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  • Author or Editor: Melinda M. MacDonald x
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Abstract

Objective—To determine whether adenosine influences the in vitro release of nitric oxide (NO) from differentiated primary equine articular chondrocytes.

Sample Population—Articular cartilage harvested from the metacarpophalangeal and metatarsophalangeal joints of 11 horses (3 to 11 years old) without history or clinical signs of joint disease.

Procedure—Chondrocytes were isolated, plated at a high density (105 cells/well), and treated with adenosine, the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), bradykinin, or other agents that modify secondary messenger pathways alone or in combination with bacterial lipopolysaccharide (LPS) or recombinant human interleukin-1α (rhIL-1α). Nitric oxide release was measured indirectly by use of the Griess reaction and was expressed as µmol of nitrite in the supernatant/µg of protein in the cell layer. Inducible nitric oxide synthase (iNOS) activity was determined by measuring the conversion of radiolabeled arginine to radiolabeled citrulline.

Results—Treatment of chondrocytes with adenosine alone had no significant effect on NO release. However, adenosine and NECA inhibited LPS- and rhIL-1α-induced NO release. This response was mimicked by forskolin, which acts to increase adenylate cyclase activity, but not by the calcium ionophore A23187. Treatment of chondrocytes with phorbol myristate acetate, which acts to increase protein kinase C activity, potentiated LPS-induced NO release. Adenosine treatment also significantly inhibited the LPS-induced increase in iNOS activity.

Conclusions and Clinical Relevance—Adenosine and the nonspecific adenosine receptor agonist NECA inhibited inflammatory mediator-induced release of NO from equine articular chondrocytes. Modulation of adenosine receptor-mediated pathways may offer novel methods for treatment of inflammation in horses with joint disease. (Am J Vet Res 2002;63:204–210)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To investigate the activities of hyaluronidases in equine sera and synovial fluid samples and sera from fetal and adult bovids and evaluate the extent to which the degradation of hyaluronan is influenced by chondrocytes.

Sample Population—Commercial and noncommercial samples of equine (n = 6) and bovine (6) sera and 16 synovial fluid samples from horses.

Procedure—Hyaluronidase activities in sera and synovial fluid samples were assessed via enzyme zymography (performed at pH 4, 5, 6, or 7). Chondrocytes were isolated from equine cartilage and cultured with or without hyaluronan (1 mg/mL); the degradation of hyaluronan was assessed via agarose gel electrophoresis.

Results—Hyaluronidase activity was detected in equine sera and synovial fluid samples at pH 4, but not at pH 7, and in bovine sera at both pH values. In all samples at pH 4, a major band of activity (molecular weight, approx 60 kd) and some additional higher molecular weight bands were detected; high- and low-molecular-weight activities were detected in bovine sera at pH 7. Hyaluronan in tissue culture medium with or without fetal calf serum was degraded in the presence, but not the absence, of equine chondrocytes.

Conclusions and Clinical Relevance—Hyaluronidase activity was detected in equine sera and synovial fluid at pH 4 and in bovine sera at pH 4 and 7. Primary chondrocytes in monolayer culture can degrade exogenous hyaluronan. Modulating native hyaluronidase activity may offer a new approach to improve the quantity and quality of hyaluronan in articular joints. ( Am J Vet Res 2005;66:984–990)

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in American Journal of Veterinary Research

Abstract

Objective—To determine rate and degree of cooling for the superficial digital flexor tendon (SDFT) during a standard cryotherapy application in horses and evaluate in vitro effects of cooling on survival of tendon cells.

Sample Population—6 limbs of 5 adult horses and cultured cells obtained from SDFT of 3 adult horses during necropsy.

Procedure—In vivo data were acquired by use of a thermocouple temperature probe inserted into the SDFT of a forelimb of each standing sedated horse. After baseline temperatures were recorded, a commercial compression splint with circulating coolant was placed on each selected limb, which was then exposed to cold treatment for 60 minutes. Temperatures were recorded at 30-second intervals. Mean minimum core temperature was calculated and used to design a protocol for in vitro cold treatment of cells. Specimens were obtained from the SDFT of horses during necropsy; tendon cells were cultured in suspension and exposed to 1-hour of cold treatment that mimicked the in vivo procedure. Viability of cells after cold treatment was compared with viability of cells maintained at body temperature.

Results—After 1 hour of cold treatment, SDFT core temperature was reduced by a mean of 21.8°C, reaching a mean minimum temperature of 10oC. Viability did not differ significantly between cold-treated and control cells.

