• View in gallery
    Figure 1—

    Mean ± SEM PGE2 concentration in synovial fluid collected from intercarpal joints that were injected intra-articularly twice with recombinant human IL-1β (10 and 100 ng in 0.5 mL of saline [0.9% NaCl] solution on days 0 and 1, respectively) or an equivalent volume of saline (0.9% NaCl) solution in horses fed a control diet (CON;n=5)oranexperimental diet (EXP; 5) that contained a DN (15 g/d) beginning at day −14 and continuing for 29 days. In each horse, 1 intercarpal joint was injected with IL-1β, and the contralateral joint was injected with saline solution. Approximately 1.5 mL of synovial fluid was aspirated from each intercarpal joint at days −14, 0 (sample collected immediately before injection), 1 (sample collected immediately before injection), and 1.3 (sample collected 8 hours after injection) and at intervals thereafter. Data from horses that received the control diet (A), horses that received the experimental diet (B), and IL-1β–injected joints in horses in both diet treatment groups (C) were plotted. *Value differs significantly (P < 0.05) from the day 0 value in the joint treatment group or diet-joint treatment group. †At a given time point, value differs from the value in the contralateral joint in the joint treatment group or diet-joint treatment group. Inj = Injection.

  • View in gallery
    Figure 2—

    Mean ± SEM GAG concentration in synovial fluid collected from intercarpal joints of the horses in Figure 1. Horses were fed a control diet (CON;n=5)oranexperimental diet (EXP; 5) that contained a DN (15 g/d) beginning at day −14, and each intercarpal joint in each horse was injected intra-articularly twice with IL-1β (10 and 100 ng on days 0 and 1, respectively) or saline solution. Data from horses that received the control diet (A), horses that received the experimental diet (B), and IL-1β–injected joints in horses in both diet treatment groups (C) were plotted. See Figure 1 for key.

  • View in gallery
    Figure 3—

    Mean ± SEM protein concentration in synovial fluid collected from intercarpal joints of the horses in Figure 1. Horses were fed a control diet (CON;n=5)oranexperimental diet (EXP; 5) that contained a DN (15 g/d) beginning at day −14, and each intercarpal joint in each horse was injected intra-articularly twice with IL-1β (10 and 100 ng on days 0 and 1, respectively) or saline solution. Data from horses that received the control diet (A), horses that received the experimental diet (B), and IL-1β–injected joints in horses in both diet treatment groups (C) were plotted. See Figure 1 for key.

  • View in gallery
    Figure 4—

    Mean ± SEM intercarpal joint circumference in the horses in Figure 1. Horses were fed a control diet (CON; n = 5) or an experimental diet (EXP; 5) that contained a DN (15 g/d) beginning at day −14, and each intercarpal joint in each horse was injected intra-articularly twice with IL-1β (10and100ngondays0 and 1, respectively) or saline solution. Data from horses that received the control diet (A), horses that received the experimental diet (B), and IL-1β–injected joints in horses in both diet treatment groups (C) were plotted. See Figure 1 for key.

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Evaluation of inflammatory responses induced via intra-articular injection of interleukin-1 in horses receiving a dietary nutraceutical and assessment of the clinical effects of long-term nutraceutical administration

Wendy PearsonDepartment of Plant Agriculture, Ontario Agriculture College, University of Guelph, Guelph, ON N1G 2W1, Canada.

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Michael W. OrthDepartment of Animal Science, College of Agriculture and Natural Resources, Michigan State University, East Lansing, MI 48824.

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Michael I. LindingerDepartment of Human Health and Nutritional Sciences, College of Biological Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada.

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Abstract

Objective—To evaluate inflammatory responses induced via intra-articular recombinant human interleukin (IL)-1β treatment in horses receiving a dietary nutraceutical (DN; composed of mussel, shark cartilage, abalone, and Biota orientalis lipid extract) and assess the clinical effects of long-term DN administration.

Animals—22 healthy horses.

Procedures—12 horses were fed 0, 15, 45, or 75 mg of DN (3 horses/treatment) daily for 84 days. General health and clinicopathologic variables were monitored at intervals. Ten other horses received 0 or 15 g of DN/d (5 horses/treatment) for 29 days (beginning day −14). One intercarpal joint in each horse was injected twice with IL-1β (10 and 100 ng on days 0 and 1, respectively), and the contralateral joint was similarly injected with saline (0.9% NaCl) solution. Synovial fluid prostaglandin E2 (PGE2), sulfated glycosaminoglycan (GAG), nitric oxide (NO), and protein concentrations and leukocyte counts were analyzed before and at intervals after injections.

Results—Administration of the DN (up to 75 g/d) to horses for 84 days did not induce any adverse effects. In the other experiment, synovial fluid PGE2, GAG, and protein concentrations and leukocyte count increased after intra-articular injections of IL-1β (compared with effects of saline solution injections) in horses that received no DN; NO concentration was not affected. In horses that were fed the DN, intra-articular IL-1β injections did not induce significant increases in synovial fluid PGE2 and GAG concentrations.

Conclusions and Clinical Relevance—Results suggested that administration of the DN may be useful in preventing inflammation associated with arthritis and degenerative joint disease in horses.

Abstract

Objective—To evaluate inflammatory responses induced via intra-articular recombinant human interleukin (IL)-1β treatment in horses receiving a dietary nutraceutical (DN; composed of mussel, shark cartilage, abalone, and Biota orientalis lipid extract) and assess the clinical effects of long-term DN administration.

Animals—22 healthy horses.

Procedures—12 horses were fed 0, 15, 45, or 75 mg of DN (3 horses/treatment) daily for 84 days. General health and clinicopathologic variables were monitored at intervals. Ten other horses received 0 or 15 g of DN/d (5 horses/treatment) for 29 days (beginning day −14). One intercarpal joint in each horse was injected twice with IL-1β (10 and 100 ng on days 0 and 1, respectively), and the contralateral joint was similarly injected with saline (0.9% NaCl) solution. Synovial fluid prostaglandin E2 (PGE2), sulfated glycosaminoglycan (GAG), nitric oxide (NO), and protein concentrations and leukocyte counts were analyzed before and at intervals after injections.

Results—Administration of the DN (up to 75 g/d) to horses for 84 days did not induce any adverse effects. In the other experiment, synovial fluid PGE2, GAG, and protein concentrations and leukocyte count increased after intra-articular injections of IL-1β (compared with effects of saline solution injections) in horses that received no DN; NO concentration was not affected. In horses that were fed the DN, intra-articular IL-1β injections did not induce significant increases in synovial fluid PGE2 and GAG concentrations.

Conclusions and Clinical Relevance—Results suggested that administration of the DN may be useful in preventing inflammation associated with arthritis and degenerative joint disease in horses.

Nutraceuticals, particularly those based on glucosamine and chondroitin sulfate, are increasingly applied to long-term management of horses with joint disease.1 However, administrations of most of these products have not been evaluated in horses, thereby presenting a risk of nutraceutical-related toxic effects or displacement of conventional allopathic medications. A dietary nutraceuticala that is intended for use in horses with arthritis or inflamed joints and in horses that are at risk of developing such conditions has been produced. The product is a proprietary mixture of bioactive lipids obtained from NZGLM (Perna canaliculus), abalone (Haliotis sp), and SKC (Galorhinus galeus) and a lipid extract from BO.b All raw ingredients are manufactured in New Zealand according to Good Manufacturing Practices2 and Hazard Analysis and Critical Control Point standards.3 The 4 constituents of the DN powder have been artificially digested in vitro, and ultrafiltrate of each simulated digest has been evaluated by use of a cartilage explant model of inflammation.4,5 Each constituent exerted unique effects on the formation of recombinant human IL-1β–induced PGE2, GAG, and NO and on chondrocyte viability in equine cartilage explants.4,5 The PGE2-inhibitory effect of the DN (after it had undergone the simulated in vitro digestion procedure) was significantly greater than that of any of its 4 constituents alone and was not significantly different from the PGE2-inhibitory effect of a simulated digest of indomethacin.5 The DN also significantly reduced the concentration of IL-1–induced NO and increased chondrocyte viability. These data provide evidence that the DN may be effective in inhibiting inflammation induced by high IL-1 concentrations in articulating joints, a condition that would typically be encountered in horses with arthritis and joint disease.1,4,6

Bioactive constituents in the DN are thought to be omega-3 fatty acids (from NZGLM7 and abalone8), GAGs (from SKC9), proteins (from NZGLM10), and an unidentified phytochemical from BO.11 Provision of these individual dietary components has been associated with reduced clinical signs of arthritis,12,13 together with improvement in pathologic changes associated with the disease.14

To our knowledge, there are no reports of in vivo studies in which the effect of the DN on IL-1–induced changes in PGE2 and NO production in synovial fluid of horses was investigated. Also, although the simulated digest of DN powder in amounts up to 5 times the manufacturer's recommended dose did not adversely affect chondrocyte viability in vitro,5 the safety of this product on whole-body metabolism of horses is not known.

