2.1

Animals

Forty healthy albino guinea pigs of both sexes (8 weeks old, 250–300 g) with normal Preyer’s reflex, provided by the animal center of the Fourth Military Medical University, Xi’an, China, were used in this study. The animals were maintained on a 12-h light/12-h dark schedule and had free access to water and a regular diet. All animal experiments were conducted in accordance with the National Institutes of Health guidelines and approved by the Committee on Animal Research of the Fourth Military Medical University.

2.2

Surgery and experimental groups

The animals were anesthetized using 2% pentobarbital sodium (P11011, 50 mg/kg; Merck, Germany) and 0.2 mg/kg atropine was administered to reduce respiratory secretions. Their body temperatures were maintained at approximately 37 °C using a homeothermic pad. After deep anesthetization, the postauricular region was shaved and cleaned for surgical manipulation. Lidocaine hydrochloride (1%) was injected subcutaneously to provide local anesthesia. A postauricular incision was made, and the muscles were separated to expose the temporal bone. The bulla was perforated using a 1-mm diamond-paste bur (NSK Ltd., Tokyo, Japan) for visualization of the round window. A 0.3-mm ³ gelatin sponge was surgically placed on the round window niche and permeated with 10 μl physiological saline or 100 mg/ml NaB (Sigma-Aldrich, St. Louis, MO, USA) diluted in physiological saline. The subcutaneous tissue and skin were then sutured.

The animals were divided into two experimental groups. Animals in group 1 ( n = 10) underwent bilateral ear surgery to place sponges (0.3 mm ³ ) permeated with NaB (10 μl, 100 mg/ml) and physiological saline (10 μl; control) in the right and left round window niches, respectively. The sponges were left in place for 15 days to evaluate the effects of NaB at the applied concentration. Animals in group 2 ( n = 30) underwent the same bilateral ear surgery described for Group 1. Three days later, the group 2 animals received intramuscular injections of GM (200 mg/kg/day) for five consecutive days. The auditory brainstem response (ABR) threshold was measured 7 days after the final GM injection. Hair cell counts and immunological studies were performed after ABR measurement to examine the protective effects of NaB.

2.3

ABR measurement

Auditory thresholds were determined by ABR under light anesthetic (40 mg/kg ketamine chlorhydrate with 5 mg/kg xylazine, IP). The body temperature of the animal was maintained at 38 ± 1 °C using an isothermic heating pad. The active needle electrode was inserted subcutaneously at the vertex; the reference electrode was inserted in the mastoid area of the test ear; and the ground electrode was inserted in the contralateral mastoid. For sound stimulus generation, presentation, and data acquisition, the TDT III System Auditory Evoked Potential workstation was used, controlled by SigGenRZ and BioSigRZ software (Tucker-Davis Technologies, Fort Lauderdale, FL, USA).

The ABR was elicited with tone bursts (4, 8, 16, 24, 32 kHz; 0.5-ms rise/fall time, no plateau, alternating phase) or broadband clicks (10 ms) presented at 21.97/s. The stimulus was played through an RZ6 D/A converter and presented through a high-frequency speaker (model: MF1 Multi-Field Magnetic Speakers) located approximately 2 cm in front of the test ear. The stimulus was decreased in 5-dB steps until the response disappeared. The differential potential was sampled over 10 ms, filtered (low-pass, 4 kHz; high-pass, 100 Hz), and averaged (512 sweeps of alternated stimulus polarity) to obtain mean traces at each intensity. The ABR threshold was defined as the lowest intensity that elicited a response.

2.4

Cochlear surface preparation

After ABR threshold measurement, the animals were anesthetized (as described above) and then sacrificed by decapitation. The cochleae were removed immediately. Cochleae were perfused locally with 4% phosphate-buffered paraformaldehyde (pH 7.4) through the open round windows and cochlear apexes to ensure efficient fixative perfusion, and postfixed in the same stationary liquid overnight. After decalcification in 10% ethylenediaminetetraacetic acid (Sigma-Aldrich) for 1 day, the bony capsule was removed and the spiral ligament, stria vascularis, and Reissner’s membrane were separated under a dissecting microscope. Each turn of the Corti organ was detached from the bony modiolus.

2.5

Hair cell counts

Each complete organ of Corti was washed three times with 0.01% phosphate-buffered saline (PBS) and incubated with fluoresceinyl-aminomethyldithiolano-phalloidin (1:60 in 0.01% PBS; Sigma-Aldrich) at room temperature (22–24 °C) for 8 min. After several rinses in 0.01% PBS, the specimens were mounted on microscopic slides. Phalloidin staining of the stereociliary bundles and circumferential F-actin rings on the cuticular plates of OHCs and IHCs allowed determination of the presence or absence of cells. Cell populations were assessed under an upright microscope (Leica Microsystems Inc., Bannockburn, IL, USA) equipped for epifluorescence, using a 40 × oil immersion objective. A 0.24-mm calibrated scale was imposed on the field of the right objective for reference.

