Twenty-four horses were randomly allocated to 3 groups. Horses were anesthetized, subjected to a ventral midline celiotomy, and the large colon was exteriorized and instrumented. Group-1 horses served as sham-operated controls. Group-2 horses were subjected to 6 hours of low-flow colonic arterial ischemia, and group-3 horses were subjected to 3 hours of ischemia and 3 hours of reperfusion. Baseline (BL) samples were collected, then low-flow ischemia was induced by reducing ventral colonic arterial blood flow to 20% of BL. All horses were monitored for 6 hours after BL data were collected. Blood samples were collected from the colonic vein and main pulmonary artery (systemic venous (SV) for measurement of plasma endotoxin, 6-keto prostaglandin F1alpha (6-kPG), thromboxane B2 (TXB2), and prostaglandin E2 (PGE2) concentrations. Tumor necrosis factor and interleukin-6 activities were measured in colonic venous (CV) serum samples. Data were analyzed, using two-was ANOVA, and post-hoc comparisons were made, using Dunnett's and Tukey's tests. Statistical significance was set at P < 0.05 Endotoxin was not detected in CV or SV plasma at any time. There was no detectable tumor necrosis factor or interleukin-6 activity in CV samples at any time. There were no differences at BL among groups for CV or SV 6-kPG, PGE2, or TXB2 concentrations, nor were there any changes across time in group-1 horses. Colonic venous 6-kPG concentration increased during ischemia in horses of groups 2 and 3; CV 6-kPG concentration peaked at 3 hours in group-3 horses, then decreased during reperfusion, but remained increased through 6 hours in group-2 horses. Systemic venous 6-kPG concentration increased during reperfusion in group-3 horses, but there were no changes in group-2 horses. Colonic venous PGE2 concentration increased during ischemia in horses of groups 2 and 3, and remained increased for the first hour of reperfusion in group-3 horses and for the 6-hour duration of ischemia in group-2 hors.

The ability of equine endometrium to release prostaglandin (GH) F, PGE2, and leukotriene (LT) B4 was studied in vitro, using endometrial tissue from diestrous mares. Because of the high cross-reactivity of the PGF antiserum with PGF1alpha and with PGF2alpha, results were quoted as total immunoreactive PGF. Significant concentrations of these arachidonate metabolites were released into tissue culture medium between 1 and 24 hours of incubation. Significantly higher concentrations of PGE, but not of PGE2 or LTB4, were released from endometria of mares with chronic endometritis than from genitally normal mares. Prostaglandin F was released only in low concentrations from the endometrium of a mare with pyometra, but concentrations of PGE2 and LTB4 were similar to those of genitally normal mares.

The metabolic responses of equine articular cartilage to incubation with bacterial lipopolysaccharide (LPS) were studied, using explant cultures of articular cartilage obtained from the metatarsophalangeal joints of 15 horses, age of which ranged from 3 months to 20 years. For comparison, explants were also established from the metatarsophalangeal joints of 3 calves. Explants were cultured for 3 days in medium containing various concentrations of LPS from 0 (control) to 100 microgram/ml. Glycosaminoglycan (GAG) released during the 3-day incubation was determined by a spectrophotometric assay, using the dye 1,9-dimethylmethylene blue. Newly synthesized GAG content was assayed by measuring [35S]sulfate incorporation during a 3-hour pulse labeling period. In addition, prostaglandin E2 (PGE2) synthesis was quantified, using a [3H]PGE2 radioimmunoassay kit and magnetic separation. Finally, explants from 3 animals were used to evaluate the effect of supplementing culture medium with 5% serum on the response of explants to LPS, and explants from 1 horse were used to compare responses to stimulation with LPS derived from 2 bacterial sources. Equine explants cultured with bacterial LPS had a dose-dependent decrease in synthesis and increase in release of GAG, and these responses were significantly (P < 0.0001) greater in explants from younger horses. In addition, equine explants had a significant (P = 0.0001) dose-dependent increase in concentration of PGE2 released into the culture medium in response to incubation with LPS. Comparison of data for GAG synthesis from equine and bovine explants revealed a significant (P = 0.025) difference in responsiveness to LPS between the 2 species. Equine explants tended to have a greater suppression of GAG synthesis in response to incubation with increasing concentrations of LPS than did age-corrected bovine samples.

