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Specific expression of substance P in synovial tissues of patients with symptomatic, non-reducing internal derangement of the temporomandibular joint: Comparison with clinical findings
British Journal of Oral and Maxillofacial Surgery 45 (2007) 372–377
Specific expression of substance P in synovial tissues of patients with symptomatic, non-reducing internal derangement of the temporomandibular joint: Comparison with clinical findings Jun Sato a,c,∗ , Natsuki Segami a , Yoshino Yoshitake b , Keiseki Kaneyama a , Hiroshi Yoshimura a , Kazuma Fujimura a , Yoshimasa Kitagawa c a
Department of Oral and Maxillofacial Surgery, Kanazawa Medical University, Daigaku, Uchinada-machi, Kahoku-gun, Ishikawa 920-0293, Japan Department of Biochemistry, Kanazawa Medical University, Japan c Oral Diagnosis and Medicine, Department of Oral Pathobiological Science, Hokkaido University Graduate School of Dental Medicine, North 13, West 7, Kita-ward, Sapporo City 060-8586, Japan b
Accepted 21 September 2006 Available online 13 November 2006
Abstract Our aim was to find out the extent of expression of substance P in synovial tissue from the human temporomandibular joints (TMJ) with symptomatic, non-reducing internal derangement, and to investigate the relationship between substance P and clinical findings. Fifty-four joints in 54 patients were examined immunohistochemically. Specimens of synovial tissue from 10 joints in 8 subjects with habitual dislocation of the TMJ with no pain were examined as controls. Cells that stained for substance P were found mainly among the endothelial cells in the blood vessels beneath the lining cells in synovial tissues from 47 of the 54 joints (87%) with internal derangement and from 5 of the 10 control joints. The extent score of cells that stained for substance P in joints with internal derangement was significantly higher than that in controls (p = 0.02). The extent score of these cells did not correlate with pain in the joint or the degree of synovitis. These results suggest that substance P may have some roles in both the physiological and pathological conditions in patients with symptomatic internal derangement of the TMJ. © 2006 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Keywords: Substance P; Synovial tissue; Neurogenic inflammation; Temporomandibular joint
Introduction Internal derangement of the temporomandibular joint (TMJ) is often accompanied by synovitis. Neuropeptides are now recognised as a key to understanding the pathophysiology of arthritis.1 These neuropeptides, including substance P and calcitonin gene-related peptide (CGRP), are present mainly
∗ Corresponding author at: Oral Diagnosis and Medicine, Department of Oral Pathobiological Science, Hokkaido University Graduate School of Dental Medicine, North 13, West 7, Kita-ward. Sapporo City 060-8586, Japan. Tel.: +81 11 716 2111; fax: +81 11 706 4280. E-mail address: [email protected] (J. Sato).
in nociceptive C-fibres, which usually correlate with nociception and the sensation of pain.2,3 These neuropeptides are released by activated peripheral nerve terminals into the surrounding tissues and cause an inflammatory response, known as neurogenic inflammation.4 Substance P is a member of the tachykinin family, and it has been suggested that it is one of the most important neuropeptides that modulate the inflammatory process of arthritis.5 Substance P acts not only as a transmitter of pain signals, “but also stimulates macrophages, neutrophils, and endothelial cells to induce phagocytosis, chemotaxis, and plasma to extravasate”.5,6 Recently, we showed that CGRP and the capsaicin receptor TRPV-1 are expressed in synovial tissues of the TMJ. Transient receptor
0266-4356/$ – see front matter © 2006 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bjoms.2006.09.011
J. Sato et al. / British Journal of Oral and Maxillofacial Surgery 45 (2007) 372–377
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Table 1 (Continued )
Table 1 Patient and substance P-staining score Case number
Age /sex (years)
VAS for pain
Synovitis score (evaluated by arthroscopy)
Extent score for number of cells stained for substance P
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
23/F 24/F 70/F 36/F 61/M 70/F 38/F 55/M 22/F 40/F 28/F 41/F 21/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 55/F 32/F 36/M 25/F 19/F 56/F 13/F 40/F 22/F
4 8 7 7 7 5 8 9 4 7 6 10 5 5 3 4 8 6 6 6 3 6 6 5 5 1 4 8 7 9 9 6 7 2 1 10 8 4 6 5 5 7 5 7 3 8 7 5 3 7 4 5 8 5
8 8 7 5 5 3 7 5 6 8 6 3 6 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 9 4 7 6 7 8 9 5 8
6 40 40 3 25 0 4 7 4 66 18 10 25 40 18 0 20 0 16 8 38 14 20 30 0 40 35 45 10 12 85 10 8 25 33 0 70 2 40 36 25 10 20 26 40 8 0 18 12 0 40 22 36 33
Control subjects with habitual dislocation:† 55† 23/M 0 0 56† 66/F 0 0 57† 27/M 0 0 58† 46/M 0 0
36 0 0 6
Case number
Age /sex (years)
59† 60† 61† 62† 63† 64†
72/M 72/M 25/M 25/F 29/F 55/F
VAS for pain
0 0 0 0 0 0
Synovitis score (evaluated by arthroscopy) 0 0 2 0 1 0
Extent score for number of cells stained for substance P 10 0 0 4 0 12
potential V1 (TRPV-1) is one of the major nociceptors and has a key role in inflammatory pain.7,8 We showed that the expression of CGRP is correlated significantly with joint pain in patients with symptomatic internal derangement,9,10 and that there is a possibility that some neuropeptides and neurogenic inflammation are involved in the pathophysiological mechanisms of pain in the TMJ. The expression and clinical relevance of substance P in synovial tissues in patients with symptomatic internal derangement of the TMJ have not been investigated fully. Before the present study, we hypothesised that substance P may be involved in pain in the joint in much the same way as CGRP in patients with non-reducing internal derangement of the TMJ. Here, we have made an immunohistochemical examination of synovial tissues of the TMJ and assessed the correlation between expression of substance P and clinical findings.
