Simulated Microgravity Promotes Angiogenesis through RhoA-Dependent Rearrangement of the Actin Cytoskeleton.

BACKGROUND/AIMS: Microgravity leads to hydrodynamic alterations in the cardiovascular system and is associated with increased angiogenesis, an important aspect of endothelial cell behavior to initiate new vessel growth. Given the critical role of Rho GTPase-dependent cytoskeleton rearrangement in cell migration, small GTPase RhoA might play a potential role in microgravity-induced angiogenesis. METHODS: We examined the organization of actin filaments by FITC-conjugated phalloidin staining, as well as the expression and activity of RhoA by quantitative PCR and Western blot, in human umbilical vein endothelial cells (HUVECs) under normal gravity and simulated microgravity. Effect of simulated microgravity on the wound closure and tube formation in HUVECs, and their dependence on RhoA, were also analyzed by cell migration and tube formation assays. RESULTS: We show that in HUVECs actin filaments are disorganized and RhoA activity is reduced by simulated microgravity. Blocking RhoA activity either by C3 transferase Rho inhibitor or siRNA knockdown mimicked the effect of simulated microgravity on inducing actin filament disassembly, followed by enhanced wound closure and tube formation in HUVECs, which closely resembled effects seen on microgravity-treated cells. In contrast, overexpressing RhoA in microgravity-treated HUVECs restored the actin filaments, and decreased wound closure and tube formation abilities. CONCLUSION: These results suggest that RhoA inactivation is involved in the actin rearrangement-associated angiogenic responses in HUVECs during simulated microgravity.

The Impact of Simulated Weightlessness on Endothelium-Dependent Angiogenesis and the Role of Caveolae/Caveolin-1.

BACKGROUND/AIMS: The potential role of caveolin-1 in modulating angiogenesis in microgravity environment is unexplored. METHODS: Using simulated microgravity by clinostat, we measured the expressions and interactions of caveolin-1 and eNOS in human umbilical vein endothelial cells. RESULTS: We found that decreased caveolin-1 expression is associated with increased expression and phosphorylation levels of eNOS in endothelial cells stimulated by microgravity, which causes a dissociation of eNOS from caveolin-1 complexes. As a result, microgravity induces cell migration and tube formation in endothelial cell in vitro that depends on the regulations of caveolin-1. CONCLUSION: Our study provides insight for the important endothelial functions in altered gravitational environments.

Clinorotation enhances autophagy in vascular endothelial cells.

Individuals exposed to extended periods of spaceflight or prolonged 6° head-down-tilt bed rest often suffer from health hazards represented by cardiovascular deconditioning. Many studies have reported that alterations in vascular endothelial cells contribute to cardiovascular dysfunction induced by microgravity. Autophagy, a lysosomal degradation pathway, serves an adaptive role for survival, differentiation, and development in cellular homeostasis, and can be triggered by various environmental stimuli. However, whether autophagy can be induced in endothelial cells by real or simulated microgravity remains to be determined. This study was designed to investigate the effects of simulated microgravity on the activation of autophagy in human umbilical vein endothelial cells (HUVECs). We report here that clinorotation, a simulated model of microgravity, enhances autophagosome formation, increases LC3 and beclin-1 expression, and promotes the conversion of LC3-I to LC3-II in HUVECs. These results demonstrate that simulated microgravity for 48 h activates autophagy of vascular endothelial cells.

Visual symptoms and G-induced loss of consciousness in 594 Chinese Air Force aircrew--a questionnaire survey.

A questionnaire survey was performed for the first time to assess the prevalence of visual symptoms and G-induced loss of consciousness (G-LOC) due to +Gz exposure in the Chinese Air Force (CAF) to determine the effectiveness of current G tolerance training. Responses were received from 594 individuals. Among them, 302 reported at least one episode of some sort of symptoms related to +Gz, including 110 (18.5%) with visual blurring, 231 (38.9%) with greyout, 111 (18.7%) with blackout, and 49 (8.2%) with G-LOC. Incidences were most common in aircrew with 250-1,000 flying hours (53.6%) and were more prevalent in those with fewer on type flying hours (p < 0.001). The most common situation was reported between +5 and 5.9 Gz. The results indicate a fairly high prevalence of visual symptoms and G-LOC among Chinese Air Force aircrew. There remains considerable scope for +Gz education, particularly in the early centrifuge training and selection of rational physical exercises.