The objective of the study reported here was to evaluate inflammatory responses to intra-articular recombinant human IL-1β treatment in horses receiving the DN and assess the clinical effects of long-term DN administration. We hypothesized that the administration of DN at doses as much as 75 g/d (5 times the manufacturer's recommended dose) for 84 days would be safe in healthy horses, as determined via clinicopathologic analyses; that intra-articular injection of IL-1β into intercarpal joints of horses would increase concentrations of PGE2, GAG, NO, and protein and would induce neutrophilic leukocytosis in synovial fluid, compared with the effects of intra-articular injection of saline (0.9% NaCl) solution in the contralateral joints; and that the inflammatory responses to intra-articular injection of IL-1β would be significantly reduced after supplemental DN was added to the diet fed to horses (15 g/d) for 29 days.

Materials and Methods

The first experiment was conducted to comply with animal care committee requirements that the product first be determined safe for use in horses. This first experiment was conducted from January 2006 through April 2006. During that period, ambient temperatures ranged from −20° to 15°C.

Horses used in the first experiment—The first experiment was approved by the University of Guelph Animal Care Committee, and the horses were cared for in accordance with the guidelines of the Canadian Council on Animal Care. Twelve horses were allocated (randomized block) to 1 of 4 treatment groups (3 horses/group) and were administered 0, 15, 45, or 75 g of DN/d (which corresponded to 0, 1, 3, and 5 times the manufacturer's recommended dose). The horses were considered healthy on the basis of results of physical examination. Horses received approximately 0.5 kg of their normal grain ration twice daily and were offered grass-alfalfa hay, water, and trace minerals ad libitum (Appendix 1). They were turned out into small paddocks during the day and stabled indoors at night.

Preparation and administration of the DN powder—The DN powder was prepared in our laboratory by combining abalone, NZGLM, SKC, and BO lipid extract according to the proprietary composition provided by the manufacturer. The daily dose was divided into 2 equal amounts; 1 portion was added to the morning grain ration, and the other was added to the afternoon grain ration, each with approximately 2 tablespoons of molasses to ensure adequate mixing.

Procedures for the first experiment—One week prior to commencement of the experiment in the other horses, procedures were commenced in 1 horse that was fed DN in an amount equivalent to 5 times the manufacturer's recommended dose. The intent was to detect possible signs of toxicosis at this highest dose.

Administration of the DN treatments was commenced at day 1. All horses were monitored daily for general health, including subjective assessment of fecal consistency and measurements of heart rate, respiratory rate, and rectal temperature. Body mass (± 0.5 kg) was recorded weekly by use of a portable scale.c For each horse, clinicopathologic analyses were performed at intervals. A urine sample and 2 blood (approx 10 mL each) samples were obtained from the horse administered the highest dose of DN as a pilot investigation prior to beginning DN treatment and then every 7 days thereafter; such samples were obtained from the other 11 horses at days −7, 0 (before commencement of DN treatments), 42, and 84. Blood samples were collected with and without anticoagulant to provide whole blood and serum samples for analysis. Urine samples were collected by use of a clean plastic cup attached to the end of a 1-m-long handle. Urine and blood samples were kept chilled (at 4°C) until analyzed. All analyses were performed on the day of collection (within 5 hours) at the Animal Health Laboratory, University of Guelph.

Serum biochemical analyses—Analyses were performed by use of an automated biochemical analyzer.d Data collected from each serum sample included calcium, phosphorus, magnesium, sodium, potassium, chloride, albumin, globulin, urea, creatinine, glucose, cholesterol, total bilirubin, conjugated bilirubin, unconjugated bilirubin, and haptoglobin concentrations; sodium-to-potassium ratio and albumin-to-globulin ratio; alkaline phosphatase, γ-glutamyltransferase, aspartate aminotransferase, creatine kinase, and glutamyl dehydrogenase activities; and calculated osmolarity.

Hematologic analyses—Analyses were performed by use of an automated analyzer.e Data collected from each whole blood sample included counts of WBCs, RBCs, platelets, neutrophils, lymphocytes, monocytes, eosinophils, and basophils; Hct and hemoglobin concentration; and mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red-cell distribution width, and mean plasma volume.

Urinalysis—Data collected from each urine sample included color and clarity; specific gravity; pH; concentrations of protein, glucose, ketones, urine bile, blood, and urobilinogen; counts of leukocytes and RBCs; and presence of squamous epithelial cells, bacteria, calcium oxalate crystals, and calcium carbonate crystals.

Horses used in the second experiment—The second experiment was approved by the University of Guelph Animal Care Committee, and horses were cared for in accordance with the guidelines of the Canadian Council on Animal Care. Ten horses were used in the second experiment. The horses were considered healthy on the basis of results of physical examination. Prior to inclusion in the study, each horse appeared sound at the trot, and the result of a intercarpal joint flexion test was negative, as determined by the attending veterinarian who was experienced in lameness assessments. Each horse had no history of lameness (determined from detailed records that were collected as part of standard operating procedures of the University of Guelph) during the preceding 4-week period and had no detectable signs of trauma (eg, cuts, scrapes, heat, or effusion) in proximity to the intercarpal joints. All horses were turned out in small paddocks during the day and housed in box-stalls overnight. In the stalls, they were bedded on wood shavings and offered hay, water, and mineral salts ad libitum.

Rationale for the use of intra-articular injections of IL-1 to induce articular inflammation—Interleukin-1 is the prototypical catabolic cytokine, which has a pivotal role in pathogenesis of joint disease. Stimulation of equine cartilage explants with IL-1 results in increased GAG release and reduced synthesis of proteoglycan.15,16 Interleukin-1 induces the synthesis of all proteases involved in matrix catabolism by chondrocytes17–19 and stimulates synthesis of PGE217,20 and NO.21 Thus, it was hypothesized that intra-articular injections of exogenous IL-1 would stimulate production of PGE2 and NO in synovial fluid, as well as increase release of GAG from the cartilage matrix. For the study, recombinant human IL-1β was used. Although recombinant equine IL-1 would likely have induced an inflammatory response of greater magnitude at a lower dose response,22 recombinant human IL-1β was administered to allow comparison of results of this investigation and previous in vitro studies involving the DN product.4,5

Procedures for the second experiment—One of the 10 horses underwent the experimental procedures prior to the others to establish the pattern of change in synovial fluid PGE2 concentration and to confirm that the IL-1β injections would not result in clinical lameness, as determined in previous experiments performed by our group (data not shown). This horse was not treated with the DN, and the data collected were included in the control group data for analysis. Once the procedures were completed for this first horse, the study was continued; 4 horses (2 treated and 2 not treated with the DN) underwent the experimental procedures, and 2 weeks later, 5 additional horses (3 treated and 2 not treated with the DN) underwent the same procedures. The study was conducted in July through August 2006.

