Further investigations into the potentiation of infection by intra-articular injection of polysulfated glycosaminoglycan and the effect of filtration and intra-articular injection of amikacin

Polysulfated glycosaminoglycan (PSGAG) recently have been reported to potentiate the infectivity of Staphylococcus aureus in horses with experimentally induced septic arthritis. Four groups of 8 horses each had 1 midcarpal joint injected with approximately 33 viable colony-forming units (CFU) of S aureus plus either 1 ml of saline solution (group 1), 250 mg of PSGAG (group 2), 250 mg of PSGAG passed through a 0.6-microns filter (group 3), or 250 mg of PSGAG plus 125 mg of amikacin (group 4). Horses that developed clinical signs consistent with sepsis were euthanatized, and samples were collected at necropsy. Horses that survived had samples obtained by use of arthroscopy at days 13 and 14 after injection. Staphylococcus aureus was isolated from 1 group-1 horse, 8 group-2 horses, and 7 of 7 group-3 horses that met protocol, but was not isolated from any group-4 horses. All 16 aforementioned horses had clinical signs, results of synovial fluid analysis, and gross pathologic and synovial membrane histopathologic findings that were consistent with septic arthritis. Polysulfated glycosaminoglycan (250 mg) increased the infectivity of 33 CFU of S aureus (P = 0.001); filtering the PSGAG had no effect. Intra-articular injection of 125 mg of amikacin immediately after inoculating the joint with 33 CFU of S aureus significantly (P = 0.001) decreased potentiation of infection by the PSGAG.     

Quantitative microanalysis of equine synovial fluid glycosaminoglycan concentration

An alcian blue precipitation method for quantifying the hyaluronic acid (HA) and sulphated glycosaminoglycan concentration (SGAG) in solutions containing both compounds was assessed. The assay was found to be rapid and reliable in solutions containing 0 to 200 mg of HA/dl and 50 to 1,000 micrograms of SGAG/dl, and was not affected by the presence of protein, hemoglobin, or methemoglobin in concentrations normally found in synovial fluid. The HA and SGAG concentrations in intercarpal synovial fluid from 13 clinically normal and 11 arthritic horses were evaluated. A relationship was not found between the concentration of HA and SGAG and any other synovial fluid variable. The SGAG concentration was found to be markedly high in several of the synovial fluid samples from arthritic horses, but did not correlate with the degree of articular cartilage erosion.     

Synovial Fluid Studies in Navicular Disease

The purpose of this study was to investigate biochemical changes in synovial fluid in navicular disease, and to establish if synovial fluid from the distal interphalangeal joint (DIP) could be used diagnostically to assess alterations in the synovial fluid of the navicular bursa. Cartilage oligomeric matrix protein (COMP), total glycosaminoglycans (GAG), hyaluronan (HA), metalloproteinases 2 and -9 (MMP-2 and MMP-9) and total protein (TP) levels were determined in synovial fluids obtained from 18 navicular bursae and 35 DIP -joints from animals suffering from navicular disease, and the same synovial structures in 16 joints of horses with no evidence of abnormalities involving the foot. To avoid dilution effects, GAG/COMP, HA/COMP, MMP-2/ COMP and MMP-9/COMP ratios were also calculated for different synovial cavities. There was a good correlation, for COMP, GAG, HA, MMP-2 and TP levels, between synovial fluid from the navicular bursa and fluid from the DIP -joint in healthy animals. However, in animals with navicular disease, only COMP levels showed no difference between the navicular bursal fluid and the DIP-joint fluid concentration. Thus, enabling the use of COMP to standardise other biochemical concentration measurements from the synovial joint fluids. In horses with navicular disease, there was a significantly lower absolute concentration of GAG, and a significantly lower GAG/COMP ratio, in the synovial fluid of the navicular bursa and the DIP-joint compared to synovial fluid from the same joints from healthy horses. In contrast, the absolute HA concentration and HA/ COMP, MMP-2/COMP and MMP-9/COMP ratios were higher in synovial fluid from the DIP-joint of horses with navicular disease, and MMP-2 and MMP-9 relative activity levels and MMP-2/COMP and MMP-9/ COMP ratios were increased in fluid from navicular bursae in horses with navicular disease when compared to a control group.     

