Simple vertebrate model development 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 highly technical conditions for radiobiology experiments.
Our main aim was to introduce and validate a vertebrate system for in vivo experiments to investigate the biological effects of novel hadron beams. The endpoints at diverse dose levels were observed during a certain time frame in order to establish the most relevant factors for relative biological effectiveness (RBE) definition.
Material/methods: Series of zebrafish embryos in 24 hour post fertilization ages in different holders like tubes and 96 well plates varying the number (n) of embryos/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 (at the plateau and at the middle of Spread Out Bragg Peak), furthermore, with reference linear accelerator photon (0, 5, 10, 15, 20 Gy) beams (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 days post irradiation (dpi). 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: The RBE was highly sensitive in this system to time, dose and endpoints. The most robust result could be revealed by survival analysis with RBE of definition on the base of LD50- s at the 5th to 7th dpi: RBE between 10 and 4.8 for the fission and = 3.5 MeV cyclotron based neutrons and around 1.1-1.4 for protons, respectively. The morphological distortions and its severity exhibited a good agreement to the survival derived RBE with a narrow time and dose frame for the different type (i.e. pericardial edema: 3 dpi 20 Gy, spine curvature 4 dpi 15 and 20 Gy). 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 radiation setup, radiation dose and observation time points for assessment of the different biological endpoints could be established. This vertebrate model proved to be highly reproducible, reliable, and seems to be well applicable for 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., The project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement no 654148 Laserlab-Europe and by the German BMBF, grant no. 03Z1N511.