Figure 6.

Elevated ROS levels cause reduced proliferation and an elongated hypertrophic zone in growth plates of Atm^−/− mice. (A) Three Atm ^−/− mice were treated subcutaneously with PBS and three were treated with NAC (1 g/kg, every 2 d) for 2 wk from the day of birth, and intracellular ROS concentration was evaluated by dihydroethidium staining in growth plates. Growth plates are shown between the dotted lines. (right) Data are mean relative concentrations ± SD of ROS in chondrocytes in mice treated with NAC compared with PBS-treated mice. Increased intracellular ROS and its reduction by NAC treatment were observed in the chondrocytes of Atm ^−/− mice. *, P < 0.01. Bar, 100 μm. (B) Three Atm ^−/− mice were treated subcutaneously with PBS and three were treated with NAC (1 g/kg, every 2 d) for 2 wk from the day of birth, and chondrocyte proliferation was evaluated by BrdU staining. (left) Representative data are shown. Growth plates are shown between the dotted lines. (right) Data are mean relative numbers ± SD of BrdU-positive chondrocytes in Atm ^−/− mice treated with NAC compared with PBS-treated Atm ^−/− mice. *, P < 0.01. Bar, 100 μm. (C) Three Atm ^−/− mice were treated subcutaneously with PBS and three were treated with NAC (1 g/kg, every 2 d) for 3 wk from the day of birth, and chondrocyte hypertrophy was analyzed by Alcian blue staining. The hypertrophic zone (H) was reduced in NAC-treated compared with PBS-treated mice. (left) Representative data are shown. (right) Data are mean relative lengths ± SD of the hypertrophic zone in Atm ^−/− growth plates treated with NAC for 3 wk compared with that of PBS-treated Atm ^−/− mice. *, P < 0.01. Bar, 50 μm. (D) Three Atm ^−/− mice were treated subcutaneously with PBS and three were treated with NAC (1 g/kg, every 2 d) for 3 wk from the day of birth, and chondrocyte apoptosis was evaluated by TOTO3 nuclear staining. The number of apoptotic cells was reduced in NAC-treated compared with PBS-treated mice. Data are mean relative numbers ± SD of apoptotic chondrocytes in Atm ^−/− mice treated with NAC compared with those of PBS-treated Atm ^−/− mice. *, P < 0.01. (E) Three Atm ^−/− mice were treated subcutaneously with PBS and three were treated with NAC (1 g/kg, every 2 d) for 2 wk from the day of birth, and angiogenesis was analyzed by PECAM-positive endothelial cells. Frozen sections were stained with rat anti-PECAM antibody, followed by Alexa Fluor 488–conjugated goat anti–rat Ig antibody, and observed by phase contrast (left) or confocal (middle and right) microscopes. Representative data are shown. Bar, 50 μm.