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Plastic pollution is increasing at an alarming rate yet the impact of this pollution on human health is poorly understood. Because human induced pluripotent stem cells (hiPSC) are frequently derived from dermal fibroblasts, these cells offer a powerful platform for the identification of molecular biomarkers of environmental pollution in human cells. Here, we describe a novel proof-of-concept for deriving hiPSC from human dermal fibroblasts deliberately exposed to polystyrene (PS) nanoplastic particles; unexposed hiPSC served as controls. In parallel, unexposed hiPSC were exposed to low and high concentrations of PS nanoparticles. Transcriptomic and epigenomic signatures of all fibroblasts and hiPSCs were defined using RNA-seq and whole genome methyl-seq, respectively. Both PS-treated fibroblasts and derived hiPSC showed alterations in expression of ESRRB and HNF1A genes and circuits involved in the pluripotency of stem cells, as well as in pathways involved in cancer, inflammatory disorders, gluconeogenesis, carbohydrate metabolism, innate immunity, and dopaminergic synapse. Similarly, the expression levels of identified key transcriptional and DNA methylation changes (DNMT3A, ESSRB, FAM133CP, HNF1A, SEPTIN7P8, and TTC34) were significantly affected in both PS-exposed fibroblasts and hiPSC. This study illustrates the power of human cellular models of environmental pollution to narrow down and prioritize the list of candidate molecular biomarkers of environmental pollution. This knowledge will facilitate the deciphering of the origins of environmental diseases.

Despite advances in implant hardware, neuroprosthetic devices in otolaryngology have sustained evolutionary rather than revolutionary changes over the past half century. Although electrical stimulation has the capacity for facile activation of neurons and high temporal resolution, it has limited spatial selectivity. Alternative strategies for neuronal stimulation are being investigated to improve spatial resolution. In particular, light-based neuronal stimulation is a viable alternative and complement to electrical stimulation. This article provides a broad overview of light-based neuronal stimulation technologies. Specific examples of active research on light-based prostheses, including cochlear implants, auditory brainstem implants, retinal implants, and facial nerve implants, are reviewed.

Commercially manufactured or generated through environmental degradation, microplastics (MPs) and nanoplastics (NPs) considerably contribute to environmental pollution. There is a knowledge gap in how exposure to MPs/NPs changes cellular function and affects animal and human health. Here, we demonstrate that after oral uptake, fluorescent polystyrene (PS) nanoparticles pass through the mouse digestive system, accumulate and aggregate in different organs, and induce functional changes in cells and organs. Using cochlear explant as a novel in vitro system, we confirmed the consequences of PS-MP/NP interaction with inner ear cells by detecting aggregates and hetero-aggregates of PS particles in hair cells. The testes of treated males accumulated MPs/NPs in the interstitial compartment surrounding the seminiferous tubules, which was associated with a statistically significant decrease in testosterone levels. Male mice showed increased secretion of interleukins (IL-12p35 and IL-23) by splenocytes while cyto- and genotoxicity tests indicated impaired cell viability and increased DNA damage in spleen tissue. Males also showed a broad range of anxiogenic responses to PS nanoparticles while hippocampal samples from treated females showed an increased expression of Bax and Nlrp3 genes, indicating a pro-apoptotic/proinflammatory effect of PS treatment. Taken together, induced PS effects are also gender-dependent, and therefore, strongly motivate future research to mitigate the deleterious effects of nanosized plastic particles.

Optogenetics is a transformative technology based on light-sensitive microbial proteins, known as opsins, that enable precise modulation of neuronal activity with pulsed radiant energy. Optogenetics has been proposed as a means to improve auditory implant outcomes by reducing channel interaction and increasing electrode density, but the introduction of opsins into cochlear spiral ganglion neurons (SGNs) in vivo has been challenging. Here we test opsin delivery using a synthetically developed ancestral adeno-associated virus (AAV) vector called Anc80L65. Wild-type C57BL/6 mouse pups were injected via the round window of cochlea with Anc80L65 carrying opsin Chronos under the control of a CAG promoter. Following an incubation of 6-22 weeks, pulsed blue light was delivered to cochlear SGNs via a cochleosotomy approach and flexible optical fiber. Optically evoked auditory brainstem responses (oABRs) and multiunit activity in inferior colliculus (IC) were observed. Post-experiment cochlear histology demonstrated opsin expression in SGNs (mean = 74%), with an even distribution of opsin along the cochlear basal/apical gradient. This study is the first to describe robust SGN transduction, opsin expression, and optically evoked auditory electrophysiology in neonatal mice. Ultimately, this work may provide the basis for a new generation of cochlear implant based on light.

The bone encasing the inner ear, known as the otic capsule, is unique because it remodels little postnatally compared to other bones in the body. Previous studies established that osteoprotegerin (OPG) in the inner ear inhibits otic capsule remodeling. OPG acts as a decoy receptor of receptor activator of nuclear factor κB ligand (RANKL) to disrupt the interaction between RANKL and RANK, the primary regulators of bone metabolism. Here we studied the expression and function of RANK and RANKL in the murine cochlea. Using a combination of in situ hybridization, real-time quantitative RT-PCR, and western blot, we demonstrate that Rankl and Rank genes and their protein products are expressed in the intracochlear soft tissues and the otic capsule in a developmentally regulated manner. Using a culture of neonatal murine cochlear neurons, we show that the interaction between RANK and RANKL inhibits neurite outgrowth in these neurons, and is associated with upregulation of NOGO-A expression. Taken together, our results suggest that, in addition to regulating otic capsule bone remodeling, RANK and RANKL expressed by intracochlear soft tissues may also regulate spiral ganglion neuron function by affecting neurite outgrowth.

Introduction: As otology enters the field of gene therapy and human studies commence, the question arises whether audiograms - the current gold standard for the evaluation of hearing function - can consistently predict cellular damage within the human inner ear and thus should be used to define inclusion criteria for trials. Current assumptions rely on the analysis of small groups of human temporal bones post mortem or from psychophysical identification of cochlear "dead regions" in vivo, but a comprehensive study assessing the correlation between audiometric thresholds and cellular damage within the cochlea is lacking. Methods: A total of 131 human temporal bones from 85 adult individuals (ages 19-92 years, median 69 years) with sensorineural hearing loss due to various etiologies were analyzed. Cytocochleograms - which quantify loss of hair cells, neurons, and strial atrophy along the length of the cochlea - were compared with subjects' latest available audiometric tests prior to death (time range 5 h-22 years, median 24 months). The Greenwood function and the equivalent rectangular bandwidth were used to infer, from cytocochleograms, cochlear locations corresponding to frequencies tested in clinical audio grams. Correlation between audiometric thresholds at clinically tested frequencies and cell type-specific damage in those frequency regions was examined by calculating Spearman's correlation coefficients. Results: Similar audiometric profiles reflected widely different cellular damage in the cochlea. In our diverse group of patients, audiometric thresholds tended to be more influenced by hair cell loss than by neuronal loss or strial atrophy. Spearman's correlation coefficient across frequencies was at most 0.7 and often below 0.5, with 1.0 indicating perfect correlation. Conclusions: Audiometric thresholds do not predict specific cellular damage in the human inner ear. Our study highlights the need for better non- or minimally-invasive tools, such as cochlear endoscopy, to establish cellular-level diagnosis and thereby guide therapy and monitor response to treatment. (C) 2016 Elsevier B.V. All rights reserved.

Background Higher intake of certain vitamins may protect against cochlear damage from vascular compromise and oxidative stress, thereby reducing risk of acquired hearing loss, but data are limited. Objective We prospectively examined the relation between carotenoids, vitamin A, vitamin C, vitamin E, and folate intake and risk of self-reported hearing loss in women. Design This prospective cohort study followed 65,521 women in the Nurses' Health Study II from 1991 to 2009. Baseline and updated information obtained from validated biennial questionnaires was used in Cox proportional hazards regression models to examine independent associations between nutrient intake and self-reported hearing loss. Results After 1,084,598 person-years of follow-up, 12,789 cases of incident hearing loss were reported. After multivariable adjustment, we observed modest but statistically significant inverse associations between higher intake of β-carotene and β-cryptoxanthin and risk of hearing loss. In comparison with women in the lowest quintile of intake, the multivariable-adjusted RR of hearing loss among women in the highest quintile was 0.88 (95% CI: 0.81, 0.94; P-trend Conclusion Higher intakes of β-carotene, β-cryptoxanthin, and folate, whether total or from diet, are associated with lower risk of hearing loss, whereas higher vitamin C intake is associated with higher risk.

Sensorineural hearing loss (SNHL) is an extraordinarily common disability, affecting 466 million people across the globe. Half of these incidents are attributed to genetic mutations that disrupt the structure and function of the cochlea. The human cochlea’s interior cannot be imaged or biopsied without damaging hearing; thus, everything known about the morphologic correlates of hereditary human deafness comes from histopathologic studies conducted in either cadaveric human temporal bone specimens or animal models of genetic deafness. The purpose of the present review is to a) summarize the findings from all published histopathologic studies conducted in human temporal bones with known SNHL-causing genetic mutations, and b) compare the reported phenotypes of human vs. mouse SNHL caused by the same genetic mutation. The fact that human temporal bone histopathologic analysis has been reported for only 22 of the nearly 200 identified deafness-causing genes suggests a great need for alternative and improved techniques for studying human hereditary deafness; in light of this, the present review concludes with a summary of promising future directions, specifically in the fields of high resolution cochlear imaging, intracochlear fluid biopsy, and gene therapy.

Sensorineural hearing loss is the most common sensory deficit worldwide, yet our understanding of the underlying pathophysiology is limited by the challenges of access to the inner ear in a safe and reliable manner. We present a novel microneedle device for trans-round window membrane liquid biopsy, which utilizes controlled depth of perforation and microliter aspiration control to safely biopsy fluids of the inner ear. Of eleven devices tested in fresh frozen human temporal bones, seven demonstrated alignment between electrical, visual and tactile detection of round window membrane perforation, and nine were successful in aspiration of meaningful diagnostic samples from the perilymphatic space. Purity of the average perilymph sample was 69% for a 5 μL sample volume, equivalent to 3.5 μL attributable to perilymph. Diagnostic success was shown both by transmastoid facial recess and transcanal tympanotomy approach. This device can enable new advances in the understanding of inner ear pathology, and brings us one step closer to liquid biopsy of the inner ear becoming a routine part of clinical care.

Mechanisms that regulate endolymphatic pH are unknown. It has long been recognized that, because of the large positive endolymphatic potential in the cochlea, a passive movement of protons would be directed out of endolymph leading to endolymphatic alkalization. However, endolymphatic pH is close to that of blood, suggesting that H+ is being secreted into endolymph. Since the kidney and the inner ear are both actively engaged in fluid and electrolyte regulation, we attempted to determine whether proteins responsible for acid secretion in the kidney also exist in the guinea pig inner ear. To that end, a monoclonal antibody against a 31 kDa subunit of a vacuolar vH+ATPase and a polyclonal, affinity purified antibody against the AE2 Cl − HCO 3 − exchanger (which can also recognize AEI under some conditions) were used. In the cochlea, the strongest immunoreactivity for the vH+ATPase was found in apical plasma membranes and apical cytoplasm of strial marginal cells. These cells were negative for the Cl − HCO 3 − exchanger. Certain cells of the inner ear demonstrated both apical staining for vH+ATPase and basolateral staining for the Cl − HCO 3 − exchanger; these included interdental cells and epithelial cells of the endolymphatic sac. Cochlear cell types with diffuse cytoplasmic staining for vH+ATPase and a basolaterally localized Cl − HCO 3 − exchanger included inner hair cells, root cells and a subset of supporting cells in the organ of Corti. Hair cells of the utricle, saccule and cristae ampullaris also expressed both vH+ATPase and the Cl − HCO 3 − exchanger, but immunostaining for the vH+ATPase was less intense and less polarized than in the cochlea. These immunocytochemical results support a role for the vH+ATPase and Cl − HCO 3 − exchanger in the regulation of endolymphatic pH and suggest that certain cells (including strial marginal cells and epithelial cells of the endolymphatic sac) may be specialized for this regulation.

Hearing loss is the most common sensory disorder worldwide. Approximately 15% of American adults report trouble hearing. Prevalence of hearing loss increases from 2% (age 45-54) to 50% (over age 75). Between 2 and 3 per 1000 live births present with hearing loss in one or both ears; 50% of hearing loss at birth is genetic in etiology. While the first cochlear gene therapy for hearing loss is underway, early results, as well as data from preclinical models, indicate a need for improved gene delivery vehicles for safe and efficient targeting of relevant cell types in the cochlea. In particular, outer hair cells (OHC) have been a challenging target for AAV. OHCs are important for auditory sensitivity and frequency selectivity, key parameters for complex and sophisticated hearing function in humans.Here, we evaluated several common AAV serotypes as well as Anc80L65, an in silico designed AAV based on evolutionary modeling, for its ability to target the neurosensory cells in the inner ear, particularly inner (IHC) and outer hair cells. Cochlear explants of both CBA/CaJ and C57Bl/6 mice were exposed to equal doses of iodixonal purified preparations of AAV1,2,6,8,9, and Anc80L65 that encoded eGFP. After 48h, explants were evaluated either immediately or after an additional 5 days of culture. In both animal model explants, Anc80L65 demonstrated highly efficient IHC and OHC transduction (60-100% at 48h, and 100% at 48h+5d). AAV2 in this setting was also efficient, although to a lesser extent. Most serotypes targeted supporting cells and the spiral limbus, to a moderate extent, with AAV8 being least efficient. GFP expression was present from apex to base, with moderately increased targeting for basal hair cells.Next, we tested Anc80L65 in vivo using a clinically relevant round window injection approach. C57Bl/6 mice received 1µl injection of vector at p0-p1 and cochlea were dissected at p6. Results demonstrate transduction from base to apex of IHC, OHC, and supporting cells at rates of close to 100% for IHC, and ranging from approximately 50-100% of OHCs, although at lesser fluorescent intensity. Animals injected at p1 retained similar levels of GFP hair cell positivity at p30 day of sacrifice. Animals in our study were monitored for function of single cells electrophysiogically, hearing function by Auditory Brainstem Response and Distortion Product Otoacoustic Emission measurements, ectopic expression, and immunological parameters. Ongoing studies are evaluating the transduction properties at later times of injection and disease models of hearing loss.In conclusion, Anc80L65's unique ability to target IHC as well as OHC following RW injection is highly desirable to restore higher level hearing function in genetic forms of deafness through gene therapy.