Low back pain is a major public health problem in our society. Degeneration of intervertebral disc (IVD) appears to be the leading cause of chronic low-back pain [1]. Mechanical stimulations including compressive and tensional forces are directly implicated in IVD degeneration. Several studies have implicated the cytoskeleton in mechanotransduction [2, 3], which is important for communication and transport between the cells and extracellular matrix (ECM). However, the potential roles of the cytoskeletal elements in the mechanotransduction pathways in IVD are largely unknown. Outer annulus fibrosus (OAF) and nucleus pulposus (NP) cells from skeletally mature bovine IVD were either seeded onto Flexcell¯ type I collagen coated plates or seeded in 3% agarose gels, respectively. OAF cells were subjected to cyclic tensile strain (10%, 1Hz) and NP cells to cyclic compressive strain (10%, 1Hz) for 60 minutes. Post-loading, cells were processed for immunofluorescence microscopy and RNA extracted for quantitative PCR analysis.Introduction
Methods
Rheumatoid arthritis (RA) is a systemic autoimmune disease affecting 350,000 people in the UK. Within synovial joints, synoviocytes form a destructive pannus that degrades articular cartilage and bone. Synovial fluid glutamate levels increase 54 fold in RA patients and are also elevated in animal models of inflammatory and osteoarthritis. To determine whether elevated glutamate levels contribute to RA pathology we investigated which synovial joint tissues express glutamate receptors and whether glutamate stimulation influences synovio-cyte phenotype. Various glutamate receptor mRNAs (NMDAR1, KA1, AMPAGluR2, AMPA GluR3, mGluR4) were expressed in tissues of the rat knee. All receptors were expressed in the patella. The fibrocartilagenous menis-cus and articular cartilage chondrocytes expressed mGluR4 and both AMPA receptor subunits. Human synoviocytes expressed NMDAR1 and KA1 mRNA. To determine whether glutamate receptors were functional in human synoviocytes, cells were preloaded with a fluorescent indicator of intracellular calcium (iCa 2+) and stimulated with glutamate or specific agonists (NMDA or kainate, 500mM). Glutamate stimulated release of iCa2+ in 25% of synoviocytes whereas NMDA and Kainate each stimulated 15% of cells. NMDA responses increased to 57% in the absence of Mg2+ consistent with the inhibitory effect of Mg2+ on this receptor. To determine whether activation of glutamate receptors can influence human synoviocyte phenotype, we cultured synoviocytes in various glutamate concentrations (50mM to 2mM) and measured effects of glutamate receptor antagonists on release of a proinflammatory cytokine (IL-6) and degradative enzymes (MMP2 and 9). In some RA patients, glutamate stimulation increased synoviocyte pro MMP-2 release. TIMP1 and TIMP2 release were not affected by glutamate stimulation or co-treatment with receptor antagonists. IL-6 expression varied greatly in human synoviocytes derived from different RA patients (0–120pg/ml media). However, the AMPA/KA receptor antagonist NBQX significantly reduced IL-6 release at all glutamate concentrations. This inhibition was greater than that by CFM2 (AMPAR antagonist), indicating that activation of kainate receptors in human synoviocytes may induce IL-6 release. We conclude that glutamate receptors are functional in human synoviocytes and regulate release of MMP-2 and IL-6 Thus glutamatergic signalling may contribute to RA pathology and represent a new therapeutic target.
The presence of the connective tissue components fibronectin and the different types of collagen was demonstrated by histological and immunohistological methods in the granulation and scar tissue of a healing injury in rat muscle. The effects of physical activity on granulation tissue production, scar formation and muscle regeneration at various stages of healing were studied. It was shown that immobilisation after injury accelerates granulation tissue production, but if continued too long, leads to contraction of the scar and to poor structural organisation of the components of regenerating muscle and scar tissue. However, a certain period of immobilisation, about five days for rat muscle, is required to allow newly-formed granulation tissue to cover the injured area and to have sufficient tensile strength to withstand subsequent mobilisation. This mobilisation, at the correct interval, seems essential for the quicker resorption of scar tissue and the better structural organisation of the muscle.
