Development of zebrafish embryo model for radiobiology research at ELI-ALPS on laser driven hadron beams

Purpose/objective: High power lasers provide the basis of particle acceleration, but at the actual status of the development, low energy, limited size beams with special properties (ultrahigh dose rate, pulsed mode) are available under technical conditions for radiobiology experiments. Our main aim was to introduce, optimize and validate a vertebrate system for in vivo experiments to study the biological effects of novel hadron beams.
Material/methods: Series of zebrafish embryos in different ages (from 1 hour post-fertilization (hpf) to 72 hpf), in different holders varying the number/well were prepared. For irradiation we used fission neutron (0, 1.25, 1.875, 2, 2.5 Gy), cyclotron-based neutron (0, 2, 4, 6.8, 8.12, 10.28 Gy) and proton (0, 5, 10, 15, 20 and 30 Gy) at two positions along the proton depth-dose curve (PDDC): at the plateau and at the middle of Spread Out Bragg Peak (mSOBP), furthermore, with reference linear accelerator photon (0, 5, 10, 15, 20 Gy) beam (n=96 in each group), repeated several times (≥3). Thereafter, survival, any type of organ developmental disturbance (pericardial edema, spine curvature, shortening of the body length and micro-opthalmia) were detected each days up to 7 post irradiation days (pid). Histological evaluation (size of the eye, brain necrosis, intestinal changes, liver vacuolization, hyper eosinophilic necrotic muscle-fibers) and molecular changes were evaluated with RT-PCR method at certain time points post irradiation.
Results: A higher vulnerability and radiation sensitivity could be observed at earlier stages of the embryogenesis (1-12 hpf). The LD50 was determined with the well reproducible survival curves, resulting in RBE between 10 and 4.8 for the 1MeV and 14MeV neutrons and the Relative Biological Effectivity (RBE) around 1.1-1.4 for proton sources at the two positions respectively. The morphological distortions shown close correlation to the dose delivered and their evaluation on the 4th pid exhibited a good agreement to the survival derived RBE. The gravity of the histopathological changes on the basis of semi-quantitative analysis corresponded well to the macro morphological abnormalities (eye layer disorganization, degree of brain necrosis, increased numbers of the goblet cells in the gastrointestinal tract, and muscle fibrosis).
Conclusion: Numerous features of the zebrafish embryo model makes it amenable for large scale of radiobiological investigations. On the basis of our experimental series the optimal embryonal age (hpf), radiation setup and observation time points for assessment of the different biological endpoints could be established. The defined parameters proved to be suitable for reliable RBE determination.

Acknowledgement: The ELI-ALPS project (GINOP-2.3.6-15-2015-00001) is supported by the European Union and co-financed by the European Regional Development Fund, and by the German BMBF, grant nos. 03ZIK445 and 03Z1N511. The project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement no 654148 Laserlab-Europe.