This work was supported by the German Research Foundation (DFG) Transregio 67 (project 387653785), the DFG SPP 2084 &#181;Bone (project KN 1102/2-1) to FK. This work was supported by the Light Microscopy Facility (DFG project 413875620), a Core Facility of the CMCB Technology Platform at TU Dresden. The work at the TU Dresden was co-financed with tax revenues based on the budget agreed by the Saxon Parliament (&#39;Landtag&#39;).

The study protocol received approval from the Landesdirektion Sachsen, permit numbers 25-5131/564/2, DD25-5131/450/4, 25-5131/496/56, DD25.1-5131/354/87. The zebrafish strains used were maintained according to national law and under standardized conditions as previously described27,28. The details of all the reagents and the equipment used in the study are listed in the Table of Materials.
1. Preparation of mater...

Laser Ablation Method to Study Bone Regeneration in Zebrafish Larvae

<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. 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(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. 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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. 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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. 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Laser ablation has been applied in various disciplines of biological research. In particular, it has been useful as a method to study tissue regeneration10,11,12. For example, the recruitment and phenotype changes of innate immune cells were recently analyzed over time using UV laser or two-photon laser ablation assays, along with the recovery of osteoblasts at the laser ablation site10. Live imaging of i...

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.

<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 (Danio rerio) have an outstanding capacity to regenerate different organs and appendages. Bone regeneration in zebrafish has been studied using different methods such as fin amputation, scale plucking, skull trepanation, and microscopic approaches. Using a confocal laser scanning setup equipped with a two-photon laser, a laser ablation method was developed as a lesion paradigm to ablate bone-forming cells (osteoblasts) in the developing opercle of zebrafish larvae. The method described here allows the ablation of cells in a precise manner, as the area, shape, and depth can be finely adjusted. In addition, this method allows imaging of the area before and just after the ablation, so that short-term effects of the injury can be analyzed. In this experimental setup, the immune response after ablation of osteoblasts in the injured area was studied. An increase in the recruitment of macrophages was observed after ablation, indicating the relevance of their presence during bone regeneration.

Author Spotlight: Immune-Mediated Bone Regeneration &#8212; The Critical Role of Macrophages and Controlled Immunomodulation in Restorative Processes

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

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.

Laser ablation was performed as indicated in the protocol above. The GFP signal of the osteoblasts in the ablated area disappeared instantaneously after ablation. To study the response of the osteoblast ablation in terms of the immune response, the presence of macrophages in 6 dpf larvae before and at 6 hpl was imaged. Before ablation, very few macrophages were observed in the opercle area10 (Figure 2). At 6 hpl, a strong accumulation of macrophages in the ablated ope...

This protocol describes a two-photon laser ablation approach carried out in zebrafish larvae, which serves as a model to study bone regeneration and the effects of the immune response to ablation.

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

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Research

JoVE Journal

Developmental Biology

Zebrafish Breeding, Embryo Collection and Larvae Embedding in Low-Melting Agarose for In Vivo Imaging

To begin, set up parental zebrafish with the desired single transgenes in standard mating cages the evening before mating. Using a divider in the mating cage, keep males and females separated until mating. Remove the divider in the morning of the next day to initiate mating.At noon, at the latest, remove parental zebrafish from the breeding tanks. Strain the water from the mating cage through a sieve to collect the embryos. Then, place the embryos in a 100-millimeter diameter dish, containing E3 media, and keep them in an incubator at 28-degrees Celsius.Afterward, remove unfertilized eggs and dead embryos from the dish using a plastic pipette and a stereo microscope. Using a three-milliliter plastic Pasteur pipette, transfer the four-day post-fertilization larvae with reporter transgene expression to a glass-bottom microwell dish. With the same pipette, remove the excess E3 media.Now, take the melted low-melting agarose from the water bath or thermo block. Add a cooling drop of low-melting agarose to the dish, covering the glass bottom where the larvae were positioned. Place the larvae into the desired lateral position before the agarose solidifies.Once the agarose solidifies, add E3 media containing a maximum of 0.02%MS-222 to the dish to avoid drying of the agarose.

Studying the Biology of Bone Regeneration Using Two-Photon Laser Ablation Approach in Zebrafish Larvae

To begin, embed the transgenic larva in low-melting agarose in a glass-bottom microwell dish. Place the dish onto the microscopic stage at the inverted microscope. Position and focus the desired region for ablation in the center of the field of view.Choose the Z-stack settings to image the entire breadth of the opercle. Image the opercle area of interest before ablation. Then, draw a circular region with a diameter of 25 micrometers in a selected 2D plane.Focus on the opercle using the region of interest, or ROI tool. To perform the ablation, apply ablation power measured without objective to 450 milliwatts for a duration of 10 seconds. Expose the selected area to the two-photon laser.After ablation, confirm the ablation structurally by imaging the same Z-stack with the same settings, as used prior to ablation. Using forceps or a dissection needle, remove the larvae carefully from the low-melting agarose. First, remove a layer of agarose covering the larvae.Second, remove the agarose in direct contact with the larvae. Place the larvae back in a dish with pure E3 media. At four or six hours post-lesion, re-image the opercle area using agarose-embedded larvae at the same settings on the same microscope.After processing the images in Fiji, quantify cells with a Nuclear label, or by measuring the area, in case cells are overlapping. To quantify the area, create a mask encompassing the macrophages. Use the Image, Adjust, Threshold Tool, Create a ROI, and analyze the particles.Then, measure the area. Generate graphs plotting the measurement results using suitable software. A significant accumulation of macrophages in the ablated opercle region was detected six hours post-ablation in six days'post-fertilization larvae.In larvae four days post-fertilization, the number of macrophages increased at four hours post-lesion. Macrophage number and area increased at four hours post-lesion compared to the un-ablated state.

137 Views.  CRTD - Center for Regenerative Therapies TU Dresden.  This protocol describes a two-photon laser ablation approach carried out in zebrafish larvae, which serves as a model to study bone regeneration and the effects of the immune response to ablation.

Laser Ablation Method to Study Bone Regeneration in Zebrafish Larvae

Summary