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

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

Agentes de contraste de ultra-som foram injetadas em embriões em estágio final de gestação do mouse e imagens de contraste não-lineares foram adquiridos para medir parâmetros de perfusão e direcionados microbolhas vinculativo. Imagiologia bem sucedida de microbolhas no seio da vasculatura embrionária era dependente de uma série de factores, a primeira sendo a viabilidade do embrião. Todos os equipamentos e aparelhos foram preparados com antecedência, a fim de minimizar o tempo necessário para o isolamento de...

Ultra-sonografia com contraste faz uso de agentes de contraste de microbolhas para visualizar e caracterizar o ambiente vascular. Estes agentes permitir a avaliação não invasiva da microcirculação, vascularização e função cardiovascular. Além disso, a modificação da superfície da bolha pode resultar em microbolhas de ligação orientada para biomarcadores endoteliais, como demonstrado em aplicações pré-clínicos de angiogénese, a aterosclerose e a inflamação 1,2 fazendo imagiologia de ultra-sons molecular de eventos vasculares possíveis. Com aumento de contraste de ultra-sons pode, portanto, ser usadas para identificar os ambientes complexos e diversos que influenciam estados vasculares saudáveis ​​e doentes 3-5. No número últimos anos, o interesse pelo utilitário de imagem de microbolhas se estendeu para o modelo do rato embrião versátil. Como um modelo de desenvolvimento de mamíferos, introdução de microbolhas para a vasculatura embrionária aumenta fisiológicoestudo do sistema circulatório em desenvolvimento (por exemplo, o fluxo de sangue, o débito cardíaco) e nos casos de transgénicos e modelos de ratos mutantes alvo de doença cardíaca 6,7, pode produzir insights sobre como os fatores genéticos que alteram a função cardiovascular. De fato, análises 2D quantitativa e qualitativa da vasculatura cerebral embrionária já foram atingidos 8. Além disso, o embrião de rato apresenta-se como um excelente modelo para examinar a ligação de microbolhas orientadas para marcadores vasculares in vivo. Bartelle et al. 9, por exemplo, introduziram microbolhas avidina em embrião ventrículos cardíacas para avaliação alvejado obrigatório em embriões transgênicos BIOTAG-Bira e examinar a anatomia vascular. A geração de modelos de heterozigotos e homozigotos do mouse pode ser também ser utilizado como um substituto para estudos de modelos tumor com o objetivo de definir a natureza quantitativa de ultra-som molecular - uma referência importante na tradução dessa técnica para a clínica. <p class = &quot;jove_content&quot;&gt; Microbubbles são mais frequentemente introduzidas para a circulação embrionária através de injeções intra-cardíaco em embriões únicos expostos através de uma laparotomia 8-10. Em injeções útero, no entanto, enfrentam uma série de desafios. Estes incluem orientação injeção, a necessidade de combater o movimento na mãe e embrião exteriorizada, a manutenção da viabilidade hemodinâmico na mãe e embriões exteriorizados, abordando os efeitos a longo prazo da anestesia e complicações devido ao sangramento 11. Portanto, o objetivo da investigação foi desenvolver uma técnica para a injeção de microbolhas em isolado de vida em estágio final de embriões 12. Esta opção oferece mais liberdade em termos de controle de injeção e posicionamento, reprodutibilidade do plano de imagem, sem obstrução, e análise de imagem simplificada e quantificação. No presente estudo, descrevemos um novo procedimento para a injeção de microbolhas em que vivem embriões murinos for o objectivo de estudar o comportamento cinético de microbolhas e de estudar a ligação a marcadores de superfície endoteliais endógenas microbolhas alvejado. Ultra-sonografia específico contraste não-linear é usado para medir de uma série de parâmetros de perfusão básicas, incluindo intensificação de pico (PE), lave-in taxa e tempo de pico (TTP) em embriões E17.5 isoladas. Nós também demonstrar a validade do modelo de embrião para avaliar a natureza quantitativa de ultra-som molecular em uma perda endoglin embrionário de rato modelo função transgênica, onde endoglin é um alvo clinicamente relevante devido à sua elevada expressão em células endoteliais vasculares em locais de angiogênese ativo 13 . A adesão de (MB E), segmentada por endoglin isótipo rato IgG 2 controle (MB C) e não segmentados (MB U) microbolhas é avaliada em endoglin heterozigotos (Eng +/-) e endoglin homozigoto (Eng + / +) expressando embriões. Análise do bindi alvejadong revela que o ultrassom molecular é capaz de diferenciar entre os genótipos endoglina e relacionando densidade do receptor de ultra-som para níveis moleculares quantificáveis.

