Indiana University-Purdue University Indianapolis (IUPUI) / Our ability to hear relies on sensory cells found in the inner ear that transduce sound into biological signals. Microvilli-like protrusions called stereocilia are bundled on the apical surfaces of these cells and allow them to respond to sound-evoked vibrations. The architecture of the stereocilia bundle is highly patterned to ensure normal hearing. Filaments of polymerized actin proteins are bundled in parallel into large cylindrical structures that define the dimensions of stereocilia. This network is then anchored to the cell by inserting into another actin-based structure called the cuticular plate, which forms a gel-like structure and facilitates the mechanical properties of the bundle. The shape of the bundle is determined through tissue-level and intrinsic polarization signaling pathways. Auditory brainstem-evoked response testing, immunofluorescence imaging, scanning electron microscopy, and biochemical labeling techniques were used to study how the ADF/cofilin family of actin filament severing and depolymerizing proteins contributes to the development of the stereocilia bundle. Loss of these proteins disrupts the normal bundle patterning process, changes the lengths and widths of stereocilia, and alters the regulation of filament ends near the ion channel at stereocilia tips that is responsible for mechanotransduction. The activity of this channel regulates ADF/cofilins and the actin at stereocilia tips. Aberrant actin growth in actin networks beneath the stereocilia bundle influences the bundle patterning process, causes dysmorphic bundles to form. This work identifies that ADF/cofilins are necessary during auditory sensory cell development to facilitate normal bundle patterning and establishes this protein family as a molecular link between mechanotransduction and stereocilia bundle maturation.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13157号 / 論医博第2144号 / 新制||医||1028(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 影山 龍一郎, 教授 別所 和久, 教授 辻川 明孝 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Regulators of G-protein signaling (RGS) are a multifunctional and highly diverse group of proteins that negatively regulate G-protein coupled receptor (GPCR) signaling pathways. The common mechanism of RGS is to act as GTPase accelerating proteins (GAP) to accelerate the hydrolysis of active GTP bound G proteins and terminate the actions of the associated GPCR. In addition to the traditional function of inhibiting G-protein signaling, recent studies have highlighted the role of RGS proteins in modulating GPCRs in GAP-independent way. There are more than 30 RGS proteins, and depending upon cell/tissue type, they interact and associate with different G proteins and GPCRs to modulate various physiological functions. RGS17, a member of the RGS-RZ subfamily, commonly targets GTP bound Gαz, Gαi, and Gαo for hydrolysis and signal termination. RGS17 is abundantly expressed in the central nervous system and is highly associated with opioid, dopamine and cannabinoid receptors in the brain. RGS17 is also upregulated in many malignant tumors such as lung, prostate and breast cancers. Analysis of whole cochlea transcriptome data from our lab revealed higher levels of RGS17 in the cochlea after cisplatin treatment. This highlights a possible role of RGS17 (and probably other RGS proteins) in cisplatin ototoxicity. Activation of endogenous, otoprotective GPCRs such as adenosine (A1AR) and cannabinoid (CB2) receptor is beneficial for promoting protection against cisplatin-induced hearing loss. Taking all this together, the underlying hypothesis for this study is that cisplatin could possibly mediate ototoxicity by increasing the expression of RGS17, which reduces the otoprotective effect of endogenous receptors such as cannabinoid receptor 2. The main objective of the study is to examine the expression and function of RGS17 in the cochlea and determine if inhibition of RGS17 could protect against cisplatin ototoxicity.The expression of RGS17 was observed in the both in vitro and in vivo models of cochlear cell types. Immunofluorescence study, western blot analysis, and RT-qPCR results showed the presence of RGS17 in UB\OC-1, as well as Wistar rat cochlea; expression levels increased after cisplatin treatment. To determine the role of RGS17 in hearing, first, it was