A single row of IHCs and all three rows of OHCs were oriented longitudinally within each 0.24-mm frame. The absence of IHCs and OHCs was evaluated in each 0.24-mm field, beginning at the apex and moving down the organ of Corti to the base. Cell counts were entered into the Cytocochleogram computer program (version 3.0.6; Kresge Hearing Research Institute, University of Michigan, Ann Arbor, MI, USA). The percentage of missing hair cells was calculated and plotted as a function of distance from the apical turn to the basal turn of the organ of Corti. The Cytocochleogram software was used to create plots for the NaB and control groups, based on results from the right and left ears of all experimental animals.

2.6

Indirect immunofluorescence staining

Complete organs of Corti were washed three times with 0.01% PBS and permeabilized for 30 min with 3% Triton X-100 in 0.01% PBS at room temperature. The specimens were washed an additional three times with 0.01% PBS and blocked with 10% normal goat serum for 30 min at room temperature. Then, they were incubated at 4 °C overnight with a primary polyclonal rabbit anti-HDAC1 antibody (1:1000 in 0.01% PBS; Sigma-Aldrich). After three washes with 0.01% PBS, a fluorescent-labeled secondary antibody (1:300 in Alexa Fluor 594, Molecular Probes; Life Technologies, Carlsbad, CA, USA) was applied at room temperature for 2 h in the dark.

For fluorescent visualization of hair cells, the specimens were incubated with fluoresceinyl-aminomethyldithiolano-phalloidin (1:60 in 0.01% PBS; Sigma-Aldrich) at room temperature for 8 min. For visualization of nuclei, the specimens were incubated with Hoechst 33342 (1:1000 in 0.01% PBS; Sigma-Aldrich) at room temperature for 5 min. After several rinses in 0.01% PBS, the specimens were mounted on a microscopic slide. Preparations were imaged with a confocal laser scanning microscope (Fluoview 500; Olympus American, Melville, NY, USA).

Immunostaining of preparations was quantified from confocal images taken under identical conditions and equal setting parameters using ImageJ software (National Institute of Health, Bethesda, MD, USA). Individual nuclei were localized and counted by outlining the nuclear membrane on blue Hoechst 33342-stained images. The fluorescence of HDAC1 staining was analyzed on corresponding red images, and the fluorescent visualization of hair cells was localized on corresponding green images.

2.7

Total protein extraction and western blotting

Complete organs of Corti were washed with PBS. Eight explants were pooled and homogenized in ice-cold ristocetin-induced platelet agglutination lysis buffer by using a glass/glass micro tissue grinder for 30 s. Subsequently, tubes were sonicated in an ultrasonic water bath for 15 s. After 30 min on ice, insoluble material was removed by centrifugation at 12,000 g at 4 °C for 10 min. Supernatants were stored at − 80 °C until analysis.

Protein concentrations were determined by using the Bio-Rad Protein Assay dye reagent (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as a standard. Samples (40 μg each) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred onto nitrocellulose membranes (Pierce, Rockford, IL, USA) and blocked with 5% nonfat dry milk in PBS with 0.1% Tween 20 (PBS-T). The membranes were incubated with primary antibodies, rabbit anti-HDAC1 (1: 1000), anti-histone H3 (1:1000), or mouse anti-GAPDH (1:10,000), in 5% skim milk in PBS-T overnight at 4 °C. They were washed three times (10 min each) with PBS-T. Membranes were incubated with an appropriate secondary antibody at a dilution of 1:1000 (Cell Signaling Technology Inc.) or 1:10,000 (Jackson Immunoresearch Laboratory) for 1 h at room temperature (22–24 °C). After the membrane was washed extensively, the immunoreactive bands were visualized by enhanced chemiluminescence.

Scans of Western blots were analyzed using AlphaEase software SpotDenso tool (Alpha Innotech, San Leandro, CA, USA). The band densities were first normalized to the background, and then the ratio of the selected proteins to GAPDH (run on the same gel) was calculated. Finally, the difference in relative densities of the control and experimental bands was tested for statistical significance using the Primer of Biostatistics software (McGraw-Hill Software, New York, NY, USA).

2.8

Statistical analyses

Data are presented as means ± standard deviations. One-way analysis of variance (ANOVA) was used to identify differences among groups. A value of P < 0.05 was considered statistically significant. Analyses were performed using the SPSS software (version 13.0; SPSS Inc., Chicago, IL, USA).

