Three-dimensional visualization of the human membranous labyrinth: The membrana limitans and its role in vestibular form

The inner ear contains the end organs for balance (vestibular labyrinth) and hearing (cochlea). The vestibular labyrinth is comprised of the semicircular canals (detecting angular acceleration) and otolith organs (utricle and saccule, which detect linear acceleration and head tilt relative to gravity). Lying just inferior to the utricle is the membranous membrana limitans (ML). Acting as a keystone to vestibular geometry, the ML provides support for the utricular macula and acts as a structural boundary between the superior (pars superior) and inferior (pars inferior) portions of the vestibular labyrinth. Given its importance in vestibular form, understanding ML morphology is valuable in establishing the spatial organization of other vestibular structures, particularly the utricular macula. Knowledge of the 3D structure and variation of the ML, however, remain elusive. Our study addresses this knowledge gap by visualizing, in 3D, the ML and surrounding structures using micro-CT data. By doing so, we attempt to clarify: (a) the variation of ML shape; (b) the reliability of ML attachment sites; and (c) the spatial relationship of the ML to the stapes footplate using landmark-based Generalized Procrustes, Principal Component and covariance analyses. Results indicate a consistent configuration of three distinct bony ML attachments including an anterolateral, medial, and posterior attachment which all covary with bony structure. Our results set the stage for further understanding into vestibular and more specifically, utricular macula spatial configuration within the human head, offering the potential to aid in clinical and evolutionary studies which rely on a 3D understanding of vestibular spatial configuration.     

Formation of the Periotic Space During the Early Fetal Period in Humans

The inner ear is a very complicated structure, composed of a bony labyrinth (otic capsule; OC), membranous labyrinth, with a space between them, named the periotic labyrinth or periotic space. We investigated how periotic tissue fluid spaces covered the membranous labyrinth three‐dimensionally, leading to formation of the periotic labyrinth encapsulated in the OC during human fetal development. Digital data sets from magnetic resonance images and phase‐contrast X‐ray tomography images of 24 inner ear organs from 24 human fetuses from the Kyoto Collection (fetuses in trimesters 1 and 2; crown—rump length: 14.4–197 mm) were analyzed. The membranous labyrinth was morphologically differentiated in samples at the end of the embryonic period (Carnegie stage 23), and had grown linearly to more than eight times in size during the observation period. The periotic space was first detected at the 35‐mm samples, around the vestibule and basal turn of the cochlea, which elongated rapidly to the tip of the cochlea and semicircular ducts, successively, and almost covered the membranous labyrinth at the 115‐mm CRL stage or later. In those samples, several ossification centers were detected around the space. This article thus demonstrated that formation of the membranous labyrinth, periotic space (labyrinth), and ossification of the OC occurs successively, according to an intricate timetable. Anat Rec, 301:563–570, 2018. © 2018 Wiley Periodicals, Inc.     

Inner ear formation during the early larval development of Xenopus laevis.

The formation of the eight independent endorgan compartments (sacculus, utricle, horizontal canal, anterior canal, posterior canal, lagena, amphibian papilla, and basilar papilla) of the Xenopus laevis inner ear is illustrated as the otic vesicle develops into a complex labyrinthine structure. The morphology of transverse sections and whole-mounts of the inner ear was assessed in seven developmental stages (28, 31, 37, 42, 45, 47, 50) using brightfield and laser scanning confocal microscopy. The presence of mechanosensory hair cells in the sensory epithelia was determined by identification of stereociliary bundles in cryosectioned tissue and whole-mounts of the inner ear labeled with the fluorescent F-actin probe Alexa-488 phalloidin. Between stages 28 and 45, the otic vesicle grows in size, stereociliary bundles appear and increase in number, and the pars inferior and pars superior become visible. The initial formation of vestibular compartments with their nascent stereociliary bundles is seen by larval stage 47, and all eight vestibular and auditory compartments with their characteristic sensory fields are present by larval stage 50. Thus, in Xenopus, inner ear compartments are established between stages 45 and 50, a 2-week period during which the ear quadruples in length in the anteroposterior dimension. The anatomical images presented here demonstrate the morphological changes that occur as the otic vesicle forms the auditory and vestibular endorgans of the inner ear. These images provide a resource for investigations of gene expression patterns in Xenopus during inner ear compartmentalization and morphogenesis.     