Hyaluronan in horses: physiological production rate, plasma and synovial fluid concentrations in control conditions and following sodium hyaluronate administration

REASONS FOR PERFORMING STUDY: Hyaluronic acid (HA) is an endogenous glycosaminoglycan used in the treatment of joint diseases, but medication control is required by horseracing authorities. Therefore, a medication control policy needs to be established. OBJECTIVES: To establish physiological plasma HA concentrations in post race horses, determine the HA endogenous production rate and document the disposition of HA after i.v. and intra-articular hyaluronic acid administration at recommended therapeutic doses. METHODS: Hyaluronan concentrations in plasma were determined using an ELISA specific test; concentrations in synovial fluid were determined using a radiometric binding assay. RESULTS: The overall mean plasma HA concentration in 120 post competition horses was 89 ng/ml. In a group of 6 experimental horses, synovial fluid control concentration was 328+/-112 microg/ml. After i.v. sodium hyaluronate administration (37.8 mg in toto), the terminal half-life was very short (43+/-29 mins) and after a delay of 3 h, the plasma concentration returned to control values. The endogenous HA production rate was 33-164 mg in toto per day, i.e. 1-4 times the recommended i.v. daily dose. Twenty-four hours after intra-articular administration, HA concentration was not significantly different from control values (328+/-112 microg/ml). CONCLUSIONS AND POTENTIAL RELEVANCE: Due to the rapid disappearance of HA from plasma after i.v. administration and from the joint after intra-articular administration, long-term detection needs a more appropriate approach to be developed.     

Cartilage oligomeric matrix protein and hyaluronan levels in synovial fluid from horses with osteoarthritis of the tarsometatarsal joint compared to a control population

REASONS FOR PERFORMING STUDY: Quantification of cartilage oligomeric matrix protein (COMP) levels within synovial fluid from the tarsometatarsal joint has not previously been reported and an effective synovial fluid marker would allow monitoring of disease progression and treatment. OBJECTIVES: To quantify levels of COMP and hyaluronan (HA) in synovial fluid from the tarsometatarsal joint, identify differences in levels from horses with osteoarthritis (OA) of the tarsometatarsal joint compared to a control population and to correlate levels with radiographic changes in horses with OA. METHODS: Synovial fluid was collected from the tarsometatarsal joint of 25 horses without hindlimb lameness (controls) and 25 lame horses, subjected to analgesia of the joint. COMP concentrations were measured using a homologous inhibition ELISA. Immunoblots of synovial fluid from 3 lame horses and 3 controls were performed to identify fragmentation of COMP. Hyaluronan (HA) concentration in synovial fluid was determined using a competition ELISA. Radiographs of the lame horses with OA were scored and correlated with levels of COMP and HA. RESULTS: Concentrations of COMP in OA of the tarsometatarsal joint were significantly lower than in the control samples. An additional fragment band of COMP (approximately 30 kDa) was identified on the immunoblots of the horses with OA and this fragment was not identified in controls. No significant difference was identified in the HA or HA:COMP ratio between lame and control horses. There was no correlation between levels of synovial fluid COMP and HA, and radiographic changes. CONCLUSIONS AND POTENTIAL RELEVANCE: Lowered levels of COMP in synovial fluid of tarsometatarsal joints correlates with the presence of osteoarthritis. However, a single value cannot be used to stage the disease process. Levels of HA may not be a useful marker for this disease. Decreased, rather than increased COMP levels, may reflect significant loss of cartilage in established osteoarthritis. A specific assay for the COMP fragment generated with osteoarthritis may allow the earlier detection of clinical cases.     

Synovial fluid and plasma kinetics of methylprednisolone and methylprednisolone acetate in horses following intra-articular administration of methylprednisolone acetate

Synovial fluid and plasma kinetics of methylprednisolone acetate (MPA) and methylprednisolone (MP) after a single intra-articular administration of MPA at a therapeutic dose (111 mg in toto) was measured in five horses. MPA was detected in synovial fluid for two to six days post injection and MP, which results from synovial MPA hydrolysis, was present in pharmacologically significant concentrations for 4.8 to 39 days, depending on the horse. MPA synovial concentration was maximal (289 +/- 284 micrograms/ml) at the first sampling time (2 h after administration) and MP synovial concentration was maximal (from 58.9 to 379.5 micrograms/ml) at the first or second sampling time (2 to 10 h after administration). Thereafter, both MP and MPA declined rapidly. From time of administration to about five days later, MP synovial fluid concentration fell progressively with a half-time of 9.95 h. Subsequently, the MP synovial fluid concentration decreased more slowly with an apparent half-time of 115 h. During the first 24 h following MPA administration, trace amounts of MP (less than 5 ng/ml) were detected in plasma. Plasma hydrocortisone levels were depressed for three to four days after administration but adrenal responsiveness to adrenocorticotrophic hormone tests remained unaffected.     