Patients and methods Patients (Table 1) We studied 54 joints in 54 patients with symptomatic nonreducing internal derangement of the TMJ. There were 7 men and 47 women, with a mean (S.D.) age of 41 (17) years (range 13–84) (Table 1). Magnetic resonance imaging showed that all of the patients had anterior disc displacement without reduction. The median duration of symptoms was 4 months (range 1–24). All of the patients had had arthroscopy after failure of non-surgical treatment—occlusal appliance, non-steroidal anti-inflammatory drugs (NSAIDS), and physiotherapy. Just before operation, the patients indicated the degree of subjective pain caused by moving the jaw on a visual analogue scale (VAS) of 0–10. This ranged from 1 to 10, with a mean (S.D.) of 5 (2). The control group consisted of 10 joints in eight subjects, four men and four women, with a mean (S.D.) age of 43 (20) years (range 23–72), who had habitual dislocation without pain. All control patients had had arthroscopic eminoplasty.11 The validity of adopting these patients as controls has been discussed in detail previously.9,10 The patients with internal derangement and the control subjects also participated in our previous studies.9,10,12 None of the subjects in either group had had arthroscopy of the TMJ before this study.
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All the participants gave their informed consent to arthroscopy, synovial biopsy, and histological examination.
Table 2 Scoring of intensity of synovitis Score
Synovial tissues and immunohistochemical staining
0
Findings Normal pale, almost translucent, synovial lining with a fine network of anastomosing small blood vessels Increased vascularity and capillary hyperaemia (mild) Increased vascularity and capillary hyperaemia (moderate) Increased vascularity and capillary hyperaemia (severe) Capillary dilatation and increasing network (mild to moderate) Capillary dilatation and increasing network (severe) Contact bleeding on palpation with probe (mild to moderate) Contact bleeding on palpation with probe (severe) Microbleeding and effusion Granulative change, effusion, and debris (mild to moderate) Granulative change, effusion, and debris (severe)
Two or three synovial tissue biopsy specimens roughly 2 mm in diameter were obtained arthroscopically from each patient from a portion of the posterior disc attachment using the triangular technique under direct visualisation.9,10,12 Immediately after resection, the specimens were fixed in 4% paraformaldehyde for 4 h and embedded in paraffin. Sections were prepared and stained immunohistochemically by the avidin–biotin–peroxidase complex method with a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) as described previously.9,10,12 The sections were treated with primary antibody to substance P (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA; sc-21715) and left overnight at 4 ◦ C. This antibody was purified monoclonal rat IgG to substance P in multiple species, and its specificity has been confirmed by the manufacturer. The following day the specimens were incubated in a solution of biotinylated anti-rat antibody (1:200 dilution; Dako, Carpinteria, CA) for 30 min at room temperature, and avidin /biotinylated horseradish peroxidase complex was added for 30 min at room temperature. The colour was developed using 3-amino-9-ethyl carbazole followed by counterstaining with heamatoxylin. The cells that reacted to substance P stained red. Negative controls in which the primary antibody was replaced with normal rat IgG were run with each specimen. The cells stained for substance P were evaluated in two to five regions of maximal immunohistochemical density under light microscopy at a magnification of ×200. The extent score of cells stained for substance P was calculated as the percentage of blood vessels containing those cells. About 10 to 100 blood vessels were evaluated in each specimen. This work was done by two of the authors (KK and JS) who were unaware of the identities of the patients from whom the specimens had been obtained.
1 2 3 4 5 6 7 8 9 10
Arthroscopy
Cells that stained for substance P were seen in synovial tissue specimens from 47 of the 54 joints (87%) in the internal derangement group (Fig. 1). All the negative control specimens showed only background staining (data not shown). The specimens from 5 of the 10 control subjects with habitual dislocation also stained for substance P (Fig. 2A and B). The mean (S.D.; median) extent score of cells that stained for substance P was 23 (19; 20) in the internal derangement specimens compared with 7 (11; 2) in the control specimens, the difference being significant (p = 0.02) (Fig. 3). Cells that stained for substance P were mostly located in the endothelial cells in the blood vessels beneath the lining cells, with some lining the synovium (Figs. 1 and 2B).
