Inhibition of interleukin-1 activity by equine synovial fluid.

The presence, in equine synovial fluid, of inhibitors of interleukin-1 (IL-1) activity has been investigated by means of an assay involving IL-1-mediated production of PGE2 by synovial cells. Inhibitors of IL-1 alpha and IL-1 beta were identified in normal synovial fluid and synovial fluid from two horses with early joint disease. Inhibitors of IL-1 alpha were also present in synovial fluid from two horses with long-standing joint disease. However, IL-1 beta inhibitory activity was not present in fluid from the horses with more chronic joint disease. The effect appeared to be specific for IL-1, and not a direct action on PGE2 production, as synovial fluid had no effect on lipopolysaccharide-mediated PGE2 production. It is suggested that the inhibitory activity may be involved physiologically in the control of IL-1 activity in the joint, and the loss of IL-1 inhibition may be at least as important biologically as increased production of IL-1.     

Plasma and synovial fluid endothelin-1 and nitric oxide concentrations in horses with and without joint disease

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, 7926 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.     

The effect of implanting gentamicin-impregnated polymethylmethacrylate beads in the tarsocrural joint of the horse

OBJECTIVE: To determine the effect of intra-articular gentamicin-impregnated polymethylmethacrylate (PMMA) beads inserted in the equine tarsocrural joint on the synovial fluid, synovial lining, and cartilage, and to determine the peak and sustainable gentamicin concentrations in synovial fluid and plasma. STUDY DESIGN: Pharmacokinetic, cytologic, and histologic study of the effect of gentamicin-impregnated PMMA on normal equine tarsocrural joints. ANIMALS: Five healthy adult horses. METHODS: Gentamicin-impregnated PMMA bead strands (3 strands each of 40 beads, with each strand containing 100 mg gentamicin) were surgically inserted into one radiographically normal tarsocrural joint in 5 horses. Each horse had both joints flushed with 1 L of lactated Ringer's solution before bead administration. Synovial fluid total protein concentration, white blood cell (WBC) count, gentamicin concentration, synovial histology, cartilage integrity, and cartilage glycosaminoglycan (GAG) concentrations were determined. RESULTS: Gentamicin concentration (mean +/- SEM peak concentration, 27.9 +/- 2.27 microg/mL) occurred in the first 24 hours and remained above 2 microg/mL for 9 days. Gentamicin concentrations in control joints and the plasma remained below detectable levels. The synovial fluid WBC count for treated joints was increased compared with control joints for 72 hours, but was similar at day 6. The synovial protein concentration in gentamicin-treated joints remained increased for 21 days. Synovium in treated joints had diffuse synovitis, whereas control joints had less fibrovascular proliferation. Superficial cartilage erosion was present in all treated joints. There was no difference in the GAG content of treated and control joint cartilage. CONCLUSIONS: Short-term implantation of gentamicin (300 mg)-impregnated PMMA beads can provide therapeutic levels of gentamicin (>2 microg/mL) in the normal tarsocrural joint for 9 days; however, gentamicin-impregnated PMMA beads induce synovitis and superficial cartilage erosion. CLINICAL RELEVANCE: Temporary intra-articular administration of antibiotic-impregnated PMMA may be an effective way to treat septic joints that require constant high concentrations of antibiotics.     

Proinflammatory cytokine activities, matrix metalloproteinase-3 activity, and sulfated glycosaminoglycan content in synovial fluid of dogs with naturally acquired cranial cruciate ligament rupture

OBJECTIVE: To measure and compare activities of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and matrix metalloproteinase-3 (MMP-3); as well as sulfated glycosaminoglycan (S-GAG) content in synovial fluid from dogs with cranial cruciate ligament rupture (CCLR) and dogs with clinically normal stifles. To determine whether correlations exist between demographic and disease-related variables and these synovial markers. STUDY DESIGN: Prospective clinical study. ANIMALS: Dogs with CCLR (n=23) and Beagles with normal stifle joints (n=21). METHODS: Synovial fluid activities of proinflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) were determined by bioassay. MMP-3 activity was measured using fluorogenic substrate. S-GAG contents were determined by dimethylmethylene blue dye-binding assay. Mann-Whitney U-test was used to compare results from CCLR joints with normal controls. Spearman's rank correlation test was used to evaluate associations between demographic and disease-related markers and synovial markers. RESULTS: Mean values for synovial markers were significantly higher in CCLR joints compared with controls. IL-1beta and MMP-3 were positively correlated with lameness duration. CONCLUSIONS: Activities of proinflammatory cytokines, MMP-3 activity and S-GAG contents were significantly elevated in synovial fluid from canine stifle joints with naturally acquired CCLR. These results indicate that there is joint inflammation and increased release of GAGs into synovial fluid, suggesting that these inflammatory changes are associated with depletion of proteoglycan from articular cartilage. CLINICAL RELEVANCE: Medical and surgical treatments designed to decrease joint inflammation and breakdown of proteoglycans may be of value in the management of CCLR in the dog.     

