Microanatomy of the human tunnel of Corti structures and cochlear partition-tonotopic variations and transcellular signaling.

Auditory sensitivity and frequency resolution depend on the optimal transfer of sound-induced vibrations from the basilar membrane (BM) to the inner hair cells (IHCs), the principal auditory receptors. There remains a paucity of information on how this is accomplished along the frequency range in the human cochlea. Most of the current knowledge is derived either from animal experiments or human tissue processed after death, offering limited structural preservation and optical resolution. In our study, we analyzed the cytoarchitecture of the human cochlear partition at different frequency locations using high-resolution microscopy of uniquely preserved normal human tissue. The results may have clinical implications and increase our understanding of how frequency-dependent acoustic vibrations are carried to human IHCs. A 1-micron-thick plastic-embedded section (mid-modiolar) from a normal human cochlea uniquely preserved at lateral skull base surgery was analyzed using light and transmission electron microscopy (LM, TEM). Frequency locations were estimated using synchrotron radiation phase-contrast imaging (SR-PCI). Archival human tissue prepared for scanning electron microscopy (SEM) and super-resolution structured illumination microscopy (SR-SIM) were also used and compared in this study. Microscopy demonstrated great variations in the dimension and architecture of the human cochlear partition along the frequency range. Pillar cell geometry was closely regulated and depended on the reticular lamina slope and tympanic lip angle. A type II collagen-expressing lamina extended medially from the tympanic lip under the inner sulcus, here named "accessory basilar membrane." It was linked to the tympanic lip and inner pillar foot, and it may contribute to the overall compliance of the cochlear partition. Based on the findings, we speculate on the remarkable microanatomic inflections and geometric relationships which relay different sound-induced vibrations to the IHCs, including their relevance for the evolution of human speech reception and electric stimulation with auditory implants. The inner pillar transcellular microtubule/actin system's role of directly converting vibration energy to the IHC cuticular plate and ciliary bundle is highlighted.

Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue.

A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at -80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (B2M, HPRT1, GAPDH and GUSB). The selected reference genes were then used to examine the effect on the expression outcome of target genes (OTOF and TECTA), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of OTOF and TECTA, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies.

Effects of Therapeutic Hypothermia on Macrophages in Mouse Cochlea Explants.

Globally, over the next few decades, more than 2.5 billion people will suffer from hearing impairment, including profound hearing loss, and millions could potentially benefit from a cochlea implant. To date, several studies have focused on tissue trauma caused by cochlea implantation. The direct immune reaction in the inner ear after an implantation has not been well studied. Recently, therapeutic hypothermia has been found to positively influence the inflammatory reaction caused by electrode insertion trauma. The present study aimed to evaluate the hypothermic effect on the structure, numbers, function and reactivity of macrophages and microglial cells. Therefore, the distribution and activated forms of macrophages in the cochlea were evaluated in an electrode insertion trauma cochlea culture model in normothermic and mild hypothermic conditions. In 10-day-old mouse cochleae, artificial electrode insertion trauma was inflicted, and then they were cultured for 24 h at 37 °C and 32 °C. The influence of mild hypothermia on macrophages was evaluated using immunostaining of cryosections using antibodies against IBA1, F4/80, CD45 and CD163. A clear influence of mild hypothermia on the distribution of activated and non-activated forms of macrophages and monocytes in the inner ear was observed. Furthermore, these cells were located in the mesenchymal tissue in and around the cochlea, and the activated forms were found in and around the spiral ganglion tissue at 37 °C. Our findings suggest that mild hypothermic treatment has a beneficial effect on immune system activation after electrode insertion trauma.

HCN channels in the mammalian cochlea: Expression pattern, subcellular location, and age-dependent changes.

Neuronal diversity in the cochlea is largely determined by ion channels. Among voltage-gated channels, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels open with hyperpolarization and depolarize the cell until the resting membrane potential. The functions for hearing are not well elucidated and knowledge about localization is controversial. We created a detailed map of subcellular location and co-expression of all four HCN subunits across different mammalian species including CBA/J, C57Bl/6N, Ly5.1 mice, guinea pigs, cats, and human subjects. We correlated age-related hearing deterioration in CBA/J and C57Bl/6N with expression levels of HCN1, -2, and -4 in individual auditory neurons from the same cohort. Spatiotemporal expression during murine postnatal development exposed HCN2 and HCN4 involvement in a critical phase of hair cell innervation. The huge diversity of subunit composition, but lack of relevant heteromeric pairing along the perisomatic membrane and axon initial segments, highlighted an active role for auditory neurons. Neuron clusters were found to be the hot spots of HCN1, -2, and -4 immunostaining. HCN channels were also located in afferent and efferent fibers of the sensory epithelium. Age-related changes on HCN subtype expression were not uniform among mice and could not be directly correlated with audiometric data. The oldest mice groups revealed HCN channel up- or downregulation, depending on the mouse strain. The unexpected involvement of HCN channels in outer hair cell function where HCN3 overlaps prestin location emphasized the importance for auditory function. A better understanding may open up new possibilities to tune neuronal responses evoked through electrical stimulation by cochlear implants.

