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Cochlear implants (CIs) are the most successful neuroprostheses: they restore hearing in deaf people to a surprisingly high degree. As direct recordings in humans are not possible, computational models that predict neuronal excitation patterns are fundamental to advance this technology. We have developed [for full text, please go to the a.m. URL]

BackgroundHuman 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 MethodsWe 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.ResultsThe 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.ConclusionExpressions 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.

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.

International audience; 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.

Due to limitations of human in vivo studies, detailed computational models enable understanding the neural signaling in the degenerated auditory system and cochlear implants (CIs). Four human cochleae were used to quantify hearing levels depending on dendritic changes in diameter and myelination thickness from type I of the auditory nerve fibers (ANFs). Type I neurons transmit the auditory information as spiking pattern from the inner hair cells (IHCs) to the cochlear nucleus. The impact of dendrite diameter and degree of myelination on neural signal transmission was simulated for (1) synaptic excitation via IHCs and (2) stimulation from CI electrodes. An accurate three-dimensional human cochlear geometry, along with 30 auditory pathways, mimicked the CI environment. The excitation properties of electrical potential distribution induced by two CI were analyzed. Main findings: (1) The unimodal distribution of control dendrite diameters becomes multimodal for hearing loss cases; a group of thin dendrites with diameters between 0.3 and 1 μm with a peak at 0.5 μm appeared. (2) Postsynaptic currents from IHCs excite such thin dendrites easier and earlier than under control conditions. However, this advantage is lost as their conduction velocity decreases proportionally with the diameter and causes increased spike latency and jitter in soma and axon. Firing probability reduces through the soma passage due to the low intracellular current flow in thin dendrites during spiking. (3) Compared with dendrite diameter, variations in myelin thickness have a small impact on spiking performance. (4) Contrary to synaptic excitation, CIs cause several spike initiation sites in dendrite, soma region, and axon; moreover, fiber excitability reduces with fiber diameter. In a few cases, where weak stimuli elicit spikes of a target neuron (TN) in the axon, dendrite diameter reduction has no effect. However, in many cases, a spike in a TN is first initiated in the dendrite, and consequently, dendrite degeneration demands an increase in threshold currents. (5) Threshold currents of a TN and co-stimulation of degenerated ANFs in other frequency regions depend on the electrode position, including its distance to the outer wall, the cochlear turn, and the three-dimensional pathway of the TN.

Expression patterns of transcription factors leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), transforming growth factor-beta-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.

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)&minus, 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-&beta, 1 (1 ng/mL TGF-&beta, 1) induced TrkB in a subpopulation of SCC-25 cells. A single 10-&micro, g/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-&beta, 1 treatment, but was restored by BDNF if it followed TGF-&beta, 1. Taken together, BDNF might be ineffective in HPV+ HNSCC patients. In HPV&minus, HNSCC patients, tumor cells did not die after chemotherapeutic challenge and BDNF with TGF-&beta, 1 could improve tumor cell survival and contribute to worse patient prognosis.

There are several limitations to assess immunohistochemical staining in the cells manually such as wide differences in the qualifications of researchers working with histological samples, infeasible to count every single cell, time consuming and experience needed. In order to bridge the gap between experienced and inexperienced researchers, save time, and optimize quantitative accuracy, automated and semi-automated methods for histologic tissue analyses have been introduced. These methods make the interpretation of protein expression in immunohistochemistry (IHC) more objective. One such tool is HistoQuest® software, a semi-automated cell analysis program (TissueGnostics GmbH, Austria) that can be used for counting the number of positive and negative-staining cells as well as for quantitatively assessing the relative staining intensity on a histological slide. The aim of this methodological study was to compare the manual method of IHC with a semi-automated method in evaluating endometriosis lesions with IHC stained with p75 receptor antibody. Methods: Two independent observers were selected, both are experienced gynecologists: Experienced and inexperienced observer. Each received a tutorial about Histoquest® software and then independently set the parameters/tools in Histoquest® prior to independent slide interpretation. The images were arranged using TissueFaxs Plus system (Tissue-Gnostics®, Vienna, Austria) software with the same settings including exposure time and signal amplifications. Quantitative immunohistochemistry analysis was performed by using Histoquest® immunohistochemistry analysis software (TissueGnostics®, Vienna, Austria). Manual counting was done for comparison. Results: Histoquest software was able to identify nuclei stained with hematoxylin perfectly if the nucleus had a ‘normal’ form which is not pale color in the center. If the nucleus appeared irregular with uneven color, the software failed to identify. The discrepancy between semi-automated cell counting and manual counting by intra-experienced observer was smaller than the discrepancy in intra- inexperienced observer in overall result (95% LoA -9.76 to 12.8 vs. 95% LoA -63.39 to 49.25). The two observers had very poor agreement. Conclusion: With IHC, cell counting using Histoquest software benefits the experienced observer/user since it gives highly consistent results compared to the manual method, with the additional benefits of being more objective and very much less time consuming. The software cannot be used by the inexperienced observer, as it cannot overcome that individual's lack of knowledge and produces erroneous results.

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.

Balance orientation depends on the precise operation of the vestibular end organs and the vestibular ganglion neurons. Previous research on the assemblage of the neuronal network in the developing fetal vestibular organ has been limited to data from animal models. Insights into the molecular expression profiles and signaling moieties involved in embryological development of the human fetal inner ear have been limited. We present an investigation of the cells of the vestibular end organs with specific focus on the hair cell differentiation and innervation pattern using an uninterrupted series of unique specimens from gestational weeks 8-12. Nerve fibers positive for peripherin innervate the entire fetal crista and utricle. While in rodents only the peripheral regions of the cristae and the extra-striolar region of the statolithic organs are stained. At week 9, transcription factors PAX2 and PAX8 were observed in the hair cells whereas PAX6 was observed for the first time among the supporting cells of the cristae and the satellite glial cells of the vestibular ganglia. Glutamine synthetase, a regulator of the neurotransmitter glutamate, is strongly expressed among satellite glia cells, transitional zones of the utricle and supporting cells in the sensory epithelium. At gestational week 11, electron microscopic examination reveals bouton contacts at hair cells and first signs of the formation of a protocalyx at type I hair cells. Our study provides first-hand insight into the fetal development of the vestibular end organs as well as their pattern of innervation by means of immunohistochemical and EM techniques, with the aim of contributing toward our understanding of balance development.