Nitric oxide synthase activity in healthy and interleukin 1beta-exposed equine synovial membrane.

OBJECTIVE: To quantitate nitric oxide synthase (NOS) activity in healthy and interleukin 1beta (IL-1beta)-exposed equine synovial membrane. ANIMALS: 6 healthy horses, 2 to 8 years old. PROCEDURE: Recombinant human IL-1beta (0.35 ng/kg of body weight) was injected intra-articularly into 1 metacarpophalangeal joint of each horse. The contralateral joint served as an unexposed control. All horses were euthanatized 6 hours after injection of IL-1beta, and synovial membrane specimens were assayed for NOS activity by measuring conversion of arginine to citrulline. Severity of inflammation was semiquantitated by analysis of synovial fluids and histologic examination of synovial membrane. RESULTS: Equine synovial membrane had minimal NOS activity. A significant difference was not detected in NOS activity between control and IL-1beta-exposed specimens. Histologic examination revealed a neutrophilic infiltrate in synovial membrane exposed to IL-1beta. Synovial fluid from IL-1beta-exposed joints had a moderate inflammatory response and significantly greater concentrations of IL-1beta and interleukin-6 than fluid from healthy joints. CONCLUSION: Healthy equine synovial membrane had low NOS activity that was not affected by exposure to IL-1beta.     

Regional limb perfusion for antibiotic treatment of experimentally induced septic arthritis.

Septic arthritis was induced in one antebrachiocarpal joint of seven horses by the intra-articular injection of 1 mL Staphylococcus aureus suspension containing a mean of 10(5) colony-forming units. Twenty-four hours after inoculation, four horses were treated by regional perfusion with 1 g of gentamicin sulfate, and three horses received 2.2 mg/kg gentamicin sulfate intravenously (IV) every 6 hours. Synovial fluid was collected for culture and cytology at regular intervals, and the synovial membranes were collected for culture and histologic examination at euthanasia 24 hours after the first treatment. Gentamicin concentration in the septic synovial fluid after three successful perfusions was 221.2 +/- 71.4 (SD) micrograms/mL; after gentamicin IV, it was 7.6 +/- 1.6 (SD) micrograms/mL. The mean leukocyte count in the inoculated joints decreased significantly by hour 24 in the successfully perfused joints. Terminal bacterial cultures of synovial fluid and synovial membranes were negative in two horses with successfully perfused joints. S. aureus was isolated from the infected joints in all three horses treated with gentamicin IV.     

Synovial fluid D-dimer concentration in foals with septic joint disease

Background: Increased synovial fibrinolytic activity (detected by increases in synovial D‐Dimer concentrations) has been observed in different joint diseases in humans and adult horses, presumably in order to minimize fibrin deposition within the joint and thus avoid its detrimental effects. Objective: To investigate fibrinolytic pathway activation in joint sepsis in foals by measuring synovial D‐Dimer concentrations. Animals: Eighteen septic foals with septic joints, 9 septic foals without septic joints, 9 systemically healthy foals with septic joint, and 3 controls are included. Methods: Prospective observational clinical study of foals admitted for septic arthritis. Synovial D‐Dimer concentration and routine synovial fluid analysis were performed. Diagnosis of joint sepsis was made whenever synovial total nucleated cell count was >30,000 cells/μL, synovial total protein >4 g/dL, and neutrophil percentage of >80%, or synovial fluid culture resulted positive. Results were compared among groups by general lineal models. Results: Synovial D‐Dimer concentration was significantly (P < .001) higher in the foals with septic joints compared with foals without joint disease (P < .001). Conclusions and Clinical Importance: Septic joint disease is associated with a marked increase of synovial D‐Dimer concentration (marked activation of the fibrinolytic activity) within the affected joint. Although further studies are needed, the measurement of synovial D‐Dimer concentration may be considered a complementary diagnostic marker of septic joint disease.     

