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Materials

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Developmental Biology

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

Published: October 18th, 2018

DOI:

10.3791/58429

1CRTD – Center for Regenerative Therapies Dresden, TU Dresden, 2Center for Healthy Aging, 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.

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.

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 s....

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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 'fish.......

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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's effects during bone regeneration can be pursued. For example, studies on bone formation and mineralization in the regenerate can be performed........

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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,

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This study was supported by a grant of the Center of Regenerative Therapies Dresden ("Zebrafish as a model to unravel the mechanisms of glucocorticoid-induced bone loss") 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, Nicole Cudak, and other members of the Knopf and Brand labs for technical assistance a....

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Name Company Catalog Number Comments
Prednisolone Sigma-Aldrich P6004
Dimethylsulfoxid (DMSO) Sigma-Aldrich D8418
Ethyl-3-aminobenzoate methanesulfonate (MS-222) Sigma-Aldrich A5040
Blunt forceps Aesculap BD027R
Fine forceps Dumont 91150-20
Scalpel Braun 5518059
Agarose Biozym 840004
Injection needle (0.3x13 mm) BD Beckton Dickinson 30400
Micro drill Cell Point Scientific 67-1000 distributed e.g. by Harvard Apparatus
Steel burrs (0.5 µm diameter) Fine Science tools 19007-05
Artemia ssp. Sanders 425GR
Pasteur pipette (plastic, Pastette) Alpha Labs LW4111
Paraformaldehyde Sigma-Aldrich 158127
Alizarin red S powder Sigma-Aldrich A5533
Alcian blue 8 GX Sigma-Aldrich A5268
Calcein Sigma-Aldrich C0875
Trypsin Sigma-Aldrich T7409
Stereomicroscope Leica MZ16 FA with QIMAGING RETIGA-SRV camera
Stereomicroscope Olympus MVX10 with Olympus DP71 or DP80 camera

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