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...

<ol>
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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=Mutations+affecting+somite+formation+and+patterning+in+the+zebrafish,+Danio+rerio.">Mutations affecting somite formation and patterning in the zebrafish, Danio rerio.</a> Development. 123, 153-164 (1996).</li><li>Walker, M. B., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-color+acid-free+cartilage+and+bone+stain+for+zebrafish+larvae.">A two-color acid-free cartilage and bone stain for zebrafish larvae.</a> Biotechnic &amp; Histochemistry. 82 (1), 23-28 (2007).</li><li>Kyritsis, 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=Acute+inflammation+initiates+the+regenerative+response+in+the+adult+zebrafish+brain.">Acute inflammation initiates the regenerative response in the adult zebrafish brain.</a> Science. 338 (6112), 1353-1356 (2012).</li><li>Oppedal, D., Goldsmith, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+screen+to+identify+novel+inhibitors+of+fin+regeneration+in+zebrafish.">A chemical screen to identify novel inhibitors of fin regeneration in zebrafish.</a> Zebrafish. 7 (1), 53-60 (2010).</li><li>Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A., Lieschke, G. J. <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>Spoorendonk, K. 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. 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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 (0.3x13 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 e.g. by Harvard Apparatus</td>
		</tr>
		<tr>
			<td>Steel burrs (0.5 &#181;m diameter)</td>
			<td>Fine Science tools</td>
			<td>19007-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Artemia ssp.</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 ...

This method can help answer key questions in the field of immunosuppressive therapeutic intervention for studying the adverse effects of drugs on tissue regeneration, in particular, in bones. The main advantage of this technique is that it combines past models of Zebrafish bone regeneration with systemic drug exposure by immersion of the injured Zebrafish in drug supplemented fish water. Before beginning the procedure, autoclave one aluminum foil covered 600 milliliter beaker per Zebrafish and an appropriate number of glass bottles of fish water for the experiment and prepare the immunosuppressive drug in experimental control solutions of interest.For an amputation injury, use blunt forceps to carefully place an anesthetized Zebrafish on its lateral side on the inversed lid of a 100 millimeter Petri dish and use a scalpel to resect 50%of the fin. Then place the fish into an autoclaved beaker containing 300 milliliters of fish water supplemented with appropriate experimental agent and cover the beaker with foil to prevent Zebrafish escape. To induce a fin fracture injury, place an anesthetized Zebrafish on its lateral side in a 100 millimeter agarose coated Petri dish under a dissecting microscope and push an injection needle slightly into a bony fin ray segment until a crack appears.It is important to introduce not too many fractures into the fin because this might greatly impair stability. Then transfer the fish into an autoclaved beaker containing 300 milliliters of fish water supplemented with the appropriate experimental agent. To generate a calvarial skull injury, hold an anesthetized Zebrafish in the upright position so the calvarial bones are easily visualized under a dissection microscope.Place a rotating micro drill equipped with a 500 micrometer drill bit over the center of the frontal bone and gently touch the bone to produce a hole the size of the micro drill burr. It is critical to not move the drill laterally while producing the injury and to stop immediately once the resistance of the tissue drops. Otherwise, the brain will be damaged.Then place the fish into an autoclaved beaker containing 300 milliliters of fish water supplemented with the appropriate experimental agent. Change the water in the beakers daily by transferring the Zebrafish and the fish water into an appropriate temporary container and refilling the beaker with fresh drug supplemented fish water. Then use a fish net to return the Zebrafish to its beaker.For treatments lasting up to two days total, do not feed the Zebrafish. During longer experiments, feed the Zebrafish with 0.5 to one milliliter of hatched Artemia SSP every second day. For fin injury analysis, harvest the fine and use fine forceps to grasp the fin at one edge of the stump.Use a second pair of forceps to grab the opposite stump edge and slightly press the tissue onto the bottom of the dish which contains cold 4%PFA for 10 to 20 seconds. Repeat fin flattening as necessary. The fin should now lay flat without curling.For long term sample storage after fixation, wash the tissue with three 20 minute PBS washes and an ascending methanol series. The samples can then be stored in 100%methanol at minus 20 degrees Celsius. For Alizarin Red staining, rehydrate methanol stored samples in a descending methanol series followed by two 20 minute washes in PBS and one five minute wash in deionized water.After the last wash, add Alizarin Red solution to the sample tissue and incubate the sample at room temperature with rocking for the appropriate staining period. To clear the samples, treat the tissue with the decreasing 1%potassium hydroxide glycerol series. Then store the samples in a one to one 0.1%potassium hydroxide to 80%glycerol solution and mount the tissues with 80%glycerol for imagining.For Calcein staining, incubate up to three Zebrafish simultaneously in 100 millimeters of Calcein solution for 20 minutes in a glass beaker covered with aluminum foil. At the end of the incubation, transfer the Zebrafish to a container of fish water and let the fish swim briefly before transferring them to a new container of fish water. After 20 minutes in the second beaker to acquire live images of fin regenerates, place an anesthetized Zebrafish onto an agarose coated Petri dish and image the fin by stereo microscopy.To acquire images of the regenerating skull bones, place the Zebrafish upright in a sponge and image the skull by fluorescence microscopy. Treatment with Prednisolone, similar to other glucocorticoids, leads to an overall inhibition of fin regeneration including bone formation as detected by Alizarin Red staining of fixed caudal fin tissue. Similarly, the drug has delaying effect on calvarial skull injury closure.Due to immunosuppression, reduced macrophage numbers can also be detected by immunohistochemistry on frozen tissue sections as evidenced by using anti-mCherry and anti-GFP antibody staining and transgenic MPEG-1 mCherry crossed with Osterix NGFP Zebrafish on Zebrafish skull tissue samples from Prednisolone treated animals. While attempting these procedures it is important to minimize the variability in the bone regeneration speed by carrying out the injury essays in a highly reproducible manner. It is also crucial to single house Zebrafish in autoclaved beakers containing autoclaved fish water in order to avoid microbial infection.This protocol will help to further address the underlying mechanisms of glucocorticoid action. For instance, in the context of glucocorticoid induced osteoporosis and may also be adapted for the use of other drugs and other Zebrafish bone.

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8.7K visualizações.  TU Dresden. Este método pode ajudar a responder a perguntas-chave no campo da intervenção terapêutica imunossupressor para estudar os efeitos adversos de drogas na regeneração tecidual, em particular, nos ossos. A principal vantagem desta técnica é que combina modelos passados de regeneração óssea de Zebrafish com exposição sistêmica de drogas pela imersão do zebrafish ferido na água de peixe suplementada. Antes de iniciar o procedimento, autoclave uma folha de alumínio coberta 600 milil...