磁共振3D-CISS与CT高分辨率成像在电子耳蜗植入前的应用

目的利用中、内耳高分辨C计算机X线断层照相术(computed tomography,CT)扫描及磁共振(magnetic resonance imaging,MRI)高分辨3D-CISS序列成像对拟行人工耳蜗植入术的患者作术前检查,分析、比较两种方法的临床价值。方法选择30例拟行电子耳蜗植入的感音性耳聋患者。行中内耳CT高分辨放大扫描,层厚1mm,层间隔1mm,放大倍数2。磁共振高分辨率三维稳态干扰序列(three dimensional constructive inference insteady state,3D-CISS)扫描(TR12.30ms,TE5.9ms),翻转角70°,层厚0.7mm,无间隔扫描,最大信号强度投影(maximum intensity projection,MIP)内耳重建。比较两种方法提供的信息。结果CT可观察中耳内耳、骨性结构;MRI可显示内耳膜迷路情况及内听道的神经。结论将中、内耳螺旋CT高分辨成像与磁共振高分辨3D-CISS序列其联合应用可为人工耳蜗植入提供更精确可靠的术前信息。     

Magnetic resonance imaging and micro-computed tomography combined atlas of developing and adult mouse brains for stereotaxic surgery

Stereotaxic atlases of the mouse brain are important in neuroscience research for targeting of specific internal brain structures during surgical operations. The effectiveness of stereotaxic surgery depends on accurate mapping of the brain structures relative to landmarks on the skull. During postnatal development in the mouse, rapid growth-related changes in the brain occur concurrently with growth of bony plates at the cranial sutures, therefore adult mouse brain atlases cannot be used to precisely guide stereotaxis in developing brains. In this study, three-dimensional stereotaxic atlases of C57BL/6J mouse brains at six postnatal developmental stages: postnatal day (P) 7, P14, P21, P28, P63 and in adults (P140–P160) were developed, using diffusion tensor imaging (DTI) and micro-computed tomography (CT). At present, most widely-used stereotaxic atlases of the mouse brain are based on histology, but the anatomical fidelity of ex vivo atlases to in vivo mouse brains has not been evaluated previously. To account for ex vivo tissue distortion due to fixation as well as individual variability in the brain, we developed a population-averaged in vivo magnetic resonance imaging adult mouse brain stereotaxic atlas, and a distortion-corrected DTI atlas was generated by nonlinearly warping ex vivo data to the population-averaged in vivo atlas. These atlas resources were developed and made available through a new software user-interface with the objective of improving the accuracy of targeting brain structures during stereotaxic surgery in developing and adult C57BL/6J mouse brains.     

Retention of progenitor cell phenotype in otospheres from guinea pig and mouse cochlea

Background  

Culturing otospheres from dissociated organ of Corti is an appropriate starting point aiming at the development of cell therapy for hair cell loss. Although guinea pigs have been widely used as an excellent experimental model for studying the biology of the inner ear, the mouse cochlea has been more suitable for yielding otospheres in vitro. The aim of this study was to compare conditions and outcomes of otosphere suspension cultures from dissociated organ of Corti of either mouse or guinea pig at postnatal day three (P3), and to evaluate the guinea pig as a potential cochlea donor for preclinical cell therapy.     

Intratympanic delivery of oligoarginine-conjugated nanoparticles as a gene (or drug) carrier to the inner ear