Diet treatments—A mixed ration containing the DN was prepared by addition of the DN powder (10 g/kg of feed), molasses (20 g/kg of feed), and flavoringf (1 g/kg of feed) to a sweet feed horse rationg (Appendix 2). The experimental ration was blended in a diet mixer in 5-kg batches until fully mixed. A control ration was prepared by blending the same sweet feed horse ration with molasses (20 g/kg of feed) and flavoring (1 g/kg of feed). The experimental ration was prepared so that 15 g of DN powder was provided to each horse daily. This amount was the dose recommended by the manufacturer; in vitro, this dose (albeit scaled down) diminished IL-1–induced increases in PGE2 and NO concentrations in cartilage explant preparations.5

Experimental design—Among the 10 horses in the second experiment, 5 were allocated to receive the experimental diet (ie, treatment with DN; 1.5 kg of feed/d), and 5 were allocated to receive the control diet (ie, no DN treatment; 1.5 kg of feed/d). Horses were fed their allocated diet for the entire duration of the experiment (29 days). Diet feeding was commenced 14 days prior to administration of an intra-articular injection of 10 ng of IL-1β (designated as day −14 and day 0, respectively). On day 1, each horse was administered a second injection of IL-1β (100 ng) in the same joint. Horses were monitored and evaluated at intervals until day 15. Samples of synovial fluid and jugular venous blood were collected from each horse on days −14, 0 (immediately prior to administration of IL-1β; baseline), 1 (immediately prior to administration of IL-1β), 1.3 (ie, 8 hours after the second IL-1β injection), 2, 4, 8, and 15.

Administration of intra-articular injections of IL-1B—Prior to injection on days 0 and 1, a lidocaineprilocaine anesthetic creamh was applied liberally to the left and right intercarpal joints of each horse. After approximately 20 minutes, the surface of each joint was aseptically prepared. Injections of 10 and 100 ng of recombinant human IL-1βi (in 0.5 mL of sterile saline solution) were administered into either the left or right intercarpal on days 0 and 1, respectively; the contralateral joint was injected with 0.5 mL of sterile saline solution. Because IL-1 is rapidly cleared from the joint capsule and does not stimulate endogenous IL-1 production,23 a double-injection procedure was used to ensure that the inflammatory response within the joint capsule was measurable but not associated with development of overt lameness.

Sample collection and monitoring during the second experiment—Synovial fluid samples (1.5 to 2 mL) were collected via aseptic arthrocentesis from IL-1β– and saline solution–injected joints at days −14, 0 (collected immediately before injection), 1 (collected immediately before injection), 1.3 (collected 8 hours after injection), 2, 4, 8, and 15. Samples were collected into evacuated tubes and placed on ice immediately after collection. Approximately 0.5 mL of synovial fluid was retained for cytologic examination; the remaining fluid was transferred to a microcentrifuge tube and centrifuged at 11,000 X g for 10 minutes at room temperature (approx 21°C) to remove cellular debris. Supernatant was transferred to another microcentrifuge tube containing 10 μg of indomethacin (to prevent possible further formation of PGE2 during storage of samples) and immediately frozen at −80°C until analyzed for PGE2, GAG, and NO.

In each horse, the circumference of each intercarpal joint was measured by use of a flexible measuring tape at each sample collection time point up to and including day 8; measurements were made prior to aseptic preparation of the injection or sample collection site. Lameness during trotting (without flexion test) was assessed by a qualified veterinarian immediately prior to administration of the IL-1β and saline solution injections on days 0 and 1 and immediately prior to preparation of the joints for sample collection at subsequent time points. Each horse was monitored for inflammatory reaction (ie, lameness during trotting and joint heat) to IL-1β or saline solution injection 4 times daily during the first 72 hours after the first intra-articular treatment and then once daily beginning 48 hours after the second intra-articular treatment.

Cytologic examination of synovial fluid—Samples of synovial fluid (approx 0.5 mL) underwent cytologic examination at the Animal Health Laboratory, University of Guelph. Assessments included total nucleated cell count,j cell differential (based on observation of 100 nucleated cells), and measurement of protein concentration (assay detection limit, 20 g of protein/L of synovial fluid).

Measurement of synovial fluid PGE2 concentration—Prostaglandin E2 was extracted from synovial fluid by use of lysis reagents provided as part of a commercially available PGE2 ELISA kitk to dissociate PGE2 from soluble membrane receptors and binding proteins. The concentration of PGE2 in synovial fluid was then quantified according to the manufacturer's protocol. In brief, each synovial fluid sample was thawed to room temperature and incubated with 20 μL of hyaluronidase (10 mg/mL) on a tube rocker for 30 minutes at 37°C to digest hyaluronic acid. The sample was then diluted (1:2 [vol/vol]) with 0.1% formic acid and centrifuged at 12,000 X g for 10 minutes.24 The supernatant was decanted and analyzed for PGE2 by use of the ELISA. Microtiter plates from the ELISA kit were coated with sheep anti-mouse PGE2 antibody. There are no known differences in arachidonic acid derivative structure between mice and horses; thus, the assay can be appropriately applied to equine samples.6,24 Plates were evaluated by use of a microplate readerl with absorbance set at 450 nm. A best-fit third-order polynomial standard curve was developed for each plate (R2 ≥ 0.99), and the equations derived from the regression analysis were used to calculate PGE2 concentrations in samples in each plate.

Measurement of synovial fluid GAG concentration—Hyaluronic acid in synovial fluid samples was digested with hyaluronidase as described for the PGE2 concentration measurements. Synovial fluid GAG concentration was determined by use of a 1,9-DMB spectrophotometric assay.25 This assay is specific for detection of sulfated GAGs and is not expected to interact with the unsulfated heparin salt that is used to coat the evacuated collection tubes. To prepare the DMB reagent, 1,9-DMB (4 mg) was dissolved in 100% ethanol (1.25 mL), combined with sodium formate (0.5 g) and formic acid (0.5 mL), and then made up to a volume of 250 mL with double-distilled water (pH 6.5). Samples were diluted 1:3 (vol/vol) with dilution buffer (4.1 mg of sodium acetate and 0.5 μL of Tween 20/mL of double-distilled H2O) and placed into a 96-well microtiter plate. Guanidine hydrochloride (275 g/L of double-distilled H2O) was added to each well, followed immediately by addition of 150 μL of the DMB reagent. Plates were incubated in the dark for 10 minutes, and absorbance was assessed by use of a microplate readerl at 530 nm. Absorbance of each sample was compared with that of a bovine chondroitin sulfate standard. A best-fit linear standard curve was generated for each plate (R2 ≥ 0.99), and the equations derived from the regression analysis were used to calculate GAG concentrations in samples in each plate.

Measurement of synovial fluid NO concentration—Nitrite (NO2−), a stable oxidation product of NO, was analyzed by use of the Griess reaction.26 Undiluted synovial fluid samples were added to 96-well plates. Sulfanilamide (0.01 g/mL) and N-(1)-napthylethylene diamine hydrochloride (1 mg/mL) dissolved in phosphoric acid (0.085 g/L) was added to all wells. Absorbance was assessed within 5 minutes by use of a microplate readerl at 530 nm. Absorbance of each sample was compared with that of a sodium nitrite standard.

Statistical analyses—Data are presented as mean ± SEM. For the first experiment, data were analyzed by use of a 2-way repeated-measures ANOVA (with respect to time and diet). When a significant F ratio was obtained, the Holm-Sidak post hoc test was used to identify significantly different means. For the second experiment, data were analyzed by use of a 2-way repeated-measures ANOVA (with respect to time and intra-articular treatment) to assess changes from baseline values in IL-1β– and saline solution–treated joints and detect differences between those treated joints within each diet group. Data were collapsed together on the basis of diet treatment (ie, n = 10), and a 3-way ANOVA (with respect to diet, intra-articular treatment, and time) was used to detect the effect of each independent variable on dependent variables. As for the first experiment analyses, when a significant F ratio was obtained, the Holm-Sidak post hoc test was used to identify significantly different means. Analyses were performed by use of computer softwarem; for all comparisons, differences were considered significant at a value of P < 0.05.