Conclusion and Clinical Relevance—Results indicated that topical application of cryotherapy significantly reduced core SDFT temperature in standing sedated horses. Temperatures achieved in vivo during cold treatment were not detrimental to the in vitro viability of tendon cells. (Am J Vet Res 2003;64:835–844)

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in American Journal of Veterinary Research

Abstract

Objective—To investigate accumulation of extracellular adenosine (ADO) by equine articular chondrocytes and to compare effects of adenosine kinase inhibition and adenosine deaminase inhibition on the amount of nitric oxide (NO) produced by lipopolysaccharide (LPS)-stimulated chondrocytes.

Sample Population—Articular cartilage from metacarpophalangeal and metatarsophalangeal joints of 14 horses.

Procedure—Chondrocytes were cultured as monolayers, and cells were incubated with LPS, the adenosine kinase inhibitor 5'-iodotubercidin (ITU), or the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3- nonyl)adenine hydrochloride (EHNA). Concentrations of ADO in cell supernatants were measured by use of reverse-phase high-performance liquid chromatography. Effect of inhibition of enzymatic metabolism of ADO on induced NO production was evaluated by exposing cells to a combination of LPS and ITU or LPS and EHNA.

Results—Articular chondrocytes accumulated extracellular ADO when exposed to LPS or ITU. Chondrocytes exposed to ITU accumulated ADO in a time-dependent manner. Unstimulated chondrocytes did not accumulate ADO. Similarly, EHNA alone did not produce detectable ADO concentrations; however, addition of EHNA and ITU resulted in a synergistic effect on accumulation of ADO. Lipopolysaccharideinduced NO production was more effectively suppressed by exposure to ITU than to EHNA

Conclusions and Clinical Relevance—Equine articular chondrocytes release ADO in response to the proinflammatory stimulus of bacterial LPS. Inhibition of the metabolism of ADO increases accumulation of extracellular ADO. Autocrine release of ADO from chondrocytes may play a role in the cellular response to tissue damage in arthritic conditions, and pharmacologic modulation of these pathways in joints of arthritic horses could be a potential method of therapy. (Am J Vet Res 2002;63:1512–1519)

Full access
in American Journal of Veterinary Research
in Journal of the American Veterinary Medical Association

Summary

The metabolic responses of equine articular cartilage to incubation with bacterial lipopolysaccharide (lps) were studied, using explant cultures of articular cartilage obtained from the metatarsophalangeal joints of 15 horses, age of which ranged from 3 months to 20 years. For comparison, explants were also established from the metatarsophalangeal joints of 3 calves. Explants were cultured for 3 days in medium containing various concentrations of lps from 0 (control) to 100 μg/ml. Glycosaminoglycan (gag) released during the 3-day incubation was determined by a spectrophotometric assay, using the dye 1,9-di-methylmethylene blue. Newly synthesized gag content was assayed by measuring [35S]sulfate incorporation during a 3-hour pulse labeling period. In addition, prostaglandin E2 (pge 2) synthesis was quantified, using a [3H]pge 2 radioimmunoassay kit and magnetic separation. Finally, explants from 3 animals were used to evaluate the effect of supplementing culture medium with 5% serum on the response of explants to lps, and explants from 1 horse were used to compare responses to stimulation with lps derived from 2 bacterial sources.

Equine explants cultured with bacterial lps had a dose-dependent decrease in synthesis and increase in release of gag, and these responses were significantly (P < 0.0001) greater in explants from younger horses. In addition, equine explants had a significant (P = 0.0001) dose-dependent increase in concentration of pge 2 released into the culture medium in response to incubation with lps.

Comparison of data for gag synthesis from equine and bovine explants revealed a significant (P = 0.025) difference in responsiveness to lps between the 2 species. Equine explants tended to have a greater suppression of gag synthesis in response to incubation with increasing concentrations of lps than did age-corrected bovine samples. However, similar analysis of data on gag release did not indicate any difference in sensitivity between the 2 species for this response. There was no evidence that the presence or absence of serum supplementation or the use of lps derived from different bacterial sources made a significant difference in the response of explants to incubation with lps.

Free access
in American Journal of Veterinary Research

Summary

Explant cultures were set up, using articular cartilage obtained from metatarsophalangeal joints of 11 horses. Explants from 2 horses were used to determine culture conditions appropriate for tissue viability. The cartilage explants maintained steady-state metabolism of proteoglycans during a 13-day evaluation period. The metabolic response of equine articular cartilage to incubation with recombinant human interleukin 1 (0.01 to 100 ng/ml) was studied, using cartilage obtained from the remaining 9 horses, age of which ranged from 3 months to 20 years. Interleukin 1 induced a dose-dependent release of glycosaminoglycan from the matrix during a 3-day incubation period. It also caused dose-dependent inhibition of glycosaminoglycan synthesis during a 3-hour pulse-labeling period. Expiants obtained from older horses were significantly (P < 0.05) less responsive to interleukin 1, with respect to synthesis and release of glycosaminoglycan.

