基于多尺度分辨率的耳蜗神经纤维三维成像

doi:10.3969/j.issn.1674-8115.2025.11.002

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (11): 1421-1431.doi: 10.3969/j.issn.1674-8115.2025.11.002

• 创新团队成果专栏 • 上一篇

基于多尺度分辨率的耳蜗神经纤维三维成像

成亚琼¹^,²^,³(), 杜一唯¹^,²^,³, 刘思迪¹^,²^,³, 经典¹^,²^,³(), 吴皓¹^,²^,³()

^1.上海交通大学医学院附属第九人民医院耳鼻咽喉头颈外科，上海 200011
^2.上海交通大学医学院耳科学研究所，上海 200125
^3.上海市耳鼻疾病转化医学重点实验室，上海 200125

收稿日期:2025-03-20 接受日期:2025-05-09 出版日期:2025-11-28 发布日期:2025-12-03
通讯作者: 吴　皓，教授，主任医师，博士；电子信箱：wuhao@shsmu.edu.cn
经　典，副研究员，博士；电子信箱：jingdian@shsmu.edu.cn。
基金资助:
上海市科学技术委员会科技创新行动计划(21JC1404000);上海市耳鼻疾病转化医学重点实验室(14DZ2260300)

Three-dimensional imaging of cochlear nerve fibers based on multi-scale resolution

CHENG Yaqiong¹^,²^,³(), DU Yiwei¹^,²^,³, LIU Sidi¹^,²^,³, JING Dian¹^,²^,³(), WU Hao¹^,²^,³()

^1.Department of Otorhinolaryngology, Head and Neck Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^2.Ear Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China
^3.Shanghai Key Laboratory of Translation Medicine on Ear and Nose Disease, Shanghai 200125, China

Received:2025-03-20 Accepted:2025-05-09 Online:2025-11-28 Published:2025-12-03
Contact: WU Hao, E-mail: wuhao@shsmu.edu.cn
JING Dian, E-mail: jingdian@shsmu.edu.cn.
Supported by:
Science and Technology Innovation Action Plan of Shanghai Municipal Science and Technology Commission(21JC1404000);Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases(14DZ2260300)

摘要/Abstract

摘要：

目的·通过多尺度分辨率三维成像构建耳蜗及周围组织整体成像的标准化研究平台，实现生理状态下耳蜗的三维可视化分析。方法·8~12周龄胸腺细胞抗原1（Thy1）驱动黄色荧光转基因小鼠（Thy1-YFP-16小鼠）经心脏灌注后，获得单个耳蜗或耳蜗及全脑样品。使用PEGASOS技术进行组织透明化，再通过激光共聚焦显微镜4×和10×物镜对耳蜗及周围组织进行整体深度直接成像。对单个耳蜗样品，采用神经丝蛋白200（NF200）抗体及碘化丙啶（PI）荧光染料进行全组织免疫荧光染色，再结合PEGASOS和TESOS透明化及包埋技术进行处理；通过石蜡切片机序列切片及激光共聚焦显微镜63×油镜对整个耳蜗分层成像；使用ImageJ进行图像拼接获得三维图像后，通过Imaris软件处理图像、追踪单根神经纤维并测量长度，MATLAB计算路径曲率，GraphPad Prism进行统计分析。结果·通过组织透明化技术成功制备耳蜗及周围组织的透明化样品。共聚焦显微镜的中、低倍镜直接成像获得耳蜗及周围组织中等分辨率三维图像，重构了耳蜗的三维形态及其与前庭神经等周围结构相邻交错的空间位置；显微镜高倍镜下的连续切拍实现了整体耳蜗样本的单细胞分辨率三维成像，显示了耳蜗内THY1⁺纤维从毛细胞侧到蜗轴内的整体路径，并通过单根神经纤维追踪展示了螺旋神经节附近耳蜗传出神经系统与听神经纤维的不同走行特点。结论·利用PEGASOS、TESOS技术可实现对耳蜗及周围整体组织的多尺度分辨率三维成像；结合全组织免疫荧光染色，可用于获取耳蜗与毗邻组织、耳蜗内细胞与纤维的三维空间关系，实现对单神经纤维的路径追踪。

关键词: 耳蜗, 神经纤维, 组织透明化, 三维成像

Abstract:

