Name: adult zebrafish injury models to study the effects of prednisolone in regenerating bone tissue
Uploaded: 2018-10-18T14:30:00
Description: Watch this Scientific Journal Video about Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue at JoVE.com

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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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. 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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 ...

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