This study was supported by a grant of the Center of Regenerative Therapies Dresden (&#34;Zebrafish as a model to unravel the mechanisms of glucocorticoid-induced bone loss&#34;) and additionally by a grant of the Deutsche Forschungsgemeinschaft (Transregio 67, project 387653785) to FK. We are very grateful to Jan Kaslin and Avinash Chekuru for their guidance and assistance on performing trepanation of the calvariae and fractures in bony fin rays. Experiments were designed, performed and analyzed by KG and FK. FK wrote the manuscript. We would also like to thank Katrin Lambert,&#160;Nicole Cudak, and other members of the Knopf and Brand labs for technical assistance and discussion. Our thanks also goes to Marika Fischer and Jitka Michling for excellent fish care and to Henriette Knopf and Josh Currie for proofreading the manuscript.

All methods described here were approved by the Landesdirektion Dresden (Permit numbers: AZ 24D-9168.11-1/2008-1, AZ 24-9168.11-1/2011-52, AZ 24-9168.11-1/2013-5, AZ 24-9168.11-1/2013-14, AZ DD24.1- 5131/354/87).
1. Preparation of Materials and Solutions
NOTE: Prednisolone, like other glucocorticoids, leads to immunosuppression. Thus, precaution must be taken to prevent infection in treated animals during the experiment. To this end, autoclave glass ware and &#39;fish water&#39; (i.e., the water that is used to rear adult zebrafish) before starting the experiment.
<ol>
	<li>Calculate the number of zebrafish to be used in the experiment. Be sure to include the individuals that serve as controls.</li>
	<li>Autoclave 600 mL glass beakers that are covered with aluminum foil with autoclave tape. Zebrafish will be treated individually, thus 1 glass beaker is required per zebrafish.</li>
	<li>Calculate the amount of fish water needed for the experiment. 300 mL are required per individual and day. Take into consideration the length of the experiment (e.g., 1 week/7 days).</li>
	<li>Fill glass bottles with fish water, close them and autoclave the bottles with autoclave tape. Ideally, use 2 L bottles, but other bottles like 1 L or 5 L can be used as well. 
	NOTE: If experiments are long (e.g., 4 weeks/28 days) and the amount of glass bottles is limited, make sure to prepare bottles for a minimum of several days at a time and repeat the process (1.3 to 1.4) throughout the experiment.</li>
	<li>Prepare a prednisolone stock solution of sterile-filtered 100 mM prednisolone in dimethylsulfoxide (DMSO). Freeze the stock in 1 mL or 2 mL microcentrifuge tube aliquots. 
	CAUTION: Always wear protective clothing when working with prednisolone (protective gloves, safety goggles, lab coat).</li>
	<li>On the first day of the experiment, prepare incubation solutions with prednisolone and DMSO, respectively. To this end, thaw the required amount of 100 mM prednisolone stock solution and add prednisolone to autoclaved fish water at a final concentration of 50 &#181;M.
	<ol>
		<li>For the control treatments, add the corresponding volume of DMSO to autoclaved fish water. As an example, to produce 2 L prednisolone containing fish water, add 1 mL of 100 mM prednisolone stock to 2 L fish water. In another 2 L bottle of fish water, add 1 mL DMSO, correspondingly.</li>
		<li>Work in an appropriate place that is designated to perform drug treatments, ideally in a room at 27 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Generation of Injuries in Zebrafish Fins
NOTE: To injure bone in zebrafish fins, perform resection of the fin (amputation, usually in the caudal fin) or fracture individual bony fin rays (fracture model). To this end anaesthetize adult zebrafish first.
<ol>
	<li>Amputation
	<ol>
		<li>To anaesthetize adult zebrafish, prepare a 100 mm diameter Petri dish containing 0.02% MS-222 (Ethyl-3-aminobenzoate methanesulfonate) in fish water (this water does not need to be autoclaved).
		<ol>
			<li>Transfer one zebrafish at a time to the MS-222 containing Petri dish by using a fish net and incubate it until it lies on its side without movement and does not respond to touch. Test the latter by touching the anal fin with blunt forceps.</li>
			<li>Wait until level 4 of anesthesia is reached which is the appropriate level for fin resection12. At this stage, the rate of opercular movement is decreased, muscle tone is lost and the fish does not move upon touch12.</li>
		</ol>
		</li>
		<li>Take blunt forceps and transfer the anaesthetized zebrafish carefully to the inversed lid of the 100 mm Petri dish. Place the zebrafish on its lateral side. Make sure the zebrafish always lie in the same orientation, for example on their right body side. With a scalpel or a razor blade, resect 50% of the fin (Figure 1A, 2A). 
		NOTE: The speed of zebrafish fin regeneration depends on the amount of resected fin tissue, i.e., the more fin tissue is resected, the faster the fin regenerates13. Thus, to minimize variations in fin regeneration make sure to always resect the fin at the same level in all zebrafish.</li>
		<li>Transfer the injured zebrafish to an autoclaved glass beaker containing 300 mL of prednisolone or DMSO containing autoclaved fish water. Cover the glass beaker with aluminum foil to avoid escape of the zebrafish.</li>
		<li>Repeat steps 2.1.2 to 2.1.3 until the required number of zebrafish for the experiment is reached.</li>
	</ol>
	</li>
	<li>Fin fracture
	<ol>
		<li>Prepare an agarose-coated 100 mm Petri dish by heating a 2% agarose solution in fish water or E3 (embryo media 3, 50 mM sodium chloride, 0.17 mM potassium chloride, 0.33 mM calcium chloride, 0.33 mM magnesium sulfate). Carefully pour roughly 10&#8211;15 mL of the solution into the Petri dish. The bottom of the dish should be completely covered. Let the agarose solidify.</li>
