the Micro-CT imaging facility (Y. Cheetah, Y-XLON, Germany). The technician performing the scans and analysis was blinded to the treatment groups. One mandible from each experimental group was dissected, cleaned, and fixed in 4% paraformaldehyde. The samples (n  =  4 per group) were analyzed by Micro-CT. The serial tomographic projections were acquired at 80 kV and 500 mA, with a voxel size of 8 μm, then corrected by the conventional X-rays (0.8 μm), and constructed 3-dimensional images for quantitative evaluation. In order to assess the histomorphometry of the subchondral bone trabeculae, a 0.5 mm × 0.5 mm × 0.5 mm cube was selected in the center of the subchondral bone central section of the condyle. Then the ratio of bone volume to tissue volume (BV/TV), trabecular thickness, trabecular number, and trabecular separation were measured in the subchondral trabecular area.

      Behavioral Tests

      Before and after UAC surgery, the thermal withdrawal latency of hind paws, the mechanical withdrawal threshold of hind paws, upper back and thigh of rats, as well the maxillofacial mechanical threshold were performed. The specific experimental flowchart is shown in Fig 1A.
      Figure 1
      Figure 1Experimental design. (A) The baselines of the thermal withdrawal latency (T), mechanical withdrawal threshold (M) and maxillofacial mechanical threshold (F) were tested before UAC. From the first day after UAC was established, the thermal withdrawal latency and mechanical withdrawal threshold were measured on the first and second days and then every 8 days. On day 3 after UAC was established, the maxillofacial mechanical threshold was tested, and then every 4 days. The measurement continued until the pain threshold returned to the baseline level. UAC, unilateral anterior crossbite. CCK1 receptor antagonist loxiglumide (B) or IL-18-binding protein (IL-18BP) (C) was intrathecally injected for 5 consecutive days at 4 weeks post UAC.

      Thermal Withdrawal Latency

      Thermal sensitivity was tested using a plantar thermal test device (Ugo Balile, Gemonio, Italy). Rats were placed in individual plexiglass enclosures on a transparent glass plate and allowed 30 minutes to adapt. The dividers between adjacent rats were non-transparent to reduce mutual interference between rats.
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      After the rat adapted (quiet, no exploratory behavior and grooming behavior), the left hind paw of the rat was stimulated from underneath with infrared radiation. The time from the start to the end of the thermal stimulation was the thermal withdrawal latency. A cut-off of 20 seconds was set to avoid tissue damage. Each rat was measured 3 times at an interval of 5 minutes, and the average value was taken as the final measurement value.

      Mechanical Withdrawal Threshold

      The measurement of mechanical withdrawal threshold was a test method to detect the sensitivity of animal paws to mechanical stimuli, which was measured with a series of calibrated von Frey filaments (Stoelting, Wood Dale, IL).
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      Rats were individually placed in a transparent plexiglass enclosure on a stainless-steel wire grid to adapt for 30 minutes. von Frey filaments with logarithmically incremental stiffness (0.41–26 g) were used to stimulate the middle plantar part of the right hindpaw perpendicularly. The mechanical withdrawal threshold was determined by gradually increasing and decreasing the intensity of the stimulus (‘‘up and down’’ method) using the threshold calculation software (JFlashDixon Calcultor, University of Arizona). Consistent with the mechanical pain threshold test of the hind paw, we examined the mechanical nociceptive threshold in the other 2 areas of the body: upper back and thigh. Each filament was placed perpendicularly to the middle upper back at T12 vertebra level and right thigh. A positive stimulus of the thigh was recorded by flinching, lifting or licking of the thigh. Flinching to stimulation of the upper back was considered a positive response.

      Maxillofacial Mechanical Threshold

      The threshold of maxillofacial masticatory muscle was measured with a series of calibrated von Frey filaments (15–100 g). Rats were constrained in the experimenter's left hand.
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      They were acclimated to the test environment by normal petting for 2 or 3 days, about half an hour per day, before UAC surgery. The mechanical threshold of bilateral masseter muscles to von Frey's filaments was measured at fixed time points every day. Stimulation was confined to the masseter muscle area within 1 cm of the left maxillofacial region from the midpoint of the connection between the outer canthus and the ear canal. A rapid “head shrinking” appearance or “calling” response is considered a positive reaction to the stimulation.
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      Consistent with the mechanical pain threshold test of the hind paw, the “up and down” method was used.

      Intrathecal Injection

      Rats were anesthetized with 2 to 3% isoflurane, the hair on the low back was shaved, and the operation area was disinfected. The rat was placed in a prone position to bend the lumbosacral vertebrae with a round tube underneath the abdomen. A 25-gauge stainless steel needle attached to a glass microsyringe was inserted into the intervertebral space between the L4 to L5 vertebrae. A quick flick of the tail indicated that the needle entered into the intrathecal space. IL-18 binding protein (IL-18BP, 1 μg) diluted in phosphate buffered saline (PBS) or CCK1 receptor antagonist loxiglumide (100 nmol) diluted in saline was intrathecally injected (i.t.; 10 μL) for 5 consecutive days at 4 weeks post UAC establishment. The vehicle group was injected with equal volume of saline or PBS. The doses of drugs were decided based on previous studies
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      and our preliminary studies. The specific experimental flowchart is shown in Fig. 1B and 1C.

