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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Discussion

Zebrafish have proven useful in skeletal research in many regards. Selected mutants mimic aspects of human disease such as osteogenesis imperfecta or osteoarthritis^{23,24,25,26,27}, and larvae as well as scales are being used to identify bone anabolic compounds in small molecule screens^7,28,

Materials

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