		<li>Anaesthetize zebrafish as described in section 2.1.</li>
		<li>Take blunt forceps and transfer the anaesthetized zebrafish carefully to the agarose-coated 100 mm Petri dish. Place the zebrafish on its lateral side. Under the dissection microscope, spread the caudal fin completely to be able to identify distinct bony fin rays and fin ray segments. Make sure the zebrafish always lie in the same orientation, for example on their right body side.</li>
		<li>With an injection needle (0.3 mm x 13 mm) introduce a fracture in the center of a bony fin ray segment (Figure 1B). To do this, the needle is pushed slightly onto the segment until a crack appears (arrow in Figure 2B). While gentle pressure leads to fracture of only one of the two opposing hemirays stronger pressure will lead to fracture of both hemiray segments.
		<ol>
			<li>Do not introduce too many fractures in fins, since this will greatly impair stability of the fin. As a suggestion, apply fractures only in 3 to 5 fin rays.</li>
			<li>Always produce fractures in corresponding segments across different fin rays and zebrafish, for example in dorsal fin rays 3, 7 (or 8) and ventral fin ray 3. The proximal-distal level of fracture is ideal at 3 segments proximal to the fin rays&#39; bifurcation point 11.</li>
		</ol>
		</li>
		<li>Transfer the injured zebrafish to an autoclaved glass beaker containing 300 mL of prednisolone or DMSO containing autoclaved fish water. Cover the glass beaker with aluminum foil to avoid escape of the zebrafish.</li>
		<li>Repeat steps 2.2.2 to 2.2.5 until the required number of zebrafish for the experiment is reached.</li>
	</ol>
	</li>
</ol>
3. Generation of Calvarial Skull Injuries (Trepanation)
NOTE: The calvariae in zebrafish are homologous to calvarial bones in mammals. Thus, these exoskeletal bones14 represent a tissue of special interest when studying the pathogenesis of GIO. To injure the skull, trepanation is performed by drilling a hole in the Os frontale and/or Os parietale (Figure 1C, 2C) with the help of a microdrill11.
<ol>
	<li>Anaesthetize the zebrafish according to section 2.1.</li>
	<li>Hold the anesthetized zebrafish upright with forceps, so that the calvarial bones are well visible under a dissection microscope from above (Figure 1C).</li>
	<li>Locate the rotating microdrill on the center of the frontal bone (Os frontale) and touch the bone. The produced hole is of the size of the microdrill burr (500 &#181;m, Figure 2C, Figure 3B). 
	NOTE: Be sure to not move the microdrill laterally while producing the injury. Also stop immediately when the resistance of the bone suddenly drops. Do not drill any further, otherwise the brain will be damaged 15.</li>
	<li>Transfer the injured zebrafish to a cage filled with fish water until it fully recovers from anesthesia. Watch it carefully.</li>
	<li>Transfer the injured zebrafish to a fish water plus prednisolone or DMSO containing glass beaker.</li>
</ol>
4. Treatment of Zebrafish During Incubation
NOTE: During application of prednisolone/DMSO, the drug containing fish water needs to be changed daily, and zebrafish need to be fed regularly.
<ol>
	<li>Water changes
	<ol>
		<li>To exchange the fish water, thaw 100 mM prednisolone stock and prepare the required amount of 50 &#181;M prednisolone in autoclaved fish water fresh every day of the treatment period. Prepare DMSO containing fish water for the control zebrafish accordingly.</li>
		<li>Take a glass beaker containing a single housed zebrafish in its incubation solution and transfer the solution including the zebrafish into an appropriate container, for example a temporal housing cage. 
		NOTE: Always use separate interim containers for prednisolone and DMSO treated zebrafish, respectively, to make sure DMSO control zebrafish do not get exposed to prednisolone and vice versa.</li>
		<li>Refill the glass beaker with fresh drug containing fish water (300 mL).</li>
		<li>Transfer the zebrafish from the interim container to the glass beaker containing fresh incubation solution using a fish net. Put back the foil to avoid escape of the zebrafish.</li>
		<li>If the time of the experiment exceeds 2 weeks, exchange the glass beakers.</li>
	</ol>
	</li>
	<li>Feeding
	<ol>
		<li>During short treatments (for up to 2 days) do not feed zebrafish. During longer experiments, feed zebrafish. Take special care to avoid pollution of the incubation solution. Thus, feed with Artemia ssp. rather than with flake food, and only on every second day. 
		NOTE: Artemia are regularly used to feed young zebrafish, and should be available in the zebrafish haltering unit.
		<ol>
			<li>Roughly 2 h before the fish water is exchanged, feed zebrafish with Artemia ssp in their glass beakers. To do so, use a plastic pipette and feed 0.5 to 1 mL of hatched Artemia solution per glass beaker.</li>
			<li>Wait for 2 h and exchange the incubation solution with fresh drug containing fish water according to section 4.1.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Analyses of Samples
NOTE: After incubation of injured zebrafish in prednisolone and DMSO containing fish water, respectively, either perform bone mineralization/calcification analyses (5.1 to 5.3) or carry out live imaging of zebrafish under the dissection microscope (5.4)10,11,16. Use live imaging to determine fin regenerate length and to detect differences in reporter gene expression in transgenic zebrafish.
<ol>
	<li>Fixation of tissues of interest
	<ol>
		<li>For fixation of fins and/or skulls prepare 4% paraformaldehyde (PFA) in PBS and freeze at -20 &#176;C for storage. Use freshly prepared 4% PFA in PBS or thaw the required amount before use. Alternatively, 2% PFA in PBS can be used. 
		CAUTION: PFA is toxic and should be handled carefully under a fume hood. Wear protective clothing.</li>