      Western Blot

      Rats were anesthetized with isoflurane (5%) and quickly decapitated at 2 and 4 weeks after UAC surgery. The spinal cord was flushed out with ice-cold saline.
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      Epigenetic upregulation of metabotropic glutamate receptor 2 in the spinal cord attenuates oestrogen-induced visceral hypersensitivity.
      The lumbar spinal segments (L4–L5) were collected, and the dorsal part of spinal cord was isolated and stored at − 80 °C until use. Tissues were lysed in radio immunoprecipitation assay lysis buffer in a mixture of phosphatase inhibitors and protease inhibitors and centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was collected and the bicinchoninic acid method was used to determine the protein concentration. The protein sample (18 μg) was separated on 4 to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene fluoride membrane after denaturation, which was blocked in blocking buffer for 2 hours, and then incubated with primary antibodies for CCK1 receptor (1:1000, Bioss, bs-11514R, Beijing, China), CCK2 receptor (1:1000, Bioss, bs-1777R), IL-18 (1:1000, Proteintech, 10663-1-AP, Wuhan, China), IL-18R (1:500, Santa Cruz Biotechnology, sc-80051, Dallas, TX) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:4000, Boster, BA2913, Wuhan, China) overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary anti-bodies (1:5000) for 2 hours at room temperature. An enhanced chemiluminescence detection system (Thermo Scientific, Waltham, MA) was used for positive immunoreaction detection. Image J software was used to analyze the density of immunoreactive bands.

      Immunohistochemistry

      Four weeks after the UAC model was established, the rats were deeply anesthetized with isoflurane (5%), and transcardially perfused with 250 mL of cold saline (4°C) followed by 500 mL of 4% paraformaldehyde. The L4 to L5 spinal segments were removed, and post-fixed in paraformaldehyde for 24 hours. Then the spinal cord was immersed in 30% sucrose until the spinal cord sank. Paraffin sections (10 μm) were obtained using a microtome and every 6 sections were collected in 0.1 M PBS. For immunofluorescence staining, depending on the type of secondary antibody host, the free-floating slices were blocked with TBS containing 10% goat serum for 2 hours, and incubated in the primary antibody overnight at 4°C. The sections were washed in 0.05 M Tris-HCl (pH 7.4; 3 times, 5 minutes each), and then incubated in the secondary antibody for 2 hours at room temperature and washed. Sections were mounted on slides and covered with glycerin for observation using a confocal microscope (Pannoramic DESK, P-MIDI, P250, 3D HISTECH, Hungary). The dilution of antibodies used included anti-IL-18 (1:50, Proteintech, 10663-1-AP), anti-IL-18R (1:200, Bioss, bs-2615R), anti-NeuN (1:5000, Servicebio, GB13138, Wuhan, China), anti-glial fibrillary acidic proteins (anti-GFAP, 1:8000, Servicebio, GB12096), and anti-Iba-1 (1:5000, Servicebio, GB131051). Double immunofluorescence showed higher-magnification photographs in the superficial dorsal horn (layer I–III). To determine the localization of IL-18/IL-18R with neurons and glia cells, we examined the colocalization between IL-18/IL-18R and several biochemical markers.49
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      Specifically, 6 sections per rat were selected randomly for each group, and 6 to 8 visual fields per section were captured. The images of the superficial dorsal horn (layer I–III) of the spinal cord were captured under the objective lens of × 20.

      Drugs

      Loxiglumide (MedChemExpress, 107097-80-3) was dissolved in saline and L-18BP (R&D systems, 119-BP-100, Abingdon, UK) was dissolved in PBS. Saline and PBS have no consequential effect on spinal cord nociceptive information transmission.
      • Hylden JL
      • Wilcox GL.
      Intrathecal morphine in mice: A new technique.

      Data Analysis

      All data are presented as mean ± standard error of mean. Statistical and figure analyses were performed using GraphPad Prism 8 software. The t-test analysis was used to compare the data of CCK1, CCK2, IL-18 and IL-18R expression in the L4 to L5 spinal dorsal horn, as well as the quantitative results of Micro-CT between the experimental groups. Two-way analysis of variance (ANOVA) was used to compare the data of the thermal withdrawal latency, mechanical withdrawal threshold, maxillofacial mechanical threshold and thickness of hypertrophic layer between the sham and UAC groups at different time points. P < .05 was considered significant.

      Results

      Establishment of TMJ-OA Animal Model

      Histological Changes

      In the sham group, the condyle cartilage showed normal tissue structure. Specifically, condyle cartilage includes 4 layers, namely the fibrous layer, the proliferative layer, the hypertrophic layer and the endochondral ossification layer (Fig 2A). However, at 2 and 4 weeks post UAC, OA-like lesions were observed, which were characterized by reduced number and size of chondrocytes in cartilage, irregular cell arrangement, nuclear pyknosis, and even cell-free areas. The thickness of the hypertrophic layer at 4 weeks post UAC was lower than that in the age-matched sham group (Fig 2C). To compare cartilage thickness changes in different portions, the thickness of the hypertrophic layer in each portion was determined (Fig 2B). The thickness of the hypertrophic layer in the middle portion at 2 and 4 weeks post UAC establishment was significantly lower than that of the sham group at the same time point (t-test: 2 weeks: P = .0059; 4 weeks: P = .0470, Fig. 2C and 2E). The hypertrophic layer at the anterior portion in the UAC group was significantly reduced at 4 weeks (2 weeks: P = .1257; 4 weeks: P = .0345, Fig 2D). However, the hypertrophic layer at the posterior portion of the UAC group was similar to the sham group at 2 and 4 weeks post UAC (2 weeks: P = .1378; 4 weeks: P = .9425, Fig 2F).
      Figure 2
      Figure 2The pathologic changes in the condyle cartilage. (A) The condyle cartilage includes 4 layers: the fibrous layer, the proliferative layer, the hypertrophic layer and the endochondral ossification layer. (B) Delineation of the anterior, middle and posterior portion of the condyle cartilage. (C) Example of condyle cartilage from sham and 2, 4 weeks post UAC, Bar = 200 µm. The boxes depict the regions of higher magnification in each section. The thickness of hypertrophic layer of anterior portion (D), middle portion (E) and posterior portion (F). *, **P < .05, .01 versus the sham group.

