For breeding, sexually mature male (6-week-old) and female (8-week-old) CBA/J mice were purchased from Harlan Sprague Dawley Incorporation (Indianapolis, IN, USA). For KM in vivo trials, male CBA/J mice at 4 weeks of age were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Male Hartley guinea pigs (200–250 g) were purchased from Charles River (Wilmington, MA, USA). All animals were kept at 22 ± 1°C under a standard 12-h light/12-h dark schedule and had free access to water and a regular mouse (Harlan 2918) or guinea pig diet (#5025; 18.5% protein; Purina, St. Louis, MO, USA). All experimental protocols and all compounds used were approved either by the University of Michigan Committee on the Use and Care of Animals (UCUCA) or the Institutional Animal Care & Use Committee at the Medical University of South Carolina (MUSC). Animal care was under the supervision of the University of Michigan’s Unit for Laboratory Animal Medicine (ULAM) or under the supervision of the Division of Laboratory Animal Resources at MUSC.

The culture procedures have been described in detail (Chen et al., 2009). In brief, postnatal day 3 (p3) CBA/J pups were euthanized after antisepsis using 70% ethanol. Inner ears were extracted from surrounding tissue and immersed in cold Hank’s balanced salt solution. The lateral wall tissues (stria vascularis and spiral ligament) and the auditory nerve bundle were micro-dissected from the organ of Corti. The explants were placed onto a prepared culture dish containing a 15-μL polymerized drop of rat tail collagen in 1 mL of culture medium consisting of Basal Medium Eagle, 1% serum-free supplement (Invitrogen, Carlsbad, CA, USA), 1% bovine serum albumin, 5 mg/mL glucose, and 10 U/mL penicillin G. After 4 h of incubation (37°C, 5% CO₂), an additional 1 mL of medium was added to submerge the explants.

Explants were incubated for 2 days to recover from dissection stress before administering HDAC inhibitors and gentamicin (GM; Sigma-Aldrich Co., St. Louis, MO, USA). The medium was then exchanged for new medium containing a final concentration of 4.5 μM GM with or without various concentrations of the HDAC inhibitors and incubated for 72 h. The stock solutions with 95 mM of SAHA/vorinostat (Cayman Chemical, Ann Arbor, MI, USA), 100 mM of belinostat (Selleckchem, Houson, TX, USA), and 100 mM of panobinostat (Cayman Chemical), were dissolved in 100% dimethyl sulfoxide (DMSO) and stored at −20°C. GM solution was made fresh from powder in culture medium at 0.2 mM and then diluted to the final concentration for each experiment.

Explants were fixed with 4% paraformaldehyde overnight at 4°C and permeabilized for 30 min with 3% Triton X-100 in phosphate-buffered saline (PBS) at room temperature (22–24°C). The specimens were then washed three times with PBS and incubated with rhodamine-phalloidin (Invitrogen) at a 1:100 dilution for 60 min at room temperature (22–24°C). After rinsing in PBS, the specimens were mounted on a slide with Gel-Mount™ (BioMeda Corp., Foster City, CA, USA). The phalloidin-stained stereociliary bundles and circumferential F-actin rings on the cuticular plate of OHCs allowed the determination of cells that were present or missing. A clear “V” shape of stereocilia indicates the presence of OHCs. If the “V” shape is not visualized, we accept this as a damaged hair cells. Cell populations were assessed on a Leitz Orthoplan upright microscope equipped for epifluorescence, using a 50× oil-immersion objective. The right objective had a 0.19-mm calibrated scale imposed on the field for reference and all three rows of OHCs were oriented longitudinally within each 0.19-mm frame. Each successive 0.19-mm field was evaluated for the absence of OHCs beginning from the apex and moving down the organ of Corti to the base. The percentage of OHC loss was calculated.

Male CBA/J mice at the age of 5 weeks received subcutaneous (SQ) injections of KM at 700 mg base/kg (dissolved in saline) twice per day (AM and PM). SAHA for administration was dissolved in 100% DMSO for stock solutions of 50 mg/mL and diluted with saline immediately before intraperitoneal (IP) injection, for a final dose of 50 mg/kg, given twice per day for 15 days. The concentration of DMSO vehicle control was 1 mL/kg body weight. All solutions were filtered with a syringe filter before injection. Body weight was measured before each injection.