		<li>For fixation of fins prepare a small Petri dish (e.g., 30 mm) with cold 4% PFA in PBS and make sure that the bottom of the dish is fully covered. For fixation of skulls, prepare a little glass jar with lid or a tube with cold 4% PFA in PBS. 
		CAUTION: Handle PFA containing solutions only under the fume hood as it is toxic. Wear protective clothing.</li>
		<li>Harvest the tissue of interest.
		<ol>
			<li>To harvest regenerating fin tissue or fins with fractures anaesthetize the zebrafish according to section 2.1 or euthanize the zebrafish according to section 5.1.5.</li>
			<li>Transfer the anaesthetized zebrafish to the lid of a Petri dish. By using a scalpel or razor blade, harvest the fin regenerate. Make sure to include stump tissue in regenerating fins to facilitate handling of the harvested tissue.</li>
		</ol>
		</li>
		<li>Fix fins flat in the PFA containing Petri dish at 4 &#176;C overnight. Flat fixation avoids curling up of the fin tissue during fixation.
		<ol>
			<li>To fix flat, grab the fin tissue with one fine forceps at the dorsal or ventral edge of the stump and immerse it in the fixation solution. Grab the opposite edge of the stump with a second fine forceps and slightly depress the tissue onto the bottom of the dish.</li>
			<li>Hold for 10 to 20 s before releasing. The fin tissue should not curl up anymore. If it does, repeat.</li>
		</ol>
		</li>
		<li>Euthanize zebrafish in order to harvest injured skull tissue. Prepare a 100 mm diameter Petri dish containing 0.1% MS-222 in fish water. Transfer one zebrafish at a time to the MS-222 containing Petri dish by using a fishing net. When level 6 of anesthesia (medullary collapse) is reached wait at least 10 min to assure death of the specimen12.</li>
		<li>Using a scalpel cut off the head plus a little trunk tissue from the rest of the body. Immerse the harvested tissue in the prepared 4% PFA solution in the glass jar or plastic tube. Fix for 24 h at 4 &#176;C with gentle rocking.</li>
		<li>To store samples long-term after fixation, wash them with PBS (3 x 20 min at RT) and methanol-treat in an increasing methanol series (1x 30%, 1x 50%, 1x 70%, 2x 100% methanol in PBS for 20 min each).
		<ol>
			<li>Store them in 100% methanol at -20 &#176;C. Otherwise, directly proceed with bone staining or other analyses after washing with PBS.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Bone staining techniques I (alizarin red staining) 
	NOTE: This staining is performed according to van Eeden et al. 199617 and Walker &#38; Kimmel 200718 with modifications.
	<ol>
		<li>To perform alizarin red staining on adult fins or skulls, harvest and fix tissue as described in sections 5.1.</li>
		<li>If samples were stored in methanol at -20 &#176;C, rehydrate samples by doing an inverse methanol series (70%, 50% and 30% in PBS, each 1x20 min) and wash twice 20 min with PBS. Wash samples for 5 min minimum with deionized water.</li>
		<li>To do alizarin red staining in skulls, treat samples with 50 mg/mL trypsin in 30% saturated Na2B4O7 at room temperature overnight. Rock samples during this time. Rinse samples in 30% saturated Na2B4O7 solution. Wash samples with deionized water 2 times for 5 min each. This step is not performed for fin tissue.</li>
		<li>Add alizarin red solution (Part B: 0.5% alizarin red S powder, 10% glycerol in deionized water) and rock the samples during incubation at RT. Check for staining after 1, 2, 4 h or overnight.</li>
		<li>To clear samples treat them with a decreasing 1% potassium hydroxide:glycerol series (3:1 overnight, 1:1 overnight, 1:3 overnight).</li>
		<li>Store the samples in a 1:1 solution of 0.1% potassium hydroxide: 80% glycerol and mount them with 80% glycerol for imaging. 
		NOTE: For combined Alcian blue/alizarin red staining treat samples with Alcian blue staining solution (Part A: final 0.02% Alcian blue, 50 mM MgCl2, 70% ethanol) after rehydration (section 5.2.2) for 2 h at RT. Treat fins with a decreasing ethanol series in 50 mM MgCl2 (70%, 50%, 30%, each 1x 15 min) and wash them for a minimum of 1h (3x 20 min) in deionized water. Proceed with alizarin red staining (section 5.2).</li>
	</ol>
	</li>
	<li>Bone staining techniques II (calcein staining) 
	NOTE: To detect calcium deposition in fin fractures and skull injuries, live fish are incubated in calcein containing solution.
	<ol>
		<li>Prepare the required amount of 0.2% calcein in deionized water (pH 7.5). Make sure the pH is neutral. Use 1 M NaOH to adjust the pH.</li>
		<li>Incubate up to 3 zebrafish simultaneously in 100 mL of calcein solution for 20 min in a glass beaker covered with aluminum foil.</li>
		<li>Transfer the zebrafish to a beaker with fish water. Let them swim briefly before transferring them to a new beaker with fish water. Let them swim in this new beaker for 20 min to wash. Take pictures (see section&#160;5.4).</li>
	</ol>
	</li>
	<li>Live imaging of zebrafish
	<ol>
		<li>Use this approach to acquire images of regenerating bone tissue such as in the fin or skull throughout the experiment without sacrificing the zebrafish. Anaesthetize the zebrafish according to section 2.1.</li>
		<li>To acquire images of fin regenerates, transfer the anaesthetized zebrafish onto an agarose-coated Petri dish (see section 2.2.1 and Figure 1B).</li>
		<li>To acquire images of regenerating skull bones, place the zebrafish upright in a sponge (Figure 1D) or place it in an agarose-coated dish that has been prepared to hold the trunk of the zebrafish.</li>
		<li>Acquire images at the desired magnification and resolution using a stereomicroscopic setup.</li>
		<li>Return the zebrafish to a container with fresh or drug containing fish water.</li>
	</ol>
	</li>
</ol>

