TMJOA that mimics the histopathologic changes both in cartilage and subchondral bone, as well as clinical symptoms, is still needed. Intra-articular injection of monosodium iodoacetate (MIA) to induce OA-like lesions is widely used to induce knee OA [16,17,18,19,20,21]. MIA mainly inhibits the activity of glyceral- dehyde-3-phosphate dehydrogenase leading to apoptosis of chondrocytes [18,20,22]. The MIA-induced OA model has the great advantage of easy modulation of the progression and severity of the articular lesions by modification of MIA concentration [19]. Although a few studies have attempted to induce OA-like lesions in rabbit TMJ by MIA injection into the lower compartment with or without surgical assistance [13,23,24], it is important to explore whether MIA could induce OA-like lesions in the rat TMJ, since rats are one of the most widely used species in experimental research and drug toxicology testing [15]. The TMJ is partitioned by a disc, which forms a larger upper and smaller lower compartment. Agent injection into the lower compartment is a difficult procedure both in humans and animals because of its limited space [25], whereas injection into the upper compartment, even in rats, is technically and manually preferable, and has often been used previously [26,27,28]. The question then arises as to whether MIA injection into the upper compartment of the rat TMJ can be used to create a comprehensive OA model. To address this question, investigations at the histopathologic, radiographic, molecular, and noceiceptive behavioral levels were performed in this study to examine whether injection of MIA into the upper compartment of the rat TMJ could induce OA-like lesions in the entire joint. Materials and Methods Ethics Statement With the approval of the Peking University Institutional Animal Care and Use Committee (NO: LA2012-59), all rats were housed under controlled temperatures in a 12 h light/dark cycle with easy access to food and water. Induction of TMJOA A total of 72 female Sprague-Dawley rats (180–200 g) were randomly assigned to either the experimental (n = 42) or control (n = 30) groups. The experimental schedule is illustrated in Fig. 1A. TMJOA was induced by injection of MIA (Sigma, Saint Louis, USA) dissolved in 50 mL saline into the upper compartment of bilateral TMJs using a 27-gauge 0.5-inch needle without surgical assistance. We first confirmed the injection site by injection of 50 mL dye into the upper compartment (Fig. 1B). Dose Course Various doses of MIA (0.05, 0.1, 0.5, 1, or 2 mg) were injected into the upper compartment of bilateral TMJs of rats in five experimental groups (n = 3/group), while 50 mL saline was injected into TMJs of rats in the control group (n = 3). All rats were sacrificed on day 28 post-injection to validate the adequate dose of MIA for further observations (Fig. 1A). Time Course After the dose course test, 0.5 mg MIA (minimum effective dose) or saline was injected into TMJs and rats were sacrificed on days 1, 3, 7, 14, 28, 56, or 84 post-injection (n = 3/group) (Fig. 1A). To determine gene expression profiles during the induction of TMJOA, an additional 12 rats, divided into two groups (n = 6/ group), were injected with 0.5 mg MIA or saline and sacrificed on day 14 post-injection. MIA Induced TMJOA in Rat Head Withdrawal Threshold (HWT) Measurements The nociceptive behavior of animals was assessed based on the HWT as described previously [27]. HWT measurements were performed pre-MIA/saline injection and on days 1, 3, 7, 14, 21, 28, 35, and 42 post-injection (Fig. 1A). The HWT was calculated as a mean value per joint of 3 rats/group. Tissue Harvesting All rats were sacrificed by pentobarbital overdose. For histopathology, the TMJs of two rats in each group were removed bilaterally en bloc, fixed in 4% paraformaldehyde, and deminer- alized in 15% EDTA. For radiographic examination, the bilateral condyles of one rat in each group were dissected. For real-time PCR analysis, the condyle heads of six rats in each group (0.5 mg MIA for 2 weeks or control) were dissected. Bilateral condyle heads of each rat were pooled for sufficient RNA extraction owing to the difficulty of isolating and acquiring enough cartilage from the small condylar head of rat and the reason that both the cartilage and subchondral bone were affected by MIA. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) TMJ discs and condylar cartilage were dissected from the rats on day 1 post-injection (n = 3/group). SEM and TEM were performed as described previously [29]. Briefly, the samples were fixed with 2.5% fluteraldehyde solution and 1% osmium tetroxide (Sigma). For SEM, the disc section was produced by tearing through the intermediate zone. For TEM, the intermediate zone of the disc or condylar cartilage was embedded in epoxy resin. Ultrathin sections (100 nm) were stained with lead citrate and uranyl acetate. Histopathologic Staining Paraffin-embedded TMJ blocs were sagittally cut in serial sections at a 5-mm thickness. Sections were stained with hematoxylin and eosin (HE) for routine histological evaluation. Safranin O-fast green (S.O) and Toluidine blue (TB) stains were used to evaluate proteoglycans in the cartilage matrix [18]. MicroCT Examination Radiographs of condyles were obtained with a high-resolution MicroCT system (Inveon, Siemens, Germany). The specimens were scanned at 60 kV, 300 mA, and 8.5 mm-effective pixel size. The images were analyzed using software provided by the manufacturer. All sagittal images were captured using the same parameters: Ct = 2550; W = 550. Real-time PCR Total RNA was isolated from the condylar heads containing cartilage and subchondral bone using TRIzol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’ s instructions. The condylar heads were ground into powder in liquid nitrogen using a cryogenic grinder (6770 Freezer/Mill, SPEX SamplePrep, NJ, USA). Reverse transcription were performed with an iScript cDNA synthesis kit (Bio-Rad) in 20 ml reaction volume containing 1 mg of total RNA as described previously [27,30]. Real-time PCR was performed with Power SYBR Green PCR Master Mix (Applied Biosystems) using a 7500 real-time PCR System (Applied Biosystems). The amplification specificity was confirmed by melting curve. The sequences of primers for rat b-actin [31], Collagen I and Aggrecan [32], Collagen II [33], ADAMTS5 (aggrecanase-2) [34], Tissue Inhibitors of Metalloproteinase (TIMP)2 [35], TNFa [36], Bax, Fas, FasL, Caspase2, Caspase3, PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45036