Figure 1.
Neuronal network growth under ground conditions.
Growth curve of the neuronal area (upper panel) within a period of 23 days with representative images at selective time points (lower panel). Values are expressed as the average of single neuron areas and bars represent standard deviation. Paired t-test was performed to determine differences between two close experimental points and obtained results showed significant differences between 1 day vs. 5 days (p = 0.046) and 5 days vs. 10 days (p = 0.041) but not between the following time points.
Figure 2.
Effects of simulated microgravity on single neurons as well as on neuronal networks.
(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 : p = 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 t-test and standard deviation bars are shown. 1, p<0.05 RPM 1 h compared to GC 1 h; 2, p<0.05 RPM 24 h compared to GC 24 h; 3, p<0.05 RPM 10 days compared to GC 10 days. GC = ground condition; RPM = Random Positioning Machine.
Figure 3.
Distribution of β-tubulin isotype 3 (β-tub 3) under simulated microgravity.
(A) Distribution of β-tub 3 between somas and neurites in neurons exposed for 1 h to RPM (neurite p = 0.029; soma p = 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 (p = 0.0012) and RPM 1 h vs. rpm 24 h vs. RPM 10 days (p<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 t-test and standard deviation bars are shown. GC = ground condition; RPM = Random Positioning Machine.
Figure 4.
Soma characteristics in neurons exposed for 1 hour, 24 hours and 10 days to the RPM compared to their respective controls.
(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 t-test and standard deviation bars are shown. 1, p<0.05 RPM 1 h compared to GC 1 h; 2, p<0.05 RPM 24 h compared to GC 24 h; 3, p<0.05 RPM 10 days compared to GC 10 days. GC = ground condition; RPM = Random Positioning Machine.
Figure 5.
Recovery in re-established ground conditions of neuronal networks and neurons previously exposed to simulated microgravity.
(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 t-test and standard deviation bars are shown.*, p<0.05 raw data 72 h RPM vs. raw data 72 h GC; 1, p<0.05 24 h vs. 0 h of neuron area after 10 days of RPM; 2, p<0.05 24 h vs 0 h of neurite area after 24 h of RPM; 3, p<0.05 24 h vs. 0 h of neurite length after 1 h of RPM; 4, p<0.05 72 h vs. 24 h of neuron area after 10 days of RPM; 5, p<0.05 72 h vs 24 h of neurite area after 24 h of RPM; 6, p<0.05 72 h vs. 24 h of neurite length after 1 h of RPM. GC = ground condition; RPM = Random Positioning Machine.
Figure 6.
Altered viability induced during and after simulated microgravity exposure.
(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. * = p<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 t-test and standard deviation bars are shown. 1, p<0.05 Ann V+-PI− RPM compared to GC; 2, p<0.05 Ann V+-PI+ RPM compared to GC; 3, p<0.05 total Ann V+ RPM compared to GC. GC = ground conditions; RPM = Random Positioning Machine.