overexpressed in the cochlea using adenoviral vector that was found to significantly increase ABR threshold shifts and decrease ABR Wave I amplitude. Conversely, knockdown of RGS17 (by siRGS17) decreased cisplatin-induced elevations in ABR thresholds along with increased wave I amplitude and latency. Furthermore, siRGS17 pretreatment prevented cisplatin-mediated synapse loss at inner hair cells. This indicates inhibition of RGS17 can preserve the functional and physiological integrity of the cochlea, which is essential for hearing. Cochleae that were treated with siRGS17, followed by cisplatin, showed fewer TUNEL-positive cells and reduced loss of Outer hair cells (OHC) as compared to cisplatin-treated rats. Moreover, overexpression of RGS17 increased the ratio of the transcription factors, pSTAT1/pSTAT3, which may indicate initiation of the apoptotic pathway. Moreover, UB\OC-1 cells treated with Celastrol, a RGS17 inhibitor, showed an increase in cell viability against cisplatin toxicity. In addition to apoptosis, overexpression of RGS17 also elevated ROS production and oxidative stress. But, the inhibition of RGS17 attenuated the cisplatin-induced increase in transcripts for oxidative and inflammatory stress markers, such as NOX3, iNOS, KIM1, TNF-α, and COX2, whereas the mRNA level of antioxidant genes such as Nrf2 and SOD2 were increased. Activation of CB2 via JWH-015 (a CB2 agonist) prior to cisplatin administration significantly reduced the cisplatin-induced elevated levels of RGS17, while knockdown of CB2 increased RGS17 expression in the cochlea. siRGS17 treatment boosted endogenous CB2R-Gα expression. Additionally, cisplatin decreased the expression of Gαi/o and Gαz in vitro, but the activation of CB2 increased the expression of these G proteins. Furthermore, JWH-015 treatment alleviated RGS17-dependent cell death. This study suggests that RGS17 could serve as a mediator of cisplatin ototoxicity by reducing the duration of active CB2R-G protein signaling, which normally suppresses cochlear oxidative stress, inflammation and hair cell apoptosis, and thereby preserves normal hearing. These data also indicate the existence of tonic reciprocal inhibition between RGS17 and CB2 mediated via the G proteins. Thus, we propose that RGS17 inhibitors could serve as an effective treatment against cisplatin ototoxicity when used alone and can potentiate the actions of CB2 agonists when used in combination therapy against cisplatin-induced hearing loss.

Eine der häufigsten Behinderungen des alternden Menschen stellt die Beeinträchtigung der Hörfunktion durch degenerative Veränderungen der Cochlea, beispielsweise am Ganglion spirale, am CORTI-Organs oder an der Stria vascularis dar. Basierend auf histologischen und funktionellen Untersuchungen, wird die Altersschwerhörigkeit (Presbycusis) beim Menschen durch SCHUKNECHT und GACEK (1993) in sechs verschiedenene Typen eingeteilt: sensorisch, strial/metabolisch, neural, cochlear-konduktiv, gemischt, unbestimmt. Die in diesem Rahmen auftretenden pathophysiologischen und pathomorphologischen Veränderungen der Cochlea sind bedingt durch hörschädigende Faktoren (u.a. Lärm, ototoxische Substanzen, Infektionen sowie genetische Ursachen), die im Laufe des Lebens im Bereich des peripheren Hörorgans und der zentral auditiven Verarbeitung gewirkt haben. Die meisten in der Literatur beschriebenen Untersuchungen, die sich mit cochleären Alterationen mit zunehmendem Alter befassen, wurden an Labortieren unternommen und beschreiben oft nur Alterationen einzelner Strukturen, nicht aber der gesamten Cochlea. Bei Hunden sind in der Literatur überwiegend ausführliche Untersuchungen bezüglich der kongenitalen vererbten Taubheit, z.B. bei Dalmatinern, beschrieben. Es existieren jedoch kaum Studien über altersbedingte morphologische und funktionelle Veränderungen der Cochlea bei Hunden. Das Ziel dieser Arbeit besteht daher in der (immun-) histologischen Untersuchung der Cochlea von 50 Hunden im Alter von 7 Tagen bis 16 Jahren, um altersbedingte morphologische und funktionelle Veränderungen in der Cochlea von Hunden, die nicht unter Laborbedingungen gehalten wurden, zu charakterisieren und sie mit den Befunden an Innenohren von anderen Tierarten und des Menschen zu vergleichen. Anhand der erhobenen Befunde sollen anschließend eventuelle Rückschlüsse auf intra vitam vorhandene Hörstörungen gezogen werden.

