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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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신경과학

光相一致性断层扫描血管造影评估的视网膜血管反应

Published: March 26, 2020
doi:
10.3791/60948
, ,
1Department of Ophthalmology,USC Roski Eye Institute, 2USC Ginsberg Institute for Biomedical Therapeutics,Keck School of Medicine of the University of Southern California

Summary

本文介绍了一种测量视网膜血管反应的方法，该方法使用气体呼吸刺激技术在获取视网膜图像的同时，为人类受试者测量体内视网膜血管反应反应。

Abstract

视网膜的血管供应已被证明通过血管收缩和血管扩张动态适应，以适应视网膜的代谢需求。这个过程，称为视网膜血管反应（RVR），由神经血管耦合进行调解，神经血管耦合在视网膜血管疾病（如糖尿病视网膜病变）中很早就受损。因此，一种临床上可行的血管功能评估方法在研究和临床环境中可能都引起很大的兴趣。最近，经FDA批准光学相干断层扫描血管造影（OCTA），这是一种非侵入性、最小风险和无染料血管造影方法，具有毛细血管水平分辨率，从而在毛细管水平上对视网膜血管图进行体内成像。同时，几位研究者也显示了RVR的生理和病理变化。本手稿中所示的方法旨在使用 OCTA 研究 RVR，无需更改临床成像程序或设备。它演示了视网膜和视网膜血管在暴露于高卡普或超氧化物条件下的实时成像。考试在 30 分钟内轻松进行，两名人员在 30 分钟内轻松进行，但主题不适或风险最小。该方法适用于其他眼科成像设备，应用可能因气体混合物和患者群的组成而异。这种方法的一个优势是，它允许在体内的人类受试者的毛细血管功能下研究视网膜血管功能。此方法的局限性主要是 OCTA 和其他视网膜成像方法（包括成像伪像和受限的动态范围）。该方法的结果为视网膜的OCT和OCTA图像。这些图像适用于在商用 OCT 或 OCTA 设备上可能进行的任何分析。然而，一般方法可以适应任何形式的眼科成像。

Introduction

视网膜的代谢需求取决于由动脉、毛细血管和静脉1调节良好的系统提供的充足和持续的氧气供应。几项研究表明，大口径人类视网膜血管的功能可以在体内评估各种生理22，3，4，53,4,5和药理学66，77刺激。此外，这种血管系统的异常功能在视网膜血管疾病中很常见，如糖尿病视网膜病变，视网膜血管反应性（RVR）即使在其早期阶段88、99通过气体刺激9和闪烁光实验5,5、10、11,11都得到衰减。视网膜血管危险因素，如吸烟也与RVR12和视网膜血流13受损有关。这些发现是重要的，因为视网膜血管疾病的临床症状发生在疾病过程相对较晚，并证明疾病的早期临床标志缺乏14。因此，评估RVR可以为早期评估可能引发或加重视网膜退行性疾病的异常提供有用的血管完整性测量。

以前的RVR实验通常依赖于激光血流计9或装有特殊滤镜15的玻璃相机等设备进行视网膜图像采集。然而，这些技术针对直径较大的血管进行了优化，如Arterioles16和静脉15，这些血管不是气体、微量营养素和分子交换发生的地方。最近的一项研究能够量化毛细血管的RVR使用自适应光学成像17，但尽管提高了空间分辨率，这些图像有一个较小的场大小，并没有FDA批准临床使用18。

光学相干断层扫描血管造影（OCTA）最近出现，为FDA提供了一种经FDA批准的非侵入性和无染料血管造影方法，用于评估人类患者和受试者体内毛细血管水平变化。OCTA在临床实践中被广泛接受，作为评估视网膜血管疾病（如糖尿病视网膜病变19，视网膜静脉闭塞20，血管炎21和许多其他22个）毛细血管灌注损伤的有效工具。因此，OCTA为评估毛细管水平变化提供了绝佳的机会，在临床环境中，毛细管水平变化可有显著的空间和时间异质性23以及病理变化。我们小组最近证明，OCTA可用于量化毛细管2级视网膜血管对灵感氧生理变化的反应，这是视网膜血管收缩刺激16、24,24和二氧化碳，这是视网膜血管扩张刺激33，5。5

本文的目的是描述一种协议，使读者能够使用OCTA评估较小的动脉和毛细管床的视网膜血管反应。这些方法与Lu等人25中介绍的方法进行了调整，后者描述了脑血管反应与磁共振成像的测量。虽然本文采用于OCTA成像2期间开发和使用的方法，但适用于具有相对简单和明显修改的其他视网膜成像设备。

Protocol

这项研究得到了南加州大学机构审查委员会的批准，并遵循了《赫尔辛基宣言》的原则。 1. 气体非再呼吸装置的安装 图1：非呼吸器图。完整设置已根据其功能和独立处理的频率分成三个独立的单元。其中包括：空气控制单元、非呼吸单元和主题/成像设?…

Representative Results

本实验的输出包括从脉冲氧仪获取的手动读数、气体暴露或OCTA扫描的定时以及原始的OCTA成像数据。OCTA 图像由 OCT B 扫描和与每个 B 扫描关联的非相关信号组成。数据参数由设备的规格给出。使用了一台集中波长为1040~1060nm的扫描源激光平台OCTA机。这些图像提供20μm的横向分辨率，光学轴向分辨率为6.3μm。通常，OCTA 数据以 2D 面带格式显示，如代表图 2所示。有许多指标可用?…

