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....

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 ....

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