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The average body weight of rats in all groups was comparable before the experiments and increased in all groups at sacrifice (Table 1). No significant difference was observed between the I, U, and C groups but the body weight of H and H+U+I group was significantly less than the C group (P < 0.01). However, no marked difference was observed between the two groups. In general, rats treated with simulated microgravity or combined-three factors exhibit significant impact on the body weight.
Group Weight before Experiments (g) Weight before Sacrifice (g) BL 194.40 ± 3.01 ₋ C 193.29 ± 4.07 230.57 ± 4.74 H 188.00 ± 3.97 206.29 ± 5.32** U 199.29 ± 4.18 234.80 ± 8.34 I 193.94 ± 3.90 228.29 ± 7.11 H+U+I 190.60 ± 2.90 200.89 ± 2.94** Note. Rats in all groups were weighed on day 0 of the experiment and day 28 before sacrifice. BL: baseline group; C: control group; H: hindlimb suspension group; U: ultradian rhythms group; I: ionizing radiation group; H+U+I: hindlimb suspension + ultradian rhythms + ionizing radiation group. Values represent the means ± SEM. **P < 0.01, compared to the control group. Table 1. Changes in Body Weight of Different Groups
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The biomechanical properties of the femurs from hindlimbs were tested by a tensile testing machine in a 3-point bending way. The elastic modulus (Figure 1A) of the H, U, and H+U+I group was significantly lower than that of the C group (P < 0.01). The elastic modulus of the U group was also significantly lower than that of the C group (P < 0.05). No significant difference was observed between the I and C group. The ultimate force (Figure 1B) of the H group and the H+U+I group was significantly lower than that of the C group (P < 0.01). We observed no significant differences between the I or U group when compared to the C group. These results indicate that the biomechanical properties of the femurs from rats treated with simulated microgravity or the combined factors are effectively decreased.
Figure 1. Comparison of biomechanical testing in femurs between the control group (C) and hindlimb suspension group (H), ultradian rhythms group (U), ionizing radiation group (Ⅰ), hindlimb suspension + ultradian rhythms + ionizing radiation group (H+U+I). A. The elastic modulus; B. The ultimate force. Values represent the means ± SEM. *P < 0.05, **P < 0.01, compared to the control group.
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The BMD of the femurs were analyzed by the DEXA (Figure 2). We found that BMD of H group and H+U+I group was much lower than that of the C group (P < 0.01) while the I group or the U group showed no significant differences when compared to the C group. These results suggest that the bone mineral density is effectively decreased in animals treated with simulated microgravity or combined-three factors.
Figure 2. Changes of femur BMD measured by dual-energy X-ray absorptiometer (DEXA). C: control group. H: hindlimb suspension group. U: ultradian rhythms group. I: ionizing radiation group. H+U+I: hindlimb suspension + ultradian rhythms + ionizing radiation group. Values represent the means ± SEM. **P < 0.01, compared to the control group.
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Histological analysis of the cancellous bone proximal of the tibial metaphysis revealed that the percentage of trabecular area (Figure 3A) of the H group and H+U+I group was significantly decreased when compared to the C group (P < 0.01), and no significant difference was observed when compared to each others. Similarly, the trabecular thickness (Figure 3B) of the H group or the H+U+I group was significantly decreased in comparison to the C group (P < 0.01), and the H group and the H+U+I group did not show significant differences. However, the trabecular separation (Figure 3C) was significantly higher in the H (P < 0.05) and the H+U+I group (P < 0.01) when compared to the C group. No significant difference was observed between the H group and the H+U+I group. The trabecular number (Figure 3D) in all experimental groups showed no significant differences in comparison to the C group. The histological results (Figure 3E) revealed that simulated microgravity or combined factors significantly deteriorated the tibial trabecular parameters.
Figure 3. Histological analysis of cancellous bone proximal of the tibial metaphysis. (A) Percentage of trabecular area; (B) Trabecular thickness; (C) Trabecular separation; (D) Trabecular number; (E) The representative pictures of the histological sections stained by Van Gieson's staining. Values represent the means ± SEM. *P < 0.05, **P < 0.01, compared to the control group.
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Micro-CT analysis of the femurs from hindlimbs indicated that the cancellous BMD (Figure 4A) was significantly decreased in the H group, I group, and H+U+I group compared to the control (P < 0.01, P < 0.05 and P < 0.01, respectively), while no difference was found in the cortical BMD (Figure 4B) between the control group and the treatment groups. Similarly to cancellous BMD results, trabecular volume fraction (BV/TV, Figure 4C) and trabecular thickness (Tb.Th, Figure 4D) in the H group and the H+U+I group (P < 0.01) were significantly lower than that in C group. However, no significant difference was found in the trabecular number (Tb.No, Figure 4E) among all experimental groups. In addition, we found that the ionizing radiation, hindlimb suspension, and combined-three factors increased the trabecular separation (Tb.Sp, Figure 4F) when compared to the control. The three-dimensional (3D) reconstruction of the trabecular bone (Figure 5) showed a similar tendency as the trabecular parameters, confirmingthat simulated microgravity and combined factors have deteriorating effects on the trabecula of the femurs. Taken together, the effects of simulated microgravity and combined factors on bone loss are significantly observed in the trabecular parameters.
Figure 4. Trabecular parameters of the distal femurs measured by micro-CT. (A) Cancellous bone mineral density (mg/cc); (B) Cortical bone mineral density (mg/cc); (C) Trabecular volume fraction (BV/TV); (D)Trabecular thickness (Tb.Th); (E) Trabecular number (Tb.No); (F) Trabecular separation (Tb.Sp). Values represent the means ± SEM. *P < 0.05, **P < 0.01, compared to the control group.
Figure 5. The three-dimensional trabecular architecture of the interest region (located under the epiphysis plate 2.88 mm of the femur). C: control group. H: hindlimb suspension group. U: ultradian rhythms group. I: ionizing radiation group. H+U+I: hindlimb suspension + ultradian rhythms + ionizing radiation group.
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Next, we measured the levels of bone turnover markers in the serum by ELISA. As shown in Figure 6, the concentration of bALP (Figure 6A), an early bone formation marker, was significantly decreased in the H group, U group and I group compared to the C group (P < 0.01). There was no significant difference between the H+U+I and the C group. The concentration of OCN (Figure 6B), a late marker of bone formation, was significantly reduced in the H group (P < 0.05) and the H+U+I group (P < 0.01) when compared to the C group, and the concentration of OCN in the H+U+I group was the lowest among all experimental groups. On the other hand, the concentration of TRAP-5b (Figure 6C), a bone resorption marker, was slightly increased in the H group, U group, and H+U+I group, but no significant difference was observed among the experimental groups. Taken together, simulated microgravity and combined factors induce bone loss mainly through the suppression of bone formation.