使用流量增强超声进行眼部深层血管成像
Summary
我们提出了一种非侵入性超声技术，用于在不使用造影剂的情况下在眼睛中生成三维血管造影。
Abstract
眼睛内的视网膜是体内对能量要求最高的组织之一，因此需要从丰富的血液供应中输送高速率的氧气。脉络膜的毛细血管层排列在视网膜的外表面，是大多数脊椎动物视网膜中氧气的主要来源。然而，这种血管床由于其位于高度吸光的视网膜后面，因此使用传统光学技术进行成像具有挑战性。在这里，我们描述了一种高频超声技术，随后的流动增强，以高时空分辨率对眼睛的深血管床（0.5-3厘米）进行成像。这种非侵入性方法在有核红细胞的物种（非哺乳动物和胎儿动物模型）中效果很好。它允许在不使用造影剂的情况下生成非侵入性三维血管造影，并且它独立于血流角，具有比基于多普勒的超声成像技术更高的灵敏度。
Introduction
脊椎动物视网膜上的高新陈代谢在两种截然不同的需求之间进行了内在的权衡;高血流速和没有血管的光路。为了避免注注红细胞的视觉障碍，所有脊椎动物的视网膜通过光感受器后面的一片毛细血管（脉络膜毛细血管）接收氧气和营养物质1，2，3。然而，这种营养和氧气的单一来源对视网膜的厚度施加了扩散限制4，5，因此许多视觉活性物种拥有各种精心设计的血管网络，为这种代谢活跃的器官提供额外的血液供应6。这些血管床包括哺乳动物和一些鱼类视网膜内部层的血管4，7，8，9，10，许多鱼类，爬行动物和鸟类中发现的视网膜内侧（面向光线）的血管4，11，12，13，以及鱼类脉络膜，脉络膜爬行动物的反流血管排列奇迹，允许产生超大气氧分压14，15，16，17，18，19，20。尽管这些额外的非脉络膜视网膜营养供应途径在促进优越视力4的代谢需求方面起着至关重要的作用，但这些血管结构的三维解剖结构知之甚少，限制了我们对脊椎动物眼睛形态进化的理解。
传统上，视网膜血液供应是使用光学技术（例如眼底镜检查）进行研究的。这类技术以高分辨率21 提供有关非脉络膜血管解剖学的高通量非破坏性信息，因此很容易用于视网膜血管结构异常的临床诊断22。然而，在这些光学技术中，视网膜色素上皮吸收透射光并限制视深，从而在不使用造影剂的情况下减少有关脉络膜结构和功能的信息21。在光学相干断层扫描（OCT）中也遇到了类似的深度限制。该技术可以使用光波生成高分辨率的眼底血管造影，但以深度穿透为代价23，而增强的深度成像OCT可以以视网膜成像质量为代价可视化脉络膜24。磁共振成像克服了眼底镜检查和OCT的光学局限性，可以绘制视网膜中的血管层，尽管分辨率较低25。组织学和微量计算断层扫描（μCT）保持了光学技术的高分辨率，并提供了有关全眼血管形态的信息4，但这两种技术都需要眼部采样，因此在临床或稀有或濒危物种中是不可能的。为了克服这些已建立的视网膜成像技术的一些局限性，这里的研究提出了一种关于麻醉动物的超声方案，其中通过应用一种可比较的技术，在一系列间隔相等的二维超声扫描 上以计算机 形式绘制血液运动，这些扫描跨越整个眼睛，如前所述用于胚胎和心血管成像26，27，28 和OCT血管造影29。这种方法允许在不使用造影剂的情况下生成非侵入性的三维深眼血管造影，并为绘制不同物种的眼睛内血流分布开辟了新的途径。
Protocol
以下协议是在丹麦食品，农业和渔业部，丹麦兽医和食品管理局的丹麦动物实验检查局的许可下执行的（许可证号为2016-15-0201-00835）。 1. 麻醉和超声介质 麻醉研究动物。注意：适当麻醉的类型和剂量高度依赖于物种。一般来说，浸渍式麻醉剂，如MS-222（3-氨基苯甲酸乙酯甲磺酸）、苯佐卡因（4-氨基苯甲酸乙酯）和丙泊酚（2，6-二异丙基苯酚）在鱼类和两栖动物中?…
Representative Results
用于成像眼睛血管床的流量增强超声技术可以应用于一系列物种，目前已用于46种不同的脊椎动物物种（图1， 表1）。非成体哺乳动物脊椎动物中有核红细胞的存在与电影记录中的静态组织相比，流动血液提供了积极的对比（补充文件2）。然而，当逐帧分析时，血液和周围组织之间的明显区别不太明显（图2A）。该协议中描述?…
Discussion
使用流量增强超声的血管成像为眼睛脉管系统的非侵入性成像提供了一种新方法，与现有技术相比具有多项优势，但具有其内在局限性。流量增强超声的主要优点是能够生成景深超过视网膜色素上皮的眼部血管造影，这限制了光学技术的景深。在超声成像中，空间分辨率和景深最终由超声换能器频率决定，其中较高的频率会增加空间分辨率，但代价是景深较浅，因此换能器频率的选择引入了图像?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作已获得嘉士伯基金会（CF17-0778;CF18-0658），伦德贝克基金会（R324-2019-1470;R346-2020-1210），Velux基金会（00022458），A.P. Møller医学科学促进基金会，Marie Skłodowska-Curie资助协议（第754513号）下的欧盟地平线2020研究和创新计划以及奥胡斯大学研究基金会。
Materials
| MS-222 | Sigma | E10521-50G | |
| Benzocaine | Sigma | E-1501 | |
| Propofol | B Braun | 12260470_0320 |
|
| Alfaxalon | Jurox | NA | |
| Isoflurane | Zoetis | 50019100 | |
| Ultrasound scanner | VisualSonics | Vevo 2100 |
References
- Yu, C. Q., Schwab, I. R., Dubielzig, R. R. Feeding the vertebrate retina from the Cambrian to the Tertiary. Journal of Zoology. 278 (4), 259-269 (2009).