In vivo kinetic study on uptake and distribution of intramuscular tritium-labeled polysulfated glycosaminoglycan in equine body fluid compartments and articular cartilage in an osteochondrial defect model

The uptake and distribution of intramuscularly (IM) administered tritium-labeled polysulfated glycosaminoglycan (³H-PSGAG) in serum, synovial fluid, and articular cartilage of eight horses was quantitated, and hyaluronic acid (HA) concentration of the middle carpal joint was evaluated in a pharmacokinetic study. A full-thickness articular cartilage defect, created on the distal articular surface of the left radial carpal bone of each horse served as an osteochondral defect model. ³H-PSGAG (500 mg) was injected IM, between 14 and 35 days after creation of the defects. Scintillation analysis of serum and synovial fluid, collected from both middle carpal joints at specific predetermined times up to 96 hours post-injection, revealed mean ³H-PSGAG concentrations peaked at 2 hours post-injection. ³H-PSGAG was detected in cartilage and subchondral bone 96 hours post-injection in samples from all eight horses. There were no statistically significant differences in ³H-PSGAG concentration of synovial fluid or cartilage between cartilage defect and control (right middle carpal) joints.

HA assay of synovial fluid revealed concentrations significantly increased at 24, 48, and 96 hours post-injection in both joints. The concentration nearly doubled 48 hours post-injection. However, no statistically significant differences were found between synovial concentrations of HA in cartilage defect and control joints.

³H-PSGAG administered IM to horses, was distributed in the blood, synovial fluid, and articular cartilage. HA concentrations in synovial fluid increased after IM administration of polysulfated glycosaminoglycan.     

Comparison of the effect of polysulfated glycosaminoglycan, corticosteroids, and sodium hyaluronate in the potentiation of a subinfective dose of Staphylococcus aureus in the midcarpal joint of horses

Four groups of 8 horses each had 1 midcarpal joint injected with 33 colony-forming units (CFU) of viable Staphylococcus aureus plus: 1 ml of saline solution (group 1, control), 250 mg of polysulfated glycosaminoglycan (PSGAG, group 2), 100 mg of methylprednisolone acetate (group 3), or 20 mg of sodium hyaulronate (group 4). Horses were euthanatized, and samples were obtained on the basis of clinical signs of septic arthritis that were nonresponsive to phenylbutazone administration. One group-1 horse, all 8 group-2 horses, 3 group-3 horses, and 4 group-4 horses were culture-positive for S aureus and had clinical signs, results of synovial fluid analysis, and histopathologic findings that were consistent with sepsis. The addition of 250 mg of PSGAG increased the development of sepsis significantly (P = 0.001), compared with results in control horses. Differences in the development of sepsis between horses injected with methylprednisolone acetate or sodium hyaluronate and control horses were not significant.     

The influence of corticosteroids on sequential clinical and synovial fluid parameters in joints with acute infectious arthritis in the horse

Infectious arthritis was induced experimentally in one tarsocrural joint of six horses by intra-articular injection of 1 ml Staphylococcus aureus-saline suspension with the addition of 200 mg methylprednisolone acetate. The corresponding contralateral joint was injected with 1 ml of saline with the addition of 200 mg methylprednisolone acetate, and served as a control. The purpose of the experiment was to examine the effect of corticosteroids on the acute clinical signs of infectious arthritis, and the associated changes in synovial fluid, to separate the effects of a steroid injection from those of infection alone. This should aid early diagnosis of infection. The progression of the infectious arthritis was assessed over nine days by clinical examination and sequential synovial fluid analysis. The corticosteroids masked the clinical signs in some horses for up to the third day although changes in the synovial fluid were present earlier. Cellular changes preceded biochemical changes initially. Leucocyte counts showed a significant increase in cell numbers after infection was established. Persistent neutrophilia, over 90 per cent, together with a pH under 6.9 were the most consistent findings in the infected synovia. Total protein values were lower in infected joints with, than those without, corticosteroids; although there was a progressive rise in total protein concentration throughout the experiment in both groups. Serum and synovial glucose difference and synovial lactate had very little diagnostic value because significant increases due to the corticosteroids were documented in the control joints.     

