Identification of neutrophils and eosinophils in upper airway mucosa with immunofluorescence multiplex image cytometry.

Characterization of inflammation in chronic rhinosinusitis with (CRSwNP) and without nasal polyps (CRSsNP) is an ongoing research process. To overcome limitations of current cytologic techniques, we investigated whether immunofluorescence multiplex image cytometry could quantify intact neutrophils, eosinophils, and other immune cells in solid upper airway mucosa. We used a four-channel immunofluorescence-microscopy technique for the simultaneous detection of the leukocyte marker CD45, the neutrophil marker myeloperoxidase, two eosinophil markers, i.e., major basic protein and eosinophil peroxidase, and DAPI (4',6-diamidin-2-phenylindole), in formalin-fixed paraffin-embedded upper airway tissue samples of patients with CRSwNP and CRSsNP, as well as of patients free of CRS with inferior turbinate hypertrophy (controls). Image acquisition and analysis were performed with TissueFAXS and StrataQuest (TissueGnostics, Vienna, Austria), respectively. Positive and negative immunostaining were differentiated with a specific fluorescence signal/background signal ratio. Isotype controls were used as negative controls. In six controls, nine patients with CRSsNP, and 11 patients with CRSwNP, the median area scanned and median cell count per patient were 14.2 mm2 and 34,356, respectively. In CRSwNP, the number of eosinophils was three times higher (23%) than that of neutrophils (7%). Three times more immune cells were encountered in CRSwNP (33%) compared to CRSsNP (11%). In controls, inflammation was balanced between the epithelial layer and lamina propria, in contrast to CRS (three times more pronounced inflammation in the lamina propria). The quantification of intact neutrophils, eosinophils, and other immune cells in solid tissue with undisrupted architecture seems feasible with immunofluorescence multiplex image cytometry.

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.

Analysis of cells of epithelial, connective tissue and immune differentiation in HPV-positive-, HPV-negative oropharyngeal carcinoma and normal oropharyngeal tissue by immunofluorescence multiplex image cytometry: a preliminary report.

BACKGROUND: Epithelial, connective tissue and immune cells contribute in various ways to the pathophysiology of HPV positive (HPV+) and HPV negative (HPV-) oropharyngeal squamous cell carcinoma (OPSCC). We aimed to investigate the abundance of these cell lineages and their coexpression patterns in patients with HPV + and HPV- OPSCC. METHODS: We used a 4-channel immunofluorescence-microscopy technique for the simultaneous detection of three direct-conjugated antibodies (pancytokeratin, vimentin and CD45/CD18) and DAPI (4',6-Diamidin-2-phenylindole) in formalin fixed paraffin-embedded tissue samples (FFPE) of patients with HPV + and HPV- OPSCC, and of control patients. Image acquisition and analysis were performed with TissueFAXS and StrataQuest (TissueGnostics, Vienna, Austria), respectively, in tumor cell clusters/stroma in OPSCC specimens and epithelial layer/lamina propria in control specimens. Cell populations were created based on antibodies' coexpression patterns. Isotype and positive controls were examined for plausibility. RESULTS: The proportion of cells of epithelial differentiation in tumor cell clusters was higher in HPV + OPSCC (55%) than in HPV- OPSCC samples (44%). The proportion of connective tissue cells in tumor cell cluster was lower in HPV + OPSCC patients (18%) than in HPV- OPSCC patients (26%). The proportion of immune cells in tumor cell clusters was higher in HPV + OPSCC patients (25%) than in HPV- OPSCC patients (18%). The percentage of anaplastic, potentially de-differentiated cells, was 2% in control patients, and it was higher in HPV- OPSCC (21%) than in HPV + OPSCC samples (6%). CONCLUSIONS: This study provided the first quantitative data for the abundance of cells of epithelial, connective tissue and immune differentiation, in patients with OPSCC and control patients. The abundance of these different crucial cell populations was consistently originating from the same tissue sample. De-differentiation of tumor cells was higher in HPV- OPSCC than in HPV + OPSCC. In tumor cells clusters, the antitumoral host immune response was higher in HPV + OPSCC than in HPV- OPSCC, whereas the fibroblast response was higher in HPV- OPSCC than in HPV + OPSCC. This study contributed to the understanding of histopathologic differences between HPV + OPSCC and HPV- OPSCC patients.

