MIA Induced TMJOA in Rat Figure 6. Changes in gene and protein expression in condyle following MIA injection were evaluated by real-time PCR and IHC, respectively. A. Two weeks after MIA injection, anabolism-associated aggrecan and collagen I and II were downregulated compared with the control group. Catabolism-associated MMP3, MMP13, and ADAMTS5 were upregulated and TIMP2, but not TIMP1, was correspondingly downregulated. B. Two weeks after MIA (0.5 mg) injection, apoptosis-associated genes of the death receptor family, such as, TNFa, Fas, FasL, caspase8, caspase3, and BAX, but not caspase2 and caspase9, were significantly elevated in the MIA injection group; PCNA and a-SMA, representing proliferation and fibrous restoration, respectively, were upregulated (mean 6 SEM; n = 6; **P,0.01; *P,0.05). C. There were very few chondrocytes left in the lesion labeled as L 2 weeks after MIA (0.5 mg) injection. MMP3 was mainly expressed in the hypertrophic layer in the control cartilage (a). Diffuse staining of MMP3 was observed in the chondrocytes adjacent to the lesion (L) at 2 weeks (b). Caspase3 was rarely expressed in the control cartilage (c). Enhanced staining of caspase3 was observed in the proliferative and hypertrophic layers adjacent to the lesion (L) at 2 weeks (d). Expression of a-SMA was mainly in the hypertrophic chondrocytes in the control group (e). Stronger staining of a-SMA was observed adjacent to the lesion (L) at 4 weeks (f). (Bar = 40 mm). doi:10.1371/journal.pone.0045036.g006 an important role [2]. However, reconstruction is rarely seen in TMJOA models induced by methods other than MIA. Although several animal models of TMJOA have been established, our multi-level data suggest that the present rat model accurately mimicked most of the clinical features of TMJOA. In conclusion, the present study demonstrated a reliable and simple rat model of TMJOA induced by intra-articular injection of MIA into the upper compartment. The histopathologic, radio- graphic, behavioral, and molecular changes of this model will help us to understand the progression of TMJOA and to facilitate future TMJOA-associated researches. References 1. Dworkin SF, LeResche L (1992) Research diagnostic criteria for temporoman- dibular disorders: review, criteria, examinations and specifications, critique. J Craniomandib Disord 6: 301–355. 2. Zarb GA, Carlsson GE (1999) Temporomandibular disorders: osteoarthritis. J Orofac Pain 13: 295–306. 3. Israel HA, Diamond B, Saed-Nejad F, Ratcliffe A (1998) Osteoarthritis and synovitis as major pathoses of the temporomandibular joint: comparison of clinical diagnosis with arthroscopic morphology. J Oral Maxillofac Surg 56: 1023–1027; discussion 1028. 4. Stegenga B, de Bont LG, Boering G (1989) Osteoarthrosis as the cause of craniomandibular pain and dysfunction: a unifying concept. J Oral Maxillofac Surg 47: 249–256. 5. Karsdal MA, Leeming DJ, Dam EB, Henriksen K, Alexandersen P, et al. (2008) Should subchondral bone turnover be targeted when treating osteoarthritis? Osteoarthritis Cartilage 16: 638–646. 6. Stegenga B (2001) Osteoarthritis of the temporomandibular joint organ and its relationship to disc displacement. J Orofac Pain 15: 193–205. 7. Imirzalioglu P, Uckan S, Guler N, Haberal A, Uckan D (2009) Synovial apoptosis in temporomandibular joint disc displacement without reduction. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 108: 693–698. 8. Aigner T, Kim HA (2002) Apoptosis and cellular vitality: issues in osteoarthritic cartilage degeneration. Arthritis Rheum 46: 1986–1996. 9. Brandt KD (2002) Animal models of osteoarthritis. Biorheology 39: 221–235. 10. Meng J, Ma X, Ma D, Xu C (2005) Microarray analysis of differential gene expression in temporomandibular joint condylar cartilage after experimentally induced osteoarthritis. Osteoarthritis Cartilage 13: 1115–1125. 11. Fujisawa T, Kuboki T, Kasai T, Sonoyama W, Kojima S, et al. (2003) A repetitive, steady mouth opening induced an osteoarthritis-like lesion in the rabbit temporomandibular joint. J Dent Res 82: 731–735. 12. Xinmin Y, Jian H (2005) Treatment of temporomandibular joint osteoarthritis with viscosupplementation and arthrocentesis on rabbit model. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 100: e35–38. 13. Cledes G, Felizardo R, Foucart JM, Carpentier P (2006) Validation of a chemical osteoarthritis model in rabbit temporomandibular joint: a compliment to biomechanical models. Int J Oral Maxillofac Surg 35: 1026–1033. 14. Wadhwa S, Embree MC, Kilts T, Young MF, Ameye LG (2005) Accelerated osteoarthritis in the temporomandibular joint of biglycan/fibromodulin double- deficient mice. Osteoarthritis Cartilage 13: 817–827. 