Contrast Imaging in muis embryo's met behulp van hoge-frequentie Ultrasound
Summary
Hier presenteren we een protocol om echografie microbellen contrastmiddelen in levende, geïsoleerde late zwangerschap stadium muizen embryo's te injecteren. Deze methode maakt de studie van perfusie parameters en vasculaire moleculaire merkers binnen het embryo met contrastmiddel hoogfrequente ultrasone beeldvorming.
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
Contrastversterkte echografie maakt gebruik van microbellen contrastmiddelen te visualiseren en te karakteriseren de vasculaire milieu. Deze agenten in staat stellen niet-invasieve evaluatie van de microcirculatie, vasculariteit en cardiovasculaire functie. Bovendien kan modificatie van het oppervlak bel leiden gerichte microbellen binden aan endotheel biomarkers, zoals aangetoond in preklinische toepassingen van angiogenese, atherosclerose en ontsteking 1,2 waardoor moleculaire ultrasone beeldvorming van vasculaire voorvallen mogelijk. Contrastechografie ultrageluid kan derhalve worden gebruikt om de complexe en diverse omgevingen die invloed gezonde en zieke bloedvaten toestanden 3-5 identificeren.
In de afgelopen aantal jaren is de belangstelling voor het nut van microbellen beeldvorming uitgebreid tot de veelzijdige muis embryo model. Als een model van de ontwikkeling van zoogdieren, introductie van microbellen in de embryonale vaatstelsel verbetert fysiologischestudie van de ontwikkeling bloedsomloop (bijvoorbeeld bloedstroom, hartminuutvolume) en in geval van transgene en gerichte mutant muismodellen van hartziekte 6,7, kan informatie krijgen over hoe genetische factoren veranderen cardiovasculaire functie. In feite, kwantitatieve en kwalitatieve analyses van 2D embryonale hersenen vasculatuur al bereikt 8. Bovendien, de muis embryo presenteert als een uitstekend model voor het onderzoeken van de binding van gerichte microbellen vasculaire markers in vivo. Bartelle et al. 9, bijvoorbeeld, hebben avidine microbellen geïntroduceerd in embryo cardiale ventrikels om gerichte binding beoordelen Biotag-BirA transgene embryo's en onderzoeken vasculaire anatomie. Het genereren van heterozygote en homozygote muismodellen kunnen ook worden gebruikt als een surrogaat voor tumormodel studies gericht op de kwantitatieve aard van moleculaire ultrageluid bepalen – een belangrijke benchmark vertaling van deze techniek om de kliniek.
<p class = "jove_content"> Microbelletjes worden het vaakst ingevoerd om de embryonale circulatie via intra-cardiale injecties in enkele embryo's blootgesteld via een laparotomie 8-10. In utero injecties worden echter geconfronteerd met een aantal uitdagingen. Deze omvatten injectie begeleiding, de noodzaak om de beweging in de moeder en geëxterioriseerde embryo tegen te gaan, het onderhoud van hemodynamische levensvatbaarheid bij de moeder en exteriorized embryo's, het aanpakken van de lange-termijn effecten van anesthesie en complicaties als gevolg van bloedingen 11. Daarom is het doel van het onderzoek was een techniek voor het injecteren van microbelletjes in geïsoleerde levende laat-stadium embryo 12 ontwikkelen. Deze optie biedt meer vrijheid in termen van injectie controle en positionering, reproduceerbaarheid van het beeldvlak zonder obstructie, en vereenvoudigde beeldanalyse en kwantificering.In de huidige studie, schetsen we een nieuwe procedure voor de injectie van microbellen in levende muizen embryo's for de toepassing van het bestuderen van microbellen kinetisch gedrag en van het bestuderen van gerichte microbellen binden aan endogene endotheeloppervlak markers. Niet-lineaire contrast specifieke echografie wordt gebruikt voor het meten van een aantal basis perfusieparameters inclusief piekverhogingsfactor (PE), wassen-in en de tijd tot piek (TTP) in geïsoleerde E17.5 embryo's. We tonen ook de geldigheid van de embryo voor de beoordeling van de kwantitatieve aard van moleculaire ultrageluid in een embryonale endoglin functieverlies transgeen muismodel, waarbij endoglin een klinisch relevant doel vanwege de hoge expressie in vasculaire endotheelcellen op plaatsen van actieve angiogenese 13 . De hechting van-endogline gerichte (MB E), rat isotype IgG 2 controlepunten (MB C) en ongerichte (MB U) microbellen wordt geëvalueerd in heterozygote endoglin (Eng +/-) en homozygote endoglin (Eng + / +) uiten van embryo's. Analyse van de gerichte binding blijkt dat moleculaire echografie te maken tussen genotypen en endoglin betrekking receptor dichtheden meetbare moleculaire ultrageluid niveaus.
Protocol
Representative Results
Discussion
Ultrageluidcontrastmiddelen werden geïnjecteerd in een laat stadium van de dracht muis embryo's en niet-lineaire contrast beelden werden verworven om perfusie parameters te meten en gerichte microbellen bindend. Succesvolle beeldvorming microbellen in embryonale vasculatuur was afhankelijk van een aantal factoren, waarvan de eerste levensvatbaar embryo. Alle apparatuur en toestellen werden voorbereid om de benodigde tijd voor isolatie van embryo van de baarmoeder naar het begin van injectie te minimaliseren. Aangez…
Declarações
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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