Effect of thigh cuffs on haemodynamic changes of the middle cerebral artery and on orthostatic intolerance induced by 10 days head-down bed rest.

Thigh cuffs are used by cosmonauts to limit fluid shift during space flight, but the appropriate level of cuff pressure and the duration of application to optimize their beneficial effects require further detailed investigations. In the present study, 10 days head-down tilt (HDT) bed rest was performed to assess the effects of thigh cuffs (40 mmHg, 10 h/day) on haemodynamic changes of the middle cerebral artery (MCA) and on orthostatic tolerance in six healthy male volunteers. Another six healthy male volunteers without thigh cuffs served as the control group. Haemodynamic parameters of the MCA were measured using transcranial Doppler. Orthostatic tolerance was assessed before and after HDT. After HDT, the mean upright time in the control and thigh cuff groups was 14.0 +/- 4.1 and 19.2 +/- 0.7 min, respectively. Compared with values before HDT, the percentage increase in heart rate from baseline in the upright position after HDT was significantly higher in the control group and the percentage change from baseline of mean diastolic arterial blood decreased more after HDT in this group. In the control group, systolic blood velocity (Vs) and mean blood velocity (Vm) of the right MCA decreased significantly during HDT. In the thigh cuffs group, the Vs of the right MCA decreased significantly on Days 3 and 7 of HDT and the Vm of the right MCA decreased significantly on Day 7 of HDT. The results indicate that daily use of thigh cuffs during 10 days of HDT does not completely prevent the decrease in haemodynamics of the right MCA, but is effective in preventing orthostatic intolerance.

Heart rate and respiration responses to real traffic pattern flight.

The purpose of this study was to observe heart rate and respiration responses to real traffic pattern flight. Nine experienced and nine less-experienced military pilots on active flying status participated in four uninterrupted traffic patterns flight missions with F-7 jet trainer. The heart rates and respiration waves were continuously recorded using a small recording device strapped around the chest. As compared with baseline values, significant increases in heart rates of the two groups (for experienced pilots group, F (11, 88) = 4.636, p = 0.000; for less-experienced, F (11, 88) = 4.437, p = 0.000) and mean respiration rates of less-experienced group (F (11, 88) = 4.488, p = 0.000) were obtained during the phases of take-off, final approach and landing. Heart rates of less-experienced pilots were significant higher than those of experienced pilots during the take-off phase (p < 0.05). There were no significant differences in respiration rates between the two groups at each phase of the whole flight. The results show that take-off, final approach and landing are the most mental workload phases in-flight, and less-experienced pilots show more mental workload than experienced pilots in take-off phase in-flight.

Acceleration after-effects on learning and memory in rats: +10 Gz or +6 Gz for 3 min.

High sustained positive Gz (+Gz) exposures have been shown to have a pathophysiological impact on the brain in rats. However, the consequence for brain function such as learning and memory remains elusive. In the present study, we investigated locomotor activity, learning ability and memory in rats over 6 days (d) following +6 Gz/3 min and +10 Gz/3 min exposures. In an open field test, rats treated with +10 Gz showed a significant increase in the time spent in the center square immediately and 2d after exposure compared with control rats. In contrast, the number of grid crosses and number of rears of rats decreased significantly immediately after +6 Gz and +10 Gz exposures. In addition, a Y-maze test revealed that +Gz exposures reduced the number of correct responses and increased total reaction time in rats, and the number of correct responses was negatively correlated with the total reaction time in all groups throughout the period of study. Moreover, the passive avoidance test exhibited that the latency increased significantly on 0 d and decreased significantly on 6 d after +Gz exposures compared with control. Thus, +6 Gz/3 min and +10 Gz/3 min exposures may transiently decrease locomotor activity, impair learning, and induce a deficit in memory retention in rats.

