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: Les procédures utilisant le poisson zèbre ont été approuvés par Institutional Animal Care UCSF et l&#39;utilisation Comité. 1. Préparation des outils <ol><li> Insérer la goupille de point caractéristique dans un porte-broche et serrer la goupille. </li><li> Utilisation pince fine pointe de, pliez la pointe de la broche pour créer un léger crochet. </li><li> Pour la manipulation et à la stabilisation de l&#39;embryon au cours de blessure, pliez la fin d&#39;une aiguille de ½ pouce 28 jauge montée sur une seringue à insuline en utilisant une pince à bec effilé. </li></ol> 2. Préparation des embryons de poissons zèbres pour blessures <ol><li> Mettre en place couples reproducteurs de poisson zèbre et de ramasser les œufs dans l&#39;eau d&#39;œuf (60ug / mL sels d&#39;aquarium) comme indiqué précédemment 22. </li><li> Ajouter 0,003% de N-phénylthiourée (PTU) à l&#39;eau d&#39;œuf lorsque les embryons sont environ après la fécondation de 8 heures (HPF) pour empêcher mélanisation. </li><li> Dechorionate deux fertilisation poste de jour (DPF) embryons avant l&#39;expérience à l&#39;aide de pince fine de pointe. <br/&gt; NOTE: Les embryons peuvent être blessés à tout moment après le début de la circulation. Les données présentées ici sont pour 2 dpf embryons, mais la technique a été appliquée avec succès dans les embryons jusqu&#39;à 5 dpf. </li><li> Anesthésier les embryons avec 0,02% tamponné acide 3-aminobenzoïque (tricaïne) environ 10 min avant de manipulations. </li></ol> 3. navire mécanique blessures d&#39;embryons <ol><li> Transfert d&#39;embryons anesthésiés à une diapositive de la dépression sur un stéréomicroscope à dissection en utilisant une pipette de transfert. </li><li> En utilisant le côté plat court de l&#39;aiguille de la seringue pour manipuler l&#39;embryon avec la main dominante, placer l&#39;embryon sur le côté avec la face ventrale tournée à l&#39;opposé de l&#39;aiguille. </li><li> Positionner la goupille de minutie avec la pointe dirigée directement contre la surface ventrale de la partie postérieure de poisson à l&#39;ouverture de urogénital. Placez la tige de minutie à un léger angle tel que l&#39;extrémité incurvée est capable de percer le périderme directement dans la veine caudale (Figure 1 &lt;/ Strong&gt;). </li><li> Utilisation de l&#39;aiguille de la seringue pour manipuler l&#39;embryon, percer la veine caudale avec la broche de point caractéristique en appuyant sur l&#39;embryon dans l&#39;axe pour raccorder la broche légèrement dans la veine. </li><li> Utilisation de l&#39;aiguille de la seringue, l&#39;embryon tirer loin de l&#39;axe de minutie pour créer une petite déchirure dans le récipient. NOTE: Une blessure réussie entraîner des saignements immédiate de la veine. </li></ol> 4. Analyse de l&#39;hémostase <ol><li> Choisissez seuls les embryons avec circulation visiblement cellules sanguines pour cette procédure. </li><li> Préparer la minuterie pour commencer dès que la broche de point caractéristique est extrait de la cuve. </li><li> Lancer la minuterie dès que la perte de sang peut être visualisé à partir de la plaie. Lorsque la perte de sang de la plaie cesse, arrêter le chronomètre et le temps total que Temps de saignement. Si la coagulation est inhibée, enregistrer le temps au moment où il n&#39;y a plus visiblement les cellules sanguines circulantes. </li></ol> 5. Analyse de la cicatrisation <ol><li> Transfert post-blessures animaux dans des plats d&#39;imagerie à fond de verre pour la microscopie. </li><li> Retirer la majorité de l&#39;eau de l&#39;oeuf. </li><li> Couvrir les embryons dans 0,3 à 1,2% bas point de fusion agarose dissous dans l&#39;eau d&#39;œuf, chauffé entre 42 et 45 ° C et additionné de 0,02% tricaïne. </li><li> embryons de position sur leurs côtés à l&#39;aide de pinces. </li><li> Après l&#39;agarose refroidit, remplir la cuve avec 0,02% de tricaïne dans l&#39;eau d&#39;œuf. </li><li> Acquérir des images à l&#39;aide de clair, épifluorescence ou microscopie confocale. </li><li> Retirer embryon à partir de l&#39;agarose en utilisant une pince et transfert retour à l&#39;eau d&#39;œuf. </li></ol>

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The authors declare that they have no competing financial interests.

Le poisson zèbre ont été utilisés avec succès en tant que modèle pour les différents types de plaies, y compris les blessures laser 13 à 15, la thrombose induite par laser 16, 10 et épithéliale blessant. Nous rapportons une méthode de blessure mécanique qui est simple à exécuter et produit une blessure contrôlée dans un modèle in vivo qui est prête très bien à la microscopie en temps réel. Résultats de blessures dans une réponse hémostatique rapide et mesur...

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.

<table border="0" cellpadding="0" cellspacing="0" width="867">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Name of Material/ Equipment</td>
			<td style="width:159px;">Company</td>
			<td style="width:159px;">Catalog Number</td>
			<td style="width:303px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Minutia Pins</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td>26002-10</td>
			<td>Tip diameter 0.0125 mm, rod diameter 0.1 mm</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Glass Depression Slide</td>
			<td>Aquatic Eco-Systems</td>
			<td style="width:159px;">M30</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Hirudin</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:159px;">H7016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass bottom imaging dishes</td>
			<td style="width:159px;">Mattek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td style="width:159px;">Olympus</td>
			<td style="width:159px;">SZH10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Fluorescence microscope</td>
			<td style="width:159px;">Zeiss</td>
			<td>Axio Observer</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Aquarium salts</td>
			<td style="width:159px;">Instant Ocean</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
	</tbody>
</table>

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.

Lésion vasculaire mécanique a été effectuée sur deux dpf embryons (Figure 2A - C). Résultats de blessures dans une réponse rapide et fiable la coagulation telle que mesurée par le temps à la cessation du saignement (figure 2D). Pour déterminer si les différences dans la réponse de coagulation peuvent être mesurés, l&#39;hirudine anticoagulant a été administré à des embryons par injection dans le canal de Cuvier immédiatement avant la blessure (10.5 nl...

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.

Blessure mécanique des bateaux dans embryons de poissons zèbres

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

Research

JoVE Journal

Developmental Biology

7.2K vues.  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. 