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.

Brain-Derived Neurotrophin and TrkB in Head and Neck Squamous Cell Carcinoma.

We hypothesized that in head and neck squamous cell carcinoma (HNSCC), the neurotrophin brain-derived neurotrophic factor (BDNF) and its high affinity receptor TrkB regulate tumor cell survival, invasion, and therapy resistance. We used in situ hybridization for BDNF and immunohistochemistry (IHC) for TrkB in 131 HNSCC samples. Brain-derived neurotrophic factor was highly expressed in normal mucosa in HNSCC tissue and in cell lines, whereas only 42.74% of HNSCC tissue was TrkB⁺. One fourth of HNSCC cases was human papilloma virus (HPV)- positive, but the TrkB IHC frequency was not different in HPV-positive (HPV⁺) and negative cases. The UPCI-SCC090 cells expressed constitutive levels of TrkB. Transforming-growth-factor-ß1 (1 ng/mL TGF-ß1) induced TrkB in a subpopulation of SCC-25 cells. A single 10-µg/mL mitomycin C treatment in UPCI-SCC090 cells induced apoptosis and BDNF did not rescue them. The SCC-25 cells were resistant to the MMC treatment, and their growth decreased after TGF-ß1 treatment, but was restored by BDNF if it followed TGF-ß1. Taken together, BDNF might be ineffective in HPV⁺ HNSCC patients. In HPV- HNSCC patients, tumor cells did not die after chemotherapeutic challenge and BDNF with TGF-ß1 could improve tumor cell survival and contribute to worse patient prognosis.

Nerve Growth Factor (NGF)-Receptor Survival Axis in Head and Neck Squamous Cell Carcinoma.

Neurotrophins and their receptors might regulate cell survival in head and neck squamous cell carcinoma (HNSCC). mRNA expression of nerve growth factor (NGF) and protein synthesis of high (NTRK1) and low affinity neurotrophin (p75 neurotrophin receptor; NTR) receptors were investigated in normal oral mucosa and in HNSCC. HNSCC cell lines were treated with mitomycin C (MMC) and cell survival was investigated. Normal and malignant epithelial cells expressed NGF mRNA. NTRK1 was upregulated in 80% of HNSCC tissue, and 50% of HNSCC samples were p75NTR positive. Interestingly, in HNSCC tissue: NTRK1 and p75NTR immunohistochemical reactions were mutually exclusive. Detroit 562 cell line contained only p75NTR, UPCI-SCC090 cells synthesized NTRK1 but not p75NTR and SCC-25 culture had p75NTR and NTRK1 in different cells. NGF (100 ng/mL) significantly improved (1.4-fold) the survival of cultured UPCI-SCC090 cells after MMC-induced cell cycle arrest, while Detroit 562 cells with high levels of p75NTR did not even get arrested by single short MMC treatment. p75NTR in HNSCC might be related with NGF-independent therapy resistance, while NTRK1 might transduce a survival signal of NGF and contribute in this way to improved tumor cell survival after cell cycle arrest.

Localization of TrkB and p75 receptors in peritoneal and deep infiltrating endometriosis: an immunohistochemical study.

BACKGROUND: The roles of the neurotrophins NGF (Neurotrophic growth factor) and BDNF (brain-derived neurotrophic factor) in neuronal growth and development are already known. Meanwhile, the neurotrophin receptors TrkA (tropomyosin related kinase A), TrkB, and p75 are important for determining the fate of cells. In endometriosis, this complex system has not been fully elucidated yet. The aim of this study was to evaluate the expression and location of these neurotrophins and their receptors in peritoneal (PE) and deep infiltrating endometriotic (DIE) tissues and to measure and compare the density of nerve fibers in the disease subtypes. METHODS: PE lesions (n = 20) and DIE lesions (n = 22) were immunostained and analyzed on serial slides with anti-BDNF, -NGF, -TrkA, -TrkB, -p75,-protein gene product 9.5 (PGP9.5, intact nerve fibers) and -tyrosine hydroxylase (TH, sympathetic nerve fibers) antibodies. RESULT: There was an equally high percentage (greater than 75 %) of BDNF-positive immunostaining cells in both PE and DIE. TrkB (major BDNF receptor) and p75 showed a higher percentage of immunostaining cells in DIE compared to in PE in stroma only (p < 0.014, p < 0.027, respectively). Both gland and stroma of DIE lesions had a lower percentage of NGF-positive immunostaining cells compared to those in PE lesions (p < 0.01 and p < 0.01, respectively), but there was no significant reduction in immunostaining of TrkA in DIE lesions. There was no difference in the mean density of nerve fibers stained with PGP9.5 between PE (26.27 ± 17.32) and DIE (28.19 ± 33.15, p = 0.8). When we performed sub-group analysis, the density of nerves was significantly higher in the bowel DIE (mean 57.33 ± 43.9) than in PE (mean 26.27 ± 17.32, p < 0.01) and non-bowel DIE (mean 14.6. ± 8.6 p < 0.002). CONCLUSIONS: While the neurotrophin BDNF is equally present in PE and DIE, its receptors TrkB and p75 are more highly expressed in DIE and may have a potential role in the pathophysiology of DIE, especially in promotion of cell growth. BDNF has a stronger binding affinity than NGF to the p75 receptor, likely inducing sympathetic nerve axonal pruning in DIE, resulting in the lower nerve fiber density seen.

