对比成像的小鼠胚胎使用高频超声
Summary
在这里，我们提出了一个协议注入超声微泡造影剂进入活的，孤立的晚期妊娠阶段小鼠胚胎。此方法使灌注参数和使用对比增强高频超声成像的胚胎内血管分子标志物的研究。
Abstract
Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or αvβ3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.
Introduction
超声造影成像是利用微泡造影剂的可视化和表征血管环境。这些药物可使微循环，血管和心血管功能的无创性评估。此外，气泡的表面的改性可以导致靶向微泡结合到内皮的生物标志物，其表现在血管生成，动脉粥样硬化和炎症1,2-使得可能血管事件的分子超声成像临床前应用中。超声造影，因此可以用来识别影响健康和患病的血管状态3-5的复杂和多样的环境。
在多年以前数，微泡成像工具的兴趣已经扩展到通用的小鼠胚胎模型。哺乳动物的发展模式，引入微泡成胚胎血管增强生理显影循环系统（ 例如 ，血流量，心输出量），并在转基因箱子和心脏疾病6,7的靶向突变体小鼠模型，研究可能会产生见解因素如何遗传改变心血管功能。事实上，定量和定性的2D胚胎脑血管分析已经实现8。另外，小鼠胚胎呈现为用于检查靶向微泡的血管标记物的结合在体内的优良模型。 Bartelle 等 9，例如，引入了抗生物素微泡成胚胎心室评估目标锁定在Biotag，生物素化酶BirA转基因胚胎具有约束力，并检查血管解剖。的杂合和纯合小鼠模型的产生也可被用作替代物肿瘤模型研究，目的来定义分子超声波的定量性质 – 在把这种技术应用到临床的重要基准。
<p cl屁股="“jove_content”">微泡是最经常通过心内注射入单个的胚胎通过剖腹8-10暴露引入到胚胎循环。 在子宫内注射，但是，面临着许多挑战。这些包括喷射引导，有必要以对抗在母亲和形象化胚胎运动，维护血流动力学生存能力在母亲和形象化胚胎，寻址麻醉和并发症引起的出血11的长期影响。因此，调查的目标是开发用于注射微泡成隔离的生活晚期胚胎12的技术。这个选项提供了更多的自由，在喷射控制和定位，所述成像平面的再现无阻碍，并简化图像分析和定量方面。在本研究中，我们概述了一种新的方法用于注射微泡到活鼠胚胎FOř研究微泡动力学行为和的目的，正在学靶向微泡结合的内源性内皮表面标记。非线性对比度特定超声成像是用于测量的一些基本灌注参数包括峰增强（PE），洗入速率和达峰时间（TTP）中分离的E17.5胚胎。我们还表明，胚胎模型用于评估的功能的转基因小鼠模型，其中内皮糖蛋白是一种临床上相关的目标胚胎内皮糖损失分子超声波的定量性质的有效性，由于其高表达在血管内皮细胞在血管生成活性的13位点。的内皮糖蛋白靶向（MB E），大鼠同种型的IgG 2控制（MB C）和不相关的（MB U）的微泡粘附在杂内皮糖蛋白（英文+/-）和纯合子内皮糖蛋白（英文+ / +）表达的胚胎进行了评价。目标宾迪分析吴透露，超声分子能够内皮糖蛋白的基因型之间的区别，并与受体密度，以量化的超分子水平。
Protocol
Representative Results
Discussion
超声造影剂注入晚期妊娠小鼠胚胎和非线性对比度的图像被收购来衡量灌注参数和有针对性的微泡结合。内胚胎脉管系统的微泡的成功的成像依赖于许多因素，第一个是胚胎的生存能力。所有设备和装置，以减少所需的胚胎的分离从子宫到注射开始时预先准备的。由于单次或重复暴露于麻醉对胚胎的小鼠的影响还没有被详细研究，在母亲的使用麻醉被避免前牺牲16。期间胚胎隔离，这是重…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Terry Fox Program of the National Cancer Institute of Canada.