Diagnostic arthroscopy was done by a standard technique throughout the whole area of the upper joint compartment.13 The degrees of synovitis and intra-articular adhesion were evaluated according to the criteria of Murakami et al. (Tables 2 and 3)14–16 by two of the authors (NS and JS) who were unaware of other information about the patients. The mean (S.D.) synovitis and adhesion scores were 5.1 (2.1) and 6.4 (1.6) in the internal derangement group, compared with 0.3 (0.7) and 0.8 (0.9) in the control group. Statistical analysis The Mann–Whitney test was used to compare the extent scores of cells stained for substance P between the internal derangement and control groups. The Spearman correlation coefficient was used to assess correlations between the
Table 3 Scoring of intensity of adhesion Score
Findings
0 1 2 3 4 5 6 7 8 9 10
No adhesion or fibrous change FiImy adhesion (mild) Filmy adhesion (moderate to severe) Fibrosynovial band (mild to moderate) Fibrosynovial band (severe) Fibrous band (mild to moderate) Fibrous band (severe) Formation of pseudowall (mild to moderate) Formation of pseudowall (severe) Capsular fibrosis (mild to moderate) Capsular fibrosis (severe)
extent score of cells stained for substance P and the clinical findings. StatView J-5.0 (Abacus Concepts, Berkeley, CA) was used for all statistical analyses. p < 0.05 were considered significant.
Results Immunohistochemical staining
Expression of substance P and clinical findings There were no significant correlations between the extent score of cells stained for substance P and the duration
J. Sato et al. / British Journal of Oral and Maxillofacial Surgery 45 (2007) 372–377
Fig. 1. Section stained for substance P from a patient with internal derangement (case 31). Immunoreactivities for substance P (arrows) can be seen in the endothelial cells in the blood vessels beneath the lining cells and lining the synovium (arrow heads) (original magnification: ×100, bar: 50 m).
375
Fig. 3. The extent score of cells stained for substance P in the synovial tissues of patients with internal derangement and controls. The extent score of these cells is significantly higher in the internal derangement group than in the control group (Mann–Whitney test; p = 0.02).
of symptoms in the internal derangement group (p = 0.14, r = −0.09) (Fig. 4), the degree of synovitis (p = 0.31, r = 0.14) (Fig. 5), the degree of adhesion (p = 0.19, r = −0.22) or the severity of pain in the joint (VAS) (p = 0.75, r = 0.01) (Fig. 6).
Fig. 4. The extent score of cells stained for substance P and the duration of the symptoms are not correlated significantly (Spearman correlation coefficient; p = 0.14, r = −0.09).
Fig. 5. The extent score of cells stained for substance P and the degree of synovitis are not correlated significantly (Spearman correlation coefficient; p = 0.31, r = 0.14).
Fig. 2. (A and B). Specimens stained for substance P from control patients (A: case 61, B: case 55). There is no immunoreactivity for substance P in the synovial tissue (A), and only a few stained cells in the blood vessels (B: arrows) (original magnification A: ×100 and B: ×200, bar: 50 m).
Discussion The contribution of neuropeptides to arthritis is currently a matter of debate.16 In the present study, we have shown
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Fig. 6. The extent score of cells stained for substance P and the VAS of pain are not correlated significantly (Spearman correlation coefficient; p = 0.75, r = 0.01).
