The authors would like to thank Drs. Stephen Wilson and Lisa Wilsbacher for helpful discussions. This work was supported in part by NIH HL054737.

NOTA: Os procedimentos que utilizam peixe-zebra foram aprovados pelo Institutional Animal Care da UCSF e Comitê de Uso. 1. Preparação de Ferramentas <ol><li> Insira pin minúcia em um suporte de pino e prenda o pino. </li><li> Utilizando uma pinça de ponta fina, dobre com cuidado a ponta do pino para criar um ligeiro gancho. </li><li> Para manipulação e estabilização do embrião durante a lesão, dobre a extremidade de um calibre 28 ½ polegadas agulha montada em uma seringa de insulina usando alicates. </li></ol> 2. Preparação de embriões Zebrafish por lesão <ol><li> Configure casais reprodutores de peixes-zebra e recolher os ovos em água ovo (60ug / sais aquário ml) como mostrado anteriormente 22. </li><li> Adicionar 0,003% de N-feniltioureia (PTU) para a água de ovos quando os embriões são aproximadamente 8 horas pós-fertilização (hpf) para impedir que a melanização. </li><li> Dechorionate dois dia após a fertilização (DPF) embriões antes da experiência com a pinça de ponta fina. <br/&gt; NOTA: Os embriões podem ser ferido a qualquer momento após a circulação começa. Os dados aqui apresentados são para 2 dpf embriões, mas a técnica foi aplicada com sucesso em embriões de até 5 dpf. </li><li> Anestesiar os embriões com 0,02% tamponado com ácido 3-aminobenzóico (tricaina) aproximadamente 10 min antes de manipulações. </li></ol> 3. Vessel Mecânica Lesão de Embriões <ol><li> Transferência de embriões anestesiados para uma lâmina de depressão em um microscópio estereoscópico de dissecação usando uma pipeta de transferência. </li><li> Usando o lado curto plana da agulha da seringa para manipular o embrião com a mão dominante, posicionar o embrião de lado, com a superfície ventral virada para fora da agulha. </li><li> Posicionar o pino minúcia com a ponta virada directamente contra a superfície ventral do peixe para a posterior abertura urogenital. Posicionar o pino minúcia com um ligeiro ângulo de tal modo que a ponta curvada é capaz de perfurar o periderme directamente na veia caudal (Figura 1 &lt;/ Strong&gt;). </li><li> Usando a agulha da seringa para manipular o embrião, perfurar a veia caudal com o pino de minúcia tocando no embrião para o pino de gancho ligeiramente o pino para dentro da veia. </li><li> Usando a agulha da seringa, puxe o embrião para longe do pino de minúcia para criar um pequeno rasgo no recipiente. NOTA: A lesão de sucesso vai resultar em sangramento imediato a partir da veia. </li></ol> 4. Análise da hemostasia <ol><li> Escolha apenas os embriões com visivelmente células circulantes no sangue para este procedimento. </li><li> Prepare-se para iniciar o temporizador assim que o pino de minúcia é puxado a partir do vaso. </li><li> Inicie o cronometro logo que a perda de sangue pode ser visualizado a partir da ferida. Quando a perda de sangue da ferida cessa, parar o temporizador e registrar o tempo total, como o tempo de sangramento. Se a coagulação é inibida, o tempo para gravar quando não há mais visivelmente células circulantes no sangue. </li></ol> 5. Análise da cicatrização de feridas <ol><li> Transferência posanimais t-lesão em placas de imagem com fundo de vidro para microscopia. </li><li> Remover a maior parte da água do ovo. </li><li> Cubra os embriões em 0,3-1,2% baixo ponto de fusão agarose dissolvidos na água do ovo, aquecido a entre 42 e 45 ºC e suplementadas com 0,02% tricaina. </li><li> Embriões posição sobre os seus lados, usando uma pinça. </li><li> Após a agarose esfria, encher o prato com 0,02% tricaina em água ovo. </li><li> Adquirir imagens usando brightfield, epifluorescência, ou microscopia confocal. </li><li> Remover embrião do agarose usando uma pinça e transferência de volta para a água ovo. </li></ol>

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The authors declare that they have no competing financial interests.