- Yu, D. Y., Cringle, S. J. Oxygen distribution and consumption within the retina in vascularised and avascular retinas and in animal models of retinal disease. Progress in Retinal and Eye Research. 20 (2), 175-208 (2001).
- Country, M. W. Retinal metabolism: A comparative look at energetics in the retina. Brain Research. 1672, 50-57 (2017).
- Damsgaard, C., et al. Retinal oxygen supply shaped the functional evolution of the vertebrate eye. Elife. , 8 (2019).
- Buttery, R. G., Hinrichsen, C. F. L., Weller, W. L., Haight, J. R. How thick should a retina be? A comparative study of mammalian species with and without intraretinal vasculature. Vision Research. 31 (2), 169-187 (1991).
- Ames, A., Li, Y., Heher, E., Kimble, C. Energy metabolism of rabbit retina as related to function: high cost of Na+ transport. The Journal of Neuroscience. 12 (3), 840-853 (1992).
- Chase, J. The Evolution of retinal vascularization in mammals: A comparison of vascular and avascular retinae. Ophthalmology. 89 (12), 1518-1525 (1982).
- Johnson, G. L. Ophthalmoscopic studies on the eyes of mammals. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 254 (794), 207-220 (1968).
- Johnson, G. L. I. Contributions to the comparative anatomy of the mammalian eye, chiefly based on ophthalmoscopic examination. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 194 (194-206), 1-82 (1901).
- Rodriguez-Ramos Fernandez, J., Dubielzig, R. R. Ocular comparative anatomy of the family Rodentia. Veterinary Ophthalmology. 16, 94-99 (2013).
- Copeland, D. E. Functional vascularization of the teleost eye. Current Topics in Eye Research. 3, 219-280 (1980).
- Meyer, D. B., Crescitelli, F. . The Visual System in Vertebrates. Handbook of Sensory Physiology. 7, (1977).
- Potier, S., Mitkus, M., Kelber, A. Visual adaptations of diurnal and nocturnal raptors. Seminars in Cell & Developmental Biology. 106, 116-126 (2020).
- Wittenberg, J. B., Wittenberg, B. A. Active secretion of oxygen into the eye of fish. Nature. 194, 106-107 (1962).
- Damsgaard, C. Physiology and evolution of oxygen secreting mechanism in the fisheye. Comparative Biochemistry and Physiology. 252, 110840 (2021).
- Damsgaard, C., et al. A novel acidification mechanism for greatly enhanced oxygen supply to the fish retina. Elife. 9, (2020).
- Wittenberg, J. B., Haedrich, R. L. The choroid rete mirabile of the fish eye. II. Distribution and relation to the pseudobranch and to the swimbladder rete mirabile. Biological Bulletin. 146 (1), 137-156 (1974).
- Wittenberg, J. B., Wittenberg, B. A. The choroid rete mirabile of the fish eye. I. Oxygen secretion and structure: comparison with the swimbladder rete mirabile. Biological Bulletin. 146 (1), 116-136 (1974).
