2.1 Animals

The asexual strain GI of the planarian Dugesia japonica (Tricladida Dugesiidae)⁴¹ was bred in artificial water (CaCl₂ 2.5 mM, NaHCO₃ 0.8 mM, MgSO₄ 0.4 mM, KCl 0.077 mM in distilled water) at 18°C.⁴² Planarians were transferred to ESA-ESTEC, in Noordwijk (The Netherlands), in boxes filled with artificial water at the temperature of about 22°C. They were used for experiments after 24 h of acclimation and 2 weeks of fasting. Planarians are non-cephalopod invertebrates, and all animal experiments were performed in agreement with Italian and European law, as well as with guidelines and recommendations of the Federation of European Laboratory Animal Science Associations (FELASA).

2.2 Nanoparticle characterization

A single source of CONPs (1406RE, Nanostructured & Amorphous Materials, Inc., Houston) was utilized throughout all experimental procedures. To characterize nanoparticle morphology, size and crystalline structure, micrographs of CONPs were taken via transmission electron microscopy (TEM), utilizing a JEM-1400Plus microscope with thermionic source (LaB₆), operated at 120 kV, equipped with a Gatan CCD camera Orius 830 (2048 × 2048 active pixels). Images were collected with two different modalities, namely in bright field (BF) and in selected area aperture diameter (SAED, approximately 5.25 μm). Water suspensions of CONPs for TEM analyses were prepared as follows: CONPs were suspended in ultra-pure water, sonicated with ultrasound, loaded onto an ultrathin carbon film on Cu grid, and dried in low vacuum at room temperature.

X-ray photoelectron spectroscopy (XPS) analysis was carried out to evaluate the chemical state of the CONPs used in the experiments, with a particular focus on assessing the Ce³⁺ and Ce⁴⁺ contents. The specimen for XPS analysis was prepared by pressing a few milligrams of finely ground CeO₂ powder onto a high purity indium pellet (Sigma-Aldrich). Measurements were conducted on a Kratos Axis Ultra^DLD spectrometer (Kratos Analytical Ltd.) using a monochromatic Al Kα source (hν = 1,486.6 eV) operated at 20 mA and 15 kV. Wide scans were collected at pass-energy of 160 eV and energy step of 1 eV, while high-resolution spectra were collected at pass-energy of 10 eV and energy step of 0.1 eV. The Kratos charge neutralizer system was used for all the measurements. Spectra were analyzed with CasaXPS software (Casa Software, Ltd., version 2.3.22).

To determine the size distribution profile of CONPs, nanoparticles were dispersed to 1 mg/ml, together with 1 mg/ml gum Arabic (GA, G9752, Sigma-Aldrich) in planarian artificial water, and gently sonicated. We then performed dynamic light scattering (DLS) and Z-potential measurements (at 20°C), using a Zeta-sizer NanoZS 90 instrument (Malvern Instruments Ltd.). Both polydispersity index (PDI) and Z-potential were measured as average ± standard deviation of three measurements, each consisting of 15 runs.

2.3 Cerium oxide nanoparticle treatment

CONPs used in this research underwent extensive characterization in previous works, for example,³⁹ and were already used successfully in planarians without any particular adverse effects.^{43, 44} CONPs at 1 mg/ml were dispersed through a mild sonication with a probe sonicator (GM mini20 Bandelin Sonopuls, Bandelin), 5 min at 8 W, in a GA 1 mg/ml dispersion in artificial water. For controls, GA-only dispersions were prepared following the same procedures. GA is the vehicle in which CONPs are dispersed and it has no effect on planarian tissue regeneration and stem cell biology.⁴⁴ CONP treatment was performed following the experimental conditions described in^{43, 44} at which its internalization in the cytoplasm of planarian cells has been demonstrated. Briefly, animals were immersed for 24 h in CONP or GA-only medium and then rinsed 3 times with 50 ml of artificial water to completely wash out CONPs and GA.

