Animals

All animal protocols were approved by the Institutional Animal Care and Use Committee of Capital Medical University, Beijing, China. Due to naturally carrying the Ahl gene, C57BL/6 J mice show high-frequency hearing loss at the age of 8 weeks, and thus are widely used for ARHL studies (Erway et al. 1993). All mice were randomly assigned four groups: (1) group 1, normal 8-week-old (8w) mice; (2) group 2, normal 16-week-old (16w) mice; (3) group 3, 16-week-old + AAV-ie-GFP mice (normal 8w mice were treated with AAV-ie empty vector via the posterior semicircular canal (PSC) injection, 8 weeks after which they were used for subsequent experiments); (4) group 4, 16-week-old + AAV-ie-UCP2 mice (normal 8w mice were treated with AAV-ie-UCP2 via the PSC, 8 weeks after which they were examined).

AAV-ie vector

The AAV-ie vector carrying UCP2, or empty vector with a CAG promoter and enhanced green fluorescent protein (AAV-ie-CAG-UCP2-eGFP or AAV-ie-CAG-eGFP), were manufactured by PackGene Biotech (Guangzhou, China). AAV-ie-CAG-eGFP was as control vector.

Surgery

The operation was performed as previously descripted in our laboratory (Guo et al. 2018; He et al. 2020). Briefly, mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection (i.p.). The PSC on the left ear was exposed and dilled a tiny hole using a needle. The polyimide tube was gently inserted into the PSC through the hole. A total of 2 μL of AAV-ie was injected into each cochlea within 4 min. AAV-ie empty vector and AAV-ie-UCP2 were transfected within the cochlea of mice in the group 3 and group 4, respectively. After the virus injection, the hole in the PSC was rapidly blocked using a small piece of muscle. And the skin incision was sutured and disinfect with povidone.

Auditory brainstem response (ABR) testing

The method of ABR testing has been described previously (Liang et al. 2021; Sergeyenko et al. 2013). Briefly, it was conducted in sound-proof room using BioSigRZ software (Tucker-Davis Technologies, USA). Mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg). The condition of the external auditory canal and the tympanic membrane (TM) was observed using an electric otoscope before testing. Mice with acute otitis externa (AOE) and otitis media were excluded. AOE is characterized by an inflamed external auditory canal (Wiegand et al. 2019); The main manifestation of otitis media is a red, swollen, or even perforated TM, and may also include purulent exudate or middle ear effusion (Sundgaard et al. 2021). ABR response was recorded by subcutaneous needle electrodes. The recording, the reference and the ground electrode were located in subcutaneous tissue of the vertex of the skull, the mastoid process of the tested ear and the mastoid process of contralateral ear, respectively. The electrical signals were amplified, filtered and averaged (1024 samples). The sound level was diminished in 5 dB steps from 90 dB SPL to the hearing threshold. The response was elicited in tone bursts at four frequencies of 4, 8, 16, and 32 kHz. The hearing threshold was determined as the lowest stimulus level at which a repeatable Wave II could be visually identified. The amplitude of ABR waves was the difference between the peak and the subsequent trough (Sergeyenko et al. 2013). Amplitudes of Waves I at 90 dB SPL were evaluated.

Histology and immunofluorescence (IF) staining

Mice were heavily anesthetized with ketamine and xylazine, and euthanized by cervical dislocation. Cochleae were fixed with 4% paraformaldehyde (PFA) overnight at 4 °C. After washed with PBS three times, parts of cochleae were decalcified in 10% EDTA for 48 h at 4 °C and then were washed with PBS. Samples were dehydrated in a gradient of 20%, and 30% sucrose (1 h each) at 4 °C. Subsequently, samples were embedded in optimal cutting temperature compound, and then were sliced at − 20 °C with a thickness of 10 μm using a Leica Cryastat (German). Cochlear frozen sections were stored at − 80 °C for IF staining. Remaining cochlea were decalcified for 2 h at room temperature. Cochleae were microdissected into three pieces: apical, middle, and basal turn.