Databases are an easy way of storing information kept together and accessible and can save time by finding the information easier. Cochleaimplant could be seen as a form of advanced hearing aid, this product consists of two parts, an external part that recieves sound and sends signals to the internal part through electromagnetism. The internal part is the implant itself and is surgically placed in to the cochlea. Örebro University hospital has just started working within the field of Cochleaimplants and are therefore in need of a database to store patient related information. The database problem was solved using Microsoft Access 2003 combined with the built-in support for Visual Basic, which is a sort of programming language. The result of the thesis work was a fully functioning database according to the given specification and is now being used at Örebro University hospital. / Databaser är ett smidigt sätt att lagra information samlat och lättåtkomligt och kan spara tid genom att informationen smidigare kan hittas. Cochleaimplantat kan ses som en slags avancerad hörapparat, denna hörapparat består av två delar, en extern del som tar emot ljud och skickar signaler vidare till den interna delen via elektromagnetism. Den interna delen är själva implantatet och sitter inopererat i hörselsnäckan(Cochlea). Universitetssjukhuset i Örebro har precis börjat arbeta inom området Cochleaimplantat och var därför i behov av en databas över patientinformation. Databasproblemet löstes med Microsoft Access 2003 kombinerat med det inbyggda stödet för programmeringsspråket Visual Basic. Resultatet av Examensarbetet blev en fungerande databas enligt uppdragsspecifikationen som just nu används på USÖ. / <p>Fredrik Reinholdsson: högskoleingenjörsexamen, Kaj Mählqvist: kandidatexamen</p>

Aus der Einleitung: "Die Ursachen für hochgradige Schwerhörigkeit bzw. Taubheit können unterschiedlich sein. Zum einen liegt eine angeborene Hörstörung bei einem von tausend Neugeborenen vor (Willenborg et al. 2009). Zum anderen kann eine erworbene Taubheit durch verschiedene Erkrankungen, wie z.B. Meningitis, Enzephalitis, Masern oder Tuberkulose hervorgerufen werden. Wenn dann die Nutzung von konventionellen Hörgeräten für ein ausreichendes Hörvermögen nicht mehr in Frage kommt, wird eine elektronische Innenohrprothese verwendet. Dieses sogenannte Cochlea-Implantat (CI) wandelt die akustischen Signale in elektronische Signale um und überträgt diese direkt auf den Hörnerv. Für die Implantation des Cochlea-Implantates muss der Chirurg einen komplizierten und risikobehafteten Eingriff durchführen. Dafür schafft er mit Hilfe verschiedener Fräsköpfe einen Zugang zur Hörschnecke (Cochlea), in der er dann einen Elektrodenträger einführt und genau platziert (siehe Abbildung 2). Entscheidend für eine erfolgreiche Operation sind vor allem das Geschick und die Erfahrung des Chirurgen. Für Ausbildungs- und Übungszwecke ist deshalb ein kontinuierliches Training notwendig."

La surdité est l'un des déficits sensoriels les plus fréquents dans nos sociétés industrialisées. Parmi les pathologies de l'audition, les surdités de perception ou neurosensorielles sont les plus répandues. Les surdités de perception sont dues à un dysfonctionnement de la cochlée impliquant l'homéostasie ionique, la perte des cellules sensorielles et des neurones ganglionnaires. Alors qu'une altération de l'homéostasie ou que la perte de cellules sensorielles entraine immanquablement la survenue d'une surdité, l'impact de la perte de neurones ganglionnaires est mal connu.L'objet de cette thèse était d'évaluer l'impact des pertes neuronales sur l'audition. Pour ce faire, nous avons développé un outil pharmacologique capable de créer une perte sélective de neurones auditifs primaires, sans endommager les structures pré-synaptiques telles que les cellules sensorielles et la strie vasculaire. Pour ce faire, nous avons appliqué des doses croissantes de ouabaïne sur la membrane de la fenêtre ronde chez la gerbille. Les tests électrophysiologiques (produits de distorsions acoustiques, potentiel endocochléaire et potentiel d'action composite du nerf auditif) ont été réalisés avant et 6 jours après l'application de ouabaïne. A la fin des explorations fonctionnelles, les cochlées étaient prélevées et préparées pour réaliser des évaluations morphologiques en microscopie confocale et en microscopie électronique à transmission.Jusqu'à 80 µM, la ouabaïne n'entrainait aucun changement