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.

D-galactose-induced mitochondrial oxidative damage and apoptosis in the cochlear stria vascularis of mice.

BACKGROUND: Age-related hearing loss, known as presbycusis, is the result of auditory system degeneration. Numerous studies have suggested that reactive oxygen species (ROS) and mitochondrial oxidative damage play important roles in the occurrence and progression of aging. The D-galactose (D-gal)-induced aging model is well known and widely utilized in aging research. Our previous studies demonstrate that administration of D-gal causes mitochondrial oxidative damage and causes subsequent dysfunction in the cochlear ribbon synapses, which in turn leads to hearing changes and early stage presbycusis. Stria vascularis (SV) cells are vital for hearing function. However, it is unclear to what extent D-gal induces oxidative damage and apoptosis in the cochlear SV of mice. In addition, the source of the causative ROS in the cochlear SV has not been fully investigated. METHODS: In this study, we investigated ROS generation in the cochlear SV of mice treated with D-gal. Hearing function was measured using the auditory brainstem response (ABR). Immunofluorescence was used to examine apoptosis and oxidative damage. Transmission electron microscopy was also used to investigate the mitochondrial ultrastructure. DNA fragmentation was determined using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Mitochondrial membrane potential (MMP) and ATP were also measured. RESULTS: We found that D-gal-treated mice exhibited a significant shift in the mean amplitude and latency of the ABR; a remarkable increase in the levels of NADPH oxidase (NOX-2), Uncoupling protein 2 (UCP2) and cleaved caspase-3 (c-Cas3) was observed, as well as an increase in the number of TUNEL-positive cells were observed in the SV of mice. Both the expression of the DNA oxidative damage biomarker 8-hydroxy-2-deoxyguanosine (8-OHdG) and a commonly occurring mitochondrial DNA deletion were markedly elevated in the SV of mice that had been treated with D-gal to induce aging. Conversely, the ATP level and MMP were significantly reduced in D-gal-induced aging mice. We also found alterations in the mitochondrial ultrastructure in the SV of aging mice, which include swollen and distorted mitochondrial shape, shortened and thickened microvilli, and the accumulation of lysosomes in the SV. CONCLUSION: Our findings suggest that the impairment of cochlear SV during presbycusis may be caused by mitochondrial oxidative damage and subsequent apoptosis.

Adenovirus-mediated SIRT1 protects cochlear strial marginal cells in a D-gal-induced senescent model in vitro.

BACKGROUND: A primary obstacle in age-related hearing loss (ARHL) study is the lack of accelerated senescent models in vitro that explore the precise underlying mechanism in different types of ARHL. The damage to strial marginal cells (SMCs) is a subset of strial presbycusis-associated pathological changes. We aimed to establish a D-galactose (D-gal)-induced SMCs senescent model and study the effect of deacetylase sirtuin 1 (SIRT1) on presbycusis in vitro. METHODS: SMCs from C57BL/6J neonatal mice were cultured and treated with D-gal to establish accelerated senescent models. And then D-gal-induced SMCs were transfected with adenovirus (Ad)-SIRT1-GFP or Ad-GFP. Oxidative stress and mitochondrial DNA (mtDNA) damage were determined by histological analysis or RT-PCR. Western blotting (WB) and RT-PCR were used to evaluate protein and mRNA levels of superoxide dismutase 2 (SOD2) and SIRT1, respectively. Additionally, apoptosis was investigated by WB and TUNEL staining. RESULTS: D-gal-induced SMCs exhibited several characteristics of senescence, including increased the level of 8-hydroxy-2'-deoxyguanosine, which is a marker of DNA oxidative damage, and elevated the amount of mtDNA 3860-bp deletion, which is a common type of mtDNA damage in the auditory system of mice. SIRT1 overexpression effectively inhibited these changes by upregulating the level of SOD2, thereby inhibiting cytochrome c translocation from mitochondria to cytoplasm, inhibiting cell apoptosis, and ultimately delaying aging in the D-gal-induced senescent SMCs. CONCLUSIONS: Altogether, the evidence suggests that the D-gal-induced SMCs accelerated aging model is successfully established, and SIRT1 overexpression protects SMCs against oxidative stress by enhancing SOD2 expression in ARHL.

AAV-ie-mediated UCP2 overexpression accelerates inner hair cell loss during aging in vivo.