Three-dimensional morphology of inner ear development in Xenopus laevis.

The three-dimensional morphology of the membranous labyrinth of Xenopus laevis is presented from embryonic through late tadpole development (stages 28 to 52, inclusive). This was accomplished by paint-filling the endolymphatic spaces of Xenopus ears at a series of stages, beginning with the embryonic otic vesicle and ending with the complex ear of the late tadpole. At stage 52, the inner ear has expanded approximately 23-fold in its anterior/posterior dimension compared with stage 28 and it is a miniature of the adult form. The paint-filling technique illustrates the dramatic changes required to convert a simple ear vesicle into the elaborate form of the adult, including semicircular canal formation and genesis of vestibular and auditory organs, and it can serve as a basis for phenotype identification in experimentally or genetically manipulated ears.     

The third vascular route of the inner ear or the canal of Cotugno: Its topographical anatomy,fetal development,and contribution to ossification of the otic capsule cartilage

Three vascular routes to the inner ear are known: (a) through the internal acoustic meatus with the vestibulocochlear nerve; (b) from the endolymphatic duct aperture; and (c) along the canal of Cotugno (CC) inserted into the vestibular part of the ear from the superior or brain side. The third is believed to contain only veins. Examinations of 33 human embryos and fetuses at 6–40 weeks demonstrated that (a) the CC appeared as a recess of epidural mesenchymal tissues at the superior aspect of the otic capsule cartilage in embryos and it was inserted deeply to issue multiple peripheral divisions inferolaterally and posteriorly at midterm; (b) the CC consistently passed through a ring of the superior or anterior semicircular canal and contained both, the arteries from the vestibulocochlear nerve origin at the midbrain and the vein draining into the sigmoid sinus or petrosal sinuses; and (c) the CC appeared not to contribute to ossification of the otic capsule cartilage but, after endochondral ossification of the internal ear, woven bone development occurred along a smooth interface of the CC with the ossified ear. In contrast, another interface between the developing bone and the residual cartilage of the otic capsule was rough and wavy with many short bony columns, called osseous globules. In addition, the endolymphatic duct accompanied veins but no arteries. Our results show that the CC is a major vascular route to the vestibular part of the otic capsule cartilage, but its role appears to be limited after ossification.     

Enlarged vestibular aqueduct (EVA) related with recurrent benign paroxysmal positional vertigo (BPPV)

The vestibular aqueduct is a bony canal related to the bony labyrinth of the inner ear and represents the non-sensory components of the endolymph-filled, closed, membranous labyrinth. The association of congenital sensorineural hearing loss with a large or enlarged vestibular aqueduct is well known as the large vestibular aqueduct syndrome (LVAS). The enlarged VA (EVA) comprises abnormalities not only in the structure of the inner ear, but also in the physiology of the auditory and vestibular systems. The clinical picture of this clinical entity is variable [Yetiser S, Kertment M, Ozkaptan Y. Vestibular disturbance in patients with Large Vestibular Aqueduct Syndrome (LVAS). Acta Otolaryngol (StochK) 1999;119: 641-646]. Signs and symptoms of the auditory impairment are more commonly described in the literature: hearing loss ranges from mild to profound, arising from fluctuating to stepwise progressive or sudden. Vestibular disturbances, ranging from mild imbalance to episodic vertigo, are rarely described in the literature. Benign paroxysmal positional vertigo (BPPV) is a labyrinthine disorder with a typical behavior: intense crises of rotational vertigo induced by postural changes of the head, with short duration and usually good responsiveness to rehabilitative maneuvers. These maneuvers are effective in about 80% of patients with BPPV. BPPV often recurs. About 1/3 of patients have a recurrence in the first year after treatment, and by five years, about half of all patients have a recurrence. Vestibular aqueduct has been demonstrated by conventional tomography and computed tomography (CT), however, CT scans cannot show the membranous labyrinth itself. On MR images it is not the vestibular aqueduct that is visualized but its contents, the endolymphatic duct and sac, and can show the abnormalities of the fluid spaces related to the membranous labyrinth. It is proposed that recurrent benign paroxysmal positional vertigo (BPPV) is related with volumetric abnormalities of vestibular aqueduct. This verifiable hypothesis tries to define this rapport and explore new diagnostic and therapeutic possibilities.     