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M., 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=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> Development. 135 (22), 3765-3774 (2008).</li><li>Gioia, 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=The+chaperone+activity+of+4PBA+ameliorates+the+skeletal+phenotype+of+Chihuahua,+a+zebrafish+model+for+dominant+osteogenesis+imperfecta.">The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta.</a> Human Molecular Genetics. 26 (15), 2897-2911 (2017).</li><li>Fiedler, I. A. K., 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=Severely+impaired+bone+material+quality+in+Chihuahua+zebrafish+resembles+classical+dominant+human+osteogenesis+imperfecta.">Severely impaired bone material quality in Chihuahua zebrafish resembles classical dominant human osteogenesis imperfecta.</a> Journal of Bone and Mineral Research. , (2018).</li><li>Fisher, S., Jagadeeswaran, P., Halpern, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiographic+analysis+of+zebrafish+skeletal+defects.">Radiographic analysis of zebrafish skeletal defects.</a> Developmental Biology. 264 (1), 64-76 (2003).</li><li>Hayes, A. J., 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=Spinal+deformity+in+aged+zebrafish+is+accompanied+by+degenerative+changes+to+their+vertebrae+that+resemble+osteoarthritis.">Spinal deformity in aged zebrafish is accompanied by degenerative changes to their vertebrae that resemble osteoarthritis.</a> PLoS One. 8 (9), e75787 (2013).</li><li>Mitchell, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tools+for+studying+osteoarthritis+genetics+in+zebrafish.">New tools for studying osteoarthritis genetics in zebrafish.</a> Osteoarthritis Cartilage. 21 (2), 269-278 (2013).</li><li>Fleming, A., Sato, M., Goldsmith, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+in+vivo+screening+for+bone+anabolic+compounds+with+zebrafish.">High-throughput in vivo screening for bone anabolic compounds with zebrafish.</a> Journal of Biomolecular Screening. 10 (8), 823-831 (2005).</li><li>de Vrieze, E., Zethof, J., Schulte-Merker, S., Flik, G., Metz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+osteogenic+compounds+by+an+ex+vivo+sp7:luciferase+zebrafish+scale+assay.">Identification of novel osteogenic compounds by an ex vivo sp7:luciferase zebrafish scale assay.</a> Bone. 74, 106-113 (2015).</li></ol>

