Introduction: Inflammation is an important feature of many joint diseases, and levels of cartilage biomarkers measured in synovial fluid may be influenced by local inflammatory status. Little is known about the magnitude and time course of inflammation-induced changes in cartilage tissue turnover as measured in vivo by synovial fluid markers. We aimed to study temporal changes in concentrations of inflammatory mediators, matrix metalloproteinase activity and cartilage biomarkers over 1 week in joints with experimentally induced inflammation. Methods: Localized inflammation was induced in the intercarpal joint of six horses by sterile injection of 0.5 ng lipopolysaccharide, and synovial fluid was collected at post-injection hours (PIH) 0, 8, 24 and 168. Concentrations of inflammatory mediators (prostaglandin E₂, substance P, and bradykinin), general matrix metalloproteinase activity and markers of collagen II turnover (CPII and C2C) as well as aggrecan turnover (CS846 and glycosaminoglycans) were measured with appropriate assays. One-way analysis of variance on repeated measures was used to analyze differences in synovial fluid marker levels over time. Results: Lipopolysaccharide-injection led to a sharp rise in prostaglandin E₂ at PIH 8, while substance P, bradykinin and matrix metalloproteinase activity showed more sustained increases at PIH 8 and 24. Glycosaminoglycan release paralleled changes in the CS846 epitope, with an increase by PIH 8, a peak at PIH 24, and return to baseline by PIH 168. For type II collagen, a parallel time course between catabolic (C2C) and anabolic (CPII) markers was also observed, but the time course differed from that seen for proteoglycan markers: collagen II markers peaked later, at PIH 24, and were still elevated over baseline at PIH 168. Conclusions: A primary intra-articular inflammatory insult, characterized by local release of peptide and lipid mediators and matrix metalloproteinase activation, can alter synovial fluid levels of proteoglycan biomarkers as early as 8 hours post-induction, and can lead to sustained rises in collagen II biomarkers during at least 1 week after onset.

Introduction: Articular tissues are capable of producing a range of eicosanoid mediators, each of which has individual biological effects and may be affected by anti-inflammatory treatment. We set out to develop and evaluate a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) approach for the simultaneous analysis of multiple eicosanoid lipid mediators in equine synovial fluid (SF), and to illustrate its use for investigation of the in vivo effects of non-steroidal anti-inflammatory drug (NSAID) treatment. Methods: Synovial fluid samples were obtained from normal joints of 6 adult horses at baseline (0 hr) and at 8, 24 and 168 hours after experimental induction of transient acute synovitis, with horses treated once daily with oral NSAID (meloxicam, 0.6 mg/kg) or placebo. Following solid-phase extraction, SF lipid mediator quantitation was based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis, and results were compared between disease states using linear discriminant analysis (LDA) and analysis of variance (ANOVA) with multiple comparisons corrections. Results: Of a total of 23 mediators targeted, 14 could be reliably identified and quantified in SF samples based on detection of characteristic fragment ions at retention times similar to those of commercial standards. LDA analysis of baseline, 8, 24 and 168 hour synovial fluid samples revealed a separation of these groups into discrete clusters, reflecting dynamic changes in eicosanoid release over the course of synovitis. Prostaglandin (PG) E₂ was significantly lower in NSAID vs. placebo treated samples at all time points; PGE₁, 11-hydroxyeicosatetraenoic acid (11-HETE) and 13,14-dihydro-15keto PGF₂α were reduced at 8 and 24 hours by NSAID treatment; while 15-HETE, 6-keto PGF₁α, PGF₂α, 13,14-dihydro-15keto PGE₂ and thromboxane B₂ (TXB₂) were reduced at the 8 hour time point only. An interesting pattern was seen for Leukotriene B₄ (LTB₄), NSAID treatment causing an initial increase at 8 hours, but a significant reduction by 168 hours. Conclusions: The described method allows a comprehensive analysis of synovial fluid eicosanoid profiles. Eicosanoid release in inflamed joints as well as differences between NSAID treated and placebo treated individuals are not limited to PGE₂ or to the early inflammatory phase.

Assessment of chronic pain is very important for monitoring and improving welfare and quality of life in donkeys. Freedom from disease and pain is one of the 'five freedoms' underlying animal welfare. The aim of the current study was to develop a pain scale for assessment of chronic pain in donkeys (Donkey Chronic Pain Scale; DCPS), including behavioural and facial expression-related parameters. The scale was applied to 77 donkeys (38 donkeys diagnosed with chronic health problems by means of clinical examination and additional diagnostic procedures and 39 healthy control animals). Animals were assessed twice daily for three consecutive days by two observers that were not masked to the condition of the animals but were unaware of the analgesic treatment regimens. Both composite, facial expression-based and combined DCPS pain scales showed excellent inter-observer reliability (Cronbach's alpha = 0.98, 0.96 and 0.98 respectively; P < 0.001). Individual composite and facial expression-based pain scores and the resulting combined DCPS showed significant differences between donkeys with chronic conditions and control donkeys at all time points (P < 0.001). A DCPS cut-off of 6 showed good sensitivity and specificity (92% and 82.5% respectively) for presence of a chronic painful condition. Facial expression-related parameters separately showed low sensitivity. In conclusion, it is possible to use a composite pain scale for assessment of chronic pain in donkeys, based on behavioural and facial expression-based parameters. Further studies are needed to validate this pain scale before it can be used in veterinary practice.