TBX1 is required for inner ear morphogenesis

TBX1 is thought to be a critical gene in the pathogenesis of del22q11/DiGeorge syndrome (DGS). Morphological abnormalities of the external ear and hearing impairment (conductive or sensorineural) affect the majority of patients. Here we show that homozygous mutation of the mouse homolog Tbx1 is associated with severe inner ear defects that prevent the formation of the cochlea and of the vestibulum. Consistent with phenotypic abnormalities, Tbx1 is expressed early in otocyst development in the otic epithelium and in the periotic mesenchyme. Tbx1 loss-of-function blocks inner ear development at early otocyst stage and after neurogenesis. Analysis of chimeras suggests that Tbx1 function is required in the otic epithelium cell autonomously, but abnormalities of the periotic mesenchyme indicate that the pathogenesis of the inner ear phenotype is complex. We propose a model where Tbx1 is required for expansion of a subpopulation of otic epithelial cells, which is required to form the vestibular and auditory organs. Our data suggest that Tbx1 deletion in del22q11 patients may cause not only external and middle ear defects but also sensorineural and vestibular phenotypes observed in these patients.     

Wnt-dependent regulation of inner ear morphogenesis is balanced by the opposing and supporting roles of Shh

The inner ear is partitioned along its dorsal/ventral axis into vestibular and auditory organs, respectively. Gene expression studies suggest that this subdivision occurs within the otic vesicle, the tissue from which all inner ear structures are derived. While the specification of ventral otic fates is dependent on Shh secreted from the notochord, the nature of the signal responsible for dorsal otic development has not been described. In this study, we demonstrate that Wnt signaling is active in dorsal regions of the otic vesicle, where it functions to regulate the expression of genes (Dlx5/6 and Gbx2) necessary for vestibular morphogenesis. We further show that the source of Wnt impacting on dorsal otic development emanates from the dorsal hindbrain, and identify Wnt1 and Wnt3a as the specific ligands required for this function. The restriction of Wnt target genes to the dorsal otocyst is also influenced by Shh. Thus, a balance between Wnt and Shh signaling activities is key in distinguishing between vestibular and auditory cell types.     

Specification of the mammalian cochlea is dependent on Sonic hedgehog

Organization of the inner ear into auditory and vestibular components is dependent on localized patterns of gene expression within the otic vesicle. Surrounding tissues are known to influence compartmentalization of the otic vesicle, yet the participating signals remain unclear. This study identifies Sonic hedgehog (Shh) secreted by the notochord and/or floor plate as a primary regulator of auditory cell fates within the mouse inner ear. Whereas otic induction proceeds normally in Shh(-/-) embryos, morphogenesis of the inner ear is greatly perturbed by midgestation. Ventral otic derivatives including the cochlear duct and cochleovestibular ganglia failed to develop in the absence of Shh. The origin of the inner ear defects in Shh(-/-) embryos could be traced back to alterations in the expression of a number of genes involved in cell fate specification including Pax2, Otx1, Otx2, Tbx1, and Ngn1. We further show that several of these genes are targets of Shh signaling given their ectopic activation in transgenic mice that misexpress Shh in the inner ear. Taken together, our data support a model whereby auditory cell fates in the otic vesicle are established by the direct action of Shh.     