The authors have nothing to disclose.

Zebrafish have proven useful in skeletal research in many regards. Selected mutants mimic aspects of human disease such as osteogenesis imperfecta or osteoarthritis23,24,25,26,27, and larvae as well as scales are being used to identify bone anabolic compounds in small molecule screens7,28,<s...

Zebrafish represent a powerful animal model to study vertebrate development and disease. This is due to the fact that they are small animals that breed extremely well and that their genome is fully sequenced and amenable to manipulation1. Other advantages include the option to perform continued live imaging at different stages, including in vivo imaging of adult zebrafish2, and the ability to perform high throughput drug screens in zebrafish larvae3. Additionally, zebrafish possess a high regenerative capacity in a variety of organs and tissues including bone, and thus serve as a useful system to study skeletal disease and repair4,5.
Glucocorticoid-induced osteoporosis (GIO) is a disease that results from long term treatment with glucocorticoids, for example in the course of autoimmune disease treatment such as of asthma or rheumatoid arthritis. GIO develops in about 30% of glucocorticoid-treated patients and represents a major health issue6; therefore, it is important to investigate the impact of immunosuppression on bone tissue in great detail. In recent years a variety of zebrafish models dealing with the pathogenesis of GIO have been developed. Glucocorticoid-mediated bone loss has been induced in zebrafish larvae, for example, which led to the identification of counteractive compounds increasing bone mass in a drug screen7. Furthermore, glucocorticoid-induced bone inhibitory effects have been mimicked in zebrafish scales both in vitro and in vivo8,9. These assays are very convincing approaches, especially when it comes to the identification of novel immunosuppressive and bone anabolic drugs. However, they only partly take into account the endoskeleton and have not been performed in a regenerative context. Thus, they do not allow the investigation of glucocorticoid-mediated effects during rapid modes of adult, regenerative bone formation.
Here, we present a protocol enabling researchers to study glucocorticoid-mediated effects on adult zebrafish bones undergoing regeneration. Injury models include partial amputation of the zebrafish caudal fin, trepanation of the skull, as well as the creation of fin ray fractures (Figure 1A-1C), and are combined with glucocorticoid exposure via incubation (Figure 1E). We have recently used a portion of this protocol to describe the consequences of exposure to prednisolone, one of the commonly prescribed corticosteroid drugs, on adult zebrafish regenerating fin and skull bone10. In zebrafish, prednisolone administration leads to decreased osteoblast proliferation, incomplete osteoblast differentiation and rapid induction of apoptosis in the monocyte/macrophage lineage10. In this protocol, we also describe how fractures can be introduced into single bony fin ray segments11, as this approach may be useful when studying glucocorticoid-mediated effects on bone occuring during fracture repair. The methods presented here will help to further address underlying mechanisms of glucocorticoid action in rapidly regenerating bone and may also be employed in other settings of systemic drug administration in the context of zebrafish tissue regeneration.