2.4 Simulated altered gravity experiments

The large diameter centrifuge (LDC)⁴⁵ (Figure 1(a)) was used at 10 g for simulated hypergravity experiments. Simulated microgravity was achieved via a random positioning machine (RPM; Dutch Space/EADS) at 30°/s (Figure 1(b)).^{46, 47} The RPM was used with a maximum random speed of 30°/s, random direction and interval. The samples were never more than 10 cm away from the center of rotation. Both kinds of experiments were invariably carried out at 18°C. For 1 g control, animals were simply left at 18°C for the appropriate amount of time in parallel with the LCD or RPM run. Groups of 10 planarians were put in 50 ml falcon tubes, filled out completely with artificial water. Those for RPM experiments were especially inspected to make sure that no visible air bubbles formed after caps were screwed on securely. Two independent runs (run 1 and run 2) were performed for each experimental condition at a distance of 2 days (experiments of exposition to altered gravity for 6 and 24 h) or 3 days (experiments of exposition to altered gravity for 60 h) (Figure 2).

2.5 Morphometric analysis of blastema size

Immediately before to enter LDC or RPM, GA-, and CONPs-treated animals were cut between auricles and pharynx, producing head (tail-regenerating), and tail (head-regenerating) fragments. Fragments were then exposed to simulated altered gravity for 6 or 60 h and fixed as previously described⁴⁸ at 60 h after cut, namely immediately at the end of a 60-h run or after 54 h from a 6-h run. Controls (1 g) were processed analogously. Samples were examined under a stereomicroscope (Stemi 305, Carl Zeiss Microscopy GmbH), equipped with a camera (Axiocam Erc 5 s, Carl Zeiss Microscopy GmbH). Digital images were quantified using Image J software.⁴⁹ For each animal, blastema and total body area were measured for at least 15 regenerating animals for each experimental group. We considered as blastema the unpigmented region below the wound epithelium; blastemal margin was manually highlighted by the operator in blind. The total body area was approximated to the area of the rectangle drawn around the body margin.

2.6 Whole-mount in situ hybridization

DNA template for NB.21.11.e, a marker for early stem cell progeny,⁵⁰ was produced as previously described.⁵¹ Digoxigenin (DIG)-labeled RNA probe was obtained by in vitro transcription of the purified template using DIG-labelling mix (Roche Applied Science). 60 h after exposure to altered gravity, intact animals were fixed and treated for whole-mount in situ hybridization (WISH) as previously described.⁵² After colorimetric development, samples were dipped in 50% glycerol/10% methanol, and analyzed under the stereomicroscope. NB.21.11.e signal was quantified as already described.⁵³ Briefly, each animal was photographed at the same magnification and exposition; pictures were then converted into grayscale mode and inverted. The mean gray value (raw intensity/animal area) was recorded by the ImageJ software. Mean gray recorded from animals (n = 10) belonging to the same experimental class was utilized to obtain the mean gray value of the group.

2.7 Terminal deoxynucleotidyl transferase dUTP nick end labeling

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed by using ApopTAg Red in situ apoptosis detection kit (EMD Millipore Corporation) as previously described.⁴⁸ Stained animals were mounted in 80% glycerol, and analyzed using a confocal microscope (TCS SP8, Leica Microsystems CMS) by tile scan function and zeta-stack optical sectioning every 2 μm, respectively. Five planarians were analyzed for each experimental condition. A single composite image was taken for each specimen. The number of apoptotic cells in intact and regenerating (60 h of simulated altered gravity regime) animals was evaluated using the ''find maxima'' option of the Image J software and normalized versus the animal body area. In the case of regenerating animals exposed to artificially altered gravity for 6 h, the number of apoptotic cells was exclusively counted in 100 μm tissue strip from the amputation site.

