No description available.

No description available.

The vertebrate inner ear is a remarkable sensory organ, harboring two different senses: the auditory system, responsible for hearing, and the vestibular system, responsible for balance. Even though the anatomical structure of the vertebrate inner ear is very complex, only three different cell types are mainly involved on a cellular level in the perception of sound as well as balance and movement: sensory hair cells that are surrounded by supporting cells receive the stimulus and transfer it via sensory neurons to the brain. Worldwide, millions of people suffer from sensorineural hearing loss, caused by the loss of sensory hair cells and/or their innervating neurons within the inner ear. In mammals, including humans, both cell types are only produced during fetal stages making loss of these cells and the resulting consequences irreversible. In contrast, it is known that zebrafish produce sensory hair cells throughout life and additionally possess the remarkable capacity to regenerate them upon lesion. However, it is unknown whether new sensory neurons are also formed throughout life in the zebrafish statoacoustic ganglion (SAG), which transduces signals from the inner ear to the brain. Moreover, it is unknown whether sensory neurons are replaced upon loss. Hence, the first aim of this study was to investigate whether new sensory neurons are produced beyond larval stages. To this end, analysis of different transgenic lines combined with immunohistochemistry against known markers for neuronal stem and progenitor cells, neurons, glia and myelinating cells as well as markers for proliferation were used to identify distinct cell populations and anatomical landmarks in the juvenile and adult SAG. In the juvenile SAG, a pool of highly proliferating Neurod/Nestin-positive neuronal progenitors produces large amounts of new sensory neurons. In contrast, at adult stages this neurogenic niche transitions to a quiescent state, in which Neurod/Nestin-positive neuronal progenitor cells are no longer proliferating and the neurogenesis rate is very low. Moreover, BrdU pulse chase experiments revealed the existence of a proliferative but otherwise marker-negative cell population that replenishes the Neurod/Nestinpositive progenitor pool throughout life, indicating a neural stem cell-like cell population upstream of the neuronal progenitor cell pool. Additionally, expression of glia markers and a switch in the myelination pattern was found to mark the peripheral and central nervous system transitional zone (PCTZ) as a prominent landmark of the SAG. To further study the nature of the proliferating but otherwise unknown stem cell-like cell population replenishing the Neurod/Nestin-positive neuronal progenitor pool, the transcriptome of proliferating cells and their progeny of the juvenile and adult SAG was analyzed via single cell RNA-sequencing using the Smart-Seq2 technology. Therefore, a pipeline including preparation of the SAG as well as cell dissociation followed by fluorescence-activated cell sorting was established to obtain single cells from the SAG. The fluorescent reporters Tg(pcna:GFP) and Tg(nestin:mCherry-CreERT2) were used to label proliferating cells (GFP-only positive), proliferating progenitors (GFP/mCherry-double positive as well as nonproliferating progenitor cells (mCherry-positive). Additionally, based on the perdurance of the fluorophores in the progeny of the cells expressing the reporter constructs, this sorting strategy also enables to sort the progeny of proliferating cells differentiating into neuronal progenitor cells (GFP/mCherry-double positive but not expressing pcna) to trace back the putative stem cell-like cell population replenishing the Neurod/Nestin-positive progenitor population. Similar, the sorting strategy also included newborn neurons as the progeny of neuronal progenitors (mCherry-positive but not expressing nestin). In the transcriptome data obtained from the juvenile SAG, the majority of the analyzed cells could be assigned to the neuronal lineage, reflecting the neuronal differentiation trajectory from neuronal progenitor cells transitioning to newborn neurons and even further differentiating into mature neurons. Additionally, two different putative neuronal stem cell-like cell clusters were identified which are currently under validation. In contrast, in the adult transcriptome data the majority of cells were identified as cells from the sensory lineage, including cells expressing markers specific for hair cells and the sensory epithelium. Only a minority of cells came from the neuronal lineage, with the group of newborn and differentiating neurons clustering together in one cluster. Very few cells were identified as neuronal progenitor cells and did not cluster together, whereas both putative stem cell-like cell populations could be identified as distinct cluster. However, validation of the putative stem cell population remains subject to further studies. The second aim of this thesis was to investigate the regenerative capacity of the adult SAG and to study whether the neurogenic progenitor cell niche can be reactivated and to give rise to new sensory neurons upon damage. Therefore, a lesion paradigm using unilateral injections into the otic capsule was established. Upon lesion, mature SAG neurons undergo apoptosis and a massive infiltration with immune cells was found. Importantly, the Neurod-positive progenitor cells reentered the cell cycle displaying a peak in proliferation at 8 days post lesion before they returned to homeostatic levels at 57 days post lesion. In parallel to reactive proliferation, an increase in neurogenesis from the Neurod-positive progenitor pool was observed. Reactive neurogenesis started at around 4 days post lesion, peaked at 8 days post lesion decreased again to low homeostatic levels at 57 days post lesion. The administration of the thymidine analog BrdU to label proliferating cells and their progeny revealed the generation of new sensory neurons from proliferating neuronal progenitor cells within 19 days post lesion. Interestingly, reactive proliferation as well as an increased neurogenesis rate were also detected in the unlesioned SAG, revealing a systemic effect of the unilateral lesions. Taken together, this study is the first to show that neurogenesis in the zebrafish SAG persists way beyond larval stages. New neurons descend from a population of Neurod/Nestin-positive neuronal progenitor cells that is highly proliferative during juvenile stages but turn quiescent at adulthood. Nevertheless, this neuronal progenitor cell pool is replenished throughout life by a currently unknown neuronal stem cell-like cell population. Additional this study reveals the regenerative capacity of the adult SAG: upon lesion Neurod/Nestin-positive progenitor cells are reactivated to re-enter the cell cycle, proliferate and give rise to new neurons leading to an increased neurogenesis rate to replace lost mature neurons. Studying the underlying genes and pathways in zebrafish compared to mammalian species will hopefully provide valuable insights that will help developing cures for auditory and vestibular neuropathies in the future.