BACKGROUND: Uncoupling protein 2 (UCP2), activated by excessive reactive oxygen species (ROS) in vivo, has the dual effect of reducing ROS to protect against oxidative stress and reducing ATP production to regulate cellular metabolism. Both the UCP2 and ROS are increased in cochleae in age-related hearing loss (ARHL). However, the role of UCP2 in sensory hair cells in ARHL remains unclear. METHODS: Male C57BL/6 J mice were randomly assigned to an 8-week-old group (Group 1), a 16-week-old group (Group 2), a 16-week-old + adeno-associated virus-inner ear (AAV-ie) group (Group 3), and a 16-week-old + AAV-ie-UCP2 group (Group 4). Mice aged 8 weeks were administrated with AAV-ie-GFP or AAV-ie-UCP2 via posterior semicircular canal injection. Eight weeks after this viral intervention, hearing thresholds and wave-I amplitudes were tested by auditory brainstem response (ABR). Subsequently, the cochlear basilar membrane was dissected for investigation. The number of hair cells and inner hair cell (IHC) synapses, the level of ROS, and the expression of AMP-activated protein kinase α (AMPKα), were assessed by immunofluorescence staining. In addition, mitochondrial function was determined, and the expression of AMPKα and UCP2 proteins was further evaluated using western blotting. RESULTS: Mice with early-onset ARHL exhibited enhanced oxidative stress and loss of outer hair cells and IHC synapses, while UCP2 overexpression aggravated hearing loss and cochlear pathophysiological changes in mice. UCP2 overexpression resulted in a notable decrease in the number of IHCs and IHC synapses, caused ATP depletion and excessive ROS generation, increased AMPKα protein levels, and promoted IHC apoptosis, especially in the apical and middle turns of the cochlea. CONCLUSION: Collectively, our data suggest that UCP2 overexpression may cause mitochondrial dysfunction via energy metabolism, which activates mitochondrion-dependent cellular apoptosis and leads to IHC loss, ultimately exacerbating ARHL.

SIRT3-mediated deacetylation protects inner hair cell synapses in a H2O2-induced oxidative stress model in vitro.

Oxidative stress is considered a driving event in the damage to inner hair cell (IHC) synapses. Mitochondrial deacetylase sirtuin 3 (SIRT3) is an important regulator of reactive oxygen species (ROS) production. However, the effect of SIRT3 on IHC synapses remains elusive. In this study, we treated cochlear basilar membrane (CBM) with hydrogen peroxide (H2O2) to establish an oxidative stress model in vitro. The H2O2-induced CBM exhibited decreased the number of IHC synapses with low levels of ATP and mitochondrial membrane potential. Additionally, H2O2-induced CBM showed markedly reduced levels of forkhead box protein O 3a (FOXO3a), superoxide dismutase 2 (SOD2), and isocitrate dehydrogenase 2 (IDH2), thereby increasing ROS generation. SIRT3 overexpression via administrating nicotinamide riboside in the H2O2-induced CBM protected IHC synapses against oxidative stress and inhibited hair cell apoptosis. We further demonstrated that SIRT3 overexpression led to upregulation of IDH2, and hypoacetylation of several proteins, such as FOXO3a and SOD2, which in turn reduced the levels of ROS and improved mitochondrial function. Collectively, these findings reveal that overexpressing SIRT3 may be a potential therapeutic approach for damaged IHC synapses induced by oxidative stress.

Autophagy-Mediated Synaptic Refinement and Auditory Neural Pruning Contribute to Ribbon Synaptic Maturity in the Developing Cochlea.

In rodents, massive initial synapses are formed in the auditory peripheral nervous system at the early postnatal stage, and one of the major phenomena is that the number of afferent synapses in the cochlea is significantly reduced in the duration of development. This raises the hypothesis that the number of cochlear ribbon synapses are dramatically changed with hearing development and maturation. In this study, several tracers identifying activities of autophagy were applied to estimate the level of autophagy activity in the process of ribbon synapse development in mice; further, changes in the synaptic number and spiral ganglion nerve (SGN) fibers were quantitatively measured. We found robust expression of LC3B and lysosomal-associated membrane protein 1 as well as LysoTracker in or near inner hair cells and cochlear ribbon synapses in the early stage of postnatal development. Moreover, we found a significant loss in the intensity of SGN fibers at ribbon synaptic development and hearing onset. Thus, this study demonstrates that ribbon synaptic refinement and SGN fibers pruning are closely associated with the morphological and functional maturation of ribbon synapses and that synaptic refinement and SGN fiber pruning are regulated by the robust activities of autophagy in the earlier stages of auditory development.

ROS-Induced Oxidative Damage and Mitochondrial Dysfunction Mediated by Inhibition of SIRT3 in Cultured Cochlear Cells.

