Determination of total protein concentration and viscosity of synovial fluid from the tibiotarsal joints of horses.

Viscosity of synovial fluid (SF) from 29 clinically normal horses was determined by use of a rotational cone and plate microviscosimeter. Total protein concentration in the SF of the 29 horses, as measured with a refractometer, was less than 2.5 g/dl. When the Coomassie brilliant blue test was used to determine total protein concentration in SF for 15 horses, the mean value was 1,088 mg/dl. Viscosity values at 60, 30, 12, 6, 3, and 1.5 revolutions/min (rpm) spindle speed were 4.41 +/- 1.54 centipoise (cp), 5.29 +/- 1.94 cp, 6.76 +/- 2.76 cp, 8.52 +/- 4.27 cp. 10.41 +/- 6.30 cp, and 13.07 +/- 9.05 cp, respectively. Synovial fluid viscosity increased with decreasing rpm and shear rate, but the shape of the curve for each horse fitted the asymptotic curve. The rotational cone and plate microviscosimeter was an accurate instrument in measuring SF viscosity at multiple rpm or shear rates in horses. The values obtained on clinically normal horses in this study will serve as a baseline for comparison in the evaluation of horses with joint disease.     

Plasma and synovial fluid pharmacokinetics of a single intravenous dose of meropenem in adult horses

The objective of this study was to determine the pharmacokinetics of meropenem in horses after intravenous (IV) administration. A single IV dose of meropenem was administered to six adult horses at 10 mg/kg. Plasma and synovial fluid samples were collected for 6 hr following administration. Meropenem concentrations were determined by bioassay. Plasma and synovial fluid data were analyzed by compartmental and noncompartmental pharmacokinetic methods. Mean ± SD values for elimination half‐life, volume of distribution at steady‐state, and clearance after IV administration for plasma samples were 0.78 ± 0.176 hr, 136.1 ± 19.69 ml/kg, and 165.2 ± 29.72 ml hr^‐1 kg^?1, respectively. Meropenem in synovial fluid had a slower elimination than plasma with a terminal half‐life of 2.4 ± 1.16 hr. Plasma protein binding was estimated at 11%. Based on a 3‐compartment open pharmacokinetic model of simultaneously fit plasma and synovial fluid, dosage simulations were performed. An intermittent dosage of meropenem at 5 mg/kg IV every 8 hr or a constant rate IV infusion at 0.5 mg/kg per hour should maintain adequate time above the MIC target of 1 μg/ml. Carbapenems are antibiotics of last resort in humans and should only be used in horses when no other antimicrobial would likely be effective.     

Ethylenediaminetetraacetic acid (EDTA) anticoagulant affects the refractometric measurement of total protein concentration in equine synovial fluid

The objectives of this study were to (i) determine the effect of ethylenediaminetetraacetic acid (EDTA) concentration on the refractometric measurement of the total protein (TP) concentration of equine synovial fluid and (ii) assess the effect of time and temperature on the refractometric measurement of TP concentration. Synovial fluid was retrieved aseptically from horses presenting to Liphook Equine Hospital either (a) immediately after euthanasia or (b) from those requiring arthrocentesis as part of their clinical investigation. Samples were aliquoted into commercially available (i) plain tubes (0.5 mL; control) and (ii) EDTA tubes (at different volumes to obtain serial dilutions of EDTA:synovial fluid). TP concentration was measured by two independent observers using a calibrated hand-held refractometer. Samples from a separate group of horses were stored for 24 h at 4°C and 20°C before analysis to assess the effects of time and temperature. Results were compared using repeated measures ANOVA. TP concentration was significantly higher at all EDTA concentrations compared to control samples, with the greatest effect seen with higher concentrations (P<0.0001 for samples with ≥3.17 mg EDTA/mL). Storage time or temperature did not significantly affect TP concentration. Refractometric measurement of small volumes of synovial fluid in commercially available EDTA tubes leads to artificially elevated TP concentrations. Delays in sample analysis and alterations in storage conditions did not significantly affect TP concentration within the parameters studied. The magnitude of overestimation of synovial total protein concentration could result in misclassification of suspect synovial sepsis cases where EDTA tubes are used.     

