Microbeam Irradiation of the C. elegans Nematode

Abstract

The understanding of complex radiation responses in biological systems, such as non-targeted effects as represented by the bystander response, can be enhanced by the use of genetically amenable model organisms. Almost all bystander studies to date have been carried out by using conventional single-cell in vitro systems, which are useful tools to characterize basic cellular and molecular responses. A few studies have been reported in monolayer explants and bystander responses have been also investigated in a three-dimensional normal human tissue system. However, despite the well-know usefulness of in vitro models, they cannot capture the complexity of radiation responses of living systems such as animal models. To carry out in vivo studies on the bystander effect we have developed a new technique to expose living organisms using proton microbeams. We report the use of a nematode C. elegans strain with a Green Fluorescent Protein (GFP) reporter for the hsp-4 heat-shock gene as an in vivo model for radiation studies. Exposing animals to heat and chemicals stressors leads to whole body increases in the hsp-4 protein reflected by enhanced fluorescence. We report here that γ-rays also can induce stress response in a dose dependent manner. However, whole body exposure to stress agents does not allow for evaluation of distance dependent response in non targeted tissues: the so-called bystander effect. We used the RARAF microbeam to site specifically deliver 3 MeV protons to a site in the tail of young worms. GFP expression was enhanced after 24 hours in a number dependent manner at distances > 100 μm from the site of irradiation.

Behavioral Resistance of Caenorhabditis elegans Against High-LET Radiation Exposure

“…These results show that similar egg-lethal effects of high energy carbon ions occur before and after hatching, and that high-LET radiation does not additionally induce lethal effects on eggs in the body of C. elegans. This may support the hypothesis that the bystander effect is little if any in the last stage of embryogenesis of germ-line in C. elegans (Sugimoto et al, 2006), although the bystander-induced expression of heat-shock protein HSP-4 was reported (Bertucci et al, 2009). Taken together, we found that eggs of C. elegans were more sensitive to high-LET radiation exposure than to low-LET radiation exposure.…”

Novel features of radiation-induced bystander signaling in Arabidopsis thaliana demonstrated using root micro-grafting

“…[12][13][14][15][16][17][18] However, the underlying mechanisms are far from clear, including the temporal and spatial course of bystander signaling in the context of whole organism.The RIBE in model plant Arabidopsis thaliana has been investigated in our group. After micro-beam irradiation of naked seed embryos and low-energy ion irradiation of intact seeds, some post-embryonic developmental phenotypes were inhibited significantly, which differentiated from bystander shoot apical meristem (SAM) cells or root apical meristem (RAM) cells.…”

Embryos of the zebrafish Danio rerio in studies of non-targeted effects of ionizing radiation

“…Ureter models [55,134], as well as commercially available skin reconstruct models containing both epidermis and dermis or the epidermal layer alone [135][136][137], are classified as tissue models here. Although tissue models can provide some valuable information regarding the non-targeted effects of ionizing radiation in the multicellular tissue environment, it remains necessary to extrapolate such results back to the biology of the whole living organism; thus, there remains a marked advantage to perform the relevant studies in whole organism models [138,139]. As remarked by Bertucci et al [139], studies using whole organisms, which target specific cells, cell groups or organs, would be needed to realistically elucidate the mechanisms underlying the RIBE.…”

“…Although tissue models can provide some valuable information regarding the non-targeted effects of ionizing radiation in the multicellular tissue environment, it remains necessary to extrapolate such results back to the biology of the whole living organism; thus, there remains a marked advantage to perform the relevant studies in whole organism models [138,139]. As remarked by Bertucci et al [139], studies using whole organisms, which target specific cells, cell groups or organs, would be needed to realistically elucidate the mechanisms underlying the RIBE. We will hereafter focus on whole organism models when discussing in vivo models.…”