The cochlea contains macrophages. These cells participate in inflammatory responses to cochlear pathogenesis. However, it is not clear how and when these cells populate the cochlea during postnatal development. The current study aims to determine the postnatal development of cochlear macrophages with the focus on macrophage development in the organ of Corti and the basilar membrane. Cochleae were collected from C57BL/6J mice at ages of postnatal day (P) 1 to P21, as well as from mature mice (1-4 months). Macrophages were identified based on their expression of F4/80 and Iba1, as well as their unique morphologies. Two sets of macrophages were identified in the regions of the organ of Corti and the basilar membrane. One set resides on the scala tympani side of the basilar membrane. These cells have a round shape at P1 and start to undergo site-specific differentiation at P4. Apical macrophages adopt a dendritic shape. Middle and basal macrophages take on an irregular shape with short projections. Basal macrophages further differentiate into an amoeboid shape. The other set of macrophages resides above the basilar membrane, either beneath the cells of the organ of Corti or along the spiral vessel of the basilar membrane. As the sensory epithelium matures, these cells undergo developmental death with the phenotypes of apoptosis. Macrophages are also identified in the spiral ligament, spiral limbus, and neural regions. Their numbers decrease during postnatal development. Together, these results suggest a dynamic rearrangement of the macrophage population during postnatal cochlear development.

Membrane transporters can be major determinants in the targeting and effectiveness of pharmaceutical agents. A large number of biologically important membrane transporters have been identified and localized to a variety of tissues, organs and cell types. However, little is known about the expression of key membrane transporters in the inner ear, a promising site for targeted therapeutics, as well as a region vulnerable to adverse drug reactions and environmental factors. In this study, we examined the levels of endogenous membrane transporters in rat cochlea by targeted PCR array analysis of 84 transporter genes, followed by validation and localization in tissues by immunohistochemistry. Our studies indicate that several members of the SLC, VDAC and ABC membrane transporter families show high levels of expression, both at the RNA and protein levels in the rat cochlea. Identification and characterization of these membrane transporters in the inner ear have clinical implications for both therapeutic and cytotoxic mechanisms that may aid in the preservation of auditory function.

Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway; it catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate and NADP+ to NADPH and is thought to be the principal source of NADPH for the cytosolic glutathione and thioredoxin antioxidant defense systems. We investigated the roles of G6PD in the cytosolic antioxidant defense in the cochlea of G6pd hypomorphic mice that were backcrossed onto normal-hearing CBA/CaJ mice. Young G6pd-deficient mice displayed a significant decrease in cytosolic G6PD protein levels and activities in the inner ears. However, G6pd deficiency did not affect the cytosolic NADPH redox state, or glutathione or thioredoxin antioxidant defense in the inner ears. No histological abnormalities or oxidative damage was observed in the cochlea of G6pd hemizygous males or homozygous females. Furthermore, G6pd deficiency did not affect auditory brainstem response hearing thresholds, wave I amplitudes or wave I latencies in young males or females. In contrast, G6pd deficiency resulted in increased activities and protein levels of cytosolic isocitrate dehydrogenase 1, an enzyme that catalyzes the conversion of isocitrate to α-ketoglutarate and NADP+ to NADPH, in the inner ear. In a mouse inner ear cell line, knockdown of Idh1, but not G6pd, decreased cell growth rates, cytosolic NADPH levels, and thioredoxin reductase activities. Therefore, under normal physiological conditions, G6pd deficiency does not affect the cytosolic glutathione or thioredoxin antioxidant defense in mouse cochlea. Under G6pd deficiency conditions, isocitrate dehydrogenase 1 likely functions as the principal source of NADPH for cytosolic antioxidant defense in the cochlea.SIGNIFICANCE STATEMENT Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway; it catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate and NADP+ to NADPH and is thought to be the principal source of NADPH for the cytosolic glutathione and thioredoxin antioxidant defense systems. In the current study, we show that, under normal physiological conditions, G6pd deficiency does not affect the cytosolic glutathione or thioredoxin antioxidant defense in the mouse cochlea. However, under G6pd deficiency conditions, isocitrate dehydrogenase 1 likely functions as the principal source of NADPH for cytosolic antioxidant defense in the cochlea.

