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Objective: To characterize the initial events in the cleavage of type II collagen mediated by cathepsin K and demonstrate the presence of the resulting products in human and equine articular osteoarthritic cartilage.Design: Equine type II collagen was digested with cathepsin K and the cleavage products characterized by mass spectrometry. Anti-neoepitope antibodies were raised against the most N-terminal cleavage products and used to investigate the progress of collagen cleavage, in vitro, and the presence of cathepsin K-derived products in equine and human osteoarthritic cartilage.Results: Six cathepsin K cleavage sites distributed throughout the triple helical region were identified in equine type II collagen. Most of the cleavages occurred following a hydroxyproline residue. The most N-terminal site was within three residues of the previously identified site in bovine type II collagen. Western blotting using anti-neoepitope antibodies showed that the initial cleavages occurred at the N-terminal sites and this was followed by more extensive degradation resulting in products too small to be resolved by SDS gel electrophoresis. Immunohistochemical staining of cartilage sections from equine or human osteoarthritic joints showed staining in lesional areas which was not observed in non-arthritic sites.Conclusions: Cathepsin K cleaves triple helical collagen by erosion from the N-terminus and with subsequent progressive cleavages. The liberated fragments can be detected in osteoarthritic cartilage and may represent useful biomarkers for disease activity. [ABSTRACT FROM AUTHOR]

Summary: Objective: To compare synovial glucosamine levels in normal and inflamed equine joints following oral glucosamine administration and to determine whether single dose administration alters standard synovial parameters of inflammation. Methods: Eight adult horses were studied. On weeks 1 and 2, all horses received 20mg/kg glucosamine hydrochloride by nasogastric (NG) intubation or intravenous injection. On weeks 3 and 4, 12h after injection of both radiocarpal joints with 0.25ng Escherichia coli lipopolysaccharide (LPS) to induce inflammation, glucosamine hydrochloride or a placebo was administered by NG intubation. Plasma samples were collected at baseline and 5, 15, 30, 60, 120, 360, 480 and 720min after dosing. Synovial fluid (SF) samples were collected within 48h before dosing and 1, 6 and 12h post-dosing. Glucosamine was analyzed by Liquid Chromatography Electrospray Tandem Mass Spectrometry (LC-ESI/MS/MS). Clinicopathological evaluation of SF parameters included white blood cell (WBC) count and total protein (TP) analyses. Results: No significant differences between groups were observed in SF baseline levels of WBC and TP at any stage of the study. SF WBC and TP significantly increased following IA LPS. The mean (±SD) maximal SF glucosamine levels (422.3±244.8ng/mL) were significantly higher (>fourfold) in inflamed joints when compared to healthy joints (92.7±34.9ng/mL). Glucosamine did not have any effect on standard SF parameters of inflammation. Conclusion: Synovial inflammation leads to significantly higher synovial glucosamine concentrations compared to levels attained in healthy joints following oral administration of glucosamine hydrochloride. Whether these higher levels are translated into a therapeutic effect on the joint tissues remains to be elucidated. [Copyright &y& Elsevier]

Summary: Objective: To compare the pharmacokinetics of glucosamine and the synovial fluid levels attained following treatment with glucosamine sulphate or glucosamine hydrochloride in a large animal model at clinically relevant doses. Methods: Eight adult female horses were used. Crystalline glucosamine sulphate (Dona®) or glucosamine hydrochloride was administered at a dose of 20mg/kg by either intravenous (IV) injection or nasogastric (NG) intubation. Plasma samples were collected before dosing and at 5, 15, 30, 60, 120, 360, 480 and 720min after dosing. Synovial fluid samples were collected from the radiocarpal joints within 48h before dosing and at 1, 6 and 12h post-dosing. Glucosamine was assayed by Liquid Chromatography Electrospray Tandem Mass Spectrometry (LC-ESI/MS/MS). Results: Plasma concentrations reached ∼50μg/mL after IV injection and ∼1μg/mL after NG administration of both types of glucosamine. The median oral bioavailability was 9.4% for glucosamine sulphate and 6.1% for glucosamine hydrochloride. Synovial fluid concentrations were significantly higher at 1 and 6h following oral treatment with glucosamine sulphate compared to glucosamine hydrochloride. Twelve hours following oral administration, glucosamine levels in the plasma and the synovial fluid were still significantly higher than baseline for the glucosamine sulphate preparation, but not for the hydrochloride preparation. Conclusion: Following oral administration of a clinically recommended dose of glucosamine sulphate (Dona®), significantly higher synovial fluid concentrations of glucosamine are attained, when compared to an equivalent dose of glucosamine hydrochloride. Whether this difference is translated into a therapeutic effect on the joint tissues remains to be elucidated. [Copyright &y& Elsevier]

