Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Summary

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

Abstract

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

Introduction

The zebrafish (Danio rerio) is a well-known animal model that has been widely used in research over the last 3 decades. It is a small freshwater fish that is easy to rear and breed under laboratory conditions. The zebrafish has been extensively used for various developmental and toxicological studies^{1,2,3,4,5,6,7,8}. The zebrafish has high fecundity and short embryonic generation; the embryos are suitable for tracking different developmental stages, are visually transparent, and are amenable to varieties of genetic manipulation and high-throughput screening platforms^{9,10,11,12,13,14}. Besides, the zebrafish provides in toto and live imaging for which its developmental process and different deformities in the presence of various toxic substances or factors can be easily studied using stereo or fluorescent microscopy^7,15,16.

Radiotherapy is one of the major therapeutic modes used in treating cancer^{17,18,19,20,21,22,23,24}. However, cancer radiotherapy demands potential radioprotectors to protect normal healthy cells from dying while killing malignant cells or safeguard human health during therapy involving high energy radiations^{25,26,27,28,29}. Conversely, potent radiosensitizers are also being investigated to increase the efficiency of radiation to kill malignant cells, especially in targeted and precision therapies^30,31,32,33. Therefore, to validate potent radioprotectors and sensitizers, a model suitable for semi-high-throughput drug screening and measurably exhibiting radiation effects is highly solicited. Several available models are used in radiation studies and involved in drug screening experiments. However, higher vertebrates and even the most commonly used in vivo model, mice, are unsuitable for large-scale drug screening because it is time-consuming, costly, and challenging to design such screening experiments with these models. Similarly, cell culture models are ideal for varieties of high-throughput drug screening experiments^34,35. However, experiments involving cell culture are not always pragmatic, highly reproducible, or reliable as cells in culture may markedly change their behavior according to the growth conditions and kinetics. Also, varieties of cell types show differential radiation sensitization. Notably, 2D and 3D cell culture systems do not represent the whole organism scenario, and, thus, the results obtained may not recapitulate the actual level of radiotoxicity^36,37. In this regard, the zebrafish provides several advantages in screening for novel radiosensitizers and radioprotectors. The ease of handling, large clutch size, short life span, rapid embryonic development, embryo transparency, and small body size make the zebrafish a suitable model for large-scale drug screening. Due to the above advantages, experiments can be readily repeated in a short time, and the effect can be observed easily under a dissecting microscope in multi-well plates. Hence, the zebrafish is gaining popularity in drug screening research involving radiation studies^38,39.

The potential of zebrafish as a bonafide model to screen radiation modifiers has been demonstrated in various studies^{40,41,42,43,44,45}. The radioprotective effect of potential radio modifiers, such as nanoparticle DF1, amifostine (WR-2721), DNA repair proteins KU80 and ATM, and transplanted hematopoietic stem cells, and the effects of radiosensitizers, such as flavopiridol and AG1478, in the zebrafish model have been reported^{19,41,42,43,44,45,46}. Using the same system, the radioprotective effect of DF-1 (fullerene nanoparticle) was assessed both at systemic and organ-specific levels, and also the use of zebrafish embryos for radioprotector screening was further explored⁴⁷. Recently, the Kelulut honey was reported as a radioprotector in zebrafish embryos and was found to increase embryo survival and prevent organ-specific damage, cellular DNA damage, and apoptosis⁴⁸.

Similarly, the radioprotective effects of polymers generated via Hantzsch's reaction were checked on zebrafish embryos in a high-throughput screening, and the protection was mainly conferred by protecting cells from DNA damage⁴⁹. In one of the previous studies, the lipophilic statin fluvastatin was found as a potential radiosensitizer using the zebrafish model with this approach⁵⁰. Similarly, gold nanoparticles are considered to be an ideal radiosensitizer and have been used in many studies^51,52.

The embryonic development in zebrafish involves cleavage in the initial 3 h in which a single-celled zygote divides to form 2 cells, 4 cells, 8 cells, 16 cells, 32 cells, and 64 cells that are easily identified with a stereomicroscope. Then, it attains the blastula stage with 128 cells (2.25 h post-fertilization, hpf), where the cells double every 15 min and proceed through these following stages: 256 cells (2.5 hpf), 512 cells (2.75 hpf), and reaching 1,000+ cells in just 3 h (Figure 1). At 4 h, the egg attains the sphere stage, followed by the formation of a dome shape in the embryonic mass^7,53,54. The gastrulation in zebrafish starts from 5.25 hpf⁵⁴, where it reaches the shield stage. The shield clearly indicates the rapid convergence movement of the cells to one side of the germ ring (Figure 1) and is a prominent and distinct phase of gastrulating embryos that can be easily identified^53,54. Although radiation exposure to embryos could be done at any stage of their development, radiation exposure during gastrulation might have more distinct morphological changes facilitating better readouts of radiation-induced toxicities⁵⁵; similarly, administration of drugs to embryos can be started as early as 2 hpf⁵⁴.