- Berenbrink, M. Historical reconstructions of evolving physiological complexity: O2 secretion in the eye and swimbladder of fishes. Journal of Experimental Biology. 210, 1641-1652 (2007).
- Berenbrink, M., Koldkjaer, P., Kepp, O., Cossins, A. R. Evolution of oxygen secretion in fishes and the emergence of a complex physiological system. Science. 307 (5716), 1752-1757 (2005).
- Keane, P. A., Sadda, S. R. Retinal imaging in the twenty-first century: State of the art and future directions. Ophthalmology. 121 (12), 2489-2500 (2014).
- Yung, M., Klufas, M. A., Sarraf, D. Clinical applications of fundus autofluorescence in retinal disease. International Journal of Retina and Vitreous. 2 (1), 12 (2016).
- Ang, M., et al. Optical coherence tomography angiography: a review of current and future clinical applications. Graefe’s Archive for Clinical and Experimental Ophthalmology. 256 (2), 237-245 (2018).
- Spaide, R. F., Koizumi, H., Pozonni, M. C. Enhanced depth imaging spectral-domain optical coherence tomography. American Journal of Ophthalmology. 146 (4), 496-500 (2008).
- Shen, Q., et al. Magnetic resonance imaging of tissue and vascular layers in the cat retina. Journal of Magnetic Resonance Imaging. 23 (4), 465-472 (2006).
- Tan, G. X., Jamil, M., Tee, N. G., Zhong, L., Yap, C. H. 3D reconstruction of chick embryo vascular geometries using non-invasive high-frequency ultrasound for computational fluid dynamics studies. Annals of Biomedical Engineering. 43 (11), 2780-2793 (2015).
- Ho, S., Tan, G. X. Y., Foo, T. J., Phan-Thien, N., Yap, C. H. Organ dynamics and fluid dynamics of the HH25 chick embryonic cardiac ventricle as revealed by a novel 4D high-frequency ultrasound imaging technique and computational flow simulations. Annals of Biomedical Engineering. 45 (10), 2309-2323 (2017).
- Dittrich, A., Thygesen, M. M., Lauridsen, H. 2D and 3D echocardiography in the Axolotl (Ambystoma Mexicanum). Journal of Visualized Experiments: JoVE. (141), e57089 (2018).
- Jia, Y., et al. Split-spectrum amplitude-decorrelation angiography with optical coherence tomography. Optics Express. 20 (4), 4710-4725 (2012).
- Clarke, K. W., Trim, C. M., Trim, C. M. . Veterinary Anaesthesia E-Book. , (2013).
- Flecknell, P. . Laboratory Animal Anaesthesia. , (2015).
- West, G., Heard, D., Caulkett, N. . Zoo Animal and Wildlife Immobilization and Anesthesia. , (2014).
- Lauridsen, H., Hansen, K., Nørgård, M. &. #. 2. 1. 6. ;., Wang, T., Pedersen, M. From tissue to silicon to plastic: three-dimensional printing in comparative anatomy and physiology. Royal Society Open Science. 3 (3), 150643 (2016).
- Lauridsen, H., et al. Inside out: Modern imaging techniques to reveal animal anatomy. PLoS One. 6 (3), 17879 (2011).
- Ruthensteiner, B., Heß, M. Embedding 3D models of biological specimens in PDF publications. Microscopy Research and Technique. 71 (11), 778-786 (2008).
- Damsgaard, C., Lauridsen, H. Deep vascular imaging in the eye with flow-enhanced ultrasound. bioRxiv. , 447055 (2021).
- Mueller, R. L., Ryan Gregory, T., Gregory, S. M., Hsieh, A., Boore, J. L. Genome size, cell size, and the evolution of enucleated erythrocytes in attenuate salamanders. Zoology. 111 (3), 218-230 (2008).
- Greis, C. Quantitative evaluation of microvascular blood flow by contrast-enhanced ultrasound (CEUS). Clinical Hemorheology and Microcirculation. 49, 137-149 (2011).
- Urs, R., Ketterling, J. A., Tezel, G., Silverman, R. H. Contrast-enhanced plane-wave ultrasound imaging of the rat eye. Experimental Eye Research. 193, 107986 (2020).
- Walls, G. L. . The vertebrate eye and its adaptive radiation. , (1942).
Cite This Article
Damsgaard, C., Lauridsen, H. Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound. J. Vis. Exp. (176), e62986, doi:10.3791/62986 (2021).