Fig. 4.

Representative Z-plane photomicrographs showing uptake of AM1-43 (red) into vestibular hair cells (HC) in macula of utricle. Nuclei of cells labeled with ToPro-3 (blue) to label nuclei and Alex Fluor 488-conjugated phalloidin (green) to identify hair cell stereocilia and cuticular plate. (A) In controls (0 μM cisplatin), robust AM1-43 labeling was present in hair cell cytoplasm, but was absent from support cells (SC) beneath the hair cells. (B) After 48 h treatment with 10 μM cisplatin, AM1-43 was reduced or absent from hair cells with damaged or missing stereocilia (arrowhead). After 48 h treatment with 50 μM cisplatin, many hair cells were damaged (dotted circles around shrunken nuclei) and only small, scattered puncta of AM1-43 were present in damaged areas of the epithelium. (D) After 48 h treatment with 1000 μM cisplatin, most hair cells were intact (large nuclei, phalloidin label on apical surface of hair cells); however, AM1-43 was absent from hair cells. (E) Cultures treated for 18 h with 50 μM cisplatin; then culture medium was replaced with standard culture medium and cultured an additional 18 h after which AM1-43 was added to medium. Note damage to stereocilia and shrunken nuclei (dotted circle) due to low-dose cisplatin. (F) Same conditions as E except cisplatin dose was 1000 μM cisplatin. Note robust AM1-43 labeling in cytoplasm and phallodin labeling of cuticular plate of hair cells, which appear normal.