MIA Induced TMJOA in Rat Figure 5. Apoptosis of chondrocytes in condyle after MIA treatment. A. TEM view of condylar chondrocytes. (a). The chondrocytes in the control group were polygonal. (b). Chondrocytes treated by MIA were shrunken with vacuolar degeneration after 1 day (dotted arrow). (c). Magnified photograph of the white frame in (a). Abundant mitochondria (arrowhead) and endoplasmic reticulum (hollow arrow) were observed around the nuclei (N) in the control chondrocyte. (d). Magnified photograph of the white frame in (b). Apoptotic bodies (black arrow) were observed in the chondrocyte following MIA induction. (Bar = 0.5 mm) B. Comparison of TUNEL assay and HE staining results. (a) There were few apoptotic chondrocytes (arrow) in the control group and the corresponding HE staining shown in (b). (c). Diffuse apoptotic chondrocytes were observed in the region corresponding to the lightly stained area with nuclear condensation of HE staining (black frame in d) at 3 days post-MIA injection. The TUNEL positive chondrocytes almost disappeared (e) due to the extreme loss of chondrocytes as shown by HE staining (black frame in f) at 1 week. (Bar = 80 mm). doi:10.1371/journal.pone.0045036.g005 The histopathologic features of MIA-induced lesions in the rat TMJ were similar to that of TMJOA. The present study revealed a typical time- and dose-dependent degeneration of TMJ tissues, showing the progress of cartilage degradation, erosion, osteophyte formation, and sclerosis in the subchondral bone, synovitis, and thinning in the disc and temporal surface. The current results are similar to the previous description of TMJOA [2]. The lesions were specifically limited to the load-bearing areas of the condyle. Although MIA was injected into the upper compartment and should have a more direct action on the surface of the temporal fossa than on the condyle, the destruction of condylar cartilage and subchondral bone was more severe than that of the temporal fossa. This feature is similar to the clinical and experimental observations that the condyle is active and undergoes greater destruction and remodeling [42,43,44]. Interestingly, the disc did not prevent MIA from penetrating into the lower compartment. In as little as 24 h the disc cells underwent apoptotic changes, such as cell body shrinkage and mitochondrial breakage, accompanied by disruptive ECM and loosened junctions between cells and between cells and the ECM. These changes facilitated the penetration of MIA through the disc to the lower compartment after injection into the upper compartment. The radiographic findings of MIA-induced lesions in the rat TMJ were similar to that of TMJOA. The typical clinical radiographic findings for the condyle are erosion, sclerosis, and osteophytes [3]. All of these features could also be observed with MicroCT in our MIA-induced rat TMJOA model. Moreover, the radiographic features of our TMJOA model corresponded well to the histopathologic changes. Therefore, this MIA model provides detailed histopathologic changes for the corresponding radio- graphic changes. In addition, this model can also be used for in vivo radiographic analysis of subchondral bone to understand the pathogenesis of TMJOA, as it already known for knee OA [17]. Nociceptive responses of MIA-induced TMJOA corresponded to the observed histopathologic changes. Pain is one of the predominant clinical features of OA and it may arise from the soft tissues around the joint or the subchondral bone undergoing destruction [45]. Therefore, a successful animal model of OA should have appropriate nociceptive responses corresponding to its histopathologic changes. The HWT is usually used evaluating TMJ nociceptive responses and is inversely associated with TMJ inflammation and pain [26]. We observed that TMJ hyperalgesia corresponded to the observed histological and radiographic changes in the MIA-induced TMJOA. Specifically, the hyperal- gesia of TMJ in the first week after MIA injection could be mainly inflammatory response, whereas in the 2–4 weeks after MIA injection, the hyperalgesia could well correspond to the subsequent pronounced destruction of condylar cartilage and subchondral bone erosion. When the synovitis was alleviated and cartilage damage was repaired by fibrous tissue and the subchondral bone underwent a sclerotic change, the nociceptive responses corre- spondingly returned to baseline. This was consistent with known clinical features. For example, patients often experience severe pain during the active destructive phase of TMJOA with synovitis [46] and feel alleviation over time [47,48]. However, the hyperalgesia in our TMJOA model recovered to the control level within 6 weeks, whereas last-long hyperalgesia was observed in the MIA-induced knee joint OA model [49]. Although the reasons for this difference are unknown, it might be related to the difference in the degree of cartilage damage induced by MIA in different joints, since the same dose of MIA induces more severe cartilage loss in the knee joint than in the TMJ [49]. It might also be related to the properties of the different types of cartilage, i.e., the TMJ is covered with fibrocartilage and the knee joint with hyaline cartilage. Since the hyperalgesia of the TMJ correspondingly reflected the degree of lesions induced by MIA, our results also suggested that MIA-induced TMJOA can be used for evaluating osteoarthritic pain in the TMJ. MIA induced TMJOA through chondrocyte apoptosis and the disturbance of cartilage and subchondral bone metabolism. MIA could sensitively induce chondrocyte apoptosis as early as 1 day after MIA injection and condylar apoptosis reached a peak on day 3, leading to hypocellular changes in the cartilage and disc. Chondrocyte apoptosis in the early stages could be an important initiator of cartilage degeneration. Genes of the death receptor family, such as Fas and FasL, have been reported to be related to chondrocyte apoptosis [50,51,52]. Gene expression of the death receptor family and IHC staining of caspase3 further showed that the apoptotic process appeared to be caspase-dependent. This is consistent with previous studies of OA in the knee [18,22] and discectomy-induced TMJOA [53]. In addition, cartilage degener- ation also results from the imbalance between anabolism and catabolism due to increased matrix degrading proteases and decreased synthesis of matrix [54]. Although the genes expression was evaluated from the condylar head containing both cartilage and subchondral bone, the results showed that the catabolic genes MMP3, MMP13, and ADAMTS5 were elevated in the condylar head, whereas the anabolic genes aggrecan and collagen I and II were decreased in the condylar head. The observed changes in gene expression were similar to previous reports of experimental OA or clinical OA [55,56]. Therefore, MIA-induced imbalances in gene expression with regard to cartilage metabolism and subchondral bone could also be an important factor contributing to condylar deterioration. The present model of TMJOA has advantages and disadvan- tages. Lack of severe histopathologic changes associated with TMJOA, such as vertical splitting in the cartilage, exposure of subchondral bone, and disc perforation, could be one of the disadvantages for MIA-induced TMJOA model. In contrast, advantages include the signs of reconstruction, including hyper- trophic reactions in the cartilage surrounding lesions, fibrous restoration as represented by a-SMA [41] overexpression in the proliferative cells, and hypertrophy of the chondrocyte layer at 4 weeks post-MIA injection. In addition, sclerosis of subchondral bone and osteophyte formation were observed in the later stages, which mimics the typical clinical features [42]. Lesion repair following MIA injection was also reported in a rabbit model [13,23]. TMJOA is a self-limiting disease and reconstruction plays PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e45036