Abstract: The purpose of this study was to compare the gait parameters recorded on the CatWalk and the mechanical sensitivity with von Frey filaments of two putative models of osteoarthritis over a one month period, and to evaluate the effect of celecoxib on these parameters. Animals underwent either a surgical sectioning of the anterior cruciate ligament with partial medial menisectomy (ACLT+pMMx) to create a joint instability model or received an intra-articular injection of monoiodoacetate (MIA) as a putative inflammatory joint pain model. Animals were assessed for four consecutive weeks and knee joints were then evaluated histologically. Spinal cord lumbar enlargements were harvested for selected neuropeptide analysis (substance P (SP) and calcitonin gene related peptide (CGRP)). With the MIA model, significant changes persisted in selected dynamic gait parameters throughout the study in the injured limb as well as with the von Frey filaments. The ACLT+pMMx model in contrast showed no clear differential response between both hind limb for both gait parameters and pain-related behavior with von Frey filaments occurred only on the last day of the study. Neuropeptide analysis of spinal cord lumbar enlargements revealed a significant increase in CGRP concentration in both models and an increase in SP concentration only in the MIA model. Histological evaluation confirmed the presence of articular cartilage lesions in both models, but they were much more severe in the MIA model. Celecoxib had an effect on all selected gait parameters at the very beginning of the study and had an important alleviating effect on mechanical allodynia. These results suggest that the MIA model may be more appropriate for the evaluation of short term pain studies and that celecoxib may modulate mechanical allodynia through central sensitization mechanisms. [ABSTRACT FROM AUTHOR]

Few studies have verified the validity of behavioral and physiological methods of pain assessment in cattle. This prospective, blinded, randomized controlled experimental study aimed to validate different methods of pain assessment during acute and chronic (up to 21 d postintervention) conditions in dairy cattle, in response to 3 analgesic treatments for traumatic reticuloperitonitis. Cerebrospinal fluid (CSF) biomarkers and mechanical sensitization were measured as indicators of centralized pain. Proteomics in the CSF were examined to detect specific (to pain intensity) and sensitive (responsive to analgesia) markers. Recordings of spontaneous behavior with video analysis, telemetered motor activity, pain scales, electrodermal activity, and plasma cortisol concentration were quantified at regular intervals. Cows were assigned to group 1 (n = 4, standard control receiving aspirin), group 2 (n = 5, test group receiving preemptive tolfenamic acid), or group 3 (n = 3, positive control receiving preemptive multimodal analgesia composed of epidural morphine, plus tolfenamic acid and butorphanol). Rescue analgesia was administered as needed. Generalized estimating equations tested group differences and the influence of rescue analgesia on the measurements. All 3 groups demonstrated a long-term decrease in a CSF protein identified as transthyretin. The decrease in transthyretin expression inversely correlated with the expected level of analgesia (group 1 < 2 < 3). Moreover, in group 1, CSF noradrenaline decreased long term, cows were hypersensitive to mechanical stimulation, and they demonstrated signs of discomfort with higher motor activity and "agitation while lying" recorded from video analysis. Decreased "feeding behavior," observer-reported pain scales, electrodermal activity, and plasma cortisol concentration were inconsistent to differentiate pain intensity between groups. In summary, changes in CSF biomarkers and mechanical sensitization reflected modulation of central pain in dairy cows. The spontaneous behavior "agitation while lying" was the only behavioral outcome validated for assessing acute and chronic pain in this visceral pain model. [ABSTRACT FROM AUTHOR]