Results

Experiment to assess the safety of long-term DN administration in horses—In the first experiment, 4 groups of 3 healthy horses were fed 0, 15, 45, or 75 g of DN/d beginning day 1 for 84 days. Breeds represented among the 12 horses included 3 Thoroughbreds, 3 Canadian Horses, and 1 each of 6 other breeds (Percheron cross, Quarter Horse, Standardbred, Standardbred-Appaloosa cross, warmblood cross, and Welsh cross). There were 5 stallions and 7 geldings aged from 2.5 to 24 years (mean age, 8.6 years). Each treatment group contained stallions and geldings and no horses of the same age (minimum age difference among horses in a group, 2.5 years). No adverse events were observed at any time in the horse that was used in the initial pilot investigation.

Effects of DN administration on aspects of general health—During the 84-day experimental period, there were no significant changes from day 1 in body mass, heart rate, respiratory rate, or rectal temperature in the horses that received 15, 45, or 75 g of DN/d. No visual changes in fecal consistency were detected. With the exception of 1 horse that was kicked in the head by another horse, all horses remained healthy throughout the experimental period.

Effects of DN administration on serum biochemical variables—All serum biochemical variables remained within reference intervals throughout the 84-day experimental period in all DN treatment groups. Significant differences in anion gap; osmolality; cholesterol, total bilirubin, and free bilirubin concentrations; and alkaline phosphatase activity were detected among groups. However, there was no effect of time on these variables; thus, the changes were considered unrelated to treatment.

Effects of DN administration on hematologic variables—All hematologic variables remained within reference intervals throughout the 84-day experimental period in all DN treatment groups. Significant differences in WBC and lymphocyte counts, hemoglobin concentration, mean corpuscular volume, and red cell distribution width were detected among groups. However, there was no effect of time on these variables; thus, the changes were considered unrelated to treatment.

Effects of DN administration on urine variables—Urine samples could not be collected at all time points from all horses because of technical difficulties. Thus, statistical analysis was not applied to these data. All variables that were assessed in successfully collected samples were within reference intervals.

Experiment to assess the effect of DN administration on inflammatory responses to inter-articular injection of IL-1β in horses—In the second experiment, 10 geldings from a research herd were used. Among the horses, there were 3 Thoroughbreds and 7 Standardbreds; their ages ranged from 5 to 12 years (mean age, 7 years).

The horses were randomly allocated to receive a diet that did or did not contain the DN (experimental and control diets, respectively) for 29 days (5 horses/group); feedings were commenced at day −14, and intra-articular injections were administered at days 0 and 1. In each horse, one intercarpal joint was injected with IL-1β and the contralateral joint was injected with saline solution on each of the 2 days; synovial fluid samples from the injected joints and blood samples were collected at days −14, 0, 1, 1.3, 2, 4, 8, and 15.

Effect of intra-articular injection of IL-1 or saline solution on synovial fluid PGE2 concentration within each diet group—In the horses that received the control diet, there was no significant change in synovial fluid PGE2 concentration in saline solution–injected joints at any time, compared with the day 0 value (Figure 1). In IL-1β–injected joints, synovial fluid PGE2 concentration was significantly (P = 0.04) increased at day 1.3, compared with the value at day 0 (498.0 ± 250.8 pg/mL vs 49.4 ± 32.3 pg/mL). Synovial fluid PGE2 concentration was significantly (P < 0.001) higher in IL-1β–injected joints than in saline solution–injected joints at day 1.3 in this group of horses.

Figure 1—
Figure 1—

Mean ± SEM PGE2 concentration in synovial fluid collected from intercarpal joints that were injected intra-articularly twice with recombinant human IL-1β (10 and 100 ng in 0.5 mL of saline [0.9% NaCl] solution on days 0 and 1, respectively) or an equivalent volume of saline (0.9% NaCl) solution in horses fed a control diet (CON;n=5)oranexperimental diet (EXP; 5) that contained a DN (15 g/d) beginning at day −14 and continuing for 29 days. In each horse, 1 intercarpal joint was injected with IL-1β, and the contralateral joint was injected with saline solution. Approximately 1.5 mL of synovial fluid was aspirated from each intercarpal joint at days −14, 0 (sample collected immediately before injection), 1 (sample collected immediately before injection), and 1.3 (sample collected 8 hours after injection) and at intervals thereafter. Data from horses that received the control diet (A), horses that received the experimental diet (B), and IL-1β–injected joints in horses in both diet treatment groups (C) were plotted. *Value differs significantly (P < 0.05) from the day 0 value in the joint treatment group or diet-joint treatment group. †At a given time point, value differs from the value in the contralateral joint in the joint treatment group or diet-joint treatment group. Inj = Injection.

Citation: American Journal of Veterinary Research 70, 7; 10.2460/ajvr.70.7.848

In the horses that received the experimental diet, there was no change in synovial fluid PGE2 concentration in saline solution–injected joints at any time point, compared with the day 0 value (Figure 1). Similarly, there was no significant change in synovial fluid PGE2 concentration in IL-1β–injected joints at any time point, compared with the day 0 value. There was no difference (P = 0.98) in synovial fluid PGE2 concentration between IL-1β– and saline-injected joints at any time point in these horses. Overall, no interaction (P = 0.52) between either intra-articular treatment and time was detected.

On day 1.3, synovial fluid PGE2 concentration was significantly (P < 0.001) lower (by 45.4%) in IL-1β–injected joints in horses that received the experimental diet, compared with those joints in horses that received the control diet (271.9 ± 138.2 pg/mL and 498.0 ± 250.8 pg/mL, respectively; Figure 1). When data from horses within each diet treatment were collapsed together (ie, data from 10 joints in 5 horses/diet), intraarticular injection of IL-1β resulted in significantly (P = 0.02) higher synovial fluid PGE2 concentration than did injection of sterile saline solution. The change in synovial fluid PGE2 was significant (P < 0.001) on day 1.3, compared with day 0.

Effect of intra-articular injection of IL-1 or saline solution on synovial fluid GAG concentration within each diet group—In the horses receiving the control diet, synovial fluid GAG concentration in saline solution–injected joints was increased (P < 0.05) at day 2, compared with the day 0 value (48.1 ± 9.6 μg/mL and 18.3 ± 6.8 μg/mL, respectively; Figure 2). The first intra-articular injection of IL-1β (10 ng) caused a marked increase (P < 0.05) in synovial fluid GAG concentration in this group at day 1 (77.6 ± 4.4 μg/mL), compared with the preinjection day 0 value (24.5 ± 7.3 μg/mL). Synovial fluid GAG concentration remained increased (P < 0.05) in IL-1β–injected joints at day 1.3 (66.0 ± 9.6 μg/mL), compared with the day 0 value. The magnitudes of change (from day 0 value) in synovial fluid GAG concentration at days 1 and 1.3 were greater (P = 0.003) in IL-1β–injected joints than in saline solution– injected joints in horses receiving the control diet.

Figure 2—
Figure 2—

Mean ± SEM GAG concentration in synovial fluid collected from intercarpal joints of the horses in Figure 1. Horses were fed a control diet (CON;n=5)oranexperimental diet (EXP; 5) that contained a DN (15 g/d) beginning at day −14, and each intercarpal joint in each horse was injected intra-articularly twice with IL-1β (10 and 100 ng on days 0 and 1, respectively) or saline solution. Data from horses that received the control diet (A), horses that received the experimental diet (B), and IL-1β–injected joints in horses in both diet treatment groups (C) were plotted. See Figure 1 for key.

