Hexosamine concentration (an index of proteoglycan content), DNA content (an index of cellularity), and [35S]sulfate incorporation (an index of proteoglycan synthesis) of articular cartilage were measured in biopsy specimens from medial proximal sesamoid bone, medial condyle of the third metacarpal bone, and proximal dorsal rim of the proximal phalanx in both metacarpophalangeal joints of 6 adult horses. One limb was then placed in a fiberglass cast that extended down from the proximal portion of the metacarpus and enclosed the hoof; the other limb was not casted. After 30 days of staff confinement, additional specimens were taken from the medial proximal sesamoid bone, medial condyle of the third metacarpal bone, midproximal portion of the proximal phalanx, distal portion of the proximal phalanx, and proximal portion of the middle phalanx of both limbs for comparison. Immobilization resulted in an apparent decrease in the hexosamine content of the cartilage when the 30-day immobilized vs 30-day mobilized specimens were analyzed. This decrease was accentuated by opposing trends in the 2 limbs. The immobilized cartilage tended to lose hexosamine, whereas the mobilized limb tended to gain hexosamine during the 30-day period; a similar trend also was seen with [31S] incorporation, but this trend was not statistically significant. The largest change was a significant increase in glycosaminoglycan synthesis in the mobilized limb, compared with little change in the immobilized joint cartilage. We concluded that contralateral limbs are unsuitable for controls in immobilization studies because of their biological response to increased weight bearing. We also concluded that the changes in articular cartilage found following simple cast immobilization of 30 days' duration are minor and probably of little clinical consequence.

Because articular chondrocytes are a target for drugs that can influence the integrity of cartilage, we investigated the effects of 3 antiarthritic drugs, glycosaminoglycan polysulfate, diclofenac-Na, and S-adenosylmethionine sulfate p-toluenesulfonate on total protein, fibronectin, and DNA synthesis, as well as on extradomain-A fibronectin and keratan sulfate content. Glycosaminoglycan polysulfate stimulated dose-dependent incorporation of [35S]methionine into protein and fibronectin, whereas incorporation of [3H]thymidine into DNA was unaffected. Total fibronectin, extradomain-A fibronectin, and keratan sulfate content were high in chondrocyte cultures treated with glycosaminoglycan polysulfate. In contrast, fibronectin and DNA synthesis, as well as extradomain-A fibronectin and keratan sulfate content were unaffected by diclofenac-Na. S-adenosylmethionine decreased dose-dependently the synthesis of fibronectin, as well as the content of fibronectin and keratan sulfate. At the highest concentration of S-adenosylmethionine tested, findings suggest that cell viability was impaired as assessed by the release of lactate dehydrogenase into the media.

Hexosamine concentration, DNA concentration, and [35S]sulfate incorporation for articular cartilage obtained from various sites in the metacarpophalangeal and carpal joints of horses were measured. The same measurements were made on the repair tissue filling full-thickness articular defects in the intermediate carpal bone and on cartilage surrounding partial-thickness defects 6 weeks after the defects were created arthroscopically. Cellularity (measured as DNA concentration), proteoglycan content (measured as hexosamine concentration), and proteoglycan synthesis (measured as [35S]sulfate incorporation) varied according to the site sampled. Cartilage from the transverse ridge of the head of the third metacarpal bone and the radial facet of the third carpal bone had the lowest hexosamine concentration, whereas rate of proteoglycan synthesis was lowest in cartilage from the transverse ridge of the head of the third metacarpal bone and the distal articular surface of the radial carpal bone. Repair tissue filling a full-thickness cartilage defect at 6 weeks was highly cellular. It was low in proteoglycan content, but was actively synthesizing these macromolecules. In contrast, the cartilage surrounding a partial-thickness defect was unchanged 6 weeks after the original defect was made.

OBJECTIVE To determine the median time to maximum concentration (tmax) of amikacin in the synovial fluid of the tarsocrural joint following IV regional limb perfusion (IVRLP) of the drug in a saphenous vein of horses. ANIMALS 7 healthy adult horses. PROCEDURES With each horse sedated and restrained in a standing position, a 10-cm-wide Esmarch tourniquet was applied to a randomly selected hind limb 10 cm proximal to the point of the tarsus. Amikacin sulfate (2 g diluted with saline [0.9% NaCl] solution to a volume of 60 mL) was instilled in the saphenous vein over 3 minutes with a peristaltic pump. Tarsocrural synovial fluid samples were collected at 5, 10, 15, 20, 25, and 30 minutes after completion of IVRLP. The tourniquet was removed after collection of the last sample. Amikacin concentration was quantified by a fluorescence polarization immunoassay. Median maximum amikacin concentration and tmax were determined. RESULTS 1 horse was excluded from analysis because an insufficient volume of synovial fluid for evaluation was obtained at multiple times. The median maximum synovial fluid amikacin concentration was 450.5 μg/mL (range, 304.7 to 930.7 μg/mL), and median tmax was 25 minutes (range, 20 to 30 minutes). All horses had synovial fluid amikacin concentrations ≥ 160 μg/mL (therapeutic concentration for common equine pathogens) at 20 minutes after IVRLP. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that, in healthy horses, maintaining the tourniquet for 20 minutes after IVRLP of amikacin in a saphenous vein was sufficient to achieve therapeutic concentrations of amikacin in the tarsocrural joint.

The aim of this study was to determine the pharmacokinetics of amikacin and penicillin G sodium when administered in combination as an intravenous regional limb perfusion (IVRLP) to horses. Seven healthy adult horses underwent an IVRLP in the cephalic vein with 2 g of amikacin sulfate and 10 mill IU of penicillin G sodium diluted to 60 mL in 0.9% saline. A pneumatic tourniquet set at 450 mmHg was left in place for 30 min. Synovial fluid was collected from the metacarpophalangeal joint 35 min and 2, 6, 12, and 24 h after infusion of the antimicrobials. Concentrations of amikacin and penicillin in synovial fluid were quantitated by liquid chromatography tandem-mass spectrometry analysis. Therapeutic concentrations of amikacin and penicillin for equine-susceptible pathogens were achieved in the synovial fluid. Maximum synovial concentrations (Cmax) (mean ± SE) for amikacin and penicillin were 132 ± 33 μg/mL and 8474 ± 5710 ng/mL, respectively. Only 3 horses had detectable levels of penicillin at 6 h and 1 at the 12 h sample. The combination of amikacin with penicillin G sodium via IVDLP resulted in reported therapeutic concentrations of both antibiotics in the synovial fluid. The Cmax:MIC (minimum inhibitory concentration) ratio for amikacin was 8:1 and Time > MIC for penicillin was 6 h. At 24 h, the mean concentration of amikacin was still above 4 μg/mL. Terminal elimination rate constants (T1/2 lambdaz) were 13.6 h and 2.8 h for amikacin and penicillin, respectively. The use of IVDLP with penicillin may therefore not be practical as rapid clearance of penicillin from the synovial fluid requires frequent perfusions to maintain acceptable therapeutic concentrations.

OBJECTIVE To evaluate the effect of volume of IV regional limb perfusion (IVRLP) on amikacin concentrations in synovial and interstitial fluid of horses. ANIMALS 8 healthy adult horses. PROCEDURES Each forelimb was randomly assigned to receive IVRLP with 4 mL of amikacin sulfate solution (250 mg/mL) plus 56 mL (total volume, 60 mL) or 6 mL (total volume, 10 mL) of lactated Ringer solution. Horses were anesthetized, and baseline synovial and interstitial fluid samples were collected. A tourniquet was placed, and the assigned treatment was administered via the lateral palmar digital vein. Venous blood pressure in the distal portion of the limb was recorded. Additional synovial fluid samples were collected 30 minutes (just before tourniquet removal) and 24 hours after IVRLP began; additional interstitial fluid samples were collected 6 and 24 hours after IVRLP began. RESULTS 30 minutes after IVRLP began, mean amikacin concentration in synovial fluid was significantly greater for the large-volume (459 μg/mL) versus small-volume (70 μg/mL) treatment. Six hours after IVRLP, mean concentration in interstitial fluid was greater for the large-volume (723 μg/mL) versus small-volume (21 μg/mL) treatment. Peak venous blood pressure after large-volume IVRLP was significantly higher than after small-volume IVRLP, with no difference between treatments in time required for pressure to return to baseline. CONCLUSIONS AND CLINICAL RELEVANCE Study findings suggested that large-volume IVRLP would deliver more amikacin to metacarpophalangeal joints of horses than would small-volume IVRLP, without a clinically relevant effect on local venous blood pressure, potentially increasing treatment efficacy.

Holstein steers were fed carbohydrate-rich, short-fiber basal diets with and without added sodium sulfate. Steers fed the high-sulfate diet developed the CNS disorder polioencephalomalacia (PEM). The onset of signs of PEM was associated with increased sulfide concentration in the rumen fluid. Over the course of the disease, anaerobic rumen bacteria were enumerated in roll tubes by use of the Hungate method to determine the effect of dietary sulfate on sulfate-reducing bacterial numbers. Media used included a general type for total counts and sulfate-containing media with and without cysteine to assess sulfate-reducing bacteria. Changes in total and sulfate-reducing bacterial numbers attributable to dietary sulfate content were not observed. The capacity to generate hydrogen sulfide from sulfate in fresh rumen fluid in vitro was substantially increased only after steers had been fed the high-sulfate diet for 10 to 12 days, which coincided with the onset of signs of PEM. The low capacity for hydrogen sulfide production of rumen fluid taken at earlier times in the feeding period suggests that rumen microorganisms must adapt to higher dietary sulfate content before they are capable of generating potentially toxic concentrations of sulfide.

