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

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.

RNA-seq Profiling and Co-expression Network Analysis of Long Noncoding RNAs and mRNAs Reveal Novel Pathogenesis of Noise-induced Hidden Hearing Loss.

Noise-induced hidden hearing loss (NIHHL), one of the family of conditions described as noise-induced hearing loss (NIHL), is characterized by synaptopathy following moderate noise exposure that causes only temporary threshold elevation. Long noncoding RNAs (lncRNAs) mediate several essential regulatory functions in a wide range of biological processes and diseases, but their roles in NIHHL remain largely unknown. In order to determine the potential roles of these lncRNAs in the pathogenesis of NIHHL, we first evaluated their expression in NIHHL mice model and mapped possible regulatory functions and targets using RNA-sequencing (RNA-seq). In total, we identified 133 lncRNAs and 522 mRNAs that were significantly dysregulated in the NIHHL model. Gene Ontology (GO) showed that these lncRNAs were involved in multiple cell components and systems including synapses and the nervous and sensory systems. In addition, a lncRNA-mRNA network was constructed to identify core regulatory lncRNAs and transcription factors. KEGG analysis was also used to identify the potential pathways being affected in NIHHL. These analyses allowed us to identify the guanine nucleotide binding protein alpha stimulating (GNAS) gene as a key transcription factor and the adrenergic signaling pathway as a key pathway in the regulation of NIHHL pathogenesis. Our study is the first, to our knowledge, to isolate a lncRNA mediated regulatory pathway associated with NIHHL pathogenesis; these observations may provide fresh insight into the pathogenesis of NIHHL and may pave the way for therapeutic intervention in the future.

D-Galactose-induced oxidative stress and mitochondrial dysfunction in the cochlear basilar membrane: an in vitro aging model.

The cochlear basilar membrane (CBM) contains inner hair cells and outer hair cells that convert sound waves into electrical signals and transmit them to the central auditory system. Cochlear aging, the primary reason of age-related hearing loss, can reduce the signal transmission capacity. There is no ideal in vitro aging model of the CBM. In this study, we cultured the CBM, which was dissected from the cochlea of the C57BL/6 mice 5 days after birth, in a medium containing 20 mg/mL, 40 mg/mL, or 60 mg/mL D-galactose (D-gal). Compared with the control group, the levels of senescence-associated ß-galactosidase were increased in a concentration-dependent manner in the CBM of the D-gal groups. In addition, levels of the mitochondrial superoxide and patterns of an age-related mitochondrial DNA3860-bp deletion were significantly increased. The ATP levels and the membrane potential of the mitochondrial were significantly decreased in the CBM of the D-gal groups compared with the control group. Furthermore, in comparison with the control group, damaged hair cell stereocilia and a loss of inner hair cell ribbon synapses were observed in the CBM of the D-gal groups. A loss of hair cells and activation of caspase-3-mediated outer hair cell apoptosis were also observed in the CBM of the high-dose D-gal group. These insults induced by D-gal in the CBM in vitro were similar to the ones that occur in cochlear natural aging in vivo. Thus, we believe that this is a successful in vitro aging model using cultured CBM. These results demonstrate the effects of mitochondrial oxidative damage on presbycusis and provide a reliable aging model to study the mechanisms of presbycusis in vitro.

Age-related insult of cochlear ribbon synapses: An early-onset contributor to D-galactose-induced aging in mice.

Presbycusis results from age-related degeneration of the auditory system. D-galactose (D-gal)-induced aging is an ideal and commonly used animal model in aging research. Previous studies demonstrate that administration of D-gal can activate mitochondria-dependent apoptosis in the cochlear stria vascularis. However, D-gal-induced changes to cochlear inner (IHCs) and outer (OHCs) hair cells, spiral ganglion cells (SGCs), and ribbon synapses connecting IHCs and SGCs have not been systematically reported. The current study investigated changes in the numbers of hair cells, SGCs, and ribbon synapses in the mouse model of aging. We found that in comparison to control mice, the numbers of ribbon synapses and their nerve fibers were significantly decreased in D-gal-treated mice, whereas the numbers of OHCs, IHCs, and SGCs were almost unchanged. Moreover, hair cell stereocilia were also not obviously influenced by D-gal administration. Although D-gal-induced aging did not significantly shift the auditory brainstem response (ABR) thresholds in the 8, 16, and 32 kHz frequency bands, the amplitude and latency of the ABR wave I, reflecting ribbon synapse functions, were abnormal in D-gal-treated mice compared to control mice. We also found that 8-hydroxy-2-deoxyguanosine, a marker of oxidative DNA damage, was significantly increased in mitochondria of cochleae from mice exposed to D-gal-induced aging in comparison to control mice. Moreover, D-gal administration increased the levels of H2O2 and mitochondrial 3860-bp common deletion, and decreased superoxide dismutase activity and ATP production in the cochlea. Furthermore, compared with control mice, the protein levels of NADPH oxidase 2 and uncoupling protein 2 were significantly increased in the cochlea of D-gal-treated mice. Taken together, these findings support that the cochlear ribbon synapse is the primary insult site in the early stage of presbycusis, and mitochondrial oxidative damage and subsequent dysfunctions might be responsible for this insult.