When the cochlea is stimulated with two primary tones (f1 and f2) some of the energy is reflected back and propagates via the middle ear into the outer ear. Due to cochlear nonlinearities, distortion product otoacoustic emissions (DPOAEs), may be detected by a probe microphone sealed in the ear canal. Reduced DPOAEs may indicate subclinical cochlear lesions. The relationship between hearing sensitivity and the strength of DPOAEs is debatable, especially in the extended high frequency (EHF) region (≥8 kHz). Monitoring cochlea function corresponding to the EHF range is important for detecting early stages of hearing loss, which typically begins above 8 kHz. Complex interactions of high-frequency pure-tones in the ear canal result in standing waves that increases test-retest variability of DPOAEs measured for f2≥6 kHz. The aim of the project was to evaluate reliability of DPOAEs measured up to 12 kHz with a system used routinely in audiology clinics. Thirty-one adults (age, 18-30 yrs) with normal middle-ear function and normal hearing thresholds in the conventional region (≤8 kHz) participated. The EHF audiometry was performed for frequencies up to 16 kHz. The DPOAE data were collected for the f2 frequency varied from 1.5 to 12 kHz, twice for each ear with the probe removed and then repositioned after the first test. The EHF audiometric data of four participants showed elevated thresholds. Their DPOAEs were reduced or absent for f2≥9 kHz, i.e., supporting the sensitivity of DPOAEs for cochlear hearing loss above the conventional audiometry frequency range. Mean and standard deviations of DPOAE levels were calculated separately for the left and the right ears of subjects with normal EHF thresholds. There were no differences between mean DPOAE values in the left and the right ears. The intersubject variability of the DPOAE levels was moderate (SD≈6 dB or lower) but it increased significantly in the 12-kHz region, per the F-test for variances, possibly due to 1. effects of standing waves on the high-frequency DPOAE reliability and/or 2. subclinical pathology in the most basal portion (i.e., high-frequency region) of the cochlea. For each ear, absolute values of differences between test/retest levels of detectable DPOAEs were calculated. ANOVA showed the main effect of frequency for the data collected in the left and the right ears. Post-hoc analyses indicated that test/retest variability of DPOAEs was rather constant for f2 frequencies up to 10 kHz, but a statistically significant increase of test/retest variability for f2 of 11 and 12 kHz was found. This aspect needs to be considered when using DPOAE tests for longitudinal monitoring of cochlear function in the basal portion. Nevertheless, combining behavioral thresholds with DPOAEs collected for the EHF range is vital for detecting the initial stage of the cochlear pathology corresponding to the high-frequency region, e.g., due to ototoxicity or aging of the cochlea.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23750号 / 医博第4796号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村川 泰裕, 教授 斎藤 通紀, 教授 藤渕 航 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Die Optimierung der Akquisitionstechniken führte zu einer verbesserten Abgrenzbarkeit klinisch relevanter Bilddetails und war gleichzeitig Voraussetzung für die Erzeugung bildqualitativ hochwertiger dreidimensionaler Bildrekonstruktionen. Die Kombination aus hochauflösenden Akquisitionstechniken und der dreidimensionalen Visualisierung mittels Volume Rendering ermöglichte die nicht-invasive endoluminale Darstellung im Sinne einer virtuellen Oto- bzw. Neuroendoskopie. Trotz weitgehender Übereinstimmung zwischen der realen und virtuell dargestellten Pathomorphologie ossikulärer Strukturen in der Mittelohr-Bildgebung zeigten sich methodische Limitationen in der Erfassung der Pathophysiologie. Die 3D-Bildgebung des Innenohrs gewährleistete eine detaillierte Darstellung pathoanatomisch komplexer Veränderungen und erlaubte dadurch Rückschlüsse auf die Pathogenese. Insbesondere in der Bildgebung des Mittelohrs und Kleinhirn-Brückenwinkels erwiesen sich die virtuell-endoskopischen Ansichten als komplementäre Darstellungstechniken, deren Ergebnisse im Zusammenhang mit den primären Schnittbilddaten und den klinischen Untersuchungsergebnissen zu interpretieren sind. Prospektiv ist aufgrund neuer Gerätetechnologien in der Bilddaten-Erfassung (z.B. 16-Schicht-Spiral-CT) sowie höheren Rechnerleistungen in der Daten-Nachverarbeitung von einer zunehmenden Verbreitung dreidimensionaler Bildgebungstechniken auszugehen. / Optimized data acquisition resulted in an improved delineation of critical image elements and was a prerequisite for generating 3D reconstructions of high image quality. Combining high-resolution data acquisition and 3D visualization by means of volume rendering allowed for the noninvasive endoluminal assessment known as virtual oto- and neuroendoscopy. Despite a high degree of agreement between the intraoperatively verified ossicular pathomorphology and that visualized by virtual otoscopy in middle ear imaging methodological limitations were encountered with respect to pathophysiological evaluation. 3D imaging provided detailed views of even complex pathoanatomical conditions, thus permitting conclusions regarding the underlying pathogenesis. As far as imaging of the middle ear and cerebellopontine angle was concerned, virtual endoscopic views proved to be complementary visualization techniques whose results have to be interpreted in the context of the primary cross-sectional data and clinical findings. Prospectively, a more widespread use of 3D imaging techniques may be expected from upcoming acquisition technologies (for example 16-slice helical CT) as well as from more powerful computers with improved image data postprocessing capacities.