Materials
| Reagents | Company | Catalog Number | Comments/Description |
| Antibodies (biotinylated, eBioscience) | Antibody choice depends on the experiment | ||
| rat isotype IgG2 control | eBioscience | 13-4321-85 | This antibody/microbubble combination is often required as experimental control |
| biotin anti-mouse CD309 | eBioscience | 13-5821-85 | |
| Biotinylated rat MJ 7/18 antibody to mouse endoglin | In house hybridoma | Outside antibodies may also be appropriate: we have used eBioscience (13-1051-85 ) in the past | |
| Distilled water | |||
| Embryo media | |||
| 500 mL Dulbecco’s Modified Eagle’s Medium with high glucose | Sigma | D5796 | |
| 50 mL Fetal Bovine Serum | ATCC | 30-2020 | lot # 7592456 |
| Hepes | Gibco | 15630 | 5mL, 1M |
| Penicillin-Streptomycin | Gibco | 15140-122 | 5 mL, 10,000 units Pen., 10,000 ug Strep |
| Ethanol, 70% | |||
| Ice | |||
| Paraformaldehyde | Sigma | 76240 | 4% |
| Phosphate Buffered Saline [1x] | Sigma | D8537 | 1x, w/o calcium chloride & magnesium chloride |
| Pregnant mouse, CD-1 | Charles River Laboratories Inc. | ||
| 0.9% sodium chloride (saline) | Hospira | 0409-7984-11 | |
| Ultrasound contrast agent, target ready and untargeted | MicroMarker; VisualSonics Inc. | ||
| Ultrasound gel (Aquasonic 100, colourless) | CSP Medical | 133-1009 | |
| Equipment | |||
| Cell culture plates (4) : 100×20 mm | Fisher Scientific | 08-772-22 | |
| Cell culture plates (12) : 60×15 mm | Sigma | D8054 | |
| Centrifuge | Sorvall Legend RT centrifuge | ||
| Conical tubes, 50 mL BD Falcon | VWR | 21008-938 | |
| Diluent | Beckman Coulter | Isoton II Diluent, 8448011 | |
| Dissection scissors (Wagner) | Fine Science Tools | Wagner 14068-12 | |
| Forceps (2), Dumont SS (0.10×0.06 mm) | Fine Science Tools | 11200-33 | |
| Forceps, splinter | VWR | 25601-134 | |
| Glass beaker, 2 L (Griffin Beaker) | VWR | 89000-216 | |
| Glass capillaries, 1×90 mm GD-1 with filament | Narishige | GD-1 | |
| Glass needle puller | Narishige | PN-30 | |
| Gloves | Ansell | 4002 | |
| Gross anatomy probe | Fine Science Tools | 10088-15 | |
| Hot plate | VWR | 89090-994 | |
| Ice bucket | Cole Parmer | RK 06274-01 | |
| Imaging Platform | VisualSonics Inc. | Integrated Rail System | |
| Light source, fiber-optic | Fisher Scientific | 12-562-36 | Ideally has adjustable arms |
| Luers (12), polypropylene barbed female ¼-28 UNF thread | Cole Parmer | 45500-30 | |
| Micro-ultrasound system, high-frequency | VisualSonics Inc. | Vevo2100 | |
| Needles, 21 gauge (1”) | VWR | 305165 | |
| Particle size analyzer | Beckman Coulter | Multisizer 3 Coulter Counter | |
| Perforated spoon (Moria) | Fine Science Tools | MC 17 10373-17 | |
| Pins (6), black anodized minutien 0.15 mm | Fine Science Tools | 26002-15 | |
| Pipettors [2-20 uL, 20-200uL, 100-1000uL] | Eppendorf | Research Plus adjustable 3120000038; 3120000054; 3120000062 | |
| Pipettor tips [2-200uL, 50-1000uL] | Eppendorf | epT.I.P.S. 22491334; 022491351 | |
| Scissors | |||
| Sylgard 184 Silicone Elastomer Kit | Dow Corning | ||
| Tubing, Tygon laboratory 1/32×3/32” | VWR | 63010-007 | |
| Wooden applicator stick (swab, cotton head) | VWR | CA89031-270 | |
| Surgical microscope 5-8x magnification | Fisher Scientific | Steromaster | |
| Syringes, 1 mL Normject | Fisher | 14-817-25 | |
| Syringes (10), 30 mL | VWR | CA64000-041 | |
| Syringe infusion pump | Bio-lynx | NE-1000 | |
| Thermometer, -20-110oC | VWR | 89095-598 | |
| Timer | VWR | 33501-418 | |
| Tubes, Eppendorf | VWR | 20170-577 | |
| Tube racks (3) | VWR | 82024-462 | |
| Ultrasound transducer, 20 MHz | VisualSonics Inc. | MS250 | |
| Vannas-Tubingen, angled up | Fine Science Tools | 15005-08 |
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