Otolaryngology, Department of Surgery, University of Melbourne, Royal Victorian Eye and Ear Hospital, East Melbourne, Australia.
Biological Optical Microscopy Platform, University of Melbourne, Parkville, Australia.
Hear Res. 2020 Jul;392:107956. doi: 10.1016/j.heares.2020.107956. Epub 2020 May 5.
Visualisation of cochlear histopathology in three-dimensions has been long desired in the field of hearing research. This paper outlines a technique that has made this possible and shows a research application in the field of hearing protection after cochlear implantation. The technique utilises robust immunofluorescent labelling followed by effective tissue clearing and fast image acquisition using Light Sheet Microscopy. We can access the health of individual components by immunofluorescent detection of proteins such as myosin VIIa to look at cochlear hair cells, NaKATPase alpha 3 to look at spiral ganglion neurons, and IBA1 to look at macrophages within a single cochlea, whilst maintaining the integrity of fine membranous structures and keeping the cochlear implant in place. This allows the tissue response to cochlear implantation to be studied in detail, including the immune reaction to the implant and the impact on the structure and health of neural components such as hair cells. This technique reduces time and labour required for sectioning of cochleae and can allow visualisation of cellular detail. Use of image analysis software allows conversion of high-resolution image stacks into three-dimensional interactive data sets so volumes and numbers of surfaces can be measured. Immunofluorescent whole cochlea labelling and Light Sheet Microscopy have the capacity to be applied to many questions in hearing research of both the cochlea and vestibular system.
在听力研究领域，长期以来一直希望能够对耳蜗组织病理学进行三维可视化。本文概述了一种能够实现这一目标的技术，并展示了该技术在耳蜗植入后的听力保护研究中的应用。该技术利用强大的免疫荧光标记，然后通过有效的组织透明化和使用光片显微镜进行快速图像采集。我们可以通过免疫荧光检测肌球蛋白 VIIa 等蛋白质来检测单个耳蜗内的毛细胞、NaKATPase alpha 3 来检测螺旋神经节神经元，以及 IBA1 来检测巨噬细胞，从而了解各个组件的健康状况，同时保持精细膜结构的完整性并使耳蜗植入物保持原位。这使得可以详细研究耳蜗植入后的组织反应，包括对植入物的免疫反应以及对毛细胞等神经成分的结构和健康的影响。该技术减少了耳蜗切片所需的时间和劳动力，并可以实现细胞细节的可视化。使用图像分析软件可以将高分辨率的图像堆栈转换为三维交互式数据集，从而可以测量体积和表面数量。免疫荧光全耳蜗标记和光片显微镜有能力应用于耳蜗和前庭系统的听力研究中的许多问题。
