<meta charset="utf8"></head><body>激光消融法研究斑马鱼幼虫的骨再生

<ol>
	<li>Marques, I. J., Lupi, E., Mercader, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+systems+for+regeneration:+Zebrafish.">Model systems for regeneration: Zebrafish.</a> Dev. 146 (18), dev167692 (2019).</li><li>Dietrich, K., Fiedler, I. A. K., Kurzyukova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+biology+and+disease+modeling+in+zebrafish.">Skeletal biology and disease modeling in zebrafish.</a> J Bone Miner Res. 36 (3), 436-458 (2021).</li><li>Geurtzen, K., Knopf, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+zebrafish+injury+models+to+study+the+effects+of+prednisolone+in+regenerating+bone+tissue.">Adult zebrafish injury models to study the effects of prednisolone in regenerating bone tissue.</a> J Vis Exp. (140), e58429 (2018).</li><li>Sousa, S., Valerio, F., Jacinto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+zebrafish+bone+crush+injury+model.">A new zebrafish bone crush injury model.</a> Biol Open. 1 (9), 915-921 (2012).</li><li>Gonz&#225;lez-Rosa, J. M., Mercader, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoinjury+as+a+myocardial+infarction+model+for+the+study+of+cardiac+regeneration+in+the+zebrafish.">Cryoinjury as a myocardial infarction model for the study of cardiac regeneration in the zebrafish.</a> Nat Protoc. 7 (4), 782-788 (2012).</li><li>Chassot, B., Pury, D., Ja&#378;wi&#324;ska, 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+fin+regeneration+after+cryoinjury-induced+tissue+damage.">Zebrafish fin regeneration after cryoinjury-induced tissue damage.</a> Biol Open. 5 (6), 819-828 (2016).</li><li>Singh, S. P., Holdway, J. E., 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=Regeneration+of+amputated+zebrafish+fin+rays+from+de+novo+osteoblasts.">Regeneration of amputated zebrafish fin rays from de novo osteoblasts.</a> Dev Cell. 22 (4), 879-886 (2012).</li><li>Curado, 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=Conditional+targeted+cell+ablation+in+zebrafish:+a+new+tool+for+regeneration+studies.">Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies.</a> Dev Dyn. 236 (4), 1025-1035 (2007).</li><li>Ando, K., Shibata, E., Hans, S., Brand, M., Kawakami, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblast+production+by+reserved+progenitor+cells+in+zebrafish+bone+regeneration+and+maintenance.">Osteoblast production by reserved progenitor cells in zebrafish bone regeneration and maintenance.</a> Dev Cell. 43 (5), 643-650.e3 (2017).</li><li>Geurtzen, K., L&#243;pez-Delgado, A. C., Duseja, A., Kurzyukova, A., Knopf, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-mediated+osteoblast+ablation+triggers+a+pro-osteogenic+inflammatory+response+regulated+by+reactive+oxygen+species+and+glucocorticoid+signaling+in+zebrafish.">Laser-mediated osteoblast ablation triggers a pro-osteogenic inflammatory response regulated by reactive oxygen species and glucocorticoid signaling in zebrafish.</a> Dev. 149 (8), 1998030 (2022).</li><li>Volpe, B. A., Fotino, T. H., Steiner, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscope-based+laser+ablation+and+regeneration+assay+in+zebrafish+interneuromast+cells.">Confocal microscope-based laser ablation and regeneration assay in zebrafish interneuromast cells.</a> J Vis Exp. (159), e60966 (2020).</li><li>Johnson, C. S., Holzemer, N. F., Wingert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+ablation+of+the+zebrafish+pronephros+to+study+renal+epithelial+regeneration.">Laser ablation of the zebrafish pronephros to study renal epithelial regeneration.</a> J Vis Exp. (54), e2845 (2011).</li><li>Goldstein, A. M., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notochord+regulates+cardiac+lineage+in+zebrafish+embryos.">Notochord regulates cardiac lineage in zebrafish embryos.</a> Dev Biol. 201 (2), 247-252 (1998).</li><li>Zhang, J., Jeradi, S., Str&#228;hle, U., Akimenko, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+ablation+of+the+sonic+hedgehog-a-expressing+cells+during+fin+regeneration+affects+ray+branching+morphogenesis.">Laser ablation of the sonic hedgehog-a-expressing cells during fin regeneration affects ray branching morphogenesis.</a> Dev Biol. 365 (2), 424-433 (2012).</li><li>Marshall, A. R., 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=Two-photon+cell+and+tissue+level+laser+ablation+methods+to+study+morphogenetic+biomechanics.">Two-photon cell and tissue level laser ablation methods to study morphogenetic biomechanics.</a> Methods Mol Biol. 2438, 217-230 (2022).</li><li>Angelo, J. R., Tremblay, 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=Laser-mediated+cell+ablation+during+post-implantation+mouse+development.">Laser-mediated cell ablation during post-implantation mouse development.</a> Dev Dyn. 242 (10), 1202-1209 (2013).</li><li>Lee, J., Lee, S., Truong, V. 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+ablation+of+pancreatic+cancer+using+a+cylindrical+light+diffuser.">Laser ablation of pancreatic cancer using a cylindrical light diffuser.</a> Lasers Med Sci. 37 (6), 2615-2621 (2022).</li><li>Luther, E., Mansour, S., Echeverry, N., 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+ablation+for+cerebral+metastases.">Laser ablation for cerebral metastases.</a> Neurosurg Clin N Am. 31 (4), 537-547 (2020).</li><li>Smutny, M., Behrndt, M., Campinho, P., Ruprecht, V., Heisenberg, C. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV+laser+ablation+to+measure+cell+and+tissue-generated+forces+in+the+zebrafish+embryo+in+vivo+and+ex+vivo.">UV laser ablation to measure cell and tissue-generated forces in the zebrafish embryo in vivo and ex vivo.</a> Methods Mol Biol. 1189, 219-235 (2015).</li><li>Vogel, A., Venugopalan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+pulsed+laser+ablation+of+biological+tissues.">Mechanisms of pulsed laser ablation of biological tissues.</a> Chem Rev. 103 (2), 577-644 (2003).</li><li>Wu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-based+tumor+ablation:+Role+of+the+immune+response.">Heat-based tumor ablation: Role of the immune response.</a> Adv Exp Med Biol. 880, 131-153 (2016).</li><li>Renvoiz&#233;, C., Biola, A., Pallardy, M., Br&#233;ard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis:+Identification+of+dying+cells.">Apoptosis: Identification of dying cells.</a> Cell Biol Toxicol. 14 (2), 111-120 (1998).</li><li>Julier, Z., Park, A. J., Briquez, P. S., Martino, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoting+tissue+regeneration+by+modulating+the+immune+system.">Promoting tissue regeneration by modulating the immune system.</a> Acta Biomater. 53, 13-28 (2017).</li><li>Petrie, T. A., Strand, N. S., Tsung-Yang, C., Rabinowitz, J. S., Moon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+modulate+adult+zebrafish+tail+fin+regeneration.">Macrophages modulate adult zebrafish tail fin regeneration.</a> Dev. 141 (13), 2581-2591 (2014).</li><li>Geurtzen, K., Vernet, A., Freidin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+suppressive+and+bone+inhibitory+effects+of+prednisolone+in+growing+and+regenerating+zebrafish+tissues.">Immune suppressive and bone inhibitory effects of prednisolone in growing and regenerating zebrafish tissues.</a> J Bone Miner Res. 32 (12), 2476-2488 (2017).</li><li>Fleischhauer, L., L&#243;pez-Delgado, A. C., Geurtzen, K., Knopf, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocorticoid+effects+in+the+regenerating+fin+reflect+tissue+homeostasis+disturbances+in+zebrafish+by+affecting+Wnt+signaling.">Glucocorticoid effects in the regenerating fin reflect tissue homeostasis disturbances in zebrafish by affecting Wnt signaling.</a> Front Endocrinol. (Lausanne). 14, 1122351 (2023).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book: A Guide for the laboratory use of zebrafish (Danio rerio). , (2002).</li><li>Brand, M., Granato, M., N&#252;sslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+and+raising+zebrafish.">Keeping and raising zebrafish.</a> Zebrafish: A Practical Approach. , (2002).</li><li>Spoorendonk, K. M., Peterson-Maduro, J., Renn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+and+Cyp26b1+are+critical+regulators+of+osteogenesis+in+the+axial+skeleton.">Retinoic acid and Cyp26b1 are critical regulators of osteogenesis in the axial skeleton.</a> Dev. 135 (22), 3765-3774 (2008).</li><li>Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A. <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>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+Breeding+and+Embryo+Handling.">Zebrafish Breeding and Embryo Handling.</a> Biology II: Mouse, Zebrafish, and Chick. , (2023).</li><li>Schindelin, J., Arganda-Carreras, I., Frise, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</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>Blanchard, G. B., Scarpa, E., Muresan, L., Sanson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stress+combines+with+planar+polarised+patterning+during+metaphase+to+orient+embryonic+epithelial+cell+divisions.">Mechanical stress combines with planar polarised patterning during metaphase to orient embryonic epithelial cell divisions.</a> Dev. 151 (10), dev202862 (2024).</li><li>Park, W., Wei, S., Kim, B. -. 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=Diversity+and+complexity+of+cell+death:+A+historical+review.">Diversity and complexity of cell death: A historical review.</a> Exp Mol Med. 55 (8), 1573-1594 (2023).</li><li>O'Connor, J., Akbar, F. B., Hutson, M. S., Page-McCaw, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zones+of+cellular+damage+around+pulsed-laser+wounds.">Zones of cellular damage around pulsed-laser wounds.</a> PLoS One. 16 (9), e0253032 (2021).</li><li>Griebel, M., Vasan, A., Chen, C., Eyckmans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+clearance+of+damaged+tissue+following+laser+ablation+in+engineered+microtissues.">Fibroblast clearance of damaged tissue following laser ablation in engineered microtissues.</a> APL Bioeng. 7 (1), 16112 (2023).</li><li>Matrone, G., Taylor, J. M., Wilson, K. 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-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></ol>