A drug delivery system to the inner ear using nanoparticles consisting of oligoarginine peptide (Arg8) conjugated to poly(amino acid) (poly(2-hydroxyethyl l-aspartamide; PHEA) was investigated to determine whether the limitations of low drug transport levels across the round window membrane (RWM) and poor transport into inner ear target cells, including hair cells and spiral ganglion, could be overcome. Three types of carrier materials, PHEA-g-C18, PHEA-g-Arg8, and PHEA-g-C18-Arg8, were synthesized to examine the effects of oligoarginine and morphology of the synthesized carriers. Nile red (NR) was used as a fluorescent indicator as well as to model a hydrophobic drug. Compared with PHEA-g-C18-NR nanoparticles, the oligoarginine-conjugated nanoparticles of PHEA-g-C18-Arg8-NR and PHEA-g-Arg8-NR entered into HEI-OC1 cells at significant levels. Furthermore, the strongest fluorescence intensity was observed in nuclei when PHEA-g-C18-Arg8 nanoparticles were used. The high uptake rates of PHEA-g-C18 and PHEA-g-C18-Arg8 nanoparticles were observed in ex vivo experiments using hair cells. After the delivery of PHEA-g-C18-Arg8 nanoparticles with reporter gene transfer, EGFP (enhanced green fluorescent protein) expression was monitored as an indicator of gene delivery. In the inner ear cells, PHEA-g-C18-Arg8 nanoparticles showed comparable or better transfection capabilities than the commercially available Lipofectamine reagent. PHEA-g-C18-Arg8 penetrated in vivo across the RWM of C57/BL6 mice with Nile red staining and GFP expression in various inner ear tissues. In conclusion, PHEA-g-C18-Arg8 nanoparticles were successfully transported into the inner ear through the intratympanic route and are proposed as promising candidates as delivery carriers to address inner ear diseases.     

Registration of Micro-Computed Tomography and Histological Images of the Guinea Pig Cochlea to Construct an Ear Model Using an Iterative Closest Point Algorithm

We present a practical and systematic method to reconstruct accurate physical models of the guinea pig ear (n = 1). The method uses a semi-automatic technique to create three-dimensional (3-D) models of the guinea pig cochlea by registration of micro-computed tomography (CT) and histological images. An iterative closest point algorithm was employed to minimize the sum of square errors with respect to the closest histological model and corresponding micro-CT model. This allowed creation of an accurate geometric ear model including external ear canal, tympanic membrane, middle ear cavity, auditory ossicles, and the cochlea. The characteristic cross-sectional areas of scala tympani, scala vestibuli, and scala media were measured. The length, thickness, and apex width of the guinea pig’s basilar membrane were compared to the data found in literature. Some shape parameters were also compared among different species. The results confirmed that the geometric model created by this method was accurate. This method provides an effective way to visualize the 3-D structure and the detailed information about ear geometry required for finite element and multibody dynamic analysis.     

Semiautomatic Segmentation of the Cochlea Using Real-Time Volume Rendering and Regional Adaptive Snake Modeling

The human cochlea in the inner ear is the organ of hearing. Segmentation is a prerequisite step for 3-dimensional modeling and analysis of the cochlea. It may have uses in the clinical practice of otolaryngology and neuroradiology, as well as for cochlear implant research. In this report, an interactive, semiautomatic, coarse-to-fine segmentation approach is developed on a personal computer with a real-time volume rendering board. In the coarse segmentation, parameters, including the intensity range and the volume of interest, are defined to roughly segment the cochlea through user interaction. In the fine segmentation, a regional adaptive snake model designed as a refining operator separates the cochlea from other anatomic structures. The combination of the image information and expert knowledge enables the deformation of the regional adaptive snake effectively to the cochlear boundary, whereas the real-time volume rendering provides users with direct 3-dimensional visual feedback to modify intermediate parameters and finalize the segmentation. The performance is tested using spiral computed tomography (CT) images of the temporal bone and compared with the seed point region growing with manual modification of the commercial Analyze software. Our method represents an optimal balance between the efficiency of automatic algorithm and the accuracy of manual work.     

Transplantation and survival of mouse inner ear progenitor/stem cells in the organ of Corti after cochleostomy of hearing-impaired guinea pigs: preliminary results