Assessment of glycosaminoglycan concentration in equine synovial fluid as a marker of joint disease.

A modification of a colorimetric assay was used to determine synovial fluid total and individual sulphated-glycosaminoglycan concentration in various clinical presentations of joint disease in horses. Concentrations of synovial fluid and serum sulphated-glycosaminoglycan (GAG) were measured by the 1,9-dimethylmethylene blue (DMMB) dye assay in normal horses (n = 49), horses with acute (n = 26) or chronic (n = 27) joint disease (defined by clinical, radiographic, and clinicopathological parameters), and horses with cartilaginous lesions at diagnostic arthroscopy, but with normal radiographs and synovial fluid (n = 9). Horses with acute joint disease were subdivided into moderate acute (n = 21) and severe acute (n = 5) joint disease on the basis of synovial fluid analysis and clinical examination. Horses with chronic joint disease were subdivided into mild chronic (n = 9), moderate chronic (n = 10), and severe chronic (n = 8) joint disease on the basis of synovial fluid analysis, clinical examination, and radiographic findings. The concentrations of chondroitin sulphate (CS) and keratan sulphate (KS) were analyzed in each sample following sequential enzymatic digestion of the sample with chondroitinase or keratanase. In addition, the concentration of hyaluronate (HA) in each sample was determined by a colorimetric assay following digestion of the sample with microbial hyaluronidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentrations of serum amyloid A in serum and synovial fluid from healthy horses and horses with joint disease

OBJECTIVE: To determine serum amyloid A (SAA) concentrations in serum and synovial fluid from healthy horses and horses with joint disease and assess the effect of repeated arthrocentesis on SAA concentrations in synovial fluid. Animals-10 healthy horses and 21 horses with various types of joint disease. PROCEDURES: Serum and synovial fluid samples were obtained from each horse. In 5 of the 10 healthy horses, arthrocentesis was repeated 9 times. Concentrations of SAA were determined via immunoturbidometry. RESULTS: Serum and synovial fluid SAA concentrations were less than the assay detection limit in healthy horses and did not change in response to repeated arthrocentesis. Synovial fluid SAA concentrations were significantly higher in horses with suspected bacterial joint contamination or infectious arthritis, or tenovaginitis than in healthy controls, and serum concentrations were significantly higher in horses with infectious conditions than in the other groups. Neither serum nor synovial fluid SAA concentrations in horses with low-inflammation joint conditions differed significantly from those in healthy controls. Concentrations of SAA and total protein in synovial fluid were significantly correlated. CONCLUSIONS AND CLINICAL RELEVANCE: Synovial fluid SAA concentration was a good marker of infectious arthritis and tenovaginitis and appeared to reflect changes in inflammatory activity. The advantages of use of SAA as a marker include the ease and speed of measurement and the fact that concentrations in synovial fluid were not influenced by repeated arthrocentesis in healthy horses. Further study of the SAA response in osteoarthritic joints to assess its usefulness in diagnosis and monitoring of osteoarthritis is warranted.     

Evaluation of the inflammatory response to two intra-articular hyaluronic acid formulations in normal equine joints

Intra-articular (IA) hyaluronic acid (HA) is commonly used to treat equine arthritis. Inflammatory response or “joint flare” is a recognized potential side effect. However, the incidence and severity of inflammation following IA HA injection in horses is not well documented. This study compared the effects of two IA HA formulations of different molecular weight (MW) and a saline control on clinical signs and synovial fluid markers of inflammation in normal equine joints. Eight adult horses each had three healthy fetlock joints randomly assigned to treatment with either 1.4 mega Dalton HA, 0.8 mega Dalton HA or saline control once weekly for three weeks. Clinical evaluation and synovial fluid analysis were performed by blinded assessors. Outcomes of interest were lameness score, joint effusion score and synovial fluid white cell count and differential, total protein, viscosity and serum amyloid A. Joints injected with HA developed significant mild-to-moderate inflammatory responses often associated with lameness and joint effusion compared with saline control joints. The higher MW HA formulation elicited a significantly greater inflammatory response than the lower MW HA after the first injection. In HA injected joints, viscosity remained poor for the entire study. Both IA HA formulations in this study induced an inflammatory response in healthy equine joints. This may have implications for the use of HA in equine joints. The findings in this study are limited to the two HA formulations used. Further investigation of different HA formulations and the use of HA in normal and arthritic equine joints is warranted.     