For IF staining, frozen sections were washed with PBS three times (10 min each). Frozen sections and cochlear turns were permeabilized and blocked with PBS consisting of 5% goat serum and 0.3% TritonX-100 for 2 h, and incubated with primary antibodies overnight at 4 °C. The primary antibodies were applied as follows: mouse anti-4 hydroxynonenal (4-HNE, diluted 1:200; Abcam, USA), a biomarker of oxidative damage; mouse anti-8-hydroxy-2′-deoxyguanosine (8-OHdG, diluted 1:300; Abcam, USA), a marker of DNA oxidative damage; rabbit anti-Myosin VIIa (diluted 1:300; Proteus Biosciences, USA), a hallmark of cochlear hair cells; mouse anti-C-terminal binding protein 2 (CtBP2, diluted 1:300; BD Biosciences, USA), which is used to label the presynaptic ribbon; mouse anti-GFP (diluted 1:100, Santa Cruz Biotechnology, USA). Additionally, samples were immunostained with primary antibody to rabbit anti-phosphorylated AMP-activated protein kinase α (pAMPKα, diluted 1:50; Cell Signaling, USA) for 48 h at 4 °C. The following day, after washing with PBS three times, samples were incubated with at appropriate Alexa-conjugated secondary antibodies for 1 h at room temperature. All secondary antibodies were diluted at 1:300 in PBS. Samples were mounted with an anti-fluorescence quenching agent containing DAPI.

Confocal microscopy and image analysis

All confocal images of three random fields per turn ('Apical', 'Middle', 'Basal') from each cochlea were captured using a Leica TCS SP8 laser microscope. Image analysis were performed as previously described with a minor modification (Kujawa and Liberman 2009; Sergeyenko et al. 2013).

To count OHCs and IHCs labeled with the anti-Myosin VIIa antibodies, images with a z-step-size of 2 μm were obtained with an oil immersion objective with 2 × digital zoom. The number of OHCs and IHCs in apical, middle, and basal turns was manually counted, respectively.

To count synapses labeled with anti-CtBP2, images with a z-step-size of 0.5 μm were obtained with 63×/1.40-NA oil objective with 2 × digital zoom. IHC synapses in apical, middle, and basal turns were quantified, respectively. And IHC synapses were divided by the total number of IHCs stained by anti-Myosin VIIa in per field under the microscope. If needed, 3D renderings were generated to avoid miscounting the number of synapses due to the image overlay process.

To quantify levels of 4-HNE and 8-OHdG, images with a z-step-size of 1 μm were obtained with oil immersion objective with 1.5 × digital zoom. The relative fluorescence intensities of 4-HNE and 8-OHdG were semi-quantified by measuring the average intensity from three random microscope fields in each cochlear.

To quantify the level of pAMPK per turn, images with a z-step-size of 1 μm were obtained with an oil immersion objective with 2 × digital zoom. The relative fluorescence intensity of pAMPK was semi-quantified by measuring the average intensity from three random microscope fields in each cochlear turn using ImageJ software (NIH, Bethesda, MD, USA), and normalized to controls.

Mitochondrial ROS measurement

Mitochondrial ROS were determined by MitoSOX™ Red (Invitrogen, USA) according to the manufacturer's instructions. Briefly, after sacrificing the mice, part of the cochlear bone shell was quickly removed under a microscope. Because the cochlear shell is relatively hard in mice over 8 weeks old, it is difficult to completely remove it without decalcification or causing damage to hair cells. Thereafter, the samples were incubated with DMEM/F12 and 5 μM MitoSOX™ Red for 35 min at 37 °C and washed thrice with warm PBS, after which they were fixed with 4% PFA at 4 °C overnight. After again washing thrice with PBS, samples were decalcified in 10% EDTA for 1 h. Cochleae were then microdissected into apical, middle, and basal turns while being protected from light. The samples were then sealed with an anti-fluorescence quenching agent with DAPI and visualized using confocal microscopy. MitoSOX™ fluorescence intensity was quantified using ImageJ (NIH, Bethesda, MD, USA), and the data were normalized to normal cochlea controls.

