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).
Conclusion: 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.
*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.
Address correspondence and reprint requests to Thomas R. Van De Water, Ph.D., Cochlear Implant Research Program, University of Miami Ear Institute, 1600 NW 10th Avenue, RMSB 3160, Miami, FL 33136-1015; E-mail: firstname.lastname@example.org
Dr. Van De Water has a preclinical grant from MED-EL Hearing Devices Company, Innsbruck, Austria.
Dr. Telischi is a paid consultant for MED-EL Hearing Devices Company and Cochlear Americas.
Dr. Eshraghi has a preclinical grant from MED-EL Hearing Devices Company, Innsbruck, Austria.
John William Wood and Esperanza Bas these authors contributed equally to this paper.
The authors disclose no conflicts of interest.