Animals

60 male C57BL/6J mice aged 5 weeks were obtained from Capital Medical University’s Experimental Animal Center. The mice were raised in an environment at a constant temperature (22–23 °C) and humidity (60 ± 5%), on a 12-h light/dark cycle, with free access to food and water. All mice were acclimated for a week before being divided into three groups (n = 20 per group): control, low-dose D-gal (D-gal-L) and high-dose D-gal (D-gal-H) groups. The mice in the control group received a subcutaneous injection of 0.9% saline (the D-gal vehicle) once daily for six weeks. The second and third groups of mice received a subcutaneous injection of D-gal (Sigma-Aldrich, St. Louis, MO, USA) once daily for 6 weeks at a dose of 500 and 1000 mg/kg body weight, respectively. In compliance with the National Institutes of Health’s Guidelines for the Care and Use of Laboratory Animals, every effort was made to minimize animal suffering and reduce the number of animals used. The Capital Medical University Committee on the Ethics of Animal Experiments approved the study’s protocol.

Auditory function evaluation

Auditory brainstem response (ABR) was used to analyze the hearing function in all animals. The method of ABR testing has been described previously [24]. Briefly, it was conducted in an anechoic room using BioSigRZ software (Tucker-Davis Technologies, Alachua, FL, USA). A total of 18 mice (n = 6 per group) were anesthetized by intraperitoneal injection of a ketamine (100 mg/kg) and xylazine (10 mg/kg) mixture. Before testing, the external auditory canal and tympanic membrane (TM) were examined with an electric otoscope. Mice with acute otitis externa (AOE) or otitis media were excluded from the study.

The sound delivery tube of an inserted earphone was tightly fitted into the external auditory canal. Subcutaneous needle electrodes were used to record the ABR response. Three stainless steel recording electrodes were subcutaneously inserted posterior to the tested pinna (recording), vertex (reference), and contralateral pinna (ground). A TDT System 3 (Tucker-Davis Technologies) was used to measure ABR responses to a tone burst stimulus at 8 kHz, 16 and 32 kHz, starting at a 90-dB sound pressure level (SPL) and decreasing in 5-dB steps. The ABR threshold for each frequency was determined, which is the lowest SPL that will reliably produce an ABR recording with one or more distinct waves that can be easily distinguished by visual inspection of the waveforms. To confirm the uniformity of the waveforms, the procedure was repeated at low SPLs close to the threshold.

Immunohistochemistry of cochlear frozen sections

Mice (18 per group, n = 6) were anesthetized with ketamine and xylazine before being euthanized via cervical dislocation. Cochleae were fixed with 4% paraformaldehyde (PFA) overnight at 4 °C, then washed three times with PBS before being decalcified in 10% EDTA for 48 h at 4 °C. Samples were dehydrated in a sucrose gradient of 20 and 30% for 1 h each. Samples were embedded in optimal cutting temperature compound before being sliced at 20 °C with a thickness of 10 μm using a Leica Cryastat (Wetzlar, Germany) and mounted on Superfrost Plus microscopic slides (KeyGEN Biotech, Nanjing, China). One side of the cochleae were used for immunohistochemistry, and the other side for TUNEL staining. The expression of NOX2, UCP2, 8-OHdG, cleaved caspase 3 (c-Cas3) were evaluated using immunohistochemistry. Cochlear sections were washed and then incubated with 0.3% Triton X-100 solution (Sigma-Aldrich) for 30 min at room temperature. They were washed again and blocked with 10% goat serum for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies: monoclonal mouse anti-8-OHdG (diluted 1:200; Abcam, USA, Cat.No#ab62623), anti-NOX2 (diluted 1:200; Invitrogen, Waltham, MA, USA, Cat.No#PA5-76034,), anti-UCP2 (diluted 1:200; Invitrogen,Cat.No#PA5-103176) and anti-C-cas3 (diluted 1:200; CST, Danvers, MA, USA, Cat.No#Asp175). The slides were then washed and incubated in the dark for 2 h with Alexa Fluor 568-, Alexa Fluor 488-, or Alexa Fluor 594-conjugated secondary antibodies at a dilution of 1:300. The sections were washed and mounted using mounting media containing 4′,6-diamidino-2-phenylindole (DAPI; ZSGB-BIO, Beijing, China). Following a wash with PBS, the sections were examined using a laser scanning confocal microscope (Leica TCS SP8). The expression levels of 8-OHdG, NOX2, UCP2 and c-Cas3 were analyzed using the Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). Sections were treated the same way in the negative control, but the primary antibody incubation step was skipped.