significatif des produits de distorsions acoustiques ce qui reflétait le bon fonctionnement des cellules ciliées externes, ni du potentiel endocochléaire témoin du fonctionnement normal de la strie vasculaire. En revanche, les mêmes concentrations de ouabaïne provoquaient une diminution dose-dépendante de l'amplitude du potentiel d'action composite du nerf auditif, étroitement associée à une perte de neurones ganglionnaires et de synapses afférentes. Si l'amplitude du potentiel d'action composite du nerf auditif constitue un bon indicateur du nombre et de l'état fonctionnel des neurones ganglionnaires et des synapses afférentes, ce n'est donc pas le cas pour les seuils audiométriques. En effet, ce n'était qu'avec une perte de 75 % des synapses afférentes et supérieure à 55 % des neurones ganglionnaires, qu'une élévation des seuils audiométriques était observée, après une perfusion de 80 µM de ouabaïne. A 100 µM de ouabaïne, l'élévation des seuils auditifs résultait de la perte cumulée des cellules sensorielles et de l'altération de la strie vasculaire, se surajoutant aux dommages neuronaux et synaptiques.L'ensemble de nos résultats montrait que l'application de ouabaïne sur la membrane de la fenêtre ronde chez la gerbille constitue un excellent modèle pour étudier l'impact de la perte sélective des neurones ganglionnaires sur la fonction auditive. Il apparaît aussi nécessaire de développer des outils d'investigation plus précis que le simple audiogramme pour évaluer les pertes neuronales chez l'homme. / Deafness is one of the most frequent sensory deficits in our industrialized societies. Among the auditory pathologies, sensorineural deafness is the most wide-spread. Sensorineural deafness is due to a dysfunction of the cochlea involving the ionic homeostasis, loss of sensory cells and spiral ganglion neurons. While an alteration of the homeostasis or the loss of sensory cells induce inevitably the appearance of deafness, the impact of spiral ganglion neuron loss is unknown.The object of this thesis was to estimate the impact of spiral ganglion neuron losses on the auditory function. We developed a pharmacological tool capable of creating a selective loss of spiral ganglion neurons, without damaging the presynaptic structures such as the sensory cells and the stria vascularis. To do this, we applied increasing doses of ouabain to the round window membrane in the gerbil. Electrophysiological evaluations such as the distortion product otoacoustic emissions, the endocochlear potential and the compound action potentials of the cochlear nerve were recorded before and 6 days after application of ouabain. At the end of the functional evaluations, the cochlea were removed and prepared for morphological evaluations using confocal microscopy and transmission electron microscopy.Our results showed that up to a concentration of 80 µM, ouabain did not induce any significant change of the amplitude of the distortion product otoacoustic emissions, which indicated a normal functional state of the outer hair cells, nor of the endocochlear potential which reflected an intact stria vascularis. On the other hand, the same concentrations of ouabain led to a dose-dependent decrease of the amplitude of the compound action potentials, which was strictly associated with a loss of spiral ganglion neurons and afferent synapses, as assessed by morpho-anatomical analyses. If the amplitude of the compound action potentials constitutes a good indicator of the number and the functional state of the spiral ganglion neurons and the afferent synapses, it is not the case for the audiometric thresholds. Indeed, a loss of 75 % of afferent synapses and more than 55 % loss of the ganglion neurons was necessary before an elevation of the audiometric thresholds was observed in the cochleae perfused with 80 µM ouabain. At 100 µM ouabain, the elevation of the auditory thresholds may result from the accumulated loss of sensory cells, damage to the stria vascularis, in addition to the loss of the spiral ganglion neurons and afferent synapses. All these results indicate that the application of ouabain onto the round window membrane in the gerbil is an excellent model to study the impact of the selective loss of the spiral ganglion neurons on hearing function. More generally, this study points towards the necessity of developing more precise tools, beyond the simple audiogram, for the investigation of auditory neuron loss in humans.