Activation of TrkB receptors by NGFß mimetic peptide conjugated polymersome nanoparticles.

Activation of tyrosine kinase receptor B (TrkB), a neurotrophin receptor, has been shown to increase neuronal cell survival and promote regeneration. Stimulation of the TrkB receptor by neurotrophic growth factors has been identified as a possible therapeutic target for the treatment of neurodegenerative disorders. However, growth factor delivery is problematic because of a short half-life in vivo. We have conjugated hNgf-EE, a short peptide mimetic of NGFß to the surface of polymersome nanoparticles and shown that they are capable of activating the TrkB receptor in vitro in the SHSY-G7 cell line. We propose that polymersomes could act as a scaffold for the delivery of TrkB activating moieties and that the polymersome size and polyethylene glycol surface have been shown to increase in vivo retention time. These multifunctional nanoparticles have potential for the treatment of neurodegenerative disorders by TrkB activation. From the ClinicaL Editor: Tyrosine kinase receptor B activation has been shown to promote regeneration and survival of neurons. However, growth factor delivery to stimulate these receptors remains problematic. The authors demonstrate that a peptide mimetic of NGFß conjugated to the surface of polymersome nanoparticles is capable of activating the TrkB receptors. These nanoparticles may offer a novel treatment strategy for a variety of neurodegenerative disorders.

Signal Transduction Regulators in Axonal Regeneration.

Intracellular signal transduction in response to growth factor receptor activation is a fundamental process during the regeneration of the nervous system. In this context, intracellular inhibitors of neuronal growth factor signaling have become of great interest in the recent years. Among them are the prominent signal transduction regulators Sprouty (SPRY) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN), which interfere with major signaling pathways such as extracellular signal-regulated kinase (ERK) or phosphoinositide 3-kinase (PI3K)/Akt in neurons and glial cells. Furthermore, SPRY and PTEN are themselves tightly regulated by ubiquitin ligases such as c-casitas b-lineage lymphoma (c-CBL) or neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4) and by different microRNAs (miRs) including miR-21 and miR-222. SPRY, PTEN and their intracellular regulators play an important role in the developing and the lesioned adult central and peripheral nervous system. This review will focus on the effects of SPRY and PTEN as well as their regulators in various experimental models of axonal regeneration in vitro and in vivo. Targeting these signal transduction regulators in the nervous system holds great promise for the treatment of neurological injuries in the future.

Effects of Degrees of Degeneration on the Electrical Excitation of Human Spiral Ganglion Neurons Based on a High-Resolution Computer Model.

