高周波超音波を用いて、マウス胚におけるコントラストイメージング
概要
ここでは、生きている、孤立した後期妊娠期のマウス胚に超音波マイクロバブル造影剤を注入するためのプロトコルを提示する。この方法は、灌流パラメータ及びコントラスト強調高周波超音波イメージングを用いて、胚内の血管の分子マーカーの研究を可能にする。
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
造影超音波イメージングは、血管環境を視覚化し、特徴付けるために、マイクロバブル造影剤を利用する。これらの物質は、微小循環、血管分布および心血管機能の非侵襲的評価を可能に。また、気泡表面の改質が可能な血管イベントの分子の超音波イメージングを行う血管形成、アテローム性動脈硬化症および炎症1,2の前臨床用途で実証されるように、内皮細胞のバイオマーカーに結合する標的化マイクロバブルをもたらすことができる。造影超音波は、したがって、健康と病気の血管の状態3-5に影響を与える複雑かつ多様な環境を識別するために使用することができる。
年の過去数には、マイクロバブルイメージングの有用性への関心は汎用性の高いマウス胚モデルに拡張しました。哺乳類の発生のモデルとして、胚の血管系へのマイクロバブルの導入は、生理的な強化開発循環系（ 例えば 、血流量、心拍出量）およびトランスジェニック例での研究と心疾患6,7の変異マウスモデルをターゲットとは、遺伝的要因が心血管機能を変化させる方法への洞察をもたらすことができる。実際には、胚の脳血管系の定量的および定性的な二次元分析は、既に8を達成している。さらに、マウス胚は、生体内での血管マーカーに標的マイクロバブルの結合を調べるための優れたモデルとして提示します。 Bartelle ら 9は 、例えば、Biotag-BirAをトランスジェニック胚に結合し、血管構造を検討対象と評価するために胚心臓の心室にアビジンマイクロバブルを導入しています。診療所にこの技術を翻訳において重要なベンチマーク – ヘテロ接合およびホモ接合性マウスモデルの生成は、分子の超音波の定量的性質を規定することを目的と腫瘍モデル研究のための代用として使用することができる。
<p clお尻= "jove_content">マイクロバブルは、最も頻繁に開腹8-10を介して露出し、単一の胚に心臓内注射を経由して胎児の血液循環に導入される。 子宮内注射では 、しかし、多くの課題に直面しています。これらは、注射の指導、母親と体外胚の動きに対抗する必要性、母親における血行動態の生存率の維持および11の出血による麻酔の合併症の長期的影響に対処体外胚を、含む。従って、調査の目的は、単離されたリビング後期胚12にマイクロバブルを注入するための技術を開発することであった。このオプションは、噴射制御やポジショニング、障害物のない撮像面の再現性、および単純化された画像解析と定量化の面でより多くの自由を提供しています。本研究では、foの生きているマウスの胚にマイクロバブルを注入するための新規な手順を概説Rマイクロバブル速度論的挙動を研究するとの目的は、内因性の内皮表面マーカーに結合するマイクロバブルをターゲットに勉強。非線形コントラスト特有の超音波イメージングは、ピークエンハンスメント（PE）、ウォッシュアウト速度および時間孤立E17.5胚において（TTP）をピークに含む基本的な灌流パラメータの数を測定するために使用される。我々はまた、活性な血管新生13の部位で血管内皮細胞におけるその高い発現をエンドグリンは、臨床的に関連する標的で機能するトランスジェニックマウスモデルの胚グリン損失分子音波の定量的性質を評価するための胚のモデルの妥当性を実証する。エンドグリンを標的（MB E）の付着、ラットアイソタイプIgGを2コントロール（MB C）および非標的（MB U）マイクロバブルは、ヘテロ接合グリン（ 工学 +/-）で評価され、ホモ接合エンドグリン（ 工 + / +）の胚を表す。目標とビンディの分析ngは、分子の超音波は、エンドグリン遺伝子型を区別し、定量化可能な分子の超音波レベルに受容体密度に関連することが可能であることを明らかにする。
Protocol
Representative Results
Discussion
超音波造影剤は、後期妊娠マウス胚および非線形コントラスト画像に注射し、灌流パラメータを測定するために取得し、結合マイクロバブルを標的とした。胚の血管系内のマイクロバブルの成功イメージングは、多くの要因、最初のもの胚の生存率に依存していた。すべての機器および装置は、注入の開始に子宮から胚を単離するために必要な時間を最小にするために、事前に調製した?…
開示
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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