<table border="0" cellpadding="0" cellspacing="0" width="1477">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:419px;">Reagents</td>
			<td style="width:215px;">Company</td>
			<td style="width:207px;">Catalog Number</td>
			<td style="width:639px;">Comments/Description</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:419px;"></td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Antibodies (biotinylated, eBioscience)</td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td>Antibody choice depends on the experiment</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">&#160;&#160;&#160;&#160;&#160; rat isotype IgG2 control</td>
			<td style="width:215px;">eBioscience</td>
			<td style="width:207px;">13-4321-85</td>
			<td>This antibody/microbubble combination is often required as experimental control&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">&#160;&#160;&#160;&#160;&#160; biotin anti-mouse CD309</td>
			<td style="width:215px;">eBioscience</td>
			<td style="width:207px;">13-5821-85</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Biotinylated rat MJ 7/18 antibody to mouse endoglin</td>
			<td style="width:215px;">In house hybridoma</td>
			<td style="width:207px;"></td>
			<td>Outside antibodies may also be appropriate: we&#160; have used eBioscience (13-1051-85 ) in the past</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Distilled water</td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Embryo media</td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">&#160;&#160;&#160;&#160; 500 mL Dulbecco&#8217;s Modified Eagle&#8217;s Medium with high glucose</td>
			<td>Sigma</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">&#160;&#160;&#160;&#160; 50 mL Fetal Bovine Serum</td>
			<td>ATCC</td>
			<td style="width:207px;">30-2020</td>
			<td>lot # 7592456</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">&#160;&#160;&#160;&#160; Hepes&#160;</td>
			<td style="width:215px;">Gibco</td>
			<td style="width:207px;">15630</td>
			<td>5mL, 1M</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">&#160;&#160;&#160;&#160; Penicillin-Streptomycin&#160;</td>
			<td style="width:215px;">Gibco</td>
			<td style="width:207px;">15140-122</td>
			<td>5 mL, 10,000 units Pen., 10,000 ug Strep</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ethanol, 70%</td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ice</td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Paraformaldehyde</td>
			<td style="width:215px;">Sigma</td>
			<td style="width:207px;">76240</td>
			<td>4%</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Phosphate Buffered Saline [1x]&#160;</td>
			<td style="width:215px;">Sigma</td>
			<td style="width:207px;">D8537</td>
			<td>1x, w/o calcium chloride &#38; magnesium chloride</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Pregnant mouse, CD-1</td>
			<td style="width:215px;">Charles River Laboratories Inc.&#160;</td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">0.9% sodium chloride (saline)</td>
			<td style="width:215px;">Hospira</td>
			<td style="width:207px;">0409-7984-11</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ultrasound contrast agent, target ready and untargeted</td>
			<td style="width:215px;">MicroMarker; VisualSonics Inc.</td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ultrasound gel (Aquasonic 100, colourless)</td>
			<td style="width:215px;">CSP Medical</td>
			<td style="width:207px;">133-1009</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;"></td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Cell culture plates (4) :&#160; 100x20 mm</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:207px;">08-772-22</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Cell culture plates (12) : 60x15 mm</td>
			<td style="width:215px;">Sigma</td>
			<td style="width:207px;">D8054</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:419px;">Centrifuge</td>
			<td>Sorvall Legend RT centrifuge&#160;</td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Conical tubes, 50 mL BD Falcon</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">21008-938</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Diluent</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:207px;">Isoton II Diluent, 8448011</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Dissection scissors (Wagner)</td>
			<td style="width:215px;">Fine Science Tools</td>
			<td style="width:207px;">Wagner 14068-12</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Forceps (2), Dumont SS (0.10x0.06 mm)</td>
			<td style="width:215px;">Fine Science Tools</td>
			<td style="width:207px;">11200-33</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Forceps, splinter</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">25601-134</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Glass beaker, 2 L (Griffin Beaker)</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">89000-216</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Glass capillaries, 1x90 mm GD-1 with filament</td>
			<td style="width:215px;">Narishige</td>
			<td style="width:207px;">GD-1</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Glass needle puller</td>
			<td style="width:215px;">Narishige</td>
			<td style="width:207px;">PN-30</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Gloves</td>
			<td style="width:215px;">Ansell</td>
			<td style="width:207px;">4002</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Gross anatomy probe</td>
			<td style="width:215px;">Fine Science Tools</td>
			<td style="width:207px;">10088-15</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Hot plate</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">89090-994</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ice bucket</td>
			<td style="width:215px;">Cole Parmer</td>
			<td style="width:207px;">RK 06274-01</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Imaging Platform</td>
			<td style="width:215px;">VisualSonics Inc.</td>
			<td style="width:207px;">Integrated Rail System</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Light source, fiber-optic</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:207px;">12-562-36</td>
			<td>Ideally has adjustable arms</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Luers (12), polypropylene barbed female &#188;-28 UNF thread</td>
			<td style="width:215px;">Cole Parmer</td>
			<td style="width:207px;">45500-30</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Micro-ultrasound system, high-frequency</td>
			<td style="width:215px;">VisualSonics Inc.</td>
			<td style="width:207px;">Vevo2100</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Needles, 21 gauge&#160; (1&#8221;)</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">305165</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Particle size analyzer</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:207px;">Multisizer 3 Coulter Counter</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Perforated spoon (Moria)</td>
			<td style="width:215px;">Fine Science Tools</td>
			<td style="width:207px;">MC 17 10373-17</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Pins (6), black anodized minutien 0.15 mm</td>
			<td style="width:215px;">Fine Science Tools</td>
			<td style="width:207px;">26002-15</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;">Pipettors [2-20 uL, 20-200uL, 100-1000uL]</td>
			<td style="width:215px;">Eppendorf</td>
			<td style="width:207px;">Research Plus&#160; adjustable 3120000038;&#160;&#160;&#160;&#160;&#160;&#160; 3120000054;&#160;&#160;&#160;&#160;&#160;&#160; 3120000062</td>
			<td style="width:639px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Pipettor tips [2-200uL, 50-1000uL]</td>
			<td style="width:215px;">Eppendorf</td>
			<td style="width:207px;">epT.I.P.S.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 22491334;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 022491351</td>
			<td style="width:639px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Scissors</td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:215px;">Dow Corning</td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Tubing, Tygon laboratory 1/32x3/32&#8221;</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">63010-007</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Wooden applicator stick (swab, cotton head)</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">CA89031-270</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Surgical microscope 5-8x magnification</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:207px;">Steromaster</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Syringes, 1 mL Normject</td>
			<td style="width:215px;">Fisher</td>
			<td style="width:207px;">14-817-25</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Syringes (10), 30 mL</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">CA64000-041</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Syringe infusion pump&#160;</td>
			<td style="width:215px;">Bio-lynx&#160;</td>
			<td style="width:207px;">NE-1000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Thermometer, -20-110oC</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">89095-598</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Timer</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">33501-418</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Tubes, Eppendorf</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">20170-577</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Tube racks (3)</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">82024-462</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ultrasound transducer, 20 MHz</td>
			<td style="width:215px;">VisualSonics Inc.</td>
			<td style="width:207px;">MS250</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vannas-Tubingen, angled up</td>
			<td style="width:215px;">Fine Science Tools</td>
			<td style="width:207px;">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.