Urea as a measure of dilution of equine synovial fluid

This paper tests the hypothesis that serum and synovial urea concentrations are similar and that urea concentration can be used as an accurate marker for synovial fluid dilution in normal equine joints. Serum and synovial fluid urea concentrations were compared in 42 horses and were equivalent for individual horses (P<0.0001). Mean +/- s.e. serum concentration was 6.1+/-0.552 mmol/l and synovial concentration 6.0+/-0.459 mmol/l. The normal range for synovial urea concentration was determined as 2.5-7.7 mmol/l. The synovial urea concentration from different synovial structures in individual horses were compared and were equivalent (P = 0.002). Known dilutions of synovial fluid with saline were made. The actual and expected synovial urea concentrations were compared and were equivalent (P<0.001). An accurate method of calculating synovial fluid dilution has been determined.     

Interaction of transforming growth factor-beta-1 with alpha-2-macroglobulin from normal and inflamed equine joints.

Binding between equine plasma alpha-2-macroglobulin (alpha 2M) and several cytokines known to participate in inflammatory reactions in other species was initially examined. Plasma was obtained from 5 horses with various abnormalities. Samples, both untreated and after reaction with methylamine, were incubated with exogenous, radiolabeled, porcine-derived transforming growth factor-beta-1 (125I-TGF-beta 1), recombinant human interleukin-1-beta (125I-IL-1 beta), and recombinant human tumor necrosis factor-alpha (125I-rhTNF-alpha). They were then subjected to nondenaturing polyacrylamide gel electrophoresis (PAGE). Binding of the native (slow) and activated (fast) forms of alpha 2M to each cytokine was subjectively evaluated with autoradiography. Equine alpha 2M bound 125I-TGF-beta 1. However, poor or no binding was observed between alpha 2M and either of 125I-rhTNF-alpha or 125I-IL-1 beta. Synovial fluid was then obtained from 6 normal horses, 6 horses with septic arthritis, and 6 horses with degenerative joint disease. Untreated and methylamine-reacted samples were quantitatively examined for binding with 125I-TGF-beta 1, using the autoradiographic techniques described above and densitometry. Native and activated alpha 2M were also quantified by densitometry of PAGE gels. Native alpha 2M was significantly elevated in septic arthritis (6.4% to 29.5% of total protein detected) and degenerative joint disease (2.8% to 12.3%), compared to normal joints (0.9% to 4.2%). Activated alpha 2M, however, was not detected in untreated synovial fluid samples. In all plasma and joint fluid samples, whether untreated or reacted with methylamine, 125I-TGF-beta 1 bound predominantly to alpha 2M, and preferentially to the activated form of alpha 2M. In synovial fluid, the amount of 125I-TGF-beta 1 binding was proportional to the quantity of alpha 2M present. These results indicate that: 1) equine alpha 2M binds TGF-beta 1; 2) the native form of alpha 2M is present in both equine plasma and synovial fluid, and 3) alpha 2M is a major binding protein for TGF-beta 1 in equine synovial fluid. Therefore, alpha 2M may play a role in regulating this mediator of inflammation in equine joints.     

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.     

Cartilage-derived retinoic acid-sensitive protein in equine synovial fluid from healthy and diseased joints