Absolute and relative cell counts for synovial fluid from clinically normal shoulder and stifle joints in cats

OBJECTIVE: To determine absolute and relative cell counts for synovial fluid from grossly, radiographically, and histologically normal shoulder and stifle joints in healthy cats. DESIGN: Clinical study. ANIMALS: 52 cats scheduled to be euthanatized for unrelated reasons. PROCEDURE: Arthrocentesis of the shoulder and stifle joints was performed bilaterally, and synovial fluid was analyzed for absolute WBC count, WBC morphology, and percentages of neutrophils and mononuclear cells. Joints were examined grossly and radiographically, and synovial membrane specimens were submitted for histologic examination. Synovial fluid samples that were contaminated with blood and samples from joints with any gross, radiographic, or histologic abnormalities were excluded. RESULTS: 82 of the 208 synovial fluid samples were excluded because abnormalities were identified during physical examination; the volume of fluid obtained was insufficient for analysis; there was evidence of blood contamination; or the joint had gross, radiographic, or histologic abnormalities. Median WBC count for the remaining 126 synovial fluid samples was 91 cells/microL (96.4% mononuclear cells and 3.6% neutrophils); WBC count was not significantly different between left and right joint samples or between shoulder and stifle joint samples. Body weight was associated with synovial fluid WBC count, with WBC count increasing as body weight increased. Sixteen of the 52 (30%) cats had radiographic evidence of osteoarthritis involving at least 1 joint. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that synovial fluid can be obtained reliably from shoulder and stifle joints in cats.     

Comparison of Synovial Fluid in Middle Carpal Joints Undergoing Needle Aspiration, Infusion with Saline, and Infusion with a Combination of N-Acetyl-d-Glucosamine, Hyaluronic Acid, and Sodium Chondroitin Sulfate

Synovial fluid white blood cell (WBC) count and total protein (TP) concentration were evaluated in the midcarpal joints of horses to not only determine the effects of needle aspiration, infusion with saline, and infusion with a combination of N-acetyl-d-glucosamine, hyaluronan, and sodium chondroitin sulfate (GHCS) at two different doses to evaluate the latter for safety, but to also provide information on saline injection as a control in joints. The midcarpal joints from 24 horses were used for this study. One midcarpal joint served as an untreated control, in which only synovial fluid was aspirated, whereas the opposite joint received either 2.5 mL isotonic saline (n = 8 horses), 2.5 mL of GHCS (n = 8 horses), or 7.5 mL of GHCS (n = 8 horses). Synovial fluid WBC and TP concentration were measured on days 1, 3, 5, 7, 14, and 21. Needle aspiration caused a transient increase in synovial fluid WBC and TP levels after 1 day. Instillation of fluid (2.5 mL), whether saline or GHCS, caused significantly higher WBC and TP concentrations. GHCS at a dose of 7.5 mL created an elevation in TP level for an additional 48 hours; however, after 48 hours, WBC and TP were at concentrations that were not statistically different from controls. Even though an increase in WBC and TP concentrations occurred because of intra-articular saline and GHCS administration, these results were transient demonstrating that GHCS is no different than saline on synovial fluid, WBC, and TP parameters and that as previously described short-term elevation in synovial fluid inflammatory parameters should be expected when saline is used as a control.     

Factors associated with prognosis for survival and athletic use in foals with septic arthritis: 93 cases (1987-1994).

