Otology & Neurotology 35:e187Ye194 Ó 2014, Otology & Neurotology, Inc.

Otoprotective Properties of Mannitol Against Gentamicin Induced Hair Cell Loss *John William Wood, *Esperanza Bas, *Chhavi Gupta, †Yamil Selman, *Adrien Eshraghi, *Fred F. Telischi, and *Thomas R. Van De Water *University of Miami Ear Institute, Department of Otolaryngology, University of Miami Miller School of Medicine, Miami, Florida; and ÞUniversity of Virginia School of Medicine, Charlottesville, Virginia, U.S.A.

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 KM 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 KM for all gene expression and CellRox experiments); 3) gentamicin +mannitol 100 mM; and 4) mannitol 100 mM for tumor necrosis factorY alpha (TNF->), TNF-> receptor (TNFR1A), interleukin-1 beta (IL-1A) 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 = j0.999 for inner hair cells and j0.972 for outer hair cells). Mannitol demonstrated dose-dependent otoprotection of IHCs and outer hair cells ( p G 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 G 0.05). TNF->, IL-1A TNFR1A, and COX-2 mRNA levels were upregulated by gentamicin ( p G 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 9 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. Key Words: AminoglycosideVHair cell lossVMannitolV OtoprotectionVOtotoxicityVReactive oxygen species.

Gentamicin is among the most commonly prescribed aminoglycosides for the treatment of gram-negative infec-

tions, tuberculosis, neonatal sepsis, and resistant bacteria (1Y3). Although the use of these medications has declined in recent decades, aminoglycoside therapy is still widely used in developing countries as they are inexpensive, highly effective, and widely available. Gentamicin is used also for the treatment of the vertigo experienced in intractable Me´nie`re’s disease by ablating the hair cells of the semicircular canals. Measurable hearing loss by audiometry after receiving gentamicin occurs in up to 25% of patients (4). The pattern of hair cell loss has been described as primarily in the outer hair cells and most severe in the basal aspect of the cochlea (5Y7). Using an energy-dependent process, aminoglycoside molecules enter cells, where they initiate the formation of free radicals using a pathway that has been

Otol Neurotol 35:e187Ye194, 2014.

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: [email protected] Disclosure: 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.

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implicated in the pathology of several inner ear disorders. The generation of reactive oxygen species involves the formation of an aminoglycoside-iron complex, which then catalyzes the production of reactive oxygen species from unsaturated fatty acids. This is noteworthy because several reports have concluded that the generation of reactive oxygen species is linked to ototoxicity of the aminoglycosides because of the ability of these free radicals to promote both apoptotic and necrotic cell death. Previous studies have focused on the effects of gentamicin on outer hair cells, whereas few have evaluated the extent of damage on inner hair cells (7Y9). A key regulator of apoptosis and the inflammatory cascade is TNF->, which is involved in the production of ototoxic levels of reactive oxygen species and has been demonstrated to be specifically upregulated in gentamicinmediated ototoxicity (10). IL-1A, a cytokine produced by macrophages, has also been demonstrated to be upregulated in organ of Corti explants challenged with gentamicin. Cycloxegenase-2 (COX-2) is a downstream inducible enzyme in the inflammatory cascade initiated by TNF-> and IL-1A (10,11). The aim of the present study is to evaluate the effects of gentamicin on inner hair cells as well as outer hair cells and to determine of the efficacy of mannitol to protect against gentamicin-initiated damage to the cochlea. Mannitol has historically been used for the reduction of intracranial pressure and the induction diuresis. However, it was noted that patients exhibited protection from organ injury when placed on mannitol, which could not be explained by its osmotic properties. Subsequently, numerous studies have demonstrated its cytoprotective properties in multiple organ systems (12Y18). Furthermore, it is effective in preserving cochlear function when challenged with anoxia. Mannitol protects against hair cell death when organ of Corti explants are challenged with TNF-> in vitro and has no deleterious effects on hair cells even at high concentrations (19,20). Although mannitol does not cross the blood-labyrinthine barrier, it does pass through the round window (21Y23). In addition to studies measuring mannitol within the cochlea after round window membrane application, its effectiveness in preserving outer hair cells function after transient ischemia has been demonstrated when applied in this manner (24). Therefore, we hypothesized that gentamicin would reduce aminoglycoside-induced hair cell loss in organ of Corti explants and that this would occur primarily through its antioxidant properties. MATERIALS AND METHODS This animal study was conducted with the approval of the Animal Care and Use Committee of the University of Miami (protocol 11-086) and fully complies with the NIH guidelines for the care and use of laboratory animals. Organ of Corti explants were harvested from 3-day-old rats. Explants were placed in serum-free media consisting of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with glucose (final conc. 6 g/L), N-1 supplement (1%), and penicillin G (500 U/mL). Organ of Corti explants were incubated at 37-C in 95% humidified atmosphere and 5% CO2 for either 96 hours (hair cell counts) or Otology & Neurotology, Vol. 35, No. 5, 2014

24 hours (gene expression analysis and imaging of total reactive oxygen species).

Protective Effect of Mannitol on Gentamicin-Induced Hair Cell Loss In vitro experiments were performed by exposing the organ of Corti explants to gentamicin and increasing concentrations of mannitol. By performing this in vitro, the entire organ of Corti is exposed to the same concentration of each chemical, reducing the possibility of anatomic differences affecting relative concentration. Previous studies have demonstrated no effect from mannitol alone on organ of Corti in vitro. Forty-five organ of Corti explants were placed into 5 experimental groups. In all groups with gentamicin, 10 KM was used. The groups include the following: 1) control (6 explants); 2) gentamicin (10KM, 6 explants); 3) gentamicin + mannitol 10 mM (7 explants); 4) gentamicin 50 mM (8 explants); and gentamicin+ mannitol 100 mM (8 explants). Explants were placed in the defined serum-free DMEM as described previously (with or without treatment). They were incubated at 37-C in 95% humidified atmosphere and 5% CO2. After 96 hours of incubation, the explants were fixed, stained with fluorescein isothiocyanate (FITC)Y labeled phalloidin and analyzed for the presence of viable hair cells (i.e., hair cells with stained intact cuticular plates and stereociliary bundles). Hair cell count analysis was designed with the primary aim of determining the otoprotective effect on outer hair cells exposed to gentamicin with or without mannitol explants. Inner hair cell effects were secondary aims. Therefore, a power analysis was performed assuming a mean population value of 50 outer hair cells with a standard deviation of 10 designed to detect at 20% reduction in hair number (based on previous work done in our lab). Alpha was set at 0.05 and beta 0.8. With this, we arrived at a sample size of 7 for each group. Given the technical difficulty of microdissecting organ of Corti explants from rat pup cochlea as well as the possibility of poor growth in culture medium, as would be manifest by supporting cell loss in addition to hair cell loss, we originally planned for 8 organ of Corti explants for each group. However, in the control group and gentamicin group, each had 2 organ of Corti explants that were unusable for analysis. At this point, we proceeded with statistical analysis to determine if further testing was needed, and given the strength of the statistics, we did not perform further testing.

Fixation and Staining The tissues were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer solution (PBS) for 48 hours at 4-C. The explants were washed 3 times in PBS and subsequently incubated in 5% normal goat serum, 1% Triton X-100 in PBS for 30 minutes at 25-C. They were then incubated with FITC-labeled phalloidin for 45 minutes at 25-C. After washing, the organ of Corti explants were transferred to glass slides with mounting medium, cover slips applied, and viewed under a Zeiss Axiovert 200 fluorescence microscope. An RT-Spot digital camera and imaging software was used for photography. Several segments of apical, middle, and basal turns were photographed, and the total hair cell count for each defined 220-Km segment was determined.

RNA Extraction and Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction Gentamicin-exposed explants demonstrate increased expression of proapoptotic genes within organ of Corti (10). We sought to evaluate the efficacy of mannitol to attenuate this upregulation.

MANNITOL PROTECTS AGAINST GENTAMICIN OTOTOXICITY TABLE 1. The 5’ and 3’ sets of primers used in the real-time RT-PCR study of gene expression level of the 6 listed genes Genes

Primer sets

TNF>

5’-AACTCGAGTGACAAGCCCGTAG-3 3’-GTACCACCAGTTGGTTGTCTTTGA-5’ TNF receptor superfamily 5’-GAACACCGTGTGTAACTGCC-3 member 1A (TNFR1A) 3’-ATTCCTTCACCCTCCACCTC-5’ Interleukin -1beta (IL-1A) 5’-ACCCAAGCACCTTCTTTTCC-3’ 3’-AGACAGCACGAGGCATTTTT-5’ IL1r1 5’-TGACCCAGGATCCACGATAC-3’ 3’-AGTCAGGAACTGGGTATAC-5’ A-actin 5’-CGTTGACATCCGTAAAGACC-3’ 3’-AGCCACCAATCCACACAGAG-5’ COX2 5’-CGTGTTGACGTCCAGATCACA-3’ 3’-GATTTAAGTCCACTCCATGGCC-5’

Sixty organ of Corti explants for each real-time RT-PCR experiment were cultured for 24 hours under the following conditions: saline (S); gentamicin (100 KM); gentamicin (100 KM) + mannitol (100 mM); and mannitol (100 mM). Four independent experiments were carried out (n = 4 explants/condition). RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA purity and concentration were determined by the absorbance at 260 and 280 nm using Nano Drop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized using iScript kit (Bio-Rad, Hercules, CA, USA). Quantitative realtime PCR was performed in duplicate using iQ SYBR Green Supermix (Bio-Rad) on iCycler Real-Time CFX96 Detection System (Bio-Rad). The mRNA level was normalized by housekeeping gene A-actin. The primers were designed based on the cDNA sequences obtained from Ensembl Genome Browser (http://www.ensembl.org), and all primers used for the genes studied (i.e., TNF->, TNFR1A, IL-1A, COX2, and A-actin) are presented in Table 1. Real-time PCR was carried out to 40 cycles at 95-C, 61-C, and 72-C, 50 seconds each. Melting curves were also performed to ensure primer specificity and evaluate for any contamination. Relative changes in mRNA levels of genes were assessed using the 2j$$CT method (25) and normalized to the housekeeping gene A-actin and then to the expression levels of control organ of Corti explants for calculation of mean fold change (MFC). As such, the MFC for all controls were standardized to equal 1.

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Aldrich) for 5 minutes at 25-C, following 3 additional washings with PBS and transferred to a glass slide with mounting medium, cover slipped, and viewed under a confocal Zeiss Axiovert 700 microscope. ImageJ 1.45h (http://imagej.nih.gov/ij) software was used for processing, analyzing and quantifying the CellRox Deep Red images. Mean values T SD from 6 images all from the basal turn area of the explants with a constant size area (i.e., 300 Km2) measured from each ROI (region of interest) for each of the groups presented in Figure 6B (n = 6/explant group).

Chemical Interaction Detection Escherichia coli were streaked onto agar plates and incubated at 37C for 24 hours under the following conditions: 1) saline, 2) gentamicin 500 KM; 3) gentamicin 500 KM + mannitol 100 mM, and 4) mannitol 100 mM. Plates were then analyzed and compared for bacterial growth.

Statistical Analysis For all hair cell count comparisons and RT-PCR, a 1-way ANOVA test followed by post hoc Tukey and Bonferroni tests for differences between means. For gene expression results, outliers were eliminated by calculations of Grubbs test (26). The correlation coefficient (Pearson´s r) was calculated to study the ototoxic effect of gentamicin along the organ of Corti. For mannitol dose response, a linear regression analysis was performed and R square calculated. For CellROX Deep Red, nonparametric analysis was performed using a Kruskal-Wallis test followed by Dunn’s multiple comparison test. All data are expressed as mean T SD, and a p G 0.05 was considered significant. All calculations were performed on GraphPad Prism v5.0 for Mac OS (La Jolla, CA, USA). Statistical methods and calculations were