Transcriptome-Wide Analysis Reveals a Role for Extracellular Matrix and Integrin Receptor Genes in Otic Neurosensory Differentiation from Human iPSCs.

We analyzed transcriptomic data from otic sensory cells differentiated from human induced pluripotent stem cells (hiPSCs) by a previously described method to gain new insights into the early human otic neurosensory lineage. We identified genes and biological networks not previously described to occur in the human otic sensory developmental cell lineage. These analyses identified and ranked genes known to be part of the otic sensory lineage program (SIX1, EYA1, GATA3, etc.), in addition to a number of novel genes encoding extracellular matrix (ECM) (COL3A1, COL5A2, DCN, etc.) and integrin (ITG) receptors (ITGAV, ITGA4, ITGA) for ECM molecules. The results were confirmed by quantitative PCR analysis of a comprehensive panel of genes differentially expressed during the time course of hiPSC differentiation in vitro. Immunocytochemistry validated results for select otic and ECM/ITG gene markers in the in vivo human fetal inner ear. Our screen shows ECM and ITG gene expression changes coincident with hiPSC differentiation towards human otic neurosensory cells. Our findings suggest a critical role of ECM-ITG interactions with otic neurosensory lineage genes in early neurosensory development and cell fate determination in the human fetal inner ear.

ß-Secretase BACE1 Is Required for Normal Cochlear Function.

Cleavage of amyloid precursor protein (APP) by ß-secretase BACE1 initiates the production and accumulation of neurotoxic amyloid-ß peptides, which is widely considered an essential pathogenic mechanism in Alzheimer's disease (AD). Here, we report that BACE1 is essential for normal auditory function. Compared with wild-type littermates, BACE1-/- mice of either sex exhibit significant hearing deficits, as indicated by increased thresholds and reduced amplitudes in auditory brainstem responses (ABRs) and decreased distortion product otoacoustic emissions (DPOAEs). Immunohistochemistry revealed aberrant synaptic organization in the cochlea and hypomyelination of auditory nerve fibers as predominant neuropathological substrates of hearing loss in BACE1-/- mice. In particular, we found that fibers of spiral ganglion neurons (SGN) close to the organ of Corti are disorganized and abnormally swollen. BACE1 deficiency also engenders organization defects in the postsynaptic compartment of SGN fibers with ectopic overexpression of PSD95 far outside the synaptic region. During postnatal development, auditory fiber myelination in BACE1-/- mice lags behind dramatically and remains incomplete into adulthood. We relate the marked hypomyelination to the impaired processing of Neuregulin-1 when BACE1 is absent. To determine whether the cochlea of adult wild-type mice is susceptible to AD treatment-like suppression of BACE1, we administered the established BACE1 inhibitor NB-360 for 6 weeks. The drug suppressed BACE1 activity in the brain, but did not impair hearing performance and, upon neuropathological examination, did not produce the characteristic cochlear abnormalities of BACE1-/- mice. Together, these data strongly suggest that the hearing loss of BACE1 knock-out mice represents a developmental phenotype.SIGNIFICANCE STATEMENT Given its crucial role in the pathogenesis of Alzheimer's disease (AD), BACE1 is a prime pharmacological target for AD prevention and therapy. However, the safe and long-term administration of BACE1-inhibitors as envisioned in AD requires a comprehensive understanding of the various physiological functions of BACE1. Here, we report that BACE1 is essential for the processing of auditory signals in the inner ear, as BACE1-deficient mice exhibit significant hearing loss. We relate this deficit to impaired myelination and aberrant synapse formation in the cochlea, which manifest during postnatal development. By contrast, prolonged pharmacological suppression of BACE1 activity in adult wild-type mice did not reproduce the hearing deficit or the cochlear abnormalities of BACE1 null mice.

Early appearance of key transcription factors influence the spatiotemporal development of the human inner ear.