2.8 Western blotting

Mitotic cells were quantified using the mitotic marker phospho-histone H3 antibody (H3P Ab, 06–570, Sigma-Aldrich) by Western blotting. Nine animals for each experimental class were amputated and immediately exposed to artificially altered gravity for 6 or 60 h. Nine intact animals were exposed to simulated altered gravity for 24 h. After exposition, three independent groups (each composed by three animals) were immediately frozen, and proteins were extracted by adding 1X Laemmli buffer (0.1% 2-mercaptoethanol, 0.0005% bromophenol blue, 10% glycerol, 2% SDS electrophoresis-grade, 63 mM Tris–HCl pH 6.8). Proteins were transferred onto nitrocellulose membrane using Mini Trans-blot electrophoretic transfer cell apparatus (Bio-Rad Laboratories Inc.) following the manufacturer's instruction. After blocking with 5% nonfat dry milk (Bio-Rad Laboratories Inc.) in PBST (0.05% Tween 20, Sigma, in PBS 1×), membranes were incubated with 1:500 of H3P antibody in 1% non-fat dry milk in PBST for 2 h. Anti-α-tubulin antibody, mouse monoclonal (T6199, Sigma-Aldrich) was used as loading control at 1:20000 dilution. Membranes were then washed in PBST and incubated with anti-mouse IgG horseradish peroxidase (HRP; 6,402–05, Biovision) or anti-rabbit IgG HRP (6401–05, Biovision) diluted 1:50000 in 1% nonfat dry milk in PBST for 1 h. The chemiluminescence detection was performed using the Immobilon Western chemiluminescent HRP substrate kit (Millipore Corp.) following the manufacturer's instruction. To visualize immunoblots, we used a luminescent image analyzer (Chemidoc XSR + System Bio-Rad) and the Image LabTM Software (Bio-Rad Laboratories). Only bands below the saturation limit were analyzed. The chemiluminescence was expressed in terms of volume of specific immunoreactive bands and, in each lane, the protein level was normalized to the α-tubulin level. Three technical replicates were performed for each Western blot.

2.9 Statistical analysis

Unpaired Student's t tests (α = 0.05) and two-way ANOVA (α = 0.05) were applied to evaluate statistical significance, respectively using the software GraphPad Prism 7.00 and Free Statistics Software v1.2.1 (https://www.wessa.net/rwasp_Two%20Factor%20ANOVA.wasp/).

3.1 Cerium oxide nanoparticle characterization

Figure 3 shows a representative TEM image of CONPs (a), the diffraction pattern of the powder (b), as well as the XPS wide scan (c), with high-resolution spectra collected on the binding energy regions typical for Ce 3d and O 1 s peaks (d).

Powder electron diffraction pattern show sharp diffraction rings indicating that the nanoparticles have a crystalline nature compatible with that one of cubic ceria.⁵⁴

Concerning XPS, From the wide scan we could confirm the high purity of the nanomaterial, as the observed peaks could all be assigned to either cerium or oxygen, a part from a low intensity carbon peak at ~285 eV, partially overlapping with Ce 4 s ones. The high-resolution spectra are shown after background subtraction, together with the outcome of the fitting procedure. The best fit on the Ce 3d spectrum was obtained with five sets of spin-orbit split doublets, three of which representative of Ce(IV) (light blue profiles in the figure) and two of Ce(III) (orange profiles) oxides, as described in.⁵⁵ The splitting between the two components of each doublet has been set to 18.6 eV, as reported in,⁵⁶ while the intensity ratio between the two components is set to 3:2, due to spin-orbit coupling. The relative content of Ce³⁺ and Ce⁴⁺ species in the sample was calculated from the ratio of the sum of the integrated areas of the XPS 3d peaks related to the two oxidation states to the total integral area for the whole Ce 3d region. The acquired XPS spectrum is consistent with a Ce³⁺ percent concentration of 19.6 ± 2.0% (Ce⁴⁺ 80.4 ± 2.0%), corresponding to a Ce³⁺/Ce⁴⁺ ratio of about 0.24. The O 1 s spectrum was fitted with three peaks. The main one, centered at 529.0 ± 0.2 eV, corresponds to lattice oxygen in CeO₂, in agreement with the literature.⁵⁷ To obtain the best fit, two low intensity peaks were centered at 530.9 ± 0.2 eV and 533. 3 ± 0.2 eV, and are assigned to hydroxides and C–OH groups at the surface of the CONPs.⁵⁷ DLS measurements (Figure 3(e)) are in line with our previous reports,⁴³ namely an hydrodynamic size of 425 ± 17 nm, a PDI of 0.366 ± 0.004, and a Z-potential of −17.9 ± 0.4 mV.