Protocol

The present study was conducted with prior approval from and following the guidelines of the Institutional Animal Ethical Committee, Institute of Life Sciences, Bhubaneswar. All zebrafish maintenance and breeding were conducted at an ambient fish culture facility at 28.5 °C, and the embryos were maintained in a biological oxygen demand (BOD) incubator at a temperature of 28.5 °C. Here, the zebrafish AB strain was used, and the staging was carried out according to Kimmel et al.⁵⁴. X-ray radiation was given at 6 hpf (shield stage), and different phenotypes were observed until 120 hpf.

1. Breeding setup and embryo collection

1. Set the breeding tanks (made up of polycarbonate, capacity 1 L, see Table of Materials). Pour system water (pH, 6.8-7.5; conductivity, 500 µS; and temperature, 28.5 °C) into the breeding tanks covering nearly 40% of its volume. Place the divider in the tank to create two chambers, one for females and the other for males.
2. From the parent tanks, carefully collect two healthy females and one healthy male with the help of a net, put them in their respective halves, and keep them in the dark overnight (minimum 10 h) at 28.5 °C.
3. The next morning, remove the divider and allow the fishes to mate without disturbing the breeding tanks.
   NOTE: The females will start spawning, and the eggs will be seen lying on the bottom of the tank within 10-15 min after the fishes are allowed to mate^56,57,58.
4. Return the fishes to their tanks after spawning, collect the embryos from the breeding tank using a strainer, wash them properly with the system water, and keep the collected eggs in a Petri plate with E-3 media (4.94 mM of NaCl, 0.17 mM of KCl, 0.43 mM CaCl₂, 0.85 mM of MgCl₂ salts, 1% w/v of methylene blue, see Table of Materials).
5. Observe the eggs under a dissecting microscope, remove the unfertilized or dead embryos using a Pasteur pipette, and keep the Petri plates containing fertilized eggs in the E-3 medium at 28.5 °C in an incubator for their proper growth and maintenance.
   NOTE: Unfertilized eggs can be identified with a milky white appearance with a coagulated chorion or with ruptured cells inside the chorion. Along with unfertilized eggs, eggs not undergoing cleavage and eggs with deformities like irregularities during cleavage, e.g., asymmetry, vesicle formation, or injuries of the chorion, or not actively developing, must be discarded to keep the collected embryos healthy and to keep the media clean^7,56.

2. Monitoring embryos and selection for radiation experiments

1. Monitor the growing embryos under the dissecting microscope, identify the proper stage^7,54, and remove any dead or unhealthy embryos. Ensure adequate embryo staging as the radiation and drug doses will be given at a particular gastrulation stage.
   NOTE: Every day, check for the level and quality of media in the culture dishes. Change the media every 24 h, along with removing dead embryos. Pasteur pipettes are preferred to be used for picking embryos or changing media.
2. Before starting the experiment, carefully distribute the healthy embryos in the experimental plates with the help of a Pasteur pipette. For each experimental group, take 15-20 embryos.
   ​NOTE: Place only healthy embryos of the desired developmental stages in the experimental plate. Suppose the drug treatment has to be done with embryos at 6 hpf, then start seeding them in experimental plates at least 30-60 min earlier.

3. Drug treatment

1. Add drugs of desired concentration to the zebrafish embryos. Prepare the drug-containing E-3 media well in advance. Ensure the stock solution of the drug has no undissolved drug before preparing the working media for treating zebrafish embryos.
2. Before adding any drug to a medium for radiation screening, check the cytotoxic effect of the drug with the grades of concentrations of the drug. Follow the OECD guidelines to evaluate the LC 50 of the drugs under evaluation^59,60,61.
   ​NOTE: Be careful while moving the plates and dishes during the irradiation or observation time. There are many chances that the plates will be disturbed during this handling, causing the media to leak out of the wells or the embryos to spill out of their respective wells, potentially contaminating nearby wells and ruining the experiment.