Reactive Oxygen Species Detection CellROX Deep Red Reagent (Life Technologies Corporation) is a fluorogenic probe for measuring cellular oxidative stress in both live a fixed cell imaging. The cell-permeant dye is nonfluorescent while in a reduced state and exhibits fluorescence upon oxidation by reactive oxygen species (http://www.lifetechnologies.com). Twelve organ of Corti explants (n = 3 explants/group) were cultured for 24 hours. The groups were as follows: 1) control; 2) gentamicin (100 KM); 3) gentamicin (100 KM) + mannitol (100 mM); and 4) mannitol (100 mM). Then CellRox Deep Red (5 KM, Invitrogen) was added to each well and incubated at 37-C for 30 minutes. The samples were then washed 3 times with PBS fixed in 4% paraformaldehyde in 0.1 M PBS at 4-C for 48 hours. The explants were washed 3 times in PBS and subsequently incubated in 5% normal goat serum (Sigma Aldrich), 1% Triton X-100 (Fluka) in PBS for 30 minutes at 25-C. They were incubated with FITC-labeled phalloidin for 45 minutes at 25-C. After washing, the organ of Corti explants were incubated in 600 nM 4’,6-diamidino-2-phenylindole (DAPI) solution (Sigma

FIG. 1. Gentamicin induces greater hair cell losses with progression from the base to the apex of the cochlea. GM 0 KM represents the control group. GM 10 KM represents the gentamicin group. Pearson test demonstrated correlation in the increase of hair cell (HC) loss of both (A) inner hair cells and (B) outer hair cells, transitioning from the apex to base of the organ of Corti explants. For the gentamicin group: inner hair cells (apex 14.5 T 3.5, middle 14.1 T 3.5, base 10.2 T 2.9). Outer hair cells (apex 40 T 5.5, middle 35 T 3.6, base 14.75 T 4.57). Otology & Neurotology, Vol. 35, No. 5, 2014

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J. W. WOOD ET AL. observed ( p G 0.05) (Fig. 2, A and B). Linear regression analysis determined a dose-dependent response to mannitol treatment on hair cell protection from gentamicin ototoxicityinduced losses and was an excellent fit for a linear model in the case of outer hair cells (in basal turns R2 = 0.948) (Fig. 3, A and B).

FIG. 2. Mannitol preserves hair cells in organ of Corti explants treated with gentamicin at 50 and 100 mM for both (A) IHCs and (B) outer hair cells. Mannitol at 50 mM protected the IHCs and outer hair cells of the apical, middle, and in less extent the basal segments of the cochlea. Mannitol at the highest concentration tested (100 mM) protected all segments of inner hair cells and outer hair cells. A lower concentration of mannitol 10 mM was not otoprotective against gentamicin. organ of Corti explants 96 hour in vitro. Columns present mean values T SD (error bars). *p G 0.05, ***p G 0.001. Mean and standard deviation values are presented in Table 2.

Gene Expression Analysis TNF-> demonstrated significant upregulation when organ of Corti explants were exposed to gentamicin (MFC = 1.65 T 0.61, p G 0.05), whereas gentamicin (100 KM) + mannitol (100 mM) was equivalent to controls (MFC = 1.02 T 0.40, p 9 0.05). Although there was a clear trend of lower expression of TNF-> when comparing gentamicin alone (100 KM) to gentamicin + mannitol, this difference was not statistically significant. Mannitol alone (100 mM) did not alter the TNF-> gene expression levels in explants ( p 9 0.05) (Fig. 4A). TABLE 2. Mannitol preserves Hair Cells in organ of Corti explants treated with gentamicin at 50 and 100 mM for both A) IHCs and B) outer hair cells Inner hair cells

reviewed by the University of Miami Miller School of Medicine Biostatistical Core.

