<meta charset="utf8"></head><body>Método de ablação a laser para estudar a regeneração óssea em larvas de peixe-zebra

<ol>
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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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Calcium chloride dihydrate, CaCl2&#183;H2O</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&#160;</td>
			<td>Biozyme</td>
			<td>840101</td>
			<td>Biozym Plaque Agarose</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate, MgSO4&#183;7H2O</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&#160;</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

Zebrafish regenerate diverse organs such as the retina, brain, heart, and pancreas1. In addition, zebrafish regenerate skeletal elements, which is why they have been used to study the regeneration of fins, scales, and calvariae (skull caps)2. Different experimental paradigms have been used to study tissue and bone regeneration, such as fin resection (amputation), fin fracture, skull trepanation3,4, cryoinjury5,6, or genetic ablation7,8,9. Recently, laser ablation approaches have been widely used in zebrafish to study the healing and regeneration response after injury10,11,12,13,14.
Laser-mediated ablation has been used in different areas of biological research, such as mechanobiology, developmental biology, regeneration research, and tumor surgery10,11,12,15,16,17,18,19. There are various methodologies of laser ablation, such as using YAG (yttrium aluminum garnet), UV (ultraviolet), or 2p (two-photon) lasers20. Laser ablation allows the removal of single cells or larger portions of tissues in a very accurate manner, enabling the study of different processes, such as the response of the immune system to tumor ablation21 or during zebrafish opercle regeneration. In the latter example, ablation was confirmed by the disappearance of the nuclear and cytoplasmic fluorophore signal and necrosis staining10,22.
It is well known that the innate immune response and its regulated kinetics are essential for appropriate tissue regeneration. Neutrophils and macrophages are the first cells to be recruited to the injury site, where they perform different roles, such as cytokine and growth factor release, cellular debris removal, and extracellular matrix remodeling23. This recruitment and subsequent macrophage functionalization have also been observed in amputated zebrafish fins24 and the developing opercle, which was subjected to laser &#39;nanodissection&#39; leading to osteoblast ablation10. In the latter experiment, recovery of the osteoblast number, normal opercle development, and recruitment of innate immune cells (neutrophils, macrophages, and osteoclast-like cells) to the injured area was observed after UV laser and two-photon laser-mediated ablation10. Experiments in zebrafish in which the immune system was pharmacologically suppressed by using glucocorticoids showed an impairment of regeneration upon misregulated immunity, supporting the functional role of the immune system in tissue repair3,10,25,26.
Here, a two-photon laser-mediated ablation methodology to study the biology of bone regeneration in developing zebrafish bones is described. In particular, the effect of the osteoblast ablation approach in the opercle is shown in terms of the immune response, which is investigated by monitoring the recruitment of macrophages to the ablation site.

<meta charset="utf8"></head><body>Autor em destaque: Regeneração óssea imunomediada - o papel crítico dos macrófagos e imunomodulação controlada em processos restauradores

Ablação mediada por laser de dois fótons de osteoblastos em larvas de peixe-zebra em desenvolvimento

Our research focuses on bone regeneration. We try to understand the mechanisms that drive bone regeneration, in particular, how the immune system is involved in the restoration of bone. The immune response is essential for bone homeostasis and regeneration.The absence or excess of an inflammatory response after injury impairs the regeneration process. For that reason, immunomodulation must be tightly controlled to allow for successful regeneration. We have shown that macrophages, which are innate immune cells, are recruited to bone injuries, and that immunosuppression impair bone regeneration.

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

176 visualizações.  CRTD - Center for Regenerative Therapies TU Dresden. Nossa pesquisa se concentra na regeneração óssea. Tentamos entender os mecanismos que impulsionam a regeneração óssea, em particular, como o sistema imunológico está envolvido na restauração do osso. A resposta imune é essencial para a homeostase e regeneração óssea.A ausência ou excesso de resposta inflamatória após a lesão prejudica o processo de regeneração. Por esse motivo, a imunomodulação deve ser rigidamente controlada para...