优化组织清除方法以改善视网膜和视神经的成像

IF 2.3 4区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Neuroscience Methods Pub Date : 2025-09-01 DOI:10.1016/j.jneumeth.2025.110563

Aubin Mutschler , Volha V. Malechka , Nikita Bagaev , Petr Baranov , Jonathan R. Soucy

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引用次数: 0

摘要

基因和细胞疗法有望恢复遗传性和晚期视神经病变患者的视力。准确评估这些疗法需要先进的成像方法，可以看到移植细胞在完整的视网膜组织。我们提出了一种针对小鼠视网膜和视神经优化的全贴装组织清除工作流程，以改善细胞移植后供体神经元整合的可视化。对五种清除方法进行了评估，并通过在鳞片中添加聚乙烯醇来改善荧光保存，从而开发了一种改进的方案ScaleH。结果在测试的方法中，视网膜的透明度（增加46 %）和免疫组织化学清晰度（增加89 %）最高。ScaleH保留了相当的清晰度，同时显著减少荧光衰减（32% %减少衰减）。ScaleH与内源性报告细胞和免疫标记兼容，能够在视网膜神经节细胞层中对移植的人干细胞来源的视网膜神经元进行详细成像，以及视神经中的神经突、小胶质细胞和细胞核的可视化。与其他清除方案相比，ScaleH在不影响光学清晰度的情况下提供了优越的荧光保留和稳定性。它与免疫染色和内源性荧光蛋白的相容性支持在成像方面的广泛应用。结论scaleh是一种可靠、高分辨率的视网膜和视神经成像方法。它有助于对再生眼科中供体细胞整合的可靠评估，并可能广泛应用于神经生物学研究。

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Optimizing tissue clearing methods for improved imaging of whole-mount retinas and optic nerves

Background

Gene and cell therapies hold promise for restoring vision in hereditary and advanced optic neuropathies. Accurate evaluation of these therapies requires advanced imaging methods that can visualize transplanted cells within intact retinal tissue.

New method

We present a whole-mount tissue-clearing workflow optimized for the mouse retina and optic nerve to improve visualization of donor neuron integration following cell transplantation. Five clearing methods were evaluated, and a modified protocol, ScaleH, was developed by adding polyvinyl alcohol to ScaleS to improve fluorescence preservation.

Results

ScaleS yielded the highest transparency (46 % increase) and immunohistochemical clarity (89 % increase) in the retina among the tested methods. ScaleH retained comparable clarity while significantly reducing fluorescence decay over time (32 % less decay). ScaleH was compatible with endogenous reporters and immunolabeling, enabling detailed imaging of transplanted human stem cell-derived retinal neurons in the retinal ganglion cell layer, as well as visualization of neurites, microglia, and cell nuclei in the optic nerve.

Comparison with existing methods

Compared to other clearing protocols, ScaleH provided superior fluorescence retention and stability without compromising optical clarity. Its compatibility with both immunostaining and endogenous fluorescent proteins supports broad application in imaging.

Conclusions

ScaleH is a reliable and high-resolution clearing method for imaging whole-mount retinas and optic nerves. It facilitates robust assessment of donor cell integration in regenerative ophthalmology and may be broadly applicable in neurobiological research.

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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.