Citation: American Journal of Veterinary Research 70, 7; 10.2460/ajvr.70.7.848

In horses receiving the experimental diet, synovial fluid GAG concentration increased from day −14 to day 0 (prior to intra-articular injection of saline solution or IL-1β), although these increases were not significant (P = 0.09; Figure 2). During this interval, values in the joints assigned to receive intra-articular injections of saline solution or IL-1β changed from 29.3 ± 5.9 μg/mL and 27.0 ± 10.8 μg/mL, respectively, to 85.5 ± 28.0 μg/mL and 83.2 ± 27.9 μg/mL, respectively. In this group of horses, consumption of the DN-supplemented diet resulted in a sustained (albeit not significant) increase in synovial fluid GAG concentration in both joint treatment groups at days 1 through 15. In contrast to findings in the horses that received the control diet, synovial fluid GAG concentration did not increase significantly in IL-1β–injected joints (compared with the day 0 value) and did not differ between IL-1β– and saline solution–injected joints in horses that received the experimental diet. Concentrations of GAG in synovial fluid samples collected from IL-1β–injected joints in horses that received the control or experimental diet did not differ significantly except at the day 15 time point.

When data from horses were collapsed together (ie, data from 10 joints in 5 horses/diet), synovial fluid GAG concentration was increased (P = 0.03) in horses that received the experimental diet (compared with findings in horses that received the control diet). Compared with the value at day −14, synovial fluid GAG concentration in horses that received the experimental diet was significantly increased (P < 0.001) at all time points except day 15 (P = 0.1).

Effect of intra-articular injection of IL-1 or saline solution on synovial fluid NO concentration within each diet group—In the horses receiving the control diet, synovial fluid NO concentration was low and variable over the study period in both the IL-1β– and saline solution–injected joints, and there was no difference in this variable between IL-1β– and saline solution–injected joints at any time point (Table 1).

Table 1—

Mean ± SEM NO concentration (μg/mL) in synovial fluid collected from intercarpal joints that were injected intra-articularly twice with recombinant human IL-1β (10 and 100 ng in 0.5 mL of saline [0.9% NaCl] solution on days 0 and 1, respectively) or an equivalent volume of saline (0.9% NaCl) solution in horses fed a control diet (n = 5) or an experimental diet (5) that contained a DN (15 g/d) beginning at day −14 and continuing for 29 days. In each horse, 1 intercarpal joint was injected with IL-1β and the contralateral joint was injected with saline solution. Approximately 1.5 mL of synovial fluid was aspirated from each intercarpal joint at days −14, 0 (sample collected immediately before injection), 1 (sample collected immediately before injection), and 1.3 (sample collected 8 hours after injection) and at intervals thereafter.

DayControl dietExperimental diet
IL-1β– injected jointsSaline solution– injected jointsIL-1β– injected jointsSaline solution– injected joints
−140.61 ± 0.130.52 ± 0.260.19 ± 0.060.49 ± 0.24
00.19 ± 0.010.35 ± 0.130.27 ± 0.080.63 ± 0.39
10.80 ± 0.310.33 ± 0.080.39 ± 0.060.46 ± 0.16
1.30.77 ± 0.260.48 ± 0.130.97 ± 0.15*1.22 ± 0.44*
20.53 ± 0.200.86 ± 0.200.79 ± 0.20*1.33 ± 0.37*
40.64 ± 0.380.27 ± 0.020.66 ± 0.161.09 ± 0.56
80.61 ± 0.420.17 ± 0.020.37 ± 0.240.29 ± 0.12

Within a diet group, value differs significantly (P < 0.05) from the day 0 value.

In the horses receiving the experimental diet, synovial fluid NO concentration increased significantly at days 1.3 (P = 0.003) and 2 (P = 0.002) in both the saline solution– and IL-1β–injected joints, compared with the respective day 0 values. However, there was no difference in synovial fluid NO concentration between IL-1β– and saline solution–injected joints at any time point. Overall, there was no effect of diet (P = 0.12) or intra-articular treatment (P = 0.48) on synovial fluid NO concentration.

Effect of intra-articular injection of IL-1 or saline solution on cytologic findings and protein concentration in synovial fluid within each diet group—In the horses receiving the control diet, the first intra-articular injection of IL-1β (10 ng) resulted in a significant (P < 0.05) increase in total cell count at day 1, compared with the day 0 value (40.16 ± 16.1 × 109 cells/L and 0.58 ± 0.16 × 109 cells/L, respectively; Table 2). Cell count was not further increased following the second IL-1β injection (100 ng); values at days 1.3 and 2 were less than half the day 1 peak value and were only slightly greater than the day 0 value for the remainder of the experiment. Other than the day 1 value, findings were not significantly different from the day 0 value. In saline solution–injected joints in horses receiving the control diet, the day 0 cell count was 0.27 ± 0.07 × 109 cells/L. A peak value of 5.98 ± 2.61 × 109 cells/L was detected on day 2; compared with the day 0 value, this difference was not significant. Total cell counts in IL-1β– and saline solution–injected joints were significantly different on day 1.

Table 2—

Mean ± SEM total cell count and percentage of neutrophils in synovial fluid collected from intercarpal joints that were injected intra-articularly twice with recombinant human IL-1β (10 and 100 ng on days 0 and 1, respectively) or saline solution in horses fed a control diet (n = 5) or an experimental diet (5) that contained a DN (15 g/d) beginning at day −14. In each horse, 1 intercarpal joint was injected with IL-1β and the contralateral joint was injected with saline solution. Approximately 1.5 mL of synovial fluid was aspirated from each intercarpal joint at days −14, 0 (sample collected immediately before injection), 1 (sample collected immediately before injection), and 1.3 (sample collected 8 hours after injection) and at intervals thereafter.

VariableDayControl dietExperimental diet
IL-1β–injected jointsSaline solution–injected jointsIL-1β–injected jointsSaline solution–injected joints
Total cell count (× 109 cells/L)−140.61 ± 0.110.58 ± 0.200.37 ± 0.040.41 ± 0.08
00.58 ± 0.160.27 ± 0.070.45 ± 0.090.39 ± 0.11
140.16 ± 16.11*3.98 ± 2.2327.49 ± 8.66*2.96 ± 1.38
1.313.00 ± 5.425.62 ± 3.1523.56 ± 9.984.03 ± 2.65
214.02 ± 7.805.98 ± 2.6119.30 ± 10.312.21 ± 0.51
41.80 ± 1.700.85 ± 0.551.47 ± 0.781.27 ± 0.26
80.72 ± 0.120.66 ± 0.220.58 ± 0.060.56 ± 0.11
151.30 ± 0.880.64 ± 0.190.65 ± 0.190.53 ± 0.12
Percentage of neutrophils−141.0 ± 0.040.0 ± 0.021.0 ± 12.020.0 ± 12.0
027.4 ± 13.38.0 ± 0.026.8 ± 12.715.3 ± 13.9
192.4 ± 2.8*81.0 ± 8.2*88.5 ± 3.5*74.0 ± 14.7*
1.387.0 ± 2.579.8 ± 9.877.2 ± 5.970.8 ± 10.0
281.0 ± 5.971.8 ± 14.283.6 ± 7.553.3 ± 10.9
410.0 ± 0.012.0 ± 2.018.7 ± 8.33.7 ± 0.9
815.0 ± 12.43.0 ± 1.552.4 ± 7.7*69.2 ± 23.2*
154.0 ± 0.48.0 ± 4.036.7 ± 20.58.3 ± 5.7

Within a diet group, value at this time point differs from the value in the IL-1β–injected joints.

See Table 1 for remainder of key.

Increases in cell counts in synovial fluid of both IL-1β– and saline solution–injected joints in horses receiving the control diet were largely attributable to changes in the percentages of neutrophils. Peak percentages of neutrophils in synovial fluid were detected in IL-1β– and saline solution–injected joints at day 1 (92.4 ± 2.8% and 81.0 ± 8.2%, respectively); these peak values were significantly different from findings at day 0. During the remainder of the experiment, neutrophil counts declined from the peak values in both IL-1β– and saline-injected joints. There was no significant difference in percentage of neutrophils in synovial fluid between IL-1β– and saline-injected joints at any time point.