the presence of cells stained for substance P in the specimens of synovial tissue in the posterior disc attachment in patients with symptomatic internal derangement of the TMJ. The extent score of these cells was significantly higher in the internal derangement group than in the control group. Substance P has been suggested as not only one of the most important neuropeptides that modulate the inflammatory process of arthritis,17 but also one of the most important to act as a local regulator of blood flow and pain.1,18 Although we expected a close relation between the expression of substance P and joint pain (the degree of synovitis), we were unable to confirm our hypothesis from the results of the present study. We know of a few previous studies of substance P in the TMJ region. In a preliminary study, Henry and Wolford3 showed that cells that stained for substance P were present mainly within the walls of the vasculature in synovial tissues of the human TMJ. As they obtained synovial tissues from only nine women, they did not investigate the relation between the expression of substance P and clinical findings. Our immunohistochemical study supports their observation that cells that stained for substance P were present in the blood vessels. Appelgren et al.18 reported that substance Plike immunoreactivity in the synovial fluids from patients with arthritic TMJs was correlated with intra-articular temperature and pain. Holmlund et al.19 detected substance P-like immunoreactivity in 17 of 19 synovial fluid samples from patients with internal derangement, osteoarthrosis (OA), and rheumatoid arthritis (RA). They concluded that there were no significant correlations between the concentrations of substance P in the synovial fluids and the arthroscopic findings, including the degrees of synovitis and OA. They also concluded that these unexpected results were the results of the relatively small number of patients with pronounced synovitis who were included in their study. Marshal et al.20 reported that the concentrations of substance P in synovial fluids in patients with RA did not differ significantly from those found in patients with OA. Mapp et al.17 found a large number of afferent fibres of substance P in normal synovial tissues, but few such fibres in patients with RA. They speculated that
large amounts of neuropeptides, including substance P, may be released from the sensory terminals in the early phases of inflammation, resulting in permanent depletion. They concluded that substance P and other neuropeptides may be involved in the pathogenesis of inflammation of the joint in the early rather than the chronic phases. However, we found no significant correlation between the extent score of cells stained for substance P and the duration of symptoms. This discrepancy may have been caused by the difference in pathogenesis between internal derangement of the TMJ and RA. As the degree of inflammation of the synovial tissues in patients with internal derangement of the TMJ is usually much less than that in patients with RA, neuropeptides may never be permanently depleted. We detected substance P at low concentrations in the synovial tissues from control subjects. This suggests that it may play some part in the maintenance of physiological conditions in the TMJ. Substance P has been suggested to regulate vascular functions (such as blood flow) under normal conditions,17 and its expression may be a protective response to tissue injury.18,21 The main limitations of the present study were the small number of observations and characteristics of the control patients. These problems are difficult to overcome, as it is impossible to obtain fresh samples of synovial tissues from healthy people.9,10 We have explained the validity of our control subjects in previous reports,9,10,12 and think that these patients with dislocations were suitable control subjects for the present study. As we performed punch biopsies on the posterior disc attachment, it was not possible to assess the distribution of substance P in the whole intracapsular structure of the TMJ. As the pathological mechanisms underlying joint pain are complex, we are aware that it is not possible to work out the mechanisms of all cases of TMJ based pain from our results. In conclusion, we have shown that substance P may be involved in the pathogenesis of symptomatic internal derangement of the TMJ. However, it is unlikely that its important physiological and pathological roles of are mediated independently. Substance P has been suggested to play an important part in the pathogenesis of arthritis by up-regulation of production of other cytokines,22 and it stimulates synovial cells and lymphocytes to produce prostaglandins23 and to release cytokines, such as IL-1.24 It is therefore important to examine the relations between substance P and other chemical mediators in more depth. Further studies are required to elucidate the possible contribution of substance P to physiological and pathological conditions in patients with internal derangement of the TMJ.
Acknowledgements This study was partially supported by a Grant for Collaborative Research from Kanazawa Medical University (C200055), Grant-in-Aid for Young Scientists (B-16791265) and for
J. Sato et al. / British Journal of Oral and Maxillofacial Surgery 45 (2007) 372–377
Scientific Researcher (17390527) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 13.
References 14. 1. Kido MA, Kiyoshima T, Kondo T, et al. Distribution of substance P and calcitonin gene-related peptide-like immunoreactive nerve fibers in the rat temporomandibular joint. J Dent Res 1993;72:592–8. 2. Pedersen-Bjergaard U, Nielsen LB, Jensen K, Edvinsson L, Jansen I, Olesen J. Calcitonin gene-related peptide, neurokinin A and substance P: effects on nociception and neurogenic inflammation in human skin and temporal muscle. Peptides 1991;12:333–7. 3. Henry CH, Wolford LM. Substance P and mast cells: preliminary histologic analysis of the human temporomandibular joint. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;92:384–9. 4. Rosenfeld MG, Mermod JJ, Amara SG, et al. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 1983;304:129–35. 5. Saito T, Koshino T. Distribution of neuropeptides in synovium of the knee with osteoarthritis. Clin Orthop Relat Res 2000;376:172–82. 6. Lembeck F, Holzer P. Substance P as neurogenic mediator of antidromic vasodilation and neurogenic plasma extravasation. Naunyn Schmiedebergs Arch Pharmacol 1979;310:175–83. 