Name: deep vascular imaging in the eye with flow-enhanced ultrasound
Uploaded: 2021-10-04T14:45:00
Description: Watch this Scientific Journal Video about Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound at JoVE.com

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。

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
<ol><li>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?...

<ol>
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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MS-222</td>
			<td>Sigma</td>
			<td>E10521-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzocaine</td>
			<td>Sigma</td>
			<td>E-1501</td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>B Braun</td>
			<td> 
			12260470_0320</td>
			<td></td>
		</tr>
		<tr>
			<td>Alfaxalon</td>
			<td>Jurox</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis</td>
			<td>50019100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound scanner</td>
			<td>VisualSonics</td>
			<td>Vevo 2100</td>
			<td></td>
		</tr>
	</tbody>
</table>

deep vascular imaging in the eye with flow-enhanced ultrasound

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。

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?...

Watch this Scientific Journal Video about Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound at JoVE.com

Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound

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。

使用流量增强超声进行眼部深层血管成像

The retina within the eye is one of the most energy-demanding tissues in the body and thus requires high rates of oxygen delivery from a rich blood ...

Flow-Enhanced Ultrasound技术使我们能够在不使用造影剂的情况下以三维方式对眼睛的脉管系统进行成像。这种技术的主要优点是它对色素沉着的视网膜后面的外脏进行成像的稳定性，这在几种光学成像技术中具有挑战性。我们在金鱼中展示了这种技术，但它可以应用于所有具有有核红细胞的物种，使研究人员能够深入了解眼睛的功能进化。该技术可以由具有超声成像和动物处理基本培训的研究人员应用。确认最佳麻醉水平后，将动物置于允许从上方直接进入眼睛的姿势。将合适的超声介质放在动物的眼睛上。如果鳞片眼睑覆盖眼睛，则用棉签轻轻移位。对于水生动物，水作为超声介质效果很好，接下来，根据所需的图像方向，将超声换能器（内侧）定位在背侧腹侧或喙尾方向。在B模式下，以最大景深对眼睛的内侧和最深处进行成像，以确保所有感兴趣的结构在图像场中都可见。慢慢地将换能器平移到每一侧，同时检查实时图像。确保所有感兴趣的结构在图像字段中都可见。如果没有，请切换到频率较低、景深较大的换能器。调整图像深度、深度偏移、图像宽度和焦点区域的位置数，以覆盖所有三个空间维度中所需的感兴趣区域。然后，将帧速率设置为每秒 50 到 120 帧。接下来，将2D增益调整到一个水平，使得解剖结构在B模式采集中仅可见，以增加后续流量增强重建中的信噪比。要在单个切片位置获取2D流动增强图像，请将换能器平移到该位置，然后继续进行流动增强图像重建。要获取整个感兴趣区域的 3D 记录，请将换能器转换为感兴趣区域的一个极端。要确定极端端的确切位置，请简要增加2D增益。正确放置传感器后，在录制前降低2D增益，以确保在随后的流量增强重建中具有最大的信噪比。对于 3D 录制中的每个步骤或切片，采集大于 100 帧的帧。然后，使用微型机械手或内置传感器电机，以25或50微米的步长将传感器平移到整个感兴趣区域，并为每个步骤重复采集超过100帧。对于流程增强的影像构建，请将录制文件导出为 DICOM 文件格式。要基于大于 100 帧的场景记录生成单个流动增强图像，请使用此公式计算像素级别的标准偏差，并对 3D 记录中的每个切片重复计算。要自动执行 3D 记录中多个切片的标准偏差计算和图像重建过程，请使用文本手稿中提供的 ImageJ 和宏脚本在批处理模式下执行此操作。使用 ImageJ 中的“图像到堆栈”命令将所有重建的图像合并到一个图像堆栈中。然后，使用“属性”命令，根据采集期间使用的步长指定切片厚度，并将图像堆栈另存为 3D TIFF 文件。与场景记录中的静态组织相比，非成年哺乳动物脊椎动物中存在有核红细胞，为流动的血液提供了积极的对比。然而，现在逐帧分析时，血液和周围组织之间的明显区别就不那么明显了。这种血流增强程序，本质上，将2D空间中的多时间点记录编译成单个图像，其中在固有的信号值波动的像素中，定位在流动的血液中，得分比周围的静态组织更高的标准偏差，产生正对比度。在 3D 采集中，可以将具有已知间距的多个平行切片组合成 3D 图像数据。它可用于三维体积渲染和解剖建模。基于多普勒的超声成像也提供了特异性成像血流的选择，但是，灵敏度低于该方法。这种流量增强的超声程序允许对一系列具有有核红细胞的物种进行血流成像。如果存在于该物种中，则可以对深部眼血管床（例如某些鱼类中的脉络膜爬行动物奇幻床）进行成像。该方法受到哺乳动物中没有有核红细胞的限制，其中流量增强程序产生一定程度的血流对比度，但不像具有有核红细胞的物种那样明显。流量增强型超声对运动噪声敏感。呼吸运动可导致图像模糊和伪影，例如组织边界增强。前瞻性或回顾性门控可用于调整运动噪声。对于2D和3D采集，由于不稳定的换能器设置的麻醉不足，限制由动物移动引起的运动伪影至关重要。该技术的发展为绝大多数脊椎动物眼中深层血管网络的非侵入性体内检查铺平了道路，这些脊椎动物拥有有核红细胞。

Research

JoVE Journal

Biology

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

科研

生物学

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

住在果蝇眼睛成虫光盘胶质细胞迁移成像

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

In vivo Imaging of Deep Cortical Layers using a Microprism

In vivo Imaging of Deep Cortical Layers using a Microprism

在使用微深皮质层的活体成像

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

功能成像在小鼠体内的棕色脂肪与FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

优化结构静电纺丝血管移植在大鼠模型中的植入

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

用迈尔斯测定法评价皮肤血管 Hyperpermeability 诱导剂的含量

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...