Discussion

刚才描述的方法是气体呼吸挑衅实验的完整协议，允许在特定时间点的受控环境中测量受试者的RVR，无需修改OCTA成像装置，并且对受试者的不适或风险最小。此设置的描述方式允许轻松修改以满足研究人员的需求。它可以容纳额外的管道，以适应不同的诊所房间和某些元素，如内部管或肘关节可能被省略或替代与其他组件。图 1显示了设置的关键部分（空气控制单元、非呼?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了NIH K08EY027006、R01EY030564、UH3NS100614、卡尔蔡司梅迪克公司（加利福尼亚州都柏林）的研究资助和预防失明研究（纽约，纽约）的无限制部门资助。

Materials

5% CO2 gas [5% CO2, 21% O2, 74% N2] (Compressed) Institution Dependent (Praxair)
Bacdown Disinfectant Detergent Decon Labs 8001 https://deconlabs.com/products/disinfectant-bdd/
Clean-Bor Tubes (35 mm Inner Diameter) Vacumed 1011-108 http://www.vacumed.com/zcom/product/Product.do?compid=27&skuid=1197
Cuff adapter for Douglas bag filling Vacumed 22254 http://www.vacumed.com/zcom/product/Product.do?compid=27&prodid=343
Douglas bag (200-liter capacity) Harvard Apparatus 500942 https://www.harvardapparatus.com/douglas-bag.html
Elbow Joint (Inner Diameter 19mm/ Outer Diameter 22 mm), Modified in House
Fingertip Pulse Oximeter (Pro-Series) CMS CMS 500DL https://www.walmart.com/ip/Pro-Series-CMS-500DL-Fingertip-Pulse-Oximeter-Blood-Oxygen-Saturation-Monitor-with-silicon-cover-batteries-and-lanyard/479049154
Gas Delivery Tube (22 mm Inner Diameter) Modified in House
Gas filling tube (1/8" for compressed gas)
Hydrogen Peroxide Cleaner Disinfectant Wipes Clorox Healthcare 30824 https://www.cloroxpro.com/products/clorox-healthcare/hydrogen-peroxide-cleaner-disinfectants/?gclid=EAIaIQobChMIk-KG4vi15QIVcRh9Ch0NNwLPEAAYASAAEgJIa_D_BwE&gclsrc=aw.ds
Lubricant Eye Drops Refresh Refresh Plus https://www.refreshbrand.com/Products/refresh-plus
Manual Directional Control Valves: Three-Way T-Shape Stopcock Type (Inner Diameter 28.6 mm, Outer Diameter 35 mm) Hans Rudolph 2100C Series www.rudolphkc.com
Medical O2 (Compressed) Institution Dependent
Mouth piece (Silicone, Model #9061) Hans Rudolph 602076 www.rudolphkc.com
OCTA Imaging Device (PLEX Elite 9000) Carl Zeiss Meditec, Dublin, CA, USA https://www.zeiss.com/meditec/int/product-portfolio/optical-coherence-tomography-devices/plex-elite-9000-swept-source-oct.html
Phenylephrine Hydrochloride Ophthalmic Solution, USP 2.5% Paragon Bioteck, Inc NDC 42702-102-15 https://paragonbioteck.com/products/diagnostics/phenylephrine-hydrochloride-ophthalmic-solution-usp-2-5/
Plastic Nose Clip Sterile Foam CS100 Sklar Sterile 96-2951 https://www.sklarcorp.com/disposables/plastic/plastic-nose-clip-sterile-foam-box-of-100.html
Proparacaine Hydrochloride Ophthalmic Solution, USP .5% Bausch + Lomb NDC 24208-730-06 https://www.bausch.com/ecp/our-products/rx-pharmaceuticals/generics
Regulator (tank dependent- 5% CO2: Fisherbrand Mulitstage Gas Cylinder Regulators) Genstar Technologies Company 10575150 https://www.fishersci.com/shop/products/fisherbrand-multistage-cylinder-regulators-22/10575150?keyword=true
Regulator (tank dependent- Oxygen: Fisherbrand Multistage Gas Cylinder Regulators) Genstar Technologies Company 10575145 https://www.fishersci.com/shop/products/fisherbrand-multistage-cylinder-regulators-22/10575145?keyword=true
Rubber Tubing (Inner diameter 19 mm, Outer diameter 27 mm), Made in House
Sealing tape- Parafilm Wrap (2" Wide) Cole Parmer PM992 https://www.coleparmer.com/i/parafilm-pm992-wrap-2-wide-250-ft-roll/0672050?PubID=VV&persist=True&ip=no&gclid=EAIaIQobChMInY3vqomz5QIVfyCtBh1VSg64EAAYASAAEgJ9n_D_BwE
Sterile Alcohol Prep Pads Medline MDS090670 https://www.medline.com/product/Sterile-Alcohol-Prep-Pads/Swab-Pads/Z05-PF03816
Tropicamide Ophthalmic Solution, USP 1% Akorn NDC 17478-102-12 http://www.akorn.com/prod_detail.php?ndc=17478-102-12
Tubing Adapter, Made in House
Two-way non-rebreathing valve (2600 Series- Inner Diameter 28.6 mm, Outer Diameter 35 mm) Hans Rudolph 2600 Series, UM-112078 www.rudolphkc.com
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Retinal Vascular ReactivityOptical Coherence Tomography Angiography (OCTA)Retinal Vascular FunctionRetinal Capillary FunctionRetinal Vascular IntegrityRetinal Vascular PathologyGas Delivery ApparatusNonrebreathing ValveAir Control UnitPulse OximeterOCTA Imaging

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Kushner-Lenhoff, S., Ashimatey, B. S., Kashani, A. H. Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography. J. Vis. Exp. (157), e60948, doi:10.3791/60948 (2020).
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