[Effects of low gravity preconditioning on rat learning and memory impairment induced by high gravity exposure].

OBJECTIVE: To study the protective effects of low gravity preconditioning against learning and memory function impairment induced by high gravity exposure in rats. METHOD: Twenty-four male SD rats were randomized equally into control group, high gravity exposure group (+10 Gz/3 min) and low gravity preconditioning group (LGP group, preconditioned by a daily exposure to +4 Gz/3 min for 3 days before +10 Gz/3 min exposure). Both the learning and memory abilities of the rats in the 3 groups were examined after +10 Gz/3 min exposure. RESULTS: In open field test, the rats exposed to +10 Gz/3 min, in comparison with the control rats, exhibited significantly declined total square-crossing and rearing (SCR) on days 0 and 2 (P<0.05), with also significantly prolonged stay in the central square at 0 d(P<0.01). In the low gravity preconditioned rats, SCR declined and length of stay in the central square increased significantly on day 0 (P<0.01). Compared with the rats in +10 Gz/3 min group, the rats in the LGP group showed decreased length of stay in the central square on day 2, and such changes was statistically significant on day 6 (P<0.05), but there was no difference in SCR at all time points. In Y-maze test, the number of times of right reaction (RR) decreased and the reaction time (RT) increased significantly in +10 Gz/3min group compared with those of the control group (P<0.01), whereas no significant differences were found between the control group and LGP group. Compared with +10 Gz/3 min group, the RR was increased (P<0.01) and RT shortened significantly (P<0.01) in LGP group. In the step-through test, the latent time (LT) increased (P<0.05) on day 0 but was then shortened significantly on day 6 in + 10 Gz/3min group (P<0.01), and no difference was found between LGP group and the control group. LT on day 6 increased significantly (P<0.01) in LGP group as compared with the +10 Gz/3 min group. CONCLUSION: High gravity exposure-induced rat learning and memory impairment can be markedly improved by lower gravity preconditioning.

[Effects of simulated weightlessness on ALP, ACP, collagen I, and biomechanics of hind-limb bones in rats].

OBJECTIVE: To investigate effects of 3 weeks simulated weightlessness on biomechanical parameters, alkaline phosphatase (ALP), acid phosphatase (ACP) and collagen I of hind-limb bones in tail-suspended rats. METHOD: Fourteen male SD rats were divided equally into control group (CON) and experimental group; tail-suspended (TS) was used to simulate weightlessness. After 3 weeks tail-suspension, biomechanical parameters of femur were measured; ALP, ACP and collagen I of tibia were observed. RESULT: Elastic load, maximum load, and bending rigidity coefficient decreased significantly (P<0.01), while the maximum deformation and bending toughness coefficient increased markedly (P<0.01 or P<0.05). Morphological results showed that ALP declines significantly in TS group, ACP and type I collagen increased significantly in TS group. CONCLUSION: After tail-suspension for 3 weeks, growth of rat's weight bearing bones are suppressed, biomechanical capability declines, and collagen I metabolism becomes disordered.

Changes of hippocampus somatostatin and learning ability in rats after +Gz exposure.

OBJECTIVE: To investigate changes of learning ability and somatostatin (SS) changes after positive acceleration (+Gz) exposures. METHOD: Eighty male SD rats were randomly divided into 3 groups: control group (Con), +6 Gz/3 min group (+6 Gz), and +10 Gz/3 min group (+10 Gz), 8 rats in each group. Changes of learning ability in rats were observed at 0 d, 2 d, 4 d and 6 d after +Gz exposure. SS in hippocampus was measured by RIA at 0 d, 2 d and 4 d after +Gz exposures (there were 8 rats every time, in each group). RESULT: In Y-maze test, number of correct response decreased significantly (P<0.01), and total reaction time increased significantly (P<0.01) in +6 Gz and +10 Gz groups as compared with control group; number of correct response and total reaction time in +10 Gz group changed significantly at 0 d (P<0.01 or P<0.05) as compared with +6 Gz group. RIA showed that, content of SS in hippocampus declined at 0 d and 2 d (P<0.05 or P<0.01) in +6 Gz and +10 Gz groups as compared with control group. CONCLUSION: +Gz exposure could impair learning ability of rats, and inhibit expression of SS in hippocampus.