<table><tbody><tr><td>Calcium chloride dihydrate, CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>5239.1</td><td></td></tr><tr><td>Cell Culture Dish, PS, 100/20 mm</td><td>Greiner Bio-one</td><td>664160</td><td></td></tr><tr><td>Dumont #55 Foceps</td><td>FST</td><td>11295-51</td><td>Tip shape straight, 11 cm, 0.05 x 0.02 mm</td></tr><tr><td>Falcon 6-well plate</td><td>Corning</td><td>353502</td><td></td></tr><tr><td>Glass-bottom microwell dish</td><td>MatTek</td><td>P35G-1.5-14-C</td><td>35 mm Dish, No. 1.5 Coverslip, 14 mm Glass Diameter, Uncoated</td></tr><tr><td>Insight X3 multiphoton laser</td><td>Spectra-Physics</td><td></td><td></td></tr><tr><td>Leica Application Suite</td><td>LAS X, Leica Microsystems</td><td></td><td></td></tr><tr><td>Low melting agarose&amp;nbsp;</td><td>Biozyme</td><td>840101</td><td>Biozym Plaque Agarose</td></tr><tr><td>Magnesium sulfate heptahydrate, MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M5921</td><td></td></tr><tr><td>Mating cages</td><td></td><td></td><td>many varieties, e.g. Tecniplast</td></tr><tr><td>Methyleneblue</td><td>Carl Roth</td><td>A514.1</td><td></td></tr><tr><td>MS-222</td><td>SIGMA Aldrich</td><td>A5040</td><td></td></tr><tr><td>Potassium Chloride, KCl</td><td>PanReac AppliChem</td><td>131494</td><td></td></tr><tr><td>Sodium chloride, NaCl</td><td>Carl Roth</td><td>3957.1</td><td></td></tr><tr><td>SP8 FALCON&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td>Equipped with a Insight X3 multiphoton laser and Leica Application Suite software</td></tr><tr><td>Stainless Steel Dissect Needle</td><td>Bochem</td><td>12010</td><td>140 mm</td></tr><tr><td>Stereo Microscope System SZX16</td><td>Olympus</td><td></td><td>Equipped with a LED illumination base SZX2-ILLTQ</td></tr><tr><td>Thermostatic Cabinets TS - WTW</td><td>xylem</td><td>TS 608/2-i</td><td>For incubation (embryo)</td></tr><tr><td>Transfer pipette, 3.5 mL</td><td>SARSTEDT</td><td>861171</td><td>155 x 15 mm, LD-PE, transparent</td></tr><tr><td>Zeiss SteREO Discovery.V12 version 4.7.1.0.</td><td>Zeiss</td><td></td><td>Equipped with Axiocam MRm camera and AxioVision sofware</td></tr></tbody></table>

two-photon laser-mediated ablation of osteoblasts in developing zebrafish larvae

<meta charset="utf8"></head><body>作者聚焦:免疫介导的骨再生 — 巨噬细胞和受控免疫调节在修复过程中的关键作用

发育中的斑马鱼幼虫的双光子激光介导的成骨细胞消融

Zebrafish (Danio rerio) have an outstanding capacity to regenerate different organs and appendages. Bone regeneration in zebrafish has been studied using ...