Coordinated molecular control of otic capsule differentiation: Functional role of Wnt5a signaling and opposition by sfrp3 activity

Wnt proteins constitute one of the major families of secreted ligands that function in developmental signaling, however, little is known of the role of Wnt5a during inner ear development. It is hypothesized that Wnt5a acts as a mediator of chondrogenesis in the developing otic capsule, a cartilaginous structure that surrounds the developing inner ear and presages the formation of the endochondral bony labyrinth. We report the pattern of expression of Wnt5a protein and mRNA in the developing mouse inner ear using immunohistochemistry, whole-mount in situ hybridization and RT-PCR, and the ability of exogenous Wnt5a to stimulate otic capsule chondrogenesis when added to high-density cultures of periotic mesenchyme containing otic epithelium (periotic mesenchyme + otic epithelium), a well-established model of otic capsule formation. We show that in the presence of secreted frizzled related protein 3 (sfrp3), a Wnt antagonist expressed in the developing inner ear, or Wnt5a-specific antisense oligonucleotide, which diminishes endogenous Wnt5a, otic capsule chondrogenesis is suppressed in culture. We determined by histological analysis and aggrecan immunoreactivity that chondrogenic differentiation is disturbed in Wnt5a null embryos, and provide evidence that the periotic mesenchyme + otic epithelium harvested from Wnt5a null mice is compromised in its ability to differentiate into cartilage when interacted in culture. We propose a model whereby sfrp3 and Wnt5a act antagonistically to ensure appropriate patterns of chondrogenesis and provide coordinated control of otic capsule formation. Our findings support Wnt5a and sfrp3 as regulators of otic capsule formation in the developing mouse inner ear.     

Coordinated molecular control of otic capsule differentiation: functional role of Wnt5a signaling and opposition by sfrp3 activity

Wnt proteins constitute one of the major families of secreted ligands that function in developmental signaling, however, little is known of the role of Wnt5a during inner ear development. It is hypothesized that Wnt5a acts as a mediator of chondrogenesis in the developing otic capsule, a cartilaginous structure that surrounds the developing inner ear and presages the formation of the endochondral bony labyrinth. We report the pattern of expression of Wnt5a protein and mRNA in the developing mouse inner ear using immunohistochemistry, whole-mount in situ hybridization and RT-PCR, and the ability of exogenous Wnt5a to stimulate otic capsule chondrogenesis when added to high-density cultures of periotic mesenchyme containing otic epithelium (periotic mesenchyme + otic epithelium), a well-established model of otic capsule formation. We show that in the presence of secreted frizzled related protein 3 (sfrp3), a Wnt antagonist expressed in the developing inner ear, or Wnt5a-specific antisense oligonucleotide, which diminishes endogenous Wnt5a, otic capsule chondrogenesis is suppressed in culture. We determined by histological analysis and aggrecan immunoreactivity that chondrogenic differentiation is disturbed in Wnt5a null embryos, and provide evidence that the periotic mesenchyme + otic epithelium harvested from Wnt5a null mice is compromised in its ability to differentiate into cartilage when interacted in culture. We propose a model whereby sfrp3 and Wnt5a act antagonistically to ensure appropriate patterns of chondrogenesis and provide coordinated control of otic capsule formation. Our findings support Wnt5a and sfrp3 as regulators of otic capsule formation in the developing mouse inner ear.     

15例成年国人内耳膜迷路及内耳道MRI最大密度投影三维重建图像的解剖学研究

目的研究成年国人内耳膜迷路及内耳道最大密度投影(MIP)三维重建图像,观测内耳主要结构磁共振成像(MRI)的正常解剖测量值,为临床耳显微外科及神经外科手术提供解剖学依据。方法选用15名健康志愿者,使用GE-signal 1．5T超导型核磁共振机,环行耳颞部线圈,三维快速自旋回波序列(3D／FSE／T2W1)(水成像)及脂肪抑制技术,对两侧耳部同时进行扫描。原始扫描图像行MIP三维重建,多角度旋转对内耳主要结构进行解剖学观测。所得数据用SPSS10．0软件进行统计学分析。结果MIP三维重建能满意显示两侧内耳膜迷路及内耳道的解剖结构,其中3个膜半规管、椭圆囊、球囊、蜗管及内耳道呈高信号。测量结果内耳主要结构均无显著的侧别差异。结论临床MIP三维重建能直观、立体地显示内耳膜迷路及内耳道的结构,为成年国人内耳主要结构MRI正常解剖测量值的确立提供了一定的基础资料。     