3.2 Altered gravity and tissue regeneration

With the aim to understand the effect of gravity on tissue regeneration, we performed a morphometric analysis of 3-day-old blastema area of planarians exposed to simulated altered gravity, with or without previous treatment with CONPs and according to the experimental design depicted in Figure 4(a). In order to normalize differences in blastema area that might depend on the use of animals of different sizes, we initially investigated the dependence of these two variables. Regression analysis of blastema area with respect to body area, performed throughout the dataset, shows a linear correlation (R = 0.7, significance F close to 0). For this reason, we chose the ratio between blastema area and total body area as the most appropriate parameter to evaluate effects on tissue regeneration. Our data indicate that 6 or 60 h exposition to simulated hypergravity produced a significant reduction in the head fragment regeneration, and this reduction was rescued by CONP treatment (Figure 4(b)-(d) and Figure S1). RPM did not affect tissue regeneration at both 6 and 60 h, and CONP supplementation slightly increases the process at 60 h (Figure 4(c)-(d), and Figure S1). No statistically significant differences were detected between groups for tail fragments (Figure S2a-b).

3.3 Altered gravity and cell proliferation rate

To assess the effect of altered gravity on cell proliferation, we analyzed the expression of phospho-histone H3, a marker of mitosis, in regenerating and intact animals treated with CONPs or GA according to the experimental design indicated in Figure 5(a). We did not detect significant changes in the expression of the mitotic marker in both intact animals (exposed to simulated altered gravity for 60 h; Figure S2c) and in regenerating fragments exposed to artificially altered gravity for only 6 h (Figure S2d). On the contrary, a significant reduction in the number of mitotic cells in regenerating fragments exposed to RPM for 60 h was detectable. A similar trend, although not statistically significant, was also detectable in regenerating fragments exposed for 60 h to artificial hypergravity (p value from 0.05 to 0.1 for the different technical replicates; Figure 5(b)-(c) and Figure S1). Pre-treatment of animals with CONPs induced a rescue in animals exposed to simulated altered gravity with respect to the control (Figure 5(b)-(c) and Figure S1).

3.4 Altered gravity and cell differentiation

To understand the effect of simulated altered gravity on cell commitment, we analyzed the expression of NB.21.11.e, a marker of early neoblast progeny towards epidermal cell lineage, by WISH experiments performed on intact planarians exposed to artificially altered gravity for 60 h with or without previous treatment with CONPs (Figure 6(a) and Figure S1). In particular, we exposed animals to altered gravity for 60 h and sacrificed them, a time point at which the effect can be evaluated on early epidermal progeny.⁵⁰ We found that NB.21.11.e expression is increased upon simulated hypergravity and reduced upon simulated microgravity (Figure 6(b)-(c) and Figure S1). These differences were not detectable in animals treated with CONPs (Figure 6(b)-(c) and Figure S1).

3.5 Altered gravity and cell death

The effect of altered gravity on cell death was analyzed by whole-mount TUNEL assay in intact planarians and regenerating fragments at 6 and 60 h after amputation. We detected a significant increase in the number of apoptotic cells in intact animal treated with GA and exposed to simulated altered gravity for 24 h (Figure 7 and Figure S1). The number of apoptotic cells clearly increased 6 h after amputation in animals exposed to artificial hypergravity with respect to controls, while it also resulted in a significant reduction in animals in the RPM (Figure 8 and Figure S1). No difference was observed in animals exposed to altered gravity after 60 h of regeneration (Figure S2e). The treatment of both intact and regenerating animals with CONPs counteracted the alteration in the number of apoptotic cells (Figure 7, 8 and Figure S1).