two-photon-laser-mediated-ablation-osteoblasts-developing-zebrafish

Research

JoVE Journal

Developmental Biology

Two-Photon Laser-Mediated Ablation of Osteoblasts in Developing Zebrafish Larvae

322 次观看.  CRTD - Center for Regenerative Therapies TU Dresden. 我们的研究重点是骨再生。我们试图了解驱动骨骼再生的机制，特别是免疫系统如何参与骨骼的修复。免疫反应对于骨稳态和再生至关重要。受伤后炎症反应的缺失或过度会损害再生过程。因此，必须严格控制免疫调节才能成功再生。我们已经表明，巨噬细胞是先天免疫细胞，被募集到骨损伤中，免疫抑制会损害骨再生。

视频: 作者聚焦:免疫介导的骨再生 — 巨噬细胞和受控免疫调节在修复过程中的关键作用

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

科研

发育生物学

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

生物学

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

斑马鱼内胚细胞中基于共体显微镜的激光消融和再生测定

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula

Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly coordinated, and accumulating evidence suggests this is achieved, at least in part, through mechanical interactions. Testing this in the embryo requires direct physical perturbations. Laser ablations are an increasingly used option that allows relieving mechanical constraints or physically isolating two cell populations from each other. However, many ablations are performed with an ultraviolet (UV) laser, which offers limited axial resolution and tissue penetration. A method is described here to ablate deep, significant, and spatially well-defined volumes using a two-photon microscope. Ablations are demonstrated in a transgenic zebrafish line expressing the green fluorescent protein in the axial mesendoderm and used to sever the axial mesendoderm without affecting the overlying ectoderm or the underlying yolk cell. Cell behavior is monitored by live imaging before and after the ablation. The ablation protocol can be used at different developmental stages, on any cell type or tissue, at scales ranging from a few microns to more than a hundred microns.

Embryonic development requires large-scale coordination of cell motion. Two-photon excitation mediated laser ablation allows the spatially controlled 3-dimensional ablation of large groups of deep cells. In addition, this technique can probe the reaction of collectively migrating cells in vivo to perturbations in their mechanical environment.

在斑马鱼胃中使用双光子显微镜进行深度和空间控制的体积消融

Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

激光烧蚀斑马鱼Pronephros研究肾小管上皮再生

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

用双光子激光消融神经种群及其对斑马鱼幼虫钙成像和行为记录的评价

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

神经科学

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.

激光伤害的斑马鱼胚胎骨骼肌

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

Two-Photon Laser Axotomy: A Method to Injure Axons in Zebrafish Embryos and Observe Axonal Recovery

Source: O&#39;Brien G. S., et al. <a href="https://www.jove.com/v/1129/" target="_blank">Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos</a>. J. Vis. Exp. (2009).
This video describes the method to injure axons in zebrafish embryos using two photon laser axotomy and observing axonal recovery from injury.

双光子激光轴索切开术:一种损伤斑马鱼胚胎轴突并观察轴突恢复的方法

Source: O&#39;Brien G. S., et al. Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos. J. Vis. Exp. (2009).
This video describes ...

发育中的斑马鱼幼虫的双光子激光介导的成骨细胞消融

副本

摘要

探索更多视频

此视频中的章节

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

在现场的斑马鱼胚胎的基于双光子Photoactivation

斑马鱼内胚细胞中基于共体显微镜的激光消融和再生测定

在斑马鱼胃中使用双光子显微镜进行深度和空间控制的体积消融

激光烧蚀斑马鱼Pronephros研究肾小管上皮再生

用双光子激光消融神经种群及其对斑马鱼幼虫钙成像和行为记录的评价

激光伤害的斑马鱼胚胎骨骼肌

双光子激光轴索切开术:一种损伤斑马鱼胚胎轴突并观察轴突恢复的方法

发育中的斑马鱼幼虫的双光子激光介导的成骨细胞消融

副本

摘要

探索更多视频

此视频中的章节

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

斑马鱼内胚细胞中基于共体显微镜的激光消融和再生测定

在斑马鱼胃中使用双光子显微镜进行深度和空间控制的体积消融

激光烧蚀斑马鱼Pronephros研究肾小管上皮再生

用双光子激光消融神经种群及其对斑马鱼幼虫钙成像和行为记录的评价

激光伤害的斑马鱼胚胎骨骼肌

双光子激光轴索切开术:一种损伤斑马鱼胚胎轴突并观察轴突恢复的方法

在现场的斑马鱼胚胎的基于双光子Photoactivation