京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19592号 / 医博第4099号 / 新制||医||1014(附属図書館) / 32628 / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 伊達 洋至, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Objectifs: Le Nystatin est un antibiotique efficace pour le traitement d’otomycose. Bien que sa sécurité au niveau de l’oreille externe soit bien établie, son utilisation n’est pas recommandée lorsqu’il y a une perforation tympanique. L’objectif de cette étude est d’évaluer le potentiel ototoxique du Nystatin lorsque celui-ci est appliqué directement au niveau de l’oreille moyenne. Méthodes: Nous avons fait une étude expérimentale avec 18 cochons d’Indes de souche Hartley que nous avons divisés en deux groupes. En exposant l’oreille moyenne de chaque animal au Nystatin (groupe I) ou à la néomycine (groupe II) et chaque oreille controlatérale à une solution physiologique (NaCl), la fonction auditive a été évaluée avec un test de potentiels évoqués auditif du tronc cérébral avant et après les injections. Une étude par microscopie électronique a permis une comparaison histologique de l’état des cellules ciliées cochléaires entre les 2 groupes. Résultats: Les pertes auditives moyennes du groupe « Nystatin » étaient de 13.0 dB et comparables aux pertes moyennes observées dans les oreilles ayant été injectées avec du NaCl (4.0 dB dans le groupe I et 15.1 dB dans le groupe II). Le groupe de contrôle « néomycine » a subi une perte auditive moyenne de 39.3 dB, ce qui représente une différence cliniquement et statistiquement significative (p<0.001). L’étude histologique avec une microscopie à balayage électronique a démontré une conservation de l’architecture des cellules ciliées cochléaires dans les groupe Nystatin et NaCl. La néomycine a causé une destruction marquée de ces structures. Conclusions: Le Nystatin ne provoque pas d’atteinte auditive ni de destruction des cellules ciliées externes après injection directe dans l’oreille moyenne chez le cochon d’Inde. / Objective: Nystatin is an effective topical antifungal agent widely used in the treatment of otomycosis. Though it is safe for external ear use, current recommendations are to avoid its use in cases of tympanic membrane perforation. The objective of our study was to test the security of Nystatin when applied directly to the middle ear of a guinea pig model. Methods: We performed an experimental study with 18 Hartley guinea pigs that were divided into two groups. Exposing middle ears from one group to Nystatin (group I) and from the other to the ototoxic neomycin (group II), we compared results of auditory brainstem response (ABR) testing at three intervals during the study. Each animal’s contralateral ear was injected with a physiological solution (NaCl). At the end of the study, we performed a histological analysis of the animals’ cochleae using a scanning electron microscope. Results: Average hearing loss in the Nystatin group was 13.0 dB which was similar to the results obtained in the NaCl-exposed ears (4.0 dB in group I and 15.1 dB in group II). Average hearing loss in the neomycin group was 39.3 dB, which represents a clinically significant difference (p<0.001). Scanning electron microscope evaluation revealed intact cochlear hair cell architecture in the Nystatin and normal saline groups, compared to important destruction in the neomycin group. Conclusion: Nystatin does not cause hearing impairment or cochlear hair cell damage when exposed directly to the middle ear of a guinea pig model.