Free access
in American Journal of Veterinary Research

Abstract

Objective—To describe the ultrasonographic and quantitative histologic effect of injecting 2% iodine in almond oil (IAO) and ethanolamine oleate (EO) in the medial and middle patellar ligaments of horses and to determine whether a difference in response exists between IAO and EO treatment.

Animals—10 healthy horses.

Procedure—In 5 horses, the medial and middle patellar ligaments of 1 limb were injected with EO, whereas IAO was injected in the medial and middle patellar ligaments of another 5 horses. Ultrasonographic evaluation was performed on the experimental and control limb before injection of IAO and EO and prior to euthanasia to determine cross-sectional area and evaluate fiber pattern. The patellar ligaments were harvested 2 weeks after injection and examined histologically to evaluate the inflammatory response, fibroplasia, and chondroid metaplasia.

Results—Injection of the patellar ligaments with IAO resulted in a greater increase in cross-sectional area on ultrasonography than EO. Both agents caused a decrease in echogenicity of the ligament. Histologically, significantly greater infiltration of inflammatory cells and fibroplasia developed after injection with IAO, compared with EO. Both agents resulted in significantly greater fibroplasia relative to control specimens.

Conclusions and Clinical Relevance—Injection of the medial and middle patellar ligaments with IAO induces more severe inflammation and fibroplasia than EO. Maturation of the inflammatory and fibrous response may contribute to resolution or attenuation of upward fixation of the patella by subsequent stiffening of the ligaments. (Am J Vet Res 2002;63:738–743)

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in American Journal of Veterinary Research

Abstract

Case Description—3 horses with penetrating wounds to the shoulder area were examined because of forelimb lameness.

Clinical Findings—All horses had physical examination findings (decreased cranial phase of the stride, swelling in the shoulder region, and signs of pain on manipulation of the shoulder) that were suggestive of problems in the upper portion of the forelimb. Injury to the biceps tendon or bursa was the primary differential diagnosis in each instance, but no abnormalities involving those structures were found. Radiographic and ultrasonographic imaging revealed injuries to the caudal eminence of the greater tubercle of the humerus, the infraspinatus tendon, and the infraspinatus bursa. Examination with ultrasound was more sensitive than radiography at detecting both osseous and soft tissue changes.

Treatment and Outcome—All 3 horses responded favorably to treatment with antimicrobials and non-steroidal anti-inflammatory drugs. Although initial response to standing lavage was favorable in 1 horse, endoscopic lavage was later required. Standing removal of fracture fragments was performed in 2 horses. Ultrasonographic imaging was helpful in monitoring the response to treatment and changes in the affected structures. All 3 horses eventually became sound after treatment.

Clinical Relevance—Infraspinatus bursitis and tendonitis should be included in the differential diagnoses of horses with shoulder lameness. Diagnosis and monitoring should include ultrasonographic monitoring. The prognosis for return to soundness after appropriate treatment appears to be good.

Full access
in Journal of the American Veterinary Medical Association

Abstract

Objective—To determine whether CT provides unique information about the treatment or prognosis for horses with ethmoid hematoma (EH).

Design—Retrospective case series.

Animals—16 horses with EH.

Procedures—Horses with a diagnosis of EH that had undergone a diagnostic CT study were included. Clinical features, treatment, outcome, radiographic and CT images, and histologic specimens were reviewed.

Results—CT provided new diagnostic information that affected treatment in 10 of 16 horses. Bilateral disease occurred in 8 of 16 horses and was undetected in 5 horses prior to CT. Paranasal sinus involvement occurred in all horses, but was incompletely defined prior to CT in 7 of 16 horses. The sphenopalatine sinus was affected in 6 of 16 horses as detected on CT; 4 of 6 of these were bilaterally affected. Medical and surgical treatments were performed. Six of 10 horses had a successful outcome, with recurrence in 4 of 10. Five of 6 patients in which treatment addressed all lesion sites identified by CT had a successful outcome. Bilateral disease did not confer a poor prognosis when all affected sites were treated. Sphenopalatine sinus involvement may have been associated with recurrence.

Conclusions and Clinical Relevance—CT provided anatomic information that may facilitate effective treatment of horses with EH, particularly in patients with bilateral disease and paranasal sinus involvement. Computed tomography is recommended for patients in which the lesion cannot be viewed endoscopically, when sinus involvement or multifocal disease are suspected, or when the lesion has been unresponsive to treatment.

Full access
in Journal of the American Veterinary Medical Association