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 w as examined in the mammalian cochlea following aminoglycoside treatment. Or ganotypic cultures were established from postnatal rats (P3) and treated wi th 1 mM neomycin sulfate for 12-48 h and analyzed using a live/dead assay u nder epifluorescence microscopy. Live hair sells were labeled with calcein, a probe whose fluorescence and cellular retention depends upon intracellul ar esterase activity and cell-membrane integrity, respectively. Hair cell d eath was determined by ethidium homodimer-1, a probe that fan enter cells w ith compromised cell membranes only. Inside the cell it binds to DNA. Hair cell morphology was also examined using phalloidin labeling, scanning elect ron microscopy sind semi-thin section analysis. Results showed that hair ce ll degeneration and hair cell death occurred in a time dependent gradient f rom base to apex. After 48 h of neomycin treatment, most apical hair cells survived while most basal hair cells died. Calcein labeling provides a sens itive functional assay for measuring hair cell survival. (C) 1999 Elsevier Science B.V. All rights reserved.