Rápidas adaptações endometriais ocorrem com a chegada do embrião no útero para criar um ambiente uterino receptivo, que é essencial para o desenvolvimento do concepto. As descrições dos eventos endometriais que ocorrem antes da chegada do embrião ao útero, são escassas. O objetivo deste experimento foi demonstrar mudanças na arquitetura e na estrutura do endométrio que ocorrem no dia 5 pós-ovulação em éguas cíclicas e inseminadas. Amostras de biopsia endometrial foram recolhidas em torno do dia 5 pós-ovulação em um grupo de 10 éguas reprodutivamente sadias, uma de cada corno, durante dois ciclos. No primeiro como ‘Cíclicas’ (n = 10) e no seguinte estas mesmas foram inseminadas para formar o grupo ‘Inseminadas’ (n = 10). As éguas inseminadas foram subdivididas em dois subgrupos: aquelas em que as amostras foram recolhidas antes do dia 5.5 e aquelas em que as amostras o foram depois do dia 5.5. As biopsias endometriais foram analisadas por microscopia de luz e microscopia eletrônica de varredura. A densidade, o diâmetro, o epitélio, o lúmen e a secreção glandular, foram submetidos à análise histomorfométrica. As células brancas foram avaliadas em uma contagem diferencial. As células ciliadas, poligonais com microvilosidades, e a presença de células planas ou ingurgitadas no epitélio, na microscopia eletrónica de varredura, foram analisados por uma contagem individual de cada um dos tipos celulares. Houve um aumento no diâmetro glandular, uma diminuição significativa nas células ciliadas e um aumento significativo na população de linfócitos nas éguas inseminadas, quando comparadas com as égua cíclicas. Na análise dos subgrupos de éguas inseminadas, não houve diferenças nas variáveis. Estes resultados levam a concluir que mudanças significativas ocorrem no endométrio no dia 5 pós ovulação, na égua inseminada. / Rapid endometrial adaptations occur as the embryo enters the uterus to create a receptive uterine environment, which is essential for the conceptus' development. Descriptions of endometrial events before the embryo reaches the uterus, are scant The aim of this experiment was to demonstrate architectural and structural changes in the endometrium on day 5 after ovulation in cyclic and inseminated mares. Endometrial biopsy samples were taken during day 5 post-ovulation from a group of 10 reproductively healthy mares, one from each uterine horn, during two cycles. The first one they were considered as ‘cyclic’ (n = 10), and in the following one, the same mares were inseminated to constitute the ‘inseminated’ group (n = 10). Inseminated mares were subdivided into two subgroups: those sampled before day 5.5 and those sampled after day 5.5. Biopsy samples were analyzed through optic microscopy and scanning electron microscopy. Glandular density, diameter, epithelial height, lumen, and secretion were analyzed through histomorphometry. White blood cells were evaluated through a differential cell count. Ciliated cells, micro-ciliated polygonal cells, and flat or protruded cells over the epithelium, on scanning electron microscopy, were analyzed through an individual cell type count. Inseminated mares presented an increase in glandular diameter, significant decrease in ciliated cell population, and a significant increase in lymphocyte population, compared to cyclic mares. No differences in any of the variables were detected between subgroups from inseminated mares. The results here presented lead to the conclusion that significant endometrial changes occur on day 5 post-ovulation in inseminated mares.

Objectifs: Le Nystatin est un antibiotique efficace pour le traitement d’otomycose. Bien que sa sécurité au niveau de l’oreille externe soit bien établie, son utilisation n’est pas recommandée lorsqu’il y a une perforation tympanique. L’objectif de cette étude est d’évaluer le potentiel ototoxique du Nystatin lorsque celui-ci est appliqué directement au niveau de l’oreille moyenne. Méthodes: Nous avons fait une étude expérimentale avec 18 cochons d’Indes de souche Hartley que nous avons divisés en deux groupes. En exposant l’oreille moyenne de chaque animal au Nystatin (groupe I) ou à la néomycine (groupe II) et chaque oreille controlatérale à une solution physiologique (NaCl), la fonction auditive a été évaluée avec un test de potentiels évoqués auditif du tronc cérébral avant et après les injections. Une étude par microscopie électronique a permis une comparaison histologique de l’état des cellules ciliées cochléaires entre les 2 groupes. Résultats: Les pertes auditives moyennes du groupe « Nystatin » étaient de 13.0 dB et comparables aux pertes moyennes observées dans les oreilles ayant été injectées avec du NaCl (4.0 dB dans le groupe I et 15.1 dB dans le groupe II). Le groupe de contrôle « néomycine » a subi une perte auditive moyenne de 39.3 dB, ce qui représente une différence cliniquement et statistiquement significative (p<0.001). L’étude histologique avec une microscopie à balayage électronique a démontré une conservation de l’architecture des cellules ciliées cochléaires dans les groupe Nystatin et NaCl. La néomycine a causé une destruction marquée de ces structures. Conclusions: Le Nystatin ne provoque pas d’atteinte auditive ni de destruction des cellules ciliées externes après injection directe dans l’oreille moyenne chez le cochon d’Inde. / Objective: Nystatin is an effective topical antifungal agent widely used in the treatment of otomycosis. Though it is safe for external ear use, current recommendations are to avoid its use in cases of tympanic membrane perforation. The objective of our study was to test the security of Nystatin when applied directly to the middle ear of a guinea pig model. Methods: We performed an experimental study with 18 Hartley guinea pigs that were divided into two groups. Exposing middle ears from one group to Nystatin (group I) and from the other to the ototoxic neomycin (group II), we compared results of auditory brainstem response (ABR) testing at three intervals during the study. Each animal’s contralateral ear was injected with a physiological solution (NaCl). At the end of the study, we performed a histological analysis of the animals’ cochleae using a scanning electron microscope. Results: Average hearing loss in the Nystatin group was 13.0 dB which was similar to the results obtained in the NaCl-exposed ears (4.0 dB in group I and 15.1 dB in group II). Average hearing loss in the neomycin group was 39.3 dB, which represents a clinically significant difference (p<0.001). Scanning electron microscope evaluation revealed intact cochlear hair cell architecture in the Nystatin and normal saline groups, compared to important destruction in the neomycin group. Conclusion: Nystatin does not cause hearing impairment or cochlear hair cell damage when exposed directly to the middle ear of a guinea pig model.

