Short-term Simulated Microgravity Research Articles

Metabolic Profiles of Encapsulated Chondrocytes Exposed to Short-Term Simulated Microgravity.

The mechanism by which chondrocytes respond to reduced mechanical loading environments and the subsequent risk of developing osteoarthritis remains unclear. This is of particular concern for astronauts. In space the reduced joint loading forces during prolonged microgravity (10-6 g) exposure could lead to osteoarthritis (OA), compromising quality of life post-spaceflight. In this study, we encapsulated human chondrocytes in an agarose gel of similar stiffness to the pericellular matrix to mimic the cartilage microenvironment. We then exposed agarose-chondrocyte constructs to simulated microgravity (SM) using a rotating wall vessel (RWV) bioreactor to better assess the cartilage health risks associated with spaceflight. Global metabolomic profiling detected a total of 1205 metabolite features across all samples, with 497 significant metabolite features identified by ANOVA (FDR-corrected p-value < 0.05). Specific metabolic shifts detected in response to SM exposure resulted in clusters of co-regulated metabolites, as well as key metabolites identified by variable importance in projection scores. Microgravity-induced metabolic shifts in gel constructs and media were indicative of protein synthesis, energy metabolism, nucleotide metabolism, and oxidative catabolism. The microgravity associated-metabolic shifts were consistent with early osteoarthritic metabolomic profiles in human synovial fluid, which suggests that even short-term exposure to microgravity (or other reduced mechanical loading environments) may lead to the development of OA.

Effects of short-term simulated microgravity on changes in extracellular space structure and substance diffusion and clearance

Substance transportation and clearance in the brain extracellular space (ECS) is crucial to brain function, but simulated microgravity disrupts substance diffusion and clearance in the brain ECS and there is limited knowledge regarding these disruptions. This study employed a tracer-based magnetic resonance imaging (MRI) technique to examine the impact of simulated microgravity on hippocampal ECS structure and the drainage of brain interstitial fluid (ISF) in a tail-suspended hindlimb-unloading rat model. The results demonstrated that under simulated microgravity conditions for 3, 7, and 14 days, the volume fraction of the hippocampal ECS increased in hindlimb unloading rats, and short-term (3 days) simulated microgravity expedited the drainage of the hippocampal ISF, but the drainage slowed under longer (7 or 14 days) simulated microgravity. These findings suggested that simulated microgravity alters the structure of the brain ECS and the speed of brain ISF drainage, which may have implications for space-flight-related brain injuries and relevant neuroprotective approaches via the brain ECS.

Short Duration Simulated Microgravity Models Improve Executive Function

PURPOSE: To determine whether executive function is altered during two variations of simulated microgravity head down tilt protocols. METHODS: 26 healthy volunteers (n=19 females and 7 males; 20.0 ± 1.7 years; 165.6 ± 24.5 cm; 71.1 ± 15.9 kg; mean±SD) took part in two randomly assigned separate simulated microgravity sessions consisting of 1.5 hours at -6° head down tilt (HDT) sessions. Each testing session lasted approximately 2.5 hours. One session consisted of the broadly used -6° HDT facing up (HDT-FU), while the other consisted of a -6° HDT facing down (HDT-FD) modification of the standard method. For each condition, the participant completed a computerized Stroop Color and Word Test during a thirty-minute horizontal position (0° tilt) prior to the interventions and again prior to the end of the 1.5-hour HDT protocols. The Stroop Color and Word Test consisted of 3 tasks (60 trials per task) including: 1) a Naming task, 2) an Inhibition task, and 3) an Inhibition/Switching task. To minimize the learning effect, both the HDT-FU and HDT-FD sessions were counterbalanced amongst all participants and were separated by a minimum of 48 hours. RESULTS: Our cognitive reaction time results demonstrate a main effect of time (pre HDT 777.60 ± 18.08ms vs post HDT 749.30 ± 19.11ms, p<0.01) and a main effect of Stroop Test (Naming < Inhibition < Inhibition/Switching, p<0.01). Additionally, we observed that the 1.5-hour protocol selectively improved the Inhibition/Switching reaction times (the most executive component) for both the HDT-FU (pre HDT-FU 945.68 ± 32.87ms vs post HDT-FU 903.16 ± 33.78ms, p=0.02) and HDT-FD (pre HDT-FD 961.25 ± 38.55ms vs post HDT-FD 868.91 ± 30.40ms, p<0.01). CONCLUSION: When executive demand is increased under short-term simulated microgravity head down tilt conditions, regardless of the position (face up or face down), cognitive reaction time is decreased, suggesting an improvement in overall cognitive function.