2.1

Animals

Forty healthy albino guinea pigs of both sexes (8 weeks old, 250–300 g) with normal Preyer’s reflex, provided by the animal center of the Fourth Military Medical University, Xi’an, China, were used in this study. The animals were maintained on a 12-h light/12-h dark schedule and had free access to water and a regular diet. All animal experiments were conducted in accordance with the National Institutes of Health guidelines and approved by the Committee on Animal Research of the Fourth Military Medical University.

2.2

Surgery and experimental groups

The animals were anesthetized using 2% pentobarbital sodium (P11011, 50 mg/kg; Merck, Germany) and 0.2 mg/kg atropine was administered to reduce respiratory secretions. Their body temperatures were maintained at approximately 37 °C using a homeothermic pad. After deep anesthetization, the postauricular region was shaved and cleaned for surgical manipulation. Lidocaine hydrochloride (1%) was injected subcutaneously to provide local anesthesia. A postauricular incision was made, and the muscles were separated to expose the temporal bone. The bulla was perforated using a 1-mm diamond-paste bur (NSK Ltd., Tokyo, Japan) for visualization of the round window. A 0.3-mm ³ gelatin sponge was surgically placed on the round window niche and permeated with 10 μl physiological saline or 100 mg/ml NaB (Sigma-Aldrich, St. Louis, MO, USA) diluted in physiological saline. The subcutaneous tissue and skin were then sutured.

The animals were divided into two experimental groups. Animals in group 1 ( n = 10) underwent bilateral ear surgery to place sponges (0.3 mm ³ ) permeated with NaB (10 μl, 100 mg/ml) and physiological saline (10 μl; control) in the right and left round window niches, respectively. The sponges were left in place for 15 days to evaluate the effects of NaB at the applied concentration. Animals in group 2 ( n = 30) underwent the same bilateral ear surgery described for Group 1. Three days later, the group 2 animals received intramuscular injections of GM (200 mg/kg/day) for five consecutive days. The auditory brainstem response (ABR) threshold was measured 7 days after the final GM injection. Hair cell counts and immunological studies were performed after ABR measurement to examine the protective effects of NaB.

2.3

ABR measurement

Auditory thresholds were determined by ABR under light anesthetic (40 mg/kg ketamine chlorhydrate with 5 mg/kg xylazine, IP). The body temperature of the animal was maintained at 38 ± 1 °C using an isothermic heating pad. The active needle electrode was inserted subcutaneously at the vertex; the reference electrode was inserted in the mastoid area of the test ear; and the ground electrode was inserted in the contralateral mastoid. For sound stimulus generation, presentation, and data acquisition, the TDT III System Auditory Evoked Potential workstation was used, controlled by SigGenRZ and BioSigRZ software (Tucker-Davis Technologies, Fort Lauderdale, FL, USA).

The ABR was elicited with tone bursts (4, 8, 16, 24, 32 kHz; 0.5-ms rise/fall time, no plateau, alternating phase) or broadband clicks (10 ms) presented at 21.97/s. The stimulus was played through an RZ6 D/A converter and presented through a high-frequency speaker (model: MF1 Multi-Field Magnetic Speakers) located approximately 2 cm in front of the test ear. The stimulus was decreased in 5-dB steps until the response disappeared. The differential potential was sampled over 10 ms, filtered (low-pass, 4 kHz; high-pass, 100 Hz), and averaged (512 sweeps of alternated stimulus polarity) to obtain mean traces at each intensity. The ABR threshold was defined as the lowest intensity that elicited a response.

2.4

Cochlear surface preparation

After ABR threshold measurement, the animals were anesthetized (as described above) and then sacrificed by decapitation. The cochleae were removed immediately. Cochleae were perfused locally with 4% phosphate-buffered paraformaldehyde (pH 7.4) through the open round windows and cochlear apexes to ensure efficient fixative perfusion, and postfixed in the same stationary liquid overnight. After decalcification in 10% ethylenediaminetetraacetic acid (Sigma-Aldrich) for 1 day, the bony capsule was removed and the spiral ligament, stria vascularis, and Reissner’s membrane were separated under a dissecting microscope. Each turn of the Corti organ was detached from the bony modiolus.

2.5

Hair cell counts

Each complete organ of Corti was washed three times with 0.01% phosphate-buffered saline (PBS) and incubated with fluoresceinyl-aminomethyldithiolano-phalloidin (1:60 in 0.01% PBS; Sigma-Aldrich) at room temperature (22–24 °C) for 8 min. After several rinses in 0.01% PBS, the specimens were mounted on microscopic slides. Phalloidin staining of the stereociliary bundles and circumferential F-actin rings on the cuticular plates of OHCs and IHCs allowed determination of the presence or absence of cells. Cell populations were assessed under an upright microscope (Leica Microsystems Inc., Bannockburn, IL, USA) equipped for epifluorescence, using a 40 × oil immersion objective. A 0.24-mm calibrated scale was imposed on the field of the right objective for reference.

