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Relationship of calcitonin gene-related peptide in synovial tissues and temporomandibular joint pain in humans
Relationship of calcitonin gene-related peptide in synovial tissues and temporomandibular joint pain in humans Jun Sato, DDS, PhD,a Natsuki Segami, DDS, PhD,b Keiseki Kaneyama, DDS, PhD,a Hiroshi Yoshimura, DDS, PhD,a Kazuma Fujimura, DDS, PhD,c and Yoshino Yoshitake, PhD,d Ishikawa, Japan KANAZAWA MEDICAL UNIVERSITY
Objective. To elucidate the expression of calcitonin gene-related peptide (CGRP) in synovial tissue taken from the human temporomandibular joint (TMJ) with internal derangement, and discuss the relationship between CGRP and joint pain. Study design. Using an immunohistochemical technique, 48 joints in 48 patients were examined. As controls, synovial tissue specimens from 7 joints with habitual dislocation without pain were also examined. Results. In all of the internal derangement and control subjects, CGRP-positive cells were observed in the connective tissues around the blood vessels beneath the lining cells. The extent score of CGRP was significantly higher in the internal derangement group than in the control group (P = .033). There was a significant positive correlation between the extent score of CGRP and joint pain (P = .036, r = 0.30). Conclusions. These results suggest that the expression of CGRP is increased in the synovial tissues from patients with internal derangement, and that CGRP seems to play an important role in the mechanism of pain production in patients with symptomatic internal derangement. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:533-40)
Although pain in the temporomandibular joint (TMJ) region is one of the major symptoms in patients with internal derangement of the TMJ,1 the cause and pathophysiological mechanisms of the local pain are not completely understood. It has been hypothesized that synovitis may be one of the causes of TMJ pain. Previous studies2,3 indicated significant correlations between joint pain and the degree of synovitis. Recent studies have explored the importance of biological changes within the synovial fluids and synovial tissues associated with joint pain.4-8 Quinn and Bazan9 detected high levels of prostaglandin E2 in synovial fluid of inflamed joints and demonstrated a strong correlation with pain. Similarly, recent studies revealed significant correlations between proinflammatory cytokines such as interleukin
This study was partially supported by a Grant for Specially Promoted Research from Kanazawa Medical University. a Lecturer, Department of Oral and Maxillofacial Surgery, Kanazawa Medical University. b Professor and Chairman, Department of Oral and Maxillofacial Surgery, Kanazawa Medical University. c Associate Professor, Department of Oral and Maxillofacial Surgery, Kanazawa Medical University. d Associate Professor, Department of Biochemistry, Kanazawa Medical University. Received for publication Nov 14, 2003; returned for revision Jan 14, 2004; accepted for publication Feb 16, 2004. 1079-2104/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.tripleo.2004.02.057
1b (IL-1b),4,7 IL-6,6 and tumor necrosis factor (TNF-a)5 in synovial fluids and joint pain. These proinflammatory cytokines are thought to contribute to the pathogenesis of synovitis and osteoarthritis in TMJs. In previous studies, however, we found no significant correlation between the concentrations of IL-1b, IL-6, and TNF-a in synovial fluids and joint pain.10 There were some discrepancies in the correlations reported between the levels of proinflammatory cytokines and joint pain among investigations. More recently, other kinds of chemical mediators, including neuropeptides such as calcitonin gene-related peptide (CGRP), substance P, and serotonin have been detected in the TMJ.1,11,12 These neuropeptides can be released from activated peripheral nerve terminals into the surrounding tissues and cause an inflammatory response.12 CGRP is a 37-amino acid peptide produced in neural cells13,14 and has been shown to coexist with substance P in mainly nociceptive C-fibers.13,15 Small afferent unmyelinated C-fibers, which are usually correlated with nociception and the sensation of pain, have been shown to contain CGRP.15,16 Moreover, CGRP is thought to be a good marker for sensory nerves.17-20 In earlier studies, some investigators11,21 detected levels of CGRP in synovial fluids of the TMJ. However, there have been few reports of CGRP expression in human synovial tissue of the TMJ. In the present study, we investigated CGRP in synovial tissue immunohistochemically and compared the CGRP levels between joints with internal derangement and those with habitual dislocation without pain. Moreover, we assessed 533
534 Sato et al the correlation between CGRP level and clinical findings, especially in joint pain. PATIENTS AND METHODS Patients Forty-eight TMJs in 48 patients with internal derangement were included in this study (Table I). Seven of the patients were men and 41 were women. The average age was 43 years old (range 17-84). MRI showed that all of the patients had anterior disk displacement without reduction. Their mean maximum interincisal opening was 33 mm and the mean duration of symptoms was 6 months. As a result of the failure of nonsurgical treatment consisting of occlusal appliance, nonsteroidal antiinflammatory drugs (NSAIDS), and physiotherapy to resolve the clinical symptoms, all of the patients had arthroscopic surgery. Twenty-two (46%) of the 48 patients were given NSAIDS within a month before the operation (Etodolac: 12 patients, 400 mg/day, range 7 to 30 days, mean 11 days and Loxoprofen sodium: 10 patients, 180 mg/day, range 