Growth Behavior and Transcriptome Profile Analysis of Proteus mirabilis Strain Under Long- versus Short-Term Simulated Microgravity Environment.

Spaceflight missions affect the behavior of microbes that are inevitably introduced into space environments and may impact astronauts’ health. Current studies have mainly focused on the biological characteristics and molecular mechanisms of microbes after short-term or long-term spaceflight, but few have compared the impact of various lengths of spaceflight missions on the characteristics of microbes. Researchers generally agree that microgravity (MG) is the most critical factor influencing microbial physiology in space capsules during flight missions. This study compared the growth behavior and transcriptome profile of Proteus mirabilis cells exposed to long-term simulated microgravity (SMG) with those exposed to short-term SMG. The results showed that long-term SMG decreased the growth rate, depressed biofilm formation ability, and affected several transcriptomic profiles, including stress response, membrane transportation, metal ion transportation, biological adhesion, carbohydrate metabolism, and lipid metabolism in contrast to short-term SMG. This study improved the understanding of long-term versus short-term SMG effects on P. mirabilis behavior and provided relevant references for analyzing the influence of P. mirabilis on astronaut health during spaceflights.

Morphofunctional, viability and antioxidant system alterations on rat primary testicular cells exposed to simulated microgravity

This study focused on effects induced by Short-term Simulated Microgravity (SMG) condition on primary cell culture from pre-pubertal Wistar rats testis. Cells were analyzed for cytoskeletal and Sex Hormone Binding Globulin (SHBG/ABP) changes by immunofluorescence technique, for antioxidant system exploiting RT-PCR and cell viability. Cells were cultured for 6 and 24h on a three-dimensional clinostat, Random Positioning Machine (RPM). At the end of each experiment, once stopped the RPM rotation, cells were either fixed in paraformaldehyde or lysed and RNA extracted. In cells exposed to SMG the cytoskeleton became disorganized, microtubules fragmented and SHBG was already undetectable after 6h of treatment. Moreover, various antioxidant systems significantly increased after 24h of SMG exposure. Initially, SMG seemed to disturb antioxidant protection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Studies on changes induced by short-term altered gravity conditions, carried out in real microgravity, could give more information on steroidogenesis and germ cell differentiation within the testis exposed to this condition and confirm the validity of simulation approach.

Interactome of miRNAs and transcriptome of human umbilical cord endothelial cells exposed to short-term simulated microgravity

Adaptation of humans in low gravity conditions is a matter of utmost importance when efforts are on to a gigantic leap in human space expeditions for tourism and formation of space colonies. In this connection, cardiovascular adaptation in low gravity is a critical component of human space exploration. Deep high-throughput sequencing approach allowed us to analyze the miRNA and mRNA expression profiles in human umbilical cord vein endothelial cells (HUVEC), cultured under gravity (G), and stimulated microgravity (MG) achieved with a clinostat. The present study identified totally 1870 miRNAs differentially expressed in HUVEC under MG condition when compared to the cells subjected to unitary G conditions. The functional association of identified miRNAs targeting specific mRNAs revealed that miRNAs, hsa-mir-496, hsa-mir-151a, hsa-miR-296-3p, hsa-mir-148a, hsa-miR-365b-5p, hsa-miR-3687, hsa-mir-454, hsa-miR-155-5p, and hsa-miR-145-5p differentially regulated the genes involved in cell adhesion, angiogenesis, cell cycle, JAK-STAT signaling, MAPK signaling, nitric oxide signaling, VEGF signaling, and wound healing pathways. Further, the q-PCR based experimental studies of upregulated and downregulated miRNA and mRNAs demonstrate that the above reported miRNAs influence the cell proliferation and vascular functions of the HUVEC in MG conditions effectively. Consensus on the interactome results indicates restricted fluctuations in the transcriptome of the HUVEC exposed to short-term MG that could lead to higher levels of endothelial functions like angiogenesis and vascular patterning.