RESULTS Chemical Interaction Analysis Escherichia coli cultures demonstrated normal bacterial colony growth when grown in the presence of either saline or mannitol. Gentamicin alone and gentamicin + mannitol inhibited bacterial growth upon visual inspection, suggesting that mannitol does not alter the antibacterial characteristics of gentamicin. Hair Cell Counts Average number of hair cells was calculated for the apex,middle, and base of the organ of Corti explants exposed to gentamicin for both IHCs and outer hair cells. The correlation coefficient test demonstrated a clear pattern of increased hair cell (HC) loss of both IHCs (j0.9989) and outer hair cells (j0.9722) transitioning from the apex to base (Fig. 1, A and B), Overall, gentamicin exposure resulted in a 51.5% outer hair cells loss and a 32.8% inner hair cells loss. Outer hair cells and inner hair cells counts were separately performed for control, gentamicin, gentamicin + mannitol (10 mM), gentamicin + mannitol (50 mM), and gentamicin + mannitol (100 mM). The lowest dose tested for mannitol (10 mM) did not protect IHCs or outer hair cells from gentamicin-induced losses in any of the segments of the organ of Corti studied (i.e., apex, middle, and base). However, in gentamicin-exposed explants treated with mannitol at 50 mM and at 100 mM, a significant increase in total outer hair cells ( p G 0.001) and IHCs was Otology & Neurotology, Vol. 35, No. 5, 2014

Apex CONTROL Mean 18.3 SD 0.942809042 Gentamicin Mean 14.3 SD 3.399346342 GM + 10 mM Mannitol Mean 15.5 SD 2.179449472 GM + 50 mM Mannitol Mean 18.3 SD 2.2 GM + 100 mM Mannitol Mean 18.0 SD 0

Middle

Base

19.7 19.7 0.745355992 0.745355992 14.2 10.2 3.236081306 2.671869924 15.2 1.9

10.0 3.3

19.5 1

14.8 2.6

19.0 0.8

19.0 0.8

Middle

Base

63.50 8.00

65.33 8.30

32.67 8.10

18.00 12.60

40.20 7.20

26.14 7.20

57.63 9.40

35.00 9.30

63.67 3.80

64.33 5.20

Outer hair cells Apex Control Mean 59.50 SD 6.20 Gentamicin Mean 40.50 SD 6.30 GM + 10 mM Mannitol Mean 56.25 SD 6.80 GM + 50 mM Mannitol Mean 68.13 SD 18.50 GM + 100 mM Mannitol Mean 64.00 SD 0.00

Mannitol at 50 mM protected the IHCs and outer hair cells of the apical, middle and in less extent the basal segments of the cochlea. Mannitol at the highest concentration tested (100 mM) protected all segments of inner hair cells and outer hair cells. A lower concentration of Mannitol 10 mM was not otoprotective against gentamicin. organ of Corti explants 96 hours in vitro.

MANNITOL PROTECTS AGAINST GENTAMICIN OTOTOXICITY

FIG. 3. Mannitol otoprotection is dose dependent. Linear regression analysis and Pearson’s correlation coefficient determined a dose-dependent response to mannitol treatment on (A) inner hair cells and (B) outer hair cells preservation and was an excellent fit for a linear model in the case of outer hair cells in the basal area.

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IL-1A gene expression demonstrated a similar pattern to TNF->. There was an associated increase in IL-1A expression when explants were exposed to gentamicin alone (MFC = 2.43 T 1.50; p G 0.05) as compared with control expression levels. Gentamicin + mannitol expression levels for IL-1A were equivalent to control explant levels (MCF = 1.81 T 1.50; p 9 0.05), but when compared with gentamicin alone, the difference was not significant. Mannitol alone did not alter the expression of IL-1A (MFC = 0.89 T 0.59; p 9 0.05; Fig. 4B). Expression levels of TNFR1A also demonstrated a significant difference when control and gentamicin-treated groups of explants were compared ( p G 0.05). Interestingly, mannitol treatment induced an increase of TNFR1A mRNA levels when compared with controls ( MFC = 2.04 T 0.63; p G 0.05); however, when the TNFR1A levels between the gentamicin and the gentamicin + mannitol explants were compared, there was no significant difference (p 9 0.05; Fig. 4C). COX-2 expression levels also did not reveal a significant difference between the gentamicin and the gentamicin + mannitol groups of explants ( p 9 0.05; Fig. 4D). Oxidative Stress Detection Organ of Corti explants exposed to gentamicin alone demonstrated an increase in reactive oxygen species (i.e.,

