Objective-To assess effects of in vitro meloxicam exposure on metabolism in articular chondrocytes from dogs with naturally occurring osteoarthritis Sample-Femoral head cartilage from 16 dogs undergoing total hip replacement Procedures-Articular cartilage samples were obtained. Tissue sulfated glycosaminoglycan (SGAG), collagen, and DNA concentrations were measured. Collagen, SGAG, chondroitin sulfate 846, NO, prostaglandin E2 (PGE2), and matrix metalloproteinase (MMP)-2, MMP-3, MMP-9, and MMP-13 concentrations in culture medium were analyzed. Aggrecan, collagen II, MMP-2, MMP-3, MMP-9, MMP-13, ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS)-4, ADAMTS-5, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, TIMP-3, interleukin-1β, tumor necrosis factor-α, cyclooxygenase-1, cyclooxygenase-2, and nducible nitric oxide synthase gene expression were evaluated. Comparisons between tissues cultured without (control) and with meloxicam at concentrations of 0.3, 3.0, and 30.0 μg/mL for up to 30 days were performed by means of repeated-measures analysis. Results-Meloxicam had no effect on chondrocyte SGAG, collagen, or DNA concentrations. Expression of ADAMTS-5 was significantly decreased in all groups on all days, compared with the day 0 value. On day 3, culture medium PGE2 concentrations were significantly lower in all meloxicam-treated groups, compared with values for controls, and values remained low. Culture medium MMP-3 concentrations were significantly lower on day 30 than on day 3 in all meloxicam-treated groups. Conclusions and Clinical Relevance-Results suggested that in vitro meloxicam treatment of osteoarthritic canine cartilage for up to 30 days did not induce matrix degradation or stimulate MMP production. Meloxicam lowered PGE2 release from this tissue, and effects on tissue chondrocyte content and matrix composition were neutral.

Objective: To develop an in vitro model of cartilage injury in full-thickness equine cartilage specimens that can be used to simulate in vivo disease and evaluate treatment efficacy. Sample: 15 full-thickness cartilage explants from the trochlear ridges of the distal aspect of the femur from each of 6 adult horses that had died from reasons unrelated to the musculoskeletal system. Procedures: To simulate injury, cartilage explants were subjected to single-impact uniaxial compression to 50%, 60%, 70%, or 80% strain at a rate of 100% strain/s. Other explants were left uninjured (control specimens). All specimens underwent a culture process for 28 days and were subsequently evaluated histologically for characteristics of injury and early stages of osteoarthritis, including articular surface damage, chondrocyte cell death, focal cell loss, chondrocyte cluster formation, and loss of the extracellular matrix molecules aggrecan and types I and II collagen. Results: Compression to all degrees of strain induced some amount of pathological change typical of clinical osteoarthritis in horses; however, only compression to 60% strain induced significant changes morphologically and biochemically in the extracellular matrix. Conclusions and Clinical Relevance: The threshold strain necessary to model injury in full-thickness cartilage specimens from the trochlear ridges of the distal femur of adult horses was 60% strain at a rate of 100% strain/s. This in vitro model should facilitate study of pathophysiologic changes and therapeutic interventions for osteoarthritis.