Effectiveness of intermittent -Gx gravitation in preventing deconditioning due to simulated microgravity.

This study was designed to compare the effectiveness of daily short-duration -Gx gravity exposure in preventing adverse changes in skeletal and cardiac muscles and bone due to simulated microgravity. Tail suspension for 28 days was used to simulate microgravity-induced deconditioning effects. Daily standing (STD) at 1 G for 1, 2, or 4 h/day or centrifugation (CEN) at 1.5 or 2.6 G for 1 h/day was used to provide -Gx gravitation as a countermeasure. The results indicate that the minimum gravity exposure requirements vary greatly in different systems. Cardiac muscle is most responsive to such treatment: 1 h/day of -Gx gravitation by STD was sufficient to prevent adverse changes in myocardial contractility; bone is most resistant: 4 h/day of -Gx gravitation only partially alleviated the adverse changes in physical and mechanical properties of the femur. The responsiveness of skeletal muscle is moderate: 4 h/day of -Gx gravitation prevented mass reduction and histomorphometric changes in the soleus muscle during a 28-day simulation period. Increasing gravitational intensity to 2.6 G showed less benefit or no additional benefit in preventing adverse changes in muscle and bone. The present work suggests that system specificity in responsiveness to intermittent gravity exposure should be considered one of the prerequisites in proposing intermittent artificial gravity as a potential countermeasure.

Effect of lower body negative pressure against orthostatic intolerance induced by 21 days head-down tilt bed rest.

BACKGROUND: Exposure to actual or simulated weightlessness is known to induce orthostatic intolerance in humans. Many different methods have been suggested to counteract orthostatic hypotension. The repetitive or prolonged application of lower body negative pressure (LBNP) has shown beneficial effects to counter orthostatic intolerance, but devoting so much time to countermeasures is not compatible with space mission objectives or costs. The purpose of the present study was to assess the effects of brief LBNP sessions against orthostatic intolerance during a 21-d head-down tilt (HDT) bed rest. METHODS: There were 12 healthy male volunteers who were exposed to -6 degrees HDT bed rest for 21 d. Six subjects received -30 mm Hg LBNP sessions for 1 h x d(-1) from day 15 to day 21 of the HDT, and six others served as control. Orthostatic tolerance was assessed by means of standard tilt test. RESULTS: Before HDT, all the subjects in the two groups completed the tilt tests. After 21 d of HDT, five subjects of the control group and one subject of the LBNP group could not complete the tilt test due to presyncopal or syncopal symptoms. The mean upright time in the control group 13.0 +/- 4.0 min) was significantly shorter (p < 0.05) than that in the LBNP group (19.0 +/- 2.2 min). Body weight decreased significantly in the control group during HDT, while increasing significantly on day 21 of HDT in the LBNP group. Urine volume increased on days 15-21 of HDT in the control group, but remained unchanged throughout HDT in the LBNP group. A significant decrease in cardiac output and cardiac index, and a significant increase in total peripheral resistance, pre-ejection period, plasma renin activity, aldosterone, and prostaglandin 12 were observed during HDT in both groups. There were no significant differences in these parameters between the two groups. CONCLUSIONS: Brief daily LBNP sessions were effective in preventing orthostatic intolerance induced by 21 d HDT bed rest. However, it did not improve cardiac pump and systolic functions and did not preserve volume regulating hormones.

[Effects of combined stress of high +Gz and noise on learning and memory in rats].

