A Novel in vitro Model Delineating Hair Cell Regeneration and Neural Reinnervation in Adult Mouse Cochlea

The study of an adult mammalian auditory system, such as regeneration, has been hampered by the lack of an in vitro system in which hypotheses can be tested efficiently. This is primarily due to the fact that the adult inner ear is encased in the toughest bone of the body, whereas its removal leads to the death of the sensory epithelium in culture. We hypothesized that we could take advantage of the integral cochlear structure to maintain the overall inner ear architecture and improve sensory epithelium survival in culture. We showed that by culturing adult mouse cochlea with the (surrounding) bone intact, the supporting cells (SCs) survived and almost all hair cells (HCs) degenerated. To evaluate the utility of the explant culture system, we demonstrated that the overexpression of Atoh1, an HC fate-determining factor, is sufficient to induce transdifferentiation of adult SCs to HC-like cells (HCLCs). Transdifferentiation-derived HCLCs resemble developmentally young HCs and are able to attract adult ganglion neurites. Furthermore, using a damage model, we showed that degenerated adult ganglions respond to regenerated HCLCs by directional neurite outgrowth that leads to HCLC-neuron contacts, strongly supporting the intrinsic properties of the HCLCs in establishing HCLC-neuron connections. The adult whole cochlear explant culture is suitable for diverse studies of the adult inner ear including regeneration, HC-neuron pathways, and inner ear drug screening.

Virus-infection in cochlear supporting cells induces audiosensory receptor hair cell death by TRAIL-induced necroptosis

Although sensorineural hearing loss (SHL) is relatively common, its cause has not been identified in most cases. Previous studies have suggested that viral infection is a major cause of SHL, especially sudden SHL, but the system that protects against pathogens in the inner ear, which is isolated by the blood-labyrinthine barrier, remains poorly understood. We recently showed that, as audiosensory receptor cells, cochlear hair cells (HCs) are protected by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker’s organ) cells (GERCs) against viral infections. Here, we found that virus-infected SCs and GERCs induce HC death via production of the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Notably, the HCs expressed the TRAIL death receptors (DR) DR4 and DR5, and virus-induced HC death was suppressed by TRAIL-neutralizing antibodies. TRAIL-induced HC death was not caused by apoptosis, and was inhibited by necroptosis inhibitors. Moreover, corticosteroids, the only effective drug for SHL, inhibited the virus-induced transformation of SCs and GERCs into macrophage-like cells and HC death, while macrophage depletion also inhibited virus-induced HC death. These results reveal a novel mechanism underlying virus-induced HC death in the cochlear sensory epithelium and suggest a possible target for preventing virus-induced SHL.

A Reversal in Hair Cell Orientation Organizes Both the Auditory and Vestibular Organs

Sensory hair cells detect mechanical stimuli with their hair bundle, an asymmetrical brush of actin-based membrane protrusions, or stereocilia. At the single cell level, stereocilia are organized in rows of graded heights that confer the hair bundle with intrinsic directional sensitivity. At the organ level, each hair cell is precisely oriented so that its intrinsic directional sensitivity matches the direction of mechanical stimuli reaching the sensory epithelium. Coordinated orientation among neighboring hair cells usually ensures the delivery of a coherent local group response. Accordingly, hair cell orientation is locally uniform in the auditory and vestibular cristae epithelia in birds and mammals. However, an exception to this rule is found in the vestibular macular organs, and in fish lateral line neuromasts, where two hair cell populations show opposing orientations. This mirror-image hair cell organization confers bidirectional sensitivity at the organ level. Here I review our current understanding of the molecular machinery that produces mirror-image organization through a regional reversal of hair cell orientation. Interestingly, recent evidence suggests that auditory hair cells adopt their normal uniform orientation through a global reversal mechanism similar to the one at work regionally in macular and neuromast organs. Macular and auditory organs thus appear to be patterned more similarly than previously appreciated during inner ear development.

Endomicroscopy of the human cochlea using a micro-optical coherence tomography catheter

Sensorineural hearing loss (SNHL) is one of the most profound public health concerns of the modern era, affecting 466 million people today, and projected to affect 900 million by the year 2050. Advances in both diagnostics and therapeutics for SNHL have been impeded by the human cochlea’s inaccessibility for in vivo imaging, resulting from its extremely small size, convoluted coiled configuration, fragility, and deep encasement in dense bone. Here, we develop and demonstrate the ability of a sub-millimeter-diameter, flexible endoscopic probe interfaced with a micro-optical coherence tomography (μOCT) imaging system to enable micron-scale imaging of the inner ear’s sensory epithelium in cadaveric human inner ears.

An Acoustic Treatment to Mitigate the Effects of the Apple Snail on Agriculture and Natural Ecosystems

Global change is the origin of increased occurrence of disturbance events in natural communities, with biological invasions constituting a major threat to ecosystem integrity and functioning. The apple snail (Pomacea maculata) is a freshwater gastropod mollusk from South America. Considered one of the 100 most harmful invasive species in the world, due to its voracity, resistance, and high reproductive rate, it has become a global problem for wetland crops. In Catalonia, it has affected the rice fields of the Ebre Delta since 2010 with significant negative impact on the local economy. As a gastropod mollusc it possesses statocysts consisting of a pair of sacs, one located on each side of the foot, that contain multiple calcium carbonate statoconia. This study shows the first ultrastructural images of pathological changes in the sensory epithelium of the statocyst of apple snail adults with an increase in the severity of the lesions over time after exposure to low frequency sounds. Sound-induced damage to the statocyst could likely result in an inhibition of its vital functions resulting in a potential reduction in the survival ability of the apple snail and lead to an effective mitigation method for reducing damage to rice fields.

miR-153/KCNQ4 axis contributes to noise-induced hearing loss in a mouse model

Damage to the cochlear sensory epithelium is a key contributor to noise-induced sensorineural hearing loss (SNHL). KCNQ4 plays an important role in the cochlear potassium circulation and outer hair cells survival. As miR-153 can target and regulate KCNQ4, we sought to study the role of miR-153 in SNHL. 12-week-old male CBA/J mice were exposed to 2–20 kHz broadband noise at 96 dB SPL to induce temporary threshold shifts and 101 dB SPL to induce permanent threshold shifts. Hearing loss was determined by auditory brainstem responses (ABR). Relative expression of miR-153 and KCNQ4 in mice cochlea were determined by Real-Time quantitative PCR. miR-153 mimics were co-transfected with wild type or mutated KCNQ4 into HEK293 cells. Luciferase reporter assay was used to validate the binding between miR-153 and KCNQ4. AAV-sp-153 was constructed and administrated intra-peritoneally 24- and 2-h prior and immediately after noise exposure to knockdown miR-153. The KCNQ4 is mainly expressed in outer hair cells (OHCs). We showed that the expression of KCNQ4 in mice cochlea was reduced and miR-153 expression was significantly increased after noise exposure compared to control. miR-153 bound to 3′UTR of KNCQ4, and the knockdown of miR-153 with the AAV-sp-153 administration restored KCNQ4 mRNA and protein expression. In addition, the knockdown of miR-153 reduced ABR threshold shifts at 8, 16, and 32 kHz after permanent threshold shifts (PTS) noise exposure. Correspondingly, OHC losses were attenuated with inhibition of miR-153. This study demonstrates that miR-153 inhibition significantly restores KNCQ4 in cochlea after noise exposure, which attenuates SNHL. Our study provides a new potential therapeutic target in the prevention and treatment of SNHL.

TRIOBP promotes bidirectional radial stiffness gradients within the organ of Corti

Hearing depends on complex mechanical properties of the inner ear sensory epithelium. Yet, the individual contributions of different cell types to the stiffness spectrum of the sensory epithelium have not been thoroughly investigated. Using sub-100 nanometer spatial resolution PeakForce Tapping Atomic Force Microscopy (PFT-AFM), we mapped the Youngs modulus (stiffness) of the apical surface of different cells of freshly-dissected cochlear epithelium from wild-type mice and mice lacking the F-actin bundling protein TRIOBP-5 or TRIOBP-4 and TRIOBP-5. Variants of the genes encoding human and mouse TRIOBP are associated with deafness. We show that TRIOBP deficiency affects formation of supporting cell apical phalangeal microfilaments and bundled cortical F-actin of hair cell cuticular plates, softening the apical surface of the sensory epithelium. Unexpectedly, high-resolution PFT-AFM-mapping also revealed previously unrecognized reticular lamina radial stiffness gradients of opposite orientations in wild-type supporting and hair cells. Deafness-associated TRIOBP deficiencies significantly modified these bidirectional radial stiffness gradients.

