内耳slow-cycling cellとして tympanic border cellを同定した

京都大学0048新制・課程博士博士(医学)甲第17422号医博第3765号新制||医||997(附属図書館)30188京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 大森 治紀, 教授 河野 憲二学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDA

Hydrogen protects auditory hair cells from cisplatin-induced free radicals.

Cisplatin is a widely used chemotherapeutic agent for the treatment of various malignancies. However, its maximum dose is often limited by severe ototoxicity. Cisplatin ototoxicity may require the production of reactive oxygen species (ROS) in the inner ear by activating enzymes specific to the cochlea. Molecular hydrogen was recently established as an antioxidant that selectively reduces ROS, and has been reported to protect the central nervous system, liver, kidney and cochlea from oxidative stress. The purpose of this study was to evaluate the potential of molecular hydrogen to protect cochleae against cisplatin. We cultured mouse cochlear explants in medium containing various concentrations of cisplatin and examined the effects of hydrogen gas dissolved directly into the media. Following 48-h incubation, the presence of intact auditory hair cells was assayed by phalloidin staining. Cisplatin caused hair cell loss in a dose-dependent manner, whereas the addition of hydrogen gas significantly increased the numbers of remaining auditory hair cells. Additionally, hydroxyphenyl fluorescein (HPF) staining of the spiral ganglion showed that formation of hydroxyl radicals was successfully reduced in hydrogen-treated cochleae. These data suggest that molecular hydrogen can protect auditory tissues against cisplatin toxicity, thus providing an additional strategy to protect against drug-induced inner ear damage

Identification of tympanic border cells as slow-cycling cells in the cochlea.

Mammalian cochlear sensory epithelial cells are believed to possess minimal regenerative potential because they halt proliferation during late stage of embryogenesis and never regenerate after birth. This means that sensorineural hearing loss caused by the death of cochlear sensory epithelial cells is a permanent condition. However, stem cells were recently identified in neonatal mice following dissociation of their inner ear organs. This suggests that regenerative therapy for sensorineural hearing loss may be possible. Unfortunately, dissociation distorts the microanatomy of the inner ear, making it difficult to determine the precise location of stem cells in unaltered specimens. To develop new therapeutic approaches based on sensory epithelial cell regeneration, the location of these stem cells must be elucidated. Stem cells normally proliferate at a slow rate in adult organs. In fact, so-called label-retaining cells, or slow-cycling cells, of the brain and skin are recognized as stem cells. In this study, using the exogenous proliferation marker, 5'-bromo-2'-deoxyuridine (BrdU) in combination with the endogenous proliferation marker Ki-67, we identified tympanic border cells. These cells, which are located beneath the basilar membrane in vivo, represent slow-cycling cells of the murine cochlea. Immunohistochemically, these cells stained positive for the immature cell marker Nestin. But it will be difficult to achieve regeneration of the cochlear function because these slow-cycling cells disappear in the mature murine cochlea

Prognosis of otitis media with effusion in pediatric patients with cleft palate during language-acquisition period treated by simultaneous tympanostomy tube placement with palatoplasty

[Objective] Cleft palate (CP) in children is frequently complicated by otitis media with effusion (OME) due to Eustachian tube dysfunction. Although tympanostomy tube (TT) placement can be beneficial in the treatment of OME to prevent short-term hearing loss, there is no consensus regarding the indications for and timing of TT insertion. The present study was performed to define the safety and effectiveness of simultaneous TT placement with palatoplasty during the language-acquisition period. [Methods] We retrospectively reviewed the medical charts of pediatric patients who underwent palatoplasty for CP in a tertiary medical center, Kyoto University Hospital, from June 2010 to October 2018. The TT retention time was estimated using the Kaplan-Meier method. The incidence of OME recurrence was compared among four Veau classification groups based on the patients' sex, type of CP, median TT retention time, and type of fluid. [Results] Seventy-six subjects (150 ears) were enrolled in the study. The median follow-up duration was 48.7 months (range, 18.2–108.0 months) after the first TT placement. A first TT retention time of <20.1 months was a significant risk factor for OME recurrence. Subjects with maxillofacial anomaly complex and subjects with cleft lip and palate and an alveolar cleft showed a significantly higher OME recurrence rate than subjects with clefts only in the hard and/or soft palate. There was no significant difference in the occurrence of sequelae between subjects with only a single TT placement and subjects with more than one TT placement. [Conclusions] Based on the findings of the present study, it is reasonable to perform TT insertion at the same time as palatoplasty on patients who meet the indications. This technique may reduce the number of times the patient requires general anesthesia and maintain good middle ear condition during the period of language acquisition between 1 and 3 years of age

Nestin-positive tympanic border cells (E18.5).