Rápidas adaptações endometriais ocorrem com a chegada do embrião no útero para criar um ambiente uterino receptivo, que é essencial para o desenvolvimento do concepto. As descrições dos eventos endometriais que ocorrem antes da chegada do embrião ao útero, são escassas. O objetivo deste experimento foi demonstrar mudanças na arquitetura e na estrutura do endométrio que ocorrem no dia 5 pós-ovulação em éguas cíclicas e inseminadas. Amostras de biopsia endometrial foram recolhidas em torno do dia 5 pós-ovulação em um grupo de 10 éguas reprodutivamente sadias, uma de cada corno, durante dois ciclos. No primeiro como ‘Cíclicas’ (n = 10) e no seguinte estas mesmas foram inseminadas para formar o grupo ‘Inseminadas’ (n = 10). As éguas inseminadas foram subdivididas em dois subgrupos: aquelas em que as amostras foram recolhidas antes do dia 5.5 e aquelas em que as amostras o foram depois do dia 5.5. As biopsias endometriais foram analisadas por microscopia de luz e microscopia eletrônica de varredura. A densidade, o diâmetro, o epitélio, o lúmen e a secreção glandular, foram submetidos à análise histomorfométrica. As células brancas foram avaliadas em uma contagem diferencial. As células ciliadas, poligonais com microvilosidades, e a presença de células planas ou ingurgitadas no epitélio, na microscopia eletrónica de varredura, foram analisados por uma contagem individual de cada um dos tipos celulares. Houve um aumento no diâmetro glandular, uma diminuição significativa nas células ciliadas e um aumento significativo na população de linfócitos nas éguas inseminadas, quando comparadas com as égua cíclicas. Na análise dos subgrupos de éguas inseminadas, não houve diferenças nas variáveis. Estes resultados levam a concluir que mudanças significativas ocorrem no endométrio no dia 5 pós ovulação, na égua inseminada. / Rapid endometrial adaptations occur as the embryo enters the uterus to create a receptive uterine environment, which is essential for the conceptus' development. Descriptions of endometrial events before the embryo reaches the uterus, are scant The aim of this experiment was to demonstrate architectural and structural changes in the endometrium on day 5 after ovulation in cyclic and inseminated mares. Endometrial biopsy samples were taken during day 5 post-ovulation from a group of 10 reproductively healthy mares, one from each uterine horn, during two cycles. The first one they were considered as ‘cyclic’ (n = 10), and in the following one, the same mares were inseminated to constitute the ‘inseminated’ group (n = 10). Inseminated mares were subdivided into two subgroups: those sampled before day 5.5 and those sampled after day 5.5. Biopsy samples were analyzed through optic microscopy and scanning electron microscopy. Glandular density, diameter, epithelial height, lumen, and secretion were analyzed through histomorphometry. White blood cells were evaluated through a differential cell count. Ciliated cells, micro-ciliated polygonal cells, and flat or protruded cells over the epithelium, on scanning electron microscopy, were analyzed through an individual cell type count. Inseminated mares presented an increase in glandular diameter, significant decrease in ciliated cell population, and a significant increase in lymphocyte population, compared to cyclic mares. No differences in any of the variables were detected between subgroups from inseminated mares. The results here presented lead to the conclusion that significant endometrial changes occur on day 5 post-ovulation in inseminated mares.