3 to 14 days, mean 8 days). Just before the operation, the degree of subjective pain from jaw movement was indicated by the patients on a visual analogue scale (VAS) of 0 to10. The VAS score ranged from 1 to 10, with a mean of 6.0. The control group comprised of 7 joints in 4 men and 3 women with a mean age of 45 years (range 23 to 72 years) who had habitual dislocation without pain. All control patients underwent arthroscopic eminoplasty.22 The validity of adopting these patients as controls has been discussed in detail previously and is discussed briefly below.23 The patients with internal derangement and the control subjects also participated in the previous study.24,25 All of the participants gave their informed consent to arthroscopy, synovial biopsy, and histological examination. Synovial tissues and immunohistochemical staining Two or 3 synovial tissue biopsy specimens approximately 2 mm in diameter were obtained arthroscopically from each patient from the portion of the posterior disk attachment using the triangular technique under direct vision.24 Immediately after resection, the specimens were fixed in 4% paraformaldehyde for 8 hours and embedded in paraffin. Sections were prepared and stained immunohistochemically by the avidin-biotin technique (Vector Laboratories, Burlingame, CA) as described previously.23-25 The sections were treated with primary antibody to CGRP (dilution rate, 1:1500, polyclonal, Affiniti Research Products Limited, Exeter, UK) and left overnight at 48C. The next day, the
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specimens were incubated in a solution of antirabbit biotinylated antibody (200 dilution; Dako, Carpinteria, CA) for 30 minutes at room temperature, and avidin/ biotinylated horseradish peroxidase complex was added for 30 minutes at room temperature. The color was developed using 3-amino-9-ethyl carbazole followed by counterstaining with hematoxylin. Negative controls in which the primary antibody was replaced with normal rabbit IgG were run with each specimen. The CGRPstained cells were evaluated in 2 to 5 regions of maximal immunohistochemical density (under light microscopy, magnification 3200). The extent score of CGRP-stained nerve fibers was calculated as the number of blood vessels surrounded by CGRP-positive cells per 10 blood vessels. About 10 to 80 blood vessels were evaluated in each specimen. This work was performed by 2 of the authors (H.Y. and J.S.) who were unaware of the identities of the patients from whom the specimens had been obtained. Moreover, the microvessel density of the tissues was determined by staining endothelial cells using a primary antibody to CD34 (dilution rate: 1:50, monoclonal, Nichirei, Tokyo, Japan), as a specific endothelial marker, according to Weidner’s method with minor modifications.26,27 The immunohistochemical method is described above.25 The microvessel density was evaluated as the total number of blood vessels in 2 areas of maximal vascularization under a light microscope (320 objective and 310 ocular, 0.74 mm2 per field). Arthroscopy Diagnostic arthroscopy was performed by the standard technique in the whole area of the upper joint compartment and all findings were recorded.28 The degree of synovitis was evaluated according to the criteria of Murakami et al.2 This evaluation was performed by 2 of the authors (N.S. and J.S.) without knowledge of other information about the patients. The mean synovitis score was 5.1. Statistical analysis The Spearman correlation coefficient was used to assess correlations between the extent score of CGRPpositive cells and the clinical symptoms. Mann-Whitney test was used to compare the extent score of CGRPpositive cells between the internal derangement and the control groups. Mann-Whitney test was also used to compare the extent of CGRP-positive cells and the distinction of sex, bone changes (osteophytes or erosion) of the condyle, and the use of NSAIDs before the operation. Stat View J-5.0 (Abacus Concepts, Berkeley, CA) was used for all statistical analysis. P values of less than .05 were considered significant.
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Table I. Patient and CGRP-staining score Patient no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49* 50* 51* 52* 53* 54* 55*
Age/Sex (yrs)
Duration (mo)
MIO (mm)
VAS
Synovitis score
MVD
CGRP score
23/F 24/F 70/F 36/F 61/M 70/F 38/F 55/M 55/M 33/F 22/F 40/F 28/F 41/F 21/F 45/F 23/F 56/F 55/F 50/M 50/F 18/F 17/F 84/F 35/M 35/F 76/F 73/F 52/F 40/F 18/F 33/F 17/F 24/F 58/F 31/F 39/M 52/F 56/F 36/F 35/F 58/F 52/F 61/F 38/F 48/M 23/F 53/F 23/M 66/F 27/M 46/M 25/F 55/F 72/M
6 1 3 3 20 8 7 1 2 16 12 6 24 7 4 8 15 5 5 2 6 11 4 4 4 5 7 7 2 1 2 2 2 2 5 7 2 2 2 9 3 6 3 2 13 7 10 6 4 7 4 3 2 60 11
27 30 35 35 34 42 31 20 33 48 30 40 37 32 32 34 36 32 27 35 37 33 33 45 33 39 30 29 36 30 30 28 40 36 30 23 33 32 38 42 28 30 25 31 24 35 30 30 44 42 52 40 50 30 50
4 8 7 7 7 5 8 9 10 3 4 7 6 10 5 7 5 3 4 8 6 6 6 3 6 6 5 5 1 4 8 7 9 9 6 5 2 1 10 8 4 6 5 5 7 5 7 3 0 0 0 0 0 0 0
8 8 7 5 5 3 7 5 3 4 6 8 6 3 6 4 5 5 4 6 10 6 5 2 5 4 2 5 6 9 2 4 4 8 4 4 9 3 2 5 6 5 2 6 3 4 6 7 0 0 0 0 0 0 0
32 15 30 33 10 10 38 16 4 12 48 45 19 5 10 17 36 58 7 3 25 25 29 6 43 33 3 25 3 19 10 6 31 20 15 35 59 14 8 56 29 75 12 65 19 75 45 66 6 20 5 5 14 15 10
5 9 4 6 8 8 7 9 3 7 5 7 8 4 5 1 6 2 1 5 1 8 4 3 5 6 6 1 3 3 9 1 9 9 3 7 3 1 3 4 2 3 5 3 2 7 3 8 7 1 3 1 2 3 2
M, male; F, female; duration, duration of the symptoms; MIO, maximum interincisal opening; VAS, visual analogue scale; synovitis score, the degree of synovitis evaluated by arthroscopy;2 MDV, number of the blood vessels/2 fields; CGRP score, extent score of CGRP-positive cells. *Control subjects with habitual dislocation.