      Morphological Parameters of Bone Tissue

      Micro-CT reflects the changes in the subchondral bone morphology of the condyle in 3 dimensions, which is a quantitative indicator of bone structure and bone density (Fig 3A). According to the Micro-CT images, the UAC group showed significant loss of subchondral trabecular bone at 4 weeks post UAC, which was characterized by an increase in trabecular bone separation (P = .0035 vs the sham group, Fig 3B) and a decrease in the bone volume fraction (bone volume/tissue volume, BV/TV, P = .0008 vs the sham group, Fig 3C). The trabecular separation (P = .4842) did not change significantly, but the BV/TV significantly decreased (P = .0027) at 2 weeks post UAC versus the sham group. There were no differences in the trabecular thickness (Fig 3D) and the number of trabeculae (Fig 3E) at 2 weeks (P = .6383 and .9311, respectively) and 4 weeks (P = .9573 and .3975, respectively) post UAC compared with the sham group.
      Figure 3
      Figure 3The Micro–CT images and bone histomorphology parameters of temporomandibular joint condyles. (A) The Micro–CT images reflect the changes in the subchondral bone morphology of the condyle in 3 dimensions at 2 and 4 weeks post UAC, respectively. Bar = 160 µm. (B) The trabecular bone separation (Tb. Sp). (C) Bone volume/total volume (BV/TV). (D) Trabecular thickness (Tb. Th). (E) Number of trabeculae (Tb. N). ** P < .01 versus sham.

      UAC Induces Maxillofacial Pain Sensitization, Thermal Hyperalgesia in Hind Paw and Mechanical Allodynia in Hind Paw, Upper Back and Thigh

      All rats were healthy, but there was a decrease in body weight in the UAC group compared to sham rats (2-way ANOVA, F13,238 = .5244, P  =  .9084 for interaction; F1,238 = 25.58, P <  .0001 for time factor; F13,238 = 14.24, P  <  .0001 for group factor, Supplemental Fig 2). UAC reduced maxillofacial mechanical pain threshold, thermal withdrawal latency and mechanical withdrawal threshold in the hind paws, upper back and thigh of rats compared to baseline. There were no statistical differences between the UAC group and the sham group at the baseline level. On the first day after the UAC was established, the maxillofacial mechanical threshold decreased significantly, which was most obvious on the 4th day post UAC, then it gradually recovered on the 8th and 12th days post UAC (left masseter: 2-way ANOVA, F13,102 = 2.596, P  =  .0038 for interaction; F1,10 = 7.946, P  = .0182 for time factor; F13,130 = 3.184, P  = .0004 for group factor, Fig 4A; right masseter: 2-way ANOVA, F13,102 = 2.108, P  =  .0196 for interaction; F1,10 = 8.430, P  = .0157 for time factor; F13,130 = 2.511, P  = .0042 for group factor, Fig 4B). There was no difference in the maxillofacial mechanical threshold between the left and right masticatory muscles (UAC: 2-way ANOVA, F13,130 = .3555, P = .9806 for interaction; F1,10 = .1020, P  = .7560 for time factor; F13,130 = 6.603, P  < .0001 for group factor, Fig 4C; sham: 2-way ANOVA, F13,104 = 1.156, P =  .3222 for interaction; F1,8 = 1.740, P  = .2236 for time factor; F13,104 = 1.029, P  = .4297 for group factor, Fig 4D).
      Figure 4
      Figure 4UAC reduced maxillofacial mechanical threshold in female rats. (A, B) Maxillofacial mechanical threshold ipsilateral (A) and contralateral (B) in the UAC and sham treated rats (n = 9-11 per group). *, **, *** P < .05, .01, .001 versus baseline (b); #, ##, ### P < .05, .01, .001 versus the sham group at the same time point. (C, D) There were no differences in the maxillofacial mechanical threshold between the left and right sides in the UAC group (C) and sham group (D).
      Following UAC thermal and mechanical sensitivity increased in the hind paws. The thermal withdrawal latency of the hind paws was significantly decreased in the UAC group compared to sham (2-way ANOVA, F9,70 = 2.955, P  =  .0049 for interaction; F1,10 = 85.69, P  <  .0001 for time factor; F9,90 = 8.348, P  <  .0001 for group factor, (Fig 5A). Likewise, the mechanical withdrawal threshold of the hind paws in the UAC group was significantly lower compared with the sham group (2-way ANOVA, F9,70 = 3.322, P =  .020 for interaction; F1,10 = 8.316, P  = .0163 for time factor; F9,90 = 2.958, P  = .0040 for group factor, Fig 5B). In order to further confirm the somatic pain hypersensitivity induced by UAC, in a separated group we examined the mechanical nociceptive threshold in other 2 areas of the body: upper back and thigh of rats. There were significant differences among groups in the mechanical withdrawal threshold in the upper back (2-way ANOVA, F9,180 =  2.559, P  =  .0086 for interaction; F1,180 = 61.48, P  <  .0001 for time factor; F9,180 = 2.631, P  = .0070 for group factor, Fig 5C) and the thigh of rats (2-way ANOVA, F9,180 = 3.998, P =  .0001 for interaction; F1,180 = 161.1, P  <  .0001 for time factor; F9,180 = 4.203, P  <  .0001 for group factor, Fig 5D). In the sham group there was no effect in the thermal withdrawal latency or mechanical withdrawal threshold compared to baseline. Taken together, these findings indicate that UAC induces widespread somatic pain hypersensitivity, a typical characteristic observed in patients with FMS.
      Figure 5
      Figure 5Thermal hyperalgesia in the hind paws and mechanical allodynia in the hind paws, the upper back and thigh of rats induced by UAC. (A) Hindpaw thermal withdrawal latency in UAC and sham-treated rats (n = 11 per group). (B) Hindpaw mechanical withdrawal threshold (n = 11). (C) Upper back mechanical withdrawal threshold (n = 10). (D) Thigh mechanical withdrawal threshold (n = 10). No mechanical allodynia was observed in rats with sham treatment. *, **, *** P < .05, .01, .001 versus baseline, respectively. #, ##, ### P < .05, .01, .0001 versus the sham group at the same time point.