CT与磁共振图像融合技术对显示正常内耳结构的应用价值

目的 探讨内耳CT与磁共振成像(MRI)图像融合的可行性以及融合图像对正常内耳结构的显示能力.方法 对20例(40耳)双侧内耳及内听道形态正常的感音神经性耳聋患者行多层螺旋CT(MSCT)和MRI扫描,并应用西门子图像融合软件将CT与MRI图像进行配准、融合,然后半定量评价融合图像对正常内耳和内听道结构的显示能力.结果 CT与MRI融合可有9种融合模式,在不同融合模式的图像中CT不反相+MRI反相对骨迷路显示最清晰;CT反相+MRI不反相、CT反相+MRI伪彩对膜迷路显示最清晰、立体感强;CT不反相+MRI反相、CT与MRI均反相对面神经、听神经及蜗神经显示最清晰、最直观、立体感最强;CT不反相+MRI反相、CT反相+MRI不反相、CT伪彩+MRI反相对蜗孔处蜗神经显示最好.结论 CT与MRI图像可以进行精确配准和融合,不同模式融合图像对内耳不同结构的显示清晰度不同.     

内耳免疫应答与内耳免疫性疾病

免疫性内耳病是指由免疫系统功能紊乱引起的或与其相关的耳蜗、前庭功能障碍。最近发现内耳虽然位于骨性包囊内并缺乏淋巴引流 ,但它并非一免疫豁免器官。含有免疫补体细胞、淋巴细胞和巨噬细胞的内淋巴囊是内耳免疫反应的主要部位 ,在内耳免疫性疾病的发生中起着重要作用。免疫性内耳疾病的诊断依靠临床表现 ,实验室免疫学检查及良好的免疫治疗反应 ,其治疗方案必须针对每个患者做到个体化。     

基于内耳体素模型的膜迷路三维可视化

目的探讨基于内耳体素模型三维可视化膜迷路。方法磁共振显微成像颞骨扫描影像数据使用3D Slicer软件进行表面模型体裁剪,将内耳体素模型通过表面绘制和体绘制混合成像三维显示膜迷路。结果通过内耳体素模型可以三维可视化膜迷路,且内耳体素模型文件较表面模型文件容量更小。结论内耳体素模型对于内耳解剖学习和研究有重要意义。     

Morphogenesis of the Inner Ear at Different Stages of Normal Human Development

This study examined the external morphology and morphometry of the human embryonic inner ear membranous labyrinth and documented its three‐dimensional position in the developing embryo using phase‐contrast X‐ray computed tomography and magnetic resonance imaging. A total of 27 samples between Carnegie stage (CS) 17 and the postembryonic phase during trimester 1 (approximately 6–10 weeks after fertilization) were included. The otic vesicle elongated along the dorso‐ventral axis and differentiated into the end lymphatic appendage and cochlear duct (CD) at CS 17. The spiral course of the CD began at CS18, with anterior and posterior semicircular ducts (SDs) forming prominent circles with a common crus. The spiral course of the CD comprised more than two turns at the postembryonic phase, at which time the height of the CD was evident. A linear increase was observed in the length of anterior, posterior, and lateral SDs, in that order, and the length of the CD increased exponentially over the course of development. Bending in the medial direction was observed between the cochlear and vestibular parts from the latero‐caudal view, with the angle decreasing during development. The position of the inner ear was stable throughout the period of observation on the lateral to ventral side of the rhombencephalon, caudal to the pontine flexure, and adjacent to the auditory ganglia. The plane of the lateral semicircular canal was approximately 8.0°–14.6° with respect to the cranial caudal (z‐)axis, indicating that the orientation of the inner ear changes during growth to adulthood. Anat Rec, 298:2081–2090, 2015. © 2015 Wiley Periodicals, Inc.     