OBJECTIVE: To identify factors affecting the prognosis for survival and athletic use in foals with septic arthritis. DESIGN: Retrospective study. ANIMALS: 93 foals with septic arthritis. PROCEDURE: Medical records were reviewed to obtain clinical findings, laboratory test results, radiographic findings, treatment method, and outcome. Race records for Thoroughbreds and Standardbreds were evaluated to determine whether foals subsequently raced and whether they raced successfully. RESULTS: 43 foals had 1 affected joint, 44 foals had multiple affected joints, and number of affected joints was not recorded for 6 foals. The femoropatellar and tarsocrural joints were most commonly affected. Osteomyelitis or degenerative joint disease were detected in 59% (46/78) of foals. Failure of passive transfer, pneumonia, and enteritis were common. Foals were treated with lavage, lavage and intra-articular administration of antibiotics, lavage and arthroscopic debridement with or without partial synovectomy, or lavage and arthrotomy to debride infected bone and systemic administration of antibiotics. Seventy-three foals survived to be discharged from hospital, and approximately a third raced. Isolation of Salmonella spp from synovial fluid was associated with an unfavorable prognosis for survival and multisystem disease was associated with an unfavorable prognosis for survival and ability to race; other variables were not significantly associated with survival and ability to race. CONCLUSIONS AND CLINICAL RELEVANCE: With treatment, the prognosis for survival of foals with septic arthritis was favorable, whereas prognosis for ability to race was unfavorable. Multisystem disease, isolation of Salmonella spp from synovial fluid, involvement of multiple joints, and synovial fluid neutrophil count > or = 95% at admission may be of prognostic value.     

Synovial fluid chondroitin sulphate indicates abnormal joint metabolism in asymptomatic osteochondritic horses

Reasons for performing study: Alternative methods to evaluate the joint condition in asymptomatic osteochondrosis dissecans (OCD) and other joint diseases may be useful. Objectives: To investigate possible changes in synovial fluid composition that may lead to joint conditions in asymptomatic OCD, in mature horses. Methods: Animals aged >2 years, of different breeds, with OCD in the intermediate ridge of distal tibia, symptomatic or not, were studied. Synovial fluid samples (10 healthy; 11 asymptomatic OCD; 25 symptomatic OCD) were collected by arthroscopy from 29 horses. Glycosaminoglycans (GAGs) were analysed by a combination of agarose gel electrophoresis and enzymatic degradation with specific GAG lyases. The viscosity, white blood cell (WBC) count, protein concentration and hyaluronic acid (HA) molecular weight were also determined. Results: The method used here to analyse synovial fluid GAGs is reliable, reproducible and specific. The main synovial fluid GAGs are HA and chondroitin sulphate (CS), 93% and 7% respectively in normal horses. In symptomatic OCD, the concentrations of both increased (expressed as GAG/urea ratios), but CS increased more. The CS increased also in asymptomatic OCD. An inflammatory reaction was suggested by the increased WBC counts in OCD. The molecular weight of the synovial fluid HA was reduced in OCD, explaining the lower viscosity observed. Conclusions: The increased CS in synovial fluid of OCD joints in mature horses suggests that the synovial fluid CS and the WBC count are good markers of the joint conditions, allowing the identification of pathological phase in joint diseases. Potential relevance: The analysis of synovial fluid GAGs shows that cartilage damage occurs even in asymptomatic OCD, implying that arthroscopic removal of osteochondral fragments should be performed even in asymptomatic OCD.     

Gentamicin concentrations in synovial fluid obtained from the tarsocrural joints of horses after implantation of gentamicin-impregnated collagen sponges

OBJECTIVE: To determine synovial fluid gentamicin concentrations and evaluate adverse effects on the synovial membrane and articular cartilage of tarsocrural joints after implantation of a gentamicin-impregnated collagen sponge. ANIMALS: 6 healthy adult mares. PROCEDURES: A purified bovine type I collagen sponge impregnated with 130 mg of gentamicin was implanted in the plantarolateral pouch of 1 tarsocrural joint of each horse, with the contralateral joint used as a sham-operated control joint. Gentamicin concentrations in synovial fluid and serum were determined for 120 hours after implantation by use of a fluorescence polarization immunoassay. Synovial membrane and cartilage specimens were collected 120 hours after implantation and evaluated histologically. RESULTS: Median peak synovial fluid gentamicin concentration of 168.9 microg/mL (range, 115.6 to 332 microg/mL) was achieved 3 hours after implantation. Synovial fluid gentamicin concentrations were < 4 microg/mL by 48 hours. Major histologic differences were not observed in the synovial membrane between control joints and joints implanted with gentamicin-impregnated sponges. Safranin-O fast green stain was not reduced in cartilage specimens obtained from treated joints, compared with those from control joints. CONCLUSIONS AND CLINICAL RELEVANCE: Implantation of a gentamicin-impregnated collagen sponge in the tarsocrural joint of horses resulted in rapid release of gentamicin, with peak concentrations > 20 times the minimum inhibitory concentration reported for common pathogens that infect horses. A rapid decrease in synovial fluid gentamicin concentrations was detected. The purified bovine type I collagen sponges did not elicit substantial inflammation in the synovial membrane or cause mechanical trauma to the articular cartilage.     

