Molecular and quantitative spatial analysis of aminoglycoside-induced hair cell death and regeneration in the avian cochlea

Sensory hair cells in the cochlea convert fluid movement resulting from sound pressure waves into neural signals to relay information about sound to the brain. Damage to these sensory receptors can result in permanent hearing loss in humans. A leading cause of sensory hair cell death is exposure to aminoglycoside antibiotics (e.g. gentamicin), which are clinically used to treat bacterial infection despite their known ototoxicity. The avian cochlea is a useful animal model to study aminoglycoside-induced sensory hair cell death due to structural similarities to the mammalian cochlea and similar susceptibility to aminoglycosides. The avian cochlea also has the capacity to regenerate lost hair cells via induction of neighboring supporting cells to re-enter the cell cycle and repopulate the damaged epithelium, unlike the mammalian cochlea. The aims of this dissertation are to study the mechanisms of aminoglycoside-induced hair cell death in the avian cochlea and the subsequent replacement of damaged hair cells via supporting cell proliferation. A subcutaneous gentamicin injection (300 mg/kg) induced a proximal to distal progression of hair cell loss in the avian cochlea. Immunohistochemical labeling of avian cochlear preparations and confocal analysis were utilized to demonstrate the primary mechanism of hair cell death was via caspase-mediated apoptosis. A novel segmentation algorithm was developed to process confocal images of the avian cochlea to assess hair cell density across the surface of the sensory epithelium. This algorithm was utilized to demonstrate the opposite patterns of hair cell loss in the avian cochlea following systemic gentamicin treatment in vivo versus exposure to gentamicin in vitro and the rapid hair cell loss along the neural edge of the sensory epithelium following placement of explanted cochleae into the culture environment. Finally, the spatio-temporal progression of hair cell regeneration following systemic gentamicin treatment was analyzed using 3D confocal image stacks. The results of this analysis indicated a proximal to distal and inferior to superior progression of hair cell replacement that mimicked the progression of hair cell loss. However, no specific pattern of initial progenitor cell location within the depth of the sensory epithelium was determined.