Artificially altered gravity elicits cell homeostasis imbalance in planarian worms, and cerium oxide nanoparticles counteract this effect

Abstract

Gravity alterations elicit complex and mostly detrimental effects on biological systems. Among these, a prominent role is occupied by oxidative stress, with consequences for tissue homeostasis and development. Studies in altered gravity are relevant for both Earth and space biomedicine, but their implementation using whole organisms is often troublesome. Here we utilize planarians, simple worm model for stem cell and regeneration biology, to characterize the pathogenic mechanisms brought by artificial gravity alterations. In particular, we provide a comprehensive evaluation of molecular responses in intact and regenerating specimens, and demonstrate a protective action from the space-apt for nanotechnological antioxidant cerium oxide nanoparticles.

Keywords: cerium oxide nanoparticles; hypergravity; microgravity; planarians; stem cells.

FIGURE 1

Ground‐based facilities at ESTEC. (a) LDC used at 10 g for simulated hypergravity. (b) RPM, used with a setting of maximum speed of 30°/s for simulated microgravity

FIGURE 2

Overall study overview. Schematic representation including all experimental groups and times of run 1 and run 2

FIGURE 3

Cerium oxide nanoparticle characterization. (a) Representative TEM image. (b) Diffraction pattern, showing crystalline structure. (c) XPS wide scan. (d) High‐resolution spectra collected on the Ce 3d and O 1 s energy regions. High resolution data are shown together with the results of the best fit procedure. (e) DLS measurements, with size distribution; PDI, Z‐average and Z‐potential indicated in the box as mean values ± standard deviation

FIGURE 4

Morphometric analysis of head blastema in animals pre‐treated with cerium oxide nanoparticles (CONPs) or gum Arabic (GA) and exposed to different gravity regimes. (a) Schematic representation of the experimental set‐up. (b) Representative head fragments regenerating a tail exposed to 1 g (GA 1 g), hypergravity (GA 10 g), or artificial microgravity (GA RPM). The blastema is marked by black dots. Scale bar is 500 μm. (c) Morphometric analysis of blastemal area after 6 h of exposure to different gravity regimes, in GA‐treated animals exposed to 1 g (GA 1 g), hypergravity (GA 10 g), simulated microgravity (GA RPM) and in CONPs‐treated animals exposed to 1 g (CONPs 1 g), hypergravity (CONPs 10 g), or simulated microgravity (CONPs RPM). Each bar is the mean ± standard deviation of 15 independent samples of run 1. Comparable results were obtained with samples of run 2. Significant differences were evaluated by unpaired Student's t‐test analysis (***p < 0.001; **p < 0.01; ns: not significant) and two‐way ANOVA. (d) Morphometric analysis of blastemal area after 60 h of exposure to different gravity regimes. Each bar is the mean ± standard deviation of 15 independent samples of run 1. Comparable results were obtained with samples of run 2. Significant differences were evaluated by unpaired Student's t test analysis (**p < 0.01; *p < 0.05; ns: not significant) and two‐way ANOVA

FIGURE 5

Quantification of the mitotic marker phospho‐histone H3 in animals pre‐treated with cerium oxide nanoparticles (CONPs) or only gum Arabic (GA) and exposed to different gravity regimes. (a) Schematic representation of the experimental set‐up. Numbers indicate hours (h). (b) Representative Western blotting. Lane 1: GA‐treated animals at 1 g; lane 2: GA‐treated animals at 10 g; lane 3: GA‐treated animals exposed to artificial microgravity; lane 4: CONPs‐treated animals at 1 g; lane 5: CONPs‐treated animals at 10 g; lane 6: CONPs‐treated animals exposed to simulated microgravity. (c) Quantification of mitotic cells. GA‐treated animals exposed to 1 g (GA 1 g), 10 g (GA 10 g), or simulated microgravity (GA RPM); CONPs‐treated animals exposed to 1 g (CONPs 1 g), 10 g (CONPs 10 g), or simulated microgravity (CONPs RPM). Each bar is the mean ± standard deviation of three independent samples, for each experimental group, of run 1, normalized versus the corresponding GA control, to which an arbitrary value of 1 was attributed. Comparable results were obtained with samples of run 2. Significant differences were evaluated by unpaired Student's t test analysis (*p < 0.05; ns: not significant) and two‐way ANOVA. Each Western blotting has been performed in triplicate

FIGURE 6

Analysis of the expression of NB‐21.11.e, a marker of early progeny, in animals pre‐treated with gum Arabic (GA) or cerium oxide nanoparticles (CONPs) and exposed for 60 h at different gravity regimes. (a) Schematic representation of the experimental set‐up. Numbers indicate hours (h). (b) Representative images of NB‐21.11.e expression by whole‐mount in situ hybridization (WISH) in a GA‐treated animal exposed to 1 g (GA 1 g), 10 g (GA 10 g) or simulated microgravity (GA RPM). Scale bar is 500 μm. (c) Quantification of signal intensity detected by WISH. Each bar is the mean value ± standard deviation of the mean gray values measured in ten independent samples, for each experimental group, of run 1. Comparable results were obtained with samples of run 2. Significant differences were evaluated by unpaired Student's t test analysis (***p < 0.001; **p < 0.01; ns: not significant) and two‐way ANOVA

FIGURE 7

Analysis of apoptotic cells by TUNEL assay in intact animals after 24 h of altered gravity. (a) Representation of the experimental set‐up. Numbers indicate hours (h). (b‐d) Confocal representative images of apoptotic cells (red dots) in animals treated with Gum Arabic (GA) and exposed to (b) 1 g (GA 1 g), (c) hypergravity (GA 10 g) or (d) simulated microgravity (GA RPM). Scale bar is 100 μm. (e) Quantification of apoptotic cells in animals exposed to altered gravity in presence of GA or cerium oxide nanoparticles (CONPs). CONPs‐treated animals exposed to 1 g (CONPs 1 g), hypergravity (CONPs 10 g) or simulated microgravity (CONPs RPM). Each bar is the mean ± standard deviation of three independent samples of run 1. Comparable results were obtained with samples of run 2. Significant differences were evaluated by unpaired Student's t test analysis (*p < 0.05; ns: not significant) and two‐way ANOVA

FIGURE 8

Analysis of apoptotic cells by TUNEL assay in regenerating planarians 6 h after amputation. (a) Representation of the experimental set‐up. Numbers indicate hours (h). (b‐d) Confocal representative images of TUNEL positive‐cells (red dots) in animals treated with Gum Arabic (GA) exposed to (b) 1 g (GA 1 g), (c) hypergravity (GA 10 g), (d) simulated microgravity (GA RPM). Scale bar is 100 μm. (e) Quantification of apoptotic cells in animals exposed to 6 h altered gravity in presence of GA or cerium oxide nanoparticles (CONPs). CONPs‐treated animals exposed to 1 g (CONPs 1 g), 10 g (CONPs 10 g) or simulated microgravity (CONPs RPM). Each bar is the mean ± standard deviation of five independent samples of run 1. Comparable results were obtained with samples of run 2. Significant differences were evaluated by unpaired Student's t test analysis (***p < 0.001; *p < 0.05; ns: not significant) and two‐way ANOVA