Growth hormone (GH) was upregulated in the zebrafish inner ear following sound exposure in a previous study. To identify the specific role of GH in hair cell regeneration and the possible cellular mechanisms of this regeneration, groups of zebrafish were divided into baseline (no sound exposure, no injection), buffer-injected and GH-injected groups. Buffer- and GH-injected fish were exposed to a 150 Hz tone at a source level of 179 dB re 1 μPa root mean squared (RMS) for 36 h. Phalloidin-staining was used to assess the effects of GH on hair cell bundle density; BrdU-labeling was used to assess the effects of GH on cellular proliferation; TUNEL-labeling was used to assess the effects of GH on apoptosis in the zebrafish inner ear following acoustic trauma. The time-course of hair cell bundle density, cell proliferation, and apoptosis was established by combining data for baseline fishes and sound-exposed fishes at post-sound exposure day 1 (psed1), psed2, and psed3. GH-injected fish exhibited greater densities of hair cells than bufferinjected controls. In addition, GH-injected fish had higher levels of cell proliferation and lower levels of apoptosis than buffer-injected controls. This suggests that GH may play an important role in zebrafish inner ear hair cell regeneration by stimulating cellular proliferation and inhibiting cellular apoptosis.

A hearing impairment is primarily diagnosed by measuring the hearing thresholds at a range of auditory frequencies (air-conduction audiometry). Although this clinical procedure is simple, affordable, reliable and fast, it does not offer differential information about origins of the hearing impairment. The main goal of this thesis is to quantitatively link specific cochlear pathologies to certain changes in the spectral and temporal characteristics of the auditory system. This can help better understand the underlying mechanisms associated with sensorineural hearing impairments, beyond what is shown in the audiogram. Here, an electromechanical signal-transmission model is devised in MATLAB where the parameters of the model convey biological interpretations of mammalian cochlear structures. The model is exploited to simulate the cell-level cochlear pathologies associated with two common types of sensorineural hearing impairments, 1: presbyacusis (age-related hearing impairment) and, 2: noise-induced hearing impairment. Furthermore, a clinical study, consisting of different psychoacoustic and physiological tests, was performed to trace and validate the model predictions in human. The results of the clinical tests were collated and compared with the model predictions, showing a reasonable agreement. In summary, the present model provides a biophysical foundation for simulating the effect of specific cellular lesions, due to different inner-ear diseases and external insults, on the entire cochlear mechanism and thereby on the whole auditory system. This is a multidisciplinary work in the sense that it connects the ‘biological processes’ with ‘acoustic modelling’ and ‘clinical audiology’ in a translational context.

