Summary

可见光光学相干断层扫描光纤图与同一小鼠视网膜的共聚焦图像的对齐

Published: June 30, 2023
doi:

Summary

本方案概述了 体内可见光光学相干断层扫描纤维成像(vis-OCTF)图像与同一小鼠视网膜的 离体 共聚焦图像对齐的步骤,以验证 在体内 图像中观察到的视网膜神经节细胞轴突束形态。

Abstract

近年来, 活体 视网膜成像提供了关于生物系统和过程的无创、实时和纵向信息,越来越多地用于客观评估眼部疾病的神经损伤。同一视网膜的 离体 共聚焦成像通常是必要的,以验证 体内 发现,尤其是在动物研究中。在这项研究中,我们展示了一种将小鼠视网膜 的离体 共聚焦图像与其 体内 图像对齐的方法。一种新的临床成像技术称为可见光光学相干断层扫描纤维成像(vis-OCTF),用于获取小鼠视网膜的 活体 图像。然后,我们对与“金标准”相同的视网膜进行了共聚焦成像,以验证 体内 与OCTF图像的对比。这项研究不仅能够进一步研究分子和细胞机制,而且为灵敏客观地评估 体内神经损伤奠定了基础。

Introduction

视网膜神经节细胞 (RGC) 在视觉信息处理中起着关键作用,通过其在内丛状层 (IPL) 中的树突树接收突触输入,并通过它们在视网膜神经纤维层 (RNFL) 中的轴突将信息传递到大脑 1,2,3,4。在青光眼等疾病中,早期 RGC 变性可能导致患者和啮齿动物模型 RNFL、神经节细胞层 (GCL)、IPL 和视神经的细微变化 5,6,7,8,9。因此,及早发现RGC的这些形态变化对于及时干预以防止RGC和视力丧失至关重要。

我们最近开发了一种新的临床成像技术,称为可见光光学相干断层扫描(vis-OCT),以满足RGC损伤的体内监测需求。Vis-OCT 提高了轴向分辨率,在视网膜中达到 1.3 μm10,11允许可视化 RNFL 中的单个 RGC 轴突束。随后,建立了 vis-OCT 纤维成像 (vis-OCTF) 来跟踪和量化小鼠111213 中单轴突束水平的 RGC 损伤。然而,通常需要对与金标准相同的视网膜进行离体共聚焦成像,以验证体内发现。因此,本研究将演示如何将 vis-OCTF 获取的体内图像与同一小鼠视网膜的离体共聚焦图像对齐。该协议旨在通过离体共聚焦成像验证体内发现,并为检查疾病条件下 RGC 损伤的分子和细胞变化奠定基础。

Protocol

所有动物程序均由弗吉尼亚大学机构动物护理和使用委员会批准,并符合美国国立卫生研究院 (NIH) 的动物使用指南。有关本协议中使用的所有材料、试剂和仪器的详细信息,请参阅 材料表 。 1. 体内 vs-OCT成像 vis-OCT 系统使用使用超连续光源的小动物 vs-OCT 系统对小鼠眼睛进行成像,该系统提供 480 nm 和 650 nm 之间的可见光照明?…

Representative Results

将复合 vs-OCT 纤维图与用 Tuj-1 免疫染色的 RGC 轴突的平面视网膜的相应共聚焦图像进行比较(图 1D,上图)。通过 vis-OCTF 成像的轴突束可以与共聚焦图像上的 Tu-j1 标记的轴突束相匹配。在纤维图图像中,与周围的轴突束相比,血管通常表现出可区分的分支结构,这可以与共聚焦图像上的ICAM-2标记的血管相匹配(图1D,下图)。 ?…

Discussion

该协议中有两个步骤需要注意。首先,有必要确保动物处于深度麻醉状态,并且在 vs-OCT 成像之前他们的眼睛完全散瞳。如果小鼠没有得到充分的麻醉,它们的快速呼吸可能导致 面部 图像的不稳定运动,这可能会对纤维图的质量产生不利影响。此外,扩张不足也会对图像质量产生负面影响,因为虹膜可能会阻挡光线,阻止光线到达视网膜。其次,在灌注后但从小鼠眼窝中取出眼球之前,标…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项研究得到了青光眼研究基金会 Shaffer Grant、4-CA Cavalier 合作奖、R01EY029121、R01EY035088 和圣殿骑士眼科基金会的支持。

Materials

Equipment
Halo 100 Opticent Health, Evanston, IL
Zeiss LSM800 microscope Carl Zeiss
Drugs and antibodies
4% paraformaldehyde (PFA) Santz Cruz Biotechnology, SC-281692 1-2 drops
Bovine serum albumin powder Fisher Scientific, BP9706-100 1:10
Donkey anti Mouse Alexa Fluor 488 dye Thermo Fisher Scientific, Cat# A-21202 1:1,000
Donkey anti rat Alexa Fluor 594 dye Thermo Fisher Scientific, Cat# A-21209 1:1,000
Euthasol (a mixture of pentobarbital sodium (390 mg/mL) and phenytoin sodium (50 mg/mL)) Covetrus, NDC 11695-4860-1 15.6 mg/mL
Ketamine Covetrus, NADA043304 114 mg/kg
Mouse anti-Tuj1 A gift from Anthony J. Spano, University of Virginia 1:200
Normal donkey serum(NDS) Millipore Sigma, S30-100 mL 1:100
Phosphate-buffered saline (PBS, 10x), pH 7.4
(Contains 1370 mM NaCl, 27 mM KCl, 80 mM Na2HPO4, and 20 mM KH2PO4)
Thermo Fisher Scientific, Cat# J62036.K3 1:10
Rat anti-ICAM-2 BD Pharmingen, Cat#553325 1:500
Tropicamide drops  Covetrus, NDC17478-102-12
Triton X-100
(Reagent Grade)
VWR, CAS: 9002-93-1 1:20
Vectashield mounting medium Vector Laboratories Inc. H2000-10
Xylazine Covetrus, NDC59399-110-20 17 mg/kg

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Cite This Article
Chang, S., Xu, W., Fan, W., McDaniel, J. A., Grannonico, M., Miller, D. A., Liu, M., Zhang, H. F., Liu, X. Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina. J. Vis. Exp. (196), e65237, doi:10.3791/65237 (2023).

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