The effect of three different doses of sodium pentosan polysulphate on haematological and haemostatic variables in adult horses

OBJECTIVE: To evaluate the effects of three different doses of sodium pentosan polysulphate (PPS) on haematological and haemostatic variables in adult horses. DESIGN: Eight adult standardbred horses were used. All horses received a single injection of 0, 3, 6, and 10 mg/kg of PPS at the beginning of each treatment week for 4 weeks so that by the end of the study all horses had received all four doses of PPS. Blood samples were collected at 0, 1, 2, 3, 4, 6, 8, 12, 24, 48, and 168 h after each weekly injection of PPS. Variables measured were packed cell volume, haemoglobin, red blood cell count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, white cell count, neutrophil count, lymphocyte count, eosinophil count, monocyte count, serum protein, fibrinogen, prothrombin time, and activated partial thromboplastin time (PTT). Data were analysed using an ANOVA. Significance was set at P < 0.05. RESULTS: There was a dose-dependent increase in PTT. A significant increase in PTT occurrred in all treatment groups when compared to horses receiving 0 mg/kg in which there was no change over time. The PTT values all returned to baseline by 48 h after treatment. The mean neutrophil count was higher 3 h after treatment when compared to time 0. Horses receiving 3 mg/kg of PPS had a higher lymphocyte count 4 h after injection, and those receiving 6 and 10 mg/kg had higher counts at 3,4,6 and 8 h after injection when compared to time 0. At 8 h after injection horses receiving 6 and 10 mg PPS had higher lymphocyte counts than horses not receiving PPS. CONCLUSIONS: PPS causes a dose-dependent prolongation of PTT in horses. At the dose rates currently recommended for treatment of joint problems in horses this increase was small and remained elevated from baseline for up to 24 h. Based on these findings doses of PPS up to 3 mg/kg should not be administered to horses within 24 h of high stress activities or where physical injury may occur.     

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.     

Variation in water disappearance,daily dose,and synovial fluid concentrations of tylvalosin and 3‐O‐acetyltylosin in commerical pigs during five day water medication with tylvalosin under field conditions

Tylvalosin (TVN) is a water soluble macrolide used in swine production to treat enteric, respiratory, and arthritic pathogens. There is limited data on its distribution to synovial fluid beyond gavage studies, which do not represent field conditions. This study measured water disappearance, TVN concentration in the medicated water, daily dose, and concentrations of TVN and 3‐O‐acetyltylosin (3AT) in the synovial fluid and plasma of treated pigs over the administration period. The study emphasized understanding variation in tissue TVN concentrations within the context of a field setting. Sixty finisher pigs were housed individually with individual waterers. Six pigs were randomly allocated to the following time points for sample collection: 0, 48, 60, 72, 84, 96, 102, 108, 114, and 120 hr on medication. TVN was administered daily in the water for 5 days. Water disappearance and medicated water concentration were measured daily. At each time point, six pigs were euthanized and plasma and synovial fluid were collected for analysis. Median TVN synovial fluid concentrations ranged between <1 ng/ml (hour 0) to 3.6 ng/ml (hour 84). There was substantial variation between individual pigs for water disappearance (mean 4.36L and range 0–7.84). Median TVN water concentration was 59 ppm (range 38–75 ppm).     

Determination of MMP-2 and -9 activities in synovial fluid of horses with osteoarthritic and arthritic joint diseases using gelatin zymography and immunocapture activity assays

REASON FOR PERFORMING STUDY: Matrix metalloproteinases (MMPs)-2 and -9 activities have been found elevated in synovial fluid from various joint diseases in man. However, in the horse few data are available. OBJECTIVES: To explore the clinical significance of MMP-2 and -9 activities in synovial fluid of horses with different forms of joint diseases. METHODS: Gelatin zymography and MMP-2 and -9 immunocapture activity assays were applied on synovial fluids from control joints and joints with aseptic joint disease (AJD) and septic arthritis (SA). Additionally, MMP-2 and -9 activities were measured in samples from SA to monitor the disease process. RESULTS: Zymographic analysis revealed that samples from AJD and SA contained significantly increased latent MMP-2 activity compared to controls. Samples from SA showed significantly increased monomeric latent MMP-9 activity compared with all other affected joints and controls. Trace amounts of MMP-9 activity, due to the active and dimer form, were detected in samples from SA; however, these bands were absent in samples from AJD and controls. Using immunocapture activity assays, MMP-2 and -9 activities were found to be significantly elevated in joints from SA compared to controls and AJD samples. MMP-2 activity in samples from AJD was significantly increased compared to controls. Both MMP activities decreased in the joints from SA in the course of successful therapy. CONCLUSIONS: Data from zymographic analysis confirmed that MMP-2 and -9 were elevated in equine joint diseases. Immunocapture activity assays have been shown to be suitable for the quantitative determination of MMP-2 and -9 activities in synovial fluid of horses. Both MMP-2 and -9 activities seem to be useful to indicate SA, and MMP-2 activity might be a suitable marker for AJD. POTENTIAL RELEVANCE: These findings encourage the potential use of MMP-2 and -9 as additional aids to clinical investigation. Further work is required to validate the clinical significance of MMP activities in the progress of different joint diseases in horses.     