ADAMTS1, MPDZ, MVD, and SEZ6: candidate genes for autosomal recessive nonsyndromic hearing impairment

Hearing impairment (HI) is a common disorder of sensorineural function with a highly heterogeneous genetic background. Although substantial progress has been made in the understanding of the genetic etiology of hereditary HI, many genes implicated in HI remain undiscovered. Via exome and Sanger sequencing of DNA samples obtained from consanguineous Pakistani families that segregate profound prelingual sensorineural HI, we identified rare homozygous missense variants in four genes (ADAMTS1, MPDZ, MVD, and SEZ6) that are likely the underlying cause of HI. Linkage analysis provided statistical evidence that these variants are associated with autosomal recessive nonsyndromic HI. In silico analysis of the mutant proteins encoded by these genes predicted structural, conformational or interaction changes. RNAseq data analysis revealed expression of these genes in the sensory epithelium of the mouse inner ear during embryonic, postnatal, and adult stages. Immunohistochemistry of the mouse cochlear tissue, further confirmed the expression of ADAMTS1, SEZ6, and MPDZ in the neurosensory hair cells of the organ of Corti, while MVD expression was more prominent in the spiral ganglion cells. Overall, supported by in silico mutant protein analysis, animal models, linkage analysis, and spatiotemporal expression profiling in the mouse inner ear, we propose four new candidate genes for HI and expand our understanding of the etiology of HI.

Sox10 Gene is Required for the Survival of Saccular and Utricular Hair Cells

Background:. Pathological changes of the cochlea and hearing loss have been well addressed in Waardenburg syndrome (WS). However, the vestibular organ malformation in WS is still largely unknown. In this study, the differentiation and development of vestibular sensory epithelium and vestibular function caused by SOX10 mutation, a critical gene induces WS, has been studied in minature pig model. Results: Degeneration of vestibular hair cells was found in this Sox10 mutation porcine model. Inner ear phenotype of the SOX10+/R109W miniature pigs showed cochlear abnormalities as well as saccular hypofunction. In the mutant pigs, no prominent dissimilarity was shown in the bone structure of the semicircular canals. However, the saccular membrane was collapsed and the infusion of stereocilia of the hair cells were observed. There was no dark cells in the uticules in th mutant pigs. The density of the utricular hair cells was also significantly lower in the mutant pigs compared to the wild type. Conclusions: Our study demonstrated that the SOX10 gene and melanocytes play important roles in the vestibular organ development. Sox10 mutation disrupts the KIT-DCT signaling pathway, affects the development of melanocytes and leads to vestibule morphogenesis.

Morphological and Histological Features of the Vomeronasal Organ in African Pygmy Hedgehog (Atelerix albiventris)

The vomeronasal organ (VNO) detects specific chemicals such as pheromones and kairomones. Hedgehogs (Eulipotyphla: Erinaceidae) have a well-developed accessory olfactory bulb that receives projections from the VNO, but little is known about the hedgehog VNO. Here, we studied the histological features of the VNO in five individual African pygmy hedgehogs by hematoxylin-eosin, periodic acid-Schiff, and Alcian blue stains. The hedgehog VNO comprises a hyaline cartilage capsule, soft tissue and epithelial lumen, and it branches from the site just before the incisive duct opening into the nasal cavity. The soft tissues contain several small mucous (or mucoserous) glands and a large serous gland, and many venous sinuses all around the lumen. The VNO lumen is round to oval throughout the hedgehog VNO, and the sensory epithelium lines almost the entire rostral part and medial wall of the middle part. These findings indicate that the VNO is functional and plays an important role in the hedgehog. Notably, the VNO apparently has a characteristic flushing mechanism with serous secretions like those of gustatory glands, which the hedgehog might frequently use to recognize the external environment.