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Fig 1. A and B, CGRP-stained sections obtained from a patient with internal derangement (A: case number 17, B: case number 34). Immunoreactivities for CGRP (arrows) can be seen in the connective tissues around the blood vessels beneath the lining cells (original magnification A: 3100, B: 3200). C, CGRP-stained sections obtained from patient number 38 with internal derangement. Immunoreactivities for CGRP (arrows) can be seen in the connective tissues around the blood vessels beneath the lining cells. No immunoreactivities for CGRP can been seen around the some blood vessels (arrow head) (original magnification 3100). D, CGRPstained specimen obtained from the control patient (case number 53). Only a few immunopositive cells for CGRP are observed around the blood vessels (arrows) (original magnification 3100).
RESULTS Immunohistochemical staining In the internal derangement and control groups, CGRP-positive cells were detected immunohistochemically in all of the synovial tissues. In the internal derangement group, cells that stained positively for CGRP were seen in the connective tissues around the blood vessels beneath the lining cells (Fig 1, A-C). All of the negative control sections showed only background staining (data not shown). The distribution of CGRPpositive cells in the control specimens was the same as in the internal derangement (Fig 1, D). The mean 6 SD (median) extent score of CGRP-positive cells was 4.8 6 2.6 (5) in the internal derangement specimens versus 2.7 6 2.1 (2) in the control specimens. The extent score of CGRP was significantly higher in the internal
derangement group than in the control group (P = .033) (Fig 2). The expression of CGRP and clinical variables, microvessel density In the internal derangement group, there were no significant correlations between the extent score of CGRP-positive cells and maximum interincisal opening (P = .17), the duration of symptoms (P = .51), the degree of synovitis (P = .50), or microvessel density (P = .56). There were also no significant correlations between the extent score of CGRP-positive cells and the distinction of sex (P = .72) or use of NSAIDs before the operation (P = .07). There was a significant positive correlation between the extent score of CGRP-positive cells and joint pain (VAS) (P = .036, r = 0.30) (Fig 3).
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Fig 2. The extent score of CGRP-positive cells in synovial tissues of patients with internal derangement and controls. The extent score CGRP-positive cells is significantly higher in the internal derangement group than in the control group (P = .033; MannWhitney test).
Fig 3. The extent score of CGRP-positive cells and VAS of pain. Significant correlation is indicated by the Spearman correlation coefficient (P = .036, r = 0.30).
Moreover, there was a significant negative correlation between the extent score of CGRP-positive cells and the age of the patients (P = .02, r = e0.34) (Fig 4). However, there was no significant correlation between age of the patients and joint pain (P = .25). In addition, there was no significant correlation between the degree of synovitis and joint pain (VAS) (P = .42) and between microvessel density and joint pain (P = .35).
DISCUSSION In this study, we demonstrated that CGRP-containing nerve fibers were present in the synovial tissues of the posterior disk attachment in patients with internal derangement. Moreover, the extent score of CGRPcontaining nerve fibers in the synovial tissues was significantly higher in the internal derangement group than in the control group. CGRP coexists with substance
538 Sato et al
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Fig 4. The extent score of CGRP-positive cells and the age of the patients. Significant negative correlation is indicated by the Spearman correlation coefficient (P = .02, r = ÿ0.34).