4. X-ray irradiation

1. While setting up a radiation experiment, include a control/non-irradiated and a radiation-only group. Similarly, while performing a drug screening, include another group where the drugs will be given with the same concentration as those administered in the screening experiment along with radiation.
   NOTE: Label both the lid and base of well plates or culture dishes so that the lids do not get misplaced.
2. Distribute the embryos in a well plate if the radiation shields can cover and protect the extra wells from radiation while the other wells are exposed to a particular radiation dose; otherwise, use individual plates or discs to seed the embryos per radiation dose.
3. Turn on the X-ray irradiator machine (see Table of Materials), and initiate the machine initialization and warm-up.
   NOTE: The source to subject distance (SSD) value must be 50 cm; one can use different SSDs yet again, which requires standardization.
4. Place the experimental plate under the irradiator inside the machine in the center, ensuring that the plate is directly below the X-ray source, and then set the dose (e.g., 5 GY) and start the X-ray.
   NOTE: Seal the plates with paraffin film to avoid any unwanted spillage or contamination during the transportation of the plates from the incubator to the irradiator and back.
5. After the completion of irradiation, take out the plates, shut down the machine program, switch off the machine, and check the plates under the microscope immediately after radiation. Remove the dead embryos and return the plates to the incubator at 28.5 °C. Record the number of dead embryos after evaluating them under the dissecting microscope.
   NOTE: Irradiate the different groups of embryos with designated radiation doses without much delay between individual groups as the effect of radiation may be significantly affected by the difference in the developmental stage.
   ​CAUTION: While operating the X-ray machine, take proper protective measures.

5. Data collection, imaging, and analysis

1. Collect data at predetermined time intervals, such as every 24 h after the radiation is given. Record all possible observations such as survival, hatching efficiency, stage of development, heartbeat count, body and tail curvature, pericardial edema, the extension of the yolk sac, microcephaly, swim bladder development, general motility or activity, etc.^62,63,64.
2. To capture images, choose representative embryos on a clean slide, check the embryos under the microscope, orient them in a particular direction, and click on images. Rename the image files according to the group and time.
   NOTE: The same magnification and illumination must be used while capturing pictures at different time intervals.

Results

The overall layout of the protocol is depicted in Figure 2. The effect of radiation and the characterization in a dose-dependent manner was evaluated with the following analyses.

Assessment of X-ray-induced toxicities
Using a stereomicroscope, the following abnormalities were assessed and characterized after the drug treatment and/or radiation. As per the OECD guidelines⁶¹, for toxicity evaluation in fi...

Discussion

Zebrafish are used as valuable models in many studies, including several types of cancer research. This model provides a useful platform for large-scale drug screening^67,68. Like any other toxicity evaluation method, the quantitative evaluation of the major biological changes upon radiation and/or drug treatment is the most crucial part of this protocol. In these kinds of studies, survival must not be the only criteria to observe toxicity; it needs to be supporte...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6 Well plates	Corning	CLS3335	Polystyrene
B.O.D Incubator	Oswald	JRIC-10
Calcium Chloride	Fisher Scientific	10101-41-4
Dissecting Microscope	Zeiss	Stemi 2000
External Tank for the 1.0 L Breeding Tank	Tecniplast	ZB10BTE	Polycarbonate
Glass petriplates	Borosil	3165A75	Glass
GraphpadPrism	GraphPad Software, Inc.		Version 5.01
Kline concavity slides	Himedia	GW092-1PK	Glass
Magnesium Chloride	Sigma-Aldrich	M8266
Methylene blue hydrate	Sigma-Aldrich	66720-100G
Parafilm	Tarsons	380020	Paraffin film
Pasteur pipettes	Himedia	PW1212-1X500NO	Polyethylene plastic
Perforated Internal Tank for the 1.0 L Breeding Tank	Tecniplast	ZB10BTI	Polycarbonate
Polycarbonate Divider for the 1.0 L Breeding Tank	Tecniplast	ZB10BTD	Polycarbonate
Polycarbonate Lid for the 1.0 L Breeding Tank	Tecniplast	ZB10BTL	Polycarbonate
Potassium Chloride	Sigma-Aldrich	P5655
Sodium Chloride	Sigma-Aldrich	S7653-5KG
Sodium hydroxide pellet	SRL	1949181
Stereo Microscope Leica M205FA	Leica	Model/PN MDG35/10 450 125
X-Rad 225 Precision X-Ray	Precision X-Ray	X-RAD 225XL