High efficiency labeling of nerve fibers in cleared tissue for light-sheet microscopy

IF 2.3 4区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Neuroscience Methods Pub Date : 2025-09-04 DOI:10.1016/j.jneumeth.2025.110567

Marta Rojas-Rodríguez , Elisa Imbimbo , Claudia Capitini , Irene Costantini , Giacomo Mazzamuto , Francesco Saverio Pavone , Ludovico Silvestri , Martino Calamai

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引用次数: 0

Abstract

Background

Tissue clearing techniques combined with light-sheet fluorescence microscopy (LSFM) enable high-resolution 3D imaging of biological structures without physical sectioning. While widely used in neuroscience to determine brain architecture and connectomics, their application for spinal cord mapping remains more limited, posing challenges for studying demyelinating diseases like multiple sclerosis. Myelin visualization in cleared tissues is particularly difficult due to the lipid-removal nature of most clearing protocols, and alternative immunolabeling approaches failed to reach satisfying results.

New method

To overcome these limitations, we developed a novel protocol named HELF -High Efficiency Labeling of Fibers- which takes advantage of a fluorescently labeled aminosterol, trodusquemine, which displays a strong affinity for cholesterol-rich membranes, and a supplementary round of fixation with glutaraldehyde.

Results and comparison with existing methods

The labeling with trodusquemine was tested in combination with various established tissue clearing techniques and compared with HELF, which resulted to be the best approach for providing high-brightness myelin staining in mouse spinal cord and brain, and in human brain samples. Finally, we demonstrated that HELF can be used to stain and image with LSFM a whole cleared mouse spinal cord.

Conclusions

Our data support the potential use of HELF coupled to LSFM as a practical tool for the evaluation of novel therapeutics for remyelination in preclinical models of CNS diseases.

查看原文本刊更多论文

薄层显微镜下清除组织中神经纤维的高效标记。

背景：组织清除技术与光片荧光显微镜（LSFM）相结合，无需物理切片即可实现生物结构的高分辨率3D成像。虽然在神经科学中广泛用于确定大脑结构和连接组学，但它们在脊髓制图中的应用仍然有限，这对研究多发性硬化症等脱髓鞘疾病提出了挑战。由于大多数清除方案的脂质去除性质，清除组织中的髓磷脂可视化特别困难，而替代的免疫标记方法未能达到令人满意的结果。新方法：为了克服这些限制，我们开发了一种名为HELF的新方案-纤维高效标记-利用荧光标记的氨基甾醇，trodusquemine，它对富含胆固醇的膜具有很强的亲和力，并与戊二醛进行补充固定。结果及与现有方法的比较：结合各种已建立的组织清除技术对trodusquemine标记进行了测试，并与HELF进行了比较，结果表明该方法是在小鼠脊髓和大脑以及人脑样品中提供高亮度髓磷脂染色的最佳方法。最后，我们证明了HELF可以用LSFM染色和成像整个清除的小鼠脊髓。结论：我们的数据支持HELF联合LSFM作为评估中枢神经系统疾病临床前模型中髓鞘再生新疗法的实用工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Neuroscience Methods 医学-神经科学

CiteScore

7.10

自引率

3.30%

发文量

226

审稿时长

52 days

期刊介绍： The Journal of Neuroscience Methods publishes papers that describe new methods that are specifically for neuroscience research conducted in invertebrates, vertebrates or in man. Major methodological improvements or important refinements of established neuroscience methods are also considered for publication. The Journal''s Scope includes all aspects of contemporary neuroscience research, including anatomical, behavioural, biochemical, cellular, computational, molecular, invasive and non-invasive imaging, optogenetic, and physiological research investigations.