<table><tbody><tr><td>Prednisolone</td><td>Sigma-Aldrich</td><td>P6004</td><td></td></tr><tr><td>Dimethylsulfoxid (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Ethyl-3-aminobenzoate methanesulfonate (MS-222)</td><td>Sigma-Aldrich</td><td>A5040</td><td></td></tr><tr><td>Blunt forceps</td><td>Aesculap</td><td>BD027R</td><td></td></tr><tr><td>Fine forceps</td><td>Dumont</td><td>91150-20</td><td></td></tr><tr><td>Scalpel</td><td>Braun</td><td>5518059</td><td></td></tr><tr><td>Agarose</td><td>Biozym</td><td>840004</td><td></td></tr><tr><td>Injection needle (0.3 mm x 13 mm)</td><td>BD Beckton Dickinson</td><td>30400</td><td></td></tr><tr><td>Micro drill</td><td>Cell Point Scientific</td><td>67-1000</td><td>distributed &lt;em&gt;e.g.&lt;/em&gt; by Harvard Apparatus</td></tr><tr><td>Steel burrs (0.5 &amp;micro;m diameter)</td><td>Fine Science tools</td><td>19007-05</td><td></td></tr><tr><td>&lt;em&gt;Artemia ssp.&lt;/em&gt;</td><td>Sanders</td><td>425GR</td><td></td></tr><tr><td>Pasteur pipette (plastic, Pastette)</td><td>Alpha Labs</td><td>LW4111</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Alizarin red S powder</td><td>Sigma-Aldrich</td><td>A5533</td><td></td></tr><tr><td>Alcian blue 8 GX</td><td>Sigma-Aldrich</td><td>A5268</td><td></td></tr><tr><td>Calcein</td><td>Sigma-Aldrich</td><td>C0875</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T7409</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>MZ16 FA</td><td>with QIMAGING RETIGA-SRV camera</td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>MVX10</td><td>with Olympus DP71 or DP80 camera</td></tr></tbody></table>

adult zebrafish injury models to study the effects of prednisolone in regenerating bone tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

The protocol presented here has been used repeatedly to induce rapid bone formation in the course of regeneration of the zebrafish fin and skull10,11,16. In combination with the presented method of prednisolone administration, studies on prednisolone&#39;s effects during bone regeneration can be pursued. For example, studies on bone formation and mineralization in the regenerate can be performed....

Watch this Scientific Journal Video about Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue at JoVE.com

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

adult-zebrafish-injury-models-to-study-effects-prednisolone

Research

JoVE Journal

Developmental Biology

8.8K Views.  TU Dresden. Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Video: Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from &#945;-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish.

Here, we present a protocol for cell transplantation of zebrafish skeletal muscle and embryonal rhabdomyosarcoma (ERMS) into adult immune compromised rag2E450fs homozygous mutant zebrafish. This protocol allows for the efficient analysis of regeneration and malignant transformation of muscle cells.

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have ...

Immunology and Infection

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.