Pigmented male guinea pigs initially weighing 200–250 g from Charles River were used in the in-vivo study. Experiments were begun 1 week after the guinea pigs arrived. To select safe doses of SAHA for protective studies, we conducted two separate sets of experiments to assess serum platelet concentrations. In the first set, pigmented guinea pigs received daily IP injections of SAHA at lower doses (LD; 2 mg/kg and 5 mg/kg) concurrent with SQ injections of 120 mg GM base/kg body weight daily for 2 weeks. In the second study, the higher doses (HD) of SAHA (15 mg/kg and 25 mg/kg) were used. The DMSO concentration was 1.1 g/kg, equal to the DMSO concentration used for the 25 mg/kg concentration of SAHA. Since the highest dose (25 mg/kg/day for 14 days) of SAHA significantly reduced serum platelet concentrations, the doses of SAHA used for the protective studies were 25 mg/kg or less. We then tested the protective ability of SAHA at LD (2 mg/kg and 5 mg/kg) and at HD of SAHA (15 mg/kg and 25 mg/kg) against GM-induced hearing loss concurrent with daily SQ injections of GM (dissolved in saline) at 120 mg base/kg for 14 days. SAHA was dissolved in 100% DMSO for stock solutions of 25 mg/mL and diluted with saline immediately before IP injections. All compound solutions were filtered with syringe filters before injection. Body weights were measured before each injection. If a guinea pig lost ~5% of its body weight, an additional 3–4 mL of saline was administrated subcutaneously.

The procedures for surface preparations of guinea pig cochlear epithelia were followed as previously described (Sha and Schacht, 1999b). After the last auditory brain stem response (ABR) measurement, guinea pigs from the second group of experiments were deeply anesthetized in a CO₂ chamber and decapitated. Cochleae were immediately removed and perfused with 4% paraformaldehyde in PBS at pH 7.4 and fixed overnight at 4°C. Micro-dissected surface preparations of the organ of Corti were permeabilized for 30 min with 0.5% Triton X-100 in PBS at room temperature (22–24°C) and stained with rhodamine phalloidin (Invitrogen) at a 1:100 dilution for 60 min at room temperature (22–24°C). After rinsing in PBS, the specimens were mounted on a slide with Gel-Mount™ (BioMeda Corp.). OHC counts were conducted with microscopy as described above for mice.

Blood was obtained by nail clipping after light anesthesia of the guinea pigs by metofane inhalation after the last drug injections. Blood cells were separated by centrifugation at 1000× g for 15 min, and sera were stored at −20°C. Blood urea nitrogen (BUN), creatinine and platelet concentrations were determined using a Kodak Ektachem700XR Clinical Chemistry Analyzer (Clinical Products Division, Eastman Kodak, Rochester, NY, USA) and analyzed by Dr. Donald Giacherio, Chemical Pathology Laboratory, University of Michigan.

ABRs were measured 3 days before and 3 weeks after drug treatments. Animals were anesthetized with IP injections of ketamine (100 mg/kg) and xylazine (10 mg/kg) for mice or ketamine (58.8 mg/kg), xylazine (2.4 mg/kg), and acepromazine (1.2 mg/kg) for guinea pigs. Body temperature was maintained near 37°C with a heating pad. ABRs were measured at 12 and 32 kHz for guinea pigs or 8, 16, and 32 kHz for mice in a sound-isolated and electrically shielded booth (Acoustic Systems, Austin, TX, USA). Sub-dermal electrodes were inserted at the vertex of the skull, under the left ear and under the right ear (ground). Tucker Davis Technology System III hardware and SigGen/Biosig software were used to present the stimuli monaurally (15 ms duration tone bursts with 1 ms rise-fall time) through a Beyer earphone attached to a customized plastic speculum inserted into the ear canal. Up to 1024 responses were averaged for each stimulus level. Thresholds (the lowest stimulus level with a reproducible response) were determined by reducing the intensity in 10-dB steps and then in 5-dB steps approaching the threshold until no organized response was detected. Thresholds were evaluated based on ABR wave I. Scores were given by an expert blinded to experimental conditions between the highest stimulus level without a response and the lowest stimulus level where an organized response was observed. All ABR measurements were conducted by the same experimenter.

Data were analyzed using SYSTAT and GraphPad software for Windows. The group size (n) in vivo was determined by the variability of measurements and the magnitude of the differences between groups. Statistical methods used include one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons and unpaired t-tests. All tests were two-tailed and a p-value < 0.05 was considered statistically significant.

