This work was supported by the Terry Fox Program of the National Cancer Institute of Canada.

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F.S. Foster acknowledges his role as consultant to VisualSonics Inc.

Ultraschallkontrastmittel wurden in der Spätphase der Trächtigkeit Mausembryonen und nichtlineare Kontrastbilder injiziert wurden erworben, um die Perfusion Parameter zu messen und zielgerichtete Mikrobläschen verbindlich. Erfolgreiche Abbildung von Mikrobläschen in embryonalen Vaskulatur war abhängig von einer Reihe von Faktoren, wobei der erste Embryo Lebensfähigkeit. Die gesamte Ausrüstung und die Vorrichtung wurden im Voraus, um die Zeit für die Trennung von Embryonen aus dem Uterus zu Beginn der Einspritzun...

Die kontrastverstärkte Ultraschallbildgebung nutzt Mikrobläschenkontrastmittel sichtbar zu machen und kennzeichnen das Gefäßumgebung. Diese Agenten ermöglichen die nichtinvasive Beurteilung der Mikrozirkulation, Vaskularität und Herz-Kreislauf-Funktion. Zusätzlich kann eine Modifikation der Blasenoberfläche in gezielter Mikrobläschen führen Bindung an Endothelzellen Biomarkern in der präklinischen Anwendungen der Angiogenese, Arteriosklerose und Entzündung 1,2 Herstellung Molekularultraschallbildgebungs vaskulärer Ereignisse möglich demonstriert. Kontrastverstärkten Ultraschall kann daher verwendet werden, um die komplexen und vielfältigen Umgebungen, die gesunden und erkrankten Gefäßzustände 3-5 beeinflussen zu identifizieren. In den vergangenen Jahren hat das Interesse an der Nützlichkeit der Mikroblasenabbildungs ​​der vielseitigen Mausembryo Modell erweitert. Als ein Modell der Entwicklung von Säugetieren, die Einführung von Mikroblasen in der embryonalen Gefäßsystem erhöht physiologischenStudie der Entwicklungskreislauf-System (zB Blutfluss, Herzzeitvolumen) und in Fällen von transgenen und gezielte Mutante Mausmodellen der Herzerkrankungen 6,7, kann Einblicke in die genetischen Faktoren Herz-Kreislauf-Funktion zu ändern ergeben. Tatsächlich Analysen von embryonalen Gehirngefäß quantitative und qualitative 2D sind bereits verwirklicht 8. Weiterhin stellt die Mausembryo als ausgezeichnetes Modell für die Untersuchung der Bindung von Zielmikrogefäßmarker in vivo. Bartelle et al. 9, zum Beispiel, haben Avidin Mikroblasen in Embryo Herzkammern eingeführt zu beurteilen, zielgerichtete Bindung in Biotag-BirA transgenen Embryonen und untersuchen Gefäßanatomie. Eine wichtige Benchmark bei der Übersetzung dieser Technik in die Klinik - Die Erzeugung von heterozygote und homozygote Maus-Modelle können auch als Ersatz für die Tumor-Modell-Studien mit dem Ziel, die quantitative Natur der molekularen Ultraschall definieren verwendet werden. 8-10 ausgesetzt eingeführt. In utero Injektionen, jedoch mit einer Reihe von Herausforderungen. Dazu gehören Injektion Führung, die Notwendigkeit, die Bewegung in der Mutter und exteriorisierten Embryo zu begegnen, die Aufrechterhaltung der hämodynamischen Lebensfähigkeit in der Mutter und außen gelegt Embryonen Bewältigung langfristigen Auswirkungen der Narkose und Komplikationen durch Blutungen 11. Daher ist das Ziel der Untersuchung war es, eine Technik zum Injizieren von Mikroblasen in isolierte Lebensspätstadium Embryonen 12 zu entwickeln. Diese Option bietet mehr Freiheit hinsichtlich der Einspritzsteuerung und die Positionierung, die Reproduzierbarkeit der Bildebene ohne Hindernis, und vereinfachte Bildanalyse und Quantifizierung. In der vorliegenden Studie haben wir in lebende Mäuse-Embryonen fo skizzieren ein neues Verfahren für die Injektion von Mikrobläschenr die Zwecke der Untersuchung von Mikrobläschen Kinetik und des Studiums gezielt Mikroblasen-Bindung an endogene endothelialen