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.

Single-incision cochlear implantation and hearing evaluation in piglets and minipigs.

OBJECTIVES: Various animal models have been established and applied in hearing research. In the exploration of novel cochlear implant developments, mainly rodents have been used. Despite their important contribution to the understanding of auditory function, translation of experimental observations from rodents to humans is limited due to the size differences and genetic variability. Large animal models with better representation of the human cochlea are sparse. For this reason, we evaluated domestic piglets and Aachen minipigs for the suitability as a cochlear implantation animal model with commercially available cochlear implants. METHODS: Four domestic piglets (two male and two female) and six Aachen minipigs were implanted with either MED-EL Flex24 or Flex20 cochlear implants respectively, after a step-by-step surgical approach was trained with pig cadavers. Electrophysiological measurements were performed before, during and after implantation for as long as 56 days after surgery. Auditory brainstem responses, electrocochleography as well as electrically and acoustically evoked compound action potentials were recorded. Selected cochleae were further analyzed histologically or with micro-CT imaging. RESULTS: A surgical approach was established using a retroauricular single incision. Baseline auditory thresholds were 27 ± 3 dB sound pressure level (SPL; auditory brainstem click responses, mean ± standard error of the mean) and ranged between 30 and 80 dB SPL in frequency-specific responses (0.5 - 32 kHz). Follow-up measurements revealed deafness within the first two weeks after surgery, but some animals partially recovered to a hearing threshold of 80 dB SPL in certain frequencies as well as in click responses. Electrically evoked compound action potential thresholds increased within the first week after surgery, which led to lower stimulation responses or increase of necessary charge input. Immune reactions and consecutive scalar fibrosis following implantation were confirmed with histological analysis of implanted cochleae and may result in increased impedances. A three-dimensional minipig micro-CT segmentation revealed cochlear volumetric data similar to human inner ear dimensions. CONCLUSIONS: This study underlines the feasibility of cochlear implantation with clinically used cochlear implants in a large animal model with representative inner ear dimensions comparable to humans. To bridge the gap between small animal models and humans in translational research and to account for the structural and size differences, we recommend the minipig as a valuable animal model for hearing research. First insights into the induced trauma in minipigs after cochlear implant surgery and a partial hearing recovery present important data of the cochlear health changes in large animal cochleae.

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.

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.

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.

KLF4, Slug and EMT in Head and Neck Squamous Cell Carcinoma.

Epithelial to mesenchymal transition (EMT) is clinically relevant in head and neck squamous cell carcinoma (HNSCC). We hypothesized that EMT-transcription factors (EMT-TFs) and an anti-EMT factor, Krüppel-like-factor-4 (KLF4) regulate EMT in HNSCC. Ten control mucosa and 37 HNSCC tissue samples and three HNSCC cell lines were included for investigation of EMT-TFs, KLF4 and vimentin at mRNA and protein levels. Slug gene expression was significantly higher, whereas, KLF4 gene expression was significantly lower in HNSCC than in normal mucosa. In the majority of HNSCC samples, there was a significant negative correlation between KLF4 and Slug gene expression. Slug gene expression was significantly higher in human papilloma virus (HPV) negative HNSCC, and in tumor samples with irregular p53 gene sequence. Transforming-growth-factor-beta-1 (TGF- ß1) contributed to downregulation of KLF4 and upregulation of Slug. Two possible regulatory pathways could be suggested: (1) EMT-factors induced pathway, where TGF-ß1 induced Slug together with vimentin, and KLF4 was down regulated at the same time; (2) p53 mutations contributed to upregulation and stabilization of Slug, where also KLF4 could co-exist with EMT-TFs.

Characterization of epithelial cells, connective tissue cells and immune cells in human upper airway mucosa by immunofluorescence multichannel image cytometry: a pilot study.

