Otoprotective properties of mannitol against gentamicin induced hair cell loss

John William Wood, Esperanza Bas Infante, Chhavi Gupta, Yamil Selman, Adrien Eshraghi, Fred F Telischi, Thomas R Van De Water

Research output: Contribution to journalArticle

15 Citations (Scopus)

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). 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.

Original languageEnglish
JournalOtology and Neurotology
Volume35
Issue number5
DOIs
StatePublished - Jan 1 2014

Fingerprint

Alopecia
Mannitol
Gentamicins
Outer Auditory Hair Cells
Inner Auditory Hair Cells
Organ of Corti
Cochlea
Oxidative Stress
Auditory Hair Cells
Tumor Necrosis Factor-alpha
Free Radical Scavengers
Tumor Necrosis Factor Receptors
Cyclooxygenase 2
Interleukin-1beta
Hearing Loss
Cell Count
RNA
Anti-Bacterial Agents

Keywords

  • Aminoglycoside
  • Hair cell loss
  • Mannitol
  • Otoprotection
  • Ototoxicity
  • Reactive oxygen species

ASJC Scopus subject areas

  • Otorhinolaryngology
  • Clinical Neurology
  • Sensory Systems

Cite this

Otoprotective properties of mannitol against gentamicin induced hair cell loss. / Wood, John William; Bas Infante, Esperanza; Gupta, Chhavi; Selman, Yamil; Eshraghi, Adrien; Telischi, Fred F; Van De Water, Thomas R.

In: Otology and Neurotology, Vol. 35, No. 5, 01.01.2014.

Research output: Contribution to journalArticle

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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). 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.",
keywords = "Aminoglycoside, Hair cell loss, Mannitol, Otoprotection, Ototoxicity, Reactive oxygen species",
author = "Wood, {John William} and {Bas Infante}, Esperanza and Chhavi Gupta and Yamil Selman and Adrien Eshraghi and Telischi, {Fred F} and {Van De Water}, {Thomas R}",
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journal = "Otology and Neurotology",
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T1 - Otoprotective properties of mannitol against gentamicin induced hair cell loss

AU - Wood, John William

AU - Bas Infante, Esperanza

AU - Gupta, Chhavi

AU - Selman, Yamil

AU - Eshraghi, Adrien

AU - Telischi, Fred F

AU - Van De Water, Thomas R

PY - 2014/1/1

Y1 - 2014/1/1

N2 - 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.

AB - 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.

KW - Aminoglycoside

KW - Hair cell loss

KW - Mannitol

KW - Otoprotection

KW - Ototoxicity

KW - Reactive oxygen species

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