Oberflächenmarker. Nicht-lineare Kontrast spezifischen Ultraschall-Bildgebung wird verwendet, um eine Reihe von Grund Perfusionsparameter einschließlich Spitzenverstärkung (PE), Wasch in Rate und Zeit (TTP) in isolierten E17.5 Embryonen Spitze messen. Wir zeigen auch die Wirksamkeit des Embryos Modell zur Bewertung der quantitativen Natur der Molekular Ultraschall in einem embryonalen Endoglin Funktionsverlust transgenes Mausmodell, in dem Endoglin ist ein klinisch relevanten Ziel aufgrund seiner hohen Expression in vaskulären Endothelzellen an Stellen aktiver Angiogenese 13 . Die Haftung der Endoglin-bezogene (MB E), Ratte Isotyp IgG 2 Steuer (MB C) und ungezielte (MB U) Mikroblasen in heterozygote Endoglin (Eng +/-) und homozygot Endoglin (Eng + / +) exprimieren Embryonen untersucht. Analyse der zielBinding zeigt, dass molekulare Ultraschall in der Lage, die Unterscheidung zwischen Endoglin Genotypen und beziehen Rezeptordichten quantifizierbare molekulare Ultraschall Ebenen ist.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Antibodies&lt;/strong&gt; (biotinylated, eBioscience) &amp;#8212; Antibody choice depends on the experiment</td></tr><tr><td>&lt;ul style=&quot;padding:0px;margin:0px&quot;&gt;&lt;li&gt;rat isotype IgG&lt;sub&gt;2&lt;/sub&gt; control&lt;/li&gt;&lt;/ul&gt;</td><td>eBioscience</td><td>13-4321-85</td><td>This antibody/microbubble combination is often required as experimental control&amp;nbsp;</td></tr><tr><td>&lt;ul style=&quot;padding:0px;margin:0px&quot;&gt;&lt;li&gt;biotin anti-mouse CD309&lt;/li&gt;&lt;/ul&gt;</td><td>eBioscience</td><td>13-5821-85</td><td></td></tr><tr><td>Biotinylated rat MJ 7/18 antibody to mouse endoglin</td><td>In house hybridoma</td><td></td><td>Outside antibodies may also be appropriate: we&amp;nbsp; have used eBioscience (13-1051-85 ) in the past</td></tr><tr><td>Distilled water</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Embryo media&lt;/strong&gt;</td></tr><tr><td>&lt;ul style=&quot;padding:0px;margin:0px&quot;&gt;&lt;li&gt;500 ml Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium with high glucose&lt;/li&gt;&lt;/ul&gt;</td><td>Sigma</td><td>D5796</td><td></td></tr><tr><td>&lt;ul style=&quot;padding:0px;margin:0px&quot;&gt;&lt;li&gt;50 ml Fetal Bovine Serum&lt;/li&gt;&lt;/ul&gt;</td><td>ATCC</td><td>30-2020</td><td>lot # 7592456</td></tr><tr><td>&lt;ul style=&quot;padding:0px;margin:0px&quot;&gt;&lt;li&gt;Hepes&lt;/li&gt;&lt;/ul&gt;</td><td>Gibco</td><td>15630</td><td>5 ml, 1 M</td></tr><tr><td>&lt;ul style=&quot;padding:0px;margin:0px&quot;&gt;&lt;li&gt;Penicillin-Streptomycin&lt;/li&gt;&lt;/ul&gt;</td><td>Gibco</td><td>15140-122</td><td>5 ml, 10,000 units Pen., 10,000 &amp;#956;g Strep</td></tr><tr><td>Ethanol, 70%</td><td></td><td></td><td></td></tr><tr><td>Ice</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>76240</td><td>4%</td></tr><tr><td>Phosphate Buffered Saline [1x]&amp;nbsp;</td><td>Sigma</td><td>D8537</td><td>1x, w/o calcium chloride &amp;amp; magnesium chloride</td></tr><tr><td>Pregnant mouse, CD-1</td><td>Charles River Laboratories Inc.&amp;nbsp;</td><td></td><td></td></tr><tr><td>0.9% sodium chloride (saline)</td><td>Hospira</td><td>0409-7984-11</td><td></td></tr><tr><td>Ultrasound contrast agent, target ready and untargeted</td><td>MicroMarker; VisualSonics Inc.