Epithelial, connective tissue and immune cells contribute in various ways to the pathophysiology of chronic rhinosinusitis (CRS). However, data of their distribution in upper airway mucosa are sparse. We aimed to provide quantitative, purely informative data on the distribution of these cell lineages and their coexpression patterns, which might help identifying, e.g., cells in the epithelium undergoing through epithelial-mesenchymal transition (EMT). For this purpose, we used immunofluorescence multichannel image cytometry (IMIC). We examined fixed paraffin-embedded tissue samples (FFPE) of six patients with chronic rhinosinusitis (CRS) and of three patients without CRS (controls). The direct-conjugated antibodies pancytokeratin, vimentin and CD45/CD18 were used for coexpression analysis in epithelial layer and lamina propria. Image acquisition and analysis were performed with TissueFAXS and StrataQuest, respectively. To distinguish positive from negative expression, a ratio between cell-specific immunostaining intensity and background was developed. Isotype controls were used as negative controls. Per patient, a 4.5-mm2 tissue area was scanned and a median of 14,875 cells was recognized. The most common cell types were cytokeratin-single-positive (26%), vimentin-single-positive (13%) and CD45/CD18-single-positive with CD45/CD18-vimentin-double-positive cells (29%). In the patients with CRS, CD45/CD18-single-positive cells were 3-6 times higher compared to the control patients. In the epithelial layer, cytokeratin-vimentin-double-positive EMT cells were observed 3-5 times higher in the patients with CRS than in the control patients. This study provided quantitative data for the distribution of crucial cell types in CRS. Future studies may focus on the distribution and coexpression patterns of different immune cells in CRS or even cancer tissue.

Expression of Na/K-ATPase subunits in the human cochlea: a confocal and super-resolution microscopy study with special reference to auditory nerve excitation and cochlear implantation.

Background: For the first time the expression of the ion transport protein sodium/potassium-ATPase and its isoforms was analyzed in the human cochlea using light- and confocal microscopy as well as super-resolution structured illumination microscopy. It may increase our understanding of its role in the propagation and processing of action potentials in the human auditory nerve and how electric nerve responses are elicited from auditory prostheses. Material and methods: Archival human cochlear sections were obtained from trans-cochlear surgeries. Antibodies against the Na/K-ATPase ß1 isoform together with α1 and α3 were used for immunohistochemistry. An algorithm was applied to assess the expression in various domains. Results: Na/K ATPase ß1 subunit was expressed, mostly combined with the α1 isoform. Neurons expressed the ß1 subunit combined with α3, while satellite glial cells expressed the α1 isoform without recognized association with ß1. Types I and II spiral ganglion neurons and efferent fibers expressed the Na/K-ATPase α3 subunit. Inner hair cells, nerve fibers underneath, and efferent and afferent fibers in the organ of Corti also expressed α1. The highest activity of Na/K-ATPase ß1 was at the inner hair cell/nerve junction and spiral prominence. Conclusion: The human auditory nerve displays distinct morphologic features represented in its molecular expression. It was found that electric signals generated via hair cells may not go uninterrupted across the spiral ganglion, but are locally processed. This may be related to particular filtering properties in the human acoustic pathway.

Drafting a Surgical Procedure Using a Computational Anatomy Driven Approach for Precise, Robust, and Safe Vestibular Neuroprosthesis Placement-When One Size Does Not Fit All.

OBJECTIVE: To design and evaluate a new vestibular implant and surgical procedure that should reach correct electrode placement in 95% of patients in silico. DESIGN: Computational anatomy driven implant and surgery design study. SETTING: Tertiary referral center. PARTICIPANTS: The population comprised 81 patients that had undergone a CT scan of the Mastoid region in the Maastricht University Medical Center. The population was subdivided in a vestibular implant eligible group (28) and a control group (53) without known vestibular loss. INTERVENTIONS: Canal lengths and relationships between landmarks were calculated for every patient. The relationships in group-anatomy were used to model a fenestration site on all three semicircular canals. Each patient's simulated individual distance from the fenestration site to the ampulla was calculated and compared with the populations average to determine if placement would be successful. MAIN OUTCOME MEASURES: Lengths of the semicircular canals, distances from fenestration site to ampulla (intralabyrinthine electrode length), and rate of successful electrode placement (robustness). RESULTS: The canal lengths for the lateral, posterior, and superior canal were respectively 12.1 mm ±â€Š1.07, 18.8 mm ±â€Š1.62, and 17.5 mm ±â€Š1.23, the distances from electrode fenestration site to the ampulla were respectively 3.73 mm ±â€Š0.53, 9.02 mm ±â€Š0.90, and 5.31 mm ±â€Š0.73 and electrode insertions were successful for each respective semicircular canal in 92.6%, 66.7%, and 86.4% of insertions in silico. The implant electrode was subsequently revised to include two more electrodes per lead, resulting in a robustness of 100%. CONCLUSIONS: The computational anatomy approach can be used to design and test surgical procedures. With small changes in electrode design, the proposed surgical procedure's target robustness was reached.

