Vulnerability to ROS-induced cell death in ageing articular cartilage: the role of antioxidant enzyme activity.
OBJECTIVE
To test the hypothesis that age-related loss of chondrocytes in cartilage is associated with impaired reactive oxygen species (ROS) homeostasis resulting from reduced antioxidant defence.


METHODS
Cell numbers: The total number of chondrocytes in the articular cartilage of the femoral head of young, mature and old rats was estimated using an unbiased stereological method. ROS quantification: Fluorescence intensity in chondrocytes was quantified using the oxygen free radical sensing probe dihydrorhodamine 123 (DHR 123), confocal laser scanning microscopy and densitometric image analysis. In order to delineate the reactive species, explants were pre-treated with N-acetylcysteine (NAC) or N(G)-nitro-l-arginine methyl ester (l-NAME) prior to ROS quantification. Induction of intracellular ROS: Explants were incubated in the redox-cycling drug menadione after which they underwent ROS quantification and cell-viability assay. Antioxidant enzyme activity: The activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPX) was measured.


RESULTS
Chondrocyte numbers: A significant and progressive loss of chondrocytes was observed with ageing. Cellular ROS levels: A significant age-related increase in cellular ROS-induced fluorescence was demonstrated. NAC significantly reduced ROS levels in old chondrocytes only. Induction of intracellular ROS: Menadione increased cellular ROS levels dose-dependently in young and old chondrocytes, with a greater effect in the latter. Old chondrocytes were more vulnerable to menadione-induced cytotoxicity. Antioxidant enzymes: Catalase activity declined significantly in aged cartilage whilst SOD and GPX activities were unaltered.


CONCLUSIONS
Substantial loss of chondrocytes occurs in rat articular cartilage which may result from increased vulnerability to elevated intracellular ROS levels, consequent upon a decline in antioxidant defence.
Department of Plastic Surgery, Royal Free Hospital, Pond Street, London, NW3 2QG, UK.
N
N
Jallali
H
H
Ridha
C
C
Thrasivoulou
C
C
Underwood
P E M
PE
Butler
T
T
Cowen
D000818:Animals; D002358:Cartilage, Articular / Q000187:drug effects* / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects*; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000378:metabolism; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism
D017382:Reactive Oxygen Species; D002374:Catalase; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase