The Italian Space Agency, in line with its scientific strategies and the National Utilization Plan for the International Space Station (ISS), contracted Thales Alenia Space Italia to design and build a spaceflight payload for rodent research on ISS: the Mice Drawer System (MDS). The payload, to be integrated inside the Space Shuttle middeck during transportation and inside the Express Rack in the ISS during experiment execution, was designed to function autonomously for more than 3 months and to involve crew only for maintenance activities. In its first mission, three wild type (Wt) and three transgenic male mice over-expressing pleiotrophin under the control of a bone-specific promoter (PTN-Tg) were housed in the MDS. At the time of launch, animals were 2-months old. MDS reached the ISS on board of Shuttle Discovery Flight 17A/STS-128 on August 28(th), 2009. MDS returned to Earth on November 27(th), 2009 with Shuttle Atlantis Flight ULF3/STS-129 after 91 days, performing the longest permanence of mice in space. Unfortunately, during the MDS mission, one PTN-Tg and two Wt mice died due to health status or payload-related reasons. The remaining mice showed a normal behavior throughout the experiment and appeared in excellent health conditions at landing. During the experiment, the mice health conditions and their water and food consumption were daily checked. Upon landing mice were sacrificed, blood parameters measured and tissues dissected for subsequent analysis. To obtain as much information as possible on microgravity-induced tissue modifications, we organized a Tissue Sharing Program: 20 research groups from 6 countries participated. In order to distinguish between possible effects of the MDS housing conditions and effects due to the near-zero gravity environment, a ground replica of the flight experiment was performed at the University of Genova. Control tissues were collected also from mice maintained on Earth in standard vivarium cages.
Research Containing: Mice
Nitric oxide affects preimplantation embryonic development in a rotating wall vessel bioreactor simulating microgravity
Microgravity was simulated with a rotating wall vessel bioreactor (RWVB) in order to study its effect on pre-implantation embryonic development in mice. Three experimental groups were used: stationary control, rotational control and clinostat rotation. Three experiments were performed as follows. The first experiment showed that compared with the other two (control) groups, embryonic development was significantly retarded after 72 h in the clinostat rotation group. The second experiment showed that more nitric oxide (NO) was produced in the culture medium in the clinostat rotation group after 72 h (P < 0.05), and the nitric oxide synthase (NOS) activity in this group was significantly higher than in the controls (P < 0.01). In the third experiment, we studied apoptosis in the pre-implantation mouse embryos after 72 h in culture and found that Annexin-V staining was negative in the normal (stationary and rotational control) embryos, but the developmentally retarded (clinostat rotation) embryos showed a strong green fluorescence. These results indicate that microgravity induced developmental retardation and cell apoptosis in the mouse embryos. We presume that these effects are related to the higher concentration of NO in the embryos under microgravity, which have cause cytotoxic consequences. (c) 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.
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Microgravity induces pelvic bone loss through osteoclastic activity, osteocytic osteolysis, and osteoblastic cell cycle inhibition by CDKN1a/p21
Bone is a dynamically remodeled tissue that requires gravity-mediated mechanical stimulation for maintenance of mineral content and structure. Homeostasis in bone occurs through a balance in the activities and signaling of osteoclasts, osteoblasts, and osteocytes, as well as proliferation and differentiation of their stem cell progenitors. Microgravity and unloading are known to cause osteoclast-mediated bone resorption; however, we hypothesize that osteocytic osteolysis, and cell cycle arrest during osteogenesis may also contribute to bone loss in space. To test this possibility, we exposed 16-week-old female C57BL/6J mice (n = 8) to microgravity for 15-days on the STS-131 space shuttle mission. Analysis of the pelvis by microCT shows decreases in bone volume fraction (BV/TV) of 6.29%, and bone thickness of 11.91%. TRAP-positive osteoclast-covered trabecular bone surfaces also increased in microgravity by 170% (p = 0.004), indicating osteoclastic bone degeneration. High-resolution X-ray nanoCT studies revealed signs of lacunar osteolysis, including increases in cross-sectional area (+17%, p = 0.022), perimeter (+14%, p = 0.008), and canalicular diameter (+6%, p = 0.037). Expression of matrix metalloproteinases (MMP) 1, 3, and 10 in bone, as measured by RT-qPCR, was also up-regulated in microgravity (+12.94, +2.98 and +16.85 fold respectively, p<0.01), with MMP10 localized to osteocytes, and consistent with induction of osteocytic osteolysis. Furthermore, expression of CDKN1a/p21 in bone increased 3.31 fold (p<0.01), and was localized to osteoblasts, possibly inhibiting the cell cycle during tissue regeneration as well as conferring apoptosis resistance to these cells. Finally the apoptosis inducer Trp53 was down-regulated by -1.54 fold (p<0.01), possibly associated with the quiescent survival-promoting function of CDKN1a/p21. In conclusion, our findings identify the pelvic and femoral region of the mouse skeleton as an active site of rapid bone loss in microgravity, and indicate that this loss is not limited to osteoclastic degradation. Therefore, this study offers new evidence for microgravity-induced osteocytic osteolysis, and CDKN1a/p21-mediated osteogenic cell cycle arrest.
Tissue-engineered conduit using urine-derived stem cells seeded bacterial cellulose polymer in urinary reconstruction and diversion
The objective of this study was to generate bacterial cellulose (BC) scaffolds seeded with human urine-derived stem cells (USC) to form a tissue-engineered conduit for use in urinary diversion. Microporous BC scaffolds were synthesized and USC were induced to differentiate into urothelial and smooth muscle cells (SMC). Induced USC (10(6) cells/cm(2)) were seeded onto BC under static and 3D dynamic (10 or 40 RPM) conditions and cultured for 2 weeks. The urothelial cells and SMC derived from USC formed multilayers on the BC scaffold surface, and some cells infiltrated into the scaffold. The urothelium derived from USC differentiation expressed urothelial markers (uroplakin la and AE1/AE3) and the SMC expressed SMC markers (a-smooth muscle actin and desmin). In addition, USC/BC scaffold constructs were implanted into athymic mice, and the cells were tracked using immunohistochemical staining for human nuclear antigen. In vivo, the cells appeared to differentiate and express urothelial and SMC markers. In conclusion, porous BC scaffolds allow 3 dimensional growth of USC, leading to formation of a multilayered urothelium and cell matrix infiltration. Thus, cell-seeded BC scaffolds hold promise for use in tissue-engineered urinary conduits for urinary reconstruction. (C) 2010 Elsevier Ltd. All rights reserved.
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Prolonged exposure to microgravity during spaceflight is thought to adversely affect the human spine because of reports that disc herniation risk is increased post-spaceflight. The increased herniation risk is highest during the first post-spaceflight year, and gradually subsides thereafter. Consequently, we hypothesized that the biomechanical properties of the intervertebral disc (IVD) deteriorate during spaceflight but then recover after acclimation to normal gravity. To test this hypothesis, we compared the compressive creep properties of caudal IVDs of murine subjects that had returned from a 13-day Shuttle mission (STS-133) to those of ground-based control mice. Spaceflight (n=6) and control (n=10) groups consisted of 13-week-old, BALB/c mice (11 weeks at launch). Mice were sacrificed +1 day, +5 days, or +7 days after the landing of STS-133. Disc height was measured in situ, and compressive creep rate was fit to a fluid transport model to determine disc biomechanical properties. Compared to controls, spaceflight mice had 12.6% lower disc height and 23.1% lower straindependence on swelling pressure. Biomechanical properties did not recover significantly over the 7-day post-flight period. Biomechanical properties of the murine caudal IVD were diminished by spaceflight, consistent with observations that prolonged exposure to microgravity increases disc herniation risk. These properties did not recover after short-term reacclimation to 1g loading.
Effect of microgravity on the biomechanical properties of lumbar and caudal intervertebral discs in mice
Prolonged exposure to microgravity has shown to have deleterious effects on the human spine, indicated by low back pain during spaceflight and increased incidence of post-spaceflight herniated nucleus pulposus. We examined the effect of microgravity on biomechanical properties of lumbar and caudal discs from mice having been on 15-day shuttle mission STS-131. Sixteen C57BL/C mice (spaceflight group, n=8; ground-based control group, n=8) were sacrificed immediately after spaceflight. Physiological disc height (PDH) was measured in situ, and compressive creep tests were performed to parameterize biomechanical properties into endplate permeability (k), nuclear swelling pressure strain dependence (D), and annular viscoelasticity (G). For caudal discs, the spaceflight group exhibited 32% lower PDH, 70% lower D and crept more compared to the control mice (p=0.03). For lumbar discs, neither PDH nor D was significantly different between murine groups. Initial modulus, osmotic pressure, k and G for lumbar and caudal discs did not appear influenced by microgravity (p>0.05). Decreases in both PDH and D suggest prolonged microgravity effectively diminished biomechanical properties of caudal discs. By contrast, differences were not noted for lumbar discs. This potentially deleterious interaction between prolonged weightlessness and differential ranges of motion along the spine may underlie the increased cervical versus lumbar disc herniation rates observed among astronauts.
Spaceflight conditions have a significant impact on a number of physiological functions due to psychological stress, radiation, and reduced gravity. To explore the effect of the flight environment on immunity, C57BL/6NTac mice were flown on a 13-day space shuttle mission (STS-118). In response to flight, animals had a reduction in liver, spleen, and thymus masses compared with ground (GRD) controls (P < 0.005). Splenic lymphocyte, monocyte/macrophage, and granulocyte counts were significantly reduced in the flight (FLT) mice (P < 0.05). Although spontaneous blastogenesis of splenocytes in FLT mice was increased, response to lipopolysaccharide (LPS), a B-cell mitogen derived from Escherichia coli, was decreased compared with GRD mice (P < 0.05). Secretion of IL-6 and IL-10, but not TNF-α, by LPS-stimulated splenocytes was increased in FLT mice (P < 0.05). Finally, many of the genes responsible for scavenging reactive oxygen species were upregulated after flight. These data indicate that exposure to the spaceflight environment can increase anti-inflammatory mechanisms and change the ex vivo response to LPS, a bacterial product associated with septic shock and a prominent Th1 response.
Spaceflight results in a number of adaptations to skeletal muscle, including atrophy and shifts toward faster muscle fiber types. To identify changes in gene expression that may underlie these adaptations, we used both microarray expression analysis and real-time polymerase chain reaction to quantify shifts in mRNA levels in the gastrocnemius from mice flown on the 11-day, 19-h STS-108 shuttle flight and from normal gravity controls. Spaceflight data also were compared with the ground-based unloading model of hindlimb suspension, with one group of pure suspension and one of suspension followed by 3.5 h of reloading to mimic the time between landing and euthanization of the spaceflight mice. Analysis of microarray data revealed that 272 mRNAs were significantly altered by spaceflight, the majority of which displayed similar responses to hindlimb suspension, whereas reloading tended to counteract these responses. Several mRNAs altered by spaceflight were associated with muscle growth, including the phosphatidylinositol 3-kinase regulatory subunit p85alpha, insulin response substrate-1, the forkhead box O1 transcription factor, and MAFbx/atrogin1. Moreover, myostatin mRNA expression tended to increase, whereas mRNA levels of the myostatin inhibitor FSTL3 tended to decrease, in response to spaceflight. In addition, mRNA levels of the slow oxidative fiber-associated transcriptional coactivator peroxisome proliferator-associated receptor (PPAR)-gamma coactivator-1alpha and the transcription factor PPAR-alpha were significantly decreased in spaceflight gastrocnemius. Finally, spaceflight resulted in a significant decrease in levels of the microRNA miR-206. Together these data demonstrate that spaceflight induces significant changes in mRNA expression of genes associated with muscle growth and fiber type.
Reinterpretation of mouse thyroid changes under space conditions: the contribution of confinement to damage
During space missions, astronauts work in a state of separation from their daily social environment and in physical confinement. It has been shown that confinement influences mood and brain cortical activity, but no data has been obtained with regard to its effect on the thyroid gland, the structure and function of which change during spaceflights. Here, we report the results of a study on the effects of confinement on mouse thyroid, which was implemented with the Mice Drawer System Facility maintained on the ground, a system used for spaceflight experiments. The results show that confinement changes the microscopic structure of the thyroid gland and that it exhibits symptoms similar to those that result from physiological and/or pathological hyperfunction. What is left unchanged, however, is the sphingomyelinase-thyrotropin receptor relationship, which is important for thyrotropin response with a consequential production of hormones that act on the metabolism of almost all tissues and reduces the production of calcitonin, a hormone involved in bone metabolism. During space missions, the overexpression of pleiotrophin, a widespread cytokine up-regulated after tissue injury that acts on bone remodeling, attenuates changes to the thyroid that are spaceflight-dependent; therefore we studied the thyroids of pleiotrophin-transgenic mice in the Mice Drawer System Facility. In confinement, pleiotrophin overexpression does not protect from the loss of calcitonin. The contribution of confinement to thyroid damage during spaceflights is discussed.
After long-term exposure to real microgravity thyroid gland in vivo undergoes specific changes, follicles are made up of larger thyrocytes that produce more cAMP and express more thyrotropin-receptor, caveolin-1, and sphingomyelinase and sphingomyelin-synthase; parafollicular spaces lose C cells with consequent reduction of calcitonin production. Here we studied four immunohistochemical tumor markers (HBME-1, MIB-1, CK19, and Galectin-3) in thyroid of mice housed in the Mouse Drawer System and maintained for 90 days in the International Space Station. Results showed that MIB-1 proliferative index and CK19 are negative whereas HBME-1 and Galectin-3 are overexpressed. The positivity of Galectin-3 deserves attention not only for its expression but also and especially for its localization. Our results highlighted that, in microgravity conditions, Galectin-3 leaves thyrocytes and diffuses in colloid. It is possible that the gravity force contributes to the maintenance of the distribution of the molecules in both basal membrane side and apical membrane side and that the microgravity facilitates slippage of Galectin-3 in colloid probably due to membrane remodelling-microgravity induced.