Zebrafish têm sido utilizados com sucesso como um modelo para vários tipos de feridas, incluindo lesão de laser 13-15, trombose induzida por laser 16, e epitelial ferindo 10. Relata-se um método de ferimento mecânico que é simples de executar e produz uma lesão controlada num modelo in vivo, que é altamente favorável à microscopia em tempo real. Resultados da lesão em um resposta hemostática rápida e mensurável e um programa de reparação ferida reprodut?...

Zebrafish have been used extensively to study a variety of topics in vascular biology, including vascular development, angiogenesis, and hematopoietic development and pathology1-3. Embryos develop a functional circulation as well as leukocytes and other components of the innate immune system by 1 day post fertilization (dpf) 1,4,5. The conservation of the inflammatory and leukocyte response to injury has made the zebrafish embryo an informative model for such diverse inflammatory processes as tuberculous infection, enterocolitis, and tissue regeneration6-9. Zebrafish embryos have been used to study injury-related inflammation particularly in the context of epithelial wounding and the neutrophil response10,11. Injury to the embryo results in a highly conserved cellular response from cells at the injury site and the innate immune cells recruited to respond to the injury and regulate its resolution11,12. Other injury models have used focused laser pulses to spatially localize injury to specific cell types including neurons, muscle cells, and cardiomyocytes13-15. 
Zebrafish embryos have been used as a model to study hemostasis and thrombosis in conditions of pharmacological and genetic manipulation, using both mechanical and laser-induced thrombus formation16-19. Components of the coagulation cascade appear to be well-conserved and transgenics have allowed for detailed studies of thrombocyte and fibrin deposition at the site of coagulation17,20,21. The procedure presented in this paper complements these methods by providing a system for studying mechanical vessel injury resulting in vessel breach, thrombus formation and resolution, and vessel repair.

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			<td style="width:159px;">Catalog Number</td>
			<td style="width:303px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:248px;">Minutia Pins</td>
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			<td>Tip diameter 0.0125 mm, rod diameter 0.1 mm</td>
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			<td height="21" style="height:21px;width:248px;">Pin Holder</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td>26016-12</td>
			<td></td>
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			<td height="21" style="height:21px;">Dumont #5 Fine Tip Forceps</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
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			<td height="21" style="height:21px;width:248px;">Glass Depression Slide</td>
			<td>Aquatic Eco-Systems</td>
			<td style="width:159px;">M30</td>
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			<td height="21" style="height:21px;">Low Melting Agarose</td>
			<td>Lonza&#160;</td>
			<td style="width:159px;">50081</td>
			<td>Preheated to 42 &#186; C</td>
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			<td height="21" style="height:21px;">N-Phenylthiourea (PTU)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:159px;">P7629</td>
			<td></td>
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			<td height="21" style="height:21px;width:248px;">3-aminobenzoic acid (Tricaine)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:159px;">E10521</td>
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			<td height="21" style="height:21px;width:248px;">Hirudin</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:159px;">H7016</td>
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			<td height="21" style="height:21px;">Glass bottom imaging dishes</td>
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			<td height="21" style="height:21px;width:248px;">Fluorescence microscope</td>
			<td style="width:159px;">Zeiss</td>
			<td>Axio Observer</td>
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			<td height="21" style="height:21px;width:248px;">Aquarium salts</td>
			<td style="width:159px;">Instant Ocean</td>
			<td style="width:159px;"></td>
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			<td height="21" style="height:21px;width:248px;">Insulin syringe with 28G1/2 needle</td>
			<td style="width:159px;">Becton Dickinson&#160;</td>
			<td style="width:159px;">329461</td>
			<td></td>
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mechanical vessel injury in zebrafish embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

A lesão mecânica vaso foi realizada em 2 dpf embriões (Figura 2A - C). Resultados da lesão em um resposta rápida e confiável de coagulação medida pelo tempo de interrupção do sangramento (Figura 2D). Para determinar se ou não diferenças na resposta de coagulação pode ser medido, o anticoagulante hirudina foi administrada aos embriões por injecção para dentro da conduta de Cuvier imediatamente antes do ferimento (5-10 nl de 1 unidade por ul de hirudina d...

Watch this Scientific Journal Video about Mechanical Vessel Injury in Zebrafish Embryos at JoVE.com

Mechanical Vessel Injury in Zebrafish Embryos

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Lesão Vessel Mecânica em embriões Zebrafish

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

mechanical-vessel-injury-in-zebrafish-embryos

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Developmental Biology

7.2K visualizações.  University of California. This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy. 