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.

Expression of trans-membrane serine protease 3 (TMPRSS3) in the human organ of Corti.

TMPRSS3 (Trans-membrane Serine Protease 3) is a type II trans-membrane serine protease that has proteolytic activity essential for hearing. Mutations in the gene cause non-syndromic autosomal recessive deafness (DFNB8/10) in humans. Knowledge about its cellular distribution in the human inner ear may increase our understanding of its physiological role and involvement in deafness, ultimately leading to therapeutic interventions. In this study, we used super-resolution structured illumination microscopy for the first time together with transmission electron microscopy to localize the TMPRSS3 protein in the human organ of Corti. Archival human cochleae were dissected out during petroclival meningioma surgery. Microscopy with Zeiss LSM710 microscope achieved a lateral resolution of approximately 80 nm. TMPRSS3 was found to be associated with actin in both inner and outer hair cells. TMPRSS3 was located in cell surface-associated cytoskeletal bodies (surfoskelosomes) in inner and outer pillar cells and Deiters cells and in subcuticular organelles in outer hair cells. Our results suggest that TMPRSS3 proteolysis is linked to hair cell sterociliary mechanics and to the actin/microtubule networks that support cell motility and integrity.

The Human "Cochlear Battery" - Claudin-11 Barrier and Ion Transport Proteins in the Lateral Wall of the Cochlea.

Background: The cochlea produces an electric field potential essential for hair cell transduction and hearing. This biological "battery" is situated in the lateral wall of the cochlea and contains molecular machinery that secretes and recycles K+ ions. Its functioning depends on junctional proteins that restrict the para-cellular escape of ions. The tight junction protein Claudin-11 has been found to be one of the major constituents of this barrier that maintains ion gradients (Gow et al., 2004; Kitajiri et al., 2004a). We are the first to elucidate the human Claudin-11 framework and the associated ion transport machinery using super-resolution fluorescence illumination microscopy (SR-SIM). Methods: Archival cochleae obtained during meningioma surgery were used for SR-SIM together with transmission electron microscopy after ethical consent. Results: Claudin-11-expressing cells formed parallel tight junction lamellae that insulated the epithelial syncytium of the stria vascularis and extended to the suprastrial region. Intercellular gap junctions were found between the barrier cells and fibrocytes. Conclusion: Transmission electron microscopy, confocal microscopy and SR-SIM revealed exclusive cell specialization in the various subdomains of the lateral wall of the human cochlea. The Claudin-11-expressing cells exhibited both conductor and isolator characteristics, and these micro-porous separators may selectively mediate the movement of charged units to the intrastrial space in a manner that is analogous to a conventional electrochemical "battery." The function and relevance of this battery for the development of inner ear disease are discussed.

Variable expressivity of TCTEX1D2 mutations and a possible pathogenic link of molar-incisor malformation to ciliary dysfunction.

OBJECTIVE: Clarification of the molecular basis of a ciliopathy associated with molar-incisor malformation in a consanguineous Turkish family. DESIGN: Full dental and clinical examinations, histologic analysis, comprehensive genetic analyses including exome sequencing, ciliary function tests and transmission electron microscopy of ciliary biopsies in the surviving patient. RESULTS: Two siblings had situs inversus and complex heart defects suggestive of ciliary dysfunction. The affected girl who died in utero showed severe chest abnormalities compatible with Jeune syndrome which were not present in the affected boy. Dental investigations in the boy showed typical signs of molar-incisor-malformation. Exome sequencing identified a homozygous intragenic deletion in TCTEX1D2 which is predicted to completely remove protein function. Ciliary function tests and electron microscopy showed mild irregularities of motile cilia such as compound cilia and loss of membranes. CONCLUSIONS: Our findings support the suggestion that TCTEX1D2 mutations have variable expressivity and may be associated with disturbances of embryonic development caused by both, ciliary signaling and motile dysfunction. The presence of molar-incisor-malformation in the living patient raises the possibility of a pathogenetic link of this rare dental anomaly to ciliary dysfunction during tooth development at least in some individuals.