Sensorineural hearing loss (SNHL) is one of the most common causes of disability worldwide. Previous evidence suggests that reactive oxygen species (ROS) may play an important role in the occurrence and development of SNHL, while its mechanism remains unclear. We cultured dissected organs of Corti in medium containing different concentrations (0, 0.25, 0.5, 0.75, 1, and 1.25 mM) of hydrogen peroxide (H2O2) and established a four-concentration model of 0, 0.5, 0.75, and 1 mM to study different degrees of damage. We examined ROS-induced mitochondrial damage and the role of sirtuin 3 (SIRT3). Our results revealed that the number of ribbon synapses and hair cells appeared significantly concentration-dependent decrease with exposure to H2O2. Outer hair cells (OHCs) and inner hair cells (IHCs) began to be lost, and activation of apoptosis of hair cells (HCs) was observed at 0.75 mM and 1 mM H2O2, respectively. In contrast with the control group, the accumulation of ROS was significantly higher, and the mitochondrial membrane potential (MMP) was lower in the H2O2-treated groups. Furthermore, the expression of SIRT3, FOXO3A, and SOD2 proteins declined, except for an initial elevation of SIRT3 between 0 and 0.75 mM H2O2. Administration of the selective SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine resulted in increased damage to the cochlea, including loss of ribbon synapses and hair cells, apoptosis of hair cells, more production of ROS, and reduced mitochondrial membrane potential. Thoroughly, our results highlight that ROS-induced mitochondrial oxidative damage drives hair cell degeneration and apoptosis. Furthermore, SIRT3 is crucial for preserving mitochondrial function and protecting the cochlea from oxidative damage and may represent a possible therapeutic target for SNHL.

Deletion of C1ql1 Causes Hearing Loss and Abnormal Auditory Nerve Fibers in the Mouse Cochlea.

Complement C1q Like 1 (C1QL1), a secreted component of C1Q-related protein, is known to play an important role in synaptic maturation, regulation, and maintenance in the central nervous system. C1ql1 is expressed in adult cochlear inner and outer hair cells (IHCs and OHCs) with preferential expression in OHCs. We generated C1ql1 null mice to examine the role of C1QL1 in the auditory periphery. C1ql1-null mice exhibited progressive hearing loss with elevated thresholds of auditory brainstem response and distortion product otoacoustic emission. Confocal microscopy showed that the number of nerve fibers innervating both IHCs and OHCs was significantly reduced. However, spiral ganglion neurons appeared to be normal under electron microscopy. IHC development and survival were not affected by deletion of C1ql1. Voltage-clamp recording and immunocytochmistry combined with confocal microscopy showed C1ql1-null IHCs showed no significant reduction of pre-synaptic proteins and synaptic vesicle release. This is in contrast to significant OHC loss in the KO mice. Our study suggests that C1ql1 is essential for development of hair cell innervation and OHC survival. But maturation of presynaptic machinery in IHCs does not depend on C1QL1.

Long-term exposure to low-intensity environmental noise aggravates age-related hearing loss via disruption of cochlear ribbon synapses.

Noise pollution is a major public hazard. Previous studies have shown that environmental noise affects the reorganization of the auditory cortex and leads to behavioral abnormality; however, the effects of long-term environmental noise exposure on the inner ear and hearing remain to be elucidated. In this study, we simulated environmental noise with a long-term 70 dB sound pressure level "white" noise, observed its effect on the inner ears of C57BL/6J mice, and developed an in vitro model for mechanistic studies. We found that environmental noise increased the hearing threshold, decreased the auditory response amplitude, and aggravated the range and extent of age-related hearing loss (ARHL), especially in the intermediate frequency band in mice. Cochlear ribbon synapse is the primary site of inner ear injury caused by environmental noise. We also verified, through an in vitro simulation of the excitatory toxicity of glutamate and aging effects, that the activation of NLRP3 inflammasome plays a vital role in the cochlear ribbon synaptic damage. Our results show that long-term exposure to low-intensity environmental noise can lead to hearing loss via the disruption of ribbon synapses, which is caused by an inflammatory reaction. Additionally, environmental noise can further aggravate the progression of ARHL. This study expounded the pathogenesis of the inner ear damage caused by environmental noise exposure and provides a new direction for the prevention and treatment of hearing loss.

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.

Nicotinamide riboside protects noise-induced hearing loss by recovering the hair cell ribbon synapses.

OBJECTIVE: Nicotinamide riboside (NR) has been proved to protect the hearing. To achieve animal models of temporary threshold shift (TTS) and permanent threshold shift (PTS) respectively, evaluate the dynamic change of ribbon synapse before and after NR administration. METHODS: Mice were divided into control group, noise exposure (NE) group and NR group. The noise was exposed to NE and NR group, and NR was injected before noise exposure. Auditory brainstem response (ABR), ribbon synapse count and cochlear morphology were tested, as well as the concentration of hydrogen peroxide (H2O2) and ATP. RESULTS: Ribbon synapse count decrease with the intensity of noise exposure, and the cochlear morphology remains stable during TTS and was damaged during PTS. NR promotes the oxidation resistance to protect the synapse and the inner ear morphology. CONCLUSION: Our findings suggest that TTS mice are more vulnerable to noise, and NR can promote the recovery of the synapse count to protect the animals' hearing.

