Qambari Hassanain, Hein Martin, Balaratnasingam Chandrakumar, Yu Paula, Yu Dao-Yi
Centre for Ophthalmology and Visual Science, The University of Western Australia, Perth, Western Australia, Australia; Lions Eye Institute, Perth, Western Australia, Australia.
Centre for Ophthalmology and Visual Science, The University of Western Australia, Perth, Western Australia, Australia; Lions Eye Institute, Perth, Western Australia, Australia.
Exp Eye Res. 2025 Aug;257:110410. doi: 10.1016/j.exer.2025.110410. Epub 2025 Apr 28.
Glial cells are one of the most numerous cell types in the vertebrate retina and they serve to support neurovascular function. The principal glial cell in the retina is the Müller cell, accounting for approximately 90 % of all retinal glial cells. Müller cells are phenotypically elongated in shape and were first described as 'radial fibers' by Heinrich Müller in 1851. Their structure spans the entire thickness of the retina, through all retinal layers from the internal to external limiting membrane. This unique three-dimensional spatial arrangement enables Müller cells' direct contact with almost all cell types in the retina to perform its function. Despite this, the current study of Müller cells has largely been limited to thin sections or in culture, which provide limited detail about its spatial arrangement and interconnection with other cell types. The novel technique described here enables the three-dimensional visualization of GFAP-positive Müller cell processes in rodent retina and is based on the isolated arterially perfused rat eye preparation. Our micro perfusion technique utilizes the microvasculature as the delivery channel to quickly and effectively preserve all retinal elements. Intravascular labelling enables visualization of the intact three-dimensional retinal microvasculature within its normal neuronal and glial confines. Additional immersion immunolabeling and subsequent clearing with RapiClear® enables the three-dimensional visualization of different retinal elements and their physical interaction. Volume rendering of confocal image stacks acquired from these specimens can facilitate the study of such interactions in normal and disease models to further our understanding. This technique may be replicated in human donor retinae for future investigations to provide insight into Müller cell form and spatial relationship with other cell types.
神经胶质细胞是脊椎动物视网膜中数量最多的细胞类型之一，它们有助于支持神经血管功能。视网膜中的主要神经胶质细胞是米勒细胞，约占所有视网膜神经胶质细胞的90%。米勒细胞在表型上呈细长形，1851年由海因里希·米勒首次描述为“放射状纤维”。它们的结构跨越视网膜的整个厚度，从内界膜到外界膜贯穿所有视网膜层。这种独特的三维空间排列使米勒细胞能够与视网膜中的几乎所有细胞类型直接接触以执行其功能。尽管如此，目前对米勒细胞的研究很大程度上局限于薄片或培养物，这提供了关于其空间排列以及与其他细胞类型相互连接的有限细节。这里描述的新技术能够对啮齿动物视网膜中GFAP阳性的米勒细胞突起进行三维可视化，并且基于分离的动脉灌注大鼠眼标本。我们的微量灌注技术利用微血管作为输送通道，快速有效地保存所有视网膜成分。血管内标记能够在其正常的神经元和神经胶质范围内对完整的三维视网膜微血管进行可视化。额外的浸入式免疫标记以及随后用RapiClear®进行的清除能够对不同的视网膜成分及其物理相互作用进行三维可视化。从这些标本获取的共聚焦图像堆栈的体绘制可以促进在正常和疾病模型中对这种相互作用的研究，以加深我们的理解。这种技术可以在人类供体视网膜中复制，用于未来的研究，以深入了解米勒细胞的形态及其与其他细胞类型的空间关系。