After hearing loss retrograde degeneration of spiral ganglion neurons (SGNs) has been described. Studies modeling the effects of degeneration mostly omitted peripheral processes (dendrites). Recent experimental observations indicated that degenerating SGNs manifested also a reduced diameter of their dendrites. We simulated populations of 400 SGNs inside a high resolution cochlear model with a cochlear implant, based on µCT scans of a human temporal bone. Cochlear implant stimuli were delivered as biphasic pulses in a monopolar configuration. Three SGN situations were simulated, based on our previous measurements of human SGN dendrites: (A) SGNs with intact dendrites (before degeneration), (B) degenerating SGNs, dendrites with a smaller diameter but original length, (C) degenerating SGNs, dendrites omitted. SGN fibers were mapped to characteristic frequency, and place pitch was estimated from excitation profiles. Results from degenerating SGNs (B, C) were similar. Most action potentials were initiated in the somatic area for all cases (A, B, C), except for areas near stimulating electrodes in the apex with intact SGNs (A), where action potentials were initiated in the distal dendrite. In most cases, degenerating SGNs had lower thresholds than intact SGNs (A) (down to -2 dB). Excitation profiles showed increased ectopic activation, i.e., activation of unintended neuronal regions, as well as similar neuronal regions excited by different apical electrodes, for degenerating SGNs (B, C). The estimated pitch showed cases of pitch reversals in apical electrodes for intact SGNs (A), as well as mostly identical pitches evoked by the four most apical electrodes for degenerating SGNs (B, C). In conclusion, neuronal excitation profiles to electrical stimulation exhibited similar traits in both ways of modeling SGN degeneration. Models showed degeneration of dendrites caused increased ectopic activation, as well as similar excitation profiles and pitch evoked by different apical electrodes. Therefore, insertion of electrodes beyond approximately 450° may not provide any benefit if SGN dendrites are degenerated.

Visualization of macrophage subsets in the development of the fetal human inner ear.

Background: Human inner ear contains macrophages whose functional role in early development is yet unclear. Recent studies describe inner ear macrophages act as effector cells of the innate immune system and are often activated following acoustic trauma or exposure to ototoxic drugs. Few or limited literature describing the role of macrophages during inner ear development and organogenesis. Material and Methods: We performed a study combining immunohistochemistry and immunofluorescence using antibodies against IBA1, CX3CL1, CD168, CD68, CD45 and CollagenIV. Immune staining and quantification was performed on human embryonic inner ear sections from gestational week 09 to 17. Results: The study showed IBA1 and CD45 positive cells in the mesenchymal tissue at GW 09 to GW17. No IBA1 positive macrophages were detected in the sensory epithelium of the cochlea and vestibulum. Fractalkine (CX3CL1) signalling was initiated GW10 and parallel chemotactic attraction and migration of macrophages into the inner ear. Macrophages also migrated into the spiral ganglion, cochlear nerve, and peripheral nerve fibers and tissue-expressing CX3CL1. The mesenchymal tissue at all gestational weeks expressed CD163 and CD68. Conclusion: Expressions of markers for resident and non-resident macrophages (IBA1, CD45, CD68, and CD163) were identified in the human fetal inner ear. We speculate that these cells play a role for the development of human inner ear tissue including shaping of the gracile structures.

Polarity Sensitivity of Human Auditory Nerve Fibers Based on Pulse Shape, Cochlear Implant Stimulation Strategy and Array.

Neural health is of great interest to determine individual degeneration patterns for improving speech perception in cochlear implant (CI) users. Therefore, in recent years, several studies tried to identify and quantify neural survival in CI users. Among all proposed techniques, polarity sensitivity is a promising way to evaluate the neural status of auditory nerve fibers (ANFs) in CI users. Nevertheless, investigating neural health based on polarity sensitivity is a challenging and complicated task that involves various parameters, and the outcomes of many studies show contradictory results of polarity sensitivity behavior. Our computational study benefits from an accurate three-dimensional finite element model of a human cochlea with realistic human ANFs and determined ANF degeneration pattern of peripheral part with a diminishing of axon diameter and myelination thickness based on degeneration levels. In order to see how different parameters may impact the polarity sensitivity behavior of ANFs, we investigated polarity behavior under the application of symmetric and asymmetric pulse shapes, monopolar and multipolar CI stimulation strategies, and a perimodiolar and lateral CI array system. Our main findings are as follows: (1) action potential (AP) initiation sites occurred mainly in the peripheral site in the lateral system regardless of stimulation strategies, pulse polarities, pulse shapes, cochlear turns, and ANF degeneration levels. However, in the perimodiolar system, AP initiation sites varied between peripheral and central processes, depending on stimulation strategies, pulse shapes, and pulse polarities. (2) In perimodiolar array, clusters formed in threshold values based on cochlear turns and degeneration levels for multipolar strategies only when asymmetric pulses were applied. (3) In the perimodiolar array, a declining trend in polarity (anodic threshold/cathodic threshold) with multipolar strategies was observed between intact or slight degenerated cases and more severe degenerated cases, whereas in the lateral array, cathodic sensitivity was noticed for intact and less degenerated cases and anodic sensitivity for cases with high degrees of degeneration. Our results suggest that a combination of asymmetric pulse shapes, focusing more on multipolar stimulation strategies, as well as considering the distances to the modiolus wall, allows us to distinguish the degeneration patterns of ANFs across the cochlea.