7. Numazaki M, Tominaga M. Molecular mechanisms of nociception and thermosensation: structures, expressions and functions of capsaicin receptor and its homologues. Seikagaku 2003;75:359–71. 8. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997;389:816–24. 9. Sato J, Segami N, Kaneyama K, Yoshimura H, Fujimura K, Yoshitake Y. Relationship of calcitonin gene-related peptide in synovial tissues and temporomandibular joint pain in humans. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:533–40. 10. Sato J, Segami N, Yoshitake Y, et al. Expression of capsaicin receptor TRPV-1 in synovial tissues of patients with symptomatic internal derangement of the temporomandibular joint and joint pain. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;100:674–81. 11. Segami N, Kaneyama K, Tsurusako S, Suzuki T. Arthroscopic eminoplasty for habitual dislocation of the temporomandibular joint: preliminary study. J Craniomaxillofac Surg 1999;27:390–7. 12. Sato J, Segami N, Yoshitake Y, Nishikawa K. Correlations of the expression of fibroblast growth factor-2, vascular endothelial growth factor,
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and their receptors with angiogenesis in synovial tissues from patients with internal derangement of the temporomandibular joint. J Dent Res 2003;82:272–7. McCain JP, de la Rua H, LeBlanc WG. Puncture technique and portals of entry for diagnostic and operative arthroscopy of the temporomandibular joint. Arthroscopy 1991;7:221–32. Murakami K, Segami N, Fujimura K, Iizuka T. Correlation between pain and synovitis in patients with internal derangement of the temporomandibular joint. J Oral Maxillofac Surg 1991;49:1159–62. Murakami K, Segami N, Moriya Y, Iizuka T. Correlation between pain and dysfunction and intra-articular adhesions in patients with internal derangement of the temporomandibular joint. J Oral Maxillofac Surg 1992;50:705–8. Sato J, Segami N, Nishimura M, et al. Expression of interleukin 6 in synovial tissues in patients with internal derangement of the temporomandibular joint. Br J Oral Maxillofac Surg 2003;41:95–101. Mapp PI, Kidd BL, Gibson SJ, et al. Substance P-, calcitonin gene-related peptide- and C-flanking peptide of neuropeptide Yimmunoreactive fibres are present in normal synovium but depleted in patients with rheumatoid arthritis. Neuroscience 1990;37:143–53. Appelgren A, Appelgren B, Kopp S, Lundeberg T, Theodorsson E. Substance P-associated increase of intra-articular temperature and pain threshold in the arthritic TMJ. J Orofac Pain 1998;12:101–7. Holmlund A, Ekblom A, Hansson P, Lind J, Lundeberg T, Theodorsson E. Concentrations of neuropeptides substance P, neurokinin A, calcitonin gene-related peptide, neuropeptide Y and vasoactive intestinal polypeptide in synovial fluid of the human temporomandibular joint. A correlation with symptoms, signs, and arthroscopic findings. Int J Oral Maxillofac Surg 1991;20:228–31. Marshall KW, Chiu B, Inman RD. Substance P and arthritis: analysis of plasma and synovial fluid levels. Arthritis Rheum 1990;33:87–90. Walsh DA, Mapp PI, Wharton J, et al. Localisation and characterisation of substance P binding to human synovial tissue in rheumatoid arthritis. Ann Rheum Dis 1992;51:313–7. Komuro H, Tanabe T, Ogushi S, et al. Participation of substance P distribution in the cytokine production of rheumatoid synovium. Mod Rheumatol 2000;10:83–7. Lotz M, Carson DA, Vaughan JH. Substance P activation of rheumatoid synoviocytes: neural pathway in pathogenesis of arthritis. Science 1987;235:893–5. Kimball ES, Persico FJ, Vaught JL. Substance P, neurokinin A, and Neurokinin B induce generation of IL-1-like activity in P388D1 cells. Possible relevant to arthritic disease. J Immunol 1988;141: 3564–9.
Specific expression of substance P in synovial tissues of patients with symptomatic, non-reducing internal derangement of the temporomandibular joint: Comparison with clinical findings Jun Sato a,c,∗ , Natsuki Segami a , Yoshino Yoshitake b , Keiseki Kaneyama a , Hiroshi Yoshimura a , Kazuma Fujimura a , Yoshimasa Kitagawa c a
Department of Oral and Maxillofacial Surgery, Kanazawa Medical University, Daigaku, Uchinada-machi, Kahoku-gun, Ishikawa 920-0293, Japan Department of Biochemistry, Kanazawa Medical University, Japan c Oral Diagnosis and Medicine, Department of Oral Pathobiological Science, Hokkaido University Graduate School of Dental Medicine, North 13, West 7, Kita-ward, Sapporo City 060-8586, Japan b
Accepted 21 September 2006 Available online 13 November 2006
Abstract Our aim was to find out the extent of expression of substance P in synovial tissue from the human temporomandibular joints (TMJ) with symptomatic, non-reducing internal derangement, and to investigate the relationship between substance P and clinical findings. Fifty-four joints in 54 patients were examined immunohistochemically. Specimens of synovial tissue from 10 joints in 8 subjects with habitual dislocation of the TMJ with no pain were examined as controls. Cells that stained for substance P were found mainly among the endothelial cells in the blood vessels beneath the lining cells in synovial tissues from 47 of the 54 joints (87%) with internal derangement and from 5 of the 10 control joints. The extent score of cells that stained for substance P in joints with internal derangement was significantly higher than that in controls (p = 0.02). The extent score of these cells did not correlate with pain in the joint or the degree of synovitis. These results suggest that substance P may have some roles in both the physiological and pathological conditions in patients with symptomatic internal derangement of the TMJ. © 2006 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Keywords: Substance P; Synovial tissue; Neurogenic inflammation; Temporomandibular joint
Introduction Internal derangement of the temporomandibular joint (TMJ) is often accompanied by synovitis. Neuropeptides are now recognised as a key to understanding the pathophysiology of arthritis.1 These neuropeptides, including substance P and calcitonin gene-related peptide (CGRP), are present mainly
∗ Corresponding author at: Oral Diagnosis and Medicine, Department of Oral Pathobiological Science, Hokkaido University Graduate School of Dental Medicine, North 13, West 7, Kita-ward. Sapporo City 060-8586, Japan. Tel.: +81 11 716 2111; fax: +81 11 706 4280. E-mail address: [email protected] (J. Sato).