Current technology used to diagnose hearing disorders is limited. This is mostly due to the fact that the auditory structures are very small and not easily accessible with existing imaging technologies. The objective of this dissertation was to investigate the potential of high-frequency ultrasound as a tool for exploring the anatomy of the auditory system. Three studies were conducted in order to demonstrate the feasibility of high-frequency ultrasound as a diagnostic technology for hearing disorders. In the first study, an in-house developed 50 MHz annular array-based ultrasound system was used to provide ex-vivo images of auditory structures in cadaveric temporal bones. It was shown that the spatial resolution was sufficient to visualize a high level of detail of the ossicular bones of the middle ear as well as intra-cochlear structures of the inner ear. In the second study, a 50 MHz 1.26? pitch phased array ultrasound transducer was designed for imaging intra-cochlear structures through the round window membrane. As this element pitch results in large grating lobe artifacts, novel transmit beamforming techniques were developed to suppress grating lobes resulting from this large-pitch array. Theoretical techniques using the impulse-response simulation method and experimental verification using high-frequency linear array ultrasound system (Vevo 2100, VisualSonics, Canada) showed that these techniques were able to suppress grating lobe levels up to 40 dB. In the third study, a needle mounted 45 MHz single-element ultrasound probe was fabricated in order to measure the vibrations of intra-cochlear structures on human cadavers. Basilar membrane velocimetry measurements were successfully performed using pulsed-wave Doppler ultrasound in the frequency range between 100 Hz-2 KHz. The measured velocity of the basilar membrane and the round window membrane showed that the middle ear resonance frequency near 1 KHz was present over multiple temporal bones. This is the first work that has explored the human auditory system with high resolution ultrasonic visualization and Doppler velocimetry.

Les cadres temporel, géographique et environnemental associés à l'émergence et l'évolution des premiers homininés en Afrique du Sud pendant le Plio-Pléistocène constituent des éléments clés dans la compréhension de l'histoire de la lignée humaine. Les cercopithécoïdes, relativement abondants dans les dépôts fossilifères du Néogène africain et quasi systématiquement associés aux restes d'homininés, représentent des marqueurs biochronologiques et des indicateurs écologiques sensibles. Toutefois, en se basant seulement sur les critères morphologiques traditionnels, la systématique du registre cercopithécoïde fossile reste encore incertaine. Afin de mieux caractériser leur paléobiodiversité, nous avons détaillé par des techniques d'imagerie à haute résolution les restes crânio-dentaires de 91 spécimens cercopithécoïdes provenant des sites de Makapansgat, Taung, Sterkfontein, Kromdraai et Swartkrans. En particulier, nous avons mesuré et comparé les variations dans l'organisation endostructurale dentaire, la conformation du labyrinthe osseux et l'architecture endocrânienne à travers l'application de méthodes statistiques et de modélisation virtuelle (e.g., morphométrie géométrique, modèles déformables). À titre comparatif, nous avons également intégré dans nos analyses un échantillon de 80 spécimens cercopithécoïdes actuels. En plus de l'identification de caractères nouveaux et pertinents pour la diagnose des taxons fossiles, nos résultats mettent en évidence des biomarqueurs utiles pour la reconstruction des contextes paléoenvironnementaux et biochronologiques, notamment en ce qui concerne la morphologie de la jonction émail-dentine et l'architecture néocorticale. L'estimation de la paléobiodiversité révèle un certain degré d'homogénéité morphologique pour quelques taxons papionines qui plaide en faveur d'une réduction du nombre d'espèces effectivement présentes dans les gisements fossilifères sud-africains. / The temporal, geographical and environmental frameworks of the early hominin emergence and evolution in South Africa during the Plio-Pleistocene are key elements for the understanding of the human lineage history. The cercopithecoids, relatively abundant in the African Neogene fossiliferous deposits and almost invariably associated with hominin remains, represent sensitive biochronological markers and ecological indicators. However, based on traditional morphological criteria only, the systematics of the cercopithecoid fossil record remains uncertain. In order to better characterize their paleobiodiversity, by means of high-resolution imaging techniques we have detailed 91 South African cercopithecoid craniodental specimens from the sites of Makapansgat, Taung, Sterkfontein, Kromdraai and Swartkrans. More specifically, we measured and compared the variation in dental endostructural organization, bony labyrinth conformation and endocranial architecture through the application of statistical and virtual modeling methods (e.g., geometric morphometrics, deformation-based models). For comparative purposes, we also integrated in our analyses a sample consisting of 80 extant cercopithecoid specimens. Besides the identification of some new characters suitable for the diagnosis of fossil taxa, our results reveal biomarkers useful for the reconstruction of paleoenvironmental and biochronological contexts, notably based on the enamel-dentine junction morphology and the neocortical architecture. The paleobiodiversity assessment highlights a certain degree of morphological homogeneity for some of the papionin taxa and suggests a reduction of the number of species actually present in the South African fossiliferous deposits.