Surviving murine experimental sepsis affects the function and morphology of the inner ear.

Severe sepsis is known to result in various neurological long-term deficits in human. Recently, a link between severe, lethal sepsis and significant hearing loss with correlating histomorphological inner ear changes in mice (C57BL/6) was observed. However, if similar observations can be made in severe, non-lethal sepsis in mice is unclear. This study evaluates mice after severe, non-lethal sepsis for analogue functional and histomorphological alterations of the inner ear.A total of 63 C57BL/6 mice were included in the study. All underwent an initial hearing test with auditory brainstem response on day 1. In 35 mice sepsis was induced by cecal ligation and puncture (CLP), in 15 sham surgery was performed and 13 served as healthy control. A second hearing test was performed on day 7. All mice were sacrificed afterwards for further histomorphological evaluation of the inner ears. Immunohistochemical analysis with apoptotic markers Cleaved-caspase 3, BAX and BCL-2 were performed to identify structural inner ear damage.Of all CLP mice, 21/35 (60.0%) died due to the induced sepsis. Of the surviving CLP mice, 14/35 (40.0%), post-treatment hearing thresholds differed significantly from the sham and control mice (P<0.001). Positive immunostaining at different inner ear structures, like the spiral ligament or the supporting cells could be observed. The percentage of the immunostained positive area in the spiral ligament significantly correlated with the grade of hearing loss for BAX (P=0.027) and Cleaved-caspase 3 (P=0.024) but not for BCL 2 (P>0.05).The present data suggests that severe, non-lethal sepsis in mice results in significantly elevated hearing thresholds. A positive labelling for the pro-apoptotic markers BAX and Cleaved-caspase 3 suggested the induction of apoptosis in inner ear.

Supernumerary human hair cells-signs of regeneration or impaired development? A field emission scanning electron microscopy study.

BACKGROUND: Current attempts to regenerate cochlear sensorineural structures motivate further inspection of the human organ of hearing. Here, we analyzed the supernumerary inner hair cell (sIHC), a possible sign of regeneration and cell replacement. METHODS: Human cochleae were studied using field emission scanning electron microscopy (FESEM; maximum resolution 2 nm) obtained from individuals aged 44, 48, and 58 years with normal sensorineural pure-tone average (PTA) thresholds (PTA <20 dB). The wasted tissue was harvested during trans-cochlear approaches and immediately fixed for ultrastructural analysis. RESULTS: All specimens exhibited sIHCs at all turns except at the extreme lower basal turn. In one specimen, it was possible to image and count the inner hair cells (IHCs) along the cochlea representing the 0.2 kHz-8 kHz region according to the Greenwood place/frequency scale. In a region with 2,321 IHCs, there were 120 scattered one-cell losses or 'gaps' (5%). Forty-two sIHCs were present facing the modiolus. Thirty-eight percent of the sIHCs were located near a 'gap' in the IHC row (±6 IHCs). CONCLUSIONS: The prevalence of ectopic inner hair cells was higher than expected. The morphology and placement could reflect a certain ongoing regeneration. Further molecular studies are needed to verify if the regenerative capacity of the human auditory periphery might have been underestimated.

Molecular organization and fine structure of the human tectorial membrane: is it replenished?

Auditory sensitivity and frequency resolution depend on the physical properties of the basilar membrane in combination with outer hair cell-based amplification in the cochlea. The physiological role of the tectorial membrane (TM) in hair cell transduction has been controversial for decades. New insights into the TM structure and function have been gained from studies of targeted gene disruption. Several missense mutations in genes regulating the human TM structure have been described with phenotypic expressions. Here, we portray the remarkable gradient structure and molecular organization of the human TM. Ultrastructural analysis and confocal immunohistochemistry were performed in freshly fixed human cochleae obtained during surgery. Based on these findings and recent literature, we discuss the role of human TMs in hair cell activation. Moreover, the outcome proposes that the α-tectorin-positive amorphous layer of the human TM is replenished and partly undergoes regeneration during life.