A injecção de agentes de contraste de ultra-som em embriões de ratinho ex útero é dependente do isolamento de embriões vivos, nas fases finais-gestacionais do útero e manutenção da viabilidade ao longo do curso da injecção e imagiologia de ultra-sons correspondente. Uma vez que o embrião foi exteriorizado e posicionado, como mostrado na Figura 1, a injecção do agente de contraste cuidado na vasculatura embrionária é possível. Uma imagem de ultra-som típico de modo B de um embr...

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

Aqui, apresentamos um protocolo para injetar agentes de contraste de microbolhas de ultra-som de estar, isoladas de fim de gestação de embriões palco murino. Este método permite o estudo de parâmetros de perfusão e de marcadores moleculares vasculares dentro do embrião usando de alta freqüência ultra-sonografia com contraste.

Imagem Contraste em embriões de camundongos Usando de alta freqüência ultra-som

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

Research

JoVE Journal

Developmental Biology

15.2K visualizações.  University of Toronto.  Aqui, apresentamos um protocolo para injetar agentes de contraste de microbolhas de ultra-som de estar, isoladas de fim de gestação de embriões palco murino. Este método permite o estudo de parâmetros de perfusão e de marcadores moleculares vasculares dentro do embrião usando de alta freqüência ultra-sonografia com contraste. 

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

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

analisando<em&gt; In Vivo</em&gt; Migração celular usando transplantes de células e de imagem Time-lapse em embriões Zebrafish

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Biologia do Desenvolvimento

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Imagem ao vivo do mouse Fusão do palato secundário

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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.

Um novo uso da ultra-sonografia tridimensional de alta frequência para caracterização de gravidez precoce no 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 ...

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.

Imagem cardiovascular fetal de camundongo usando um sistema de ultrassom de alta frequência (30/45MHZ)

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

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

Visualizando o nó e placa Notochordal em Gastrulating embriões de rato usando microscopia eletrônica e imunofluorescência de montagem inteira

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

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.