</td><td></td><td></td></tr><tr><td>Ultrasound gel (Aquasonic 100, colourless)</td><td>CSP Medical</td><td>133-1009</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Cell culture plates (4) :&amp;nbsp; 100&amp;nbsp;x&amp;nbsp;20&amp;nbsp;mm</td><td>Fisher Scientific</td><td>08-772-22</td><td></td></tr><tr><td>Cell culture plates (12) : 60&amp;nbsp;x&amp;nbsp;15&amp;nbsp;mm</td><td>Sigma</td><td>D8054</td><td></td></tr><tr><td>Centrifuge</td><td>Sorvall Legend RT centrifuge&amp;nbsp;</td><td></td><td></td></tr><tr><td>Conical tubes, 50 ml BD Falcon</td><td>VWR</td><td>21008-938</td><td></td></tr><tr><td>Diluent</td><td>Beckman Coulter</td><td>Isoton II Diluent, 8448011</td><td></td></tr><tr><td>Dissection scissors (Wagner)</td><td>Fine Science Tools</td><td>Wagner 14068-12</td><td></td></tr><tr><td>Forceps (2), Dumont SS (0.10 x 0.06 mm)</td><td>Fine Science Tools</td><td>11200-33</td><td></td></tr><tr><td>Forceps, splinter</td><td>VWR</td><td>25601-134</td><td></td></tr><tr><td>Glass beaker, 2 L (Griffin Beaker)</td><td>VWR</td><td>89000-216</td><td></td></tr><tr><td>Glass capillaries, 1 x 90&amp;nbsp;mm GD-1 with filament</td><td>Narishige</td><td>GD-1</td><td></td></tr><tr><td>Glass needle puller</td><td>Narishige</td><td>PN-30</td><td></td></tr><tr><td>Gloves</td><td>Ansell</td><td>4002</td><td></td></tr><tr><td>Gross anatomy probe</td><td>Fine Science Tools</td><td>10088-15</td><td></td></tr><tr><td>Hot plate</td><td>VWR</td><td>89090-994</td><td></td></tr><tr><td>Ice bucket</td><td>Cole Parmer</td><td>RK 06274-01</td><td></td></tr><tr><td>Imaging Platform</td><td>VisualSonics Inc.</td><td>Integrated Rail System</td><td></td></tr><tr><td>Light source, fiber-optic</td><td>Fisher Scientific</td><td>12-562-36</td><td>Ideally has adjustable arms</td></tr><tr><td>Luers (12), polypropylene barbed female &amp;frac14;-28 UNF thread</td><td>Cole Parmer</td><td>45500-30</td><td></td></tr><tr><td>Micro-ultrasound system, high-frequency</td><td>VisualSonics Inc.</td><td>Vevo2100</td><td></td></tr><tr><td>Needles, 21 gauge&amp;nbsp; (1&amp;rdquo;)</td><td>VWR</td><td>305165</td><td></td></tr><tr><td>Particle size analyzer</td><td>Beckman Coulter</td><td>Multisizer 3 Coulter Counter</td><td></td></tr><tr><td>Perforated spoon (Moria)</td><td>Fine Science Tools</td><td>MC 17 10373-17</td><td></td></tr><tr><td>Pins (6), black anodized minutien 0.15 mm</td><td>Fine Science Tools</td><td>26002-15</td><td></td></tr><tr><td>Pipettors [2-20 &amp;#956;l, 20-200 &amp;#956;l, 100-1,000 &amp;#956;l]</td><td>Eppendorf</td><td>Research Plus&amp;nbsp; adjustable 3120000038;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 3120000054;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 3120000062</td><td></td></tr><tr><td>Pipettor tips [2-200&amp;nbsp;&amp;#956;l, 50-1,000&amp;nbsp;&amp;#956;l]</td><td>Eppendorf</td><td>epT.I.P.S.&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 22491334;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 022491351</td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>Tubing, Tygon laboratory 1/32&amp;rdquo; x 3/32&amp;rdquo;</td><td>VWR</td><td>63010-007</td><td></td></tr><tr><td>Wooden applicator stick (swab, cotton head)</td><td>VWR</td><td>CA89031-270</td><td></td></tr><tr><td>Surgical microscope 5-8X magnification</td><td>Fisher Scientific</td><td>Steromaster</td><td></td></tr><tr><td>Syringes, 1 ml Normject</td><td>Fisher</td><td>14-817-25</td><td></td></tr><tr><td>Syringes (10), 30 ml</td><td>VWR</td><td>CA64000-041</td><td></td></tr><tr><td>Syringe infusion pump&amp;nbsp;</td><td>Bio-lynx&amp;nbsp;</td><td>NE-1000</td><td></td></tr><tr><td>Thermometer, -20-110&amp;nbsp;&amp;#176;C</td><td>VWR</td><td>89095-598</td><td></td></tr><tr><td>Timer</td><td>VWR</td><td>33501-418</td><td></td></tr><tr><td>Tubes, Eppendorf</td><td>VWR</td><td>20170-577</td><td></td></tr><tr><td>Tube racks (3)</td><td>VWR</td><td>82024-462</td><td></td></tr><tr><td>Ultrasound transducer, 20 MHz</td><td>VisualSonics Inc.</td><td>MS250</td><td></td></tr><tr><td>Vannas-Tubingen, angled up</td><td>Fine Science Tools</td><td>15005-08</td><td></td></tr></tbody></table>

contrast imaging in mouse embryos using high-frequency ultrasound

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 &#945;v&#946;3) 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.

Die Injektion von Ultraschallkontrastmitteln in ex utero Mausembryonen ist abhängig von der erfolgreichen Isolierung von lebenden spät Gestationsalter Embryonen aus dem Uterus und Wartung der Lebensfähigkeit im Verlauf der Injektion und ähnliche Ultraschallbildgebung. Nachdem der Embryo wurde nach außen verlagert und so positioniert, wie in Figur 1 gezeigt, ist eine sorgfältige Injektion von Kontrastmittel in den embryonalen Gefßsystems möglich. Eine typische B-Mode-Ultraschallbild eine...

Watch this Scientific Journal Video about Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound at JoVE.com

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Hier präsentieren wir ein Protokoll zur Ultraschall Mikrobläschenkontrastmittel in Wohn-, isoliert späten Schwangerschaftsphase murine Embryonen zu injizieren. Dieses Verfahren ermöglicht die Untersuchung der Perfusion Parameter und vaskulärer molekularer Marker innerhalb des Embryos mit kontrastverstärkten hochfrequenten Ultraschall-Bildgebung.

Contrast Imaging in Maus Embryonen Verwendung von Hochfrequenz-Ultraschall

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

contrast-imaging-in-mouse-embryos-using-high-frequency-ultrasound

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

15.3K Aufrufe.  University of Toronto.  Hier präsentieren wir ein Protokoll zur Ultraschall Mikrobläschenkontrastmittel in Wohn-, isoliert späten Schwangerschaftsphase murine Embryonen zu injizieren. Dieses Verfahren ermöglicht die Untersuchung der Perfusion Parameter und vaskulärer molekularer Marker innerhalb des Embryos mit kontrastverstärkten hochfrequenten Ultraschall-Bildgebung. 

Serie: Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

Ultraschall-gesteuerte Mikroinjektion in der Maus Vorderhirnneuronen In Utero In E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

Forschung

Neurowissenschaften

Murine Fetal Echocardiography

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development 1-3. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death 4. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies 5,6. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available 6-10. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures 11.

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

Murine fetale Echokardiographie

Transgenic mice displaying abnormalities in  cardiac development and function represent a powerful tool for the understanding  the molecular mechanisms ...

Medizin

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

Eine neue Verwendung des dreidimensionalen Hochfrequenz-Sonographie zur frühen Schwangerschaft Charakterisierung in der Maus

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Entwicklungsbiologie

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Fetale kardiovaskuläre Bildgebung mit einem Hochfrequenz-Ultraschall (30 / 45MHZ) Maus

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

In utero Messung der Herzfrequenz in Mouse von Nichtinvasive M-Mode-Echokardiographie

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

Hochfrequenz-Ultraschall für die Analyse der fetalen und plazentaren Entwicklung In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to assessing pathology post-mortem in animals euthanized at multiple time points in different cohorts, which increases the number of mice needed for a study. Imaging mice in longitudinal studies can track the progression of disease in individual animals, reducing the number of mice needed. Advances in ultrasound technology have allowed for the detection of micrometer-level changes in tissues. Ultrasound has been used to study follicle maturation in ovaries and xenograft growth but has not been applied to morphological changes in the mouse uterus. This protocol examines the juxtaposition of pathology with in vivo imaging comparisons in an induced endometrial cancer mouse model. The features observed by ultrasound were consistent with the degree of change seen by gross pathology and histology. Ultrasound was found to be highly predictive of the observed pathology, supporting the incorporation of ultrasonography into longitudinal studies of uterine diseases such as cancer in mice.

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes.