Objective: To compare the effects of autologous equine serum (AES) and autologous conditioned serum (ACS) on equine articular chondrocyte metabolism when stimulated with recombinant human (rh) interleukin (IL)-1β. Sample: Articular cartilage and nonconditioned and conditioned serum from 6 young adult horses. Procedures: Cartilage samples were digested, and chondrocytes were isolated and formed into pellets. Chondrocyte pellets were treated with each of the following: 10% AES, 10% AES and rhIL-1β, 20% AES and rhIL-1β, 10% ACS and rhIL-1β, and 20% ACS and rhIL-1β, and various effects of these treatments were measured. Results: Recombinant human IL-1β treatment led to a decrease in chondrocyte glycosaminoglycan synthesis and collagen II mRNA expression and an increase in medium matrix metalloproteinase-3 activity and cyclooxygenase-2 mRNA expression. When results of ACS and rhIL-1β treatment were compared with those of AES and rhIL-1β treatment, no difference was evident in glycosaminoglycan release, total glycosaminoglycan concentration, total DNA content, or matrix metalloproteinase-3 activity. A significant increase was found in chondrocyte glycosaminoglycan synthesis with 20% AES and rhIL-1β versus 10% ACS and rhIL-1β. The medium from ACS and rhIL-1β treatment had a higher concentration of IL-1β receptor antagonist, compared with medium from AES and rhIL-1β treatment. Treatment with 20% ACS and rhIL-1β resulted in a higher medium insulin-like growth factor-I concentration than did treatment with 10% AES and rhIL-1β. No difference in mRNA expression was found between ACS and rhIL-1β treatment and AES and rhIL-1β treatment. Conclusions and Clinical Relevance: Minimal beneficial effects of ACS treatment on proteoglycan matrix metabolism in equine chonrocytes were evident, compared with the effects of AES treatment.

Objective: To identify proteins with differential expression between healthy dogs and dogs with stifle joint osteoarthritis secondary to cranial cruciate ligament (CCL) disease. Sample: Serum and synovial fluid samples obtained from dogs with stifle joint osteoarthritis before (n = 10) and after (8) surgery and control dogs without osteoarthritis (9) and archived synovial membrane and articular cartilage samples obtained from dogs with stifle joint osteoarthritis (5) and dogs without arthritis (5). Procedures: Serum and synovial fluid samples were analyzed via liquid chromatography–tandem mass spectrometry; results were compared against a nonredundant protein database. Expression of complement component 3 in archived tissue samples was determined via immunohistochemical methods. Results: No proteins had significantly different expression between serum samples of control dogs versus those of dogs with stifle joint osteoarthritis. Eleven proteins (complement component 3 precursor, complement factor I precursor, apolipoprotein B-100 precursor, serum paraoxonase and arylesterase 1, zinc-alpha-2-glycoprotein precursor, serum amyloid A, transthyretin precursor, retinol-binding protein 4 precursor, alpha-2-macroglobulin precursor, angiotensinogen precursor, and fibronectin 1 isoform 1 preproprotein) had significantly different expression (> 2.0-fold) between synovial fluid samples obtained before surgery from dogs with stifle joint osteoarthritis versus those obtained from control dogs. Complement component 3 was strongly expressed in all (5/5) synovial membrane samples of dogs with stifle joint osteoarthritis and weakly expressed in 3 of 5 synovial membrane samples of dogs without stifle joint arthritis. Conclusions and Clinical Relevance: Findings suggested that the complement system and proteins involved in lipid and cholesterol metabolism may have a role in stifle joint osteoarthritis, CCL disease, or both.

Objective: To determine the effects of hypoglossal nerve block and electrical stimulation of the thyrohyoideus muscles on position of the larynx and hyoid apparatus in resting horses. Animals: 16 healthy horses that underwent hypoglossal nerve block and 5 healthy horses that underwent electrical stimulation of the thyrohyoideus muscles. Procedures: Horses underwent bilateral hypoglossal nerve block or electrical stimulation of the thyrohyoideus muscles. Positions of the basihyoid bone, ossified part of the thyroid cartilage, and articulations of the thyrohyoid bones and thyroid cartilage were determined in radiographic images obtained before and after performance of hypoglossal nerve blocks or during thyrohyoideus muscle stimulation. Radiographic images were obtained with the heads of horses in neutral (thyrohyoideus muscle stimulation) or neutral and extended (hypoglossal nerve block) positions. Radiographic images of horses obtained after performance of hypoglossal nerve blocks were also evaluated to detect dorsal displacement of the soft palate. Results: Hypoglossal nerve blocks did not induce significant changes in the positions of evaluated anatomic sites in radiographic images obtained in neutral or extended head positions. Hypoglossal nerve block did not induce dorsal displacement of the soft palate in horses at rest. Bilateral thyrohyoideus muscle stimulation induced significant dorsal movement (mean ± SD change in position, 18.7 ± 6.8 mm) of the ossified part of the thyroid cartilage; rostral movement of evaluated anatomic structures was small and not significant after thyrohyoideus muscle stimulation. Conclusions and Clinical Relevance: Bilateral electrical stimulation of the thyrohyoideus muscles in horses in this study induced dorsal laryngeal movement.

