The random positioning machine (RPM) is a method used to generate a simulated-microgravity environment at approximately 0 g. Using an RPM, we analyzed the global gene expression of A8 cells derived ...from the liver of adult Xenopus laevis. A range of genes on a Xenopus 44K-scale microarray were up- or downregulated two-fold or more: 43 genes (up, 36 genes; down, 7 genes) on culture day 5 in RPM, 74 genes (up, 48 genes; down, 26 genes) on day 8, 105 genes (up, 71 genes; down, 34 genes) on day 10, and 132 genes (up, 98 genes; down, 34 genes) on day 15. Five genes were upregulated two-fold or more throughout culturing in RPM, while only one gene was downregulated over the entire time. We then compared the expression patterns of the RPM-dependent genes in the A8 cells with those in A6 cells established from the kidney of adult Xenopus laevis. Six upregulated genes and three downregulated genes showed the same expression patterns throughout the culturing of A6 and A8 cells in RPM. Such globally responsive genes may play a common role in the cell response to simulated microgravity. We were particularly interested in the downregulation of SPARC in both cell types in RPM, which supported previous observations from simulated-microgravity experiments on earth or microgravity in space. We conclude that SPARC is plays a key role in the response of a cell to microgravity.
Paramecium is known to proliferate faster under microgravity conditions, and slower under hypergravity. Experiments using axenic culture medium have demonstrated that hypergravity affected directly ...on the proliferation of
Paramecium itself. In order to assess the mechanisms underlying the physiological effects of gravity on cell proliferation,
Paramecium tetraurelia was grown under clinorotation (2.5
rpm) and the time course of the proliferation was investigated in detail on the basis of the logistic analysis. On the basis of the mechanical properties of
Paramecium, this slow rate of the rotation appears to be enough to simulate microgravity in terms of the randomization of the cell orientation with respect to gravity.
P. tetraurelia was cultivated in a closed chamber in which cells were confined without air bubbles, reducing the shear forces and turbulences under clinorotation. The chamber is made of quartz and silicone rubber film; the former is for the optically-flat walls for the measurement of cell density by means of a non-invasive laser optical-slice method, and the latter for gas exchange. Because of the small dimension for culture space,
Paramecium does not accumulate at the top of the chamber in spite of its known negative gravitactic behavior. We measured the cell density at regular time intervals without breaking the configuration of the chamber, and analyzed the proliferation parameters by fitting the data to a logistic equation. As a result,
P. tetraurelia showed reduced proliferation under slow clinorotation. The saturation of the cell density as well as the maximum proliferation rate decreased, although we found no significant changes on the half maximal time for proliferation. We also found that the mean swimming velocity decreased under slow clinorotation. These results were not consistent with those under microgravity and fast rotating clinostat. This may suggest that randomization of the cell orientation performed by slow rotating clinostat has not the same effect on
Paramecium as that under microgravity that may affect the proliferation as the result of the reduced cost of propulsion.
This study tested the hypothesis that head-down tilt bed rest (HDBR) reduces adrenergic and nonadrenergic cutaneous vasoconstrictor responsiveness. Additionally, an exercise countermeasure group was ...included to identify whether exercise during bed rest might counteract any vasoconstrictor deficits that arose during HDBR. Twenty-two subjects underwent 14 days of strict 6 degrees HDBR. Eight of these 22 subjects did not exercise during HDBR, while 14 of these subjects exercised on a supine cycle ergometer for 90 min a day at 75% of pre-bed rest heart rate maximum. To assess alpha-adrenergic vasoconstrictor responsiveness, intradermal microdialysis was used to locally administer norepinephrine (NE), while forearm skin blood flow (SkBF; laser-Doppler flowmetry) was monitored over microdialysis membranes. Nonlinear regression modeling was used to identify the effective drug concentration that caused 50% of the cutaneous vasoconstrictor response (EC(50)) and minimum values from the SkBF-NE dose-response curves. In addition, the effects of HDBR on nonadrenergic cutaneous vasoconstriction were assessed via the venoarteriolar response of the forearm and leg. HDBR did not alter EC(50) or the magnitude of cutaneous vasoconstriction to exogenous NE administration regardless of whether the subjects exercised during HDBR. Moreover, HDBR did not alter the forearm venoarteriolar response in either the control or exercise groups during HDBR. However, HDBR significantly reduced the magnitude of cutaneous vasoconstriction due to the venoarteriolar response in the leg, and this response was similarly reduced in the exercise group. These data suggest that HDBR does not alter cutaneous vasoconstrictor responses to exogenous NE administration, whereas cutaneous vasoconstriction of the leg due to the venoarteriolar response is reduced after HDBR. It remains unclear whether attenuated venoarteriolar responses in the lower limbs contribute to reduced orthostatic tolerance after bed rest and spaceflight.
Oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). A baseline in which cellular changes in oxygen ...sensitive molecular processes occur during micro-g conditions would be important to pursue this question. The objective of this research is to analyze oxidation-sensitive gene expression in a model cell line rat pheochromocytoma (PC12) under simulated micro-g conditions. The PC12 cell line is well characterized in its response to oxygen, and is widely recognized as a sensitive model for studying the responses of oxygen-sensitive molecular and cellular processes. This study uses the rotating wall vessel bioreactor (RWV) designed at NASA to simulate micro-g. Gene expression in PC12 cells in response to micro-g was analyzed by DNA microarray technology. The microarray analysis of PC12 cells cultured for 4 days under simulated micro-g under standardized oxygen environment conditions revealed more than 100 genes whose expression levels were changed at least twofold (up-regulation of 65 genes and down-regulation of 39 genes) compared with those from cells in the unit gravity (unit-g) control. This study observed that genes involved in the oxidoreductase activity category were most significantly differentially expressed under micro-g conditions. Also, known oxidation-sensitive transcription factors such as hypoxia-inducible factor-2α, c-myc, and the peroxisome proliferator-activated receptor-γ were changed significantly. Our initial results from the gene expression microarray studies may provide a context in which to evaluate the effect of varying oxygen environments on the background of differential gene regulation of biological processes under variable gravity conditions.
Human islet allografts are well known to induce full and sustained remission of hyperglycemia, with complete normalization of key metabolic parameters. Nevertheless, acquiring human islets, even from ...cadaveric human donor pancreases, remains a significant impediment to successful transplantation therapy for diabetes. To overcome this difficulty, neonatal porcine cell clusters (NPCCs) have been considered for human islet substitutes because they are easily obtained by collagenase digestion of the neonatal piglet pancreas. Currently, the major hurdle in using NPCCs for xenograft is the delay (time lag) in achieving the posttransplant normalization of blood glucose levels in animal diabetic recipients. The present work is the first attempt to evaluate whether incubation of NPCCs in simulated microgravity, in the presence or absence of Sertoli cells (SC), may reduce the maturation time lag of β-cells by differentiation acceleration in vitro, thereby expediting production, viability, and acquisition of functional competence of pretransplantation β-cell-enriched islets. Following a 3-day incubation period, NPCCs maintained in conventional culture, NPCCs incubated in simulated microgravity in the HARV biochamber, and NPCCs plus co-incubated SC in simulated microgravity were examined for viability, morphology, and insulin secretion. Results show that NPCCs grown alone in the HARV biochamber are superior in quality, both in terms of viability and functional competence, when compared to other culture pretreatment protocols. This finding strongly suggests that NPCC pretreatment in simulated microgravity may enhance the transplantation success of NPCCs in the diabetic recipient.
The Golgi tendon organs (GTOs) are encapsulated mechano-receptors that, in normal conditions, monitor via Ib afferent fibers the contractile force. A 14-day period of hypodynamia, absence of weight ...bearing and hypokinesia, and reduction of motor activity (HH) is known to induce changes in postural muscles such as the soleus. At present, there is no data available regarding the Ib afferent feedback in normal rats (CONT group) and in rats after a hypodynamia–hypokinesia (HH group) period. Consequently, the aim of our study was to determine the HH effects on the morphological (histochemistry on gross morphology) and electrophysiological properties of the GTOs in rat soleus muscle. In the histological study, nine CONT and nineteen HH GTOs of the soleus muscle were identified. The results demonstrated that HH GTOs were morphologically similar to the CONT GTOs. Regarding the electrophysiological study, a L
2–L
6 laminectomy was performed under deep anesthesia (sodium pentobarbital, 60 mg kg
−1). Responses in single Ib fibers from the L
5 dorsal root to the isometric twitch and tetanic fused contractions of “in-series” motor units (MUs) were recorded. Twenty-three and twenty-eight GTO/MU pairs were studied in the CONT and HH groups, respectively. In the HH group, the Ib afferent response exhibited a decrease in dynamic peak for the high stimulation frequencies and an increase in static sensitivity for all stimulation frequencies. Our results suggest that after an HH period, the GTOs continue to fulfil their mechano-sensory function to signal the contractile force but with a higher static sensitivity.
The aim of this study was to investigate whether stimulation of the cutaneous mechanoreceptors of the rat foot sole could partially or totally prevent the soleus muscle atrophy developed after 14 ...days in hindlimb unloading conditions. Final experiments were achieved under deep anesthesia using pentobarbital sodium (60 mg/kg, ip injection). Atrophy was characterized by a significant decrease in muscle wet weight, fiber size, maximal twitch and tetanic tensions, contraction kinetics, and histochemical and electrophoretical changes. Our data demonstrate that the stimulation of these mechanoreceptors partially prevents the decrease in muscle weight (53%) and cross-sectional area of the soleus muscle (36%) and in all fiber types (type I: 31%; type Ic: 40%; type IIc: 49%; and type IIa: 44%) and also prevented the reductions in strength (peak twitch tension: 31%; peak tetanic tension: 25%). However, the decrease in contraction kinetics was not counteracted. Moreover, histochemical and electrophoretical changes were partially slowed. Thus our results suggest that stimulation of the sole mechanoreceptors can be used, in part, as a countermeasure to the muscular atrophy observed after a period of hindlimb unloading.
In situ proton stray-field (STRAFI) nuclear magnetic resonance (NMR) imaging of diamagnetic samples in simulated microgravity is described. The magnetic field gradient at the edge of the ...superconductive coil in the magnet of a NMR spectrometer is exploited to levitate diamagnetic samples and, at the same time, obtain their corresponding one-dimensional NMR images. Proton
T
2
(spin-spin relaxation time)/ diffusion-weighted profiles of samples under simulated microgravity conditions are acquired with a solid-echo radiofrequency pulse sequence. Since water is a diamagnetic main constituent substance of living systems, the methodology described could be useful to monitor non-destructively changes in the NMR properties of water protons induced by simulated microgravity and magnetic forces. Furthermore, this approach may be used to observe other NMR-sensitive nuclei of interest, e.g., fluor-19, phosphorous-31, carbon-13, etc.
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