In the horses receiving the control diet, synovial fluid protein concentration increased (P < 0.05) following the first intra-articular injection of IL-1β (10 ng); at day 0, the value was 20 ± 0.0 g/L (detection limit of the analyzer), whereas at day 1, the value was 39.4 ± 4.0 g/L (Figure 3). Synovial fluid protein concentration was not further increased following the second intraarticular injection of IL-1β (100 ng); at day 2 (24 hours after the 100 ng injection), the value had declined significantly (P < 0.05) from the peak day 1 value but remained significantly (P < 0.05) greater than the day 0 value. Thereafter, values continued to decrease and were not significantly different from the day 0 value. Intra-articular injection of saline solution also resulted in increased (P < 0.05) synovial fluid protein concentration at days 1 and 1.3; the value had essentially returned to day 0 concentrations by day 2 (25.5 ± 1.5 g/L; baseline value was 20 ± 0.0 g/L [detection limit of the analyzer]). At day 1, values in the 2 joint treatment groups differed significantly. The magnitudes of change (from the day 0 value) in protein concentration of synovial fluid during the experiment were greater (P = 0.01) in IL-1β–injected joints than in saline solution–injected joints in horses receiving the control diet.

Figure 3—
Figure 3—

Mean ± SEM protein concentration in synovial fluid collected from intercarpal joints of the horses in Figure 1. Horses were fed a control diet (CON;n=5)oranexperimental diet (EXP; 5) that contained a DN (15 g/d) beginning at day −14, and each intercarpal joint in each horse was injected intra-articularly twice with IL-1β (10 and 100 ng on days 0 and 1, respectively) or saline solution. Data from horses that received the control diet (A), horses that received the experimental diet (B), and IL-1β–injected joints in horses in both diet treatment groups (C) were plotted. See Figure 1 for key.

Citation: American Journal of Veterinary Research 70, 7; 10.2460/ajvr.70.7.848

In the horses receiving the experimental diet, the first intra-articular injection of IL-1β (10 ng) resulted in a significant (P < 0.05) increase in total cell count at day 1, compared with the day 0 value (27.49 ± 8.66 × 109 cells/L and 0.45 ± 0.09 × 109 cells/L, respectively; Table 2). Cell count was not further increased following the second IL-1β injection (100 ng). Other than the day 1 value, findings in IL-1β–injected joints were not significantly different from the day 0 value. In saline solution–injected joints, the day 0 cell count was 0.39 ± 0.11 × 109 cells/L. A peak value of 4.03 ± 2.65 × 109 cells/L was detected on day 1.3, although the difference was not significant (P = 0.24); cell counts progressively decreased thereafter. Compared with findings in saline solution–injected joints, total cell counts were significantly higher in IL-1–injected joints on days 1, 1.3, and 2.

Peak percentages of neutrophils in synovial fluid were detected in IL-1β– and saline solution–injected joints of horses that received the experimental diet at day 1 (88.5 ± 3.5% and 74.0 ± 14.7%, respectively); these peak values were significantly different from findings at day 0 (Table 2). Subsequently, values declined markedly in both IL-1β– and saline-injected joints until day 8, when dramatically increased values were detected. Percentages of neutrophils IL-1β– and saline-injected joints at day 8 (52.4 ± 7.7% and 69.2 ± 23.2%, respectively) were not as great as the values at day 1; however, day 8 values were also significantly different from the day 0 values in each joint treatment group. In horses receiving the experimental diet, there was no significant difference in percentage of neutrophils in synovial fluid between IL-1β– and saline-injected joints at any time point.

In the horses receiving the experimental diet, synovial fluid protein concentration increased (P < 0.05) following the first intra-articular injection of IL-1β (10 ng); at day 0, the value was 20 ± 0.0 g/L (detection limit of the analyzer), whereas at day 1, the value was 38.7 ± 4.9 g/L (Figure 4). Synovial fluid protein concentration was not further increased following the second intra-articular injection of IL-1β (100 ng). At days 1.3 and 2, the value was 36.2 ± 4.4 g/L and 27.8 ± 3.8 g/L, respectively; these values were also significantly different from the day 0 value. Thereafter, values continued to decrease and were not significantly different from the day 0 value. In contrast to findings in the horses that received the control diet, intra-articular injections of saline solution had no significant effect on synovial fluid protein concentration in horses that received the experimental diet. Synovial fluid protein concentration was greater in IL-1β–injected joints than in saline solution–injected joints in horses that received the experimental diet at day 1 (P = 0.002) and at day 1.3 (P = 0.004); there were no time-dependent differences in this variable between dietary groups.

Figure 4—
Figure 4—

Mean ± SEM intercarpal joint circumference in the horses in Figure 1. Horses were fed a control diet (CON; n = 5) or an experimental diet (EXP; 5) that contained a DN (15 g/d) beginning at day −14, and each intercarpal joint in each horse was injected intra-articularly twice with IL-1β (10and100ngondays0 and 1, respectively) or saline solution. Data from horses that received the control diet (A), horses that received the experimental diet (B), and IL-1β–injected joints in horses in both diet treatment groups (C) were plotted. See Figure 1 for key.

Citation: American Journal of Veterinary Research 70, 7; 10.2460/ajvr.70.7.848

With regard to findings in IL-1β–injected joints, synovial fluid total cells counts and percentages of neutrophils did not differ between horses that received the experimental diet and horses that received the control diet. Similarly, synovial fluid protein concentration in IL-1β–injected joints of horses that were and were not administered the DN did not differ at any time point (Figure 3).

Effect of intra-articular injection of IL-1 or saline solution on joint circumference within each diet group—In the horses receiving the control diet, there was no significant effect of intra-articular injection of IL-1β or saline solution on joint circumference. Furthermore, there was no significant difference in joint circumference between IL-1β– and saline solution– injected joints at any time point (Figure 4).

In the horses receiving the experimental diet, there was a significant decrease in joint circumference in IL-1β–injected joints between day 1 (31.9 ± 0.6 cm) and day 4 (30.6 ± 0.3 cm) and between day 1 and day 8 (30.7 ± 0.2 cm; Figure 4). The same pattern was evident in the saline solution–injected joints. However, changes in joints undergoing either treatment were not significant, compared with day 0 values; also, there was no significant difference in joint circumference between IL-1β– and saline solution–injected joints at any time point in horses receiving the experimental diet. With regard to the IL-1β–injected joints, joint circumference was significantly (P < 0.001) lower in horses receiving the experimental diet, compared with findings in horses receiving the control diet.

Discussion

In a previous in vitro study5 involving the DN, a simulated digest of the product significantly inhibited the inflammatory response of equine cartilage explants to exogenous IL-1. To correlate those in vitro data with in vivo data, IL-1β was injected into intercarpal joints of horses to simulate an inflammatory challenge in the present study. Our intention was to induce a self-limiting inflammation that altered the biochemical profile of synovial fluid in a manner comparable to a mild, naturally occurring inflammatory response. It was anticipated that this experimental method would simulate conditions that are typical in joints of horses considered suitable candidates for treatment with nutraceuticals.1,27 Intra-articular injection of IL-1β (175 ng) has been used successfully to induce inflammatory responses in joints of horses and caused increased synovial fluid concentrations of substance P, IL-6, and PGE2 in anesthetized horses23 and of IL-6 in standing horses.28 Because of the rapid clearance of intra-articularly injected IL-1β from equine joints and the absence of a stimulation of endogenous IL-1 formation,23 we adopted a double-injection protocol involving administration of low doses of IL-1β to induce a mild intercarpal inflammation without concurrent lameness in horses. The main findings of the present study were that intra-articular injection of recombinant human IL-1β in horses resulted in a self-limiting inflammatory response in the joint within 24 to 48 hours and that the PGE2 component of this effect was attenuated by provision of the DN.