In mammals, damage to sensory receptor cells (hair cells) of the inner ear results in permanent sensorineural hearing loss. Here, we investigated whether postnatal mouse inner ear progenitor/stem cells (mIESCs) are viable after transplantation into the basal turns of neomycin-injured guinea pig cochleas. We also examined the effects of mIESC transplantation on auditory functions. Eight adult female Cavia porcellus guinea pigs (250-350g) were deafened by intratympanic neomycin delivery. After 7 days, the animals were randomly divided in two groups. The study group (n=4) received transplantation of LacZ-positive mIESCs in culture medium into the scala tympani. The control group (n=4) received culture medium only. At 2 weeks after transplantation, functional analyses were performed by auditory brainstem response measurement, and the animals were sacrificed. The presence of mIESCs was evaluated by immunohistochemistry of sections of the cochlea from the study group. Non-parametric tests were used for statistical analysis of the data. Intratympanic neomycin delivery damaged hair cells and increased auditory thresholds prior to cell transplantation. There were no significant differences between auditory brainstem thresholds before and after transplantation in individual guinea pigs. Some mIESCs were observed in all scalae of the basal turns of the injured cochleas, and a proportion of these cells expressed the hair cell marker myosin VIIa. Some transplanted mIESCs engrafted in the cochlear basilar membrane. Our study demonstrates that transplanted cells survived and engrafted in the organ of Corti after cochleostomy.     

Effect of Bone Marrow-Derived Mesenchymal Stem Cells on Cochlear Function in an Experimental Rat Model

Mesenchymal stem cell (MSC) therapy is an emerging treatment modality for various human diseases. Although induced pluripotent stem cells have been explored for the restoration of hearing, the potential of MSCs as a therapeutic strategy for various cochlear insults is not precisely known. MSCs possess anti-inflammatory, anti-apoptotic and neuroprotective properties, making them an attractive target for the treatment of inner ear disorders such as hair cell damage in response to inflammation. Most of the previous studies have used immunosuppression or the complex surgical techniques to deliver stem cells into the cochlea. However, no information is available regarding the biocompatibility and safety of MSCs in the inner ear in immunocompetent cochlea. The aim of the present study was to determine the effect of non-surgical administration of rodent bone marrow derived MSCs (BM-MSCs) through transtympanic delivery on the cochlear function and to assess any adverse effects on the auditory system employing a rat model without immunosuppression. We observed that the transtympanic administration of BM-MSCs has no significant effect on the hearing thresholds as determined by auditory brainstem response and distortion product otoacoustic emissions. Histopathological examination revealed no recruitment of inflammatory leukocytes and edema in the cochlea of BM-MSCs administrated rats. The results of this study suggest that transtympanic administration of BM-MSCs is safe and can be explored in providing otoprotection against cochlear insults. Anat Rec, 303:487–493, 2020. © 2019 American Association for Anatomy     

花样结构白蛋白缓释载体的制备及在豚鼠内耳跨膜给药的应用

目的制备花样结构白蛋白(flower-shaped bovine serum albumin,FBSA)微纳材料作为载体进行内耳跨圆窗膜给药研究,为临床寻找新的药物缓释载体奠定基础。方法应用改良的去溶剂法制备FBSA微纳材料,并用荧光显微镜、电子显微镜、粒径分析仪等对其进行系统的表征。通过体外药物释放实验、MTT法来评估其细胞相容性和细胞毒性。通过小动物活体成像观察FBSA在豚鼠听泡内的扩散及在圆窗膜上的附着。结果蛋白基微纳米材料为放射状花样结构,大小约为50⁸0μm。空白FBSA微纳材料的zeta电位是-16.2 mV,其最高的载药量和包封率分别是21.4%和40.0%,具有缓释效果。通过L929细胞的毒性实验测试提示经热变性处理固定后的材料具有更低的毒性和更好的细胞相容性。小动物活体实验可见药物在内耳中扩散及在圆窗膜表面附着。结论成功构建FBSA微纳材料载体,在治疗内耳病的局部给药方面有着良好的应用前景。     

In-vitro perforation of the round window membrane via direct 3-D printed microneedles

