Gal-1-treated chondrocytes was analyzed with RT-qPCR. Cell culture supernatants were subjected to ELISA for determination of MMP levels. For microarray analyses, RNA was isolated from chondrocytes cultured in absence or presence of Gal-1 (n ¼ 5). Bioinformatic analyses were performed using Gene Set Enrichment Analyses (GSEA) and subsequent NF-kB analyses were carried out using quantitative Western blot and RT-qPCR. Results: Immunohistochemical staining revealed increasing amounts of Gal-1-positive chondrocytes in regions of degenerated cartilage. Correlation analysis indicated a significant positive correlation between Gal-1 presence (% chondrocyte positivity) and cartilage degeneration (p<0.0001, Wilcoxon's signed rank test). In vitro, Gal-1 was secreted by chondrocytes, whereby this secretion was not inducible by proinflammatory cytokines. Binding of Gal-1 to chondrocyte surfaces and resulting effects were inhibitable by cognate sugar (lactose). Gal-1 treatment of chondrocytes increased the expression of matrix degrading enzymes (ADAMTS4, MMP1, MMP3, MMP13) and pro-inflammatory cytokines (IL1B, TNFA), while the expression of matrix components (AGC1, COL2A1) was decreased. In agreement, elevated secretion levels of pro-MMP-1, MMP-3 and pro-MMP-13 were detected. Analyses of the microarray data revealed an overexpression of chemokines and cytokines in Gal-1-treated chondrocytes. GSEA showed an overrepresentation of NF-kB DNA binding motifs in the promoters of most significantly induced genes prompting further investigation of the NF
Purpose: Several experimental animal models have been developed for human osteoarthritis (OA) and used to study the preclinical efficacy of disease and symptom modifying OA drug candidates. The preclinical efficacy has been determined by various microscopic scoring systems and joint pain assessments. The histopathology initiative of Osteoarthritis Research Society International (OARSI) has presented recommendations for OA assessment in order to standardize preclinical efficacy studies. Recommendations for rat samples include the histological analysis of cartilage degeneration and extracellular matrix (ECM) loss, osteophytes, calcified cartilage and subchondral bone, synovium, joint capsule, and growth plate. As subchondral bone has been identified to play an important role in the development and progression of OA, treatment effects on bone tissue have been studied even in more detailed. In this study, we performed a systemic characterization of epiphyseal bone, subchondral bone plate and epiphyseal trabecular bone together with the assessment of knee joint discomfort and pain and degenerative changes in articular cartilage and synovium in four rat OA models. Methods: The study was conducted using male Lewis rats (body weight range 330-380 g). Unilateral OA was induced in their knee joints by applying the following models: 1) intra-articular injection of monoiodoacetate (MIA) at the dose of 1 mg, 2) medial meniscal tear (MMT) combined with medial collateral ligament transection (MCLT), 3) anterior cruciate ligament transection (ACLT) combined with partial medial meniscectomy (pMMx), 4) ACLT. Body weight and OA symptoms were followed in each model during the study. Knee joint discomfort and pain were used as the symptoms of OA. Knee joint discomfort was analyzed as static weight bearing using Incapacitance Tester and knee joint pain as static mechanical allodynia using von Frey monofilaments. Knee joints were harvested at two different time points in each model as follows: in the MIA model at 2 and 4 weeks, in the MMT þ MCLT model at 3 and 6 weeks, in the ACLT þ pMMx model at 4 and 8 weeks, and in the ACLT model at 5 and 10 weeks. The structure of epiphyseal bone, subchondral bone plate and epiphyseal trabecular bone were analyzed in coronal sections of medial tibial plateau followed by the histological OA assessment as recommended by the OARSI histopathology initiative. This experimental protocol was approved by National Animal Experiment Board, Regional State Administrative Agency for Southern Finland, H€ ameenlinna, Finland. Results: Knee joint discomfort was observed in operated hind limbs as decreased static weight bearing during the first week of the study. Knee joint pain was identified in operated and MIA-injected hind limbs as decreased paw withdrawal threshold during the first week and at the end of in-life phase of each model. In the rat MIA model, this knee joint pain was associated with mild synovial inflammation at 2 weeks, cartilage degeneration and ECM loss in superficial layer at 2 weeks and exacer...
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