The influence of anticoagulants on the measurement of total protein concentration in equine peritoneal fluid

The aim of this study was to evaluate the influence of two commonly used anticoagulants (K3EDTA and lithium heparin) on refractometric and spectrophotometric measurement of total protein (TP) concentration in equine peritoneal fluid samples. The influence of a commercial solution of K3EDTA, a solution of K3EDTA in distilled water and lithium heparin on the refractometric and spectrophotometric (biuret) quantification of TP content in peritoneal fluid samples was assessed. Total protein concentration measured by refractometry was consistently overestimated in samples with commercial K3EDTA. The solution of K3EDTA in distilled water only caused TP overestimation at high K3EDTA concentrations (>5 micromol/ml). By contrast, lithium heparin did not influence the refractometric values of TP. Neither anticoagulant modified TP values when measured by the biuret method. In conclusion, the use of K3EDTA as anticoagulant may result in a significant overestimation of TP values of peritoneal fluid samples measured by refractometry.     

Synovial fluid and plasma concentrations of ceftiofur after regional intravenous perfusion in the horse

OBJECTIVE: To determine radiocarpal (RC) joint synovial fluid and plasma ceftiofur concentrations after regional intravenous perfusion (RIP) and systemic intravenous (IV) administration. STUDY DESIGN: Experimental cross-over study. ANIMALS: Five normal adult horses. METHODS: One RC joint was randomly selected for RIP and the contralateral RC joint was sampled to determine intrasynovial ceftiofur concentrations after IV administration. Wash-out between IV and RIP was > or = 14 days. After surgical introduction of an intraarticular catheter, ceftiofur (2 g) was administered under general anesthesia either IV or by RIP after tourniquet application. Plasma and synovial fluid were collected over 24 hours. Samples were analyzed using high-performance liquid chromatography with ultraviolet detection and the results were statistically analyzed using a linear mixed effect model. RESULTS: Mean synovial fluid ceftiofur concentrations were consistently higher after RIP than after IV administration and were > 1 mug/mL (minimal inhibitory concentration [MIC] for common pathogens) for >24 hours. Mean synovial fluid peak concentration of ceftiofur after RIP and IV administration was 392.7+/-103.29 microg/mL at 0.5 hours postinjection (HPI) and 2.72+/-0.31 mug/mL at 1 HPI, respectively. Large variations in synovial fluid and plasma ceftiofur concentrations were observed between horses regardless of administration technique. RIP did not cause adverse effects. CONCLUSIONS: Under the present experimental conditions RIP with ceftiofur (2 g) induced significantly higher intraarticular antibiotic concentrations in the RC joint in comparison with IV administration. Moreover, after RIP, synovial fluid ceftiofur concentrations remain above the MIC for common pathogens (1 microg/mL) for > 24 hours. No adverse effects from the technique or the antibiotic were observed. CLINICAL RELEVANCE: RIP with high doses of ceftiofur may be a beneficial adjunctive therapy when treating equine synovial infections which are caused by cephalosporin susceptible microorganisms.     

