Rat disc organ culture was carried out as we previously described (35 (link)). Ex vivo, discs were pretreated with 100 μM RV for 24 h or 2 mM NAC for 12 h or 100 μM PTIO for 12 h, then treated with or without 1 mM SNP for 18 h. Then the harvested discs were fixed in 4% paraformaldehyde, and then decalcified with EDTA for 2 weeks. After embedding with paraffin, 5-μm thick serial mid-sagittal sections of discs were made for slides. Mid-sagittal sections of discs were analyzed for apoptosis using in situ cell death detection kit according to manufacturer's instructions. DAPI staining was conducted for indication of total cells. TUNEL-positive apoptotic cells and DAPI-positive total cells of NP area on mid-sagittal sections of discs were identified by fluorescent microscope (IX71; Olmypus), and apoptosis rate was calculated as the percentage of numbers of TUNEL-positive cells to the numbers of total cells using IPP version 6.0 software (Media Cybernetics). The quantitative analysis was performed on three ×200 fields/section (three sections/disc and three discs for each kinds of treatment).