Name: mechanical vessel injury in zebrafish embryos
Uploaded: 2015-02-17T14:00:00.000+00:00
Duration: 6 min 38 s
Description: Watch this Scientific Journal Video about Mechanical Vessel Injury in Zebrafish Embryos at JoVE.com

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>

<ol>
	<li>Gore, A. V., Monzo, K., Cha, Y. R., Pan, W., Weinstein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+development+in+the+zebrafish.">Vascular development in the zebrafish.</a> Cold Spring Harb Perspect Med. 2 (5), a006684 (2012).</li><li>Boatman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+hematopoiesis+using+zebrafish.">Assaying hematopoiesis using zebrafish.</a> Blood Cells Mol Dis. 51 (4), 271-276 (2013).</li><li>Ellett, F., Lieschke, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+for+vertebrate+hematopoiesis.">Zebrafish as a model for vertebrate hematopoiesis.</a> Curr Opin Pharmacol. 10 (5), 563-570 (2010).</li><li>Liu, J., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+in+the+study+of+early+cardiac+development.">Zebrafish in the study of early cardiac development.</a> Circ Res. 110 (6), 870-874 (2012).</li><li>Traver, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+organism+to+study+development+of+the+immune+system.">The zebrafish as a model organism to study development of the immune system.</a> Adv Immunol. 81, 253-330 (2003).</li><li>Lieschke, G. J., Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+immunology.">Fish immunology.</a> Curr Biol. 19 (16), R678-R682 (2009).</li><li>Lesley, R., Ramakrishnan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+early+mycobacterial+pathogenesis+from+the+zebrafish.">Insights into early mycobacterial pathogenesis from the zebrafish.</a> Curr Opin Microbiol. 11 (3), 277-283 (2008).</li><li>Oehlers, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+enterocolitis+model+in+zebrafish+larvae+that+is+dependent+on+microbiota+and+responsive+to+pharmacological+agents.">A chemical enterocolitis model in zebrafish larvae that is dependent on microbiota and responsive to pharmacological agents.</a> Dev Dyn. 240 (1), 288-298 (2011).</li><li>Gemberling, M., Bailey, T. J., Hyde, D. R., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+complex+tissue+regeneration.">The zebrafish as a model for complex tissue regeneration.</a> Trends Genet. 29 (11), 611-620 (2013).</li><li>LeBert, D. C., Huttenlocher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+wound+repair.">Inflammation and wound repair.</a> Semin Immunol. , (2014).</li><li>Henry, K. M., Loynes, C. A., Whyte, M. K., Renshaw, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+for+the+study+of+neutrophil+biology.">Zebrafish as a model for the study of neutrophil biology.</a> J Leukoc Biol. 94 (4), 633-642 (2013).</li><li>Niethammer, P., Grabher, C., Look, A. T., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tissue-scale+gradient+of+hydrogen+peroxide+mediates+rapid+wound+detection+in+zebrafish.">A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish.</a> Nature. 459 (7249), 996-999 (2009).</li><li>Rosenberg, A. F., Wolman, M. A., Franzini-Armstrong, C., Granato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+nerve-macrophage+interactions+following+peripheral+nerve+injury.">In vivo nerve-macrophage interactions following peripheral nerve injury.</a> J Neurosci. 32 (11), 3898-3909 (2012).</li><li>Otten, C., Abdelilah-Seyfried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-inflicted+injury+of+zebrafish+embryonic+skeletal+muscle.">Laser-inflicted injury of zebrafish embryonic skeletal muscle.</a> J Vis Exp. (71), e4351 (2013).</li><li>Matrone, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-targeted+ablation+of+the+zebrafish+embryonic+ventricle:+a+novel+model+of+cardiac+injury+and+repair.">Laser-targeted ablation of the zebrafish embryonic ventricle: a novel model of cardiac injury and repair.</a> Int J Cardiol. 168 (4), 3913-3919 (2013).</li><li>Jagadeeswaran, P., Carrillo, M., Radhakrishnan, U. P., Rajpurohit, S. K., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+thrombosis+in+zebrafish.">Laser-induced thrombosis in zebrafish.</a> Methods Cell Biol. 101, 197-203 (2011).</li><li>Vo, A. H., Swaroop, A., Liu, Y., Norris, Z. G., Shavit, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+fibrinogen+in+zebrafish+results+in+symptoms+consistent+with+human+hypofibrinogenemia.">Loss of fibrinogen in zebrafish results in symptoms consistent with human hypofibrinogenemia.</a> PLoS One. 8 (9), e74682 (2013).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+mutagenesis+of+zebrafish+antithrombin+III+triggers+disseminated+intravascular+coagulation+and+thrombosis,+revealing+insight+into+function.">Targeted mutagenesis of zebrafish antithrombin III triggers disseminated intravascular coagulation and thrombosis, revealing insight into function.</a> Blood. , (2014).</li><li>Jagadeeswaran, P., Liu, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hemophilia+model+in+zebrafish:+analysis+of+hemostasis.">A hemophilia model in zebrafish: analysis of hemostasis.</a> Blood Cells Mol Dis. 23 (1), 52-57 (1997).</li><li>Jagadeeswaran, P., Gregory, M., Day, K., Cykowski, M., Thattaliyath, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+genetic+model+for+hemostasis+and+thrombosis.">Zebrafish: a genetic model for hemostasis and thrombosis.</a> J Thromb Haemost. 3 (1), 46-53 (2005).</li><li>Lin, H. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+thrombocyte+development+in+CD41-GFP+transgenic+zebrafish.">Analysis of thrombocyte development in CD41-GFP transgenic zebrafish.</a> Blood. 106 (12), 3803-3810 (2005).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. (25), (2009).</li><li>Benard, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+zebrafish+embryos+with+intracellular+bacterial+pathogens.">Infection of zebrafish embryos with intracellular bacterial pathogens.</a> J Vis Exp. (61), (2012).</li><li>Jagadeeswaran, P., Sheehan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+blood+coagulation+in+the+zebrafish.">Analysis of blood coagulation in the zebrafish.</a> Blood Cells Mol Dis. 25 (3-4), 3-4 (1999).</li><li>Jin, S. W., Beis, D., Mitchell, T., Chen, J. N., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+analyses+of+vascular+tube+and+lumen+formation+in+zebrafish.">Cellular and molecular analyses of vascular tube and lumen formation in zebrafish.</a> Development. 132 (23), 5199-5209 (2005).</li><li>Traver, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nat Immunol. 4 (12), 1238-1246 (2003).</li><li>Lawson, N. D., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+embryonic+vascular+development+using+transgenic+zebrafish.">In vivo imaging of embryonic vascular development using transgenic zebrafish.</a> Dev Biol. 248 (2), 307-318 (2002).</li><li>Renshaw, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+zebrafish+model+of+neutrophilic+inflammation.">A transgenic zebrafish model of neutrophilic inflammation.</a> Blood. 108 (13), 3976-3978 (2006).</li><li>Hall, C., Flores, M. V., Storm, T., Crosier, K., Crosier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+lysozyme+C+promoter+drives+myeloid-specific+expression+in+transgenic+fish.">The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish.</a> BMC Dev Biol. 7, 48 (2007).</li><li>Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A., Lieschke, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mpeg1+promoter+transgenes+direct+macrophage-lineage+expression+in+zebrafish.">mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish.</a> Blood. 117 (4), e49-e56 (2011).</li><li>Gray, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+intravital+imaging+of+macrophage+and+neutrophil+behaviour+during+inflammation+using+a+novel+transgenic+zebrafish.">Simultaneous intravital imaging of macrophage and neutrophil behaviour during inflammation using a novel transgenic zebrafish.</a> Thromb Haemost. 105 (5), 811-819 (2011).</li><li>Bellido-Martin, L., Chen, V., Jasuja, R., Furie, B., Furie, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+fibrin+formation+and+platelet+and+endothelial+cell+activation+in+vivo.">Imaging fibrin formation and platelet and endothelial cell activation in vivo.</a> Thromb Haemost. 105 (5), 776-782 (2011).</li><li>Bielczyk-Maczynska, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+loss+of+function+screen+of+identified+genome-wide+association+study+Loci+reveals+new+genes+controlling+hematopoiesis.">A loss of function screen of identified genome-wide association study Loci reveals new genes controlling hematopoiesis.</a> PLoS Genet. 10 (7), e1004450 (2014).</li></ol>