DNA isolation and mitochondrial DNA (mtDNA) 3860-bp deletion assay

The method of DNA isolation and mtDNA deletion has been described previously [25, 26]. The accumulation of mtDNA was evaluated in 12 mice (n = 4 per group) using real-time PCR assay. DNA extraction by using DNA isolation kit (Tiangen Biotech Co., Beijing, China). GeneQuant pro DNA/RNA Calculator (Amersham Pharmacia Biotech, Staffanstorp, Sweden) was used to measure DNA concentration. 12 S rRNA gene was used as control (GenBank: NC_006914). The primers used to amplify the mtDNA 3860-bp deletion and 12 S rRNA were previously described [25]. PCR amplification was performed on a StepOne Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The cycling conditions were as follows: 95 °C for 10 min, 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The cycle number at which a significant increase in normalized fluorescence was first detected was designated as the threshold cycle number (Ct). The ratio of the mtDNA 3860-bp deletion to total mtDNA was calculated as ΔCt = (Ct3860-bp deletion − Ct12S rRNA), and the relative expression was calculated as 2 − ΔΔCt. The PCR products of the mtDNA 3860-bp deletion were cloned and verified with ABI Prism 377XL sequencer (Applied Biosystems).

Transmission electron microscopy

The ultrastructure of the mitochondria in the cochlea SV was observed using TEM. Twelve mice (n = 4 per group) were anesthetized with ketamine and xylazine before being euthanized via cervical dislocation. Then cochleae from each mouse were removed, treated with 2.5% glutaraldehyde and fixed overnight at 4 °C. The following day, the cochleae were washed with PBS and placed in 10% EDTA for decalcification for 72 h. The SV was carefully dissected and harvested from the lateral wall of the cochlea. After fixation in 1% osmium tetroxide (Maijin Biotechnology, Nanjing, China) for 2 h, the SV was dehydrated using graded ethanol or acetone, followed by a 2-h immersion in acetone/Epon 812 mixture (1:1), and a 10-h immersion in Epon 812 at 80 °C. Ultrathin Sect. (50 nm) were taken serially on copper grids and were stained with uranyl acetate and lead citrate., A FEI TecnaiG212transmission electron microscope (Philips, Amsterdam, Netherlands) was used to examine the ultrastructure of the stained sections.

TUNEL assay

A TUNEL staining kit (Roche Molecular Biochemicals, Mannheim, Germany, Cat No#11,684,817,910) was used to identify apoptotic cells in situ. The labeling reaction, which contained terminal deoxynucleotidyl transferase, was carried out at 37 °C for 60 min in a humidity chamber after treatment with 10% goat serum in 0.1% Triton X-100 for 3 min. DAPI staining solution was used to counterstain the nuclei for 5 min at room temperature. The sections were cleaned with PBS and then examined with a laser scanning confocal microscope (Leica TCS SP8).

ATP and MMP measurement

The luciferin-luciferase system was employed to quantify the ATP levels in these specimens according to the manufacturer’s instructions (Beyotime, Jiangsu, China, Cat.No#S0027). The relative ATP content of each specimen was determined by measuring the bioluminescent intensity with a microplate reader. A mitochondria isolation kit (Beyotime, Cat.No#C2006) was used to isolate mitochondria from each specimen in order to measure the MMP. Then, in accordance with the manufacturer’s instructions, the JC-1 probe was used to evaluate changes in MMP.

Statistical analysis

Data are presented as mean ± standard deviation. One-way analysis of variance was used to determine statistical significance, and a post hoc least significant difference test was used to assess statistical differences between groups. SPSS 13.0 software was used to conduct the analyses (SPSS, Inc., Chicago, IL, USA). A statistically significant difference was defined as P < 0.05.