NADPH Oxidase 2-Mediated Insult in the Auditory Cortex of Zucker Diabetic Fatty Rats.

Clinical data has confirmed that auditory impairment may be a secondary symptom of type 2 diabetes mellitus (T2DM). However, mechanisms underlying pathologic changes that occur in the auditory system, especially in the central auditory system (CAS), remain poorly understood. In this study, Zucker diabetic fatty (ZDF) rats were used as a T2DM rat model to observe ultrastructural alterations in the auditory cortex and investigate possible mechanisms underlying CAS damage in T2DM. The auditory brainstem response (ABR) of ZDF rats was found to be markedly elevated in low (8 kHz) and high (32 kHz) frequencies. Protein expression of NADPH oxidase 2 (NOX2) and its matching subunits P22phox, P47phox, and P67phox was increased in the auditory cortex of ZDF rats. Expression of 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker of DNA oxidative damage, was also increased in the neuronal mitochondria of the auditory cortex of ZDF rats. Additionally, decreases in the mitochondrial total antioxidant capabilities (T-AOC), adenosine triphosphate (ATP) production, and mitochondrial membrane potential (MMP) were detected in the auditory cortex of ZDF rats, suggesting mitochondrial dysfunction. Transmission electron microscopy results indicated that ultrastructural damage had occurred to neurovascular units and mitochondria in the auditory cortex of ZDF rats. Furthermore, cytochrome c (Cyt c) translocation from mitochondria to cytoplasm and caspase 3-dependent apoptosis were also detected in the auditory cortex of ZDF rats. Consequently, the study demonstrated that T2DM may cause morphological damage to the CAS and that NOX2-associated mitochondrial oxidative damage and apoptosis may be partly responsible for this insult.

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea.

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear "synaptopathy", which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

Mitochondrial DNA 3,860-bp Deletion Increases with Aging in the Auditory Nervous System of C57BL/6J Mice.

BACKGROUND/AIMS: Mitochondrial DNA (mtDNA) is sensitive to oxidative damage during aging, which can result in mtDNA mutations. A previous study reported that a 3,860-bp mtDNA deletion, similar to a 4,977-bp mtDNA deletion in humans, is also common occurrence in murine tissues, and increases in the brain and liver with aging. However, no previous study evaluated both topics in the murine auditory nervous system. METHODS: We compared mtDNA oxidative damage, mitochondrial ultrastructural changes, and the frequency of the 3,860-bp deletion in the peripheral (spiral ganglion, SG) and central (auditory cortex, AC) auditory nervous system of C57BL/6J mice aged 2, 12, and 18 months. RESULTS: We found that the threshold of auditory brainstem response increased with age along with the signal of 8-hydroxy-2'-deoxyguanosine - a marker of DNA oxidative damage - in the mitochondria of SG and AC neurons. The mitochondrial ultrastructural damage also increased with aging in the SG and AC neurons. Moreover, the relative amount of mtDNA 3,860-bp deletion in 12- and 18-month-old mice was significantly higher in the SG and AC as compared to 2-month-old mice. CONCLUSION: These results suggest that the mtDNA 3,860-bp deletion is common in the auditory nervous system of mice and increases with age and may contribute to age-related hearing loss.

NADPH oxidase inhibitor apocynin decreases mitochondrial dysfunction and apoptosis in the ventral cochlear nucleus of D-galactose-induced aging model in rats.

Presbycusis has become a common sensory deficit in humans. Oxidative damage to mitochondrial DNA and mitochondrial dysfunction is strongly associated with the aging of the auditory system. A previous study established a mimetic rat model of aging using D-galactose (D-gal) and first reported that NADPH oxidase-dependent mitochondrial oxidative damage and apoptosis in the ventral cochlear nucleus (VCN) might contribute to D-gal-induced central presbycusis. In this study, we investigated the effects of apocynin, an NADPH oxidase inhibitor, on mitochondrial dysfunction and mitochondria-dependent apoptosis in the VCN of D-gal-induced aging model in rats. Our data showed that apocynin decreased NADPH oxidase activity, H2O2 levels, mitochondrial DNA common deletion, and 8-hydroxy-2-deoxyguanosine (8-OHdG) expression and increased total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) activity in the VCN of D-gal-induced aging model in rats. Moreover, apocynin also decreased the protein levels of phospho-p47phox (p-p47phox), tumor necrosis factor alpha (TNFα), and uncoupling protein 2 (UCP2) in the VCN of D-gal-induced aging model in rats. Meanwhile, apocynin alleviated mitochondrial ultrastructure damage and enhanced ATP production and mitochondrial membrane potential (MMP) levels in the VCN of D-gal-induced aging model in rats. Furthermore, apocynin inhibited cytochrome c (Cyt c) translocation from mitochondria to the cytoplasm and suppressed caspase 3-dependent apoptosis in the VCN of D-gal-induced aging model in rats. Consequently, our findings suggest that neuronal survival promoted by an NADPH oxidase inhibitor is a potentially effective method to enhance the resistance of neurons to central presbycusis.