Nicht-invasive Ultraschalluntersuchung der Progression des Endometriumkarzinoms bei Pax8-gerichteter Deletion der Tumorsuppressoren Arid1a und Pten in Mäusen

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to ...

Krebsforschung

The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ultrasound pulsing schemes, or novel transducers for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. The reasons for this are the accessibility of the embryo and vessel network of the CAM as well as the low costs of the model. An important step to get access to the embryo and CAM vessels is to take the egg content out of the eggshell. In this protocol, three methods for taking the content out of the eggshell between day 5 and&#160;8 of incubation are described thus allowing the embryos to develop inside the eggshell up to these days. The described methods only require simple tools and equipment and yield a higher survival success rate of 90% for 5-day, 75% for 6-day, 50% for 7-day, and 60% for 8-day old incubated eggs in comparison to ex ovo cultured embryos (~50%). The protocol also describes how to inject cavitation nuclei, such as microbubbles, into the CAM vascular system, how to separate the membrane containing the embryo and CAM from the rest of the egg content for optically transparent studies, and how to use the chicken embryo and CAM in a variety of short-term ultrasound experiments. The in vivo&#160;chicken embryo and CAM model is extremely relevant to investigate novel imaging protocols, ultrasound contrast agents, and ultrasound pulsing schemes for contrast-enhanced ultrasound imaging, and to unravel the mechanisms of ultrasound-mediated drug delivery.

This protocol describes three methods on how to obtain and use 5 to 8-day old chicken embryos and their chorioallantoic membrane (CAM) as an in vivo&#160;model to study contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery.

Die Herstellung von Hühner-Ex-Ovo-Embryonen und chorioallantoischen Membrangefäßen als In-vivo-Modell für kontrastmittelverstärkte Ultraschallbildgebung und Mikroblasen-vermittelte Arzneimittelabgabestudien

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ...

Biotechnik

High-frequency Ultrasound Imaging of the Abdominal Aorta

Source: Amelia R. Adelsperger, Evan H. Phillips, and <a href="https://jove.unicartagenaproxy.elogim.com/author/Craig+J._Goergen">Craig J. Goergen</a>, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
High-frequency ultrasound systems are used to acquire high resolution images. Here, the use of a state-of-the-art system will be demonstrated to image the morphology and hemodynamics of small pulsatile arteries and veins found in mice and rats. Ultrasound is a relatively inexpensive, portable, and versatile method for the noninvasive assessment of vessels in humans as well as large and small animals. These are several key advantages that ultraound offers compared to other techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), and near-infrared fluorescence tomography (NIRF). CT requires ionizing radiation and MRI can be prohibitively expensive and even impractical in some scenarios. NIRF, on the other hand, is limited by the penetration depth of light required to excite the fluorescent contrast agents.
Ultrasound has limitations in terms of imaging depth; however, this may be overcome by sacrificing resolution and using a lower frequency transducer. Abdominal gas and excess body weight can severely diminish image quality. In the first case, the propagation of sound waves is limited, while in the latter case, they are attenuated by overlying tissues, such as fat and connective tissue. As a result, no contrast or faint contrast may be observed. Finally, ultrasound is a highly user-dependent technique, requiring the sonographer to be familiar with anatomy and to be able to work around issues, such as the appearance of imaging artifacts or acoustic interference.

High-frequency Ultrasound Imaging and Image Acquisition

Hochfrequenz-Ultraschall-Bildgebung der Bauchaorta

Source: Amelia R. Adelsperger, Evan H. Phillips, and Craig J. Goergen, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, 
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Lehre

JoVE Science Education

Ingenieurwesen

Biomedizinische Technik

Injection of Microbubbles into Isolated Mouse Embryos: A Technique to Deliver Microbubble Contrast Agents into the Vasculature of Living Murine Embryos

Source: Denbeigh, J. M. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/52520" target="_blank">Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound</a>. J. Vis. Exp. (2015)
This video demonstrates a procedure to inject microbubble contrast agents into an isolated late gestation stage mouse embryo. The microbubble contrast agent-based ultrasound imaging helps visualize and characterize the vascular environment of the embryo.

Injektion von Mikrobläschen in isolierte Mausembryonen: Eine Technik zur Verabreichung von Mikrobläschen-Kontrastmitteln in das Gefäßsystem lebender Mausembryonen

Source: Denbeigh, J. M. et al. Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound. J. Vis. Exp. (2015)
This video demonstrates a procedure ...