Rápidas adaptações endometriais ocorrem com a chegada do embrião no útero para criar um ambiente uterino receptivo, que é essencial para o desenvolvimento do concepto. As descrições dos eventos endometriais que ocorrem antes da chegada do embrião ao útero, são escassas. O objetivo deste experimento foi demonstrar mudanças na arquitetura e na estrutura do endométrio que ocorrem no dia 5 pós-ovulação em éguas cíclicas e inseminadas. Amostras de biopsia endometrial foram recolhidas em torno do dia 5 pós-ovulação em um grupo de 10 éguas reprodutivamente sadias, uma de cada corno, durante dois ciclos. No primeiro como ‘Cíclicas’ (n = 10) e no seguinte estas mesmas foram inseminadas para formar o grupo ‘Inseminadas’ (n = 10). As éguas inseminadas foram subdivididas em dois subgrupos: aquelas em que as amostras foram recolhidas antes do dia 5.5 e aquelas em que as amostras o foram depois do dia 5.5. As biopsias endometriais foram analisadas por microscopia de luz e microscopia eletrônica de varredura. A densidade, o diâmetro, o epitélio, o lúmen e a secreção glandular, foram submetidos à análise histomorfométrica. As células brancas foram avaliadas em uma contagem diferencial. As células ciliadas, poligonais com microvilosidades, e a presença de células planas ou ingurgitadas no epitélio, na microscopia eletrónica de varredura, foram analisados por uma contagem individual de cada um dos tipos celulares. Houve um aumento no diâmetro glandular, uma diminuição significativa nas células ciliadas e um aumento significativo na população de linfócitos nas éguas inseminadas, quando comparadas com as égua cíclicas. Na análise dos subgrupos de éguas inseminadas, não houve diferenças nas variáveis. Estes resultados levam a concluir que mudanças significativas ocorrem no endométrio no dia 5 pós ovulação, na égua inseminada. / Rapid endometrial adaptations occur as the embryo enters the uterus to create a receptive uterine environment, which is essential for the conceptus' development. Descriptions of endometrial events before the embryo reaches the uterus, are scant The aim of this experiment was to demonstrate architectural and structural changes in the endometrium on day 5 after ovulation in cyclic and inseminated mares. Endometrial biopsy samples were taken during day 5 post-ovulation from a group of 10 reproductively healthy mares, one from each uterine horn, during two cycles. The first one they were considered as ‘cyclic’ (n = 10), and in the following one, the same mares were inseminated to constitute the ‘inseminated’ group (n = 10). Inseminated mares were subdivided into two subgroups: those sampled before day 5.5 and those sampled after day 5.5. Biopsy samples were analyzed through optic microscopy and scanning electron microscopy. Glandular density, diameter, epithelial height, lumen, and secretion were analyzed through histomorphometry. White blood cells were evaluated through a differential cell count. Ciliated cells, micro-ciliated polygonal cells, and flat or protruded cells over the epithelium, on scanning electron microscopy, were analyzed through an individual cell type count. Inseminated mares presented an increase in glandular diameter, significant decrease in ciliated cell population, and a significant increase in lymphocyte population, compared to cyclic mares. No differences in any of the variables were detected between subgroups from inseminated mares. The results here presented lead to the conclusion that significant endometrial changes occur on day 5 post-ovulation in inseminated mares.