Sestrin 2 (SESN2) is a stress-inducible protein that protects tissues from oxidative stress and delays the aging process. However, its role in maintaining the functional and structural integrity of the cochlea is largely unknown. Here, we report the expression of SESN2 protein in the sensory epithelium, particularly in hair cells. Using C57BL/6J mice, a mouse model of age-related cochlear degeneration, we observed a significant age-related reduction in SESN2 expression in cochlear tissues that was associated with early onset hearing loss and accelerated age-related sensory cell degeneration that progressed from the base toward the apex of the cochlea. Hair cell death occurred by caspase-8 mediated apoptosis. Compared to C57BL/6J control mice, Sesn2 KO mice displayed enhanced expression of proinflammatory genes and activation of basilar membrane macrophages, suggesting that loss of SESN2 function provokes the immune response. Together, these results suggest that Sesn2 plays an important role in cochlear homeostasis and immune responses to stress.

Glutathione reductase (GSR), a key member of the glutathione antioxidant defense system, converts oxidized glutathione (GSSG) to reduced glutathione (GSH) and maintains the intracellular glutathione redox state to protect the cells from oxidative damage. Previous reports have shown that Gsr deficiency results in defects in host defense against bacterial infection, while diquat induces renal injury in Gsr hypomorphic mice. In flies, overexpression of GSR extended lifespan under hyperoxia. In the current study, we investigated the roles of GSR in cochlear antioxidant defense using Gsr homozygous knockout mice that were backcrossed onto the CBA/CaJ mouse strain, a normal-hearing strain that does not carry a specific Cdh23 mutation that causes progressive hair cell degeneration and early onset of hearing loss. Gsr-/- mice displayed a significant decrease in GSR activity and GSH/GSSG ratios in the cytosol of the inner ears. However, Gsr deficiency did not affect ABR (auditory brainstem response) hearing thresholds, wave I amplitudes or wave I latencies in young mice. No histological abnormalities were observed in the cochlea of Gsr-/- mice. Furthermore, there were no differences in the activities of cytosolic glutathione-related enzymes, including glutathione peroxidase and glutamate-cysteine ligase, or the levels of oxidative damage markers in the inner ears between WT and Gsr-/- mice. In contrast, Gsr deficiency resulted in increased activities of cytosolic thioredoxin and thioredoxin reductase in the inner ears. Therefore, under normal physiological conditions, GSR is not essential for the maintenance of antioxidant defenses in mouse cochlea. Given that the thioredoxin system is known to reduce GSSG to GSH in multiple species, our findings suggest that the thioredoxin system can support GSSG reduction in the mouse peripheral auditory system.

Trimethyltin (TMT) is an occupational and environmental health hazard behaving as a potent neurotoxin known to affect the central nervous system as well as the peripheral auditory system. However, the mechanisms underlying TMT-induced ototoxicity are poorly understood. To elucidate the effects of TMT on the cochlea, a single injection of 4 or 8 mg/kg TMT was administered intraperitoneally to adult rats. The compound action potential (CAP) threshold was used to assess the functional status of the cochlea and histological techniques were used to assess the condition of the hair cells and auditory nerve fibers. TMT at 4 mg/kg produced a temporary CAP threshold elevation of 25-60 dB that recovered by 28 d post-treatment. Although there was no hair cell loss with the 4 mg/kg dose, there was a noticeable loss of auditory nerve fibers particularly beneath the inner hair cells. TMT at 8 mg/kg produced a large permanent CAP threshold shift that was greatest at the high frequencies. The CAP threshold shift was associated with the loss of outer hair cells and inner hair cells in the basal, high-frequency region of the cochlea, considerable loss of auditory nerve fibers and a significant loss of spiral ganglion neurons in the basal turn. Spiral ganglion neurons showed evidence of soma shrinkage and nuclear condensation and fragmentation, morphological features of apoptotic cell death. TMT-induced damage was greatest in the high-frequency, basal region of the cochlea and the nerve fibers beneath the inner hair cells were the most vulnerable structures.

The acoustic startle reflex (ASR) amplitude can be enhanced or suppressed by noise-induced hearing loss or age-related hearing loss; however, little is known about how the ASR changes when ototoxic drugs destroy outer hair cells (OHCs) and inner hair cells (IHCs). High doses of 2-hydroxypropyl-beta-cyclodextrin (HPβCD), a cholesterol-lowering drug used to treat Niemann-Pick Type disease type C1, initially destroy OHCs and then the IHCs 6-8 weeks later. Adult rats were treated with doses of HPβCD designed to produce a diversity of hair cell lesions and hearing losses. When HPβCD destroyed OHCs and IHCs in the extreme base of the cochlea and caused minimal high-frequency hearing loss, the ASR amplitudes were enhanced at 4-, 8- and 16 kHz. Enhanced ASR occurred during the first few weeks post-treatment when only OHCs were missing; little change in the ASR occurred 6-8-WK post-treatment. If HPβCD destroyed most OHCs and many IHCs in the basal half of the cochlea, high-frequency thresholds increased ∼50 dB, and ASR amplitudes were reduced ∼50% at 4-, 8- and 16-kHz. The ASR amplitude reduction occurred in the first few weeks post-treatment when the OHCs were degenerating. The ASR was largely abolished when most of the OHCs were missing over the basal two-thirds of the cochlea and a 40-50 dB hearing loss was present at most frequencies. These results indicate that high-doses of HPβCD generally lead to a decline in ASR amplitude as OHCs degenerate; however, ASR amplitudes were enhanced in a few cases when hair cell loss was confined to the extreme base of the cochlea.

Noise-induced hearing loss generally induces loudness recruitment, but sometimes gives rise to hyperacusis, a debilitating condition in which moderate intensity sounds are perceived abnormally loud. In an attempt to develop an animal model of loudness hyperacusis, we exposed rats to a 16-20 kHz noise at 104 dB SPL for 12 weeks. Behavioral reaction time-intensity functions were used to assess loudness growth functions before, during and 2-months post-exposure. During the exposure, loudness recruitment (R) was present in the region of hearing loss, but subtle evidence of hyperacusis (H) started to emerge at the border of the hearing loss. Unexpectedly, robust evidence of hyperacusis appeared below and near the edge of the hearing loss 2-months post-exposure. To identify the neural correlates of hyperacusis and test the central gain model of hyperacusis, we recorded population neural responses from the cochlea, auditory cortex and lateral amygdala 2-months post-exposure. Compared to controls, the neural output of the cochlea was greatly reduced in the noise group. Consistent with central gain models, the gross neural responses from the auditory cortex and amygdala were proportionately much larger than those from the cochlea. However, despite central amplification, the population responses in the auditory cortex and amygdala were still below the level needed to fully account for hyperacusis and/or recruitment. Having developed procedures that can consistently induce hyperacusis in rats, our results set the stage for future studies that seek to identify the neurobiological events that give rise to hyperacusis and to develop new therapies to treat this debilitating condition.

Acoustic trauma, one of the leading causes of sensorineural hearing loss, induces sensory hair cell damage in the cochlea. Identifying the molecular mechanisms involved in regulating sensory hair cell death is critical towards developing effective treatments for preventing hair cell damage. Recently, microRNAs (miRNAs) have been shown to participate in the regulatory mechanisms of inner ear development and homeostasis. However, their involvement in cochlear sensory cell degeneration following acoustic trauma is unknown. Here, we profiled the expression pattern of miRNAs in the cochlear sensory epithelium, defined miRNA responses to acoustic overstimulation, and explored potential mRNA targets of miRNAs that may be responsible for the stress responses of the cochlea. Expression analysis of miRNAs in the cochlear sensory epithelium revealed constitutive expression of 176 miRNAs, many of which have not been previously reported in cochlear tissue. Exposure to intense noise caused significant threshold shift and apoptotic activity in the cochleae. Gene expression analysis of noise-traumatized cochleae revealed time-dependent transcriptional changes in the expression of miRNAs. Target prediction analysis revealed potential target genes of the significantly downregulated miRNAs, many of which had cell death- and apoptosis-related functions. Verification of the predicted targets revealed a significant upregulation of Taok1, a target of miRNA-183. Moreover, inhibition of miR-183 with morpholino antisense oligos in cochlear organotypic cultures revealed a negative correlation between the expression levels of miR-183 and Taok1, suggesting the presence of a miR-183/Taok1 target pair. Together, miRNA profiling as well as the target analysis and validation suggest the involvement of miRNAs in the regulation of the degenerative process of the cochlea following acoustic overstimulation. The miR-183/Taok1 target pair is likely to play a role in this regulatory process.

Isocitrate dehydrogenase (IDH) 2 participates in the TCA cycle and catalyzes the conversion of isocitrate to α-ketoglutarate and NADP+ to NADPH. In the mitochondria, IDH2 also plays a key role in protecting mitochondrial components from oxidative stress by supplying NADPH to both glutathione reductase (GSR) and thioredoxin reductase 2 (TXNRD2). Here, we report that loss of Idh2 accelerates age-related hearing loss, the most common form of hearing impairment, in male mice. This was accompanied by increased oxidative DNA damage, increased apoptotic cell death, and profound loss of spiral ganglion neurons and hair cells in the cochlea of 24-month-old Idh2-/- mice. In young male mice, loss of Idh2 resulted in decreased NADPH redox state and decreased activity of TXNRD2 in the mitochondria of the inner ear. In HEI-OC1 mouse inner ear cell lines, knockdown of Idh2 resulted in a decline in cell viability and mitochondrial oxygen consumption. This was accompanied by decreased NADPH redox state and decreased activity of TXNRD2 in the mitochondria of the HEI-OC1 cells. Therefore, IDH2 functions as the principal source of NADPH for the mitochondrial thioredoxin antioxidant defense and plays an essential role in protecting hair cells and neurons against oxidative stress in the cochlea of male mice.

Mitochondrial DNA (mtDNA) mutations are thought to have a causal role in a variety of age-related neurodegenerative diseases, including age-related hearing loss (AHL). In the current study, we investigated the roles of mtDNA deletions and point mutations in AHL in mitochondrial mutator mice (Polgmut/mut) that were backcrossed onto CBA/CaJ mice, a well-established model of late-onset AHL. mtDNA deletions accumulated significantly with age in the inner ears of Polgmut/mut mice, while there were no differences in mtDNA deletion frequencies in the inner ears between 5 and 17 months old Polg+/+ mice or 5 months old Polg+/+ and Polgmut/mut mice. mtDNA deletions also accumulated significantly in the inner ears of CBA/CaJ mice during normal aging. In contrast, 5 months old Polgmut/mut mice displayed a 238-fold increase in mtDNA point mutation frequencies in the inner ears compared to age-matched Polg+/+ mice, but there were no differences in mtDNA point mutation frequencies in the inner ears between 5 and 17 months old Polgmut/mut mice. Seventeen-month-old Polgmut/mut mice also displayed early-onset severe hearing loss associated with a significant reduction in neural output of the cochlea, while age-matched Polg+/+ mice displayed little or no hearing impairment. Consistent with the physiological and mtDNA deletion test result, 17-month-old Polgmut/mut mice displayed a profound loss of spiral ganglion neurons in the cochlea. Thus, our data suggest that a higher burden of mtDNA point mutations from a young age and age-related accumulation of mtDNA deletions likely contribute to early-onset AHL in mitochondrial mutator mice.

2-Hyroxypropyl-beta-cyclodextrin (HPβCD) is being used to treat Niemann-Pick C1, a fatal neurodegenerative disease caused by abnormal cholesterol metabolism. HPβCD slows disease progression, but unfortunately causes severe, rapid onset hearing loss by destroying the outer hair cells (OHC). HPβCD-induced damage is believed to be related to the expression of prestin in OHCs. Because prestin is postnatally upregulated from the cochlear base toward the apex, we hypothesized that HPβCD ototoxicity would spread from the high-frequency base toward the low-frequency apex of the cochlea. Consistent with this hypothesis, cochlear hearing impairments and OHC loss rapidly spread from the high-frequency base toward the low-frequency apex of the cochlea when HPβCD administration shifted from postnatal day 3 (P3) to P28. HPβCD-induced histopathologies were initially confined to the OHCs, but between 4- and 6-weeks post-treatment, there was an unexpected, rapid and massive expansion of the lesion to include most inner hair cells (IHC), pillar cells (PC), peripheral auditory nerve fibers, and spiral ganglion neurons at location where OHCs were missing. The magnitude and spatial extent of HPβCD-induced OHC death was tightly correlated with the postnatal day when HPβCD was administered which coincided with the spatiotemporal upregulation of prestin in OHCs. A second, massive wave of degeneration involving IHCs, PC, auditory nerve fibers and spiral ganglion neurons abruptly emerged 4-6 weeks post-HPβCD treatment. This secondary wave of degeneration combined with the initial OHC loss results in a profound, irreversible hearing loss.