Reasons for performing study: More sensitive and specific diagnostic methods for early detection of changes in the joint cartilage are needed. Cartilage‐derived retinoic acid‐sensitive protein (CD‐RAP) is a potential marker of cartilage synthesis and regeneration. This is the first study on equine CD‐RAP. Objectives: To evaluate the ability of a commercially available human sandwich ELISA assay to detect equine CD‐RAP in synovial fluid from healthy and diseased joints. Methods: Synovial fluid was collected from 28 horses with no signs of joint disease and from 5 with induced inflammatory arthritis. CD‐RAP concentrations were measured using a human CD‐RAP ELISA. Intra‐ and interassay imprecision of the assay were evaluated by multiple measurements on pools of equine synovial fluid. Assay inaccuracy was determined by linearity under dilution. Results: The assay showed moderate to large intra‐ and interassay variation when applied to equine synovial fluid. Equine CD‐RAP was detected in synovial fluid from healthy horses ranged at 8.2–52 ng/ml. Repeated arthrocentesis (after injection of isotonic saline), age, joint or gender did not significantly affect CD‐RAP concentrations. Twelve hours after intra‐articular injection of lipopolysaccharide, concentrations of CD‐RAP were significantly lower than after injection of isotonic saline and remained significantly lower until the end of the study at 144 h. Conclusion and potential relevance: The assay is suitable for longitudinal monitoring of CD‐RAP concentration in individual horses. Disease significantly influenced CD‐RAP levels. Similar to previous results obtained in man, CD‐RAP seems to be a marker of cartilage synthesis and/or regeneration in horses.     

Effects of intra-articular administration of dimethylsulfoxide on chemically induced synovitis in immature horses

The effects of intra-articular administration of dimethylsulfoxide (DMSO) on chemically induced synovitis in the middle carpal joint of 6 weanling horses were evaluated. Following aseptic collection of synovial fluid, the middle carpal joint of each forelimb was injected with 50 mg of Na-monoiodoacetate to induce synovitis. Eight days after injection, synovial fluid was obtained and the right middle carpal joints were injected with 2 ml of 40% DMSO in lactated Ringer solution. The corresponding joints of the left limb (control) were injected with 2 ml of lactated Ringer solution. Sampling and treatments were repeated on post-injection days 11 and 14, for a total of 3 treatments. Horses were visually evaluated daily for lameness and joint effusion. Synovial fluid was evaluated for color and clarity, differential and total WBC count, total protein content, and hyaluronic acid concentration. The Kaegi gait analysis system provided an objective assessment of lameness prior to inducing synovitis, again on day 7, and on day 17. At necropsy (day 17), synovial fluid, synovial membrane, and articular cartilage specimens were collected. Joint effusion was evident 12 hours after injection of Na-monoiodoacetate in all joints. Mild lameness was evident at 24 hours; however, the lameness resolved by 72 hours. Objective assessment of lameness did not reveal significant differences between treatment or control limbs. Hyaluronic acid concentrations increased significantly (P = 0.023) above baseline values in most joints over the study period. Synovial fluid WBC counts increased significantly (P = 0.002) following Na-monoiodoacetate injection and remained significantly (P = 0.002) above baseline values throughout the study.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of two indirect techniques for local delivery of a high dose of an antimicrobial in the distal portion of forelimbs of horses

OBJECTIVE: To compare isolated limb retrograde venous injection (ILRVI) and isolated limb infusion (ILI) for delivery of amikacin to the synovial fluid of the distal interphalangeal and metacarpophalangeal joints and to evaluate the efficacy of use of an Esmarch tourniquet in standing horses. ANIMALS: 6 healthy adult horses. PROCEDURES: Horses were randomly assigned in a crossover design. In ILRVI, the injection consisted of 1 g of amikacin diluted to a total volume of 60 mL administered during a 3-minute period. In ILI, the infusion consisted of 1 g of amikacin diluted to 40 mL administered during a 3-minute period followed by administration of boluses of diluent (82 mL total) to maintain vascular pressure. During ILI, the infusate and blood were circulated from the venous to the arterial circulation in 5-mL aliquots. Synovial fluid and serum samples were obtained to determine maximum amikacin concentrations and tourniquet leakage, respectively. RESULTS: Both techniques yielded synovial concentrations of amikacin > 10 times the minimum inhibitory concentration (MIC) for 90% of isolates (80 microg/mL) and > 10 times the MIC breakpoint (160 microg/mL) of amikacin-susceptible bacteria reported to cause septic arthritis in horses. These values were attained for both joints for both techniques. Esmarch tourniquets prevented detectable loss of amikacin to the systemic circulation for both techniques. CONCLUSIONS AND CLINICAL RELEVANCE: Both techniques reliably achieved synovial fluid concentrations of amikacin consistent with concentration-dependent killing for bacteria commonly encountered in horses with septic arthritis. Esmarch tourniquets were effective for both delivery techniques in standing horses.     