A maioria das perdas auditivas adquiridas ou congênitas decorre de dano ou perda das células ciliares da cóclea ou dos seus neurônios associados. A irreversibilidade da surdez em mamíferos ocorre devido à incapacidade de substituição das células perdidas, seja por divisão celular ou por regeneração de células endógenas no epitélio da orelha interna. Com isso o objetivo deste trabalho foi obtenção de linhagens de células progenitoras do epitélio coclear de fetos de cães com 40 dias de gestação, colaborando com futuras pesquisas relacionadas a trabalhos de tratamento para surdez neurossensorial. Foram utilizados oito fetos caninos com idade compreendidos a 40 dias de gestação, nos quais, foi realizada uma dissecação no crânio, expondo a cóclea para a retirada do epitélio coclear, visando sua analise morfológica, e obtenção de suas células. Para analise morfológica do tecido colear realizou-se as técnicas macroscópica, microscópica e de imunohistoquímica. As células obtidas da cóclea foram fotodocumentadas, e submetidas às analises de método colorimétrico MTT (3-(4,5-Dimethylthiazol-2-&#1091;l)-2,5-Diphenyltetrazolium Bromide), análise do ciclo celular, análise da imunofenotipagem e da diferenciação celular. Em cultivo as células apresentaram formato fibroblastóide. Na caracterização imunofenotipica apresentaram marcação positiva para marcadores de células-tronco mesenquimais e de pluripotência e marcação negativa para células hematopoiéticas. Apresentaram ainda a capacidade de diferenciação para linhagens celulares osteogênicas, adipogênicas e condrogênicas. Essas análises sugeriram resultados satisfatórios na obtenção, quantificação e caracterização dessas células, as quais foram adquiridas a partir de células do epitélio coclear de feto de cão, as quais poderão constituir fontes de células a serem utilizadas na terapia celular da espécie canina destinada ao tratamento de surdez causada por lesões ou danos do epitélio coclear / Most acquired or congenital hearing loss results from damage or loss of hair cells of the cochlea or their associated neurons. The irreversibility of deafness in mammals is due to the lost cells replacement inability, either by cell division or by regeneration of endogenous cells in the epithelium of the inner ear. Therefore, the objective of this work was to increase knowledge about getting progenitor cell lines of the cochlear epithelium from dogs&rsquo; fetuses with 40 days of gestation, collaborating with future research related to treatment work for sensorineural deafness. Eight canine fetuses were used aged as mentioned above, in which, a dissection was performed in the skull, exposing the cochlea to the withdrawal of the cochlear epithelium, for their morphological analysis and cells obtainment. For morphological analysis of the cochlear tissue was held the macroscopic techniques, microscopy and immunohistochemistry. The cochlea cells obtained were photo documented and analyzed for the colorimetric method MTT ( 3- ( 4,5- dimethylthiazol -2- &#1091;l ) -2,5 - Diphenyltetrazolium Bromide), cell cycle analysis, immunophenotyping analysis and cell differentiation. In culture, the cells showed fibroblast format. In immunophenotype characterization, they presented positive staining for mesenchymal stem cell markers and pluripotency and negative marking for hematopoietic cells. They also exhibit the differentiation capacity for cell osteogenic lineages, adipogenic and chondrogenic. These analyzes suggested satisfactory results in obtainment, quantification and characterization of these cells, which were acquired from the dog fetal cells&rsquo; cochlear epithelium, which may be cells of fonts to be used in cellular therapy of canine species for the treatment of deafness caused by injury or damage to the cochlear epithelium

Thesis. 1976. Ph.D.--Massachusetts Institute of Technology. Dept. of Electrical Engineering and Computer Science. / Microfiche copy available in Archives and Engineering. / Bibliography: leaves 377-385. / Ph.D.

Acquiring the motions of the inner ear sensory tissues provides insight to how the cochlea works. For this purpose, Spectral Domain Optical Coherence Tomography (SDOCT) is an ideal tool as it has a penetration depth of several millimeters. SDOCT can not only image inside the cochlear partition, but also measure the sample structures’ simultaneous displacements. We customized a commercial Spectral Domain Optical Coherence Tomography system for such functions and detailed the software and hardware steps so this powerful system could be more accessible to auditory researchers. The cochlea is surrounded by bones and tissues, and damage to it would make it passive. For this reason, cochlear vibrometry measuring locations have been limited to either the basal or apical regions. That is why I fabricated a two-dimensional scanning SDOCT-based probe, to access more cochlear locations through a small hand-drilled hole. What is exciting about the probe is that an electrode can be attached to its side to acquire spatially and temporally coincident voltage and displacement data. This would help us better understand the cochlear mechano-electrical feedback process. Lastly, I investigated how the SDPM-reported displacement could be influenced by its neighboring signals and demonstrated this signal competition phenomenon experimentally and theoretically.