Figure 2
Distribution of gentamicin-conjugated Texas Red (GTTR) in cochlear explants after treatment in vitro. (A) Whole cochlear explants on a collagen matrix were treated with (a) Texas Red (TR; 1.8 μM) or (b) GTTR (500 μM total including unconjugated gentamicin) for 30 min and fixed. The explants were embedded in paraffin and cut into 4-μm-thick sections. Specimens were deparaffinized and incubated with 4′,6-diamidino-2-phenylindole (DAPI) to observe the nucleus. Inner hair cells (IHCs) and outer hair cells (OHCs) displayed strong GTTR fluorescence intensity in the cytosol (IHCs: arrowhead, OHCs: arrow). Weak diffuse GTTR fluorescence was observed in the IHCs and OHCs nuclei. However, supporting cells displayed faint GTTR fluorescence intensity: Hensen’s cell (h), cells of Claudius (c), Deiter’s cells (d), pillar cells (p) and basilar membrane (large arrow). (B) Cochlear explants were cultivated on cover glasses and treated for 30 min with 500 μM GTTR (a, b, e), 1.8 μM TR (c) and 500 μM gentamicin plus 1.8 μM TR (d). After fixation, the explants were stained with fluorescein isothiocyanate (FITC)–phalloidin (1:1000) and observed under a fluorescent microscope. Whole cochlear explants were obtained from postnatal day 3 (P3) rats to further examine this base-to-apex gradient of gentamicin uptake in cochlea (e). After removing the modiolus, the whole cochlear explant was incubated with 500 μM GTTR for 120 min. The specimens were observed under a fluorescent microscope after fixation.