P in nociceptive C-fibers, the number of which usually correlates with the sensation of pain.13,16,29 CGRP and substance P can induce an inflammatory response by the activation of proinflammatory cytokines and arachidonic acid catabolism.12 In the present study, there was a weak, but significant positive correlation between the extent score of CGRP-positive nerve fiber and joint pain in the internal derangement group (P = .036, r = 0.30). These results may support 2 possibilities with regard to the morphological and biological changes in the synovial tissues. The first is that the density of the CGRP-positive sensory nerve fibers may be increased in the synovial tissues in patients with painful internal derangement. The second is that the release of neuropeptides such as CGRP from the nerve tissues may be increased in the synovial tissues in patients with symptomatic internal derangement. On the other hand, CGRP-positive nerve fibers were also present in the control synovial tissues without pain. Previous studies indicated that sensory joint nerve fibers are indispensable for control of joint movement and for protection of the joint structures.30,31In our study, the controls were 7 patients with habitual dislocation rather than nonsymptomatic healthy control subjects. We think that these patients were suitable controls because they were basically nonsymptomatic except for occasional difficulty of closing their mouths. They felt no pain in their jaw even before the operation. The ages of the control patients were similar to those of the internal derangement patients and their MRI revealed a normal disk position in a closed mouth position. All of the arthroscopic synovitis scores of the control group were 0. Unfortunately this is a huge challenge, as it is impossible to obtain healthy fresh samples of synovial tissues. Previous studies have demonstrated TMJ innervation using immunohistochemical methods in various animal
models.17-20,32 The innervation of the synovial tissues in human TMJ is, however, largerly unknown. Previous studies on human TMJ were based on the gold or silver impregnation technique, which may give inconstant and nonspecific results.29,33,34 Wink et al33 demonstrated neural elements within the articular disk of the human TMJ using the gold chloride technique. They reported that the concentration of neural elements was greatest at the posterior margin of the disk. Morani et al30 investigated innervation of the human TMJ capsule and articular disk using neurospecific markers S-100 protein and protein gene product 9.5 (PGP 9.5). They detected perivascular fibers in the capsule and the bilaminar part of the disk. Although CGRP is found in the sensory nerves, PGP 9.5 has a wide distribution in the sensory, motor, and autonomous nerves. Although S-100 protein was thought to be a neurospecific marker, it has been found in other types of cells, including melanocytes, epithelial cells, chondrocytes, and Langerhans cells. In these previous studies, human TMJ tissues were obtained from cadavers. Thus, it was possible that the antigens used as markers and nerve structures may have disappeared or changed.30 Moreover, the authors did not refer to the relationships between the innervation pattern and the clinical findings. Therefore, we used fresh synovial tissues to ensure maintenance of the antigens and nerve structures. We investigated the extent of the nerve fibers immunohistochemically using antiserum to CGRP, which is thought to be the most reliable maker for the sensory nerves. Holmlund et al11 detected the levels of CGRP in the synovial fluids from patients with TMJ arthropathy. These patients consisted of those with internal derangement, osteoarthrosis, and rheumatoid arthritis. CGRP was found in all of the 19 joints examined.
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However, no significant correlations were found between the concentration of CGRP in the synovial fluid and clinical or arthroscopic findings (the degree of synovitis). CGRP has a strong vasodilatory effect in the joints.35 In the present study, however, no significant correlation was observed between the extent score of CGRP and the degree of synovitis seen by arthroscopy. This result suggests that the degree of synovitis was mainly regulated by other factors, such as inflammatory cytokines, rather than CGRP. Although CGRP was frequently located along the blood vessels, no correlation was observed between the extent score of CGRP and microvessel density in the present study. In some synovial specimens, CGRP-positive cells were not observed around the increased blood vessels (Fig 1, C). This result indicates that the increase in number of blood vessels in the synovial tissues was not directly connected with an increase in CGRP. Westesson et al36 reported that joint pain has 4 probable sources: (1) compression of the highly vascularized and innervated posterior disk attachment, (2) stretching and pulling in the capsule and disk attachment, (3) inflammatory changes in the joint capsule with synovitis, and (4) distension of the joint space due to large joint effusions. Sano et al37 demonstrated that joint pain was correlated with bone marrow alterations of the condyle as seen on MRI. Moreover, it has been suggested that the central nervous system is involved not only in sensory reception but also in regulating the activities of peripheral tissues under both physiologic and pathologic conditions.30 As we performed punch biopsy from the posterior attachment, we were not able to demonstrate innervation of the whole synovial tissues and TMJ structures. As the pathological mechanisms underlying joint pain are complex, we are aware of the possibility that the CGRP-positive nerve fibers in the synovial tissues are only associated with certain mechanisms of pain. This background may support the absence of a strong significant correlation between CGRP and joint pain in the present study. In previous studies, however, we found no significant correlation between joint pain and the levels of IL-1b, IL-6, TNF-a, fibroblast growth facor-2 (FGF-2) and vascular endothelial growth factor (VEGF) in synovial fluids or synovial tissues.10,23, 24,38 Moreover, in the present study we could not detect significant correlations between synovitis , microvessel density, and joint pain. Although the significant correlation between the CGRP and joint pain was not strong, we expected that CGRP may be the important regulator of joint pain rather than other proinflammatory cytokines and growth factors. In the present study, there was a significant negative correlation between the extent score of CGRP-positive cells and the age of the patients. The changes in
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innervation of the human synovial tissues of the TMJ with age are unclear. In the rat TMJ, Ichikawa et al18 reported that the number of CGRP-positive nerve fibers in the articular disk decreased with age. In contrast, there were no significant correlations between the age of the patients and the expression of other cytoines, such as IL1b, IL-6, FGF-2, and VEGF, in previous studies.8,23,24,38 To our regret, we are not able to explain clearly the negative correlation between the age of the patients and CGRP. It may be related to aging changes of the synovial tissues. Further study is needed to elucidate this phenomenon. In conclusion, innervation of the synovial tissues of the human TMJ with neuropeptides seems to play an important role in the mechanism of pain production in patients with highly symptomatic TMJs and internal derangement. These results should provide further insight into potential treatments designed to interfere with pain pathways in this patient population.
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Reprint requests: Jun Sato, DDS Department of Oral and Maxillofacial Surgery Kanazawa Medical University Daigaku, Uchinada, Ishikawa Pref. 920-0293, Japan [email protected]
Objective. To elucidate the expression of calcitonin gene-related peptide (CGRP) in synovial tissue taken from the human temporomandibular joint (TMJ) with internal derangement, and discuss the relationship between CGRP and joint pain. Study design. Using an immunohistochemical technique, 48 joints in 48 patients were examined. As controls, synovial tissue specimens from 7 joints with habitual dislocation without pain were also examined. Results. In all of the internal derangement and control subjects, CGRP-positive cells were observed in the connective tissues around the blood vessels beneath the lining cells. The extent score of CGRP was significantly higher in the internal derangement group than in the control group (P = .033). There was a significant positive correlation between the extent score of CGRP and joint pain (P = .036, r = 0.30). Conclusions. These results suggest that the expression of CGRP is increased in the synovial tissues from patients with internal derangement, and that CGRP seems to play an important role in the mechanism of pain production in patients with symptomatic internal derangement. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:533-40)
Although pain in the temporomandibular joint (TMJ) region is one of the major symptoms in patients with internal derangement of the TMJ,1 the cause and pathophysiological mechanisms of the local pain are not completely understood. It has been hypothesized that synovitis may be one of the causes of TMJ pain. Previous studies2,3 indicated significant correlations between joint pain and the degree of synovitis. Recent studies have explored the importance of biological changes within the synovial fluids and synovial tissues associated with joint pain.4-8 Quinn and Bazan9 detected high levels of prostaglandin E2 in synovial fluid of inflamed joints and demonstrated a strong correlation with pain. Similarly, recent studies revealed significant correlations between proinflammatory cytokines such as interleukin
This study was partially supported by a Grant for Specially Promoted Research from Kanazawa Medical University. a Lecturer, Department of Oral and Maxillofacial Surgery, Kanazawa Medical University. b Professor and Chairman, Department of Oral and Maxillofacial Surgery, Kanazawa Medical University. c Associate Professor, Department of Oral and Maxillofacial Surgery, Kanazawa Medical University. d Associate Professor, Department of Biochemistry, Kanazawa Medical University. Received for publication Nov 14, 2003; returned for revision Jan 14, 2004; accepted for publication Feb 16, 2004. 1079-2104/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.tripleo.2004.02.057
1b (IL-1b),4,7 IL-6,6 and tumor necrosis factor (TNF-a)5 in synovial fluids and joint pain. These proinflammatory cytokines are thought to contribute to the pathogenesis of synovitis and osteoarthritis in TMJs. In previous studies, however, we found no significant correlation between the concentrations of IL-1b, IL-6, and TNF-a in synovial fluids and joint pain.10 There were some discrepancies in the correlations reported between the levels of proinflammatory cytokines and joint pain among investigations. More recently, other kinds of chemical mediators, including neuropeptides such as calcitonin gene-related peptide (CGRP), substance P, and serotonin have been detected in the TMJ.1,11,12 These neuropeptides can be released from activated peripheral nerve terminals into the surrounding tissues and cause an inflammatory response.12 CGRP is a 37-amino acid peptide produced in neural cells13,14 and has been shown to coexist with substance P in mainly nociceptive C-fibers.13,15 Small afferent unmyelinated C-fibers, which are usually correlated with nociception and the sensation of pain, have been shown to contain CGRP.15,16 Moreover, CGRP is thought to be a good marker for sensory nerves.17-20 In earlier studies, some investigators11,21 detected levels of CGRP in synovial fluids of the TMJ. However, there have been few reports of CGRP expression in human synovial tissue of the TMJ. In the present study, we investigated CGRP in synovial tissue immunohistochemically and compared the CGRP levels between joints with internal derangement and those with habitual dislocation without pain. Moreover, we assessed 533
534 Sato et al the correlation between CGRP level and clinical findings, especially in joint pain. PATIENTS AND METHODS Patients Forty-eight TMJs in 48 patients with internal derangement were included in this study (Table I). Seven of the patients were men and 41 were women. The average age was 43 years old (range 17-84). MRI showed that all of the patients had anterior disk displacement without reduction. Their mean maximum interincisal opening was 33 mm and the mean duration of symptoms was 6 months. As a result of the failure of nonsurgical treatment consisting of occlusal appliance, nonsteroidal antiinflammatory drugs (NSAIDS), and physiotherapy to resolve the clinical symptoms, all of the patients had arthroscopic surgery. Twenty-two (46%) of the 48 patients were given NSAIDS within a month before the operation (Etodolac: 12 patients, 400 mg/day, range 7 to 30 days, mean 11 days and Loxoprofen sodium: 10 patients, 180 mg/day, range 3 to 14 days, mean 8 days). Just before the operation, the degree of subjective pain from jaw movement was indicated by the patients