Does Pain at an Earlier Stage of Chondropathy Protect Female Mice Against Structural Progression After Surgically Induced Osteoarthritis?

OBJECTIVE: Female C57BL/6 mice exhibit less severe chondropathy than male mice. This study was undertaken to test the robustness of this observation and explore underlying mechanisms. METHODS: Osteoarthritis was induced in male and female C57BL/6 or DBA/1 mice (n = 6-15 per group) by destabilization of the medial meniscus (DMM) or partial meniscectomy (PMX). Some mice were ovariectomized (OVX) (n = 30). In vivo repair after focal cartilage defect or joint immobilization (sciatic neurectomy) following DMM was assessed. Histologic analysis, evaluation of gene expression in whole knees, and behavioral analysis using Laboratory Animal Behavior Observation Registration and Analysis System (LABORAS) and Linton incapacitance testing (n = 7-10 mice per group) were performed. RESULTS: Female mice displayed less severe chondropathy (20-75% reduction) across both strains and after both surgeries. Activity levels after PMX were similar for male and female mice. Some repair-associated genes were increased in female mouse joints after surgery, but no repair differences were evident in vivo. Despite reduced chondropathy, female mice developed pain-like behavior at the same time as male mice. At the time of established pain-like behavior (10 weeks after PMX), pain-associated genes were significantly up-regulated in female mice, including Gdnf (mean ± SEM fold change 2.54 ± 0.30), Nrtn (6.71 ± 1.24), Ntf3 (1.92 ± 0.27), and Ntf5 (2.89 ± 0.48) (P < 0.01, P < 0.01, P < 0.05, and P < 0.001, respectively, versus male mice). Inflammatory genes were not regulated in painful joints in mice of either sex. CONCLUSION: We confirm strong structural joint protection in female mice that is not due to activity or intrinsic repair differences. Female mice develop pain at the same time as males, but induce a distinct set of neurotrophins. We speculate that heightened pain sensitivity in female mice protects the joint by preventing overuse.

Nociceptive Sensitizers Are Regulated in Damaged Joint Tissues, Including Articular Cartilage, When Osteoarthritic Mice Display Pain Behavior.

OBJECTIVE: Pain is the most common symptom of osteoarthritis (OA), yet where it originates in the joint and how it is driven are unknown. The aim of this study was to identify pain-sensitizing molecules that are regulated in the joint when mice subjected to surgical joint destabilization develop OA-related pain behavior, the tissues in which these molecules are being regulated, and the factors that control their regulation. METHODS: Ten-week-old mice underwent sham surgery, partial meniscectomy, or surgical destabilization of the medial meniscus (DMM). Pain-related behavior as determined by a variety of methods (testing of responses to von Frey filaments, cold plate testing for cold sensitivity, analgesiometry, incapacitance testing, and forced flexion testing) was assessed weekly. Once pain-related behavior was established, RNA was extracted from either whole joints or microdissected tissue samples (articular cartilage, meniscus, and bone). Reverse transcription-polymerase chain reaction analysis was performed to analyze the expression of 54 genes known to regulate pain sensitization. Cartilage injury assays were performed using avulsed immature hips from wild-type or genetically modified mice or by explanting articular cartilage from porcine joints preinjected with pharmacologic inhibitors. Levels of nerve growth factor (NGF) protein were measured by enzyme-linked immunosorbent assay. RESULTS: Mice developed pain-related behavior 8 weeks after undergoing partial meniscectomy or 12 weeks after undergoing DMM. NGF, bradykinin receptors B1 and B2, tachykinin, and tachykinin receptor 1 were significantly regulated in the joints of mice displaying pain-related behavior. Little regulation of inflammatory cytokines, leukocyte activation markers, or chemokines was observed. When tissue samples from articular cartilage, meniscus, and bone were analyzed separately, NGF was consistently regulated in the articular cartilage. The other pain sensitizers were also largely regulated in the articular cartilage, although there were some differences between the 2 models. NGF and tachykinin were strongly regulated by simple mechanical injury of cartilage in vitro in a transforming growth factor ß-activated kinase 1-, fibroblast growth factor 2-, and Src kinase-dependent manner. CONCLUSION: Damaged joint tissues produce proalgesic molecules, including NGF, in murine OA.

Sulforaphane represses matrix-degrading proteases and protects cartilage from destruction in vitro and in vivo.

OBJECTIVE: Sulforaphane (SFN) has been reported to regulate signaling pathways relevant to chronic diseases. The aim of this study was to investigate the impact of SFN treatment on signaling pathways in chondrocytes and to determine whether sulforaphane could block cartilage destruction in osteoarthritis. METHODS: Gene expression, histone acetylation, and signaling of the transcription factors NF-E2-related factor 2 (Nrf2) and NF-κB were examined in vitro. The bovine nasal cartilage explant model and the destabilization of the medial meniscus (DMM) model of osteoarthritis in the mouse were used to assess chondroprotection at the tissue and whole-animal levels. RESULTS: SFN inhibited cytokine-induced metalloproteinase expression in primary human articular chondrocytes and in fibroblast-like synovial cells. SFN acted independently of Nrf2 and histone deacetylase activity to regulate metalloproteinase expression in human articular chondrocytes but did mediate prolonged activation of JNK and p38 MAPK. SFN attenuated NF-κB signaling at least through inhibition of DNA binding in human articular chondrocytes, with decreased expression of several NF-κB-dependent genes. Compared with cytokines alone, SFN (10 µM) abrogated cytokine-induced destruction of bovine nasal cartilage at both the proteoglycan and collagen breakdown levels. An SFN-rich diet (3 µmoles/day SFN versus control chow) decreased the arthritis score in the DMM model of osteoarthritis in the mouse, with a concurrent block of early DMM-induced gene expression changes. CONCLUSION: SFN inhibits the expression of key metalloproteinases implicated in osteoarthritis, independently of Nrf2, and blocks inflammation at the level of NF-κB to protect against cartilage destruction in vitro and in vivo.

Joint immobilization prevents murine osteoarthritis and reveals the highly mechanosensitive nature of protease expression in vivo.

OBJECTIVE: Mechanical joint loading is critical for the development of osteoarthritis (OA). Although once regarded as a disease of cartilage attrition, OA is now known to be controlled by the expression and activity of key proteases, such as ADAMTS-5, that drive matrix degradation. This study was undertaken to investigate the link between protease expression and mechanical joint loading in vivo. METHODS: We performed a microarray analysis of genes expressed in the whole joint following surgical induction of murine OA (by cutting the medial meniscotibial ligament). Gene expression changes were validated by reverse transcriptase-polymerase chain reaction in whole joints and microdissected tissues of the joint, including the articular cartilage, meniscus, and epiphysis. Following surgery, mouse joints were immobilized, either by prolonged anesthesia or by sciatic neurectomy. RESULTS: Many genes were regulated in the whole joint within 6 hours of surgical induction of OA in the mouse. These included Arg1, Ccl2, Il6, Tsg6, Mmp3, Il1b, Adamts5, Adamts4, and Adamts1. All of these were significantly regulated in the articular cartilage. When joints were immobilized by prolonged anesthesia, regulation of the vast majority of genes was abrogated. When joints were immobilized by sciatic neurectomy, regulation of selected genes was abrogated, and OA was prevented up to 12 weeks postsurgery. CONCLUSION: These findings indicate that gene expression in the mouse joint following the induction of OA is rapid and highly mechanosensitive. Regulated genes include the known pathogenic protease ADAMTS-5. Targeting the mechanosensing mechanisms of joint tissue may offer new strategies for disease modification.