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  • Author or Editor: Anthony M. Tesch 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)

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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