A model to simulate effects of microgravity on skeletal muscle mass and function in humans has been developed. Unilateral lower limb unloading that allowed ankle, knee, and hip joint mobility was ...conducted in six healthy men by suspending one lower limb and having the subjects walk on crutches. They performed maximal unilateral concentric or eccentric quadriceps actions at different angular velocities before and after 4 wk of suspension and after 4 days and after 7 wk of uncontrolled recovery. Peak torque (PT) and angle-specific torque (AST) were measured. Muscle cross-sectional area (CSA) and radiological density (RD) of the thigh were assessed by means of computerized tomography. Concentric and eccentric PT and AST across speeds decreased (P less than 0.05) by 22 and 16%, respectively, in response to unloading. At 4 days of recovery PT (-11%) and AST (-7%) were still lower (P less than 0.05) than before. Muscle CSA and RD decreased (P less than 0.05) by 7 and 6%, respectively. After 7 wk of recovery PT, AST, CSA, and RD had returned to normal. The control limb showed no changes over the experimental period except for a 6% decrease (P less than 0.05) in RD. It is suggested that this human model of unloading could serve to simulate effects of microgravity on skeletal muscle mass and function because reductions in muscle mass and strength were of similar magnitude to those produced by bed rest.
Adipose- derived stem cells (ADSCs) are widely used for tissue engineering and regenerative medicine. The beneficial effects of ADSCs on wound healing have already been reported. Remodeling of ...extracellular matrix (ECM) is the most important physiological event during wound healing. ECM is sensitive to mechanical stresses and the expression of its components can be therefore influenced. The aim of this study was to investigate the effect of simulated microgravity on gene expression of some ECM and adhesion molecules in human ADSCs. After isolation and characterization of ADSCs, cells were exposed to simulated microgravity for 1, 3 and 7 days. Real-time PCR, fluorescence immunocytochemistry, and MTT assay were performed to evaluate the alterations of integrin subunit beta 1 (ITGB1), collagen type 3 (ColIII), matrix metalloproteinase-1 (MMP1), CD44, fibrillin (FBN1), vimentin (VIM) genes, and ColIII protein levels as well as cells viability. Microgravity simulation increased the expression of ITGB1, ColIII, MMP1, and CD44 and declined the expression of FBN1 and VIM genes. ColIII protein levels also increased. There were no significant changes in the viability of cells cultured in microgravity. Since the high expression of ECM components is known as one of the fibroblast markers, our data suggest that pretreatment of ADSCs by simulated microgravity may increase their differentiation capacity towards fibroblastic cells. Microgravity had not adversely affected the viability of ADSCs, and it is likely to be used alone or in combination with biochemical inducers for cell manipulation.
Cell transplantation therapy for Parkinson's disease (PD) has received much attention as a potential treatment protocol for this neurodegenerative condition. Although there have been promising ...successes with this approach, it remains problematic, especially regarding the inability to provide immediate trophic support to the newly grafted cells and the inability to prevent acute and/or long-term graft rejection by the host. To address these issues of cell graftability, we have created a novel tissue construct from isolated rat Sertoli cells (SC) and the NTerra-2 immortalized human neuron precursor cell line (NT2) utilizing NASA-developed simulated microgravity technology. The two cell types were cocultured at a 1:4 (SC/NT2) ratio in the High Aspect Rotating Vessel (HARV) biochamber for 3 days, after which a disc-shaped aggregate (1–4 mm diameter) was formed. Sertoli neuron aggregated cells (SNAC) were collected by gravity sedimentation and processed either for light and electron microscopy or for fluorescent immunocytochemistry. Intra-SNAC clusters of SC and NT2 cells were identified by anti-human mitochondrial protein (huMT—specific for NT2 cells) and cholera toxin subunit B (CTb—specific for SC). There was little evidence of cell death throughout the aggregate and the absence of central necrosis, as might be expected in such a large aggregate in vitro. Ultrastructurally, SC did not express junctional modifications with NT2 cells nor with adjacent SC as is typical of SC in vivo and, in some protocols, in vitro. NT2 cells, however, showed distinct intercellular junction-like densities with adjacent NT2 cells, often defining canaliculi-like channels between the microvillus borders of the cells. The results show that the use of simulated microgravity coculture provides a culture environment suitable for the formation of a unique and viable Sertoli-NT2 (i.e., SNAC) tissue construct displaying intra-aggregate cellular organization. The structural integration of SC with NT2 cells provides a novel transplantable tissue source, which can be tested to determine if SC will suppress rejection of the grafted NT2 cells and provide for their short- and long-term trophic support in situ in the treatment of experimental PD.
Microgravity induces stress, and the brain is one of the targets that is more influenced in this environment. Alteration in transcription factors can have enormous effect because of discrepancy in ...the signaling process of the cells. Activator protein-1 (AP-1) is a stress-regulated transcription factor and is involved in the regulation of physiological and pathological stimuli that include cytokines, growth factors, and stress signals. In the present study, an attempt has been made to observe the effect of a microgravity environment on the activation of AP-1 in the mouse brain. Our results show that AP-1 transcription factor is activated in simulated microgravity conditions in different regions of the brain. The activation of the AP-1 is dependent upon the increased kinase activity of c-Jun NH-terminal2 kinase-1. These results suggest that microgravity stress in the brain can elicit AP-1 activity.
Both microgravity conditions in space and simulated microgravity using a 3-dimensional clinostat resulted in: (1) automorphosis of etiolated pea seedlings, (2) epicotyls bending ca. 45° from the ...vertical line to the direction away from cotyledons, (3) inhibition of hook formation and (4) alternation of growth direction of roots. These facts indicate that the growth and development of etiolated pea seedlings on earth is under the influence of gravistimulation. Lanthanum and gadolinium ions, blockers of stretch-activated mechanosensitive ion channels, induced automorphosis-like epicotyl bending. Cantharidin, an inhibitor of protein phosphatase, also phenocopied automorphosis-like growth. On the other hand, cytochalasin B, cytochalasin D and brefeldin A did not induce automorphological epicotyl bending and inhibition of hook formation, although these compounds strikingly inhibited elongation of etiolated pea epicotyls. These results strongly suggest that stretch-activated mechanosensitive ion channels are involved in the perception of signals of gravistimuli in plants, and they are transduced by protein phosphorylation and dephosphorylation cascades by changing levels of calcium ions. Possible mechanisms to induce automorphosis-like growth in relation to gravity signals in etiolated pea seedlings are discussed.
Although many physiological changes after space flight have been reported, it is not clear how microgravity influences our bodies. The focus of the present study was to clarify the changes in ...G-protein-coupled receptor-mediated intracellular signaling, especially Gs-adenylyl cyclase (AC)-adenosine 3', 5'-cyclic monophosphate (cyclic AMP) pathway, under simulated microgravity. Human astrocytoma 1321N1 cells were cultivated under vector-averaged microgravity conditions generated by clinostat rotation (20 rpm) for 24 hr. Isoproterenol, a β-adrenergic agonist and forskolin, a direct AC stimulant, increased intracellular cyclic AMP level in concentration dependent manners, however, both of which response were decreased in cells cultivated in clinostat rotation. While the level of Gαs or intracellular ATP, a substrate for AC, was not changed, the AC activity was significantly low in the membranes of clinostat-rotated cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that AC type 3 (AC3), AC6, and AC9 and to a lesser extent AC7 and AC8 were expressed in 1321N1 cells. Among them, the expression of AC6 mRNA was significantly decreased by clinostat rotation. These results indicate that intracellular cyclic AMP production by agonists may be decreased via a reduction in AC6 expression under simulated microgravity conditions.
Troponin C (TnC) plays a key role in the regulation of muscle contraction, thereby modulating the Ca(2+)-activation characteristics of skinned muscle fibers. This study was performed to assess the ...effects of a 15-day hindlimb unloading (HU) period on TnC expression and its functional behavior in the slow postural muscles of the rat. We investigated the TnC isoform expression in whole soleus muscles and in single fibers. The latter were also checked for their Ca(2+) activation characteristics and sensitivity to bepridil, a Ca(2+) sensitizer molecule. This drug has been described as exerting a differential effect on slow and fast fibers, depending on the TnC isoform. With regard to TnC expression, three populations were found in control muscle fibers: slow, hybrid slow, and hybrid fast fibers, with the TnC fast being always coexpressed with TnC slow. In the whole muscle, TnC fast expression increased after HU because of the increase in the proportion of hybrid fast fibers. The HU hybrid fast fibers had properties similar to those of control hybrid fast fibers. The fibers that remained slow after HU exhibited similar bepridil and Sr(2+) properties as control slow fibers. Therefore, in these fibers, the changes could not be related to the TnC molecule.
This study investigated changes in skeletal muscle cross-sectional area (CSA) evoked by fluid shifts that accompany short-term 6 degrees head-down tilt (HDT) or horizontal bed rest, the time course ...of the resolution of these changes after resumption of upright posture, and the effect of altered muscle CSA, in the absence of increased contractile activity, on proton transverse relaxation time (T2). Average muscle (CSA and T2 were determined by standard spin-echo magnetic resonance imaging. Analyses were performed on contiguous transaxial images of the neck and calf. After a day of normal activity, 24 h of HDT increased neck muscle CSA 19 +/- 4(SE)% (P < 0.05) while calf muscle CSA decreased 14 +/- 3% (P < 0.05). The horizontal posture (12 h) induced about one-half of these responses: an 11 +/- 2% (P < 0.05) in the neck muscle CSA and an 8 +/- 2% decrease (P < 0.05) in the calf. Within 2 h after resumption of upright posture, neck and calf muscle CSA returned to within 0.5% (P > 0.05) of the values assessed after a day of normal activity, with most of the change occurring within the first 30 min. No further change in muscle CSA was observed through 6 h of upright posture. Despite these large alterations in muscle CSA, T2 was not altered by more than 1.1 +/- 0.6% (P > 0.05) and did not relate to muscle size. These results suggest that postural manipulations and subsequent fluid shifts modeling micro-gravity elicit marked changes in muscle size. Because these responses were not associated with alterations in muscle T2, it does not appear that simple movement of water into muscle can explain the contrast shift observed after exercise.
We are studying microenvironmental cues which contribute to neuroendocrine organ assembly and tissue-specific differentiation. As our in vitro model, we cultured rat adrenal medullary PC12 ...pheochromocytoma cells in a novel cell culture system, the NASA rotating wall vessel (RWV) bioreactors. This "simulated microgravity" environment in RWV bioreactors, characterized by randomizing gravitational vectors and minimizing shear stress, has been shown to favor macroscopic tissue assembly and to induce tissue-specific differentiation. We hypothesized that the unique culture conditions in the RWV bioreactors might enhance the in vitro formation of neuroendocrine organoids. To test our hypothesis, we evaluated the expression of several markers of neuroendocrine differentiation in cultures of PC 12 cells maintained for up to 20 d in the slow turning lateral vessel (STLV) type RWV. PC 12 cell differentiation was assessed by morphological, immunological, biochemical and molecular techniques. PC 12 cells, cultured under "simulated microgravity" conditions, formed macroscopic, tissue-like organoids several millimeters in diameter. Concomitantly, the expression of phenylethanolamine-N-methyl transferase (PNMT), but not of other catecholamine synthesizing enzymes, was enhanced. Increased PNMT expression, as verified on both the gene and protein level, was accompanied by an increase in the specific activity of the enzyme. Furthermore, after 20 d in culture in the STLV, we observed altered patterns of protein tyrosine phosphorylation and prolonged activation of c-fos, a member of the AP-1 nuclear transcription factor complex. We conclude that culture conditions in the RWV appear to selectively activate signal transduction pathways leading to enhanced neuroendocrine differentiation of PC 12 cells.
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