A study was performed to determine the effect of proadifen hydrochloride on prostacyclin (prostaglandin I2 [PGI2]) and thromboxane A2 (TxA2) synthesis by equine peritoneal macrophages and the effect of proadifen on endotoxin-induced synthesis of PGI2 and TxA2 by equine macrophages. Peritoneal macrophages (2.5 X 10(6)/ml) were incubated for 6 hours in tissue culture media containing 1) nothing (nontreated control), 2) proadifen hydrochloride (20, 100, 250, and 500 micromol/L, 3) endotoxin (5 ng/ml), or 4) the calcium ionophore A23187 (0.95 micromol/L). In a second series of experiments, peritoneal macrophages were incubated with endotoxin (5 ng/ml) and proadifen (250 micromol/L), for 6 hours. Concentrations of 6-keto-prostaglandin F 1alpha (6-keto-PGF 1alpha) and thromboxane B2, the stable metabolites of PGI2 and TxA2, were determined in the incubation media by radioimmunoassay. Proadifen caused increased synthesis of PGI2 by equine macrophages, without affecting TxA2 production. The increased PGI2 production was similar to that induced by endotoxin and calcium ionophore; however, the latter 2 agents significantly stimulated TxA2 production as well (P less than 0.05). There were no significant differences among mean concentrations of 6-keto-PGF 1alpha in media from macrophages treated with 100, 250, or 500 micromol/L proadifen, but there was a significant curvilinear regression between their concentrations. The ratio of thromboxane B2 to 6-keto-PGF 1alpha was significantly lower than baseline in incubation media from macrophages exposed to proadifen, endotoxin, and calcium ionophore. Proadifen hydrochloride did not significantly change equine peritoneal macrophage production of PGI2 or TxA2 in response to endotoxin.

Four intrauterine treatment strategies were evaluated for effectiveness in mares that were confirmed to be susceptible to chronic uterine infection. Pretreatment samples were obtained at detection of estrus, and a genital strain of Streptococcus zooepidemicus was infused into the uterus when a preovulatory (> 35 mm) follicle was detected. At 12 hours after inoculation, mares were assigned to 1 of 4 selected treatment groups: autologous plasma, 100 ml (n = 5); potassium penicillin, 5 million U in 100 ml of phosphate-buffered saline solution (PBSS; n = 5); 10 mg of prostaglandin F2alpha in 100 ml of PBSS (n = 5); and large-volume lavage with normal saline solution (1,000 ml increments). A fifth group, treated with vehicle alone (100 ml of PBSS), served as a negative control (n = 7). All treatments were administered into the uterus. To assess the effectiveness of the treatment, samples for culture and cytologic examination were collected at 96 hours after bacterial inoculation. An effect of treatment was observed on the number of uterine neutrophils (P = 0.02) and growth of S zooepidemicus (P < 0.01). Intrauterine treatment with potassium penicillin, prostaglandin F2alpha, and large-volume uterine lavage significantly reduced the growth of S zooepidemicus (P < 0.01) as well as the number of neutrophils (P < 0.02). Autologous plasma reduced the number of neutrophils (P < 0.05), but not growth of S zooepidemicus. There was significant correlation between the number of uterine neutrophils and growth of S zooepidemicus for each treatment group (r = 0.57; P < 0.05).

Multicomponent nutraceuticals are becoming increasingly popular treatments or adjunctive therapies for osteoarthritis in veterinary medicine despite lack of evidence of efficacy for many products. The objective of this study was to evaluate the anti-inflammatory and antioxidant activities of a commercially available C-phycocyanin-based nutraceutical and select constituent ingredients in an in-vitro model of canine osteoarthritis. Normal canine articular chondrocytes were used in an in-vitro model of osteoarthritis. Inflammatory conditions were induced using interleukin-1β. The nutraceutical preparation as a whole, its individual constituents, as well as carprofen were evaluated at concentrations of 0 to 250 μg/mL for reduction of the following inflammatory mediators and indicators of catabolism of the extracellular matrix: prostaglandin E2 (PGE2), tumor necrosis factor-α (TFN-α), interleukin-6 (IL-6), metalloproteinase-3 (MMP-3), nitric oxide, and sulfated glycosaminoglycans (sGAGs). Validated, commercially available assay kits were used for quantitation of inflammatory mediators. The antioxidant capacities, as well as cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and lipoxygenase (LOX) inhibitory activities of the whole nutraceutical preparation and select constituents, were also assessed using validated commercially available assay kits. The antioxidant capacity of the nutraceutical and constituents was concentration-dependent. The nutraceutical and constituents appear to display anti-inflammatory activity primarily through the inhibition of COX-2. The nutraceutical displayed similar strength to carprofen in reducing TNF-α, IL-6, MMP-3, nitric oxide, and sGAGs at select concentration ranges. The C-phycocyanin (CPC)-based nutraceutical and constituents may be able to mediate 3 primary pathogenic mechanisms of osteoarthritis: inflammation, chondral degeneration, and oxidative stress in vitro. The nutraceutical may be clinically useful in veterinary medicine and its efficacy should be further investigated in vivo.

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.