The spread of cell death from impact damaged cartilage: lack of evidence for the role of nitric oxide and caspases.
Over 21 days in culture, cell death spreads, both radially and transversely, from loaded to surrounding cartilage. This spread was prevented by physical separation and separate culture post-impact.


OBJECTIVE
One aim was to determine if nitric oxide (NO) is the intercellular signal mediating cell death. Another aim was to clarify the nature of the cell death, whether caspase mediated apoptosis or necrosis.


METHODS
Cyclic impacts were applied to the central 2 mm core of 4 mm canine articular cartilage discs. Post-impact culturing was for 21 days in the presence or absence of the iNOS inhibitor, L-NAME, or the broad-spectrum caspase inhibitor, Z-VAD FMK. Cell death was quantified using the TUNEL assay. Culture media were collected every 2 days for measurements of glycosaminoglycan (GAG) and NO release.


RESULTS
Cell death spread from the loaded core into the surrounding ring over 21 days in culture. Although L-NAME significantly reduced nitrite release into the culture media of both loaded and control cartilage, the spread of cell death was not prevented. Neither was the spread of cell death prevented by Z-VAD FMK.


CONCLUSIONS
These data indicate that NO is not acting as an intercellular signalling factor in this in vitro system and that the cell death post-impact is not caspase mediated.
Clements
Kristen M
KM
Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Hungerford Hill Road, Ithaca, NY 14853, USA.
Burton-Wurster
Nancy
N
Lust
George
G
D000590:Amino Acid Chloromethyl Ketones / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002358:Cartilage, Articular / Q000293:injuries / Q000473:pathology / Q000502:physiology*; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000502:physiology*; D002478:Cells, Cultured; D003470:Culture Media; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D004285:Dogs; D004791:Enzyme Inhibitors / Q000378:metabolism; D006025:Glycosaminoglycans / Q000032:analysis; D020287:In Situ Nick-End Labeling; D019331:NG-Nitroarginine Methyl Ester / Q000378:metabolism; D009336:Necrosis; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000032:analysis
D000590:Amino Acid Chloromethyl Ketones; D003470:Culture Media; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D006025:Glycosaminoglycans; D009573:Nitrites; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D020169:Caspases; D019331:NG-Nitroarginine Methyl Ester