Name: zebrafish larvae as a model to evaluate potential radiosensitizers or protectors
Uploaded: 2022-08-25T14:15:00
Description: Watch this Scientific Journal Video about Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors at JoVE.com

SS&#39;s lab and RKS&#39;s lab are funded by grants from DBT and SERB, India. APM is a recipient of the ICMR fellowship, Government of India. DP is a recipient of the CSIR fellowship, Government of India. UN is a recipient of the DST-Inspire fellowship, Government of India. Figure 2 was generated using Biorender (https://biorender.com).

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 &#176;C, and the embryos were maintained in a biological oxygen demand (BOD) incubator at a temperature of 28.5 &#176;C. Here, the zebrafish AB strain was used, and the staging was carried out according to Kimmel et al.54. X-ray r...

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 screening67,68. 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...

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 studies1,2,3,4,5,6,7,8. 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 platforms9,10,11,12,13,14. 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 microscopy7,15,16.
Radiotherapy is one of the major therapeutic modes used in treating cancer17,18,19,20,21,22,23,24. 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 radiations25,26,27,28,29. Conversely, potent radiosensitizers are also being investigated to increase the efficiency of radiation to kill malignant cells, especially in targeted and precision therapies30,31,32,33. 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 experiments34,35. 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 radiotoxicity36,37. 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 studies38,39.
The potential of zebrafish as a bonafide model to screen radiation modifiers has been demonstrated in various studies40,41,42,43,44,45. 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 reported19,41,42,43,44,45,46. 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&#39;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 studies51,52.
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 mass7,53,54. 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 identified53,54. 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.

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			<td>Pasteur pipettes</td>
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			<td>PW1212-1X500NO</td>
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			<td>Perforated Internal Tank for the 1.0 L Breeding Tank</td>
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			<td>Sigma-Aldrich</td>
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zebrafish larvae as a model to evaluate potential radiosensitizers or protectors

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.

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 ...

Watch this Scientific Journal Video about Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors at JoVE.com

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

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.

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

This protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the treatment of different drugs and radiation. This will help readers to set a semi-high throughput drug screening involving x-ray exposure using zebrafish embryos, which will guide in the evaluation of radiotoxicity and the effectiveness of different drugs used. This procedure can be used to screen and validate or evaluate different drugs as potential radiosensitizers or protectors, which can be further explored or used in the field of cancer radiotherapy or radiobiology.To begin, monitor the growing embryos under the dissecting microscope. Identify the proper stage and remove any dead or unhealthy embryos. Ensure adequate embryo staging is the radiation and drug doses will be given at a particular gastrulation stage.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 to 20 embryos. While setting up a radiation experiment, include a control non-irradiated and a radiation-only group.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 see the embryos per radiation dose. Turn on the x-ray irradiator machine and initiate the machine initialization and warmup.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 and start the x-ray. 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 record their number after evaluating them under the dissecting microscope. Return the plates to the incubator at 28.5 degrees Celsius. Collect data at predetermined time intervals 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, and extension of the yolk sac, microcephaly, swimbladder development, general motility or activity, et cetera. To capture images, choose representative embryos on a clean slide. Check the embryos under the microscope, orient them in a particular direction.The survival curve for embryos irradiated at six hours post-fertilization depicted that in the control group and embryos exposed to 2 gray and 5 gray, there was no significant death in embryos. The heartbeats per minute count suggested that the heart rate decreased enormously with increased doses of x-ray radiation. In the control group at every 24-hour interval, the heart rate was seen to increase.Severe cardiovascular deformation is suspected in embryos exposed to 15 gray and 20 gray radiation as the heartbeat dropped enormously. Different phenotypic and developmental defects are depicted and evaluated for embryos exposed to varying doses of radiation at different time points. Morphological observations for spine or tail bending, head malformation and microcephaly, defects in development, pericardial edema, yolk sac deformities, swimbladder deformities, and changes in eye structure were also carried out For the protocol that we have demonstrated, choosing healthy embryos, proper placement of the embryos in the irradiator and proper evaluation of the cross-abnormalities are the key to this procedure.

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