<p>Panels A and B are from the same section. Panels C-H are from a second section. Double immunostaining (A and B) for Nestin and Myosin7a (Myo7a, hair cell marker), or triple immunostaining (C-H) for BrdU (C, D, and H), Ki-67 (E, F, and H), and Nestin (G and H) was performed. Nuclei were stained using DAPI (B, D, and F). Tympanic border cells positive for Nestin (arrows in A and B). Most of the BrdU-Ki-67-double positive tympanic border cells are also positive for Nestin (arrows in C-H). The white arrowheads indicate a cochlear spiral modiolar artery. Asterisks in A and B indicate Nestin-positive cells in GER and LER. The scale bar indicates 50 µm.</p

Localization of slow-cycling cells within tympanic border cell region.

<p>We counted the numbers of double-positive cells within three areas of the basilar membrane (A): from the region under the tympanic lip to under the medial side of the inner hair cells (medial region); from the region under the inner hair cells to the area under the outer hair cells (region below the HCs); and the region under the Hensen's and Claudius cells (lateral region) (B). Statistical analyses were performed using one-way analysis of variance (ANOVA) and multiple comparisons (Tukey-Kramer method). Roughly two-thirds of the observed slow-cycling cells (41/63) were located in the region below the hair cells where a cochlear spiral modiolar artery exists. The number of slow-cycling cells in the area below the HCs was significantly greater than was detected in other regions (A). * : p<0.01. Bars indicate the standard error of the mean. IHC: inner hair cell, OHC: outer hair cells.</p

Slow-cycling cells in the supporting cell layer of the vestibular organ.

<p>All panels are from one section. Triple immunostaining was performed for BrdU (A, B, F), Ki-67 (C, D, F), and Myosin7a (Myo7a, hair cell marker) (F), and nuclei were stained using DAPI (B, D, E, F). We detected double-positive cells (arrows) in the supporting cell layer (below Myosin7a-positive hair cells) in the E18.5 mouse vestibular organ (BrdU administered at E13.5). The scale bar indicates 50 µm.</p

Anatomy of the cochlea.

<p>Illustrations of a whole cochlea and cochlear sections (A and B). B is an illustration of a section through dotted line in A. The cochlea exhibits spiral structure with several turns (A). The maturation of its sensory epithelia occurs in the basal to apical direction (A). The cochlea includes three spaces within its duct; scala tympani (ST), scala media (SM), and scala vestibuli (SV) (B). Hematoxylin and eosin stained section of an E18.5 cochlear basal turn (C) and an illustration (D) are presented. These are enlarged images of the orange dotted box in panel B. On the SM side of basilar membrane (BM in D) there are sensory epithelia composed of inner hair cells (IHC in C and D) and outer hair cells (OHC in C and D). The organ of Corti includes one row of IHCs and three rows of OHCs (red cells ind D). Supporting cells (SC) including Claudius and Hensen's cells exist lateral to (yellow cells in D) or under the hair cells (pale orange cells in D). Tympanic border cells (green cells in B) cover the ST side of the basilar membrane. The lateral and medial regions of the organ of Corti are referred to as lesser epithelial ridge (LER) (yellow cells) and greater epithelial ridge (GER) (orange cells), respectively. Below the organ of Corti, there is a spiral modiolar artery (C and D) surrounded by the tympanic border cells. Tympanic lip is the medial border of BM. The scale bar in C indicates 50 µm.</p

Nestin-positive tympanic border cells (P4).

<p>All panels are from one section. This section is a representative result of the ten cochleae studied. Triple immunostaining was performed for BrdU (A, B, F, and H), Ki-67(C, D, F, and H) and Nestin (E, F and H). Nuclei were stained using DAPI (B, D, F, and H). The difference interference contrast image (G) and fluorescent signal detection image were taken at the same time and merged (H). We observed triple-positive cells in the tympanic border zone (arrows) in the P4 mouse cochlea (BrdU administered at E18.5). IHC: inner hair cell, OHC: outer hair cells, DIC: difference interference contrast image. The white arrowheads indicate a cochlear spiral modiolar artery. The scale bar indicates 50 µm.</p

Section through an E13.5 murine cochlear duct one hour after BrdU injection.

<p>All panels are from one section. Double immunostaining was performed for BrdU (A, C, D, and F) and Ki-67 (B, C, E, and F) simultaneously, and nuclei were stained using DAPI (D, E, and F). C is a merged image of A and B. F is a merged image of D and E. We observed many BrdU and Ki-67-positive cells in the cochlear ducts (A and B) and almost all the BrdU-positive cells were Ki-67-positive (C). Both staining patterns clearly indicate a zone of non-proliferating cells (F) representing future sensory epithelial cells. The scale bar indicates 50 µm.</p