Effects of equine recombinant interleukin-1alpha and interleukin-1beta on proteoglycan metabolism and prostaglandin E2 synthesis in equine articular cartilage explants

OBJECTIVES: To evaluate the effects of equine recombinant interleukin-1alpha (rEqIL-1alpha) and recombinant interleukin-1beta (rEqIL-1beta) on proteoglycan metabolism and prostaglandin E2 (PGE2) synthesis by equine articular chondrocytes in explant culture. SAMPLE POPULATION: Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse. PROCEDURE: Expression constructs containing cDNA sequences encoding EqIL-1alpha and EqIL-1beta were generated, prokaryotically expressed, and the recombinant protein purified. Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse were separately randomized to receive rEqIL-1alpha or rEqIL-1beta treatments 10 to 500 ng/ml). Proteoglycan release was evaluated by 1,9-dimethylmethylene blue spectrophotometric analysis of explant media glycosaminoglycan (GAG) concentration and release of 35S-sulfate-labeled GAG to explant media. Proteoglycan synthesis was assessed by quantification of 35S-sulfate incorporation into proteoglycan. Explant media PGE2 concentrations were evaluated using a PGE2-specific enzyme-linked immunoassay. Data were collected at 48-hour intervals and normalized by DNA content. RESULTS: Proteoglycan release was induced by rEqIL-1alpha and rEqIL-1beta at concentrations > or =0.1 ng/ml, with 38 to 76% and 88 to 98% of total GAG released by 4 and 6 days, respectively. Inhibition of proteoglycan synthesis (42 to 64%) was observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days. Increased PGE2 concentrations were observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days. CONCLUSIONS AND CLINICAL RELEVANCE: The rEqIL-1 induced potent concentration-dependent derangement of equine chondrocyte metabolism in vitro. These findings suggest this model may be suitable for the in vitro study of the pathogenesis and treatment of joint disease in horses.     

Influence of repeated arthrocentesis and exercise on synovial fluid concentrations of nitric oxide, prostaglandin E2 and glycosaminoglycans in healthy equine joints

REASONS FOR PERFORMING STUDY: The importance of osteoarthritis (OA) in the horse and the difficulty in its early diagnosis have led to a search for potential biomarkers of joint disease. If the levels of such markers are to be interpreted accurately, clinicians and researchers need to know whether they are influenced by environmental factors and/or interventions such as exercise and repeated arthrocentesis. OBJECTIVE: To investigate the influence of repeated arthrocentesis and exercise on nitric oxide (NO), prostaglandin E2 (PGE2) and glycosaminoglycan (GAG) concentrations in synovial fluid (SF) from normal equine joints. METHODS: SF was collected from the left metacarpophalangeal (MCP), radiocarpal and tarsocrural joints of 16 horses. Half of the horses were exercised and arthrocentesis was repeated 14, 14.5, 17 and 24 days after the start of the exercise programme, in both exercised and control horses. Nitric oxide was determined in SF from the MCP joint only and PGE2 and GAG concentrations were determined in SF from all joints. RESULTS: Repeated arthrocentesis caused an increase in NO concentration in the MCP joint on Day 145, in PGE2 concentrations in the radiocarpal and tarsocrural joints on Day 145 and the release of GAGs into SF of the MCP and radiocarpal joints on Day 17. Exercise resulted in an increase in PGE2 levels in all joints but did not influence the other parameters measured. POTENTIAL RELEVANCE: Repeated arthrocentesis is a potential confounding factor for the use of synovial NO, PGE2 and GAG concentrations as markers of joint disease. Based on this study, such a confounding effect can be avoided if one week or more separates arthrocentesis procedures. Moderate exercise causes a transient rise in PGE2 in SF.     