Effects of continuous intra-articular infusion of gentamicin on synovial membrane and articular cartilage in the tarsocrural joint of horses

OBJECTIVE: To determine the effects of a continuous intra-articular infusion of gentamicin on the synovial membrane and articular cartilage in the tarsocrural joint of horses. ANIMALS: 6 healthy adult horses. PROCEDURE: A balloon infusion system attached to a catheter placed in the plantarolateral pouch of both tarsocrural joints in each horse was used for continuous gentamicin solution (GM) or balanced electrolyte solution (BES) delivery for 5 days. Cartilage and synovial membrane specimens were collected on day 5 from 3 horses and on day 14 from the remaining 3 horses. Both infused joints from each horse were assessed, using gross evaluation and histologic scoring systems. RESULTS: Significant differences in the histologic scores of synovial membrane specimens between the GM- and BES-treated joints at either 5 or 14 days were not observed. Safranin-O-fast green staining scores were similar between cartilage specimens from GM- and BES-treated joints. Although the synovial membrane histologic scores and safranin-O-fast green staining scores improved from day 5 to 14, the changes in scores were not significant. Loss of synovial intimal cells from villi was found more commonly in sections of synovial membrane from GM-treated joints, compared with BES-treated joints. CONCLUSIONS AND CLINICAL RELEVANCE: Continuous infusion of GM into the tarsocrural joint of horses does not have significant effects on histologic scores of articular cartilage or synovial membrane, compared with those infused with BES. Continuous infusion of GM into the tarsocrural joint of horses for 5 days is an acceptable method for the treatment of septic arthritis.     

Effects of collecting serial tracheal aspirate and bronchoalveolar lavage samples on the cytological findings of subsequent fluid samples in healthy Standardbred horses

Objective To evaluate the effect of collecting serial tracheal aspirate (TA) and bronchoalveolar lavage (BAL) samples on the cytological findings of subsequent fluid samples obtained from horses without clinical signs of respiratory disease. Study design Experimental. Study population Six healthy Standardbred horses. Methods Endoscopically‐guided TA samples, and BAL samples collected using the blind field technique were obtained from the six horses on days 1, 2, 3, 4, 5, 12, and 17. On day 17, horses were sampled three times: at baseline and at 2.5 h and 4 h apart. The differential cytology of the fluid samples collected at each time point was expressed as percentages and compared statistically. Results There was a significant increase in neutrophil percentage in the TA samples taken at day 17 (at 2.5 h but not at 4 h apart). There was no significant change in the neutrophil percentages in the TA samples when repeated samples were taken ≥24 h apart. There was no significant change in the neutrophil percentages in the BAL fluid at any collection point. There were inconsistent changes in the percentages of lymphocytes and macrophages in the BAL fluid over time, but these remained within normal reference ranges and were considered clinically insignificant. Conclusions Serial TA and BAL samples can be taken at 24 h intervals without affecting the cytological findings of subsequent fluid samples collected using the techniques described.     

Comparison of high-performance liquid chromatography with a radiometric assay for determination of the effect of intra-articular administration of corticosteroid and saline solution on synovial fluid hyaluronate concentration in horses.

Two recently developed direct methods, radioassay-125I-labeled hyaluronic acid binding protein (125I-HABP)- and high-performance liquid chromatography (HPLC), were used to assess and compare the concentration of hyaluronate (HA) in synovial fluid of horses. Also determined were changes in the HA concentration in an experimental treatment model involving physiologic saline solution (PSS)-irrigated or methylprednisolone acetate-injected tarsocrural joints of clinically normal horses. Serum HA concentration was determined simultaneously, using the 125I-HABP assay. Synovial fluid HA concentration values obtained by use of the HPLC method were approximately double the values obtained by use of 125I-HABP assay. Correlation (r = 0.819) between the 2 methods was highly significant (P less than 0.001; linear regression analysis) for all samples studied and for various experimental subgroups. When pure HA standards were used, correlation between the 2 methods was close to 1 (r = 0.965; P less than 0.001), with higher values obtained by use of the 125I-HABP assay. It is suggested that the HA binding protein derived from endogenous cartilage proteoglycan interferes with the 125I-HABP assay on synovial fluid, resulting in excessively low values, compared with those obtained using the HPLC procedure. Intra-articular injection of methylprednisolone acetate significantly (P less than 0.01) increased synovial fluid HA concentration at 24 hours after injection. Increase was also detected after PSS irrigation, but owing to wide intersubject variation, this increase was not significant. The HPLC procedure, which provides simultaneous information about the concentration and degree of polymerization of HA, is recommended for the study of synovial fluid, whereas the 125I-HABP assay is more suitable for serum HA analysis.