Expression patterns of transcription factors leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), transforming growth factor-ß-activated kinase-1 (TAK1), SRY (sex-determining region Y)-box 2 (SOX2), and GATA binding protein 3 (GATA3) in the developing human fetal inner ear were studied between the gestation weeks 9 and 12. Further development of cochlear apex between gestational weeks 11 and 16 (GW11 and GW16) was examined using transmission electron microscopy. LGR5 was evident in the apical poles of the sensory epithelium of the cochlear duct and the vestibular end organs at GW11. Immunostaining was limited to hair cells of the organ of Corti by GW12. TAK1 was immune positive in inner hair cells of the organ of Corti by GW12 and colocalized with p75 neurotrophic receptor expression. Expression for SOX2 was confined primarily to the supporting cells of utricle at the earliest stage examined at GW9. Intense expression for GATA3 was presented in the cochlear sensory epithelium and spiral ganglia at GW9. Expression of GATA3 was present along the midline of both the utricle and saccule in the zone corresponding to the striolar reversal zone where the hair cell phenotype switches from type I to type II. The spatiotemporal gradient of the development of the organ of Corti was also evident with the apex of the cochlea forming by GW16. It seems that highly specific staining patterns of several transcriptions factors are critical in guiding the genesis of the inner ear over development. Our findings suggest that the spatiotemporal gradient in cochlear development extends at least until gestational week 16.

Age-Dependent Changes in the Cochlea.

Age-related hearing loss is a progressive, bilateral, and symmetrical sensorineural hearing loss due to degeneration of inner ear structures and it is considered a multifactorial complex disorder. Changes in tissue performance act from the cellular level to the molecular level. Histological visible damage in different structures of the inner ear signifies that the high frequency region is more sensitive and earlier affected. For many years it has been known that, with advancing age, there is an increasing loss of outer and inner hair cells and peripheral and central neurons. Furthermore, it comes to an atrophy of the stria vascularis. In recent years, investigations of the inner ear have shown that a loss of cochlear nerve synapses also plays an important role in age-related hearing loss. Degeneration of cochlear nerve synapses at the inner hair cell leads to a worsening of complex auditory tasks, such as understanding speech in difficult listening environments with minimal effects on pure-tone thresholds. This phenomenon has been called "hidden hearing loss." Recent research has shown that senescence of the immune system plays an important role in age-related degeneration of the inner ear, termed inflammaging. A mild form of chronic systemic inflammatory response is thought to activate cochlear macrophages.

Growth and cellular patterning during fetal human inner ear development studied by a correlative imaging approach.

BACKGROUND: Progressive transformation of the otic placode into the functional inner ear during gestational development in humans leads to the acquisition of hearing perception via the cochlea and balance and spatial orientation via the vestibular organ. RESULTS: Using a correlative approach involving micro-computerized tomography (micro-CT), transmission electron microscopy and histological techniques we were able to examine both the morphological and cellular changes associated with human inner ear development. Such an evaluation allowed for the examination of 3D geometry with high spatial and temporal resolution. In concert with gestational progression and growth of the cochlear duct, an increase in the distance between some of the Crista ampullaris is evident in all the specimens examined from GW12 to GW36. A parallel increase in the distances between the macular organs - fetal utricle and saccule - is also evident across the gestational stages examined. The distances between both the utricle and saccule to the three cristae ampullares also increased across the stages examined. A gradient in hair cell differentiation is apparent from apex to base of the fetal cochlea even at GW14. CONCLUSION: We present structural information on human inner ear development across multiple levels of biological organization, including gross-morphology of the inner ear, cellular and subcellular details of hearing and vestibular organs, as well as ultrastructural details in the developing sensory epithelia. This enabled the gathering of detailed information regarding morphometric changes as well in realizing the complex developmental patterns of the human inner ear. We were able to quantify the volumetric and linear aspects of selected gestational inner ear specimens enabling a better understanding of the cellular changes across the fetal gestational timeline. Moreover, these data could serve as a reference for better understanding disorders that arise during inner ear development.

Age-Dependent Calcium-Binding Protein Expression in the Spiral Ganglion and Hearing Performance of C57BL/6J and 129/SvJ Mice.

BACKGROUND/AIMS: Calcium-binding proteins in neurons buffer intracellular free Ca2+ ions, which interact with proteins controlling enzymatic and ion channel activity. The heterogeneous distribution of calretinin, calbindin, and parvalbumin influences calcium homeostasis, and calcium-related neuronal processes play an important role in neuronal aging and degeneration. This study evaluated age-related changes in calretinin, calbindin, and parvalbumin immune reactivity in spiral ganglion cells. METHODS: A total of 16 C57BL/6J and 16 129/SvJ mice at different ages (2, 4, 7, and 12 months) were included in the study. Hearing thresholds were assessed using auditory brainstem response before inner ears were excised for further evaluation. Semiquantitative immunohistochemistry for the aforementioned calcium-binding proteins was performed at the cellular level. RESULTS: The hearing thresholds of C57BL/6J and 129/SvJ mice increased significantly by 7 months of age. The average immune reactivity of calbin-din as well as the relative number of positive cells increased significantly with aging, but no significant alterations in calretinin or parvalbumin were observed. CONCLUSIONS: Upregulation of calbindin could serve as a protection to compensate for functional deficits that occur with aging. Expression of both calretinin and parvalbumin seem to be stabilizing factors in murine inner ears up to the age of 12 months in C57BL/6J and 129/SvJ mice.