FIG. 4. Gene expression studies. organ of Corti explants 24 hour in vitro (vontrol-untreated; gentamicin, 100KM; gentamicin, 100 KM + mannitol, 100 mM; mannitol, 100 mM). Gentamicin exposure upregulates (A) TNF-> and (B) IL-1A mRNA levels. Mannitol treatment reduces the mRNA levels of these cytokines, but the difference does not achieve statistical significance. Gentamicin-induced TNFR1A upregulation (C), treatment with mannitol increased these levels but not significantly (p 9 0.05). However, mannitol only treatment induced an increase of TNFR1A mRNA levels when compared with the controls. Mannitol treatment did not alter the increased mRNA levels of the inducible enzyme COX-2 observed in organ of Corti treated with gentamicin (D). Columns represent mean values T SD (error bars). *p G 0.05, **p G 0.01. Mean and standard deviation values presented in Table 3. Otology & Neurotology, Vol. 35, No. 5, 2014

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TABLE 3. Gene expression studies. organ of Corti explants 24 hour in vitro (Control-untreated; gentamicin, 100mM; gentamicin, 100 KM + mannitol, 100 mM; mannitol, 100 mM) Gene

Group

Mean fold change

SD

IL-1B

Control GM GM+MT MT Control GM GM+MT MT Control GM GM+MT MT Control GM GM+MT MT

1.000 2.588 1.568 0.890 1.000 1.654 1.020 1.339 1.000 1.434 2.137 1.715 1.000 1.683 1.278 1.747

0.000 0.313 0.568 0.297 0.000 0.205 0.126 0.260 0.000 0.154 0.211 0.225 0.000 0.190 0.113 0.156

TNFa

TNFR1

COX-2

Gentamicin exposure upregulates A) TNF-> and B) IL-1A mRNA levels, mannitol treatment reduces the mRNA levels of these cytokines, but the difference does not achieve statistical significance. C) Gentamicin induced TNFR1A upregulation, treatment with mannitol increased these levels but not significantly (p 9 0.05). However, mannitol only treatment induced an increase of TNFR1A mRNA levels when compared with the controls D) Mannitol treatment did not alter the increased mRNA levels of the inducible enzyme COX-2 observed in organ of Corti treated with gentamicin.

CellRox Deep Red signal levels) when compared with the control group levels. Cells exposed to gentamicin-alone had increased perinuclear staining with swollen nuclei and apoptotic bodies. The addition of mannitol (gentamicin + mannitol) to the gentamicin-exposed organ of Corti explants resulted in decreased reactive oxygen species levels (i.e., Deep Red signal surrounding nuclei) and an absence of apoptotic bodies (Fig. 5A). Quantification of the CellRox Deep Red signal levels supports the confocal observations that gentamicin exposure results in a significant increase of reactive oxygen species ( p G 0.05) and that treatment of the gentamicin-exposed explants with mannitol causes a significant reduction in reactive oxygen species levels in the treated explants ( p G 0.05). Treatment of control explants with mannitol alone did not significantly reduce the level of reactive oxygen species when compared with control explant levels of reactive oxygen species, that is, CellRox Deep Red staining ( p 9 0.05; Fig. 5B). DISCUSSION Mannitol is a hyperosmolar organic molecule, primarily used for its ability to alter fluid dynamics. It also has been shown to have powerful intracochlear effects, such as increased blood flow, decreased microvascular resistance, and increased oxygenation (17,18,24). Changes in fluid dynamics were originally thought to be the mechanism by which mannitol reduced cell death in various organ systems. However, mannitol was later shown to be a potent free radical scavenger. This was observed in models of brain injury as mannitol demonstrated cytoprotection in many organ systems among patients receiving the drug for traumatic Otology & Neurotology, Vol. 35, No. 5, 2014

