1. Animals
All experiments were performed on Sprague Dawley rats (200 g, 8-9 weeks) obtained from the Laboratory Animal Center of Fourth Military Medical University (FMMU), Xi’an Shaanxi Province, China. The animals were maintained under standard laboratory conditions (12 h dark/light cycle, temperature 22–26 °C, air humidity 40–60%) with food and water available ad libitum. The experimental protocols were approved by the Institutional Animal Care and Use Committee of FMMU. The rats were randomly divided into two groups (n = 10 per group): ① Control group, experimentally naive rats that were exposed to ambient sound levels (50-60 dB SPL, measured with a sound level meter) without noise exposure; ② Noise group, rats that were exposed to intense noise. Hearing tests were performed on Days 1, 3, 5 and 7, and the rats were anesthetized and killed on day
2. Noise exposure
The noise exposure was conducted in a noise sound box. The animals were placed in metal wire cages with free access to food and water for one week to acclimate to the noise sound box. During noise exposure, water bottles were located on the side of the cages, and there were no objects between the speakers and the rats’ ears. The noise (white noise, 110 dB SPL) was generated by a RadioShack Supertweeter located above the cages and was amplified with a power amplifier (Yamaha AX-500U, Japan) and a loudspeaker. The noise level was monitored with a sound level meter (Bruel and Kjaer, Typer 2606), with a variation less than 5 dB across the space available to animals. The animals were exposed to noise 2 h per day for 7 days.
3. Auditory brainstem response (ABR)
The ABR thresholds in rats were measured at 1 day, 3 days, 5 days and 7 days after noise exposure. Under light anesthesia with 10% chloral hydrate (400 mg/kg), the active, reference and ground needle electrodes were inserted beneath the skin at the vertex, the mastoid area of the test ear and the contralateral mastoid, respectively. The TDT III auditory-evoked potential workstation was used for sound generation and presentation, and data acquisition was performed with SigGenRZ and BioSigRZ software (Tucker Davis Technologies, Fort Lauderdale, FL, USA). The ABR was elicited by tone bursts (8, 16, 32 kHz; 0.5-millisecond rise/fall time, no plateau, alternating phase) or broadband clicks (10 milliseconds) presented at 21.97 s−1. The stimulus was played through a high-frequency speaker (model: MF1 Multi-Field Magnetic Speakers) located approximately 2 cm in front of the test ear. The intensity of the stimulus was decreased in 5-dB steps until the evoked responses disappeared. The differential potential was sampled over 10 milliseconds, filtered (low pass, 4 kHz; high pass, 100 Hz) and averaged (512 sweeps of alternated stimulus polarity) to obtain mean traces at each intensity [5]. The lowest intensity that was able to elicit a two-phase waveform from 5 to 15 ms after signal onset was considered the ABR threshold.
4. Immunofluorescence staining
Slips of SGNs were fixed in 4% paraformaldehyde for 30 min and permeabilized in 0.1% Triton X-100 for 15 min. After washing with PBS, the samples were incubated in a blocking solution of bovine serum albumin (BSA, 5%, Sigma, USA) for 20 min, followed by incubation with antibodies against AIF (1:200, rabbit, Abcam, USA) and β-tubulin (1:200, mouse, Abcam, USA) for 24 h overnight at 4 °C. Alexa 488-conjugated goat anti-rabbit (1:200, Invitrogen, USA) and Alexa 594-conjugated donkey anti-mouse (1:200, Invitrogen, USA) were used to label the primary antibodies by incubation for 40 min at 37 °C. Stained SGNs were observed under a fluorescence microscope (Olympus, Japan), and three photos of different groups were taken by the microscope.
5. Mitochondrial isolation and purification
Neurons were lysed with a lysis buffer containing protease inhibitors. The cell lysate was centrifuged for 10 min at 750 g at 4 ℃, and the pellets containing the nuclei and unbroken cells were discarded. The supernatant was then centrifuged at 15 000 × g for 15 min. The resulting supernatant was removed and used as the cytosolic fraction. The pellet fraction containing the mitochondria was further incubated with PBS containing 0.5% Triton X-100 for 10 min at 4 °C. After centrifugation at 16 000 × g for 10 min, the supernatant was collected as the mitochondrial fraction.
6. Measurement of ATP synthesis
A luciferase/luciferin-based system was used to measure ATP synthesis in isolated mitochondria as described elsewhere (Parone et al., 2013). Thirty milligrams of mitochondria-enriched pellets was resuspended in 100 ml of buffer A (150 mM KCl, 25 mM Tris HCl, 2 mM potassium phosphate, 0.1 mM MgCl2, pH 7.4) with 0.1% BSA, 1 mM malate, and 1 mM glutamate and buffer B (containing 0.8 mM luciferin and 20 mg/ml luciferase in 0.5 M Tris-acetate pH 7.75). The reaction was initiated by the addition of 0.1 mM ADP and monitored for 120 min using a microplate reader at 530 nm. There were six samples in each group, and the experiment was repeated a minimum of 3 times.
7. Western blot analysis
Total protein concentrations were measured using the Pierce BCA method (Sigma, USA). Equivalent amounts of protein (40 mg per lane) were loaded and separated on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% skim milk solution in Tris-buffered saline with 0.1% Triton X-100 (TBST) for 1 h and then incubated overnight at 4 ℃ with primary AIF OXPH, complex I ND1, ND3, ND6, GluRA, or b-actin (1:500, #4970, Cell Signaling, USA) antibody dilutions in TBST. Then, the membranes were washed and incubated with secondary antibodies (Santa Cruz, USA) for 1 h at room temperature. Immunoreactivity was detected with Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). ImageJ (Scion Corporation) was used to quantify the optical density of each band. The expression of each protein was calculated from the optical density of each band normalized against the optical density of b-actin and expressed as the fold change compared to the control levels.