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

Neuroscience

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae

Chemically induced bone loss due to lead (Pb) exposure could trigger an array of adverse impacts on both human and animal skeletal systems. However, the specific effects and mechanisms in zebrafish remain unclear. Alizarin red has a high affinity for calcium ions and can help visualize the bone and illustrate skeletal mineral mass. In this study, we aimed to detect lead acetate (PbAc)-induced bone loss in zebrafish larvae by using alizarin red staining. Zebrafish embryos were treated with a series of PbAc concentrations (0, 5, 10, 20 mg/L) between 2 and 120 h post fertilization. Whole-mount skeletal staining was conducted on larvae at 9 days post fertilization, and the total stained area was quantified using ImageJ software. The results indicated that the mineralized tissues were stained in red, and the stained area decreased significantly in the PbAc-exposure group, with a dose-dependent change in bone mineralization. This paper presents a staining protocol for investigating skeletal changes in PbAc-induced bone defects. The method can also be used in zebrafish larvae for the detection of bone loss induced by other chemicals.

Here, we have used alizarin red staining to show that lead acetate exposure causes a bone mass change in zebrafish larvae. This staining method can be adapted to the investigation of bone loss in zebrafish larvae loss induced by other hazardous toxicants.

Chemically induced bone loss due to lead (Pb) exposure could trigger an array of adverse impacts on both human and animal skeletal systems. However, the ...

Laser Ablation Method to Study Bone Regeneration in Zebrafish Larvae

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

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 ...

Removing Zebrafish Scales for Culturing and Bone Biology Studies

In Vitro Culturing Technique for Studying Cellular Dynamics in Zebrafish Scales

Zebrafish scales offer a variety of advantages for use in standard laboratories for teaching and research purposes. Scales are easily collected without the need for euthanasia, regenerate within a couple of weeks, and are translucent and small, allowing them to be viewed using a standard microscope. Zebrafish scales are especially useful in educational environments, as they provide a unique opportunity for students to engage in hands-on learning experiences, particularly in understanding cellular dynamics and in vitro culturing methods. The main objective of this protocol is to describe a method for collecting and maintaining zebrafish scales in culture for use in a variety of biological studies using basic laboratory equipment. Additionally, the protocol details their use in understanding bone homeostasis by examining the activity of bone cells involved in bone resorption and deposition. It also includes additional protocols for general techniques, such as the visualization of nuclei and apoptotic cells. The in vitro culturing protocol produces reliable results with minimal reagents and equipment. This article discusses the benefits of using in vitro cultures of zebrafish scales to foster scientific inquiry and outlines the resources needed to support their integration into educational settings.

Author Spotlight: Advancing Bone Cell Activity Research with Zebrafish Scales 

This protocol offers a method to study cellular dynamics using a simple in vitro culture technique. It provides an opportunity for zebrafish researchers and educators to study cellular processes, such as those related to bone homeostasis and basic cell biology, by visualizing fluorescent nuclei and apoptotic cells within the scales.

Zebrafish scales offer a variety of advantages for use in standard laboratories for teaching and research purposes. Scales are easily collected without ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

Medicine

Caudal Peduncle Cryoinjury in Adult Zebrafish

A Cryoinjury Model for Studying Skeletal Muscle Regeneration of the Caudal Peduncle in Adult Zebrafish

Skeletal muscle undergoes renewal and restoration after minor injury through the activation of satellite-like stem cells. Severe injuries of the musculature often lead to fibrosis in humans. In comparison to mammals, zebrafish possess a higher innate capacity for organ regeneration, providing a powerful model for studying tissue restoration after extensive damage to the organ. Here, a cryoinjury model is described to induce profound damage to four myomeres of the caudal peduncle in adult zebrafish. A custom-made cryoprobe was designed to fit the body shape and reproducibly injure the lateral musculature from the skin to the midline. Importantly, the body integrity remained intact, and the fish continued their swimming activity. Changes to the skeletal muscle were assessed by histological staining and fluorescence staining of sarcomeric proteins on tissue sections. This method will open up new avenues of research aiming to understand how the degeneration of the skeletal muscle induces reparative responses and, thus, the reactivation of the myogenic program in adult zebrafish.

Author Spotlight: A Cryoinjury Model for Studying Skeletal Muscle Regeneration of the Caudal Peduncle in Adult Zebrafish  

This protocol describes a cryoinjury model to induce profound damage of several caudal myomeres in adult zebrafish. This method provides a new approach for studying skeletal muscle regeneration after severe loss of tissue in non-mammalian vertebrates.