Figure 5.

Detection of superoxide by DHE fluorescent labeling. Specimens labeled with To-Pro-3 to label nuclei (blue), DHE (red) or Alexa 488-conguated phalloidin (green) to label the stereocilia and cuticular plate of hair cells. (A) Z-plane section from untreated cochlear explant cultured for 24 h shows negligible DHE fluorescent labeling in support cell layer (SC, bracket) and outer hair cells (OHCs) and inner hair cells (IHCs). (B) Treatment for 6 h with 200 μM induces DHE fluorescence (fuchsia color represents overlap of red DHE and blue To-Pro-3, white arrowhead) mainly in SC layer beneath the hair cells including cells beneath the basilar membrane. (C) Treatment for 12 h with 200 μM PQ induces strong DHE fluorescence in SC layer (bracket) including cells beneath the OHC and IHC and cells beneath the basilar membrane. (D) Treatment for 24 h with 200 μM PQ induces DHE fluorescence in OHC region, but little DHE fluorescence in the SC layer; destruction of support cells in deeper layers of the epithelium eliminates fluorescence from this region. (E) Mean gray level measures of superoxide in cochlear explants in untreated group cultured for 24 h, 200 μM PQ treatment for 6 h, 12 h, and 24 h groups. Horizontal lines identify between hair cells and supporting cells differences that were statistically significant (Newman-Keuls, p<0.05).