in nociceptive C-fibres, which usually correlate with nociception and the sensation of pain.2,3 These neuropeptides are released by activated peripheral nerve terminals into the surrounding tissues and cause an inflammatory response, known as neurogenic inflammation.4 Substance P is a member of the tachykinin family, and it has been suggested that it is one of the most important neuropeptides that modulate the inflammatory process of arthritis.5 Substance P acts not only as a transmitter of pain signals, “but also stimulates macrophages, neutrophils, and endothelial cells to induce phagocytosis, chemotaxis, and plasma to extravasate”.5,6 Recently, we showed that CGRP and the capsaicin receptor TRPV-1 are expressed in synovial tissues of the TMJ. Transient receptor
0266-4356/$ – see front matter © 2006 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bjoms.2006.09.011
J. Sato et al. / British Journal of Oral and Maxillofacial Surgery 45 (2007) 372–377
373
Table 1 (Continued )
Table 1 Patient and substance P-staining score Case number
Age /sex (years)
VAS for pain
Synovitis score (evaluated by arthroscopy)
Extent score for number of cells stained for substance P
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
23/F 24/F 70/F 36/F 61/M 70/F 38/F 55/M 22/F 40/F 28/F 41/F 21/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 55/F 32/F 36/M 25/F 19/F 56/F 13/F 40/F 22/F
4 8 7 7 7 5 8 9 4 7 6 10 5 5 3 4 8 6 6 6 3 6 6 5 5 1 4 8 7 9 9 6 7 2 1 10 8 4 6 5 5 7 5 7 3 8 7 5 3 7 4 5 8 5
8 8 7 5 5 3 7 5 6 8 6 3 6 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 9 4 7 6 7 8 9 5 8
6 40 40 3 25 0 4 7 4 66 18 10 25 40 18 0 20 0 16 8 38 14 20 30 0 40 35 45 10 12 85 10 8 25 33 0 70 2 40 36 25 10 20 26 40 8 0 18 12 0 40 22 36 33
Control subjects with habitual dislocation:† 55† 23/M 0 0 56† 66/F 0 0 57† 27/M 0 0 58† 46/M 0 0
36 0 0 6
Case number
Age /sex (years)
59† 60† 61† 62† 63† 64†
72/M 72/M 25/M 25/F 29/F 55/F
VAS for pain
0 0 0 0 0 0
Synovitis score (evaluated by arthroscopy) 0 0 2 0 1 0
Extent score for number of cells stained for substance P 10 0 0 4 0 12
potential V1 (TRPV-1) is one of the major nociceptors and has a key role in inflammatory pain.7,8 We showed that the expression of CGRP is correlated significantly with joint pain in patients with symptomatic internal derangement,9,10 and that there is a possibility that some neuropeptides and neurogenic inflammation are involved in the pathophysiological mechanisms of pain in the TMJ. The expression and clinical relevance of substance P in synovial tissues in patients with symptomatic internal derangement of the TMJ have not been investigated fully. Before the present study, we hypothesised that substance P may be involved in pain in the joint in much the same way as CGRP in patients with non-reducing internal derangement of the TMJ. Here, we have made an immunohistochemical examination of synovial tissues of the TMJ and assessed the correlation between expression of substance P and clinical findings.
Patients and methods Patients (Table 1) We studied 54 joints in 54 patients with symptomatic nonreducing internal derangement of the TMJ. There were 7 men and 47 women, with a mean (S.D.) age of 41 (17) years (range 13–84) (Table 1). Magnetic resonance imaging showed that all of the patients had anterior disc displacement without reduction. The median duration of symptoms was 4 months (range 1–24). All of the patients had had arthroscopy after failure of non-surgical treatment—occlusal appliance, non-steroidal anti-inflammatory drugs (NSAIDS), and physiotherapy. Just before operation, the patients indicated the degree of subjective pain caused by moving the jaw on a visual analogue scale (VAS) of 0–10. This ranged from 1 to 10, with a mean (S.D.) of 5 (2). The control group consisted of 10 joints in eight subjects, four men and four women, with a mean (S.D.) age of 43 (20) years (range 23–72), who had habitual dislocation without pain. All control patients had had arthroscopic eminoplasty.11 The validity of adopting these patients as controls has been discussed in detail previously.9,10 The patients with internal derangement and the control subjects also participated in our previous studies.9,10,12 None of the subjects in either group had had arthroscopy of the TMJ before this study.
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All the participants gave their informed consent to arthroscopy, synovial biopsy, and histological examination.