The cochlea, or inner ear, is a space fully enclosed within the temporal bone of the skull, except for two membrane-covered portals connecting it to the middle ear space. One of these portals is the round window, which is covered by the Round Window Membrane (RWM). A longstanding clinical goal is to reliably and precisely deliver therapeutics into the cochlea to treat a plethora of auditory and vestibular disorders. Standard of care for several difficult-to-treat diseases calls for injection of a therapeutic substance through the tympanic membrane into the middle ear space, after which a portion of the substance diffuses across the RWM into the cochlea. The efficacy of this technique is limited by an inconsistent rate of molecular transport across the RWM. A solution to this problem involves the introduction of one or more microscopic perforations through the RWM to enhance the rate and reliability of diffusive transport. This paper reports the use of direct 3D printing via Two-Photon Polymerization (2PP) lithography to fabricate ultra-sharp polymer microneedles specifically designed to perforate the RWM. The microneedle has tip radius of 500 nm and shank radius of 50 μ m, and perforates the guinea pig RWM with a mean force of 1.19 mN. The resulting perforations performed in vitro are lens-shaped with major axis equal to the microneedle shank diameter and minor axis about 25% of the major axis, with mean area 1670 μ m². The major axis is aligned with the direction of the connective fibers within the RWM. The fibers were separated along their axes without ripping or tearing of the RWM suggesting the main failure mechanism to be fiber-to-fiber decohesion. The small perforation area along with fiber-to-fiber decohesion are promising indicators that the perforations would heal readily following in vivo experiments. These results establish a foundation for the use of Two-Photon Polymerization lithography as a means to fabricate microneedles to perforate the RWM and other similar membranes.     

Comparative assessment of Fgf's diverse roles in inner ear development: A zebrafish perspective

Progress in understanding mechanisms of inner ear development has been remarkably rapid in recent years. The research community has benefited from the availability of several diverse model organisms, including zebrafish, chick, and mouse. The complexity of the inner ear has proven to be a challenge, and the complexity of the mammalian cochlea in particular has been the subject of intense scrutiny. Zebrafish lack a cochlea and exhibit a number of other differences from amniote species, hence they are sometimes seen as less relevant for inner ear studies. However, accumulating evidence shows that underlying cellular and molecular mechanisms are often highly conserved. As a case in point, consideration of the diverse functions of Fgf and its downstream effectors reveals many similarities between vertebrate species, allowing meaningful comparisons the can benefit the entire research community. In this review, I will discuss mechanisms by which Fgf controls key events in early otic development in zebrafish and provide direct comparisons with chick and mouse.     

基于胞外囊泡治疗诊断学智能药物递送工具

胞外囊泡 （EVs） 作为细胞外的膜颗粒,在细胞间信号传导中起着至关重要的作用。研究表明,EVs有可能用作特定器官 （如内耳） 的生物标志物或药物递送系统。在压力与健康状态下,EVs释放的分子不同,这些活性分子能够识别机体的患病状态。研究表明,可以利用EVs将药物输送到难以到达的器官,如耳蜗感觉毛细胞和大脑,因为它们能够穿过血迷路和血脑屏障。在本综述中,我们总结了有关EVs的生物组成和发生、分离技术、表征手段、作为药物输送载体的潜在治疗应用、药物加载方法及在听觉系统内耳治疗的应用潜力。     

Ontogenetic Variation in the Bony Labyrinth of Monodelphis domestica (Mammalia: Marsupialia) Following Ossification of the Inner Ear Cavities

Ontogeny, or the development of an individual from conception to death, is a major source of variation in vertebrate morphology. All anatomical systems are affected by ontogeny, and knowledge of the ontogenetic history of these systems is important to understand when formulating biological interpretations of evolutionary history and physiology. The present study is focused on how variation affects the bony labyrinth across a growth series of an extant mammal after ossification of the inner ear chambers. Digital endocasts of the bony labyrinth were constructed using CT data across an ontogenetic sequence of Monodelphis domestica, an important experimental animal. Various aspects of the labyrinth were measured, including angles between the semicircular canals, number of turns of the cochlea, volumes of inner ear constituents, as well as linear dimensions of semicircular canals. There is a strong correlation between skull length and age, but from 27 days after birth onward, there is no correlation with age among most of the inner ear measurements. Exceptions are the height of the arc of the lateral semicircular canal, the angular deviation of the lateral canal from planarity, the length of the slender portion of the posterior semicircular canal, and the length of the canaliculus cochleae. Adult dimensions of several of the inner ear structures, such as the arcs of the semicircular canals, are achieved before the inner ear is functional, and the non‐ontogenetic variation in the bony labyrinth serves as an important source for behavioral, physiological, and possibly phylogenetic information. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

MARS: a mouse atlas registration system based on a planar x-ray projector and an optical camera

