Evaluation of oral n-3 polyunsaturated fatty acid effect on the inflammatory response to a novel experimental model of synovitis in the horse

Osteoarthritis (OA) is a debilitating disease resulting in irreversible cartilage loss that can influence mobility and quality of life in multiple species. The addition of n-3 long chain polyunsaturated fatty acids (LCPUFA), specifically eicosapentaneoic acid (EPA) and docosahexanoic acid (DHA), has been reported to have beneficial effects on joint inflammation in human and mouse in-vivo and in-vitro models. Little to no information exists regarding oral n-3 supplementation and effects on inflammatory markers in the equine joint. Additionally the development of reversible in vivo models of synovitis is valuable for studying OA pathophysiology, and testing therapeutic modalities. A pilot study conducted by our laboratory concluded that a 90 day treatment of an oral EPA/DHA supplement resulted in significantly higher serum and synovial fluid concentrations of these specific fatty acids in healthy horses receiving the supplement compared to non-supplemented healthy horses. Therefore, a two-phase experiment was designed to 1) validate the use of an equine recombinant cytokine for inducing a humane, reversible bout of synovitis in the horse and 2) determine if oral supplementation of a n-3 LCPUFA supplement would influence the inflammatory response in an experimental model of equine synovitis.;In Phase One of the experiment twelve skeletally-mature mares, were utilized in a randomized block design. Synovitis was induced by an intra-articular injection of 100 ng of recombinant equine interleukin 1 beta (reIL-1beta) or 0.5 ng of lipopolysaccharide (LPS) into a middle carpal joint in 1 ml volumes. One ml of phosphate buffered saline (PBS) was injection into the contra-lateral joint to serve as a control. Lameness evaluations were conducted throughout the study trial starting at post injection hour 0 (PIH) through PIH 240. Synovial fluid samples were repeatedly taken at PIH 0, 4, 8, 24 and 48. Synovial fluid samples were analyzed for inflammatory biomarker concentrations prostaglandin E2 (PGE2), general matrix metalloproteinase (MMP) activity, glycosaminoglycan (GAG) and routine cytology. Synovial tissue and articular cartilage was collected at PIH 8 via arthroscopic biopsy procedures. Joint tissues were analyzed for gene expression of MMP 1 and 13; the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta); cyclooxygenase 2 (COX-2) and the aggrecanases ADAMTS-4 (a disintegrin and metalloproteinase with a thrombospondin motif 4) and ADAMTS-5 (a disintegrin and metalloproteinase with a thrombospondin motif 5).;In the second phase of the research (Phase Two), twelve skeletally-mature mares, free of lameness, were randomly assigned to one of two treatment groups: a CONT (control diet) group and a N3FA (control diet + a daily EPA/DHA rich supplement) group. All horses were individually fed their assigned diet, divided into two equal feedings daily and received dietary treatment for 90 days. Serum samples and synovial fluid samples collected on days 0, 30, 60, 91 and days 0 and 91 were processed and analyzed for lipid composition. On day 91, inflammation was experimentally-induced in a single carpal joint of each horse using a reIL-1beta solution and collection procedures as outlined by Phase One of experimental studies.;Results from the Phase One study indicated that a single injection of reIL-1beta or LPS increased synovial white blood cell, neutrophil count, total protein, prostaglandin E2 concentrations and general matrix metalloproteinase activity relative to control joints through PIH 8. Injections of either reIL-1beta or LPS increased mRNA expression for MMP-1 and ADAMTS-4 in synovium and for MMP-1, ADAMTS-4, and ADAMTS-5 in articular cartilage collected at PIH 8 compared to saline injected joint tissue. Synovium collected from LPS-treated joints exhibited higher expression for IL-1beta and ADAMT-4. The use of reIl-1beta was sufficient in presenting a robust, yet temporary bout of inflammation in the horse.;In Phase Two, a 90 day supplementation of approximately 36g of EPA/DHA per day to horses resulted in significantly higher serum and synovial fluid EPA and DHA compared to non-supplemented horses. Serum arachidonic acid (ARA) was significantly higher in the N3FA horses compared to CONT horses at day 30-day 90; likely due to the presence of dietary ARA in n-3 fatty acid product. Synovial fluid analysis indicated that a reIL-1beta injection increased synovial white blood cell, neutrophil count, total protein, prostaglandin E2 concentrations and general matrix metalloproteinase activity relative to control joints through PIH 8 regardless of dietary treatment. Gene expression for MMP-13, IL-1beta, ADAMTS-4, and ADAMTS-5 was higher in treated cartilage samples compared to saline injections, regardless of dietary treatment. A single injection of reIL-1beta resulted in increased expression for MMP-1, COX-2 and IL-1beta in synovium collected from all horses with no significant difference between dietary groups. Gene expression in synovial tissue for ADAMTS-4 was significantly lower in N3FA horses compared to CONT horses.;Injections of reIL-1beta into equine carpal joints resulted in a transient inflammatory response and an increase in mRNA expression of certain deleterious mediators in joint tissues that was similar in severity to the LPS injection. Given that IL-1beta is a known critical mediator of traumatic arthritis and OA, this humane and temporary model may be useful in evaluating therapeutics that act against early stages of joint disease. A follow up study utilizing the reIL-1beta model to evaluate dietary n-3 LCPUFA treatment resulted in a robust, yet temporary inflammatory response characterized by significant increases in cytology parameters, biological markers of inflammation and gene expression of degradative enzymes associated with cartilage degradation. A 90 day dietary treatment was successful in significantly altering serum phospholipid and synovial fluid lipid concentrations of EPA and DHA. Dietary supplementation of n-3 LCPUFAs did not result in significant difference from control diet for clinical response (lameness), white blood cell counts, eicosanoid production, matrix metalloproteinase activity. The discovery of significantly lower expression of ADAMTS-4 in N3FA synovium supports further inquiry into the capability of n-3 LCPUFAs, in particular, EPA and DHA as potential therapies for equine OA.