Determination of hair cell degeneration and hair cell death in neomycin treated cultures of the neonatal rat cochlea

The spatial-temporal course of hair cell degeneration and hair cell death was examined in the mammalian cochlea following aminoglycoside treatment. Organotypic cultures were established from postnatal rats (P3) and treated with 1 mM neomycin sulfate for 12-48 h and analyzed using a live/dead assay under epifluorescence microscopy. Live hair sells were labeled with calcein, a probe whose fluorescence and cellular retention depends upon intracellular esterase activity and cell-membrane integrity, respectively. Hair cell death was determined by ethidium homodimer-1, a probe that fan enter cells with compromised cell membranes only. Inside the cell it binds to DNA. Hair cell morphology was also examined using phalloidin labeling, scanning electron microscopy sind semi-thin section analysis. Results showed that hair cell degeneration and hair cell death occurred in a time dependent gradient from base to apex. After 48 h of neomycin treatment, most apical hair cells survived while most basal hair cells died. Calcein labeling provides a sensitive functional assay for measuring hair cell survival. (C) 1999 Elsevier Science B.V. All rights reserved.