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Effects of stimulus with proinflammatory mediators on nitric oxide production and matrix metalloproteinase activity in explants of cranial cruciate ligaments obtained from dogs

Marco C. Riitano Dr med vet1, Hedi Pfister2, Petra Engelhardt3, Ulf Neumann PhD4, Martin Reist Dr med vet, Dr sc nat5, Andreas Zurbriggen Dr med vet6, Michael Stoffel Dr med vet7, John Peel BVSc8, Thomas Jungi Dr rer nat9, Peter Schawalder Dr med vet10, and David E. Spreng PD, Dr med vet11
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  • 1 Division of Small Animal Surgery and Orthopedics, Faculty of Veterinary Medicine, University of Bern, Längassstrasse 128, 3012, Bern, Switzerland.
  • | 2 Division of Immunology, Faculty of Veterinary Medicine, University of Bern, Längassstrasse 128, 3012, Bern, Switzerland.
  • | 3 Novartis Pharma AG, WSJ-386.5.09, 4002 Basel, Switzerland.
  • | 4 Novartis Pharma AG, WSJ-386.5.09, 4002 Basel, Switzerland.
  • | 5 Institute of Animal Science, Swiss Federal Institute of Technology, ETH Zentrum CLU C5, 8092 Zürich, Switzerland.
  • | 6 Division of Neurology, Faculty of Veterinary Medicine, University of Bern, Längassstrasse 128, 3012, Bern, Switzerland.
  • | 7 Division of Anatomy, Faculty of Veterinary Medicine, University of Bern, Längassstrasse 128, 3012, Bern, Switzerland.
  • | 8 Novartis Centre de Recherche, Sante Animale SA, 1566 St Aubin, Switzerland.
  • | 9 Division of Immunology, Faculty of Veterinary Medicine, University of Bern, Längassstrasse 128, 3012, Bern, Switzerland.
  • | 10 Division of Small Animal Surgery and Orthopedics, Faculty of Veterinary Medicine, University of Bern, Längassstrasse 128, 3012, Bern, Switzerland.
  • | 11 Division of Small Animal Surgery and Orthopedics, Faculty of Veterinary Medicine, University of Bern, Längassstrasse 128, 3012, Bern, Switzerland.

Abstract

Objective—To evaluate the origin and degree of activity of nitric oxide (NO) and matrix metalloproteinase (MMP) in explants of cranial cruciate ligaments (CCLs) obtained from dogs and cultured with and without inflammatory activators.

Sample Population—Tissue specimens obtained from 7 healthy adult Beagles that were (mean ± SD) 4.5 ± 0.5 years old and weighed 12.5 ± 0.8 kg.

Procedure—The CCLs were harvested immediately after dogs were euthanatized, and specimens were submitted for explant culture. Cultures were stimulated by incubation with a combination of interluekin-1, tumor necrosis factor-α, and lipopolysaccharide, or they were not stimulated. Culture supernatants were examined for production of NO nitrite-nitrate metabolites (NOts) and activity of MMP. Cultured specimens were evaluated by use of immunohistochemical analysis to detect activity of inducible NO synthase (iNOS).

Results—All ligament explants produced measurable amounts of NOts. Stimulated cultures produced significantly more NOts after incubation for 24 and 48 hours, compared with nonstimulated cultures. Production of MMP in supernatants after incubation for 48 hours was significantly higher in stimulated cultures than in nonstimulated cultures. Cells with positive staining for iNOS were detected on all slides. Positively stained cells were predominantly chondroid metaplastic. There was a significant difference in intensity of cell staining between stimulated and nonstimulated cultures.

Conclusion and Clinical Relevance—Explant cultures of intact CCLs obtained from dogs produce iNOS-induced NO. Stimulation of chondroid metaplastic cells in CCL of dogs by use of inflammatory activators can increase production of iNOS, NOts, and MMP. (Am J Vet Res 2002;63:1423–1428)

Abstract

Objective—To evaluate the origin and degree of activity of nitric oxide (NO) and matrix metalloproteinase (MMP) in explants of cranial cruciate ligaments (CCLs) obtained from dogs and cultured with and without inflammatory activators.

Sample Population—Tissue specimens obtained from 7 healthy adult Beagles that were (mean ± SD) 4.5 ± 0.5 years old and weighed 12.5 ± 0.8 kg.

Procedure—The CCLs were harvested immediately after dogs were euthanatized, and specimens were submitted for explant culture. Cultures were stimulated by incubation with a combination of interluekin-1, tumor necrosis factor-α, and lipopolysaccharide, or they were not stimulated. Culture supernatants were examined for production of NO nitrite-nitrate metabolites (NOts) and activity of MMP. Cultured specimens were evaluated by use of immunohistochemical analysis to detect activity of inducible NO synthase (iNOS).

Results—All ligament explants produced measurable amounts of NOts. Stimulated cultures produced significantly more NOts after incubation for 24 and 48 hours, compared with nonstimulated cultures. Production of MMP in supernatants after incubation for 48 hours was significantly higher in stimulated cultures than in nonstimulated cultures. Cells with positive staining for iNOS were detected on all slides. Positively stained cells were predominantly chondroid metaplastic. There was a significant difference in intensity of cell staining between stimulated and nonstimulated cultures.

Conclusion and Clinical Relevance—Explant cultures of intact CCLs obtained from dogs produce iNOS-induced NO. Stimulation of chondroid metaplastic cells in CCL of dogs by use of inflammatory activators can increase production of iNOS, NOts, and MMP. (Am J Vet Res 2002;63:1423–1428)