Dynamic Foot Stimulations During Short-Term Hindlimb Unloading Prevent Dysregulation of the Neurotransmission in the Hippocampus of Rats.

Spaceflight and simulated microgravity both affect learning and memory, which are mostly controlled by the hippocampus. However, data about molecular alterations in the hippocampus in real or simulated microgravity conditions are limited. Adult Wistar rats were recruited in the experiments. Here we analyzed whether short-term simulated microgravity caused by 3-day hindlimb unloading (HU) will affect the glutamatergic and GABAergic systems of the hippocampus and how dynamic foot stimulation (DFS) to the plantar surface applied during HU can contribute in the regulation of hippocampus functioning. The results demonstrated a decreased expression of vesicular glutamate transporters 1 and 2 (VGLUT1/2) in the hippocampus after 3days of HU, while glutamate decarboxylase 67 (GAD67) expression was not affected. HU also significantly induced Akt signaling and transcriptional factor CREB that are supposed to activate the neuroprotective mechanisms. On the other hand, DFS led to normalization of VGLUT1/2 expression and activity of Akt and CREB. Analysis of exocytosis proteins revealed the inhibition of SNAP-25, VAMP-2, and syntaxin 1 expression in DFS group proposing attenuation of excitatory neurotransmission. Thus, we revealed that short-term HU causes dysregulation of glutamatergic system of the hippocampus, but, at the same time, stimulates neuroprotective Akt-dependent mechanism. In addition, most importantly, we demonstrated positive effect of DFS on the hippocampus functioning that probably depends on the regulation of neurotransmitter exocytosis.

Label-free quantitative proteomics of rat liver exposed to simulated microgravity

Microgravity affects the content of carbohydrates, lipids and proteins in liver. Since liver is the major metabolic organ of endogenous and exogenous compounds, a deep understanding of microgravity-induced effects on liver could be of great importance. Therefore, livers from tail-suspension simulated microgravity (SM) rat model were analyzed using a label-free quantitative proteomic method in the present study. The results indicated that hepatic metabolic functions were obviously disrupted by SM. After short-term SM, enzymes for oxidizing nutrients were greatly increased and thus glucose, non-ester fatty acids and amino acids in liver were heavily consumed. Meanwhile, proteins for oxidative phosphorylation were also remarkably regulated. Therefore, the vast electrons generated from oxygenolysis could not be fully transferred to dioxygen and subsequently strong oxidative stress was triggered in liver. On the other hand, antioxidants were increased compensatorily. With the restoration of most energy-metabolic proteins and the continuous up-regulation of antioxidants, hepatic oxidative status returned to physiological level after long-term SM. Moreover, several enzymes involved in xenobiotic metabolism, such as cytochrome P450s and UDP-glucuronosyltransferases were also regulated by SM in a duration-dependent way. Our data might be helpful in understanding the effects of microgravity on liver and would support the design of protective measures for manned space missions.