      CCK1 Receptors are Involved in the Somatic Pain Hypersensitivity Induced by UAC

      CCK is a neuropeptide with abundant content and wide distribution in the CNS. It has been shown that CCK can promote pain sensitization by activating descending facilitation from the periaqueductal gray matter (PAG) and RVM.
      • Lovick TA.
      Pro-nociceptive action of cholecystokinin in the periaqueductal grey: A role in neuropathic and anxiety-induced hyperalgesic states.
      To explore whether spinal CCK receptors contribute to UAC-induced widespread pain hypersensitivity in the hind paws, we investigated the protein expression of CCK receptors in the L4 to L5 spinal dorsal horn, the site of spinal nociceptive signal processing related to the hind limbs. The Western blot data showed that the expression of CCK1 receptors significantly increased in the UAC group compared with the sham group at 4 weeks, but not 2 weeks, post UAC (P = .0497 at 4 weeks, P = .1307 at 2 weeks, Fig. 6A and 6B). There were no significant differences in the expression of CCK2 receptors between those 2 groups (P = .4642 at 4 weeks, P = .5390 at 2 weeks, Fig. 6A and 6B), suggesting that CCK1 receptors, but not CCK2 receptors, in the spinal cord were up-regulated in the UAC rats.
      Figure 6
      Figure 6CCK1 receptors were involved in the somatic hyperalgesia induced by UAC. A, B: The expression of CCK1 and CCK2 receptors in the L4 to L5 spinal dorsal horn at 4 weeks (A) and 2 weeks (B) post UAC, * P < .05 (n = 5 for all groups). C: The CCK1 receptor antagonist loxiglumide (n = 9), but not vehicle (saline, n = 8), blocked the development of thermal hyperalgesia in female rats. D: Loxiglumide (n = 9), but not vehicle (n = 8), blocked mechanical allodynia induced by UAC. *, **, *** P  <  .05, .01, .001 versus baseline (b). #, ##, ### P <  .05, .01, .001 versus the UAC + saline group at the same time point.
      To confirm whether the increased CCK1 receptors contribute to UAC-induced widespread pain hypersensitivity in female rats, the CCK1 receptor antagonist loxiglumide was intrathecally injected at 4 weeks post UAC. Compared with the saline treated group, loxiglumide blocked the reduction of thermal withdrawal latency (2-way ANOVA, F10,165 = 1.975, P = .0390 for interaction; F1,165 = 94.39, P < .0001 for time factor; F10,165 = 3.831, P = .0001 for group factor, Fig 6C) and mechanical withdrawal threshold (2-way ANOVA, F10,160 = 2.516, P  =  .0077 for interaction; F1,16 = 11.78, P = .0034 for time factor; F10,160 = 1.221, P =  .0281 for group factor, Fig 6D) in the UAC group. These results indicate that the up-regulation of CCK1 receptors in the spinal cord plays a critical role in UAC-induced widespread pain hypersensitivity. To further evaluate the effects of CCK1 receptor antagonist on the sham-operated animals, loxiglumide was administered to sham treated rats and tested for thermal and mechanical hyperalgesia. The data showed that loxiglumide did not change the pain thresholds in sham rats (Supplemental Fig 3).

      Up-Regulated IL-18 in Microglia and IL-18 Receptors in Astrocytes Mediate Microglia-Astrocytic Interaction During UAC-Induced Widespread Pain Hypersensitivity

      Recently, IL-18-mediated signaling pathways have been extensively studied in the occurrence and development of chronic pain by regulating the interaction of microglia and astrocytes.
      • Liu S
      • Liu YP
      • Lv Y
      • Yao JL
      • Yue DM
      • Zhang MY
      • Qi DY
      • Liu GJ.
      IL-18 contributes to bone cancer pain by regulating glia cells and neuron interaction.
      ,
      • Miyoshi K
      • Obata K
      • Kondo T
      • Okamura H
      • Noguchi K.
      Interleukin-18-mediated microglia/astrocyte interaction in the spinal cord enhances neuropathic pain processing after nerve injury.
      ,
      • Pilat D
      • Piotrowska A
      • Rojewska E
      • Jurga A
      • Slusarczyk J
      • Makuch W
      • Basta-Kaim A
      • Przewlocka B
      • Mika J.
      Blockade of IL-18 signaling diminished neuropathic pain and enhanced the efficacy of morphine and buprenorphine.
      ,
      • Yang Y
      • Li H
      • Li TT
      • Luo H
      • Gu XY
      • Lu N
      • Ji RR
      • Zhang YQ.
      Delayed activation of spinal microglia contributes to the maintenance of bone cancer pain in female Wistar rats via P2X7 receptor and IL-18.
      It has been shown that IL-18 is mainly expressed in microglia in different animal pain models. Therefore, in order to explore the role of IL-18 in UAC-induced widespread pain hypersensitivity, we examined the expression of IL-18 and IL-18R in the spinal cord. Western blot analysis showed that the expression levels of IL-18 and IL-18R in the spinal cord significantly increased at 4 weeks post UAC compared with the sham group (IL-18: P = .0403; IL-18R: P = .0341; Fig 7A), but there were no significant differences in the expression of IL-18 and IL-18R at 2 weeks post UAC (IL-18: P = .2027; IL-18R: P = .1727; Fig 7B). Biochemical markers for neurons (NeuN), microglia (Iba-1) and astrocytes (GFAP) were used to determine the expression locations of IL-18 and IL-18R using immunohistochemistry. Double immunostaining further confirmed that IL-18 was mainly expressed in microglia in the dorsal horn (Fig 8A), which was consistent with previous observations.
      • Liu S
      • Liu YP
      • Lv Y
      • Yao JL
      • Yue DM
      • Zhang MY
      • Qi DY
      • Liu GJ.
      IL-18 contributes to bone cancer pain by regulating glia cells and neuron interaction.
      IL-18 had little and no colocalization with GFAP and NeuN (Fig. 8B and 8C), respectively. However, in contrast to IL-18 expression in microglia, IL-18R was mainly expressed in astrocytes and a lesser extent in neurons (Fig. 9A and 9B), but not in microglia (Fig 9C).
      Figure 7
      Figure 7(A) The expression of IL-18 and IL-18R in the spinal cord increased at 4 weeks post UAC compared to sham (n = 5 per group). (B) The expression of IL-18 and IL-18R at 2 weeks post UAC (n = 5 per group). * P <.05.
      Figure 8
      Figure 8IL-18 was co-localized with microglia in the spinal cord post UAC. At 4 weeks post UAC, double immunofluorescence showed that IL-18 (green) was co-localized with Iba-1 (red) (A). IL-18 did not co-localized with GFAP (red) (B) and NeuN (red) (C). Arrows indicate the co-localized cells. Bar = 50 μm.
      Figure 9
      Figure 9IL-18R was co-localized with astrocytes in the spinal cord post UAC, but not with microglia. Double immunofluorescence showed that IL-18R (green) was co-localized with GFAP (red) (A) and very few with NeuN (red) (B) in the dorsal horn but not with Iba-1 (red) (C) at 4 weeks post UAC. Arrows indicate the co-localized cells. Bar = 50 μm.
      In order to further determine the role of IL-18 in somatic pain hypersensitivity induced by UAC, IL-18-binding protein (IL-18BP), which functions as an IL-18 antagonist by binding to IL-18R and blocking its biological activity, was intrathecally injected for 5 consecutive days at 4 weeks post UAC. IL-18BP significantly blocked the decrease in the thermal withdrawal latency (2-way ANOVA, F9,60 = 3.262, P  =  .0027 for interaction; F1,60 = 17.78, P < .0001 for time factor; F9,80 = 4.297, P  = .0001 for group factor, Fig 10A) and mechanical withdrawal threshold (2-way ANOVA, F9,150 = 1.139, P  =  .3392 for interaction; F1,150 = 15.26, P = .0001 for time factor; F9,150 = 2.019, P  = .0408 for group factor, Fig 10B). The behavioral results combined with the Western blot data indicate that IL-18 activation in the spinal cord contributes to somatic pain hypersensitivity induced by UAC.
      Figure 10
      Figure 10IL-18 receptor antagonist IL-18BP blocked the thermal hyperalgesia (A, n = 8 per group) and mechanical allodynia (B, n = 8 per group) in the hind paws caused by UAC. *, **, *** P  <  .05, .01, .001 versus baseline (b) (UAC + PBS). +, ++ P <  .05, .01 versus baseline (b) (UAC + IL-18BP). #, ### P  <  .05, .001 versus the UAC + PBS group at the same time point.

      Spinal CCK1 Receptors are Involved in the Activation of IL-18 and its Receptors, Leading to UAC-Induced Somatic Pain Hypersensitivity

      It has been shown that IL-1β can up-regulate CCK receptor signaling and participate in the activation of the NF-кB cascade related to CCK receptor signaling.
      • Thamsermsang O
      • Akarasereenont P
      • Laohapand T
      • Panich U.
      IL-1beta-induced modulation of gene expression profile in human dermal fibroblasts: The effects of Thai herbal Sahatsatara formula, piperine and gallic acid possessing antioxidant properties.
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      • Tripathi S
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      The gastrin and cholecystokinin receptors mediated signaling network: A scaffold for data analysis and new hypotheses on regulatory mechanisms.
      These effects are thought to be related to pain sensitization.
      • Shavit Y
      • Wolf G
      • Goshen I
      • Livshits D
      • Yirmiya R.
      Interleukin-1 antagonizes morphine analgesia and underlies morphine tolerance.
      Therefore, we tested the relationship between CCK1 receptors and IL-18 in the spinal cord. Western blot data showed that intrathecal injection of CCK1 receptor antagonist loxiglumide for 5 consecutive days significantly reduced the expression of IL-18 (P = .0483, Fig 11A) and IL-18R (P = .0336, Fig 11B) in the spinal dorsal horn at 4 weeks post UAC establishment compared with the saline group. Therefore, the release of IL-18 and the activation of IL-18R on glial cells in the spinal cord may be involved in the CCK-dependent descending facilitation post UAC establishment.
      Figure 11
      Figure 11The effect of intrathecal injection of CCK1 receptor antagonist loxiglumide on the expression of IL-18/IL-18R in the L4 to L5 spinal cord at 4 weeks post UAC. The expression of IL-18 (A) and IL-18R (B) in the L4 to L5 spinal dorsal horn in the UAC + loxiglumide group significantly decreased compared to the UAC + saline group. n = 5 for each group. * P < .05.

      Discussion

      In the present study we demonstrated that UAC induced long-term somatic pain hypersensitivity, establishing a new animal model of TMD and FMS comorbidity. We report that spinal CCK1 and IL-18 receptors are involved in central sensitization in the development of comorbid pain. In addition, our findings demonstrate that a neuron-glia signaling cascade is involved in the mechanisms underlying spinal CCK1 receptor-mediated hypersensitivity.
      In the past few decades, CPPs such as FMS
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      Fibromyalgia.
      and TMD
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      Painful temporomandibular disorders (TMD) and comorbidities in primary care: Associations with pain-related disability.
      have been increasingly viewed as a serious health problem in clinical practice and comorbidity of these diseases frequently occur. However, the mechanism underlying this comorbidity is unclear, leading to unsatisfactory treatment outcomes. Therefore, it is essential to investigate coexisting mechanisms of comorbid pain and develop targeted drugs to effectively prevent and treat these pain syndromes.
      Central sensitization underlies chronic pain conditions, including OA and FMS.
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      Abnormal pain modulation in patients with spatially distributed chronic pain: Fibromyalgia.
      In general, central sensitization is an adaptive process that reverts back to normal after nociceptive afferent input has ceased. However, persistent, intense noxious stimuli can lead to persistent central sensitization.
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      Evidence for shared pain mechanisms in osteoarthritis, low back pain, and fibromyalgia.
      It has been reported that OA patients experience decreased pain sensitivity following successful arthroplasty,
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      Abnormalities of somatosensory perception in patients with painful osteoarthritis normalize following successful treatment.
      suggesting that the dependence of central sensitization and pain on peripheral tissue impulse input in these patients. Recently, a few studies have reported the impact of occlusion on maxillofacial tissues, and the possible impact of malocclusion on the CNS has gained much attention.
      • Cao Y
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      Experimental occlusal interference induces long-term masticatory muscle hyperalgesia in rats.
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      • Jinwu Chen
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      Hyperalgesia in response to traumatic occlusion and GFAP expression in rat parabranchial nucleus: Modulation with fluorocitrate.
      In the previous study, Wang et al found that at the 2 and 4 weeks after UAC was established, OA-like lesions were observed in the cartilage, becoming more severe over time.
      • Wang YL
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      Cartilage degradation in temporomandibular joint induced by unilateral anterior crossbite prosthesis.
      Our data show that UAC caused a long-term, recoverable decrease in maxillofacial mechanical threshold, which occurred before the widespread somatic pain hypersensitivity. Therefore, we propose that UAC acts as an injury stimulus in the maxillofacial area that is signaled into the trigeminal-subnuclei caudalis (Vc) further triggering persistent central sensitization in the brain areas and activates descending pathways that facilitate pain processing.
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      Stress caused by the malocclusion itself and alterations in feeding might contribute to the widespread pain hypersensitivity. In a previous study, Liu et al
      • Liu X
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      Malocclusion generates anxiety-like behavior through a putative lateral habenula-mesencephalic trigeminal nucleus pathway.
      found that UAC, as an unpleasant external stimulus, generated anxiety-like behavior 2 weeks after surgery. As the main component of the descending pain modulatory system, the midbrain PAG
      • Mokhtar M
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      Neuroanatomy, Periaqueductal Gray.
      and the RVM
      • Liu X
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      Selective ablation of descending serotonin from the rostral ventromedial medulla unmasks its pro-nociceptive role in chemotherapy-induced painful neuropathy.
      regulate nociceptive transmission and processing in the spinal dorsal horn.
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      Descending control mechanisms and chronic pain.
      The findings indicate that central sensitization underlying the development of pain hypersensitivity depends on peripheral nerve injury increasing afferent drive activating a descending facilitation arising from the RVM that likely requires the maintenance of descending CCK receptor-mediated facilitation in the spinal cord.
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      Spinal and supraspinal mechanisms of neuropathic pain.
      CCK signaling is part of the descending facilitation system,
      • Heinricher MM
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      Neural basis for the hyperalgesic action of cholecystokinin in the rostral ventromedial medulla.
      which drives the development of pain hypersensitivity.
      • Lovick TA.
      Pro-nociceptive action of cholecystokinin in the periaqueductal grey: A role in neuropathic and anxiety-induced hyperalgesic states.
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      Acquisition of analgesic properties by the cholecystokinin (CCK)/CCK2 receptor system within the amygdala in a persistent inflammatory pain condition.
      Some studies have shown that CCK located in the PAG, RVM, and spinal cord plays a pro-nociceptive role in pain modulation.
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      Role of cholecystokinin-B receptor in the maintenance of thermal hyperalgesia induced by unilateral constriction injury to the sciatic nerve in the rat.
      For example, the RVM promotes the activation of CCK in the spinal cord.
      • Ossipov MH
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      Spinal and supraspinal mechanisms of neuropathic pain.
      CCK acts through CCK1 and CCK2 receptors, both of which are coupled to the Gq/PLC/DAG signaling pathway, which induces calcium efflux from intracellular storage.
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      • Lee SY
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      Cell-type-specific CCK2 receptor signaling underlies the cholecystokinin-mediated selective excitation of hippocampal parvalbumin-positive fast-spiking basket cells.
      Under stress stimulation, ON-cells in the RVM can be directly activated by CCK2 receptors promoting pain sensitization, while inhibiting the activity of CCK2 receptors in the RVM can effectively reverse the pain hypersensitivity caused by chronic stress.
      • Jiang M
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      Anxiety-induced hyperalgesia in female rats is mediated by cholecystokinin 2 receptor in rostral ventromedial medulla and spinal 5-hydroxytryptamine 2B receptor.
      ,
      • Lovick TA.
      Pro-nociceptive action of cholecystokinin in the periaqueductal grey: A role in neuropathic and anxiety-induced hyperalgesic states.
      ,
      • Roca-Lapirot O
      • Fossat P
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      • Trigilio G
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      • Covita J
      • Bouali-Benazzouz R
      • Favereaux A
      • Gundlach AL
      • Landry M.
      Acquisition of analgesic properties by the cholecystokinin (CCK)/CCK2 receptor system within the amygdala in a persistent inflammatory pain condition.
      Studies have reported that CCK1 receptors are expressed by both viscero- and somato-sensory primary sensory neurons, acting as a mediator in sensory processing at the spinal level.
      • Broberger C
      • Holmberg K
      • Shi TJ
      • Dockray G
      • Hokfelt T.
      Expression and regulation of cholecystokinin and cholecystokinin receptors in rat nodose and dorsal root ganglia.
      Therefore, we propose that CCK receptors are closely related to the widespread somatic pain hypersensitivity induced by UAC. In a previous study, we confirmed that CCK2 receptors-dependent descending pain modulation at the spinal level was involved in somatic pain hypersensitivity induced by orofacial inflammatory pain combined with stress.
      • Duan LL
      • Qiu XY
      • Wei SQ
      • Su HY
      • Bai FR
      • Traub RJ
      • Zhou Q
      • Cao DY.
      Spinal CCK contributes to somatic hyperalgesia induced by orofacial inflammation combined with stress in adult female rats.
      However, the mechanism by which these events are sequentially activated through neuron-astrocytic interactions is novel and unpredicted. Interestingly, we noticed that the expression of CCK1 receptors, but not CCK2 receptors, significantly increased in the L4 to L5 spinal dorsal horn at 4 weeks post UAC and the blockade of spinal CCK1 receptors totally blocked UAC-induced thermal hyperalgesia and mechanical allodynia in the hind paws. A recent study showed that CCK-positive neurons distributed in layers III-IV of the spinal dorsal horn highly overlap with protein kinase Cγ-positive neurons at the layers II/III border, playing an important role in mechanical allodynia.
      • Peirs C
      • Williams S
      • Zhao X
      • Arokiaraj CM
      • Ferreira DW
      • Noh MC
      • Smith KM
      • Halder P
      • Corrigan KA
      • Gedeon JY.
      Mechanical allodynia circuitry in the dorsal horn is defined by the nature of the injury.
      Some studies showed that CCK2 receptors, but not CCK1 receptors, play a major role in pain perception, opioid dependence and other processes.
      • Xie JY
      • Herman DS
      • Stiller CO
      • Gardell LR
      • Ossipov MH
      • Lai J
      • Porreca F
      • Vanderah TW.
      Cholecystokinin in the rostral ventromedial medulla mediates opioid-induced hyperalgesia and antinociceptive tolerance.
      ,
      • Zhang W
      • Shannon G
      • Zhang D
      • Xie JY
      • Agnes RS
      • Hamid B
      • Hruby VJ
      • Naomi R
      • Ossipov MH
      • Vanderah TW.
      Neuropathic pain is maintained by brainstem neurons co-expressing opioid and cholecystokinin receptors.
      Multiple studies have found that CCK1 and CCK2 have opposite effects on behavior actions.
      • Hao L
      • Wen D
      • Gou H
      • Yu F
      • Cong B
      • Ma C.
      Over-expression of CCK1 receptor reverse morphine dependence.
      ,
      • Lin L
      • Huang M
      • Lan M
      • Jing L.
      Different role of cholecystokinin (CCK)-A and CCK-B receptors in relapse to morphine dependence in rats.
      ,
      • Potter RM
      • Harikumar KG
      • Wu SV
      • Miller LJ.
      Differential sensitivity of types 1 and 2 cholecystokinin receptors to membrane cholesterol.
      Therefore, the various subtypes of CCK receptors and their different sensitivity levels are not consistent, determining the diversity of CCK's biological functions. Our present data (Fig 6) may give us a clue that this phenomenon is related to the different functions of CCK1 and CCK2 receptors. These findings suggest that UAC-induced widespread somatic pain hypersensitivity is maintained by neurons in the spinal cord which are sensitive to CCK via CCK1, but not CCK2 receptors.
      It has been shown that the central pain mechanism of FMS in the induction and maintenance of chronic pain not only relies on neuronal activation, but also glial activation.
      • Staud R.
      Evidence of involvement of central neural mechanisms in generating fibromyalgia pain.
      Although spinal glial hyperactivity has been reported in studies of TMD and FMS,
      • Xin Z
      • Hartung JE
      • Bortsov AV
      • Seungtae K
      • O'Buckley SC
      • Julia K
      • Nackley AG
      Sustained stimulation of β2- and β3-adrenergic receptors leads to persistent functional pain and neuroinflammation.
      ,
      • Zhang ZJ
      • Jiang BC
      • Gao YJ.
      Chemokines in neuron-glial cell interaction and pathogenesis of neuropathic pain.
      few studies investigated the involvement of spinal CCK receptors in spinal glial hyperactivity, as well as the function of IL-18, especially in the pain model of TMD and FMS comorbidity. In the dorsal horn of the spinal cord, the unique expression of IL-18 in microglia and its receptors mainly presenting in astrocytes plays a key role in the development and maintenance of mechanical allodynia.
      • Miyoshi K
      • Obata K
      • Kondo T
      • Okamura H
      • Noguchi K.
      Interleukin-18-mediated microglia/astrocyte interaction in the spinal cord enhances neuropathic pain processing after nerve injury.
      The present study showed that UAC caused a significant increase in the expression of IL-18 and IL-18R in the spinal dorsal horn at 4 weeks post UAC. However, IL-18BP only temporarily relieved the somatic pain hypersensitivity compared with the analgesic effect of CCK1 receptor antagonists (Fig 10). The reason may be that IL-18BP as a secreted glycoprotein cannot be anchored to the cell membrane due to the lack of a transmembrane domain.
      • Dinarello CA
      • Novick D
      • Rubinstein M
