Distribution of β-tubulin isotype 3 (β-tub 3) under simulated microgravity.

<p>(A) Distribution of β-tub 3 between somas and neurites in neurons exposed for 1 h to RPM (neurite <i>p</i> = 0.029; soma <i>p</i> = 0.038). (B) Soma intensity vs. neurite intensity ratios in neurons exposed for 1 hour, 24 hours and 10 days to the RPM compared to the respective ground condition controls. Statistical analysis show a difference between GC 1 h vs. RPM 1 h (<i>p</i> = 0.0012) and RPM 1 h vs. rpm 24 h vs. RPM 10 days (<i>p</i><0.05). (C 1-2-3) Higher magnification of neurons show the morphological and fluorescence intensity differences at the soma and neurite levels between exposed and non-exposed samples. One way Anova, Paired two-tailed Student’s <i>t</i>-test and standard deviation bars are shown. GC = ground condition; RPM = Random Positioning Machine.</p

Neuronal morphology is altered after chronic RPM and irradiation exposure, and after ground recovery.

<p><b>(A, B, C)</b> Morphometric analyses on βIII tubulin stained neurons immediately after 5-day chronic exposure conditions unveiled a significantly decreased neurite area <b>(A)</b> and neurite length <b>(B)</b> after RPM exposure and after combined RPM and radiation exposure. Analysis of the soma area revealed a strong decrease in soma area after all exposure conditions (RPM, irradiation and co-exposure) <b>(C)</b>. <b>(D, E, F)</b> Morphometric analyses after a 24 h ground recovery period showed a persistent and similar reduction in neurite area <b>(D)</b> and length <b>(E)</b> after radiation and/or RPM exposure, while the soma area was shown to be mostly recovered to control levels in RPM or radiation exposed neurons after 24 h recovery <b>(F)</b>. <b>(G)</b> Fluorescent images of neuronal network cultured exposed to ground conditions (GC), RPM, 1 mGy and 1 mGy combined to RPM. RPM = random positioning machine, GC = ground conditions, Gy = Gray. Values are represented as mean±SEM. N = 3, * p<0.05, *** p<0.001. Asterisks represent differences between RPM/irradiation groups and ground controls (GC).</p

Schematic overview of the experimental set-up.

<p>RPM = random positioning machine, GC = ground condition, Gy = Gray.</p

Neuronal cell death is increased after exposure to simulated microgravity and acute X-ray irradiation.

<p><b>(A</b>) Representative image of fragmented/apoptotic <i>vs</i>. healthy nuclei in mature cultured neurons. <b>(B)</b> Analysis of the percentage of fragmented nuclei revealed a strong increase in apoptotic nuclei 6 h and 24 h after exposure to 1 Gy of acute X-rays (full blue line), as opposed to 0 and 0.1 Gy (full red and green line). Under RPM conditions, a similar dose-dependent trend was observed, with non-irradiated and 0.1 Gy irradiated neurons also showing an increased percentage of fragmented nuclei from 6 h onward (dotted lines). When kinetically comparing ground conditions with RPM exposure for the respective doses, a clear increase in the percentage of fragmented nuclei could be observed after 6 h and 24 h of RPM exposure for all doses. RPM = random positioning machine, GC = ground conditions, Gy = Gray. Values are represented as mean±SEM. N = 3, * p<0.05, *** p<0.001. Scale bar: 20 μm. Asterisks represent changes between RPM and ground conditions for the different radiation doses.</p

Soma characteristics in neurons exposed for 1 hour, 24 hours and 10 days to the RPM compared to their respective controls.

<p>(A) Size of somas in neuron cultures exposed to RPM for 1, 24 hours or 10 days and their respective controls. (B) Roundness of somas in neuron cultures exposed to RPM for 1, 24 hours or 10 days and their respective controls. Paired two-tailed Student’s <i>t</i>-test and standard deviation bars are shown. 1, <i>p</i><0.05 RPM 1 h compared to GC 1 h; 2, <i>p</i><0.05 RPM 24 h compared to GC 24 h; 3, <i>p</i><0.05 RPM 10 days compared to GC 10 days. GC = ground condition; RPM = Random Positioning Machine.</p

Recovery in re-established ground conditions of neuronal networks and neurons previously exposed to simulated microgravity.

<p>(A) Recovery dynamics of area of single neurons after RPM as expressed in ratios of RPM vs. ground control exposed cultures. (B) Recovery dynamics of neurite area per neuron after RPM as expressed in ratios of RPM vs. ground control exposed cultures. (C) Recovery dynamics of neurite length per neuron after RPM as expressed in ratios of RPM vs. ground control exposed cultures. (D) Recovery dynamics of neurite network area per image after RPM as expressed in ratios of RPM vs. ground control exposed cultures. (E) Recovery dynamics of neurite metwork length per image after RPM as expressed in ratios of RPM vs. ground control exposed cultures. (F) Size of somas in neuron cultures previously exposed to RPM and having recovered for 24 hours in ground conditions and their respective controls. (G) Roundness of somas in neuron cultures previously exposed to RPM and having recovered for 24 hours in ground conditions and their respective controls. Paired two-tailed Student’s <i>t</i>-test and standard deviation bars are shown.*, <i>p</i><0.05 raw data 72 h RPM vs. raw data 72 h GC; 1, <i>p</i><0.05 24 h vs. 0 h of neuron area after 10 days of RPM; 2, <i>p</i><0.05 24 h vs 0 h of neurite area after 24 h of RPM; 3, <i>p</i><0.05 24 h vs. 0 h of neurite length after 1 h of RPM; 4, <i>p</i><0.05 72 h vs. 24 h of neuron area after 10 days of RPM; 5, <i>p</i><0.05 72 h vs 24 h of neurite area after 24 h of RPM; 6, <i>p</i><0.05 72 h vs. 24 h of neurite length after 1 h of RPM. GC = ground condition; RPM = Random Positioning Machine.</p

Co-exposure to chronic radiation and RPM results in a synergistically increased percentage of late-apoptotic neurons.

<p><b>(A)</b> Analysis of the percentage of fragmented nuclei revealed a significant increase in apoptosis after RPM and combined exposure, but not after chronic exposure to radiation alone. <b>(B)</b> An Annexin V/PI apoptosis assay unveiled an increase in the total number of apoptotic cells (AnnV⁺/PI⁺ + AnnV⁺/PI^-) in all conditions. However, when separately investigating early and late apoptosis, AnnV⁺/PI⁺ and AnnV⁺/PI^- labeled neurons were increased only after RPM and after combined exposure. Finally, only AnnV⁺/PI⁺ labeled neurons, representing late apoptotic neurons, were shown to be synergistically increased after combined exposure. <b>(C)</b> Nuclei, Annexin V, propidium iodide (PI), differential interference contrast (DIC) and merge representative images of neuron culture exposed to ionizing radiation and RPM. GC = Ground controls, RPM = random positioning machine, Gy = Gray. Values are represented as mean±SEM. N = 3, * p<0.05, ** p<0.01, *** p<0.001. Asterisks represent differences with non-exposed controls (GC).</p

Altered viability induced during and after simulated microgravity exposure.

<p>(A) Percentage of Ann V⁺-PI⁻ and Ann V⁺-PI⁺ cells in neuronal network induced after 1 hour, 24 hours or 10 days of simulated microgravity. (B) RPM/GC ratios of total Ann V⁺ percentages of neuron cultures exposed and not exposed to simulated microgravity. (C) Percentages of Ann V⁺-PI⁻ and Ann V⁺-PI⁺ cells in neuron cultures exposed to RPM and having recovered for 24 h in ground conditions. (D) Percentages of Ann V⁺-PI⁻ and Ann V⁺-PI⁺ cells in neuron cultures exposed to RPM and having recovered for 72 h in ground conditions. (E) RPM/GC ratios of total Ann V⁺ percentages of neuron cultures recovered for 24 and 72 hours after simulated microgravity exposure. * = <i>p</i><0.05 24 h compared to 72 h. (F) Neurons stained with Annexin V-FITC/PI/Hoechst (green/red/blue) and observed under fluorescence microscope. Ann V-FITC^−/PI^−/Hoechst⁺ are considered healthy cells, V-FITC⁺/PI^−/Hoechst⁺ are considered early apoptotic cells and Ann V-FITC⁺/PI⁺/Hoechst⁺ are considered late apoptotic or necrotic cells. Paired two-tailed Student’s <i>t</i>-test and standard deviation bars are shown. 1, <i>p</i><0.05 Ann V⁺-PI⁻ RPM compared to GC; 2, <i>p</i><0.05 Ann V⁺-PI⁺ RPM compared to GC; 3, <i>p</i><0.05 total Ann V⁺ RPM compared to GC. GC = ground conditions; RPM = Random Positioning Machine.</p