The effect of interleukin 1 (IL-1) on equine articular cartilage was investigated, using a cartilage explant culture system. Measurement of [35S]O4 incorporation revealed synthesis of matrix proteoglycan by cartilage to be decreased 45, 59.7, and 37.5% after 1, 3, and 5 days, respectively, in culture in the presence of 5 U of IL-1/ml. There was no change in proteoglycan degradation as determined by measurement of [35S]O4 release into the culture medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cartilage-conditioned medium indicated that exposure of cartilage to IL-1 caused a decrease in total protein synthesis by 45, 68, and 87% after 1, 3, and 5 days, respectively, in culture while selectively inducing synthesis of the 57-kd neutral metalloproteinase stromelysin (matrix metalloproteinase-3) in young and adult horses. Identification of stromelysin was confirmed by functional characterization and immunoprecipitation. Baseline total protein synthesis, as well as specific synthesis of stromelysin in cartilage from adult and aged horses, was markedly less than that of young horses. The IL-1-induced induced reduction in total protein synthesis may not be a characteristic of equine articular cartilage from affected joints of horses with naturally acquired osteoarthritis as indicated by an overall increase in protein synthesis by osteoarthritic explants. Introduction of IL-1 into an equine articular cartilage explant culture system resulted in decrease of matrix component synthesis and increase in specific degradative enzyme synthesis and activity. Articular cartilage from aged horses had markedly less overall metabolic activity, compared with cartilage from young horses. Articular cartilage from affected joints of horses with naturally acquired osteoarthritis did not have metabolic alterations identical to those of IL-1-stimulated normal articular cartilage from the same individual, necessitating reevaluation of the validity of the IL-1-induced model of osteoarthritis. Osteoarthritis is a common, naturally acquired disease of horses, and tissue from animals of all ages and stages of osteoarthritis is available. The equine model of osteoarthritis may afford an important means of studying the alterations in articular cartilage metabolism as a function of age and disease severity.

Two commercially available tests, an antigen-capture ELISA for use on fecal samples, and a peroral nylon string test for use in dogs, were compared with a zinc sulfate fecal concentration technique (ZSCT) for detection of giardiasis in dogs. Of 77 dogs and 164 fecal samples (from these dogs), 33 and 52, respectively were found to be Giardia-positive on the basis of results of the ZSCT. The ELISA gave false-negative results for 10 and 14% of ZSCT-positive dogs and fecal samples, respectively, and false-positive results (relative to the ZSCT test results) in 13 and 10% of ZSCT-positive dogs and fecal samples, respectively. Of the 18 string-tested dogs, 14 were positive by results of the ZSCT. Of the 4 dogs that were Giardia-negative by ZSCT, 2 were Giardia-positive by ELISA. Dogs were sedated and given water and metoclopramide to aid passage into the duodenum of the capsule containing a nylon string. Of the 21 string tests performed on the 18 dogs, only 5 strings reached the duodenum, and 0 of the 5 yielded positive results for Giardia sp. Because the string broke in 1 dog (leaving most in the gastrointestinal tract and, therefore, producing a risk of string foreign body) further string tests were not done.

Using arthroscopic technique, identical diameter defects were created in the proximal articular surface of both intermediate carpal bones of 6 horses. One of each pair of defects was deepened to penetrate the subchondral plate. Removed cartilage was assayed for [35S] sulfate incorporation, total hexosamine content, and DNA content. Six weeks later, cartilage was harvested and similarly analyzed from the distolateral portion of the radius directly opposite the created lesions and the distomedial portion of the radius distant from the lesion. The repair tissue filling the full-thickness defect and the cartilage at the periphery of the partial-thickness lesion also were analyzed. There was a marked increase in synthetic activity (35S sulfate incorporation) opposite the full-thickness defect, compared with the cartilage opposite the partial-thickness defect. A marked decrease in glycosaminoglycan content in the cartilage opposite the full-thickness defect was found as compared with that opposite the partial-thickness defect. The repair tissue filling the full-thickness defect was highly cellular, high in synthetic activity, but low in glycosaminoglycan content. Insignificant changes occurred in the cartilage adjacent to the partial-thickness defect. On the basis of these results, we suggest that full-thickness defects at 6 weeks result in more detrimental change to the cartilage opposite it than do partial-thickness lesions of the same diameter.