Ultra-som de alta frequência para a análise do desenvolvimento Fetal e placentário na 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 ...

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes for CHD are still poorly understood. The developing mouse constitutes a valuable model for the study of CHD, because cardiac developmental programs between mice and humans are highly conserved. The protocol describes in detail how to produce mouse embryos of the desired gestational stage, methods to isolate and preserve the heart for downstream processing, quantitative methods to identify common types of CHD by histology (e.g., ventricular septal defects, atrial septal defects, patent ductus arteriosus), and quantitative histomorphometry methods to measure common muscular compaction phenotypes. These methods articulate all the steps involved in sample preparation, collection, and analysis, allowing scientists to correctly and reproducibly measure CHD.

In this protocol, we describe procedures to qualitatively and quantitatively analyze developmental phenotypes in mice associated with congenital heart defects.

Análise de defeitos cardíacos congênitos em embriões de camundongos usando métodos histológicos qualitativos e quantitativos

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes ...

High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its embryonic transparency, genetic tractability, and amenity to rapid, high-throughput studies. However, the loss of transparency limits heart function analysis at the adult stage, which complicates modeling of age-related heart conditions. To overcome such limitations, high-frequency ultrasound echocardiography in zebrafish is emerging as a viable option. Here, we present a detailed protocol to assess cardiac function in adult zebrafish by non-invasive echocardiography using high-frequency ultrasound. The method allows visualization and analysis of zebrafish heart dimension and quantification of important functional parameters, including heart rate, stroke volume, cardiac output, and ejection fraction. In this method, the fish are anesthetized and kept underwater and can be recovered after the procedure. Although high-frequency ultrasound is an expensive technology, the same imaging platform can be used for different species (e.g., murine and zebrafish) by adapting different transducers. Zebrafish echocardiography is a robust method for cardiac phenotyping, useful in the validation and characterization of disease models, particularly late-onset diseases; drug screens; and studies of heart injury, recovery, and regenerative capacity.

We describe a protocol to assess heart morphology and function in adult zebrafish using high-frequency echocardiography. The method allows visualization of the heart and subsequent quantification of functional parameters, such as heart rate (HR), cardiac output (CO), fractional area change (FAC), ejection fraction (EF), and blood inflow and outflow velocities.

Ecocardiografia de ultrassom de alta frequência para avaliar a função cardíaca de zebrafish

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its ...

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin rods are a hallmark of a conserved, inducible Actin Stress Response (ASR) that accompanies human pathologies, including neurodegenerative disease. Previously, we showed that the ASR contributes to morphogenesis failures and reduced viability of developing embryos. This protocol allows the continued study of mechanisms underlying actin rod assembly and the ASR in a model system that is highly amenable to imaging, genetics and biochemistry. Embryos are collected and mounted on a coverslip to prepare them for injection. Rhodamine-conjugated globular actin (G-actinRed) is diluted and loaded into a microneedle. A single injection is made into the center of each embryo. After injection, embryos are incubated at elevated temperature and intranuclear actin rods are then visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) experiments may be performed on the actin rods; and other actin-rich structures in the cytoplasm can also be imaged. We find that G-actinRed polymerizes like endogenous G-actin and does not, on its own, interfere with normal embryo development. One limitation of this protocol is that care must be taken during injection to avoid serious injury to the embryo. However, with practice, injecting G-actinRed into Drosophila embryos is a fast and reliable way to visualize actin rods and can easily be used with flies of any genotype or with the introduction of other cellular stresses, including hypoxia and oxidative stress.

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress.

Imagens Intranuclear Actin Rods em Embriões de Drosophila estressados de calor vivo

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...

Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant mice to determine evolving processes at various timepoints. Moreover, optimal and efficient breeding of model mice requires an assessment of timed pregnancies. Most commonly, mice are mated overnight, and the presence of a vaginal plug is determined; however, the positive predictive value of this technique is suboptimal, and one needs to wait to know if the mouse is truly pregnant. High-resolution ultrasound biomicroscopy is an effective and efficient tool for imaging: 1) Whether a mouse is pregnant; 2) What gestational stage the mouse has reached; and 3) Whether there are intrauterine losses. In addition to the embryos and fetuses, the investigator must also recognize common artifacts in the abdominal cavity so as not to mistake these for a gravid uterus. This article provides a protocol for imaging along with illustrative examples.

High-resolution ultrasound can help streamline experiments requiring timed-pregnant mice by determining the state of pregnancy, gestational age, and pregnancy losses. Presented here is a protocol to illustrate methods to assess mouse pregnancies as well as potential pitfalls (image artifacts) that may mimic pregnancy.

Meu rato está grávida? Avaliação de ultrassom de alta frequência

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant ...