Due to the critical role of cochlear ion channels for hearing, the focus of the present study was to examine age-related changes of Na, K-ATPase (NKA) subunits in the lateral wall of mouse cochlea. We combined qRT-PCR, western blot and immunocytochemistry methodologies in order to determine gene and protein expression levels in the lateral wall of young and aged CBA/CaJ mice. Of the seven NKA subunits, only the mRNA expressions of α1, β1 and β2 subunit isoforms were detected in the lateral wall of CBA/CaJ mice. Aging was accompanied by dys-regulation of gene and protein expression of all three subunits detected. Hematoxylin and eosin (H&E) staining revealed atrophy of the cochlear stria vascularis (SV). The SV atrophy rate (20%) was much less than the ∼80% decline in expression of all three NKA isoforms, indicating lateral wall atrophy and NKA dys-regulation are independent factors and that there is a combination of changes involving the morphology of SV and NKA expression in the aging cochlea which may concomitantly affect cochlear function. Immunoprecipitation assays showed that the α1-β1 heterodimer is the selective preferential heterodimer over the α1-β2 heterodimer in cochlea lateral wall. Interestingly, in vitro pathway experiments utilizing cultured mouse cochlear marginal cells from the SV (SV-K1 cells) indicated that decreased mRNA and protein expressions of α1, β1 and β2 subunit isoforms are not associated with reduction of NKA activity following in vitro application of ouabain, but ouabain did disrupt the α1-β1 heterodimer interaction. Lastly, the association between the α1 and β1 subunit isoforms was present in the cochlear lateral wall of young adult mice, but this interaction could not be detected in old mice. Taken together, these data suggest that in the young adult mouse there is a specific, functional selection and assembly of NKA subunit isoforms in the SV lateral wall, which is disrupted and dys-regulated with age. Interventions for this age-linked ion channel disruption may have the potential to help diagnose, prevent, or treat age-related hearing loss.

Age-related hearing loss (ARHL) -presbycusis - is the most prevalent neurodegenerative disease and number one communication disorder of our aged population; and affects hundreds of millions of people worldwide. Its prevalence is close to that of cardiovascular disease and arthritis, and can be a precursor to dementia. The auditory perceptual dysfunction is well understood, but knowledge of the biological bases of ARHL is still somewhat lacking. Surprisingly, there are no FDA-approved drugs for treatment. Based on our previous studies of human subjects, where we discovered relations between serum aldosterone levels and the severity of ARHL, we treated middle age mice with aldosterone, which normally declines with age in all mammals. We found that hearing thresholds and suprathreshold responses significantly improved in the aldosterone-treated mice compared to the non-treatment group. In terms of cellular and molecular mechanisms underlying this therapeutic effect, additional experiments revealed that spiral ganglion cell survival was significantly improved, mineralocorticoid receptors were upregulated via post-translational protein modifications, and age-related intrinsic and extrinsic apoptotic pathways were blocked by the aldosterone therapy. Taken together, these novel findings pave the way for translational drug development towards the first medication to prevent the progression of ARHL.

Advances in protective and restorative biotherapies have created new opportunities to use site-directed, programmable drug delivery systems to treat auditory and vestibular disorders. Successful therapy development that leverages the transgenic, knock-in, and knock-out variants of mouse models of human disease requires advanced microsystems specifically designed to function with nanoliter precision and with system volumes suitable for implantation. Here we present results for a novel biocompatible, implantable, scalable, and wirelessly controlled peristaltic micropump. The micropump configuration included commercially available catheter microtubing (250 μm OD, 125 μm ID) that provided a biocompatible leak-free flow path while avoiding complicated microfluidic interconnects. Peristaltic pumping was achieved by sequentially compressing the microtubing via expansion and contraction of a thermal phase-change material located in three chambers integrated adjacent to the microtubing. Direct-write micro-scale printing technology was used to build the mechanical components of the micropump around the microtubing directly on the back of a printed circuit board assembly (PCBA). The custom PCBA was fabricated using standard commercial processes providing microprocessor control of actuation and Bluetooth wireless communication through an Android application. The results of in vitro characterization indicated that nanoliter resolution control over the desired flow rates of 10-100 nL/min was obtained by changing the actuation frequency. Applying 10× greater than physiological backpressures and ± 3 °C ambient temperature variation did not significantly affect flow rates. Three different micropumps were tested on six mice for in vivo implantation of the catheter microtubing into the round window membrane niche for infusion of a known ototoxic compound (sodium salicylate) at 50 nL/min for 20 min. Real-time shifts in distortion product otoacoustic emission thresholds and amplitudes were measured during the infusion. There were systematic increases in distortion product threshold shifts during the 20-min perfusions; the mean shift was 15 dB for the most basal region. A biocompatibility study was performed to evaluate material suitability for chronic subcutaneous implantation and clinical translational development. The results indicated that the micropump components successfully passed key biocompatibility tests. A micropump prototype was implanted for one month without development of inflammation or infection. Although tested here on the small murine cochlea, this low-cost design and fabrication methodology is scalable for use in larger animals and for clinical applications in children and adults by appropriate scaling of the microtubing diameter and actuator volume.