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.

<table><tbody><tr><td>Minutia Pins</td><td>Fine Science Tools</td><td>26002-10</td><td>Tip diameter 0.0125 mm, rod diameter 0.1 mm</td></tr><tr><td>Pin Holder</td><td>Fine Science Tools</td><td>26016-12</td><td></td></tr><tr><td>Dumont #5 Fine Tip Forceps</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>Glass Depression Slide</td><td>Aquatic Eco-Systems</td><td>M30</td><td></td></tr><tr><td>Low Melting Agarose</td><td>Lonza&amp;nbsp;</td><td>50081</td><td>Preheated to 42 &amp;ordm; C</td></tr><tr><td>N-Phenylthiourea (PTU)</td><td>Sigma Aldrich</td><td>P7629</td><td></td></tr><tr><td>3-aminobenzoic acid (Tricaine)</td><td>Sigma Aldrich</td><td>E10521</td><td></td></tr><tr><td>Hirudin</td><td>Sigma Aldrich</td><td>H7016</td><td></td></tr><tr><td>Glass bottom imaging dishes</td><td>Mattek</td><td>P35G-1.5-14-C</td><td></td></tr><tr><td>Dissecting microscope</td><td>Olympus</td><td>SZH10</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Zeiss</td><td>Axio Observer</td><td></td></tr><tr><td>Aquarium salts</td><td>Instant Ocean</td><td></td><td></td></tr><tr><td>Insulin syringe with 28.5 G needle</td><td>Becton Dickinson&amp;nbsp;</td><td>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

Research

JoVE Journal

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. 

Video: Mechanical Vessel Injury in Zebrafish Embryos

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Microperla impianto nel zebrafish

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Ricerca

Biologia dello sviluppo

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

L&#39;induzione di infarto miocardico in Zebrafish adulti usando Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

Medicina

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.

L'analisi dello spostamento della deformazione meccanica miocardiale (DIAMOND) rivela l'eterogeneità segmentale della funzione cardiaca nel pesce zebra embrionale

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Stab Ferita Lesione del Zebrafish adulti Telencefalo: Un metodo per Indagare vertebrati cervello neurogenesi e rigenerazione

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Neuroscienze

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

Un dispositivo di trapianto semplice ed efficace per embrioni di zebrafish

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

<meta charset="utf8"></head><body>Studio di meccanotrasduzione in vivo utilizzando biglie magnetiche nel cuore del pesce zebra

Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads

Mechanical forces continuously provide feedback to heart valve morphogenetic programs. In zebrafish, cardiac valve development relies on heart contraction and physical stimuli generated by the beating heart. Intracardiac hemodynamics, driven by blood flow, emerge as fundamental information shaping the development of the embryonic heart. Here, we describe an effective method to manipulate mechanical forces in vivo by grafting a 30 &#181;m to 60 &#181;m diameter magnetic bead in the cardiac lumen. The insertion of the bead is conducted through microsurgery in anesthetized larvae without perturbing heart function and enables artificial alteration of the boundary conditions, thereby modifying flow forces in the system. As a result, the presence of the bead amplifies the mechanical forces experienced by endocardial cells and can directly trigger mechanical stimulus-dependent calcium influx. This approach facilitates the investigation of mechanotransduction pathways that govern heart development and can provide insights into the role of mechanical forces in cardiac valve morphogenesis.

<meta charset="utf8"></head><body>Autore in primo piano: Comprendere le forze meccaniche coinvolte nella formazione del cuore del pesce zebra

This protocol outlines a method for grafting a magnetic bead into the developing zebrafish heart through microsurgery, enabling the manipulation of mechanical forces in vivo and triggering mechanical stimulus-dependent calcium influx in endocardial cells.

Manipolazione delle forze meccaniche nel cuore del pesce zebra in via di sviluppo utilizzando perle magnetiche

Mechanical forces continuously provide feedback to heart valve morphogenetic programs. In zebrafish, cardiac valve development relies on heart contraction ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Laser-lesioni del muscolo scheletrico embrionale Zebrafish

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Biologia

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

Imaging 3D e ricostruzione di strutture cerebrovascolari in Zebrafish embrionali

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Mechanical Injury Vessel in embrioni di zebrafish

Riepilogo

Esplora Altri Video

Capitoli in questo video

Microperla impianto nel zebrafish

L'induzione di infarto miocardico in Zebrafish adulti usando Cryoinjury

L'analisi dello spostamento della deformazione meccanica miocardiale (DIAMOND) rivela l'eterogeneità segmentale della funzione cardiaca nel pesce zebra embrionale

Stab Ferita Lesione del Zebrafish adulti Telencefalo: Un metodo per Indagare vertebrati cervello neurogenesi e rigenerazione

Un dispositivo di trapianto semplice ed efficace per embrioni di zebrafish

Manipolazione delle forze meccaniche nel cuore del pesce zebra in via di sviluppo utilizzando perle magnetiche

Manipolazione delle forze meccaniche nel cuore del pesce zebra in via di sviluppo utilizzando perle magnetiche

Manipolazione delle forze meccaniche nel cuore del pesce zebra in via di sviluppo utilizzando perle magnetiche

Laser-lesioni del muscolo scheletrico embrionale Zebrafish

Imaging 3D e ricostruzione di strutture cerebrovascolari in Zebrafish embrionali