Objective: To use microarray analysis to identify genes that are differentially expressed in horses with experimentally induced osteoarthritis. Animals: 24 horses. Procedures: During arthroscopic surgery, a fragment was created in the distal aspect of the radiocarpal bone in 1 forelimb of each horse to induce osteoarthritis. At day 14 after osteoarthritis induction, horses began exercise on a treadmill. Blood and synovial fluid samples were collected before and after surgery. At day 70, horses were euthanized and tissues were harvested for RNA analysis. An equine-specific microarray was used to measure RNA expression in peripheral WBCs. These data were compared with mRNA expression (determined via PCR assay) in WBCs, cartilage, and synovium as well as 2 protein biomarkers of cartilage matrix turnover in serum and synovial fluid. Results: A metalloproteinase domain-like protein decysin-1 (ADAMDEC1), glucose-regulated protein (GRP) 94, hematopoietic cell signal transducer (HCST), Unc-93 homolog A (hUNC-93A), and ribonucleotide reductase M2 polypeptide (RRM2) were significantly differentially regulated in WBCs of horses with osteoarthritis, compared with values prior to induction of osteoarthritis. There was correlation between the gene expression profile in WBCs, cartilage, and synovium and the cartilage turnover proteins. Gene expression of ADAMDEC1, hUNC-93A, and RRM2 in WBCs were correlated when measured via microarray analysis and PCR assay. Conclusions and Clinical Relevance: Expression of ADAMDEC1, GRP94, HCST, hUNC-93A, and RRM2 was differentially regulated in peripheral WBCs obtained from horses with experimentally induced osteoarthritis. Gene expression of ADAMDEC1, hUNC-93A, and RRM2 in peripheral WBCs has the potential for use as a diagnostic aid for osteoarthritis in horses.

Objective-To investigate histomorphometric changes in the cartilage and subchondral bone of the third carpal bone associated with conditioning exercise in young Thoroughbreds. Animals-Nine 18-month-old Thoroughbreds. Procedures-Both third carpal bones of 9 horses (4 exercised spontaneously at pasture only and 5 given additional conditioning exercise beginning at a mean age of 3 weeks) were evaluated. Histomorphometric variables (hyaline and calcified cartilage thickness and collagen orientation; vascular channel area, number, and orientation; and osteochondral junction rugosity) of the third carpal bone, sampled at 4 dorsopalmar sites in the radial facet, were compared between the exercised and nonexercised groups. Results-The vascular channel area measured at the 4 dorsopalmar sites was larger in the exercised group than in the control group, but none of the variables were significantly different between groups. Both groups had significant site-specific variations in all measured variables. Most importantly, the vascular channel area was highest in the most dorsal aspect. Conclusions and Clinical Relevance-Results suggested that the mild exercise imposed in both groups during the developmental period appeared to be associated with an increase in the vascular channel area beneath the calcified cartilage layer in the third carpal bone. This increased vascular channel area could also be associated with high stress in the dorsal aspect of the radial facet, a region that is known to be vulnerable to osteochondral fragmentation.

