Abstract
Glutathione (GSH) is the major scavenger of reactive oxygen species (ROS) inside cells. We used live confocal imaging in order to clarify the role of GSH in the biology of the organ of Corti, the sensory epithelium of the cochlea, before, during and after the onset of hearing and in ~1 year old mice. GSH content was measured using monochlorobimane (MCB), a non-fluorescent cell permeant bimane that reacts with GSH, forming a fluorescent adduct through a reaction catalyzed by glutathione-S-transferase. GSH content increased significantly in inner hair cells during maturation in young adult animals, whereas there was no significant change in the outer hair cells. However, the GSH content in inner hair cells was significantly reduced in ~1 year old mice. The GSH content of supporting cells was comparatively stable over these ages. To test whether the GSH content played a significant protective role during ototoxicity, GSH synthesis was inhibited by buthionine sulfoximine (BSO) in organotypic cochlear explant cultures from immature mice. BSO treatment alone, which reduced GSH by 65 and 85% in inner hair cells and outer hair cells respectively, did not cause any significant cell death. Surprisingly, GSH depletion had no significant effect on hair cell survival even during exposure to the ototoxic aminoglycoside neomycin. These data suggest that the involvement of ROS during aminoglycoside-induced hair cell death is less clear than previously thought and requires further investigation.
Highlights
The tri-peptide gamma-glutamyl-cysteinyl-glycine is the major cellular antioxidant to combat reactive oxygen species (ROS)
Live images of MCBGSH fluorescence were acquired using multiphoton imaging in bullae from mice of different ages ranging from an immature stage prior to hearing onset (P4–6), at hearing onset (P14–16), in young adults (P33–47) and in ∼1 year old mice (Figures 2A–D)
The reduced glutathione (GSH) distribution varied across the different cell types in the organ of Corti with the most obvious differences being between supporting cells and hair cells, consistent with our recent work in neonatal cochlear explants (Blacker et al, 2014)
Summary
The tri-peptide gamma-glutamyl-cysteinyl-glycine (glutathione) is the major cellular antioxidant to combat reactive oxygen species (ROS). A major contributor to the oxidative damage inside the cell is H2O2, which can be converted to superoxide (O−2 ) and the hydroxyl radical (OH). Oxidative stress has long been associated with cellular damage that results from extracellular insults such as noise-induced hearing loss, aging and ototoxic drugs e.g. the aminoglycoside antibiotics and the cancer therapeutics, carboplatin and cisplatin (Yamane et al, 1995; Clerici et al, 1996). We used live imaging in ex vivo auditory bullae preparations to quantify the fluorescent product of a reaction between monochlorobimane (MCB) and endogenous GSH This allowed direct assessment of identifiable cells in a native, accessible environment in which the normal cell-cell interactions are maintained. In order to explore the role of GSH during aminoglycoside toxicity, we exposed organotypic cochlear explant cultures to the gammaglutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO) to deplete cellular GSH (Meister, 1983; Drew and Miners, 1984), challenged with neomycin and evaluated hair cell survival
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