Interleukin-6 and tumour necrosis factor in synovial fluid from horses with carpal joint pathology

The carpal joints are common sites of traumatic arthritis and osteoarthritis (OA) in athletic horses. The pro-inflammatory cytokines interleukin (IL)-6 and tumour necrosis factor (TNF) may be of great importance in the development of intra-articular lesions. The aim of the present study was to investigate possible associations between synovial fluid levels of bioactive IL-6 and TNF and different types of joint lesions seen in traumatic arthritis and OA. Synovial fluid was collected from horses with carpal lameness immediately before arthroscopic surgery. Articular cartilage, synovial membranes and intra-articular ligaments were assessed macroscopically at arthroscopy. Synovial fluid levels of IL-6 and TNF were determined by bioassays, and the cytokine levels between different grades of morphologic changes in each type of assessed tissue were compared. The highest levels of IL-6 were detected in joints with chip fractures. All joints with chip fractures also showed some degree of synovitis. Tumour necrosis factor bioactivity was low and not associated with any joint lesion. Hence, TNF is not useful as a biomarker indicating a specific joint lesion in equine traumatic arthritis or OA. We conclude that a dramatic increase of IL-6 in synovial fluid indicates the presence of osteochondral fragmentation, although low or undetectable levels of IL-6 do not exclude chip fractures. The role of IL-6 in the disease process of osteochondral fragmentation needs further investigation.     

Effects of proinflammatory cytokines on canine articular chondrocytes in a three-dimensional culture

OBJECTIVE: To determine the effects of interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha on canine chondrocytes cultured in an agarose-based 3-dimensional (3-D) system. SAMPLE POPULATION: Humeral head articular cartilage chondrocytes obtained from 6 adult dogs. PROCEDURE: Chondrocytes were cultured in a 3-D system for < or = 12 days in serum-free medium with IL 1alpha, IL-1beta, or TNF-alpha at concentrations of 20, 50, or 100 ng/mL. After 1, 3, 6, and 12 days, glycosaminoglycan (GAG) concentrations in 3-D constructs; nitric oxide and prostaglandin E2 (PGE2) concentrations in media samples; and relative expressions of selected genes, including metalloproteinase (MMP)-13 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, were evaluated. Control specimens were comprised of chondrocytes cultured without proinflammatory cytokines. RESULTS: In control 3-D constructs, GAG content was significantly higher than for all other constructs. Compared with control values, relative expressions of MMP-13, TIMP-1, and TIMP-2 genes in the IL-1beta (50 ng/mL) group were significantly higher at day 1; at all evaluations, media concentrations of nitric oxide were significantly higher in all TNF-alpha-treated cultures; and concentrations of PGE2 in media samples were significantly higher in the IL-1beta (50 ng/mL) and IL-1beta (100 ng/mL) groups at days 1 and 3, in the IL-1beta (100 ng/mL) group at day 6, and in all TNF-alpha groups at days 1, 3, and 6. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that TNF-alpha more readily induces production of nitric oxide and PGE2 by canine chondrocytes, compared with IL-1beta. In vitro, IL-1alpha appeared to have a minimal effect on canine chondrocytes.     

Direct and indirect markers of cartilage metabolism in synovial fluid obtained from dogs with hip dysplasia and correlation with clinical and radiographic variables

OBJECTIVE: To compare activities of interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF)-alpha, and matrix metalloproteinase (MMP)-3 and contents of sulfated glycosaminoglycan (S-GAG) in joint fluid obtained from dogs with hip dysplasia (HD) and clinically normal dogs, evaluate correlations among these markers in joint fluid obtained from dogs with HD, and evaluate correlations between each marker and clinical and radiographic variables. Animals-26 dogs with HD (clinical group) and 43 clinically normal Beagles (control group). PROCEDURE: Joint fluid was aseptically collected from the hip joints of all dogs. For each dog in the clinical group, age, duration of lameness, radiographic osteoarthritis (OA) score, and Norberg angle in each affected joint were recorded. Activities of IL-1beta, IL-6, TNF-alpha, and MMP-3 and S-GAG contents were measured. Values were compared between groups by use of Mann-Whitney U tests, and the Spearman rank correlation test was used to evaluate correlations among markers and between each marker and clinical or radiographic variables. RESULTS: Values of all markers were significantly higher for the clinical group, compared with values for the control group. There was a moderate positive correlation between lameness duration and IL-6 activity and a strong negative correlation between the Norberg angle and IL-1beta activity. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of our results indicated that there was a significant increase in markers of OA in dogs with HD. Activities of IL-1beta and IL-6 in joint fluid of dogs with HD may be influenced by the severity of laxity in the hip joint and lameness duration, respectively.