Objective-To evaluate the origin and degree of activity of nitric oxide (NO) and matrix metalloproteinase (MMP) in explants of cranial cruciate ligaments (CCLs) obtained from dogs and cultured with and without inflammatory activators. Sample Population-Tissue specimens obtained from 7 healthy adult Beagles that were (mean +/- SD) 4.5 +/- 0.5 years old and weighed 12.5 +/- 0.8 kg. Procedure-The CCLs were harvested immediately after dogs were euthanatized, and specimens were submitted for explant culture. Cultures were stimulated by incubation with a combination of interluekin-1, tumor necrosis factor-α, and lipopolysaccharide, or they were not stimulated. Culture supernatants were examined for production of NO nitrite-nitrate metabolites (NOts) and activity of MMP. Cultured specimens were evaluated by use of immunohistochemical analysis to detect activity of inducible NO synthase (iNOS). Results-All ligament explants produced measurable amounts of NOts. Stimulated cultures produced significantly more NOts after incubation for 24 and 48 hours, compared with nonstimulated cultures. Production of MMP in supernatants after incubation for 48 hours was significantly higher in stimulated cultures than in nonstimulated cultures. Cells with positive staining for iNOS were detected on all slides. Positively stained cells were predominantly chondroid metaplastic. There was a significant difference in intensity of cell staining between stimulated and nonstimulated cultures. Conclusion and Clinical Relevance—Explant cultures of intact CCLs obtained from dogs produce iNOS-induced NO. Stimulation of chondroid metaplastic cells in CCL of dogs by use of inflammatory activators can increase production of iNOS, NOts, and MMP.

Objective-To investigate accumulation of extracellular adenosine (ADO) by equine articular chondrocytes and to compare effects of adenosine kinase inhibition and adenosine deaminase inhibition on the amount of nitric oxide (NO) produced by lipopolysaccharide (LPS)-stimulated chondrocytes. Sample Population-Articular cartilage from metacarpophalangeal and metatarsophalangeal joints of 14 horses. Procedure-Chondrocytes were cultured as monolayers, and cells were incubated with LPS, the adenosine kinase inhibitor 5'-iodotubercidin (ITU), or the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3- nonyl)adenine hydrochloride (EHNA). Concentrations of ADO in cell supernatants were measured by use of reverse-phase high-performance liquid chromatography. Effect of inhibition of enzymatic metabolism of ADO on induced NO production was evaluated by exposing cells to a combination of LPS and ITU or LPS and EHNA. Results-Articular chondrocytes accumulated extracellular ADO when exposed to LPS or ITU. Chondrocytes exposed to ITU accumulated ADO in a time-dependent manner. Unstimulated chondrocytes did not accumulate ADO. Similarly, EHNA alone did not produce detectable ADO concentrations; however, addition of EHNA and ITU resulted in a synergistic effect on accumulation of ADO. Lipopolysaccharideinduced NO production was more effectively suppressed by exposure to ITU than to EHNA Conclusions and Clinical Relevance-Equine articular chondrocytes release ADO in response to the proinflammatory stimulus of bacterial LPS. Inhibition of the metabolism of ADO increases accumulation of extracellular ADO. Autocrine release of ADO from chondrocytes may play a role in the cellular response to tissue damage in arthritic conditions, and pharmacologic modulation of these pathways in joints of arthritic horses could be a potential method of therapy.

Objective-To compare plasma and synovial fluid endothelin-1 (ET-1) and nitric oxide (NO) concentrations in clinically normal horses and horses with joint disease. Animals-36 horses with joint disease, and 15 horses without joint disease. Procedure-Horses with joint disease were assigned to 1 of the 3 groups (ie, synovitis, degenerative joint disease [DJD], or joint sepsis groups) on the basis of findings on clinical and radiographic examination and synovial fluid analysis. Endothelin-1 and NO concentrations were measured in plasma from blood samples, collected from the jugular vein and ipsilateral cephalic or saphenous vein of the limb with an affected or unaffected joint, as well as in synovial fluid samples obtained via arthrocentesis from the involved joint. Results-Plasma ET-1 concentrations between affected and unaffected groups were not significantly different. Median concentration and concentration range of ET-1 in synovial fluid obtained from the joint sepsis group (35.830 pg/mL, 7.926 to 86.614 pg/mL; n = 7) were significantly greater than values from the synovitis (17.531 pg/mL, 0.01 to 46.908 pg/mL; 18), DJD (22.858 pg/mL, 0.01 to 49.990 pg/mL; 10), and unaffected (10.547 pg/mL, 0.01 to 35.927 pg/mL; 10) groups. Plasma and synovial fluid NO concentrations between affected and unaffected groups were not significantly different. Conclusions and Clinical Relevance-Endothelin-1 is locally synthesized in the joints of horses with various types of joint disease. Synovial fluid concentrations of ET-1 varied among horses with joint disease, with concentrations significantly higher in the synovial fluid of horses with joint sepsis. These results indicate that ET-1 may play a role in the pathophysiologic mechanism of joint disease in horses.