Table 2 Scoring of intensity of synovitis Score
Synovial tissues and immunohistochemical staining
0
Findings Normal pale, almost translucent, synovial lining with a fine network of anastomosing small blood vessels Increased vascularity and capillary hyperaemia (mild) Increased vascularity and capillary hyperaemia (moderate) Increased vascularity and capillary hyperaemia (severe) Capillary dilatation and increasing network (mild to moderate) Capillary dilatation and increasing network (severe) Contact bleeding on palpation with probe (mild to moderate) Contact bleeding on palpation with probe (severe) Microbleeding and effusion Granulative change, effusion, and debris (mild to moderate) Granulative change, effusion, and debris (severe)
Two or three synovial tissue biopsy specimens roughly 2 mm in diameter were obtained arthroscopically from each patient from a portion of the posterior disc attachment using the triangular technique under direct visualisation.9,10,12 Immediately after resection, the specimens were fixed in 4% paraformaldehyde for 4 h and embedded in paraffin. Sections were prepared and stained immunohistochemically by the avidin–biotin–peroxidase complex method with a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) as described previously.9,10,12 The sections were treated with primary antibody to substance P (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA; sc-21715) and left overnight at 4 ◦ C. This antibody was purified monoclonal rat IgG to substance P in multiple species, and its specificity has been confirmed by the manufacturer. The following day the specimens were incubated in a solution of biotinylated anti-rat antibody (1:200 dilution; Dako, Carpinteria, CA) for 30 min at room temperature, and avidin /biotinylated horseradish peroxidase complex was added for 30 min at room temperature. The colour was developed using 3-amino-9-ethyl carbazole followed by counterstaining with heamatoxylin. The cells that reacted to substance P stained red. Negative controls in which the primary antibody was replaced with normal rat IgG were run with each specimen. The cells stained for substance P were evaluated in two to five regions of maximal immunohistochemical density under light microscopy at a magnification of ×200. The extent score of cells stained for substance P was calculated as the percentage of blood vessels containing those cells. About 10 to 100 blood vessels were evaluated in each specimen. This work was done by two of the authors (KK and JS) who were unaware of the identities of the patients from whom the specimens had been obtained.
1 2 3 4 5 6 7 8 9 10
Arthroscopy
Cells that stained for substance P were seen in synovial tissue specimens from 47 of the 54 joints (87%) in the internal derangement group (Fig. 1). All the negative control specimens showed only background staining (data not shown). The specimens from 5 of the 10 control subjects with habitual dislocation also stained for substance P (Fig. 2A and B). The mean (S.D.; median) extent score of cells that stained for substance P was 23 (19; 20) in the internal derangement specimens compared with 7 (11; 2) in the control specimens, the difference being significant (p = 0.02) (Fig. 3). Cells that stained for substance P were mostly located in the endothelial cells in the blood vessels beneath the lining cells, with some lining the synovium (Figs. 1 and 2B).
Diagnostic arthroscopy was done by a standard technique throughout the whole area of the upper joint compartment.13 The degrees of synovitis and intra-articular adhesion were evaluated according to the criteria of Murakami et al. (Tables 2 and 3)14–16 by two of the authors (NS and JS) who were unaware of other information about the patients. The mean (S.D.) synovitis and adhesion scores were 5.1 (2.1) and 6.4 (1.6) in the internal derangement group, compared with 0.3 (0.7) and 0.8 (0.9) in the control group. Statistical analysis The Mann–Whitney test was used to compare the extent scores of cells stained for substance P between the internal derangement and control groups. The Spearman correlation coefficient was used to assess correlations between the
Table 3 Scoring of intensity of adhesion Score
Findings
0 1 2 3 4 5 6 7 8 9 10
No adhesion or fibrous change FiImy adhesion (mild) Filmy adhesion (moderate to severe) Fibrosynovial band (mild to moderate) Fibrosynovial band (severe) Fibrous band (mild to moderate) Fibrous band (severe) Formation of pseudowall (mild to moderate) Formation of pseudowall (severe) Capsular fibrosis (mild to moderate) Capsular fibrosis (severe)
extent score of cells stained for substance P and the clinical findings. StatView J-5.0 (Abacus Concepts, Berkeley, CA) was used for all statistical analyses. p < 0.05 were considered significant.
Results Immunohistochemical staining
Expression of substance P and clinical findings There were no significant correlations between the extent score of cells stained for substance P and the duration
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Fig. 1. Section stained for substance P from a patient with internal derangement (case 31). Immunoreactivities for substance P (arrows) can be seen in the endothelial cells in the blood vessels beneath the lining cells and lining the synovium (arrow heads) (original magnification: ×100, bar: 50 m).
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Fig. 3. The extent score of cells stained for substance P in the synovial tissues of patients with internal derangement and controls. The extent score of these cells is significantly higher in the internal derangement group than in the control group (Mann–Whitney test; p = 0.02).
of symptoms in the internal derangement group (p = 0.14, r = −0.09) (Fig. 4), the degree of synovitis (p = 0.31, r = 0.14) (Fig. 5), the degree of adhesion (p = 0.19, r = −0.22) or the severity of pain in the joint (VAS) (p = 0.75, r = 0.01) (Fig. 6).
