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.