OBJECTIVE: To study the effect of combined stress of noise and + Gz on learning and memory in rats. METHOD: Thirty-two male SD rats were randomly divided into 4 groups: control group, +10 Gz/3 min group, 90 dB(A)/30 min noise group, combined stress group (+10 Gz/3 min and 90 dB(A)/30 min), 8 rats in each group. Changes of learning and memory after stresses were measured by means of Y-maze test, open field test and step-through test. RESULT: As compared with control group and noise group, the number of correct reactions decreased significantly (P<0.01) and the reaction time increased significantly (P<0.01) in combined stress group at all time after stress; time stay in center grille (TSCG) increased significantly immediately after stress (P<0.01). When compared with control group, total time (TT) and number of error (NE) increased significantly (P<0.01) while latent time (LT) decreased significantly immediately and 6 d after stress (P<0.01). As compared with +10 Gz group, number of correct reactions in combined stress group decreased significantly (P<0.05) and reaction time increased significantly (P<0.01) immediately after stress. CONCLUSION: It is suggested that combined stress of noise and +Gz may impair learning and memory of rats, and +Gz seems to contribute more to the combined effects.

[Effects of +Gz exposure time on memory and behavior in rats].

OBJECTIVE: To explore the effect of +Gz exposure time on memory and behavior in rats. METHOD: Twenty-four male SD rats were randomly divided into control group (A), +10 Gz/3 min group (B) and +10 Gz/5 min group (C). Rats in group A and B were exposed to +10 Gz for 3 min or 5 min respectively. Changes of memory and behavior in rats were observed after +Gz exposure. RESULT: As compared with control, percentage of right reflex decreased significantly and reaction time lengthened significantly immediately, 1 d, 2 d, 4 d and 6 d after +10 Gz/5 min exposure (P<0.01); reaction time lengthened significantly immediately and 2 d after +10 Gz/3 min exposure (P< 0.01). As compared with +10 Gz/3 min, percentage of right reflex decreased significantly and reaction time lengthened significantly at 1 d, 2 d, 4 d and 6 d after +10 Gz/5 min exposure (P<0.01). As compared with control and +10 Gz/3 min, time stayed in center grille lengthened significantly immediately after +10 Gz/5 min exposure (P<0.01); balancing function decreased significantly immediately and 2 d after +10 Gz/5 min exposure (P< 0.01). CONCLUSION: It is suggested that +10 Gz/ 3 min exposure may induce a provisional disturbance in memory function and a change in behavior in rats, and +10 Gz/5 min exposure may induce a sustained disturbance in memory function and an obvious change in behavior in rats.

[Effects of repeated lower g exposures on high-G induced memory and balance changes in rats].

OBJECTIVE: To study the protective effects of repeated +4 Gz/3 min exposures on memory and balance changes induced by +10 Gz/5 min exposure in rats. METHOD: 32 male SD rats were randomly divided into 4 groups: control group (C) ; +10 Gz/5 min group (10 Gz); 3 d training group (3 d) ; 5 d training (5 d) group (exposed to +4 Gz/3 min per day for 3 or 5 days before +10 Gz/5 min exposure). Changes of memory and balance in rats of all the 4 groups were observed after +10 Gz/5 min exposure. RESULT: In +10 Gz group, the percentage of correct reaction (CR) decreased significantly at all times after +10 Gz exposure (P<0.01), and the reaction time (RT) lengthened significantly at all times after +10 Gz exposure (P<0.01); as compared with control group. In 3 d training group, CR decreased significantly at 1 d, 2 d and 6 d after +10 Gz exposure (P<0.05), RT lengthened significantly at all times after +10 Gz exposure (P<0.05); as compared with control group. CR increased significantly at 2 d, 4 d and 6 d after +10 Gz exposure (P<0.01), RT shortened significantly at 1 d, 2 d, 4 d and 6 d after +10 Gz exposure (P<0.01) as compared with +10 Gz group. In 5 d training group, there were no apparent changes compared to control group; but CR increased significantly and RT shortened significantly at the time of 1 d, 2 d, 4 d and 6 d after +10 Gz exposure (P<0.01) as compared with +10 Gz group. Balancing function (BF) of +10 Gz group decreased significantly immediately and 2 d after +10 Gz exposure (P<0.01) as compared with control group. BF in 3 d and 5 d training group improved significantly immediately and 2 d after +10 Gz exposure as compared with +10 Gz group (P<0.01). CONCLUSION: It is suggested that repeated low +Gz exposures could provide protective effect on memory and balance changes induced by high +Gz exposure in rats.