Connections between the facial, vestibular and cochlear nerve bundles within the internal auditory canal

The vestibular, cochlear and facial nerves have a common course in the internal auditory canal (IAC). In this study we investigated the average number of nerve fibres, the average cross-sectional areas of the nerves and nerve fibres, and the apparent connections between the facial, cochlear and vestibular nerve bundles within the IAC, using light and scanning electron microscopy. The anatomical localization of the nerves within the IAC was not straightforward. The general course showed that the nerves rotated anticlockwise in the right ear from the inner ear end towards the brainstem end and vice versa for the left ear. The average number of fibres forming vestibular, cochlear, and facial nerves was not constant during their courses within the IAC. The superior and the inferior vestibular nerves showed an increase in the number of nerve fibres from the inner ear end towards the brainstem end of the IAC, whereas the facial and the cochlear nerves showed a reduction in the number of fibres. This suggests that some of the superior and inferior vestibular nerve bundles may receive fibres from the facial and/or cochlear nerves. Scanning electron microscopic evaluations showed superior vestibular-facial and inferior vestibular-cochlear connections within the IAC, but no facial-cochlear connections were observed. Connections between the nerves of the IAC can explain the unexpected vestibular disturbances in facial paralysis or persistence of tinnitus after cochlear neurectomy in intractable tinnitus cases. The present study offers morphometric and scanning electron microscopic data on the fibre connections of the nerves of the IAC.     

A subpopulation of dorsal raphe nucleus neurons retrogradely labeled with cholera toxin-B injected into the inner ear

Previous studies have shown that: (1) raphe neurons respond to acoustic and vestibular stimuli, some with a latency of 10–15 ms; (2) alterations of the raphe nuclei alter the acoustic startle reflex; (3) the dorsal raphe nucleus (DRN) is the major source of serotonergic neurons; and (4) approximately 57% of the DRN neurons are nonserotonergic. In the present study, cholera toxin subunit-B (CTB) was injected into cat cochleas, and the brain tissue was examined after a survival period of 5–7 days. Aside from neurons which were known to project to the inner ear, i.e., olivocochlear and vestibular efferent neurons, a surprising new finding was made that somata of a subpopulation of DRN neurons were intensely labeled with CTB. These CTB-labeled neurons were densely distributed in a dorsomedian part of the DRN with some in a surrounding area outside the DRN. The present results suggest that a novel raphe-labyrinthine projection may exist. A future study of anterograde labeling with injections of a tracer in the DRN will be needed to establish the existence of a raphe-labyrinthine projection more thoroughly. A raphe-labyrinthine descending input, together with an ascending input from the inner ear to the DRN through intervening neurons, such as the juxta-acousticofloccular raphe neurons (JAFRNs) described by Ye and Kim, may mediate a brain stem reflex whereby a salient multisensory (including auditory and vestibular) stimulus may alter the sensitivity of the inner ear. As a mammal responds to a biologically important auditory-vestibular multisensory event, the raphe projections to the inner ear and other auditory and vestibular structures may enhance the mammal's ability to localize and recognize the sound and respond properly. The raphe-labyrinthine projection may also modulate the inner ear's sensitivity as a function of the sleep–wake arousal state of an organism on a slower time course.     

DNA content,mitotic activity,and incorporation of tritiated thymidine in the developing inner ear of the rat

The rat inner ear is ectodermally derived from a region adjacent to the developing hindbrain. Beginning on day 8 of a 22-day gestational period, this zone of ectoderm first forms the otic placode, then the otocyst, and ultimately the definitive membranous labyrinth. This report provides an estimation of total DNA content of the developing inner ear, and hence an estimation of the total number of cells that comprise the inner ear at each developmental stage. The incorporation of ³H-thymidine indicates that most cells of the inner ear undergo DNA synthetic activity during gestational days 13 to 15. Radioautographic observations indicate a zone of DNA synthetic activity at the base of the outpocketing cochlear duct during early development. At the later stages of development, DNA synthesis is restricted to the cristae ampullares of the semicircular canals and the maculae of the utricle and the saccule. In contradistinction to the findings of other investigators, the statoacoustic ganglion complex undergoes terminal mitosis during gestational days 17 and 18. The gestational period between days 13 and 15 may prove to be a critical stage in normal otic development. The normal values of total DNA content and the number of cells that comprise the inner ear during development, established by these methods, can be compared with pathologic inner ears to provide quantitative means of assessing the damage in malformed inner ears. These values also form the baseline for future experimental studies of inner ear development.