In the horses that received the control diet in the present study, intra-articular injection of IL-1β resulted in increased synovial fluid PGE2 concentration (at day 1.3) and marked neutrophilic leukocytosis (at day 1), compared with the effects of intra-articular injection of saline solution. These findings were consistent with the effect of intra-articular injection of IL-1β in isolated, innervated equine joints.23 The increase in synovial fluid GAG concentration at 24 through 48 hours after intraarticular injection of IL-1β was also consistent with increased proteoglycan degradation.23 The magnitude of increase in synovial fluid PGE2 concentration induced by the 2 low-dose injections of IL-1β was similar to that induced by a single injection of 175 ng of IL-1β23 and, importantly, did not result in clinical signs of lameness. Although the absence of lameness prohibits an assessment of clinical efficacy, this model of joint inflammation may prove useful as a tool for assessment of potential anti-inflammatory interventions for which testing in clinically affected horses is not ethically warranted because of the early stage of product development.

Although synovial fluid PGE2 concentration was significantly increased at day 1.3 in joints injected with IL-1β, compared with the value in joints injected with saline solution, the wide variability about the mean suggests that the sample collection time points may not have coincided with individual horse peak PGE2 formation. In future studies, more frequent sample collections over the first 8 hours after injection of IL-1β should be used to detect the time at which synovial fluid PGE2 concentration is maximally increased in each study horse. Nevertheless, the increase in synovial fluid protein concentration was consistent with increased permeability of the synovial membrane following IL-1β administration.29 These data provide good evidence for a mild inflammatory response of joints to IL-1β administered via intra-articular injection in horses.

In the horses that received the control diet, intraarticular injection of IL-1 did not induce an increase in synovial fluid NO concentration. This finding was consistent with reports that equine synovial membrane has virtually no measurable inducible NO synthase activity30 and that synovial fluid NO concentration is not increased in horses with certain joint diseases.31 These features of joint inflammation may be unique to this species because this is in contrast to high inducible NO synthase activity in inflamed human32 and rodent33 synovia. Equine chondrocytes do, however, produce NO after exposure to IL-1 in vitro.4,34 Therefore, the lack of increase in synovial fluid NO concentration in the present study may be associated with IL-1β–induced activation of endogenous antioxidant systems that scavenged peroxynitrite from synovial fluid and prevented marked accumulation of synovial fluid NO over the duration of the experiment.

A mild inflammatory response was evident in saline solution–injected joints in horses that received the control diet; significant increases in synovial fluid GAG and protein concentrations and in percentage of neutrophils resulted from intra-articular injection of saline solution. With the exception of the percentage of neutrophils, the peak increases were significantly lower than those in IL-1β–injected joints. There was no significant increase in joint circumference following IL-1β or saline solution injection. Although the measurement of joint circumference provides an indication of the extent of joint effusion, more specific indicators of joint effusion, such as injection of a high–molecular-weight dextran conjugated to a fluorescent probe (eg, fluorescein isothiocyanate) to measure synovial fluid volume,29 are available and should be used in future studies.

Compared with horses that received the control diet, the response to intra-articular injection of IL-1β in horses that received the DN was inhibited; the IL-1β–induced increases in synovial fluid PGE2 and GAG concentrations were not significant (compared with values at day 0 and with values for saline solution–injected joints). Also, although synovial fluid NO concentration was significantly increased from day 0 values at days 1.3 and 2 in IL-1β–injected joints, these values did not differ from values in the saline solution–injected joints. The total cell counts in IL-1β–injected joints were significantly increased, compared with values in saline solution–injected joints, at days 1, 1.3, and 2; however, those increases differed significantly from the day 0 value only at day 1. The percentage of neutrophils in synovial fluid was significantly increased from the day 0 value at days 1 and 8, but at both of those time points, the values did not differ from findings in the saline solution–injected joints. Unlike findings in horses that received the control diet, synovial fluid protein concentration did not increase in saline solution–injected joints in horses that received the experimental diet. Overall, the results of DN administration indicated that the product had a preventative effect against IL-1β–induced joint inflammation in horses. Further investigation is warranted to ascertain whether the product would also be a useful agent for treatment of horses with naturally occurring inflammation.

The inhibitory effect of DN administration on the IL-1β–induced increase in synovial fluid PGE2 concentration in the present study was consistent with the effect of a simulated digest of this product5 and its individual constituents4,5 in IL-1–stimulated equine cartilage explants. The mechanism by which the DN exerts this inhibitory effect on IL-1–induced PGE2 production in joints is not known and requires further research. We have conducted pilot studies to investigate alterations in expressions of genes in response to IL-1 in explants that have been exposed to simulated digest of the DN; data from those pilot studies suggested that the DN may inhibit chondrocyte mRNA expressions of both isoforms of the cyclooxygenase-1 and -2 that occur subsequent to IL-1 exposure, which provides some preliminary evidence for a direct inhibitory effect on production of cyclooxygenases. Because of the notable amounts of omega-3 fatty acids and GAGs in the individual constituents of the DN,4 its mechanism of action may include displacement of arachidonic acid in cell membranes.

At day −14 in the present study, synovial fluid GAG concentrations in horses that received the control and experimental diets were similar. However, in horses that received the DN, synovial fluid GAG concentration increased substantially during the 14 days prior to administration of intra-articular injections of IL-1β or saline solution (although the increase was not significant). This change may have occurred because of increased release of endogenous GAG fragments from cartilage proteoglycan as a result of increased cartilage degradation or because of postabsorptive accumulation of dietary GAGs in synovial fluid. Although it is possible that the DN stimulated proteoglycan degradation, currently available evidence does not support this hypothesis. Shark cartilage inhibits the expression and activity of matrix metalloproteinase-9 in the lungs of mice under inflammatory challenge with ovalbumin35 and inhibits activities of a range of matrix metalloproteinases in vitro.36 Abalone and NZGLM decrease GAG release from cartilage explants,4 and BO protects unstimulated porcine cartilage explants from passive GAG loss.5 The apparent increase in synovial fluid GAG concentration attributable to feeding a diet containing the DN in the present study was also consistent with results of feeding purified chondroitin sulfate to horses.37 Although the absolute magnitude of synovial fluid GAG concentration in that other study was higher because of different experimental methods, the increase in synovial fluid GAG concentration that resulted from feeding 2.5 g of purified low–molecular-weight chondroitin sulfate/d to horses was 2.2 times the initial prefeeding concentration,37 compared with an increase of 2.6 times the initial prefeeding concentration in our study. The duration of that other study was longer than that of the present study, and the earlier study's findings indicated that supplementation of the horses' diet with low–molecular-weight chondroitin sulfate resulted in a biphasic GAG response; synovial fluid GAG concentration was lower in treated horses than it was in control horses after 6 through 8 weeks of feeding the supplemented diet. This suggests that further studies involving the DN should be of longer duration to determine whether a biphasic response is also associated with this product. In future studies, it will be important to directly quantify plasma and synovial fluid sulfur 35 (35S)–labeled GAG concentrations after dietary provision of the 35S-labeled DN to determine whether labeled GAGs derived from the DN are preferentially sequestered in synovial fluid or whether they are evenly distributed throughout the body of an animal.

In horses that received the experimental diet, there was no effect of the supplemental DN on total cell count in synovial fluid, compared with findings in horses that received the control diet. The percentage of neutrophils in synovial fluid was increased at day 8 in horses that received the experimental diet, compared with horses that received the control diet; however, total cell count at that time point remained slightly lower, compared with the value in horses that received the control diet. Thus, the biological relevance of the increase in percentage of neutrophils in synovial fluid in DN-treated horses is unclear, but should be investigated in future research.

After the peak value of joint circumference induced by the first intra-articular injection of IL-1β was detected in horses that received the supplemental DN, it is interesting that joint circumference appeared to decrease; at day 8, the value in horses that received the experimental diet was significantly different from the value in IL-1β–injected joints in horses that received the control diet. This may be preliminary evidence of the ability of the DN to control joint effusion following an inflammatory stimulus. However, it must be noted that measurement of joint circumference by use of a flexible tape is not a sensitive indicator of joint effusion, and use of an intra-articular indicator of synovial fluid dilution would be more appropriate in future studies.