brain injury. Although this protective action was originally thought to be secondary to the abundant hydroxyl groups of mannitol, this was later demonstrated to be incorrect as mannitol through the free radical scavenging action of its hydroxyl groups prevents the formation of reactive oxygen species and therefore, the initiation of mitochondrial damage and subsequent activation of a cell death program. This is in contrast to dexamethasone, which reduces the endtarget effects of the inflammatory cascade by mediating the effects of hydroperoxide byproduct and blocks the initiation of multiple cell death pathways (12,16). When applied at the round window, mannitol has been shown to improve DPOE’s and compound action potential thresholds in animal models of ischemia and hypoxia (19,24). These studies demonstrated 2 significant properties of mannitol with respect to hearing preservation. First, the clinical application of mannitol at the round window is a feasible approach for intracochlear drug delivery. This has been confirmed by further studies measuring the mannitol within the cochlea after RW application (21Y23). Second, these reports suggest that mannitol otoprotective activity may be independent of its osmotic/vascular effects. We have previously demonstrated in organ of Corti explants that mannitol is safe for HC viability in doses up to 100 mM (20). In the current preliminary study, we observed that mannitol prevents gentamicin-induced cell death of both IHCs and outer hair cells in a dose-dependent manner. This otoprotection seems to be mainly through mediation of reactive oxygen species pathways, rather than a direct physical interaction between mannitol and reactive oxygen species molecules. gentamicin, in contradistinction to cisplatin, has been shown to cause ototoxicity by formation of reactive oxygen species through the superoxide radical. The effects of gentamicin-induced reactive oxygen species in the organ of Corti explants are of increasing severity from the base to apex of outer hair cells (1). The sensitivity of basal turn hair cells has been linked to lower levels of glutathione as compared with levels of this antioxidant in the apex. The formation of reactive oxygen species results in the upregulation of inducible inflammation cascadeYassociated enzymes such as TNF-> and IL-A and, ultimately, in increased levels of apoptosis of affected HCs. TNF-> activates the intrinsic pathway for programmed cell death through multiple pathways. This may occur through several pathways including the caspase cascade, upregulation of proapoptotic Bax gene expression levels and c-Jun-N-terminal kinase (JNK) signaling, and downregulation of antiapoptotic genes, for example, Bcl-2. Infante-Bas and colleagues previously demonstrated that HCs from organ of Corti explants exposed TNF-> were preserved by the addition of mannitol, and this occurred by the inhibition of TNF-> induced JNK signaling (20). The current study demonstrates that both TNF-> and IL-1A upregulation by gentamicin were not significantly reduced by mannitol treatment ( p 9 0.05) and that the observed reduction in their expression level therefore represents only a trend. All statistical measures were reviewed with the University of Miami Biostatistics Core. We discussed eliminating

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FIG. 5. Mannitol reduces oxidative stress in hair cells. organ of Corti explants 24 hour in vitro (control-untreated; gentamicin, 100KM; gentamicin, 100 KM + mannitol, 100 mM; mannitol, 100 mM). A, Cells form organ of Corti explants exposed to gentamicin-alone had increased CellRox Deep Red perinuclear staining with swollen nuclei and apoptotic bodies. The addition of mannitol (gentamicin+mannitol) resulted in decreased red staining surrounding nuclei and absence of apoptotic bodies. B, Quantification of the red signal intensity (arbitrary units) in a ROI of 300 Km2. There is an increase of the red signal in gentamicin (ROI 19.56 T 4.3 ) treated group compared with controls (ROI 7.0 T 3.8) (p G 0.001), which is reversed by treatment with mannitol (gentamicin + mannitol ROI 16.77 T 8.1) to control levels (control ROI 4.6 T 2.1). Columns represent mean values T SD (error bars). *p G 0.05. ***p G 0.001.

groups within each test such that the comparison would only be between ‘‘gentamicin with mannitol 100 mmol’’ or ‘‘gentamicin alone.’’ This would have increased the sensitivity of the test to detect a difference between means by allowing use of a Student’s t test or Wilcoxon rank sum for nonparametric data. However, this would have reduced the rigor of the experiment, which showed that only as dose increased to sufficient level was a difference observed. Although ANOVA demonstrates that difference exists between some of the groups in the experiment alone, it cannot determine between which groups there is difference (i.e., the only difference could be between the control and gentamicin alone). A standard tool for determining between which groups the difference exists is post hoc testing. After discussion with the Biostatistics Core, this method was confirmed to be a correct and rigorous manner of comparison between means. It was suggested to confirm the previously performed Bonferroni tests with nonparametric testing (Kruskal Wallace), which was conducted with the same results.