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.

Autophagy is Required for Remodeling in Postnatal Developing Ribbon Synapses of Cochlear Inner Hair Cells.

Cochlear ribbon synapses formed between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) are immature at birth and they require dramatic morphological and functional developments to achieve auditory maturation in postnatal mice. However, the mechanism underlying this remodeling process of cochlear ribbon synapse remains elusive. Here, we report that autophagy is necessary for the development and maturation of cochlear ribbon synapses in mice. In this study, significantly high levels of LC3B (a widespread marker of autophagy) were found in the cochlea from postnatal day 1 (P1) to P15, which then decreased at P28 to P30. Treatment of mice at P7 with rapamycin or 3-methyladenine (activator and inhibitor of autophagy, respectively) for 7 days led to the significant elevations of hearing threshold across frequencies from P15 to P30. Moreover, abnormal morphology of cochlear ribbon synapses and reduced IHC exocytosis function were detected from P15 to P30, which were likely associated to hearing impairment. Thus, our study demonstrated that autophagy was required for remodeling of cochlear ribbon synapses and provided a new insight into autophagy-related hearing disorder during auditory development. Furthermore, we implicated a novel therapeutic target for sensorineural hearing loss.

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.

Dynamic Changes of Functional Neuronal Activities Between the Auditory Pathway and Limbic Systems Contribute to Noise-Induced Tinnitus with a Normal Audiogram.

Tinnitus is thought to be triggered by aberrant neural activity in the central auditory pathway and is often accompanied by comorbidities of emotional distress and anxiety, which imply maladaptive functional connectivity to limbic structures, such as the amygdala and hippocampus. Tinnitus patients with normal audiograms can also have accompanying anxiety and depression, clinically. To test the role of functional connectivity between the central auditory pathway and limbic structures in patients with tinnitus with normal audiograms, we developed a murine noise-induced tinnitus model with a temporary threshold shift (TTS). Tinnitus mice exhibited reduced auditory brainstem response wave I amplitude, and an enhanced wave IV amplitude and wave IV/I amplitude ratio, as compared with control and non-tinnitus mice. Resting-state functional magnetic resonance imaging (fMRI) was used to identify abnormal connectivity of the amygdala and hippocampus and to determine the relationship with tinnitus characteristics. We found increased fMRI responses with amplitude of low-frequency fluctuation (ALFF) in the auditory cortex and decreased ALFF in the amygdala and hippocampus at day 1, but decreased ALFF in the auditory cortex and increased ALFF in the amygdala at day 28 post-noise exposure in tinnitus mice. Decreased functional connectivity between auditory brain regions and limbic structures was demonstrated at day 28 in tinnitus mice. Therefore, aberrant neural activities in tinnitus mice with TTS involved not only the central auditory pathway, but also limbic structures, and there was maladaptive functional connectivity between the central auditory pathway and limbic structures, such as the amygdala and hippocampus.

Long-Term Conductive Auditory Deprivation During Early Development Causes Irreversible Hearing Impairment and Cochlear Synaptic Disruption.

Conductive hearing loss is a prevalent condition globally. It remains unclear whether conductive hearing loss that occurs during early development disrupts auditory peripheral systems. In this study, a mouse model of conductive auditory deprivation (CAD) was achieved using external auditory canal closure on postnatal day 12, which marks the onset of external ear canal opening. Short-term (2 weeks) and long-term (6 weeks) deprivations involving external ear canal closure were conducted. Mice were examined immediately, 4 weeks, and 8 weeks after deprivation. Short-term deprivation induced reversible auditory brainstem response (ABR) threshold and latencies of ABR wave I, whereas long-term deprivation caused irreversible ABR thresholds and latencies of ABR wave I. Complete recovery of ribbon synapses and latencies of ABR wave I was observed in the short-term group. In contrast, we observed irreversible ABR thresholds, latencies of ABR wave I, and quantity of ribbon synapses in the long-term deprivation group. Positive 8-hydroxy-2'-deoxyguanosine signals were noted in cochlear hair cells in the long-term group, suggesting that long-term auditory deprivation could disrupt auditory maturation via mitochondrial damage in cochlear hair cells. Conversely, no significant changes in cellular morphology were observed in cochlear hair cells and spiral ganglion cells in either short- or long-term groups. Collectively, our findings suggest that long-term conductive hearing deprivation during early stages of auditory development can cause significant and irreversible disruption that persists into adulthood.

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.