Planarians as an In Vivo Experimental Model for the Study of New Radioprotective Substances

Abstract

Ionising radiation causes the death of the most actively dividing cells, thus leading to depletion of the stem cell pool. Planarians are invertebrate flatworms that are unique in that their stem cells, called neoblasts, constantly replace old, damaged, or dying cells. Amenability to efficient RNAi treatments, the rapid development of clear phenotypes, and sensitivity to ionising radiation, combined with new genomic technologies, make planarians an outstanding tool for the discovery of potential radioprotective agents. In this work, using the well-known antioxidant N-acetylcysteine, planarians are, for the first time, shown to be an excellent model system for the fast and effective screening of novel radioprotective and radio-sensitising substances. In addition, a panel of measurable parameters that can be used for the study of radioprotective effects on this model is suggested.

Keywords: irradiation; model animal; planarians; radioprotection; regeneration.

Figure 1

Inhibition of planarian head blastema growth on the 3rd day after X-ray irradiation (1—30 Gy). Data are shown as mean values ± standard error, n = 90, * p < 0.001.

Figure 2

The radioprotective effect of NAC under X-ray exposure. (a) Radioprotective effect of N-acetylcysteine (10 mM) under 10 and 15 Gy irradiation; (b) radioprotective effect of N-acetylcysteine (15 mM) under 10 and 15 Gy irradiation, * p < 0.001 via ANOVA.

Figure 3

Number of mitotic cells in the planarian body 7 days after X-ray irradiation (10 and 15 Gy) with NAC (10 mM). (a,b) Determination of the total number of mitotic cells in the planarian body stained by immunohistochemistry; (c) distribution of mitotic cells in the planarian body 10 days after irradiation at a dose of 15 Gy. # p < 0.001 (from control), * p < 0.001 (from 15 Gy). Arrows indicate the absence of mitotic cells after X-ray irradiation.

Figure 4

Gene expression of three classes of neoblast markers in regenerating planarians treated with NAC (10 mM) for 3 h (a) and 6 h (b) after irradiation. The intensity scale of the standardised expression values range from −3 (green: low expression) to +3 (red: high expression), with a 1:1 intensity value (black) representing the control (unirradiated).

Figure 5

Genomic template stability (GTS) of planarians after X-ray irradiation (DNA was isolated 2 h after irradiation). GTS—genomic stability coefficient.

Figure 6

Inhibition of ROS formation by N-acetylcysteine, measured by H2DCFDA fluorescence. Quantitative determination of the fluorescence intensity in the body of planarians (a); fluorescence micrographs of planarians after irradiation (b). Standard deviation * p < 0.001.