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We determined the relative expression levels of the receptors TrkA , TrkB , TrkC , and p75 NTR and ligands BDNF , NT-3 , NGF , and NT-4 with RNAseq analysis on fetal human inner ear samples, located TrkB and TrkC proteins, and quantified BDNF with in situ hybridization on histological sections between gestational weeks (GW) 9 to 19. Spiral ganglion neurons (SGNs) and satellite glia appear to be the main source of BDNF and synthesis peaks twice at GW10 and GW15-GW17. Tonotopical gradients of BDNF revert between GW8 and GW15 and follow a maturation and innervation density gradient in SGNs. NT-3/TrkC follows the same time course of expression as BDNF/TrkB . Immunostaining reveals that TrkB signaling may act mainly through satellite glia, Schwann cells, and supporting cells of Kölliker's organ, while TrkC signaling targets SGNs and pillar cells in humans. The NT-4 expression is upregulated when BDNF/NT-3 is downregulated, suggesting a balancing effect for sustained TrkB activation during fetal development. The mission of neurotrophins expects nerve fiber guidance, innervation, maturation, and trophic effects. The data shall serve to provide a better understanding of neurotrophic regulation and action in human development and to assess the transferability of neurotrophic regenerative therapy from animal models.

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.

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Steinacher, Chacko, Liu, Rask-Andersen, Bader, Dudas, Sergi, Dhanaseelan, Moreno, Glueckert, Hoermann and Schrott-Fischer.)

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.

Objective: Minimal invasive surgical techniques have gained high acceptance in thoracic surgery during the last 10 years. However, up to now, only scant information exists on chronic postoperative pain and discomfort in patients who underwent video-assisted thoracoscopy. Therefore, a retrospective study was performed with the aid of a self-reported questionnaire., Methods: Two hundred and thirteen patients (of whom 79 females) with a mean age of 48 (range 15-88) years were operated on for a total of 225 procedures. Thoracoscopy was performed for pneumothorax (n=70), pulmonary nodules (n=44), interstitial lung diseases (n=20), pleural effusion (n=20), and empyema (n=19). Various indications included therapeutic or diagnostic procedures in bullous disease, mediastinal tumors, carcinoma, inflammatory lung disease, hyperhidrosis mani and bronchiectasis., Results: Mean drainage time was 6.0+/-4.7 days and hospital stay 8.4+/-6.6 days. One patient died on the ninth postoperative day after lobectomy for bronchial carcinoma due to cardiac failure, five patients needed a short period of reintubation due to acute respiratory failure. In two patients, thoracoscopic reoperation was necessary for closure of bronchopleural fistula. The self-reported questionnaire was returned by 173 (81%) of all patients within a mean follow-up of 18 (3-38) months. More than half of the patients (53%) reported no thoracic pain as early as 2 weeks after the procedure. At 2 weeks after the operation, 13% of patients suffered from localized pain and 31% from diffuse discomfort. Twelve percent needed pain medication regularly, and 3% occasionally. At 6 months postoperatively, three quarters of the patients had no complaints, 5% suffered from scar pain, and 20% had diffuse chest discomfort. One year after the procedure, 86% of the patients had no complaints, 9% suffered from minimal pain, and 5% from moderate pain. Two years after the procedure, 96% of the patients had no complaints at all. One hundred and twenty-five of the 140 patients (89%) working preoperatively went back to work within 2 weeks after the operation. Fifteen patients did not work between 3 and 16 weeks; 14 due to chest pain, one due to shoulder pain., Conclusion: Video-assisted thoracoscopy permits very early recovery with rapid reintegration into the working process. Long-term complaints after videothoracoscopy are rare.