Gene expression and gene ontology enrichment analysis of neurons chronically exposed to RPM and irradiation.

<p><b>(A)</b> A microarray analysis revealed differentially expressed genes in mature neurons immediately after chronic exposure to simulated space conditions (1394 genes in co-exposed neurons, 557 genes in RPM exposed neurons) as compared to non-exposed control neurons. Of those, 390 common genes were detected. No differential genes could be detected in irradiated neurons as compared to control cells. <b>(B)</b> A gene ontology enrichment analysis showed an enrichment of the 390 overlapping genes in pathways related to e.g. synaptic transmission, axonogenesis or biogenesis. <b>(C)</b> A hierarchical clustering of all genes differentially expressed between co-exposed and non-exposed neurons (1394) showed a large gene cluster that was upregulated in co-exposed neurons (yellow square), and a smaller cluster of downregulated genes (green square). <b>(D)</b> A gene ontology enrichment analysis of the downregulated gene cluster showed the involvement of few processes, which were not linked to the observed histological phenotype, whereas a similar analysis of the upregulated gene cluster predicted the involvement of e.g. synaptic, apoptotic, cell redox or metabolic processes. <b>(E-G)</b> qPCR experiments for neurite guidance and synaptic transmission genes confirmed an upregulation of the repulsive gene <i>Slit2</i> in co-exposed neurons (E), as well as an upregulation in excitatory synapses in RPM and co-exposed neurons (<i>Slc17a6</i>) (F) and in inhibitory synapses in RPM (borderline significant) and co-exposed neurons (G). RPM = random positioning machine, IR = irradiation, GC = ground condition. Values are represented as mean±SEM. N = 3, # p = 0.05, * p<0.05, ** p<0.01. Asterisks represent differences with non-exposed controls (GC).</p

Effects of simulated microgravity on single neurons as well as on neuronal networks.

<p>(A) First line: neuronal networks cultured in ground control conditions (GC 1 h - 24 h - 10 days); second line: neuronal networks exposed to simulated microgravity (RPM 1 h - 24 h - 10 days). (B) Neuron area (soma+neurites) in neuronal network cultures exposed to RPM for 1 h, 24 h or 10 days and the respective controls. (C) Neurite area per neuron in neuronal network cultures exposed to RPM for 1 h, 24 h or 10 days and the respective controls. (D) Neurite length per neuron in neuronal network cultures exposed to RPM for 1 h, 24 h or 10 days and the respective controls. (E) Neurite area per image in cultures exposed to RPM for 1 h, 24 h or 10 days and the respective controls. (F) Neurite length per image in cultures exposed to RPM for 1 h, 24 h or 10 days and the respective controls. (G) Neuronal density per cm²; 10 day RPM vs. 1 h GC : <i>p = </i>0.055. (H) RPM vs. ground condition. (H) Rratios of neuron area, neurite area and neurite length show how neurons adapt to simulated microgravity throughout the exposure time. Paired two-tailed Student’s <i>t</i>-test and standard deviation bars are shown. 1, <i>p</i><0.05 RPM 1 h compared to GC 1 h; 2, <i>p</i><0.05 RPM 24 h compared to GC 24 h; 3, <i>p</i><0.05 RPM 10 days compared to GC 10 days. GC = ground condition; RPM = Random Positioning Machine.</p