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  • Author or Editor: Melinda H. MacDonald x
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Abstract

Objective—To measure apolipoproteins in cerebrospinal fluid (CSF) from healthy mares and to determine whether CSF concentrations of apolipoproteins change during pregnancy and lactation.

Animals—5 healthy pregnant mares.

Procedure—2 sets of CSF samples were obtained; initial samples were obtained 10 to 30 days before parturition (mean, 18 days; median, 17 days), and second samples were obtained 19 to 26 days after parturition (mean, 23 days; median, 23 days). Cerebrospinal fluid was collected from the lumbosacral subarachnoid space of standing horses by use of routine collection techniques. Cerebrospinal fluid cholesterol concentrations were measured by use of a sensitive enzymatic assay. Ultracentrifugal fractions of CSF lipoproteins were characterized by determining the distribution of apolipoproteins, using polyacrylamide gel electrophoresis.

Results—Analyses of isolated ultracentrifugal fractions by polyacrylamide gel electrophoresis revealed 2 apolipoproteins, with the expected molecular weights for apolipoprotein E and apolipoprotein A-I. No significant differences were observed between pre- and postpartum values in mares. The concentration of cholesterol in CSF fluid of mares was comparable to values reported in other mammals.

Conclusion and Clinical Relevance—Apolipoprotein E in CSF of horses is a major apolipoprotein associated with high-density lipoproteins, which is similar to findings in other mammals. Additional characterization of the role of apolipoproteins in mammalian CSF may provide critical insight into various degenerative neurologic disease processes. (Am J Vet Res 2002;63:886–889)

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in American Journal of Veterinary Research

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)

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

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)

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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

Objective—To characterize effects of IV administration of pirfenidone on clinical, biochemical, and hematologic variables and circulating tumor necrosis factor (TNF)-α concentrations in horses after infusion of a low dose of endotoxin.

Animals—18 healthy adult horses.

Procedures—Horses were randomly assigned to 3 groups (n = 6 horses/group) and administered an IV infusion of 30 ng of endotoxin/kg or saline (0.9% NaCl) solution during a 30-minute period. Lipopolysaccharide-pirfenidone horses received endotoxin followed by pirfenidone (loading dose of 11.6 mg/kg and then constant rate infusion [CRI] at 9.9 mg/kg/h for 3 hours). Lipopolysaccharide-saline horses received endotoxin followed by infusion (loading dose and CRI for 3 hours) of saline solution. Saline-pirfenidone horses received saline solution followed by pirfenidone (loading dose and then CRI for 3 hours). Physical examination variables were recorded and blood samples collected at predetermined intervals throughout the 24-hour study period. Blood samples were used for CBCs, biochemical analyses, and determinations of TNF-α concentrations.

Results—IV infusion of pirfenidone after administration of a low dose of endotoxin failed to attenuate the clinical, clinicopathologic, or cytokine alterations that developed secondary to endotoxin exposure. Intravenous infusion of pirfenidone after administration of saline solution induced mild transient clinical signs, but associated clinicopathologic changes were not detected.

Conclusions and Clinical Relevance—IV administration of pirfenidone was tolerated with only mild transient clinical adverse effects during infusion. However, administration of pirfenidone did not protect horses from the systemic effects of experimentally induced endotoxemia. Further studies of related, but more potent, drugs may be warranted.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To characterize the plasma pharmacokinetics and clinical effects of pirfenidone administered IV in healthy horses.

Animals—6 adult horses.

Procedures—A 15 mg/kg dose of pirfenidone was administered IV over 5 minutes. Physical variables were recorded and blood samples collected prior to infusion; 2.5 minutes after beginning infusion; at the end of infusion; and at 3, 6, 9, 12, 15, 20, 25, 30, 40, 50, 60, 75, and 90 minutes and 2, 2.5, 3, 4, 6, 8, 12, and 24 hours after completion of infusion. Plasma concentrations of pirfenidone and its metabolites were determined.

Results—Mild clinical effects, including tachycardia and muscle fasciculations, were observed during drug administration but stopped at the end of the infusion. Pirfenidone and 2 metabolites, hydroxypirfenidone and carboxypirfenidone, were detected by the end of the 5-minute infusion. Mean peak plasma concentration of pirfenidone was 182.5 μmol/L, detected at the end of the infusion. Mean peak plasma concentrations of hydroxypirfenidone and carboxypirfenidone were 1.07 and 3.4 μmol/L, respectively, at 40 minutes after infusion. No parent drug or metabolites were detected at 24 hours. Distribution of pirfenidone best fit a 2-compartment model, and the drug had mean ± SEM elimination half-life of 86.0 ± 4.7 minutes, mean body clearance of 6.54 ± 0.45 mL/kg/min, and apparent volume of distribution at steady state of 0.791 ± 0.056 L/kg.

Conclusions and Clinical Relevance—Intravenous administration of pirfenidone was tolerated with transient adverse affects during infusion, and drug clearance was rapid.

Full access
in American Journal of Veterinary Research

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.

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in Journal of the American Veterinary Medical Association