title: Otoprotective properties of mannitol against gentamicin induced hair cell loss.
abstract: BACKGROUND
  Gentamicin is a widely used antibiotic, which causes hearing loss because of destruction of auditory hair cells. Mannitol has been shown to have cytoprotective properties in the cochlea both in vitro and in vivo. Mannitol has been shown to be safe in concentrations up to 100 mM in organ of Corti explants. It is proposed as an otoprotective agent against gentamicin ototoxicity.
  
  
  METHODS
  Organ of Corti were dissected from P-3 rat pups and cultured under the following conditions for 96 hours: 1) control, 2) gentamicin (10 μM for all hair cell count experiments), 3) gentamicin + mannitol 10 mM, 4) gentamicin + mannitol 50 mM, and 5) gentamicin + mannitol 100 mM. The tissues were then fixed and stained, and hair cells were counted for segments of the apex, middle, and basal turns. Quantitative RT-PCR (qRT-PCR) was performed on organ of Corti explant extracted RNA after 24 hours in vitro: 1) control; 2) gentamicin (100 μM for all gene expression and CellRox experiments); 3) gentamicin +mannitol 100 mM; and 4) mannitol 100 mM for tumor necrosis factor-alpha (TNF-α), TNF-α receptor (TNFR1A), interleukin-1 beta (IL-1β) and cyclooxygenase-2 (COX-2). In vitro examination of oxidative stress was performed for the same test groups at 24 hours using CellRox Deep Red assay.
  
  
  RESULTS
  Gentamicin induced loss of both inner hair cells and outer hair cells with increasing severity from apex to middle to basal segments (Pearson r = -0.999 for inner hair cells and -0.972 for outer hair cells). Mannitol demonstrated dose-dependent otoprotection of IHCs and outer hair cells (p < 0.001 for mannitol at 100 mM). CellRox demonstrated increased oxidative stress induced by gentamicin exposure, and this effect was attenuated by treatment of gentamicin-exposed explants with mannitol (p < 0.05). TNF-α, IL-1β TNFR1A, and COX-2 mRNA levels were upregulated by gentamicin (p < 0.05). Mannitol treatment of gentamicin explants downregulated the gene expression of the proinflammatory cytokines, but this difference did not achieve significance. Interestingly, in gentamicin-challenged organ of Corti explants, Mannitol upregulated the expression of TNFR1A, but this increase did not achieve significance (p > 0.05).
  
  
  CONCLUSIONS
  Gentamicin ototoxicity is increasingly severe from the apex to basal turn of the cochlea. Treatment with mannitol prevents gentamicin-induced hair cell loss in a dose-dependent manner, protecting both IHCs and outer hair cells. Mannitol appears to act as a free radical scavenger to reduce the cytotoxic effects of gentamicin by reducing the level of oxidative stress.
authors.affiliation: *University of Miami Ear Institute, Department of Otolaryngology, University of Miami Miller School of Medicine, Miami, Florida; and †University of Virginia School of Medicine, Charlottesville, Virginia, U.S.A.
authors.forename: John William
authors.initials: JW
authors.lastname: Wood
authors.forename: Esperanza
authors.initials: E
authors.lastname: Bas
authors.forename: Chhavi
authors.initials: C
authors.lastname: Gupta
authors.forename: Yamil
authors.initials: Y
authors.lastname: Selman
authors.forename: Adrien
authors.initials: A
authors.lastname: Eshraghi
authors.forename: Fred F
authors.initials: FF
authors.lastname: Telischi
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Van De Water
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D051546:Cyclooxygenase 2 / Q000378:metabolism; D005839:Gentamicins / Q000494:pharmacology*; D006198:Hair Cells, Auditory / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D053583:Interleukin-1beta / Q000378:metabolism; D008353:Mannitol / Q000494:pharmacology*; D009925:Organ of Corti / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha
chemical_list: D000900:Anti-Bacterial Agents; D005839:Gentamicins; D053583:Interleukin-1beta; D017382:Reactive Oxygen Species; D047888:Receptors, Tumor Necrosis Factor, Type I; C506235:TNFRSF1A protein, human; D014409:Tumor Necrosis Factor-alpha; D008353:Mannitol; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human