15. Bendele AM (2001) Animal models of osteoarthritis. J Musculoskelet Neuronal Interact 1: 363–376. 16. Schuelert N, Zhang C, Mogg AJ, Broad LM, Hepburn DL, et al. (2010) Paradoxical effects of the cannabinoid CB2 receptor agonist GW405833 on rat osteoarthritic knee joint pain. Osteoarthritis Cartilage 18: 1536–1543. 17. Mohan G, Perilli E, Kuliwaba JS, Humphries JM, Parkinson IH, et al. (2011) Application of in vivo micro-computed tomography in the temporal character- isation of subchondral bone architecture in a rat model of low-dose monosodium iodoacetate induced osteoarthritis. Arthritis Res Ther 13: R210. 18. Bar-Yehuda S, Rath-Wolfson L, Del Valle L, Ochaion A, Cohen S, et al. (2009) Induction of an antiinflammatory effect and prevention of cartilage damage in rat knee osteoarthritis by CF101 treatment. Arthritis Rheum 60: 3061–3071. Acknowledgments We would like to thank Professor Yan Gao for the assistance with histopathologic evaluation. Author Contributions Conceived and designed the experiments: XDW XXK YHG YHZ. Performed the experiments: XDW XXK MMZ DQH ZM RYB YL JNZ. Analyzed the data: XDW XXK YHG YHZ. Contributed reagents/ materials/analysis tools: XDW XXK YHG YHZ. Wrote the paper: XDW YHG YHZ. 19. Guingamp C, Gegout-Pottie P, Philippe L, Terlain B, Netter P, et al. (1997) Mono-iodoacetate-induced experimental osteoarthritis: a dose-response study of loss of mobility, morphology, and biochemistry. Arthritis Rheum 40: 1670– 1679. 20. Kalbhen DA (1987) Chemical model of osteoarthritis–a pharmacological evaluation. J Rheumatol 14 Spec No: 130–131. 21. Nam J, Perera P, Liu J, Rath B, Deschner J, et al. (2011) Sequential alterations in catabolic and anabolic gene expression parallel pathological changes during progression of monoiodoacetate-induced arthritis. PLoS One 6: e24320. 22. Grossin L, Cournil-Henrionnet C, Pinzano A, Gaborit N, Dumas D, et al. (2006) Gene transfer with HSP 70 in rat chondrocytes confers cytoprotection in vitro and during experimental osteoarthritis. FASEB J 20: 65–75. 23. Guler N, Kurkcu M, Duygu G, Cam B (2011) Sodium iodoacetate induced osteoarthrosis model in rabbit temporomandibular joint: CT and histological study (Part I). Int J Oral Maxillofac Surg. 24. Duygu G, Guler N, Cam B, Kurkcu M (2011) The effects of high molecular weight hyaluronic acid (Hylan G-F 20) on experimentally induced temporo- mandibular joint osteoartrosis: part II. Int J Oral Maxillofac Surg. 25. Li C, Zhang Y, Lv J, Shi Z (2012) Inferior or double joint spaces injection versus superior joint space injection for temporomandibular disorders: a systematic review and meta-analysis. J Oral Maxillofac Surg 70: 37–44. 26. Ren K (1999) An improved method for assessing mechanical allodynia in the rat. Physiol Behav 67: 711–716. 27. Wu YW, Bi YP, Kou XX, Xu W, Ma LQ, et al. (2010) 17-Beta-estradiol enhanced allodynia of inflammatory temporomandibular joint through upregu- lation of hippocampal TRPV1 in ovariectomized rats. J Neurosci 30: 8710– 8719. 28. Wang XD, Kou XX, Mao JJ, Gan YH, Zhou YH (2012) Sustained Inflammation Induces Degeneration of the Temporomandibular Joint. J Dent Res. 29. Liu Y, Mai S, Li N, Yiu CK, Mao J, et al. (2011) Differences between top-down and bottom-up approaches in mineralizing thick, partially demineralized collagen scaffolds. Acta Biomater 7: 1742–1751. 30. Kou XX, Wu YW, Ding Y, Hao T, Bi RY, et al. (2011) 17beta-estradiol aggravates temporomandibular joint inflammation through the NF-kappaB pathway in ovariectomized rats. Arthritis Rheum 63: 1888–1897. 31. Tian YF, Zhang PB, Xiao XL, Zhang JS, Zhao JJ, et al. (2007) The quantification of ADAMTS expression in an animal model of cerebral ischemia using real-time PCR. Acta Anaesthesiol Scand 51: 158–164. 32. Wang XD, Kou XX, Mao JJ, Gan YH, Zhou YH (2012) Sustained inflammation induces degeneration of the temporomandibular joint. J Dent Res 91: 499–505. 33. Kinkel MD, Horton WE (2003) Coordinate down-regulation of cartilage matrix gene expression in Bcl-2 deficient chondrocytes is associated with decreased SOX9 expression and decreased mRNA stability. Journal of Cellular Biochemistry 88: 941–953. 34. Bao JP, Chen WP, Feng J, Hu PF, Shi ZL, et al. (2010) Leptin plays a catabolic role on articular cartilage. Molecular Biology Reports 37: 3265–3272. 35. Deschner J, Rath-Deschner B, Agarwal S (2006) Regulation of matrix metalloproteinase expression by dynamic tensile strain in rat fibrochondrocytes. Osteoarthritis and Cartilage 14: 264–272. PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e45036