Noise exposure is a major cause of hearing loss. Classical methods of studying protein involvement have provided a basis for understanding signaling pathways that mediate hearing loss and damage repair but do not lend themselves to studying large networks of proteins that are likely to increase or decrease during noise trauma. To address this issue, antibody microarrays were used to quantify the very early changes in protein expression in three distinct regions of the chinchilla cochlea 2h after exposure to a 0.5-8 kHz band of noise for 2h at 112 dB SPL. The noise exposure caused significant functional impairment 2h post-exposure which only partially recovered. Distortion product otoacoustic emissions were abolished 2h after the exposure, but at 4 weeks post-exposure, otoacoustic emissions were present, but still greatly depressed. Cochleograms obtained 4 weeks post-exposure demonstrated significant loss of outer hair cells in the basal 60% of the cochlea corresponding to frequencies in the noise spectrum. A comparative analysis of the very early (2h post-exposure) noise-induced proteomic changes indicated that the sensory epithelium, lateral wall and modiolus differ in their biological response to noise. Bioinformatic analysis of the cochlear protein profile using "The Database for Annotation, Visualization and Integrated Discovery 2008" (DAVID - http://david.abcc. ncifcrf.gov) revealed the initiation of the cell death process in sensory epithelium and modiolus. An increase in Fas and phosphorylation of FAK and p38/MAPK in the sensory epithelium suggest that noise-induced stress signals at the cell membrane are transmitted to the nucleus by Fas and focal adhesion signaling through the p38/MAPK signaling pathway. Up-regulation of downstream nuclear proteins E2F3 and WSTF in immunoblots and microarrays along with their immunolocalization in the outer hair cells supported the pivotal role of p38/MAPK signaling in the mechanism underlying noise-induced hearing loss.

Because cyclodextrins are capable of removing cholesterol from cell membranes, there is growing interest in using these compounds to treat diseases linked to aberrant cholesterol metabolism. One compound, 2-hydroxypropyl-beta-cyclodextrin (HPβCD), is currently being evaluated as a treatment for Niemann-Pick Type C1 disease, a rare, fatal neurodegenerative disease caused by the buildup of lipids in endosomes and lysosomes. HPβCD can reduce some debilitating symptoms and extend life span, but the therapeutic doses used to treat the disease cause hearing loss. Initial studies in rodents suggested that HPβCD selectively damaged only cochlear outer hair cells during the first week post-treatment. However, our recent in vivo and in vitro studies suggested that the damage could become progressively worse and more extensive over time. To test this hypothesis, we treated rats subcutaneously with 1, 2, 3 or 4 g/kg of HPβCD and waited for 8-weeks to assess the long-term histological consequences. Our new results indicate that the two highest doses of HPβCD caused extensive damage not only to OHC, but also to inner hair cells, pillar cells and other support cells resulting in the collapse and flattening of the sensory epithelium. The 4 g/kg dose destroyed all the outer hair cells and three-fourths of the inner hair cells over the basal two-thirds of the cochlea and more than 85% of the nerve fibers in the habenula perforata and more than 80% of spiral ganglion neurons in the middle of basal turn of the cochlea. The mechanisms that lead to the delayed degeneration of inner hair cells, pillar cells, nerve fibers and spiral ganglion neurons remain poorly understood, but may be related to the loss of trophic support caused by the degeneration of sensory and/or support cells in the organ of Corti. Despite the massive damage to the cochlear sensory epithelium, the blood vessels in the stria vascularis and the vestibular hair cells in the utricle and saccule remained normal.