Objective-To determine whether oxidative stress could be induced in canine chondrocytes in vitro. Sample-Chondrocytes obtained from healthy adult mixed-breed dogs. Procedures-Harvested chondrocytes were maintained at 37°C with 5% CO2 for 24 hours. To assess induction of oxidative stress, 2 stimuli were used: hydrogen peroxide and a combination of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). To determine the effect of hydrogen peroxide, a set of chondrocyte-seeded plates was incubated with control medium alone or hydrogen peroxide (100, 200, or 300μM) for 24 hours. For inhibition of oxidative stress, cells were incubated for 24 hours with N-acetylcysteine (NAC; 10mM) before exposure to hydrogen peroxide. Another set of chondrocyte-seeded plates was incubated with control medium alone or with IL-1β (10 ng/mL) and TNF-α (1 ng/mL) for 24 hours. Supernatants were obtained for measurement of prostaglandin E2 production, and cell lysates were used for measurement of superoxide dismutase (SOD) activity and reduced-glutathione (GSH) concentration. Results-Chondrocytes responded to the oxidative stressor hydrogen peroxide with a decrease in SOD activity and GSH concentration. Exposure to the antioxidant NAC caused an increase in SOD activity in hydrogen peroxide-stressed chondrocytes to a degree comparable with that in chondrocytes not exposed to hydrogen peroxide. Similarly, NAC exposure induced significant increases in GSH concentration. Activation with IL-1β and TNF-α also led to a decrease in SOD activity and increase in prostaglandin E2 production. Conclusions and Clinical Relevance-Canine chondrocytes responded to the oxidative stress caused by exposure to hydrogen peroxide and cytokines. Exposure to oxidative stress inducers could result in perturbation of chondrocyte and cartilage homeostasis and could contribute to the pathophysiology of osteoarthritis. Use of antioxidants, on the other hand, may be helpful in the treatment of arthritic dogs.

There is evidence that vitamin E (VE) has anti-inflammatory and analgesic properties in human osteoarthritis (OA). This double-blinded and randomized pilot study used a broad spectrum of clinical and laboratory parameters to investigate whether such beneficial effects could be detected in a canine experimental OA model. Dogs were divided into 2 groups: control (n = 8), which received a placebo, and test group (n = 7), which received 400 IU/animal per day of VE for 55 d, starting the day after transection of the cranial cruciate ligament. Lameness and pain were assessed using a visual analogue scale (VAS), numerical rating scale (NRS), and electrodermal activity (EDA) at day 0, day 28, and day 55. Cartilage and synovial inflammation lesions were assessed. One-side comparison was conducted at an alpha-threshold of 10%. At day 56, dogs were euthanized and concentrations of prostaglandin E2 (PGE2), nitrogen oxides (NOx), and interleukin-1 beta (IL-1β) were measured in synovial fluid. Concentrations of NOx and PGE2 in synovial fluid were lower in the test group (P < 0.0001 and P = 0.03, respectively). Values of VAS, NRS, and EDA showed a consistent trend to be lower in the test group than in the control, while statistical significance was reached for VAS at day 55 and for EDA at day 28 (adjusted P = 0.07 in both cases). Histological analyses of cartilage showed a significant reduction in the scores of lesions in the test group. This is the first time that a study in dogs with OA using a supplement with a high dose of vitamin E showed a reduction in inflammation joint markers and histological expression, as well as a trend to improving signs of pain.

Objective-To evaluate the use of CT and MRI for guidance of osteochondral sample collection for histologic detection of early osteoarthritic lesions in centrodistal (distal intertarsal) joints of horses. Sample -Right tarsal joints from the cadavers of 24 Icelandic horses aged 29 to 31 months. Procedures-CT and MRI were used to evaluate the extent of suspected osteoarthritic changes in centrodistal joints, which were graded with a semiquantitative system. The anatomic regions with the highest grade of change were identified, and osteochondral samples were obtained from these regions. Samples were also obtained from the same centrodistal joints at predetermined sites. Histologic examination of all samples was performed, with samples classified as negative or positive for osteoarthritis, and results were compared between sample collection methods. Results-Histologic examination revealed osteoarthritic lesions in 29% (7/24) of centrodistal joints with the predetermined method and in 63% (15/24) with the image-guided method. Significant associations were identified between histologic osteoarthritis detection and the summed image-guided sample collection site image grades, central osteophytes, articular cartilage thickness abnormalities, grade 2 articular mineralization front defects, and grade 2 marginal osteophytes. Conclusions and Clinical Relevance-CT and MRI aided the detection of focal changes suggestive of early-stage osteoarthritis in the centrodistal joints of equine cadavers and may be useful for detection of similar disease in live horses. The first morphological changes of centrodistal joint osteoarthritis were suspected to be in the articular cartilage and the articular mineralization front regions.