Substance P in the synovial membrane and fluid of the equine middle carpal joint.

This preliminary study was designed to determine whether the neurotransmitter substance P was present in the middle carpal synovial membrane of the normal horse and whether the neuropeptide could be identified in the synovial fluid of normal horses and those with joint diseases. Immunocytochemistry on middle carpal synovial membrane biopsies from fresh cadavers was used to demonstrate substance P-containing neural elements. Substance P was most abundant in the subintimal portion of the membrane, with occasional filaments coursing via synovial fronds to the intimal portion. Radioimmunoassay techniques were used on acidified acetonitrile-preserved synovial fluid samples to measure substance P concentrations. Fluid from 9 joints of 5 normal horses and 6 joints of 4 horses with joint diseases were analysed. Disease conditions included acute and chronic osteoarthritis and osteochondrosis. Synovia from normal horses contained a mean concentration of substance P significantly less than that of horses with joint diseases (P less than 0.05). Elevated concentrations of neurotransmitters in diseased joints suggests a potential contribution to the pathophysiology of joint disorders in horses.     

Determination of synovial fluid and serum concentrations, and morphologic effects of intraarticular ceftiofur sodium in horses

OBJECTIVES: To determine the serum and synovial fluid concentrations of ceftiofur sodium after intraarticular (IA) and intravenous (IV) administration and to evaluate the morphologic changes after intraarticular ceftiofur sodium administration. STUDY DESIGN: Strip plot design for the ceftiofur sodium serum and synovial fluid concentrations and a split plot design for the cytologic and histopathologic evaluation. ANIMALS: Six healthy adult horses without lameness. METHODS: Stage 1: Ceftiofur sodium (2.2 mg/kg) was administered IV. Stage 2: 150 mg (3 mL) of ceftiofur sodium (pHavg 6.57) was administered IA into 1 antebrachiocarpal joint. The ceftiofur sodium was reconstituted with sterile sodium chloride solution (pH 6.35). The contralateral joint was injected with 3 mL of 0.9% sterile sodium chloride solution (pH 6.35). Serum and synovial fluid samples were obtained from each horse during each stage. For a given stage, each type of sample (serum or synovial fluid) was collected once before injection and 12 times after injection over a 24-hour period. All horses were killed at 24 hours, and microscopic evaluation of the cartilage and synovium was performed. Serum and synovial fluid concentrations of ceftiofur sodium were measured by using a microbiologic assay, and pharmacokinetic variables were calculated. Synovial fluid was collected from the active joints treated during stage 2 at preinjection and postinjection hours (PIH) 0 (taken immediately after injection of either the ceftiofur sodium or sodium chloride), 12, and 24, and evaluated for differential cellular counts, pH, total protein concentration, and mucin precipitate quality. RESULTS: Concentrations of ceftiofur in synovial fluid after IA administration were significantly higher (P = .0001) than synovial fluid concentrations obtained after IV administration. Mean peak synovial fluid concentrations of ceftiofur after IA and IV administration were 5825.08 microg/mL at PIH .25 and 7.31 microg/mL at PIH 4, respectively. Mean synovial fluid ceftiofur concentrations at PIH 24 after IA and IV administration were 4.94 microg/mL and .12 microg/mL, respectively. Cytologic characteristics of synovial fluid after IA administration did not differ from cytologic characteristics after IA saline solution administration. White blood cell counts after IA ceftiofur administration were < or =3,400 cells/ML. The mean synovial pH of ceftiofur treated and control joints was 7.32 (range, 7.08-7.5) and 7.37 (range, 7.31-7.42), respectively. Grossly, there were minimal changes in synovium or cartilage, and no microscopic differences were detected (P = .5147) between ceftiofur-treated joints and saline-treated joints. The synovial half-life of ceftiofur sodium after IA administration joint was 5.1 hours. CONCLUSIONS: Synovial concentrations after intraarticular administration of 150 mg of ceftiofur sodium remained elevated above minimal inhibitory concentration (MIC90) over 24 hours. After 2.2 mg/kg IV, the synovial fluid ceftiofur concentration remained above MIC no longer than 8 hours. CLINICAL RELEVANCE: Ceftiofur sodium may be an acceptable broad spectrum antimicrobial to administer IA in septic arthritic equine joints.