In order to evaluate the protective potential of the three HDAC inhibitors on aminoglycoside-induced ototoxicity, we first determined their safety on auditory sensory hair cells using p3 murine organ of Corti explants. Explants were incubated for 2 days for recovery from the stress of dissection, followed by 72 h of incubation with or without inhibitors. Toxic effects were assessed by OHC loss. Control samples showed no loss of cells (Figure 1A) but all three HDAC inhibitors damaged OHCs in a dose-dependent manner. OHCs in the basal turn show changes in the orientation of the stereociliary bundles beginning with concentrations of 4 μM for SAHA, 3 μM for belinostat, and 20 nM for panobinostat. The toxic effects increased significantly with increasing concentrations and loss of OHCs reached 100% at 8 μM of SAHA, 5 μM of belinostat, and 50 nM of panobinostat (p < 0.0001). Statistical analysis using one-way ANOVA showed a significant dose-dependence of the toxic effect for SAHA (F_(4,12) = 840.8, p < 0.0001), as well as for belinostat (F_(3,9) = 11,856, p < 0.0001) and panobinostat (F_(3,9) = 13,972, p < 0.0001; Figures 1B–D; for detailed Tukey’s multiple comparison test values see Table 1). The median toxic doses (TD50), defined as the concentrations leading to 50% OHC loss, for SAHA, belinostat and panobinostat were 5.5 μM, 3.5 μM, and 35 nM, respectively.

After having established the safe concentration range for the HDAC inhibitors, we explored their efficacy as protective agents against GM-induced OHC loss. In the 72-h incubations of explants, GM with the vehicle alone (DMSO) caused loss of OHCs in a base-to-apex gradient with a 40% overall loss at 4.5 μM. Co-incubation with SAHA (Figure 2A) proved protective, beginning at 0.5 μM SAHA and increasing in efficacy with increased doses, such that 2 μM SAHA completely protected from OHC loss (F_(3,24) = 59.95, p < 0.001; Figure 2D). Similarly, co-administration of belinostat (Figure 2B) or panobinostat (Figure 2C) with GM also reduced GM-induced OHC loss in a dose-dependent manner. GM-induced OHC loss was attenuated by co-administration of 0.1 μM belinostat and was totally prevented by 0.4 μM (F_(4,21) = 29.2, p < 0.001; Figure 2E). Additionally, with administration of panobinostat protection started at 5 nM and OHC loss was completely blocked by 15 nM panobinostat (F_(3,8) = 81.19, p < 0.001; Figure 2F). From the assessment of safe doses and the median effective (protective) doses (ED50) of the three HDAC inhibitors, we calculated the therapeutic index (TI = TD50/ED50) to be highest (safest) for belinostat and lowest for panobinostat (for detailed values see Table 2).

In order to assess the protective potential of SAHA in vivo in a chronic ototoxicity model, we tested if SAHA treatment had protective effects against KM (700 mg base/kg body weight twice daily)-induced hearing loss in CBA/J mice. Consistent with our previous studies (Wu et al., 2001), treatment with KM for 15 days caused auditory threshold shifts of an average of 55 dB at 16 kHz (p < 0.0001) and 32 kHz (p < 0.0001). Treatment with SAHA at 50 mg/kg body weight twice daily did not attenuate KM-induced auditory threshold shifts (Figure 3). Typical ABR waveforms for each treated group are illustrated in Supplementary Figure S1.

While the CBA/J mouse is a well-established model for chronic KM-induced ototoxicity, we wished to ascertain whether the lack of protection by SAHA might be species-dependent. We therefore tested if SAHA treatment had protective effects against GM-induced hearing loss in guinea pigs. To find safe doses to be used in guinea pigs in vivo, we first assessed serum platelet concentrations. Guinea pigs tolerated injections of SAHA at all concentrations of 5–25 mg/kg body weight concurrent with SQ injections of 120 mg GM base/kg body weight daily for 2 weeks with normal appearing fur and steadily increasing body weight. Blood samples were collected after the last injection. Renal function, assessed by BUN and creatinine (Cr) concentrations, was similar to that of control guinea pigs (detailed values see Table 3). However, the platelet concentration was significantly lower with the highest concentration of SAHA (25 mg/kg; t₆ = 2.682, p = 0.037) compared to GM alone treated groups (Figure 4).

Based on the experiments to assess safe doses of SAHA, we tested several doses of SAHA below 25 mg/kg body weight against 120 mg GM base/kg body weight daily for 2 weeks in guinea pigs in vivo. In agreement with our previous results, GM treatment induced significant auditory threshold shifts at high frequencies (Figures 5A,B) with an average shift of 40 dB at 12 kHz (t₁₀ = 3.128, p = 0.011) and 60 dB at 32 kHz (p < 0.0001) and caused significant OHC loss in the basal turn (F_(1,4) = 8.013, p = 0.047; Figures 5C,D). Confirming the results from CBA/J mice, treatment with SAHA at a range of concentrations (LD: 2–5 mg/kg body weight or HD: 15–25 mg/kg body weight) did not attenuate GM-induced auditory threshold shifts at 12 (F_(2,19) = 0.9708, p = 0.397) and 32 kHz (F_(2,19) = 0.03664, p = 0.964) or loss of OHCs (F_(1,6) = 0.849, p = 0.392). DMSO (1.1 g/kg body weight) used as the solvent vehicle for SAHA did not affect GM-induced auditory threshold shifts (Figure 5A).