Fig. 4. The extent score of cells stained for substance P and the duration of the symptoms are not correlated significantly (Spearman correlation coefficient; p = 0.14, r = −0.09).
Fig. 5. The extent score of cells stained for substance P and the degree of synovitis are not correlated significantly (Spearman correlation coefficient; p = 0.31, r = 0.14).
Fig. 2. (A and B). Specimens stained for substance P from control patients (A: case 61, B: case 55). There is no immunoreactivity for substance P in the synovial tissue (A), and only a few stained cells in the blood vessels (B: arrows) (original magnification A: ×100 and B: ×200, bar: 50 m).
Discussion The contribution of neuropeptides to arthritis is currently a matter of debate.16 In the present study, we have shown
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Fig. 6. The extent score of cells stained for substance P and the VAS of pain are not correlated significantly (Spearman correlation coefficient; p = 0.75, r = 0.01).
the presence of cells stained for substance P in the specimens of synovial tissue in the posterior disc attachment in patients with symptomatic internal derangement of the TMJ. The extent score of these cells was significantly higher in the internal derangement group than in the control group. Substance P has been suggested as not only one of the most important neuropeptides that modulate the inflammatory process of arthritis,17 but also one of the most important to act as a local regulator of blood flow and pain.1,18 Although we expected a close relation between the expression of substance P and joint pain (the degree of synovitis), we were unable to confirm our hypothesis from the results of the present study. We know of a few previous studies of substance P in the TMJ region. In a preliminary study, Henry and Wolford3 showed that cells that stained for substance P were present mainly within the walls of the vasculature in synovial tissues of the human TMJ. As they obtained synovial tissues from only nine women, they did not investigate the relation between the expression of substance P and clinical findings. Our immunohistochemical study supports their observation that cells that stained for substance P were present in the blood vessels. Appelgren et al.18 reported that substance Plike immunoreactivity in the synovial fluids from patients with arthritic TMJs was correlated with intra-articular temperature and pain. Holmlund et al.19 detected substance P-like immunoreactivity in 17 of 19 synovial fluid samples from patients with internal derangement, osteoarthrosis (OA), and rheumatoid arthritis (RA). They concluded that there were no significant correlations between the concentrations of substance P in the synovial fluids and the arthroscopic findings, including the degrees of synovitis and OA. They also concluded that these unexpected results were the results of the relatively small number of patients with pronounced synovitis who were included in their study. Marshal et al.20 reported that the concentrations of substance P in synovial fluids in patients with RA did not differ significantly from those found in patients with OA. Mapp et al.17 found a large number of afferent fibres of substance P in normal synovial tissues, but few such fibres in patients with RA. They speculated that
large amounts of neuropeptides, including substance P, may be released from the sensory terminals in the early phases of inflammation, resulting in permanent depletion. They concluded that substance P and other neuropeptides may be involved in the pathogenesis of inflammation of the joint in the early rather than the chronic phases. However, we found no significant correlation between the extent score of cells stained for substance P and the duration of symptoms. This discrepancy may have been caused by the difference in pathogenesis between internal derangement of the TMJ and RA. As the degree of inflammation of the synovial tissues in patients with internal derangement of the TMJ is usually much less than that in patients with RA, neuropeptides may never be permanently depleted. We detected substance P at low concentrations in the synovial tissues from control subjects. This suggests that it may play some part in the maintenance of physiological conditions in the TMJ. Substance P has been suggested to regulate vascular functions (such as blood flow) under normal conditions,17 and its expression may be a protective response to tissue injury.18,21 The main limitations of the present study were the small number of observations and characteristics of the control patients. These problems are difficult to overcome, as it is impossible to obtain fresh samples of synovial tissues from healthy people.9,10 We have explained the validity of our control subjects in previous reports,9,10,12 and think that these patients with dislocations were suitable control subjects for the present study. As we performed punch biopsies on the posterior disc attachment, it was not possible to assess the distribution of substance P in the whole intracapsular structure of the TMJ. As the pathological mechanisms underlying joint pain are complex, we are aware that it is not possible to work out the mechanisms of all cases of TMJ based pain from our results. In conclusion, we have shown that substance P may be involved in the pathogenesis of symptomatic internal derangement of the TMJ. However, it is unlikely that its important physiological and pathological roles of are mediated independently. Substance P has been suggested to play an important part in the pathogenesis of arthritis by up-regulation of production of other cytokines,22 and it stimulates synovial cells and lymphocytes to produce prostaglandins23 and to release cytokines, such as IL-1.24 It is therefore important to examine the relations between substance P and other chemical mediators in more depth. Further studies are required to elucidate the possible contribution of substance P to physiological and pathological conditions in patients with internal derangement of the TMJ.
Acknowledgements This study was partially supported by a Grant for Collaborative Research from Kanazawa Medical University (C200055), Grant-in-Aid for Young Scientists (B-16791265) and for
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Scientific Researcher (17390527) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 13.
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