La neteja del moc i dels patògens dels pulmons, i el transport de gàmets i embrions en els òrgans reproductius de les femelles són funcions clau en els epitelis ciliats, tals com aquells que es troben presents en les vies respiratòries i l'oviducte. La taxa de transport mucociliar és funció de la freqüència de batut ciliar (CBF) i aquesta freqüència és augmentada per increments en la concentració de Ca2+ intracelul·lar. El canal catiònic "transient potential vanilloid 4" (TRPV4) intervé en l'entrada de Ca2+ en resposta a estímuls mecànics i osmòtics. L'expressió del TRPV4 en l'epiteli ciliat de les vies respiratòries i de l'oviducte és confirmada mitjançant la localització per immunofluorescència del canal iònic a la membrana apical de l'epiteli ciliat i polaritzat, allà on la senyalització de Ca2+ és requerida per la regulació de la CBF. Cèl·lules ciliades de la tràquea de ratolins TRPV4-/- no expressen el canal TRPV4, no responen a l'activador específic del TRPV4, el 4&#945;-phorbol 12,13-didecanoate (4&#945;-PDD) i presenten respostes de Ca2+ reduïdes a temperatures mitjanes (~25ºC- 8ºC), un altre estímul dels canals TRPV4. L'activació dels canals TRPV4 per solucions altament viscoses i per hypotonicitat depèn de l'activació de la via de la fosfolipasa A2(PLA2)i la subseqüent producció de àcid epoxieicosatrienoic (EET). En condicions de baixa activació de la PLA2, estímuls mecànics i hipotònics alliberen ATP per a l'activació de la via de la fosfolipasa C (PLC)-inositol trifosfat (IP3) per contribuir a l'activació dels canals TRPV4. Descrivim que el metabòlit IP3 sense ser un agonista per ell mateix, sensibilitza el TRPV4 per a l'activació de EET, essent aquest un mecanisme general. L'acoblament funcional entre els canals TRPV4 de la membrana plasmàtica i els receptors de IP3 (IP3R) és necessari tant per iniciar com mantenir la senyalització oscil·latòria del Ca2+ desencadenada per estímuls viscosos i hipotònics. Un dels principals activadors de la CBF, la adenosina-5'-trifosfat (ATP), desencadena una resposta cel·lular mediada per Ca2+ en la que es desencadena tant l'alliberament de Ca2+ des dels dipòsits intracel·lulars com l'entrada de Ca2+. És destacable la contribució de el TRPV4 en l'augment de la CBF mediada per ATP. És més, el nostre treball implica als canals TRPV4 exclusivament en l'entrada de Ca2+ activada per receptor (ROCE). Tot plegat, aquesta tesi doctoral mostra el paper dels canals TRPV4 en l'acoblament d'estímuls fisiològics tipus mecànic, osmòtic i químic a la regulació de la CBF en l'epiteli ciliat destinat al transport mucociliar. / Clearance of mucus and pathogenic agents from lungs and the transport of gametes and embryos in the female reproductive organs are key functions of ciliated epithelia such as those present in the airways and the oviduct. The rate of mucociliary transport is a function of ciliary beat frequency (CBF) and this, in turn, is increased by increases in intracellular calcium. Transient potential vanilloid 4 (TRPV4)cation channel mediates Ca2+ influx in response to mechanical and osmotic stimuli. TRPV4 expression in ciliated epithelia from airways and oviduct is confirmed by immunofluorescence localization of the channel at the apical membrane of the polarized ciliated epithelia, where the Ca2+ signalling is required for CBF regulation. Ciliated tracheal cells from TRPV4-/-mice show no TRPV4 expression, neither increases in intracellular Ca2+ and CBF in response to the TRPV4-specific activator 4&#945;- phorbol 12,13- idecanoate (4&#945;-PDD), and reduced responses to mild temperatures (~25ºC - 38ºC), another TRPV4-activating stimulus. TRPV4 gating by high viscous loads and hypotonicity depends on phospholipase A2 (PLA2) pathway activation and subsequent production of epoxyeicosatrienoic acid (EET). Under conditions of low PLA2 activation, mechanical and hypotonic stimuli use extracellular ATP release-mediated activation of phospholipase C (PLC)-inositol triphosphate(IP3)signalling to support TRPV4 gating. We describe that IP3, without being an agonist itself, sensitizes TRPV4 to EET activation. Besides, the functional coupling between plasma membrane TRPV4 channels and IP3 receptors (IP3R) is required to initiate and maintain the cellular oscillatory Ca2+ signal triggered by high viscous loads and hypotonic stimuli. One of the main CBF activators, adenosine-5'-triphosphate (ATP), triggers both Ca2+ release from intracellular Ca2+ stores and Ca2+ entry. Interestingly, TRPV4 contributes to ATP-induced increase in CBF. Furthermore, our work implicates TRPV4 channel exclusively in receptor-operated Ca2+ entry. Collectively, this PhD thesis shows the role of TRPV4 channels coupling physiologically relevant mechanical, hypotonic and chemical stimuli to CBF regulation in motile ciliary epithelia.