Effect of simulated microgravity on the photopic negative response of flash electroretinogram in adult mice

Objective To observe the effect of simulated microgravity on the photopic negative response (PhNR) of full-field flash ERG in adult mice. Methods In an experimental study, forty-eight adult male C57BL/6J mice (48 eyes) were randomly divided into model and control groups. Model mice were further divided into three subgroups of 8 each: tail-suspended for 15 days (subgroup A), tail-suspended for 30 days (subgroup B), and tail-suspended for 30 days followed by returning to normal position for 30 days (subgroup C). The three control subgroups were similarly fixed with a harness but kept in the normal position for corresponding periods of 15, 30, and 60 days. The mice were immediately examined using ERG-PhNR, flash VEP, OCT and visually-guided behavior in vivo, and subsequently sacrificed to analyze the retinal histology in vitro. PhNR amplitude was measured from baseline to PhNR trough. N1 peak-time and N1-P1 amplitude of VEP was analyzed. The escape duration was used to quantitatively evaluate the visual function of mice. In addition, inner retinal thickness was analyzed by OCT imaging. Data were compared by the independent sample t-test. Results PhNR amplitude in the model subgroup A was obviously lower than the corresponding control subgroup, the difference was statistically significant (t=−3.196, P 0.05). There was no significant difference in FVEP N1 peak-time or N1-P1 amplitude between any of the three model subgroups and the corresponding control subgroup (P>0.05). There was no significant difference in OCT-measured inner retinal thickness between any of the three model subgroups and the corresponding control subgroup (t=−0.461, 2.073, −0.402; P>0.05). The three model subgroups showed almost normal retinal structure, including the retinal ganglion cell, inner pexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, ellipsoid zone and RPE. There was no significant difference in visually-guided escape time between any of the three model subgroups and the corresponding control subgroup (t=−0.637, −0.955, 1.297; P>0.05). Conclusion Via tail-suspension, short-term simulated microgravity can affect the PhNR of flash ERG; however, the change is reversible and does not affect visual function of mice. Key words: Weightlessness simulation; Electroretinography; Animal experimentation

Altered regional homogeneity with short-term simulated microgravity and its relationship with changed performance in mental transformation.

In order to further the insight into the explanation of changed performance in mental transformation under microgravity, we discuss the change of performance in mental transformation and its relationship with altered regional homogeneity (ReHo) in resting-state brain by using simulated weightlessness model. Twelve male subjects with age between 24 and 31 received resting-state fMRI scan and mental transformation test both in normal condition and immediately after 72 hours −6° head down tilt (HDT). A paired sample t-test was used to test the difference of behavior performance and brain activity between these two conditions. Compare with normal condition, subjects showed a changed performance in mental transformation with short term simulated microgravity and appeared to be falling. Meanwhile, decreased ReHo were found in right inferior frontal gyrus (IFG) and left inferior parietal lobule (IPL) after 72 hours −6° HDT, while increased ReHo were found in bilateral medial frontal gyrus (MFG) and left superior frontal gyrus (SFG) (P<0.05, corrected). Particularly, there was a significant correlation between ReHo values in left IPL and velocity index of mental transformation. Our findings indicate that gravity change may disrupt the function of right IFG and left IPL in the resting-state, among of which functional change in left IPL may contribute to changed abilities of mental transformation. In addition, the enhanced activity of the bilateral MFG and decreased activity of right IFG found in the current study maybe reflect a complementation effect on inhibitory control process.

ICAM-1 expression and organization in human endothelial cells is sensitive to gravity

Transendothelial migration (TEM) of immune cells is a crucial process during a multitude of physiological and pathological conditions such as development, defense against infections and wound healing. Migration within the body tissues and through endothelial barriers is strongly dependent and regulated both by cytoskeletal processes and by expression of surface adhesion molecules such as ICAM-1 and VCAM-1. Space flight experiments have confirmed that TEM will be inhibited and may cause astronauts’ immune function decreased and make them easy for infection. We used NASA RCCS to provide a simulated microgravity environment; endothelial cells were cultured on microcarrier beads and activated by TNF-α. Results demonstrate after clinorotation ICAM-1 expression increased, consistent with the notion in parabolic flights. However, VCAM-1 showed no significant change between activated or inactivated cells. Depolymerization of F-actin and clustering of ICAM-1 on cell membrane were also observed in short-term simulated microgravity, and after 24 h clinorotation, actin fiber rearrangement was initiated and clustering of ICAM-1 became stable. ICAM-1 mRNA and VCAM-1 mRNA were up-regulated after 30 min clinorotation, and returned to the same level with controls after 24 h clinorotation.