The implications of the current study are significant. This is the first study to demonstrate the ability of mannitol to prevent inner and outer hair cell losses induced by a widely used clinical pharmacologic agent (i.e., gentamicin) through its antioxidant properties, that is, through a significant reduction ( p G 0.05) of reactive oxygen species levels in gentamicin-exposed organ of Corti explants compared with gentamicin only explant reactive oxygen species levels. Overall, gentamicin causes a significant increase in reactive oxygen species levels in the gentamicin-exposed explants and treatment of the gentamicin explants with the addition of mannitol results in a significant reduction in reactive oxygen species levels. The action of mannitol treatment on gentamicin-exposed explants did not appear to have a significant effect on the expression levels of inflammation related genes, for example, TNF>. Gentamicin use has recently declined in the United States, but it continues to be a necessary treatment option in many clinical scenarios and is widely used in the developing world. Up to 25% of patients receiving gentamicin Otology & Neurotology, Vol. 35, No. 5, 2014

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experience hearing loss, making this a major cause of hearing loss worldwide. Mannitol is an inexpensive and widely available medication that may reduce the ototoxic effects of this antibacterial medication. The site of action of mannitol on a reduction in insultinitiated reactive oxygen species as well as its osmotic properties may broaden its clinical applicability for other types of otologic insults (cisplatin, cochlear implant insertion trauma, sudden hearing loss, etc). Mannitol may prove to be an alternative in patients who fail intratympanic steroid therapy. Furthermore, because of the fact that mannitol appears to work through different mechanisms than dexamethasone, one may speculate that they may work synergistically or that specific conditions may be identified for which mannitol is more advantageous. Future studies are in process to assess the ability of mannitol to provide improved hearing outcomes in vivo. This has significant clinical applicability as gentamicin is a widely used antibiotic associated with hearing loss. Furthermore, mannitol may prove to be beneficial with other etiologies of hearing loss such as sudden sensorineural hearing loss, cochlear implantation insertion trauma and against HC loss that can result from exposure of the cochlea to other ototoxic medications that impact the level of oxidative stress. This study has 3 main limitations. This is an in vitro study that does not take into consideration all of the physiological mechanisms involved in vivo. In this study, organ of Corti explants are bathed in culture media, and the access of both the gentamicin and the mannitol added to these explants may be greater than would be expected to occur in vivo where these compounds would have to pass through the round window membrane and then from the perilymph of the scala tympani into the fluid spaces of the organ of Corti. The second point is that the explant tissue is derived from 3-day-old rats and, therefore, does not represent mechanisms involved in adult tissue of another species. Finally, this is only a preliminary study using a limited number of organ of Corti; these promising results need to be reproduced on a larger number of organ of Corti and an in vivo animal model before definitive conclusions can be drawn (27,28). CONCLUSION Mannitol seems to protect auditory hair cells against gentamicin ototoxicity. This otoprotection is in a dosedependent manner within organ of Corti explants. This effect is accomplished, although its antioxidant properties, which reduces the formation of reactive oxygen species that are induced by exposure to gentamicin. REFERENCES 1. Bertolaso L, Bindini D, Previati M, et al. Gentamicin-induced cytotoxicity involves protein kinase C activation, glutathione extrusion and malondialdehyde production in an immortalized cell line from the organ of Corti. Audiol Neurootol 2003;8:38Y48. 2. Nordang L, Anniko M. Nitro-L-arginine methylester: a potential protector against gentamicin ototoxicity. Acta Otolaryngol 2005;125:1033Y8. Otology & Neurotology, Vol. 35, No. 5, 2014

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