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

NOT: Zebra balığı kullanarak Prosedürleri UCSF Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. Araçlar 1. Hazırlık <ol><li> Bir iğne tutucu içine minutia pimini takın ve pin kelepçe. </li><li> Ince uçlu forseps kullanarak, dikkatli bir hafif kanca oluşturmak için pimin ucunu bükün. </li><li> Manipülasyon ve yaralanma sırasında embriyonun stabilizasyonu için, iğne-burun pense kullanarak bir insülin şırınga üzerine monte edilmiş bir 28 guage ½ inç iğne ucunu bükün. </li></ol> Sakatlık için Zebra balığı Embriyolar 2. Hazırlık <ol><li> Zebra balığı yetiştiriciliği çiftleri kurmak ve daha önce 22 gösterildiği gibi yumurta su (60ug / ml akvaryum tuzları) yumurta toplamak. </li><li> Embriyolar yaklaşık melanization önlemek için 8 saat sonra döllenme (hpf) olduğunda yumurta suya% 0.003 N-feniltiyoüre (PTU) ekleyin. </li><li> Ince uçlu forseps kullanarak deney öncesinde dechorionate iki gün sonrası fertilizasyon (dpf) embriyolar. <br/&gt; Not: dolaşım başladıktan sonra Embriyolar her zaman yaralı olabilir. Burada sunulan veriler embriyolar dpf&#39;e 2 için, ama tekniği başarıyla 5 dpf&#39;e kadar embriyolarda uygulanmıştır. </li><li> % 0.02 tamponlanmış 3-aminobenzoik asit (Tricaine), önceki kontroller için yaklaşık 10 dakika ile embriyolar anestezisi. </li></ol> 3. Mekanik Gemi Embriyolar yaralanması <ol><li> Bir transfer pipeti kullanarak bir diseksiyon stereomikroskopta bir depresyon slayt anestezi embriyolar transfer. </li><li> Dominant elin ile embriyo işlemek için şırınga iğnesi kısa düz tarafını kullanarak, uzak iğne bakan ventral yüzeyi ile yan embriyo yerleştirin. </li><li> Ucu ile minutia pimi ürogenital açılış balık posterior ventral yüzeyine doğrudan işaret yerleştirin. Hafif bir açıyla minutia pimini yerleştirin böyle kavisli ucu kaudal ven (Şekil 1 &lt;doğrudan periderm delip mümkün olduğunu/ Strong&gt;). </li><li> Embriyo işlemek için şırınga iğnesi kullanarak, hafifçe damar içine iğne kanca pin içine embriyo dokunarak minutia iğne ile kaudal ven delmek. </li><li> Şırınga iğnesi kullanarak, kap içinde küçük bir gözyaşı oluşturmak için uzak minutia pin embriyo çekin. NOT: Başarılı bir yaralanma damarından derhal kanama neden olur. </li></ol> Hemostazın 4. Analizi <ol><li> Gözle görülür bu işlem için kan hücreleri dolaşımdaki sadece embriyolar seçin. </li><li> En kısa sürede minutia pimi kabından çekilir gibi zamanlayıcı başlamak için hazırlayın. </li><li> En kısa sürede kan kaybı yaradan görüntülenmiştir edilebilir Sayacı başlatmak. Yaradan kan kaybı kesildiğinde, Sayacı durdurmak ve Zaman Kanama gibi toplam süreyi kaydetmek. Pıhtılaşma engellenir ise, artık gözle görülür dolaşan kan hücreleri varken zaman kaydedin. </li></ol> Yara Şifa 5. Analizi <ol><li> Aktarım posmikroskopi için cam alt görüntüleme yemekleri üzerine t-yaralanma hayvanlar. </li><li> Yumurta suyun çoğunluğu çıkarın. </li><li> ºC 42 ila 45 kadar ısıtılır ve% 0.02 Tricaine ile takviye agaroz yumurta suda çözünmüş 0,3-1,2% düşük erime embriyolar, örtün. </li><li> Forseps kullanarak kendi yüzüne Pozisyon embriyolar. </li><li> Agaroz soğuduktan sonra, yumurta, su içinde% 0,02 Tricaine ile çanak doldurun. </li><li> Aydınlık, Epifloresans veya konfokal mikroskopi kullanılarak görüntü elde. </li><li> Forseps kullanarak agaroz embriyo çıkarın ve yumurta suya geri aktarın. </li></ol>