Head-down tilt posture elicits transient lymphocyte mobilization from the iliac, but not mesenteric, lymph nodes of rats

The effects of short-term simulated microgravity on the lymph dynamics of rat lymph nodes were investigated using a combination of Bollman's cage and head-down tilt (HDT). Efferent lymphatics of the iliac and mesenteric lymph nodes were cannulated for the collection of lymph. There was no significant difference in lymph flow rate from the iliac lymph nodes between non-HDT (control) and HDT rats. Lymph flow rate from the mesenteric lymph nodes in HDT rats was slightly higher than that obtained with the control. The cell count obtained from the iliac lymph nodes in HDT rats was significantly larger than those of the controls, while no significant difference in the number of cells from the mesenteric lymph nodes was observed between the control and HDT groups. The cells from the iliac lymph nodes in the control and HDT rats were mostly lymphocytes. The distribution of subsets of lymphocytes (CD3+, CD4+, CD8a+, and CD45R+) from the iliac lymph nodes in HDT rats was not significantly different from the subsets of lymphocytes in the control. Immunization did not affect the distribution of lymphocyte subsets from the iliac lymph nodes in the control and HDT groups. There was no significant difference in the concentrations of lymph albumin in iliac afferent or efferent lymphatics between the control and HDT groups. These findings suggest that HDT posture in Bollman's cage induces transient output of lymphocytes from the iliac lymph nodes of rats in vivo without changing the flow rate, lymphocyte subsets, or concentration of albumin.

Abnormal pituitary-gonadal axis may be responsible for rat decreased testicular function under simulated microgravity

Space flight and simulated microgravity lead to suppression of mammalian spermatogenesis and decreased plasma testosterone level. In order to explain the mechanism behind the depression, we used rat tail-suspended model to simulate weightless conditions. To prevent cryptorchidism caused by tail-suspension, some experimental animals received inguinal canal ligation. The results showed that mass of testis decreased significantly and seminiferous tubules became atrophied in rats after tail-suspension. The levels of plasma testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in tail-suspended rats with or without inguinal canal ligation decreased significantly compared with controls, and an increased level of plasma estradiol ( E 2 ) was revealed in tail-suspended rats. The results indicate that besides the direct influence of fluid shift upon testis under short-term simulated microgravity, the pituitary function is also disturbed as a result of either immobilization stress or weight loss during tail-suspension treatment, which is responsible to some extent for the decreased testosterone secretion level and the atrophia of testis. The conversion of testosterone into E 2 under simulated microgravity is another possible cause for the decline of plasma testosterone.

Effects of clinorotation on COL1A1-EGFP gene expression

Bone-formation related gene plays a critical role in bone loss induced by space microgravity, however the exact mechanism is unclear. In this study, we aim to investigate the effect of microgravity on the activity of alpha 1(I) collagen (COL1A1) gene promoter and the expression of osteoblast-related genes. COL1A1 promoter was digested by restriction enzymes resulting in three DNA fragments. The fragments were ligated with the enhanced green fluorescent protein report gene, and subcloned into expression vectors. ROS17/2.8 cells transfected by these vectors were screened by G418, and enhanced green fluorescent protein (EGFP) positive colonies were isolated and cultured under clinostat condition. EGFP and Collagen type I expression level were detected by fluorescence intensity analysis and immunocytochemistry methods respectively. The results showed that the expression of EGFP and collagen type I was increased 24 h, 48 h after the cells were cultured under stimulated microgravity, illustrating that the activity of COL1A1 promoter might be increased. In conclusion, osteoblasts can compensatively increase the expression of type I collagen by enhancing the activity of COL1A1 promoter under short-term simulated microgravity conditions.

