ROS-induced oxidative stress and mitochondrial dysfunction: a possible mechanism responsible for noise-induced ribbon synaptic damage

Abstract

Evidence suggests that damage to the ribbon synapses (RS) may be the main cause of auditory dysfunction in noise-induced hearing loss (NIHL). Oxidative stress is implicated in the pathophysiology of synaptic damage. However, the relationship between oxidative stress and RS damage in NIHL remains unclear. To investigate the hypothesis that noise-induced oxidative stress is a key factor in synaptic damage within the inner ear, we conducted a study using mice subjected to single or repeated noise exposure (NE). We assessed auditory function using auditory brainstem response (ABR) test and examined cochlear morphology by immunofluorescence staining. The results showed that mice that experienced a single NE exhibited a threshold shift and recovered within two weeks. The ABR wave I latencies were prolonged, and the amplitudes decreased, suggesting RS dysfunction. These changes were also demonstrated by the loss of RS as evidenced by immunofluorescence staining. However, we observed threshold shifts that did not return to baseline levels following secondary NE. Additionally, ABR wave I latencies and amplitudes exhibited notable changes. Immunofluorescence staining indicated not only severe damage to RS but also loss of outer hair cells. We also noted decreased T-AOC, ATP, and mitochondrial membrane potential levels, alongside increased hydrogen peroxide concentrations post-NE. Furthermore, the expression levels of 4-HNE and 8-OHdG in the cochlea were notably elevated. Collectively, our findings suggest that the production of reactive oxygen species leads to oxidative damage in the cochlea. This mitochondrial dysfunction consequently contributes to the loss of RS, precipitating an early onset of NIHL.

Keywords: Noise-induced hearing loss; mitochondrial dysfunction; oxidative stress; reactive oxygen species; ribbon synapses.

Figure 1

Noise exposure (NE) causes a negative effect on auditory function in mice. A. NE protocol. During the course of the experiment, mice were given frequent exposure to 100 dB SPL white noise for 2 hours at each point marked in the image. ABR tests were performed within 24 hours or 2 weeks after each NE. B-F. ABR thresholds change at Click, 4 k, 8 k, 16 k, and 32 kHz frequencies. Compared with the control group, mice showed a significant hearing threshold shift after NE at all frequencies, with hearing recovery after two weeks. However, after a second NE, the hearing threshold could not return to normal levels. G. Analysis of ABR wave I latencies changes after NE at each frequency. Compared with the control group, the wave I lantencies were significantly prolonged at each frequency within 24 h or 2 weeks after single or repeated NE. H. Analysis of ABR wave I amplitudes changes after NE at each frequency. Compared with the control group, a significant decrease in wave I amplitudes were shown at each frequency within 24 h or 2 weeks after single or repeated NE. *P < 0.05; **P < 0.01 versus the control group. NE, noise exposure; ABR, auditory brainstem response.

Figure 2

Repeated noise exposure (NE) exacerbates outer hair cells (OHCs) damage but has no effect on spiral ganglion cells (SGCs). A. Representative image of immunofluorescence staining of OHCs after second NE. Scale bar = 20 μm. B. Representative image of SGCs after NE in different groups. Scale bar = 40 μm. C. Survival percentage of OHCs in three turns (Apical, Middle, and Basal) of the cochlea. D. Quantification of SGCs stained by β-Tubulin III and DAPI. *P < 0.05; **P < 0.01 versus the control group. NE, noise exposure; OHCs, outer hair cells; SGCs, spiral ganglion cells.

Figure 3

Noise exposure (NE) leads to the loss of ribbon synapses (RS). A. Representative images of immunolabeled RS (red) in IHCs with Myosin VIIIa staining (gray) after NE in the different groups. Scale bar = 5 μm. B. IHCs survival percentage in three turns (Apical, Middle, and Basal) of the cochlea. C. Quantification of RS stained by CtBP2. *P < 0.05; **P < 0.01 versus the control group. NE, noise exposure; RS, ribbon synapses; IHCs, inner hair cells.

Figure 4

NE had an effect on mitochondrial function in the cochlea. A. Changes of T-AOC in the cochlea after NE of different time points. B. The accumulation of H₂O₂ in the cochlea after NE of different time points. C. Changes in MMP were detected by the JC-1 kit under flow cytometry. D. Percentage of JC-1 aggregates cells in the different time points after NE. E. Percentage of JC-1 monomer cells in the different time points after NE. F. The levels of ATP significantly decreased as the NE frequency increased. *P < 0.05; **P < 0.01 versus the control group. NE, noise exposure; MMP, mitochondrial membrane potential.

Figure 5

Noise exposure (NE) induced oxidative stress in the inner hair cells (IHCs). A. Representative images of the expression of 4-HNE (red) and 8-OHdG (red) on IHCs in different groups following immunohistochemical staining of frozen cochlea sections. Scale bars = 10 μm. B. Relative 4-HNE expression in IHCs of different time points after NE. C. Relative 8-OHdG expression in IHCs of different time points after NE. *P < 0.05; **P < 0.01 versus the control group. NE, noise exposure; IHCs, inner hair cells.

Figure 6

Noise exposure (NE) induced oxidative stress in the spiral ganglion cells (SGCs). A. Representative images of the expression of 4-HNE (red) and 8-OHdG (red) on SGCs in different groups following immunohistochemical staining of frozen cochlea sections. Scale bar = 40 μm. B. Relative 4-HNE expression in SGCs of different time points after NE. C. Relative 8-OHdG expression in SGCs of different time points after NE. *P < 0.05; **P < 0.01 versus the control group. NE, noise exposure; SGCs, spiral ganglion cells.