Objective ·To establish a standardized research platform for comprehensive imaging of the cochlea and its surrounding tissues through multi-scale resolution three-dimensional (3D) imaging, thereby enabling 3D visualization and analysis of the cochlea under physiological conditions. Methods ·The cochlea or combined cochlea and whole brain samples were obtained from 8‒12-week-old thymus cell antigen 1 (Thy1) -driven yellow fluorescent transgenic mice (Thy1-YFP-16 mice) after cardiac perfusion. Tissue clearing was performed using PEGASOS, followed by direct imaging of the cochlea and surrounding tissues through their entire depth using a laser confocal microscope with 4× and 10× objectives. For individual cochlear samples, whole tissue immunofluorescence staining was performed in combination with a neurofilament-200 (NF200) antibody and propidium iodide (PI) fluorescent dye, in the process of tissue clearing and embedding using PEGASOS and TESOS techniques. The entire cochlea was then subjected to serial sectioning with a paraffin microtome, and each section was imaged using a laser confocal microscope equipped with a 63× oil immersion objective. 3D reconstruction was performed using ImageJ for image stitching, followed by image processing, single-nerve-fiber tracking, and length measurement conducted in Imaris software. Fiber path curvature was calculated using MATLAB, and statistical analyses were performed with GraphPad Prism. Results ·Transparent cochlear and surrounding tissue samples were successfully prepared using tissue clearing techniques. The cochlea and its surrounding tissues were directly imaged by low- and medium-magnification objectives on the confocal microscope, which reconstructed the 3D morphology of the cochlea and delineated its spatial relationship with adjacent structures such as the vestibular nerve. Serial sectioning and imaging with a high-magnification objective enabled 3D imaging of the entire cochlear sample at single-cell resolution. This approach revealed the overall pathway of THY1⁺ fibers within the cochlea, extending from the hair cell region to the modiolus. Single nerve fiber tracing revealed distinct trajectory characteristics of the cochlear efferent nervous system and auditory nerve fibers near the spiral ganglion. Conclusion ·The PEGASOS and TESOS techniques enable multi-scale resolution 3D imaging of the cochlea and its surrounding tissues as an integrated whole. When combined with whole-tissue immunofluorescence staining, this methodology can be used to delineate the 3D spatial relationships between the cochlea and adjacent tissues, as well as between cells and nerve fibers within the cochlea itself. Furthermore, it allows for the tracing of individual nerve fiber pathways.

Key words: cochlea, nerve fiber, tissue clearing, 3D imaging

中图分类号:

R764.35

成亚琼, 杜一唯, 刘思迪, 经典, 吴皓. 基于多尺度分辨率的耳蜗神经纤维三维成像[J]. 上海交通大学学报（医学版）, 2025, 45(11): 1421-1431.

CHENG Yaqiong, DU Yiwei, LIU Sidi, JING Dian, WU Hao. Three-dimensional imaging of cochlear nerve fibers based on multi-scale resolution[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(11): 1421-1431.

图/表 4

图1 双侧耳蜗及其周围组织透明化流程和2.88 μm×2.88 μm×10.00 μm分辨率三维成像Note： A. The process of tissue clearing. B. Observation of the cochleae and whole brain sample before and after tissue clearing. Each square grid has a side length of 2 mm. C. 3D imaging of the bilateral cochleae and whole brain tissues.

Fig 1 Clearing process of bilateral cochleae and surrounding tissues and 3D imaging at a resolution of 2.88 μm×2.88 μm×10.00 μm

图2 单侧耳蜗及其周围组织1.00 μm×1.00 μm×5.00 μm分辨率三维成像Note： A. 3D imaging of the cochlea and its surrounding tissues. The regions were segmented using different colours, including the cochlea (cyan), spiral ganglions and auditory nerves (off-white), cochlear nucleus (gold), vestibule and vestibular nerves (pink), and brainstem tissues (green). B. Cochlea-cochlear nucleus neural pathway. The circle indicates cochlear root neurons. C. Three subregions of the cochlear nucleus, including AVCN (red), PVCN (green), and DCN (gold).

Fig 2 3D imaging of the unilateral cochlea and its surrounding tissues at a resolution of 1.00 μm×1.00 μm×5.00 μm

图3 耳蜗荧光染色及0.24 μm×0.24 μm×1.50 μm分辨率的三维整体成像Note： A. 3D image of the cochlea. B. Distribution of outer hair cells (marked by *) in relation to THY1+ nerve fibers (green). C. Distribution of inner hair cells (marked by *) and supporting cells (marked by +) in relation to THY1+ nerve fibers (green). D. Position of nerve fiber bundles (green) in relation to the cochlear aperture. E‒H. Paths of THY1+ nerve fibers (green) in the cochlea. I/J. Distribution of NF200 expression (blue) in different parts of the cochlea. Red represents PI; green represents THY1-YFP;blue represents NF200.

Fig 3 Fluorescent staining of the cochlea and 3D imaging at a resolution of 0.24 μm×0.24 μm×1.50 μm

图4 耳蜗内单根神经纤维追踪结果及2种神经纤维的路径差异Note： A. 3D images of local spiral ganglia and fibers at a resolution of 0.24 μm×0.24 μm×1.50 μm. B/C. Two types of fiber pathways obtained by Imaris software tracing, an auditory nerve fiber (B) and MOC/LOC fibers (C). D. Local magnification of the 3D imaging of cochlear and labelling of fiber tracing at high resolution. E/F. Comparison of fiber length (E) and path curvature (F) between localized auditory nerve fibers and the MOC/LOC fibers within the cochlea. Neuronal designations were used to represent their corresponding fibers, with SGNs referring to auditory nerve fibers and OGNs (olivocochlear neurons) referring to MOC/LOC fibers. The orange/yellow dots represent the cell bodies of the tracked auditory nerves in B and D. ①P=0.002, ②P=0.113.

Fig 4 Single nerve fiber tracing in the cochlea and differences in pathways characteristics between two types of neural fibers

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基于多尺度分辨率的耳蜗神经纤维三维成像

Three-dimensional imaging of cochlear nerve fibers based on multi-scale resolution

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