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 system to study skeletal disease and repair^4,5.

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 vivo^8,9. 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.

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 water' (i.e., the water that is used to rear adult zebrafish) before starting the experiment.

1. Calculate the number of zebrafish to be used in the experiment. Be sure to include the individuals that serve as controls.
2. 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.
3. 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).
4. 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.
5. 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).
6. 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 µM.
   1. 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.
   2. Work in an appropriate place that is designated to perform drug treatments, ideally in a room at 27 °C.

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.

1. Amputation
   1. 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).
      1. 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.
      2. 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.
   2. 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.
   3. 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.
   4. Repeat steps 2.1.2 to 2.1.3 until the required number of zebrafish for the experiment is reached.
2. Fin fracture
   1. 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 10–15 mL of the solution into the Petri dish. The bottom of the dish should be completely covered. Let the agarose solidify.
   2. Anaesthetize zebrafish as described in section 2.1.
   3. 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.
   4. 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.
      1. 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.
      2. 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' bifurcation point ¹¹.
   5. 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.
   6. Repeat steps 2.2.2 to 2.2.5 until the required number of zebrafish for the experiment is reached.

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.

1. Anaesthetize the zebrafish according to section 2.1.
2. Hold the anesthetized zebrafish upright with forceps, so that the calvarial bones are well visible under a dissection microscope from above (Figure 1C).
3. 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 µm, 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.
4. Transfer the injured zebrafish to a cage filled with fish water until it fully recovers from anesthesia. Watch it carefully.
5. Transfer the injured zebrafish to a fish water plus prednisolone or DMSO containing glass beaker.

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.

1. Water changes
   1. To exchange the fish water, thaw 100 mM prednisolone stock and prepare the required amount of 50 µM prednisolone in autoclaved fish water fresh every day of the treatment period. Prepare DMSO containing fish water for the control zebrafish accordingly.
   2. 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.
   3. Refill the glass beaker with fresh drug containing fish water (300 mL).
   4. 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.
   5. If the time of the experiment exceeds 2 weeks, exchange the glass beakers.
2. Feeding
   1. 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.
      1. 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.
      2. Wait for 2 h and exchange the incubation solution with fresh drug containing fish water according to section 4.1.

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.

1. Fixation of tissues of interest
   1. For fixation of fins and/or skulls prepare 4% paraformaldehyde (PFA) in PBS and freeze at -20 °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.
   2. 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.
   3. Harvest the tissue of interest.
      1. 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.
      2. 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.
   4. Fix fins flat in the PFA containing Petri dish at 4 °C overnight. Flat fixation avoids curling up of the fin tissue during fixation.
      1. 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.
      2. Hold for 10 to 20 s before releasing. The fin tissue should not curl up anymore. If it does, repeat.
   5. 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.
   6. 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 °C with gentle rocking.
   7. 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).
      1. Store them in 100% methanol at -20 °C. Otherwise, directly proceed with bone staining or other analyses after washing with PBS.
2. Bone staining techniques I (alizarin red staining)
   NOTE: This staining is performed according to van Eeden et al. 1996¹⁷ and Walker & Kimmel 2007¹⁸ with modifications.
   1. To perform alizarin red staining on adult fins or skulls, harvest and fix tissue as described in sections 5.1.
   2. If samples were stored in methanol at -20 °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.
   3. 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.
   4. 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.
   5. To clear samples treat them with a decreasing 1% potassium hydroxide:glycerol series (3:1 overnight, 1:1 overnight, 1:3 overnight).
   6. 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).
3. 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.
   1. 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.
   2. Incubate up to 3 zebrafish simultaneously in 100 mL of calcein solution for 20 min in a glass beaker covered with aluminum foil.
   3. 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 5.4).
4. Live imaging of zebrafish
   1. 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.
   2. To acquire images of fin regenerates, transfer the anaesthetized zebrafish onto an agarose-coated Petri dish (see section 2.2.1 and Figure 1B).
   3. 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.
   4. Acquire images at the desired magnification and resolution using a stereomicroscopic setup.
   5. Return the zebrafish to a container with fresh or drug containing fish water.

Results

The protocol presented here has been used repeatedly to induce rapid bone formation in the course of regeneration of the zebrafish fin and skull^10,11,16. 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. Pre...

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

List of materials used in this article

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 (0.3 mm x 13 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