A single row of IHCs and all three rows of OHCs were oriented longitudinally within each 0.24-mm frame. The absence of IHCs and OHCs was evaluated in each 0.24-mm field, beginning at the apex and moving down the organ of Corti to the base. Cell counts were entered into the Cytocochleogram computer program (version 3.0.6; Kresge Hearing Research Institute, University of Michigan, Ann Arbor, MI, USA). The percentage of missing hair cells was calculated and plotted as a function of distance from the apical turn to the basal turn of the organ of Corti. The Cytocochleogram software was used to create plots for the NaB and control groups, based on results from the right and left ears of all experimental animals.

2.6

Indirect immunofluorescence staining

Complete organs of Corti were washed three times with 0.01% PBS and permeabilized for 30 min with 3% Triton X-100 in 0.01% PBS at room temperature. The specimens were washed an additional three times with 0.01% PBS and blocked with 10% normal goat serum for 30 min at room temperature. Then, they were incubated at 4 °C overnight with a primary polyclonal rabbit anti-HDAC1 antibody (1:1000 in 0.01% PBS; Sigma-Aldrich). After three washes with 0.01% PBS, a fluorescent-labeled secondary antibody (1:300 in Alexa Fluor 594, Molecular Probes; Life Technologies, Carlsbad, CA, USA) was applied at room temperature for 2 h in the dark.

For fluorescent visualization of hair cells, the specimens were incubated with fluoresceinyl-aminomethyldithiolano-phalloidin (1:60 in 0.01% PBS; Sigma-Aldrich) at room temperature for 8 min. For visualization of nuclei, the specimens were incubated with Hoechst 33342 (1:1000 in 0.01% PBS; Sigma-Aldrich) at room temperature for 5 min. After several rinses in 0.01% PBS, the specimens were mounted on a microscopic slide. Preparations were imaged with a confocal laser scanning microscope (Fluoview 500; Olympus American, Melville, NY, USA).

Immunostaining of preparations was quantified from confocal images taken under identical conditions and equal setting parameters using ImageJ software (National Institute of Health, Bethesda, MD, USA). Individual nuclei were localized and counted by outlining the nuclear membrane on blue Hoechst 33342-stained images. The fluorescence of HDAC1 staining was analyzed on corresponding red images, and the fluorescent visualization of hair cells was localized on corresponding green images.

2.7

Total protein extraction and western blotting

Complete organs of Corti were washed with PBS. Eight explants were pooled and homogenized in ice-cold ristocetin-induced platelet agglutination lysis buffer by using a glass/glass micro tissue grinder for 30 s. Subsequently, tubes were sonicated in an ultrasonic water bath for 15 s. After 30 min on ice, insoluble material was removed by centrifugation at 12,000 g at 4 °C for 10 min. Supernatants were stored at − 80 °C until analysis.

Protein concentrations were determined by using the Bio-Rad Protein Assay dye reagent (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as a standard. Samples (40 μg each) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred onto nitrocellulose membranes (Pierce, Rockford, IL, USA) and blocked with 5% nonfat dry milk in PBS with 0.1% Tween 20 (PBS-T). The membranes were incubated with primary antibodies, rabbit anti-HDAC1 (1: 1000), anti-histone H3 (1:1000), or mouse anti-GAPDH (1:10,000), in 5% skim milk in PBS-T overnight at 4 °C. They were washed three times (10 min each) with PBS-T. Membranes were incubated with an appropriate secondary antibody at a dilution of 1:1000 (Cell Signaling Technology Inc.) or 1:10,000 (Jackson Immunoresearch Laboratory) for 1 h at room temperature (22–24 °C). After the membrane was washed extensively, the immunoreactive bands were visualized by enhanced chemiluminescence.

Scans of Western blots were analyzed using AlphaEase software SpotDenso tool (Alpha Innotech, San Leandro, CA, USA). The band densities were first normalized to the background, and then the ratio of the selected proteins to GAPDH (run on the same gel) was calculated. Finally, the difference in relative densities of the control and experimental bands was tested for statistical significance using the Primer of Biostatistics software (McGraw-Hill Software, New York, NY, USA).

2.8

Statistical analyses

Data are presented as means ± standard deviations. One-way analysis of variance (ANOVA) was used to identify differences among groups. A value of P < 0.05 was considered statistically significant. Analyses were performed using the SPSS software (version 13.0; SPSS Inc., Chicago, IL, USA).