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: Le procedure utilizzando zebrafish sono stati approvati dalla Institutional Animal Care del UCSF and Use Committee. 1. Preparazione di strumenti <ol><li> Inserire pin minuzia in un supporto del perno e bloccare il perno. </li><li> Utilizzando pinze punta fine, piegare attentamente la punta del perno di creare un leggero uncino. </li><li> Per la manipolazione e la stabilizzazione dell&#39;embrione durante lesioni, piegate la fine di un 28 calibro ½ pollice ago montato su una siringa da insulina con pinze ad ago. </li></ol> 2. Preparazione di embrioni di zebrafish per Injury <ol><li> Impostare zebrafish coppie nidificanti e raccogliere le uova in acqua uovo (60ug / ml sali acquario), come indicato in precedenza 22. </li><li> Aggiungere 0,003% N-feniltiourea (PTU) all&#39;acqua uovo quando gli embrioni sono circa 8 ore dopo la fecondazione (HPF) per impedire melanizzazione. </li><li> Dechorionate due giorni dopo la fecondazione (dpf) embrioni prima dell&#39;esperimento con una pinza a punta fine. <br/&gt; Nota: Gli embrioni possono essere feriti in qualsiasi momento dopo l&#39;inizio circolazione. I dati qui presentati sono per 2 dpf embrioni, ma la tecnica è stata applicata con successo in embrioni fino a 5 dpf. </li><li> Anestetizzare gli embrioni con il 0,02% tamponato acido 3-aminobenzoico (Tricaine) circa 10 minuti prima di manipolazioni. </li></ol> 3. Vessel meccanica Injury di embrioni <ol><li> Trasferimento embrioni anestetizzati a una diapositiva depressione su uno stereomicroscopio dissezione utilizzando una pipetta di trasferimento. </li><li> Usando il lato corto piatto dell&#39;ago della siringa per manipolare l&#39;embrione con la mano dominante, posizionare l&#39;embrione su un lato con la superficie ventrale rivolto verso l&#39;ago. </li><li> Posizionare il perno minuzia con la punta puntato direttamente contro la superficie ventrale del posteriore pesce all&#39;apertura urogenitale. Posizionare il perno minuzia con una leggera angolazione tale che l&#39;estremità ricurva è in grado di penetrare attraverso la periderm direttamente nella vena caudale (Figura 1 &lt;/ Strong&gt;). </li><li> Utilizzando l&#39;ago della siringa per manipolare l&#39;embrione, perforare la vena caudale con il perno minutia toccando l&#39;embrione nel perno di agganciare leggermente il perno nella vena. </li><li> Utilizzando l&#39;ago della siringa, estrarre l&#39;embrione dal perno minuzia per creare un piccolo strappo nel recipiente. NOTA: Una lesione di successo si tradurrà in sanguinamento immediato dalla vena. </li></ol> 4. Analisi di Emostasi <ol><li> Scegliere solo embrioni con visibilmente circolanti globuli per questa procedura. </li><li> Preparare per iniziare il temporizzatore non appena il perno minutia è tirato dal recipiente. </li><li> Avviare il temporizzatore non appena la perdita di sangue può essere visualizzata dalla ferita. Quando la perdita di sangue dalla ferita cessa, fermare il timer e registrare il tempo totale come tempo di sanguinamento. Se la coagulazione è inibita, registrare il tempo quando non ci sono più visibilmente circolanti globuli. </li></ol> 5. Analisi di guarigione delle ferite <ol><li> Trasferimento posanimali t-lesioni su piatti di imaging con fondo di vetro per la microscopia. </li><li> Rimuovere la maggior parte dell&#39;acqua uovo. </li><li> Coprire gli embrioni a 0,3-1,2% basso punto di fusione agarosio disciolti in acqua uovo, riscaldata a tra 42 e 45 ° C e integrati con il 0,02% Tricaine. </li><li> Posizione embrioni sui loro lati con pinze. </li><li> Dopo l&#39;agarosio si raffredda, riempire il piatto con il 0,02% Tricaine in acqua uovo. </li><li> Acquisire le immagini utilizzando chiaro, epifluorescenza, o microscopia confocale. </li><li> Rimuovere embrione dal agarosio con pinze e trasferire di nuovo in acqua uovo. </li></ol>

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The authors declare that they have no competing financial interests.

Zebrafish sono stati utilizzati con successo come modello per i diversi tipi di ferite comprese lesioni laser 13-15, indotta da laser trombosi 16, e ferendo 10 epiteliale. Descriviamo un metodo di ferimento meccanica che è semplice da eseguire e produce una lesione controllata in un modello in vivo che è altamente suscettibile di microscopia in tempo reale. Risultati lesioni in una risposta rapida emostatico e misurabile e un programma di riparazione ferita riproducibile che p...

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 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>
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		<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>
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		<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.

Lesioni vaso meccanica è stata eseguita su 2 dpf embrioni (Figura 2A - C). Risultati lesioni in una rapida ed affidabile risposta coagulazione misurata come tempo per la cessazione di sanguinamento (Figura 2D). Per determinare se le differenze nella risposta della coagulazione possono essere misurati, l&#39;irudina anticoagulante è stato somministrato alle embrioni per iniezione nel condotto di Cuvier immediatamente prima ferendo (5-10 nl di 1 unità per ml irudina di...

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.

Mechanical Injury Vessel in embrioni di 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 Views.  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. 