on a visual analogue scale (VAS) of 0 to10. The VAS score ranged from 1 to 10, with a mean of 6.0. The control group comprised of 7 joints in 4 men and 3 women with a mean age of 45 years (range 23 to 72 years) who had habitual dislocation without pain. All control patients underwent arthroscopic eminoplasty.22 The validity of adopting these patients as controls has been discussed in detail previously and is discussed briefly below.23 The patients with internal derangement and the control subjects also participated in the previous study.24,25 All of the participants gave their informed consent to arthroscopy, synovial biopsy, and histological examination. Synovial tissues and immunohistochemical staining Two or 3 synovial tissue biopsy specimens approximately 2 mm in diameter were obtained arthroscopically from each patient from the portion of the posterior disk attachment using the triangular technique under direct vision.24 Immediately after resection, the specimens were fixed in 4% paraformaldehyde for 8 hours and embedded in paraffin. Sections were prepared and stained immunohistochemically by the avidin-biotin technique (Vector Laboratories, Burlingame, CA) as described previously.23-25 The sections were treated with primary antibody to CGRP (dilution rate, 1:1500, polyclonal, Affiniti Research Products Limited, Exeter, UK) and left overnight at 48C. The next day, the
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specimens were incubated in a solution of antirabbit biotinylated antibody (200 dilution; Dako, Carpinteria, CA) for 30 minutes at room temperature, and avidin/ biotinylated horseradish peroxidase complex was added for 30 minutes at room temperature. The color was developed using 3-amino-9-ethyl carbazole followed by counterstaining with hematoxylin. Negative controls in which the primary antibody was replaced with normal rabbit IgG were run with each specimen. The CGRPstained cells were evaluated in 2 to 5 regions of maximal immunohistochemical density (under light microscopy, magnification 3200). The extent score of CGRP-stained nerve fibers was calculated as the number of blood vessels surrounded by CGRP-positive cells per 10 blood vessels. About 10 to 80 blood vessels were evaluated in each specimen. This work was performed by 2 of the authors (H.Y. and J.S.) who were unaware of the identities of the patients from whom the specimens had been obtained. Moreover, the microvessel density of the tissues was determined by staining endothelial cells using a primary antibody to CD34 (dilution rate: 1:50, monoclonal, Nichirei, Tokyo, Japan), as a specific endothelial marker, according to Weidner’s method with minor modifications.26,27 The immunohistochemical method is described above.25 The microvessel density was evaluated as the total number of blood vessels in 2 areas of maximal vascularization under a light microscope (320 objective and 310 ocular, 0.74 mm2 per field). Arthroscopy Diagnostic arthroscopy was performed by the standard technique in the whole area of the upper joint compartment and all findings were recorded.28 The degree of synovitis was evaluated according to the criteria of Murakami et al.2 This evaluation was performed by 2 of the authors (N.S. and J.S.) without knowledge of other information about the patients. The mean synovitis score was 5.1. Statistical analysis The Spearman correlation coefficient was used to assess correlations between the extent score of CGRPpositive cells and the clinical symptoms. Mann-Whitney test was used to compare the extent score of CGRPpositive cells between the internal derangement and the control groups. Mann-Whitney test was also used to compare the extent of CGRP-positive cells and the distinction of sex, bone changes (osteophytes or erosion) of the condyle, and the use of NSAIDs before the operation. Stat View J-5.0 (Abacus Concepts, Berkeley, CA) was used for all statistical analysis. P values of less than .05 were considered significant.
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Table I. Patient and CGRP-staining score Patient no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49* 50* 51* 52* 53* 54* 55*
Age/Sex (yrs)
Duration (mo)
MIO (mm)
VAS
Synovitis score
MVD
CGRP score
23/F 24/F 70/F 36/F 61/M 70/F 38/F 55/M 55/M 33/F 22/F 40/F 28/F 41/F 21/F 45/F 23/F 56/F 55/F 50/M 50/F 18/F 17/F 84/F 35/M 35/F 76/F 73/F 52/F 40/F 18/F 33/F 17/F 24/F 58/F 31/F 39/M 52/F 56/F 36/F 35/F 58/F 52/F 61/F 38/F 48/M 23/F 53/F 23/M 66/F 27/M 46/M 25/F 55/F 72/M
6 1 3 3 20 8 7 1 2 16 12 6 24 7 4 8 15 5 5 2 6 11 4 4 4 5 7 7 2 1 2 2 2 2 5 7 2 2 2 9 3 6 3 2 13 7 10 6 4 7 4 3 2 60 11
27 30 35 35 34 42 31 20 33 48 30 40 37 32 32 34 36 32 27 35 37 33 33 45 33 39 30 29 36 30 30 28 40 36 30 23 33 32 38 42 28 30 25 31 24 35 30 30 44 42 52 40 50 30 50
4 8 7 7 7 5 8 9 10 3 4 7 6 10 5 7 5 3 4 8 6 6 6 3 6 6 5 5 1 4 8 7 9 9 6 5 2 1 10 8 4 6 5 5 7 5 7 3 0 0 0 0 0 0 0
8 8 7 5 5 3 7 5 3 4 6 8 6 3 6 4 5 5 4 6 10 6 5 2 5 4 2 5 6 9 2 4 4 8 4 4 9 3 2 5 6 5 2 6 3 4 6 7 0 0 0 0 0 0 0
32 15 30 33 10 10 38 16 4 12 48 45 19 5 10 17 36 58 7 3 25 25 29 6 43 33 3 25 3 19 10 6 31 20 15 35 59 14 8 56 29 75 12 65 19 75 45 66 6 20 5 5 14 15 10
5 9 4 6 8 8 7 9 3 7 5 7 8 4 5 1 6 2 1 5 1 8 4 3 5 6 6 1 3 3 9 1 9 9 3 7 3 1 3 4 2 3 5 3 2 7 3 8 7 1 3 1 2 3 2
M, male; F, female; duration, duration of the symptoms; MIO, maximum interincisal opening; VAS, visual analogue scale; synovitis score, the degree of synovitis evaluated by arthroscopy;2 MDV, number of the blood vessels/2 fields; CGRP score, extent score of CGRP-positive cells. *Control subjects with habitual dislocation.
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Fig 1. A and B, CGRP-stained sections obtained from a patient with internal derangement (A: case number 17, B: case number 34). Immunoreactivities for CGRP (arrows) can be seen in the connective tissues around the blood vessels beneath the lining cells (original magnification A: 3100, B: 3200). C, CGRP-stained sections obtained from patient number 38 with internal derangement. Immunoreactivities for CGRP (arrows) can be seen in the connective tissues around the blood vessels beneath the lining cells. No immunoreactivities for CGRP can been seen around the some blood vessels (arrow head) (original magnification 3100). D, CGRPstained specimen obtained from the control patient (case number 53). Only a few immunopositive cells for CGRP are observed around the blood vessels (arrows) (original magnification 3100).
RESULTS Immunohistochemical staining In the internal derangement and control groups, CGRP-positive cells were detected immunohistochemically in all of the synovial tissues. In the internal derangement group, cells that stained positively for CGRP were seen in the connective tissues around the blood vessels beneath the lining cells (Fig 1, A-C). All of the negative control sections showed only background staining (data not shown). The distribution of CGRPpositive cells in the control specimens was the same as in the internal derangement (Fig 1, D). The mean 6 SD (median) extent score of CGRP-positive cells was 4.8 6 2.6 (5) in the internal derangement specimens versus 2.7 6 2.1 (2) in the control specimens. The extent score of CGRP was significantly higher in the internal
derangement group than in the control group (P = .033) (Fig 2). The expression of CGRP and clinical variables, microvessel density In the internal derangement group, there were no significant correlations between the extent score of CGRP-positive cells and maximum interincisal opening (P = .17), the duration of symptoms (P = .51), the degree of synovitis (P = .50), or microvessel density (P = .56). There were also no significant correlations between the extent score of CGRP-positive cells and the distinction of sex (P = .72) or use of NSAIDs before the operation (P = .07). There was a significant positive correlation between the extent score of CGRP-positive cells and joint pain (VAS) (P = .036, r = 0.30) (Fig 3).
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Fig 2. The extent score of CGRP-positive cells in synovial tissues of patients with internal derangement and controls. The extent score CGRP-positive cells is significantly higher in the internal derangement group than in the control group (P = .033; MannWhitney test).
Fig 3. The extent score of CGRP-positive cells and VAS of pain. Significant correlation is indicated by the Spearman correlation coefficient (P = .036, r = 0.30).
Moreover, there was a significant negative correlation between the extent score of CGRP-positive cells and the age of the patients (P = .02, r = e0.34) (Fig 4). However, there was no significant correlation between age of the patients and joint pain (P = .25). In addition, there was no significant correlation between the degree of synovitis and joint pain (VAS) (P = .42) and between microvessel density and joint pain (P = .35).
DISCUSSION In this study, we demonstrated that CGRP-containing nerve fibers were present in the synovial tissues of the posterior disk attachment in patients with internal derangement. Moreover, the extent score of CGRPcontaining nerve fibers in the synovial tissues was significantly higher in the internal derangement group than in the control group. CGRP coexists with substance
538 Sato et al
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Fig 4. The extent score of CGRP-positive cells and the age of the patients. Significant negative correlation is indicated by the Spearman correlation coefficient (P = .02, r = ÿ0.34).