Neuregulin-1 (NRG1) ligand and its epidermal growth factor receptor (EGFR)/ERBB family regulate normal cellular proliferation and differentiation in many tissues including the cochlea. Aberrant NRG1 and ERBB signaling cause significant hearing impairment in mice. Dysregulation of the same signaling pathway in humans is involved in certain types of cancers such as breast cancer or non-small cell lung cancer (NSCLC). A new irreversible pan-ERBB inhibitor, canertinib, has been tested in clinical trials for the treatment of refractory NSCLC. Its possible ototoxicity was unknown. In this study, a significant dose-dependent canertinib ototoxicity was observed in a zebrafish model. Canertinib ototoxicity was further confirmed in two mouse models with different genetic backgrounds. The data strongly suggested an evolutionally preserved ERBB molecular mechanism underlying canertinib ototoxicity. Thus, these results imply that clinical monitoring of hearing loss should be considered for clinical testing of canertinib or other pan-ERBB inhibitors.

Ouabain is a common tool to explore the pathophysiological changes in adult mammalian cochlea in vivo. In prior studies, locally administering ouabain via round window membrane demonstrated that the ototoxic effects of ouabain in vivo varied among mammalian species. Little is known about the ototoxic effects in vitro. Thus, we prepared cochlear organotypic cultures from postnatal day-3 rats and treated these cultures with ouabain at 50, 500, and 1000 μM for different time to elucidate the ototoxic effects of ouabain in vitro and to provide insights that could explain the comparative ototoxic effects of ouabain in vivo. Degeneration of cochlear hair cells and spiral ganglion neurons was evaluated by hair-cell staining and neurofilament labeling, respectively. Annexin V staining was used to detect apoptotic cells. A quantitative RT-PCR apoptosis-focused gene array determined changes in apoptosis-related genes. The results showed that ouabain-induced damage in vitro was dose and time dependent. 500 μM ouabain and 1000 μM ouabain were destructively traumatic to both spiral ganglion neurons and cochlear hair cells in an apoptotic signal-dependent pathway. The major apoptotic pathways in ouabain-induced spiral ganglion neuron apoptosis culminated in the stimulation of the p53 pathway and triggering of apoptosis by a network of proapoptotic signaling pathways.

The ahl locus, shown to be a strain-specific Cdh23 dimorphism, contributes to age-related hearing loss in many inbred mouse strains. A/J mice begin to lose hearing by 4 weeks of age, much earlier than C57BL/6J (B6) mice, although both strains have the same Cdh23(ahl) variant. Here, we use recombinant inbred strains, chromosome substitution strains, and a linkage backcross to map a locus on distal Chromosome 10, designated ahl4, that contributes to the early-onset hearing loss of A/J mice. Cochleae of 9-week-old A/J mice exhibit inner and outer hair cell loss from the basal turn through the apical turn, with outer hair cell loss at the base being severest. To quantify the progression of hair cell loss, cytocochleograms were evaluated from 0 to 20 weeks of age. A/J mice showed evidence of hair cell loss in the base of the cochlea as early as 14 days of age and the magnitude and extent of loss increased rapidly during the following 2-5 months. Hair cell loss occurred earlier and was much more severe and widespread in A/J mice than in B6 mice during the first 5 months of age. Spiral ganglion neurons, cells of the stria vascularis, and vestibular hair cell densities, however, appeared normal in 20-week-old A/J mice.

Occupational exposure to high atmospheric levels of Mn produces a severe and debilitating disorder known as manganism characterized by extrapyramidal disturbances similar to that seen in Parkinson's disease. Epidemiological and case studies suggest that persistent exposures to Mn may have deleterious effects on other organs including the auditory system and hearing. Mn accumulates in the inner ear following acute exposure raising the possibility that it can damage the sensory hair cells that convert sound into neural activity or spiral ganglion neurons (SGN) that transmit acoustic information from the hair cells to the brain via the auditory nerve. In this paper we demonstrate for first time that Mn causes significant damage to the sensory hair cells, peripheral auditory nerve fibers (ANF) and SGN in cochlear organotypic cultures isolated from postnatal day three rats. The peripheral ANF that make synaptic contact with the sensory hair cells were particularly vulnerable to Mn toxicity; damage occurred at concentrations as low 0.01 mM and increased with dose and duration of Mn exposure. Sensory hair cells, in contrast, were slightly more resistant to Mn toxicity than the ANF. Mn induced an atypical pattern of sensory cell damage; Mn was more toxic to inner hair cells (IHC) than outer hair cells (OHC) and in addition, IHC loss was relatively uniform along the length of the cochlea. Mn also caused significant loss and shrinkage of SGN soma. These findings are the first to demonstrate that Mn can produce severe lesions to both neurons and hair cells in the postnatal inner ear.