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<li>Jagadeeswaran, P., Carrillo, M., Radhakrishnan, U. P., Rajpurohit, S. K., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laser-induced+thrombosis+in+zebrafish%5BTitle%5D)+AND+%22Methods+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((Loss+of+fibrinogen+in+zebrafish+results+in+symptoms+consistent+with+human+hypofibrinogenemia%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Targeted+mutagenesis+of+zebrafish+antithrombin+III+triggers+disseminated+intravascular+coagulation+and+thrombosis%2C+revealing+insight+into+function%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((A+hemophilia+model+in+zebrafish%3A+analysis+of+hemostasis%5BTitle%5D)+AND+%22Blood+Cells+Mol+Dis%22%5BJournal%5D)">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/pubmed?term=((Zebrafish%3A+a+genetic+model+for+hemostasis+and+thrombosis%5BTitle%5D)+AND+%22J+Thromb+Haemost%22%5BJournal%5D)">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/pubmed?term=((Analysis+of+thrombocyte+development+in+CD41-GFP+transgenic+zebrafish%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Microinjection+of+zebrafish+embryos+to+analyze+gene+function%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Infection+of+zebrafish+embryos+with+intracellular+bacterial+pathogens%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Analysis+of+blood+coagulation+in+the+zebrafish%5BTitle%5D)+AND+%22Blood+Cells+Mol+Dis%22%5BJournal%5D)">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/pubmed?term=((Cellular+and+molecular+analyses+of+vascular+tube+and+lumen+formation+in+zebrafish%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">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/pubmed?term=((In+vivo+imaging+of+embryonic+vascular+development+using+transgenic+zebrafish%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">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/pubmed?term=((A+transgenic+zebrafish+model+of+neutrophilic+inflammation%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((The+zebrafish+lysozyme+C+promoter+drives+myeloid-specific+expression+in+transgenic+fish%5BTitle%5D)+AND+%22BMC+Dev+Biol%22%5BJournal%5D)">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/pubmed?term=((mpeg1+promoter+transgenes+direct+macrophage-lineage+expression+in+zebrafish%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Simultaneous+intravital+imaging+of+macrophage+and+neutrophil+behaviour+during+inflammation+using+a+novel+transgenic+zebrafish%5BTitle%5D)+AND+%22Thromb+Haemost%22%5BJournal%5D)">Simultaneous intravital imaging of macrophage and neutrophil behaviour during inflammation using a novel transgenic zebrafish.</a> Thromb Haemost. 105 (5), 811-819 (2011).</li>
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<li>Bielczyk-Maczynska, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+loss+of+function+screen+of+identified+genome-wide+association+study+Loci+reveals+new+genes+controlling+hematopoiesis%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">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.

Balığı 10 yaralama lazer yaralanması 13-15, lazer kaynaklı tromboz 16 ve epiteliyal da dahil olmak üzere yaraların farklı türleri için bir model olarak başarılı bir şekilde kullanılmıştır. Bu çalıştırmak için basit ve gerçek zamanlı mikroskopi için son derece müsait olan bir in vivo modeli olarak kontrollü bir yaralanma üreten mekanik yaralama için bir yöntem sunulmaktadır. Hızlı ve ölçülebilir hemostatik tepki ve görüntü ve timelapse mikr...

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.

Mekanik damar hasarı embriyolar (- C Şekil 2A) dpf, 2 gerçekleştirilmiştir. Hızlı ve güvenilir bir pıhtılaşma yanıt olarak yaralanma sonucu (Şekil 2D) kanamanın durdurulması zaman ile ölçülmüştür. Pıhtılaşma karşılık olarak fark ölçülebilir olup olmadığını belirlemek için, pıhtılaşma önleyici Hirudin (için hemen önce (5-10 nl su içinde çözülmüş hirudin ul başına 1 ünite) yaralama ile Cuvier&#39;in gösterilen kanal içine enjekte embriyo...

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.

Zebra balığı Embriyolar Mekanik Gemi Sakatlık

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

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

7.2K Görüntüleme.  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.

Zebra balığı Embriyo microbead İmplantasyonu

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 ...

Gelişim Biyolojisi

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.

Yetişkin Zebra balığı Myokard İnfarktüsü İndüksiyon Cryoinjury kullanma

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

Tıp

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Larva Zebra balığı Omurilik Kesisi

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Nörobilim

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.

Miyokardiyal Mekanik Deformasyonun Yer Değiştirme Analizi (DIAMOND) Embriyonik Zebrabalığında Kardiyak Fonksiyonun Segmental Heterojenliğini Ortaya Çıkarır

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.

Zebra balığı Yetişkin telencephalon stab Yara Yaralanma: Omurgalı Beyin nöron ve Rejenerasyon Soruşturma Bir Yöntem

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

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.

Zebra Balığı Embriyoları İçin Basit ve Etkili Bir Transplantasyon Cihazı

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>Zebra Balığı Kalbinde Manyetik Boncuklar Kullanılarak İn Vivo Mekanotransdüksiyon Çalışması

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>Yazar Spotlight: Zebra Balığı Kalbinin Şekillenmesinde Rol Oynayan Mekanik Kuvvetleri Anlamak

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.

Gelişmekte Olan Zebra Balığı Kalbindeki Mekanik Kuvvetlerin Manyetik Boncuklar Kullanılarak Manipüle Edilmesi

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.

Zebra balığı Embriyonik İskelet Kası Lazer-yaralama

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

Zebra balığı Embriyolar Mekanik Gemi Sakatlık

Özet

Daha Fazla Video Keşfet

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Yetişkin Zebra balığı Myokard İnfarktüsü İndüksiyon Cryoinjury kullanma

Larva Zebra balığı Omurilik Kesisi

Miyokardiyal Mekanik Deformasyonun Yer Değiştirme Analizi (DIAMOND) Embriyonik Zebrabalığında Kardiyak Fonksiyonun Segmental Heterojenliğini Ortaya Çıkarır

Zebra balığı Yetişkin telencephalon stab Yara Yaralanma: Omurgalı Beyin nöron ve Rejenerasyon Soruşturma Bir Yöntem

Zebra Balığı Embriyoları İçin Basit ve Etkili Bir Transplantasyon Cihazı

Gelişmekte Olan Zebra Balığı Kalbindeki Mekanik Kuvvetlerin Manyetik Boncuklar Kullanılarak Manipüle Edilmesi

Gelişmekte Olan Zebra Balığı Kalbindeki Mekanik Kuvvetlerin Manyetik Boncuklar Kullanılarak Manipüle Edilmesi

Gelişmekte Olan Zebra Balığı Kalbindeki Mekanik Kuvvetlerin Manyetik Boncuklar Kullanılarak Manipüle Edilmesi

Zebra balığı Embriyonik İskelet Kası Lazer-yaralama