Distribution of Immune Cells Including Macrophages in the Human Cochlea.

Background: The human cochlea was earlier believed to lack capacity to mount specific immune responses. Recent studies established that the human cochlea holds macrophages. The cells appear to surveil, dispose of, and restore wasted cells to maintain tissue integrity. Macrophage activities are believed to be the central elements in immune responses and could swiftly defuse invading microbes that enter via adjacent infection-prone areas. This review updates recent human studies in light of the current literature and adds information about chemokine gene expression. Materials and Methods: We analyzed surgically obtained human tissue using immunohistochemistry, confocal microscopy, and multichannel super-resolution structured illumination microscopy. The samples were considered representative of steady-state conditions. Antibodies against the ionized calcium-binding adaptor molecule 1 were used to identify the macrophages. CD68 and CD11b, and the major histocompatibility complex type II (MHCII) and CD4 and CD8 were analyzed. The RNAscope technique was used for fractalkine gene localization. Results: Many macrophages were found around blood vessels in the stria vascularis but not CD4 and CD8 lymphocytes. Amoeboid macrophages were identified in the spiral ganglion with surveilling "antennae" projecting against targeted cells. Synapse-like contacts were seen on spiral ganglion cell bodies richly expressing single CXC3CL gene transcripts. Branching neurite-like processes extended along central and peripheral axons. Active macrophages were occasionally found near degenerating hair cells. Some macrophage-interacting T lymphocytes were observed between the scala tympani wall and Rosenthal's canal. CD4 and CD8 cells were not found in the organ of Corti. Conclusions: The results indicate that the human cochlea is equipped with macrophages and potentially lymphocytes, suggesting both an innate and adaptive immune capacity. A rich expression of fractalkine gene transcripts in spiral ganglion neurons suggest an essential role for auditory nerve protection, as has been demonstrated experimentally. The findings provide further information on the important role of the immune machinery present in the human inner ear and its potential to carry adverse immune reactions, including cytotoxic and foreign body responses. The results can be used to form a rationale for therapies aiming to modulate these immune activities.

Spike Generators and Cell Signaling in the Human Auditory Nerve: An Ultrastructural, Super-Resolution, and Gene Hybridization Study.

Background: The human auditory nerve contains 30,000 nerve fibers (NFs) that relay complex speech information to the brain with spectacular acuity. How speech is coded and influenced by various conditions is not known. It is also uncertain whether human nerve signaling involves exclusive proteins and gene manifestations compared with that of other species. Such information is difficult to determine due to the vulnerable, "esoteric," and encapsulated human ear surrounded by the hardest bone in the body. We collected human inner ear material for nanoscale visualization combining transmission electron microscopy (TEM), super-resolution structured illumination microscopy (SR-SIM), and RNA-scope analysis for the first time. Our aim was to gain information about the molecular instruments in human auditory nerve processing and deviations, and ways to perform electric modeling of prosthetic devices. Material and Methods: Human tissue was collected during trans-cochlear procedures to remove petro-clival meningioma after ethical permission. Cochlear neurons were processed for electron microscopy, confocal microscopy (CM), SR-SIM, and high-sensitive in situ hybridization for labeling single mRNA transcripts to detect ion channel and transporter proteins associated with nerve signal initiation and conductance. Results: Transport proteins and RNA transcripts were localized at the subcellular level. Hemi-nodal proteins were identified beneath the inner hair cells (IHCs). Voltage-gated ion channels (VGICs) were expressed in the spiral ganglion (SG) and axonal initial segments (AISs). Nodes of Ranvier (NR) expressed Nav1.6 proteins, and encoding genes critical for inter-cellular coupling were disclosed. Discussion: Our results suggest that initial spike generators are located beneath the IHCs in humans. The first NRs appear at different places. Additional spike generators and transcellular communication may boost, sharpen, and synchronize afferent signals by cell clusters at different frequency bands. These instruments may be essential for the filtering of complex sounds and may be challenged by various pathological conditions.