The glutathione transferase (GST) detoxification system converts exogenous and endogenous toxins into a less toxic form by conjugating the toxic compound to reduced glutathione (GSH) by a variety of GST enzymes. Of the ~20 GST isoforms, GSTA4 exhibits high catalytic efficiency toward 4-hydroxynonenal (4-HNE), one of the most abundant end products of lipid peroxidation that contributes to neurodegenerative diseases and age-related disorders. Conjugation to GSH by GSTA4 is thought to be a major route of 4-HNE elimination. In the current study, we investigated the effects of Gsta4 deficiency on age-related cochlear pathology and hearing loss using young (3-5 months old) and old (24-25 months old) Gsta4+/+ and Gsta4-/- mice that were backcrossed onto the CBA/CaJ mouse strain, a well-established model of age-related hearing loss (AHL). At 3-5 months of age, loss of Gsta4 resulted in decreased total GSTA activity toward 4-HNE in the inner ears of young mice. However, there were no differences in the levels of 4-HNE in the inner ears between Gsta4+/+ and Gsta4-/- mice at 3-5 or 24-25 months of age. No histological abnormalities were observed in the cochlea and no hearing impairments were observed in young Gsta4-/- mice. At 24-25 months of age, both Gsta4+/+ and Gsta4-/- mice showed elevated ABR thresholds compared to 3-month-old mice, but there were no differences in ABR thresholds, cochlear spiral ganglion neuron densities, or stria vascularis thickness between Gsta4+/+ and Gsta4-/- mice. Together, these results suggest that under normal physiological conditions or during normal aging, GSTA4 is not essential for removal of 4-HNE in mouse inner ears.

Niemann-Pick C1 (NPC1) is a fatal neurodegenerative disease caused by aberrant cholesterol metabolism. The progression of the disease can be slowed by removing excess cholesterol with high-doses of 2-hyroxypropyl-beta-cyclodextrin (HPβCD). Unfortunately, HPβCD causes hearing loss; the initial first phase involves a rapid destruction of outer hair cells (OHCs) while the second phase, occurring 4-6 weeks later, involves the destruction of inner hair cells (IHCs), pillar cells, collapse of the organ of Corti and spiral ganglion neuron degeneration. To determine whether the first and/or second phase of HPβCD-induced cochlear damage is linked, in part, to excess oxidative stress or neuroinflammation, rats were treated with a single-dose of 3000 mg/kg HPβCD alone or together with one of two combination therapies. Each combination therapy was administered from 2-days before to 6-weeks after the HPβCD treatment. Combination 1 consisted of minocycline, an antibiotic that suppresses neuroinflammation, and HK-2, a multifunctional redox modulator that suppresses oxidative stress. Combination 2 was comprised of minocycline plus N-acetyl cysteine (NAC), which upregulates glutathione, a potent antioxidant. To determine if either combination therapy could prevent HPβCD-induced hearing impairment and cochlear damage, distortion product otoacoustic emissions (DPOAE) were measured to assess OHC function and the cochlear compound action potential (CAP) was measured to assess the function of IHCs and auditory nerve fibers. Cochleograms were prepared to quantify the amount of OHC, IHC and pillar cell (PC) loss. HPβCD significantly reduced DPOAE and CAP amplitudes and caused significant OHC, IHC and OPC losses with losses greater in the high-frequency base of the cochlea than the apex. Neither minocycline + HK-2 (MIN+ HK-2) nor minocycline + NAC (MIN+NAC) prevented the loss of DPOAEs, CAPs, OHCs, IHCs or IPCs caused by HPβCD. These results suggest that oxidative stress and neuroinflammation are unlikely to play major roles in mediating the first or second phase of HPβCD-induced cochlear damage. Thus, HPβCD-induced ototoxicity must be mediated by some other unknown cell-death pathway possibly involving loss of trophic support from damaged support cells or disrupted cholesterol metabolism.