MIA Induced TMJOA in Rat Figure 3. Dose- and time-dependent histopathologic changes in TMJ tissues. TMJ was sectioned in saggital for HE, TB and S.O staining. A. Dose course (0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2mg per joint) of MIA 4 weeks post-injection. Black frames are magnified. Condylar cartilage in the controls stained purple blue by TB and red by S.O; (F: fibrous layer; P: proliferative layer; H: hypertrophy layer; C: calcified layer; B: subchondral bone.). Typical OA-like lesion induced by 0.5 mg MIA, including regional loss of chondrocytes (arrow), chondrocyte cluster formation (arrowhead), horizontal cleft (dotted arrow), peripheral chondrocyte proliferation (red frame), and subchondral bone erosion with adjacent bone marrow full of fibroblast-like cells (yellow frame). Lesions staining by TB and S.O was uneven. (Bar = 200 mm) B. Time-dependent changes in the condyle following MIA injection (0.5 mg/joint; 3 days to 12 weeks). Black frames were magnified. After three days, chondrocytes in the anterior and central areas of the cartilage were lightly stained with nuclear condensation (arrowhead). At 12 weeks, thin cartilage (double arrow) and sclerotic subchondral bone (arrowhead) replaced the lesion. (Bar = 200 mm) C. Time-dependent changes in the synovium, disc, and temporal fossa following MIA injection. Time-dependent changes in synovitis (fibrin-like exudates: arrow; proliferative villi of the synovium: dotted arrow), hypo-cellular change and thinning of the disc (arrowhead), and destruction of temporal fossa cartilage (black frame) following MIA induction are shown. (Bar = 300 mm). doi:10.1371/journal.pone.0045036.g003 bone surrounding the lesion became sclerotic. By 12 weeks, the lesion was replaced with smooth but sclerotic bone. Osteophytes began to present from 4 weeks until 12 weeks after MIA injection. Hyperalgesia of TMJ after Induction of TMJOA To understand the relationship between the nociceptive response and histopathological changes, the HWT was measured at different time points after MIA injection (0.5 mg/joint). (Fig. 4B). The HWT significantly decreased 24 h after MIA injection (P,0.01), remained at a decreased level until 3 weeks (P,0.05), but then gradually recovered to baseline by 4 weeks, as compared with the control group. Induction of Condylar Chondrocyte Apoptosis To understand the mechanism underlying MIA-induced chondrocyte loss in the condylar cartilage, TEM examinations and a TUNEL assay were performed following MIA injection (0.5 mg/joint). TEM showed that the chondrocytes in the control group were polygonal with abundant mitochondria and endoplas- mic reticulum, whereas chondrocytes in the group treated with MIA showed typical apoptotic features, including cell shrinkage, nuclear condensation, vacuolar degeneration, and apoptotic bodies, after 1 day (Fig. 5A). Three days after MIA injection, TUNEL-positive chondrocytes were observed diffusely in the area corresponding to the region with HE unstained nuclei, but not in the control group. However, 1 week after MIA injection, the TUNEL positive chondrocytes almost disappeared in the same region due to the extreme loss of chondrocytes as shown by HE staining (Fig. 5B). Expression of Metabolism and Apoptosis Related Genes of Condyle after MIA Injection To further understand the molecular events underlying condylar destruction following MIA induction (0.5 mg/joint), the expressions of genes related to the metabolism of cartilage and bone and apoptosis were examined from the condylar head contain both cartilage and subchondral bone by real-time PCR and IHC 2 weeks after MIA injection. As compared with the control group, mRNA expression of main matrix components, including aggrecan and collagen I and II, were significantly downregulated. However, mRNA expression of the matrix degrading proteases MMP3, MMP13, and ADAMTS5 were significantly upregulated in the MIA group. In contrast, TIMP2, but not TIMP1, was correspondingly downregulated (Fig. 6A). MIA induction resulted in a significant increase in the expression of the proapoptotic genes of the death receptor family, such as Fas, FasL, caspase8, caspase3, and BAX, but not caspase2 and caspase9 (Fig. 6B). PCNA and a-SMA, markers of proliferation and fibrosis [40,41], respectively, were also upregulated in the MIA group (Fig. 6B). Moreover, IHC showed that MMP3 was mainly expressed in the hypertrophic layer in the control cartilage, but diffuse staining of MMP3 was observed in the chondrocytes adjacent to the lesion. Stronger staining of caspase3 was observed diffusely in the proliferative and hypertrophic layers adjacent to the lesion as compared with the control group. Expression of a- SMA was mainly in the hypertrophic chondrocytes in the control group, whereas it was enhanced in the chondrocytes of the proliferative and hypertrophic layers adjacent to the OA-like lesion by 4 weeks after MIA injection (Fig. 6C). PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e45036