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


METHODS
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.


METHODS
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 interleukin-1, tumor necrosis factor-alpha, 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 non-stimulated cultures.


CONCLUSIONS
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.
Riitano
Marco C
MC
Division of Small Animal Surgery and Orthopedics, University of Berne, Switzerland.
Pfister
Hedi
H
Engelhardt
Petra
P
Neumann
Ulf
U
Reist
Martin
M
Zurbriggen
Andreas
A
Stoffel
Michael
M
Peel
John
J
Jungi
Thomas
T
Schawalder
Peter
P
Spreng
David E
DE
D000818:Animals; D019902:Chondrocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004285:Dogs; D006614:Hindlimb; D066298:In Vitro Techniques; D007249:Inflammation / Q000139:chemically induced / Q000201:enzymology / Q000378:metabolism; D007375:Interleukin-1 / Q000494:pharmacology; D007596:Joints; D008022:Ligaments / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D008070:Lipopolysaccharides / Q000494:pharmacology; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D008854:Microscopy, Electron; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D007375:Interleukin-1; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D020782:Matrix Metalloproteinases