      • Lonnemann G.
      Interleukin 18 and interleukin 18 binding protein: Possible role in immunosuppression of chronic renal failure.
      In the present study intrathecal injection of a CCK1 receptor antagonist lowered IL-18 expression levels in the spinal dorsal horn, implying that IL-18 may be one of the important cytokines activated by CCK1 receptors and subsequently participates in the development of somatic pain sensitization. Double immunostaining verified that IL-18 in the spinal cord was mainly co-expressed with microglia, and IL-18R was mainly co-expressed with astrocytes, but not with microglia. Therefore, how CCK neurons produce IL-18 by activating microglia, and then bound to IL-18 receptors on astrocytes to participate in the occurrence of somatic hyperalgesia aroused our interest. A few studies focused on the neuron-glial cell-neuron interaction as the driving force to induce and maintain the pain process.
      • Guo W
      • Miyoshi K
      • Dubner R
      • Gu M
      • Li M
      • Liu J
      • Yang J
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      • Wei F.
      Spinal 5-HT3 receptors mediate descending facilitation and contribute to behavioral hypersensitivity via a reciprocal neuron-glial signaling cascade.
      ,
      • Scholz J
      • Woolf CJ.
      The neuropathic pain triad: Neurons, immune cells and glia.
      ,
      • Vanderwall AG
      • Milligan ED.
      Cytokines in pain: Harnessing endogenous anti-inflammatory signaling for improved pain management.
      ,
      • Zhang ZJ
      • Jiang BC
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      Chemokines in neuron-glial cell interaction and pathogenesis of neuropathic pain.
      Microglial cells in the spinal cord have an established role in sensitization of pain processing.
      • Donnelly CR
      • Andriessen AS
      • Chen G
      • Wang K
      • Jiang C
      • Maixner W
      • Ji RR.
      Central nervous system targets: Glial cell mechanisms in chronic pain.
      Under normal and pathological conditions, glial cells in the spinal cord act as immune effector cells and play a key role in promoting persistent pain states.
      • Donnelly CR
      • Andriessen AS
      • Chen G
      • Wang K
      • Jiang C
      • Maixner W
      • Ji RR.
      Central nervous system targets: Glial cell mechanisms in chronic pain.
      ,
      • Gao YJ
      • Ji RR.
      Targeting astrocyte signaling for chronic pain.
      ,
      • Nam Y
      • Kim JH
      • Kim JH
      • Jha MK
      • Jung JY
      • Lee MG
      • Choi IS
      • Jang IS
      • Lim DG
      • Hwang SH
      • Cho HJ
      • Suk K.
      Reversible induction of pain hypersensitivity following optogenetic stimulation of spinal astrocytes.
      ,
      • Schomberg D
      • Olson JK.
      Immune responses of microglia in the spinal cord: Contribution to pain states.
      ,
      • Ying YL
      • Wei XH
      • Xu XB
      • She SZ
      • Zhou LJ
      • Lv J
      • Li D
      • Zheng B
      • Liu XG.
      Over-expression of P2X7 receptors in spinal glial cells contributes to the development of chronic postsurgical pain induced by skin/muscle incision and retraction (SMIR) in rats.
      When microglia and astrocytes in the spinal cord are activated, they synthesize and release factors that promote neuronal excitability and nociceptive transmission,
      • Denk F
      • Crow M
      • Didangelos A
      • Lopes DM
      • McMahon SB.
      Persistent alterations in microglial enhancers in a model of chronic pain.
      ,
      • Nieto FR
      • Clark AK
      • Grist J
      • Hathway GJ
      • Chapman V
      • Malcangio M.
      Neuron-immune mechanisms contribute to pain in early stages of arthritis.
      ,
      • Salter MW
      • Beggs S.
      Sublime microglia: Expanding roles for the guardians of the CNS.
      thus contributing to the occurrence and development of pain. Guo et al
      • Guo W
      • Miyoshi K
      • Dubner R
      • Gu M
      • Li M
      • Liu J
      • Yang J
      • Zou S
      • Ren K
      • Noguchi K
      • Wei F.
      Spinal 5-HT3 receptors mediate descending facilitation and contribute to behavioral hypersensitivity via a reciprocal neuron-glial signaling cascade.
      found that 5-HT3 receptor-expressing neurons in the spinal cord induced chemokine production, activated microglia to release IL-18 and bind to IL-18R on astrocytes. Then the combination further phosphorylated N-methyl-D-aspartic acid receptors in the spinal cord and ultimately led to hyperalgesia. In animal models of bone cancer pain, IL-18 in the spinal cord regulates N-methyl-D-aspartic acid receptor phosphorylation by binding to IL-18R.
      • Liu S
      • Liu YP
      • Lv Y
      • Yao JL
      • Yue DM
      • Zhang MY
      • Qi DY
      • Liu GJ.
      IL-18 contributes to bone cancer pain by regulating glia cells and neuron interaction.
      Furthermore, IL-18 signal activation may lead to the secretion of a variety of cytokines, including tumor necrosis factor-α and IL-1β. The involvement of IL-18R/NF-κB signaling pathway in astrocytes in hyperalgesia has also been confirmed. IL-18 binds to the ligand receptor IL-18Rα, recruiting the co-receptor IL-18Rβ to form a high-affinity complex, which activates downstream signals. IL-18 triggers the intracellular signal cascade and induces the dependent expression of NF-κB and AP-1 pro-inflammatory cytokines, chemokines, and secondary mediators of the inflammatory response.
      • Pilat D
      • Piotrowska A
      • Rojewska E
      • Jurga A
      • Slusarczyk J
      • Makuch W
      • Basta-Kaim A
      • Przewlocka B
      • Mika J.
      Blockade of IL-18 signaling diminished neuropathic pain and enhanced the efficacy of morphine and buprenorphine.
      ,
      • Rex DAB
      • Agarwal N
      • Prasad TSK
      • Kandasamy RK
      • Subbannayya Y
      • Pinto SM.
      A comprehensive pathway map of IL-18-mediated signalling.
      Overall, our current study suggests that active CCK1 receptor-dependent descending facilitation after TMD may mediate central mechanisms underlying the development of widespread somatic pain in our comorbid pain model via a reciprocal neuron-glial signaling cascade.

      Conclusion

      In conclusion, activation of CCK1 receptors in the spinal cord contributes to widespread somatic pain hypersensitivity induced by TMD. The activation of glial cells in the spinal cord promotes the release of IL-18 to further mediate this pain hypersensitivity. Neuron and glial cell cascade signals play an important role in the development of the somatic pain hypersensitivity. These results provide novel therapeutic targets for the clinical treatment of TMD and FMS comorbidities.

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