This paper introduces a mouse atlas registration system (MARS), composed of a stationary top-view x-ray projector and a side-view optical camera, coupled to a mouse atlas registration algorithm. This system uses the x-ray and optical images to guide a fully automatic co-registration of a mouse atlas with each subject, in order to provide anatomical reference for small animal molecular imaging systems such as positron emission tomography (PET). To facilitate the registration, a statistical atlas that accounts for inter-subject anatomical variations was constructed based on 83 organ-labeled mouse micro-computed tomography (CT) images. The statistical shape model and conditional Gaussian model techniques were used to register the atlas with the x-ray image and optical photo. The accuracy of the atlas registration was evaluated by comparing the registered atlas with the organ-labeled micro-CT images of the test subjects. The results showed excellent registration accuracy of the whole-body region, and good accuracy for the brain, liver, heart, lungs and kidneys. In its implementation, the MARS was integrated with a preclinical PET scanner to deliver combined PET/MARS imaging, and to facilitate atlas-assisted analysis of the preclinical PET images.     

基于VIP-Man图谱配准的腹部器官分割方法

提出了一种基于图谱配准的腹部器官分割方法.首先将一套预标记图谱向个体图像进行配准,建立二者之间器官的基本对应关系,同时完成对感兴趣器官的识别,其中配准包含全局配准和器官配准.然后,借助已配准的图谱,采用模糊连接方法对感兴趣器官进行分割.腹PCT和MR实验测试结果证明:这种方法实现了模糊连接分割方法中各项参数的自动指定,减轻了人工负担,提高了结果的可靠性.     

Inner ear abnormalities in Kabuki make-up syndrome: report of three cases

Three patients, a female and two males, 28, 15, and 14 years of age, with Kabuki make-up syndrome (KMS) were studied for middle and inner ear abnormalities by using CT scanning of the petrous bones. All three patients had bilateral dysplasia of the inner ear, i.e., hypodysplasia of the cochlea, vestibule, and semicircular canals (so-called Mondini dysplasia), whereas their middle ears had no abnormalities. Audiometry demonstrated a sharp decrease in hearing of the high tone range, bilateral in one and unilateral in another, while the third patient was noncooperative. In view of these findings, it would be advisable to study each individual with KMS and hearing impairment for possible inner ear abnormalities.     

Gene expression in the peripheral leukocytes and association analysis of PDLIM5 gene in schizophrenia

The terminal mitosis of hair cells (HCs) and supporting cells (SCs) in mammalian cochlea occurred during middle embryonic development. Most hearing loss results from the incapacity of the cochlear sensory epithelium to replace lost hear cells. Deafness due to hair cells loss is normally irreversible. The present study showed that cells acutely dissociated from the cochlea of young rat, cultured with EGF and FGF2, developed into otospheres that showed expression of nestin and incorporation of 5'-Bromo-2-deoxyuridine (BrdU). The subcultured otospheres maintained for up to 10 passages. In addition, the cochlea sphere-derivatives contributed to a variety of cell types. They were found to differentiate to neuron, glia, hair cell and supporting cell phenotypes. The results suggest that the young rat inner ear cells have self-renewal capability and multipotent differentiation potential. This work raises the possibility that inner ear cells in the early post-natal rat have the character of pluripotent stem cells and might be a source for cell replacement therapy in the inner ear.     

Inner ear abnormalities in Kabuki make‐up syndrome: Report of three cases

Three patients, a female and two males, 28, 15, and 14 years of age, with Kabuki make‐up syndrome (KMS) were studied for middle and inner ear abnormalities by using CT scanning of the petrous bones. All three patients had bilateral dysplasia of the inner ear, i.e., hypodysplasia of the cochlea, vestibule, and semicircular canals (so‐called Mondini dysplasia), whereas their middle ears had no abnormalities. Audiometry demonstrated a sharp decrease in hearing of the high tone range, bilateral in one and unilateral in another, while the third patient was noncooperative. In view of these findings, it would be advisable to study each individual with KMS and hearing impairment for possible inner ear abnormalities. Am. J. Med. Genet. 92:87–89, 2000. © 2000 Wiley‐Liss, Inc.