Animal experiments in this study were approved by the Animal Care Committee of Mackay Medical College. Eight-week-old Male C57BL/6J mice were purchased from the National Laboratory Animal Center (New Taipei City, Taiwan). All mice were bred and housed in accordance with the standard animal protocol.

1. Induction of NIHL in mice

1. Prepare the cage for the experimental mice
   1. To do so, use a rat trap cage with dimensions of 14 cm × 17 cm × 24 cm. Cut four pieces of corrugated plastic boards into appropriate sizes, making them fit into the cage (13 cm × 23 cm and 13 cm × 16 cm).
   2. To prevent the mice from getting their feet cut by the mesh grid, place two of the pieces at the bottom and on the back side, respectively. Place the other two pieces perpendicularly interlocked with each other, to divide the space within the cage into quarters.
2. Carry out this study in a soundproof box. Place a speaker 8.5 cm in front of the cage, and place both the speaker and the cage in a soundproof box.
3. Open the CLIO application software
4. Move the cursor to the speaker icon, then click on TwoSin. Enter and change the value of Freq 1 para 1000 Hz, Freq 2 para 6000 Hz, and click on OK to start playing the sound.
5. Under the Leq tab, change dBV to dBSPL. After setting the time on the software interface, click the green triangle button to play the sound.
6. Place a microphone in front of the speaker at a distance of 8.5 cm to calibrate the noise level (see Supplementary File 1). Adjust the noise level to 125 dB SPL-A and continuously monitor for no less than 3 min to ensure that the noise level is sufficiently stable. Use a generator, an analyzer, and an amplifier to create and control the noise. (Figure 1)
   NOTE: Do this step in a soundproof box to avoid damage to the operator's hearing.
7. Place four male C57BL/6J mice into the cage (one for each quarter) for noise exposure. Randomly assign the mice in each quarter during the noise exposure. Place a microphone onto the top of the cage to monitor the noise level during the noise exposure (Figure 2).
   NOTE: Do this step in a soundproof box to avoid damage to the operator's hearing.
8. Expose the mice to the noise at frequencies of 1 kHz and 6 kHz continuously for 6 h per day, for 5 consecutive days.
9. Measure the hearing thresholds of the mice 1 day after the noise exposure (i.e., on the 6^th day) by measuring the ABR. Repeat these ABR measurements again 1 week after the noise exposure (i.e., on the 13^th day). After the ABR measurements, sacrifice all the involved mice and harvest their cochleae (Figure 3).

2. Auditory brainstem response (ABR)-based assessment of hearing threshold

1. Use a commercial ABR testing system specifically designed for small animals¹⁰.
2. Intraperitoneally inject a mixture of tiletamine and zolazepam (40 mg/kg) and xylazine (9.3 mg/kg) into the mice for general anesthesia.
   NOTE: ABR assessment takes ~2 h. Make sure to provide thermal support via a heating pad and apply eye ointment to prevent dryness while the mouse is under anesthesia.
3. Measure the ABR in the mouse under general anesthesia. Place subdermal needle electrodes (12 mm) at the vertex, behind the pinna of the left ear, and back near the tail to measure the hearing threshold.
4. Present acoustic stimuli using a speaker placed 1 cm from the animal's left ear.
5. Use an oscilloscope to control the acoustic stimuli. Choose Sine Wave for the stimuli and 10 k for the window scale. Turn the Frequency knob to obtain the desired frequency of the acoustic stimuli.
6. Adjust the stimulus intensity by turning the AMLP knob on the function generator. Obtain the desired stimulus intensity by turning the AMLP knob to a suitable voltage, calculated from calibration.
7. Collect the ABR measurements under a series of stimulus intensities, from 10 dB SPL to 100 dB SPL, with a 10 dB step size.
   NOTE: Measure the hearing threshold in a soundproof box. The minimum stimulus intensity level that could give rise to a discernable Wave V in the collected signal was assumed to be the ABR threshold^10,11 (Figure 4). Post-ABR assessment, monitor the animal until it recovers from anesthesia (~1 h).

3. Microscopic examination

1. Fixing the harvested tissue
   1. After the ABR measurements, sacrifice the mouse by intraperitoneally injecting a mixture of tiletamine and zolazepam (100 mg/kg) and xylazine (23.25 mg/kg) for the microscopic examination.
   2. Harvest the cochleae from the mouse and immediately immerse them in 10% formaldehyde (FA) for fixation (two cochlea/mL) for at least 8 h at 4 °C.
   3. Following fixation, replace the FA solution with a 10% ethylenediaminetetraacetic acid (EDTA) solution for decalcification for 3-4 days at 4 °C. Then, check each cochlea with tweezers to confirm that the cochleae are sufficiently softened.
   4. Place the cochleae in a Petri dish filled with phosphate-buffered saline (PBS), and cut the spiral-shaped organ of Corti (OC) (each decalcified cochlea) into three sections-the basal turn, middle turn, and apical turn-for tissue staining.
   5. Under a dissecting microscope (magnification: 8x-35x), remove the bony structures (scala vestibuli, scala tympani, and modiolus) of the decalcified cochleae and obtain the soft tissue, including the OC (thickness: about 40 µm).
2. Cochlea immunofluorescence staining
   1. Preparation of the blocking buffer: Prepare 2% bovine serum albumin (BSA) and 0.2% Triton X-100 solutions in PBS.
   2. Transfer the tissue to be stained from the Petri dish to a microcentrifuge tube, and immerse the tissue in 0.1 mL of the blocking buffer for 2 h at room temperature (RT).
   3. Preparation of the Myo7A primary antibody buffer: Dilute the Myo7A primary antibody in the blocking buffer at a volume ratio of 1:200.
   4. Treat the tissue in the microcentrifuge tube with 100 µL of Myo7A primary antibody buffer for 2 h at RT.
   5. Rinse the tissue three times in PBS for 5 min each.
   6. Preparation of the Myo7A secondary antibody (Myo7A-Rb-488) and phalloidin antibody (phalloidin-594) buffer: Dilute Myo7A secondary antibody (1:400) and phalloidin antibody (1:200) in the blocking buffer.
   7. Treat the tissue with 100 µL of Myo7A secondary antibody and phalloidin antibody buffer for 2 h at RT.
   8. After antibody + phalloidin antibody treatment, rinse the tissue in PBS three times, for 5 min each.
   9. Transfer the rinsed tissue from the microcentrifuge tube to a Petri dish filled with PBS using a 1 mL plastic transfer pipette with a cut tip.
   10. To prepare for the microscopic examination, unfold the tissue, place it onto a glass slide, and add 15 µL of 4',6-diamidino-2-phenylindole (DAPI) fluoromount medium onto the tissue to fully cover it. Place a glass coverslip gently over the tissue. Leave the slide overnight at RT before sealing it with nail polish.
3. Image acquisition
   1. Acquire 2D images using an upright fluorescence microscope and image acquisition software.
   2. Before performing the microscopic examination, center the tissue in the field of view and adjust the sensitivity to ISO 100. Adjust the exposure time initially by clicking on the Automatic Mode button of the software, and then fine-tune manually by clicking on the Adjust button to optimize the signal-to-noise ratio.
   3. Adjust the wavelength of the incident light to excite the fluorophores by rotating the fluorescence cube. Pseudo colors were used to mark the emission from different fluorescent labels (blue light: DAPI; green light: Myo7A; red light: phalloidin).
   4. By scanning the tissue sample, generate and collect the imaging data as .tif and .jpg image files.