P in nociceptive C-fibers, the number of which usually correlates with the sensation of pain.13,16,29 CGRP and substance P can induce an inflammatory response by the activation of proinflammatory cytokines and arachidonic acid catabolism.12 In the present study, there was a weak, but significant positive correlation between the extent score of CGRP-positive nerve fiber and joint pain in the internal derangement group (P = .036, r = 0.30). These results may support 2 possibilities with regard to the morphological and biological changes in the synovial tissues. The first is that the density of the CGRP-positive sensory nerve fibers may be increased in the synovial tissues in patients with painful internal derangement. The second is that the release of neuropeptides such as CGRP from the nerve tissues may be increased in the synovial tissues in patients with symptomatic internal derangement. On the other hand, CGRP-positive nerve fibers were also present in the control synovial tissues without pain. Previous studies indicated that sensory joint nerve fibers are indispensable for control of joint movement and for protection of the joint structures.30,31In our study, the controls were 7 patients with habitual dislocation rather than nonsymptomatic healthy control subjects. We think that these patients were suitable controls because they were basically nonsymptomatic except for occasional difficulty of closing their mouths. They felt no pain in their jaw even before the operation. The ages of the control patients were similar to those of the internal derangement patients and their MRI revealed a normal disk position in a closed mouth position. All of the arthroscopic synovitis scores of the control group were 0. Unfortunately this is a huge challenge, as it is impossible to obtain healthy fresh samples of synovial tissues. Previous studies have demonstrated TMJ innervation using immunohistochemical methods in various animal
models.17-20,32 The innervation of the synovial tissues in human TMJ is, however, largerly unknown. Previous studies on human TMJ were based on the gold or silver impregnation technique, which may give inconstant and nonspecific results.29,33,34 Wink et al33 demonstrated neural elements within the articular disk of the human TMJ using the gold chloride technique. They reported that the concentration of neural elements was greatest at the posterior margin of the disk. Morani et al30 investigated innervation of the human TMJ capsule and articular disk using neurospecific markers S-100 protein and protein gene product 9.5 (PGP 9.5). They detected perivascular fibers in the capsule and the bilaminar part of the disk. Although CGRP is found in the sensory nerves, PGP 9.5 has a wide distribution in the sensory, motor, and autonomous nerves. Although S-100 protein was thought to be a neurospecific marker, it has been found in other types of cells, including melanocytes, epithelial cells, chondrocytes, and Langerhans cells. In these previous studies, human TMJ tissues were obtained from cadavers. Thus, it was possible that the antigens used as markers and nerve structures may have disappeared or changed.30 Moreover, the authors did not refer to the relationships between the innervation pattern and the clinical findings. Therefore, we used fresh synovial tissues to ensure maintenance of the antigens and nerve structures. We investigated the extent of the nerve fibers immunohistochemically using antiserum to CGRP, which is thought to be the most reliable maker for the sensory nerves. Holmlund et al11 detected the levels of CGRP in the synovial fluids from patients with TMJ arthropathy. These patients consisted of those with internal derangement, osteoarthrosis, and rheumatoid arthritis. CGRP was found in all of the 19 joints examined.
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However, no significant correlations were found between the concentration of CGRP in the synovial fluid and clinical or arthroscopic findings (the degree of synovitis). CGRP has a strong vasodilatory effect in the joints.35 In the present study, however, no significant correlation was observed between the extent score of CGRP and the degree of synovitis seen by arthroscopy. This result suggests that the degree of synovitis was mainly regulated by other factors, such as inflammatory cytokines, rather than CGRP. Although CGRP was frequently located along the blood vessels, no correlation was observed between the extent score of CGRP and microvessel density in the present study. In some synovial specimens, CGRP-positive cells were not observed around the increased blood vessels (Fig 1, C). This result indicates that the increase in number of blood vessels in the synovial tissues was not directly connected with an increase in CGRP. Westesson et al36 reported that joint pain has 4 probable sources: (1) compression of the highly vascularized and innervated posterior disk attachment, (2) stretching and pulling in the capsule and disk attachment, (3) inflammatory changes in the joint capsule with synovitis, and (4) distension of the joint space due to large joint effusions. Sano et al37 demonstrated that joint pain was correlated with bone marrow alterations of the condyle as seen on MRI. Moreover, it has been suggested that the central nervous system is involved not only in sensory reception but also in regulating the activities of peripheral tissues under both physiologic and pathologic conditions.30 As we performed punch biopsy from the posterior attachment, we were not able to demonstrate innervation of the whole synovial tissues and TMJ structures. As the pathological mechanisms underlying joint pain are complex, we are aware of the possibility that the CGRP-positive nerve fibers in the synovial tissues are only associated with certain mechanisms of pain. This background may support the absence of a strong significant correlation between CGRP and joint pain in the present study. In previous studies, however, we found no significant correlation between joint pain and the levels of IL-1b, IL-6, TNF-a, fibroblast growth facor-2 (FGF-2) and vascular endothelial growth factor (VEGF) in synovial fluids or synovial tissues.10,23, 24,38 Moreover, in the present study we could not detect significant correlations between synovitis , microvessel density, and joint pain. Although the significant correlation between the CGRP and joint pain was not strong, we expected that CGRP may be the important regulator of joint pain rather than other proinflammatory cytokines and growth factors. In the present study, there was a significant negative correlation between the extent score of CGRP-positive cells and the age of the patients. The changes in
Sato et al 539
innervation of the human synovial tissues of the TMJ with age are unclear. In the rat TMJ, Ichikawa et al18 reported that the number of CGRP-positive nerve fibers in the articular disk decreased with age. In contrast, there were no significant correlations between the age of the patients and the expression of other cytoines, such as IL1b, IL-6, FGF-2, and VEGF, in previous studies.8,23,24,38 To our regret, we are not able to explain clearly the negative correlation between the age of the patients and CGRP. It may be related to aging changes of the synovial tissues. Further study is needed to elucidate this phenomenon. In conclusion, innervation of the synovial tissues of the human TMJ with neuropeptides seems to play an important role in the mechanism of pain production in patients with highly symptomatic TMJs and internal derangement. These results should provide further insight into potential treatments designed to interfere with pain pathways in this patient population.
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Reprint requests: Jun Sato, DDS Department of Oral and Maxillofacial Surgery Kanazawa Medical University Daigaku, Uchinada, Ishikawa Pref. 920-0293, Japan [email protected]