Up-regulation of the adrenomedullin system mediates hypotension and hypoaldosteronism induced by simulated microgravity

We recently demonstrated that prolonged simulated microgravity (SMG) induced hypotension and hypoaldosteronism in rats, and gathered preliminary evidence for an involvement of circulating adrenomedullin (AM). Thus, we aimed to investigate whether short-term SMG elicits the same effects, and whether up-regulation of adrenal AM system plays a relevant role. Rats were exposed for 8 days to SMG in the form of hindlimb unweighting, and then, along with control animals, were given an intraperitoneal injection of AM22-52 and/or angiotensin-II (Ang-II) (100 nmoles/kg) or the saline vehicle. Systolic blood pressure (SBP) was measured by tail-cuff sphygmomanometry. The adrenal expression of AM was assayed by semiquantitative RT-PCR. The plasma concentrations of aldosterone (PAC) and AM, and adrenal AM content were measured by RIA. Short-term SMG induced significant decreases in SBP and PAC. Conversely, both the plasma and adrenal levels of AM, and adrenal AM mRNA were enhanced in SMG-exposed animals. The SMG-induced hypotension and hypoaldosteronism were reversed by AM22-52, an AM-receptor antagonist, thereby demonstrating a causal link between these effects and the up-regulation of AM system. SMG hampered SBP and PAC responses to Ang-II; the co-administration of AM22-52 restored these responses. These findings accord well with the known ability of AM to counteract the effects of Ang-II on both blood vessels and adrenocortical cells. Taken together, our findings allow us to conclude that up-regulation of the adrenal AM system i) occurs early and takes part in the adaptative changes occurring during SMG conditions; and ii) may account for both hypotension and hypoaldosteronism on returning to the normogravitational environment.

Specific Features of Metabolism in Humans Performing a Physical Exercise Test after 7-Day Dry Immersion

Healthy male volunteers (n = 5) aged 27–42 years participated in experimental studies (7-day immersion) designed to simulate the effects of microgravity. To study the metabolic changes caused by decreased weight bearing on the musculoskeletal system and a change in the position of the body relative to the gravity vector, a 15-min load test before and after immersion was used. A wide set of biochemical parameters characterizing the state of the energy metabolism, substrate levees, and enzyme activities, as well as the blood level of hormones, was measured in the blood plasma. Multifactor analysis was used in processing the experimental data. After immersion, a significant decrease in the blood plasma activity of isocitrate dehydrogenase, creatine phosphokinase, and lactate dehydrogenase was noted, whereas the growth hormone and insulin levels exceeded the baseline values. The physical exercise test increased the differences in the metabolic status before and after 7 days of immersion. The factor analysis allowed us to reveal the most significant biochemical variables for identifying a new metabolic state of the physiological systems after exposure to short-term simulated microgravity. Changes in the creatine phosphokinase activity and the human blood plasma levels of cortisol, triglycerides, insulin, and inorganic phosphate made the most significant contributions to these differences, and the direction of biochemical shifts in response to exercise was different before and after immersion. The results obtained are indicate that energy and substrate metabolism changes in response to a decrease in weight bearing and an altered body position relative to the gravity vector and that these changes are especially pronounced when an exercise test is used.

Spectral indices of cardiovascular adaptations to short-term simulated microgravity exposure.

We investigated the effects of exposure to microgravity on the baseline autonomic balance in cardiovascular regulation using spectral analysis of cardiovascular variables measured during supine rest. Heart rate, arterial pressure, radial flow, thoracic fluid impedance and central venous pressure were recorded from nine volunteers before and after simulated microgravity, produced by 20 hours of 6 degrees head down bedrest plus furosemide. Spectral powers increased after simulated microgravity in the low frequency region (centered at about 0.03 Hz) in arterial pressure, heart rate and radial flow, and decreased in the respiratory frequency region (centered at about 0.25 Hz) in heart rate. Reduced heart rate power in the respiratory frequency region indicates reduced parasympathetic influence on the heart. A concurrent increase in the low frequency power in arterial pressure, heart rate, and radial flow indicates increased sympathetic influence. These results suggest that the baseline autonomic balance in cardiovascular regulation is shifted towards increased sympathetic and decreased parasympathetic influence after exposure to short-term simulated microgravity.