The first experiment performed in the present study in healthy horses did not identify any adverse effects related to administration of the DN in amounts as much as 5 times the manufacturer's recommended dose during a 12-week period. Because of the wide age range of horses available for our study, we randomized them in a block design to ensure that each group was approximately balanced for age, breed, and sex. Three horses per group was the maximum number permitted by the University of Guelph Animal Care Committee for a safety assessment of an investigational product. Significant differences in dependent variables between diets were not dose or time dependent and were not related to treatment.

There are limitations to the present study. The dose of the DN that was administered in feed to the horses in the 2 experiments was calculated from the lowest dose that was efficacious in vitro.5 However, this assumes 100% bioavailability of the active compounds, which is unlikely. There is little known about the bioavailability of each component of the DN and sparse data on bioavailabilities of chondroitin sulfate38,39 and glucosamine.39 However, these studies did not take into account the fact that glucosamine is rapidly bound to plasma globulins, such that < 1% of glucosamine remains free in plasma.40 Therefore, when plasma proteins are precipitated prior to analysis of a supernatant for glucosamine content, the glucosamine may effectively be precipitated along with the protein, thereby resulting in net analysis of only free glucosamine. Thus, it may be erroneously concluded that the bioavailability of glucosamine in horses is very low (2.5%39 or 5.9%38). Thus, the bioavailability of chondroitin may be higher in horses than the estimates reported to date. Furthermore, the presence of feed and other DN constituents may influence the bioavailability of chondroitin and other constituents of the DN.

Collection of urine samples in the first experiment to determine safety of the DN product in horses was difficult because of the collection method used. Thus, the data set for urinalysis was incomplete. Future studies should use either horses that are trained to urinate on command or urine collection bags that can be attached to the horses. The number of horses used in the second experiment to assess efficacy of the DN was low; thus, the data from that experiment should be viewed as preliminary. Further studies with larger sample sizes are necessary to confirm the PGE2-inhibiting effect of the DN in horses.

The second experiment in the present study was designed so that the joint inflammation that developed as a result of intra-articular injection of IL-1β was mild and self-limiting and so that the inflammation would not cause clinical signs of lameness. The intention was to establish a model of joint inflammation in horses that could be used as a preclinical screening tool for experimental dietary anti-inflammatory products. However, there are a number of limitations of the inflammation induction method that should be noted. The IL-1β double-injection protocol remains to be optimized, both with respect to dose and timing of injections; this should be further investigated. A single intra-articular injection of 100 ng of recombinant equine IL-1β induces lameness and pronounced suppurative inflammation in horses,22 but given that equine IL-1β41 shares only 66% genetic identity with human IL-1β,42 it is expected that the inflammatory response of horses to human IL-1β would be comparatively muted. Further research to investigate the inflammatory response to a single intraarticular dose (100 ng) of human IL-1β is warranted to confirm whether the priming injection of 10 ng of IL-1β used in the present study is indeed necessary. Additionally, a more detailed determination of the temporal aspects of the PGE2 and GAG responses to intra-articular injection of IL-1β would help to reduce variability about the peak mean values; as a result, milder anti-inflammatory effects would be detectable.

The method used in the present study did not appear to induce changes in synovial fluid NO concentration. Experiments in which more frequent sampling schedules are applied or the IL-1 injection procedures are modified with regard to dose or timing may facilitate detection of changes in synovial fluid NO concentration. Also, it may be important to acidify synovial fluid immediately after sample collection to promote immediate conversion of NO to nitrite, which is a stable oxidation product. Also, the dilution effect of joint effusion was not accounted for in the present study; measurement of joint circumference provided only a crude estimate of effusion. This could be compensated for by use of an intra-articular indicator of synovial fluid dilution (eg, dextran bound to fluorescein isothiocyanate).

On the basis of the results of the present study, there was an apparent preventative effect associated with feeding a diet with supplemental DN to horses in which IL-1β–induced inflammatory responses in an intercarpal joint were induced. It is possible that the DN could be of benefit in horses that are predisposed to increased risk of articular injury such as those undergoing high-intensity exercise training. It is not yet known whether the product would also be useful as a treatment for horses with preexisting, naturally occurring joint inflammation, and further research is warranted.

ABBREVIATIONS

BO

Biota orientalis

DMB

Dimethylmethylene blue

DN

Dietary Biota orientalis nutraceutical

GAG

Glycosaminoglycan

IL

Interleukin

NO

Nitric oxide

NZGLM

New Zealand green-lipped mussel

PGE2

Prostaglandin E2

SKC

Shark cartilage

a.

Sasha's EQ powder, Interpath Pty Ltd, Ballarat West, VIC, Australia.

b.

Epiitalis, Interpath Pty Ltd, Ballarat West, VIC, Australia.

c.

March Instruments, Kitchener, ON, Canada.

d.

Hitachi 911 biochemical analyzer, Boehringer Mannheim, Laval, QC, Canada.

e.

Advia 120, Bayer Corp, Etobicoke, ON, Canada.

f.

Essential Sweet Horse Essence D 2344, Essentials Inc, Abbotsford, BC, Canada.

g.

Purina Check-R-Mix, 12% sweet horse feed (coarse; contains added selenium [0.2 mg/kg of feed]), Nestlé Purina PetCare Co, St Louis, Mo.

h.

EMLA cream (2.5% lidocaine; 2.5% prilocaine), AstraZeneca, Mississauga, ON, Canada.

i.

Biosource, Camarillo, Calif.

j.

Coulter Z2 counter, Beckman Coulter Canada Inc, Mississauga, ON, Canada.

k.

Prostaglandin E2 ELISA, GE Amersham, Baie D'Urfé, QC, Canada.

l.

Victor 3 microtiter plate reader, Perkin Elmer, Woodbridge, ON, Canada.

m.

Sigma Plot, version 11, Systat Software Inc, San Jose, Calif.

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Appendix 1

Composition of grass-alfalfa hay fed to horses that were included in an experiment to assess the safety of long-term administration of a DN.

ComponentContent
Protein components
   Protein (%)9.61
   Soluble protein (%)3.43
   Soluble protein (% of CP)35.73
   Acid detergent fiber – CP (%)0.75
   Undegradable intake protein32.13
   bypass estimate (% of CP)
Fiber components
   Acid detergent fiber (%)32.70
   Neutral detergent fiber (%)55.91
   Lignin (%)3.95
Mineral components
   Calcium (%)0.50
   Phosphorus (%)0.17
   Potassium (%)1.53
   Magnesium (%)0.19
   Sodium (%)0.02
   Sulfur (%)0.10
   Chloride (%)0.27
   Zinc (ppm)17.55
   Manganese (ppm)27.48
   Copper (ppm)3.05
Energy
   Total digestible nutrients (estimated %)41.21
   Digestible energy1.82
Other variables
   Relative feed value88.89
   Zn-Cu ratio5.75
   Dietary cation-anion balance (mEq/kg)322.19

CP = Crude protein.

Appendix 2

Composition of a sweet feed rationf used to prepare experimental and control diets fed to horses that were included in an experiment to assess the effects of administration of a DN on inflammatory responses to intra-articular injection of IL-1β.

ComponentContent
Crude protein (%)Minimum12.5
Crude fat (%)Minimum2.5
Crude fiber (%)Maximum8.5
Sodium (%)Actual0.3
Calcium (%)Actual0.65
Phosphorus (%)Actual0.55
Copper (mg/kg)Actual30
Vitamin A (U/kg)Minimum4,550
Vitamin D3 (U/kg)Minimum840
Vitamin E (U/kg)Minimum70

Contributor Notes

Supported by Interpath Pty Ltd, Australia, and the Natural Sciences and Engineering Research Council (NSERC) of Canada.

Dr. Pearson was supported by a scholarship from NSERC.

The study sponsor did not participate in the study design; collection, analysis, or interpretation of data; writing of the manuscript; or the decision to submit the manuscript for publication.

The authors thank Drs. Dan Kenney and Antonio Cruz for technical assistance regarding arthrocentesis procedures.

Address correspondence to Dr. Pearson.