Estradiol (E) is a multitasking hormone that plays a prominent role in the reproductive system, and also contributes to physiological and growth mechanisms throughout the body. Frisina and colleagues have previously demonstrated the beneficial effects of this hormone, with E-treated subjects maintaining low auditory brainstem response (ABR) thresholds relative to control subjects (Proceedings of the National Academy of Sciences of the United States of America, 2006;103:14246; Hearing Research, 2009;252:29). In the present study, we evaluated the functionality of the peripheral and central auditory systems in female CBA/CaJ middle-aged mice during and after long-term hormone replacement therapy (HRT) via electrophysiological and molecular techniques. Surprisingly, there are very few investigations about the side effects of HRT in the auditory system after it has been discontinued. Our results show that the long-term effects of HRT are permanent on ABR thresholds and ABR gap-in-noise (GIN) amplitude levels. E-treated animals had lower thresholds and higher amplitude values compared to other hormone treatment subject groups. Interestingly, progesterone (P)-treated animals had ABR thresholds that increased but amplitude levels that remained relatively the same throughout treatment. These results were consistent with qPCR experiments that displayed high levels of IGF-1R in the stria vascularis (SV) of both E and P animal groups compared to combination treatment (E + P) animals. IGF-1R plays a vital role in mediating anti-apoptotic responses via the PI3K/AKT pathway. Overall, our findings gain insights into the neuro-protective properties of E hormone treatments as well as expand the scientific knowledge base to help women decide whether HRT is the right choice for them.

Sensorineural Hearing Loss (SNHL) is a highly prevalent disorder involving permanent damage or loss to the inner ear's mechano-sensory hair cells and nerve fibers. Major contributing causes are ototoxic drugs, loud noises, and aging. Drug-induced hearing loss (DIHL), affects over 25% of patients treated with common therapeutics such as aminoglycoside antibiotics, loop diuretics or chemotherapeutics. A commonly used chemotherapeutic agent, cisplatin, is very effective for treating malignant tumors, but results in a majority of patients experiencing irreversible hearing loss and/or tinnitus. Additionally, since there is currently no FDA-approved treatments for SNHL, attenuation of ototoxicity is a major area of investigation in oncology, otolaryngology and hearing research. Several potential otoprotective agents have been investigated at the clinical trial stage, but none have progressed to a full FDA-approval. In this study, we investigated a combinatorial approach comprised of an antioxidant, a p53 inhibitor and a neurotrophin, as a multifactorial otoprotective treatment for cisplatin exposure. In vitro, HEI-OC1 cells, an immortalized organ of Corti epithelial cell line, pre-treated with this biotherapeutic cocktail had significantly reduced cisplatin-induced cell death, DNA fragmentation, and apoptotic activation. In an ex vivo study, rat pup D2-D3 organ of Corti explants, significant protection against cisplatin-based hair cell and neuronal loss was achieved by delivery of the same combinatorial pretreatment. Interestingly, the hair cell protection was localized to the basal and middle regions of the organ of Corti. Together, these findings highlight a novel approach to attenuate cisplatin ototoxicity and potentially prevent DIHL by addressing biological mechanisms of cisplatin ototoxicity.

Age-related hearing loss - presbycusis - is the number one neurodegenerative disorder and top communication deficit of our aged population. Like many aging disorders of the nervous system, damage from free radicals linked to production of reactive oxygen and/or nitrogen species (ROS and RNS, respectively) may play key roles in disease progression. The efficacy of the antioxidant systems, e.g., glutathione and thioredoxin, is an important factor in pathophysiology of the aging nervous system. In this investigation, relations between the expression of antioxidant-related genes in the auditory portion of the inner ear - cochlea, and age-related hearing loss was explored for CBA/CaJ mice. Forty mice were classified into four groups according to age and degree of hearing loss. Cochlear mRNA samples were collected and cDNA generated. Using Affymetrix® GeneChip, the expressions of 56 antioxidant-related gene probes were analyzed to estimate the differences in gene expression between the four subject groups. The expression of Glutathione peroxidase 6, Gpx6; Thioredoxin reductase 1, Txnrd1; Isocitrate dehydrogenase 1, Idh1; and Heat shock protein 1, Hspb1; were significantly different, or showed large fold-change differences between subject groups. The Gpx6, Txnrd1 and Hspb1 gene expression changes were validated using qPCR. The Gpx6 gene was upregulated while the Txnrd1 gene was downregulated with age/hearing loss. The Hspb1 gene was found to be downregulated in middle-aged animals as well as those with mild presbycusis, whereas it was upregulated in those with severe presbycusis. These results facilitate development of future interventions to predict, prevent or slow down the progression of presbycusis.

How asymmetries in motor behavior become established normally or atypically in mammals remains unclear. An established model for motor asymmetry that is conserved across mammals can be obtained by experimentally inducing asymmetric striatal dopamine activity. However, the factors that can cause motor asymmetries in the absence of experimental manipulations to the brain remain unknown. Here, we show that mice with inner ear dysfunction display a robust left or right rotational preference, and this motor preference reflects an atypical asymmetry in cortico-striatal neurotransmission. By unilaterally targeting striatal activity with an antagonist of extracellular signal-regulated kinase (ERK), a downstream integrator of striatal neurotransmitter signaling, we can reverse or exaggerate rotational preference in these mice. By surgically biasing vestibular failure to one ear, we can dictate the direction of motor preference, illustrating the influence of uneven vestibular failure in establishing the outward asymmetries in motor preference. The inner ear-induced striatal asymmetries identified here intersect with non-ear-induced asymmetries previously linked to lateralized motor behavior across species and suggest that aspects of left-right brain function in mammals can be ontogenetically influenced by inner ear input. Consistent with inner ear input contributing to motor asymmetry, we also show that, in humans with normal ear function, the motor-dominant hemisphere, measured as handedness, is ipsilateral to the ear with weaker vestibular input.

Recent evidence suggests that modulation of the large-conductance, calcium-activated potassium (BK) channel regulates auditory processing in the brain. Because ion channel expression often changes during aging, this could be a factor in age-related hearing loss. The current study explored how the novel BK channel modulator LS3 shapes central auditory processing in young and old adult mice. In vivo extracellular recordings in the auditory midbrain demonstrated that LS3 differentially modulates neural processing along the tonotopic axis. Though sound-evoked activity was reduced in the mid and ventral tonotopic regions, LS3 enhanced excitatory drive and sound-evoked responses for some neurons in the dorsal, low-frequency region. Behavioral assessment using acoustic reflex modification audiometry indicated improved tone salience following systemic LS3 administration. Moderation of these responses with aging correlated with an age-related decline in BK channel expression. These findings suggest that targeting the BK channel enhances responsivity to tonal sounds, providing the potential to improve hearing acuity and treat hearing loss.

Parkinson's disease (PD) exhibits significant clinical heterogeneity, presenting challenges in the identification of reliable electroencephalogram (EEG) biomarkers. Machine learning techniques have been integrated with resting-state EEG for PD diagnosis, but their practicality is constrained by the interpretable features and the stochastic nature of resting-state EEG. The present study proposes a novel and interpretable deep learning model, graph signal processing-graph convolutional networks (GSP-GCNs), using event-related EEG data obtained from a specific task involving vocal pitch regulation for PD diagnosis. By incorporating both local and global information from single-hop and multi-hop networks, our proposed GSP-GCNs models achieved an averaged classification accuracy of 90.2%, exhibiting a significant improvement of 9.5% over other deep learning models. Moreover, the interpretability analysis revealed discriminative distributions of large-scale EEG networks and topographic map of microstate MS5 learned by our models, primarily located in the left ventral premotor cortex, superior temporal gyrus, and Broca's area that are implicated in PD-related speech disorders, reflecting our GSP-GCN models' ability to provide interpretable insights identifying distinctive EEG biomarkers from large-scale networks. These findings demonstrate the potential of interpretable deep learning models coupled with voice-related EEG signals for distinguishing PD patients from healthy controls with accuracy and elucidating the underlying neurobiological mechanisms.