Mitochondrial isolation

A commercial tissue mitochondria isolation kit (Beyotime Biotechnology, China) was applied to extract mitochondria from mouse cochlea. Briefly, after washing with ice-cold PBS, cochlear tissue was cut into very small pieces, after which it was suspended in 10 volumes of PBS. Thereafter, the samples were incubated on ice for 3 min and centrifuged for 10–20 s. After removing the supernatant, the samples were added to 8 volumes of PBS containing 0.25 mg/mL trypsin and incubated on ice for 20 min. After removing the supernatant, samples were re-suspended in 2 volumes of mitochondrial extraction buffer to remove residual trypsin, and then samples were centrifuged for 10–20 s at 600×g. Subsequently, the supernatant was removed, the samples were added to 8 volumes of mitochondrial extraction buffer, and then homogenized 20–30 times. The samples were centrifuged at 600×g for 5 min. Next, the supernatant was transferred to a clean tube and centrifuged at 11,000×g for 10 min. The centrifuged deposit was the mitochondria.

ATP and MMP measurement

According to the manufacturer's instructions, ATP and MMP levels were measured with a commercial ATP and JC-1assay kit (Beyotime, China), respectively. The levels of ATP were quantified with relative luminescent units using a luminometer. ATP results were shown as nmol/mg protein. MMP levels were defined as the ratio of optical density between JC-1 aggregates to JC-1 monomers.

TUNEL assay

The apoptosis of hair cells was measured with a TUNEL assay kit (Beyotime, China). After being permeabilized and blocked, samples were incubated with rabbit anti-Myosin VIIa (diluted 1:300; Proteus Biosciences, USA) and mouse anti-GFP (diluted 1:100, Santa Cruz Biotechnology, USA) overnight at 4 °C. The next day, after washing with PBS, samples were incubated with secondary Alexa Fluor 647 goat anti-rabbit IgG (1:300, Invitrogen, USA) and TUNEL red solution at room temperature for 1 h. Slides were mounted and imaged by confocal microscopy. The apoptosis rate refers to the proportion of TUNEL+ cells in the number of IHCs in three random microscopic fields per turn in each sample.

Western blot (WB)

Mitochondria were extracted from cochlear tissues as described above. Briefly, the mitochondria from mouse cochlea were isolated with a tissue mitochondria isolation kit (Beyotime, China) according to the manufacturer’s instructions. The protein from mouse cochlea tissues was extracted with strong RIPA lysis buffer containing PMSF (Sigma, USA) and protease inhibitor cocktail (Thermo Fisher Scientific, USA). Then, the protein was quantified. The protein was separated using 10% SDS-PAGE gels and then blotted onto PVDF membranes. After being blocked, the membranes were exposed to primary antibodies overnight at 4 °C: anti-UCP2 (diluted 1:1000; Abcam, USA); anti-superoxide dismutase 2 (SOD2, diluted 1:1000; Cell Signaling, USA); anti-AMPK (diluted 1:1000; Cell Signaling, USA); anti-pAMPK (diluted 1:1000; Cell Signaling, USA); β-actin (diluted 1:1000; Cell Signaling, USA); α-tubulin (diluted 1:1000; Cell Signaling, USA); and GAPDH (diluted 1:1000; Cell Signaling, USA). Following day, the membranes were incubated with appropriate secondary antibodies. The protein signals were measured by enhanced chemiluminescence, and quantified using the Fujifilm LAS 400 imaging system.

Statistical analysis

SPSS (version 24.0, Chicago, IL, USA) was used for statistical analyses. Normality was analyzed with the Kolmogorov–Smirnov test. Data conforming to normal distribution are expressed as mean ± standard error of the mean (SEM). Comparisons between two groups were analyzed with a Student’s t test (two way); multiple comparisons were analyzed with the one-way analysis of variance (ANOVA) followed by the LSD’s post hoc test. A value of p < 0.05 was judged as significant. *p < 0.05; **p < 0.01.