[Changes of bone morphogenesis proteins and transforming growth factor-beta in hind-limb bones of 21 d tail-suspended rats].

OBJECTIVE: To investigate changes of bone morphogenesis proteins (BMP), transforming growth [correction of grouth] factors-beta (TGF-beta) in tibia and the growing of femur in tail-suspended rats after 21 d simulated weightlessness. METHOD: Fourteen male SD rats were randomly divided into control group (CON) and tall-suspension group (TS). After 21 d tail-suspension, basic physical parameters of the femur were measured; changes of BMP, TGF-beta in tibia were assayed by immunohistochemical method. RESULT: During 21 d tail-suspension, all rats grew well without apparent stress reaction. After 21 d weightlessness simulation, wet weight, dried weight, ash, diameter and density of femur in TS group declined significantly (P<0.01). Immunohistochemical results of tibia showed that both BMP and TGF-beta declined in tissues from all regions of the tibia. CONCLUSION: After 21 d tall-suspension, the growth of rat's weight bearing bones were suppressed, production and secretion of BMP and TGF-beta was holdback.

Acoustical stimulus changes the expression of stromal cell-derived factor-1 in the spiral ganglion neurons of the rat cochlea.

Neural stem cell (NSC) transplantation into the cochlea has been tested as a treatment for spiral ganglion neuron (SGN) degenerative disease and injury in various animal models. A recent study has shown evidence of functional recovery after transplantation of the stem cells into a degenerated-SGN model. Chemokine stromal cell-derived factor-1 (SDF-1, or known as CXC chemokine ligand-12, CXCL-12) signaling through CXCR4 has previously been identified as a key step in the homing of the stem cells within the injury areas; meanwhile, studies have revealed that the SDF-1/CXCR4 axis is also involved in axon guidance and pathfinding. A study found that transplanted neural precursor cells can migrate to the root of the auditory nerve when animals are subjected to an augmented acoustic environment (AAE). In accordance with these studies, we hypothesize that AAE will up-regulate the expression of SDF-1 in acoustic nerves. We tested our hypothesis by examining the expression of SDF-1 in different acoustic environments, and the results were confirmed by the auditory brainstem response (ABR), immunohistochemical and RT-PCR analyses. The results showed that SDF-1 was expressed at a relatively low level in the SGNs under normal animal unit acoustic conditions (40-50 dB). Moreover, it was significantly up-regulated in the SGNs under the 75 dB (augmented physiological process without hearing loss) and 90 dB AAE (pathological process with light hearing loss) conditions; however, under the 115 dB AAE (pathological process with severe hearing loss) condition, the expression of SDF-1 was not up-regulated. The results confirmed that appropriately augmented acoustical stimuli lead to the up-regulation of SDF-1, which may assist in the migration of the transplanted cells and the subsequent establishment of essential synaptic contacts between the exogenous cells and the host auditory pathway.

Simulated weightlessness aggravates hypergravity-induced impairment of learning and memory and neuronal apoptosis in rats.

It has been demonstrated that altered gravity may lead to impairments in cognitive functions. However, the effect of a combination of hypergravity and weightlessness on cognitive functions is not well understood. In the present study, we report the effects of high sustained hypergravity after 7 days' simulated weightlessness on learning and memory abilities and neuronal apoptosis in rats. In the Y-maze tests, hypergravity (HG) or simulated weightlessness (SW) significantly decreases accuracy, and increases reaction time of rats compared to that of controls. On the contrary, in the passive avoidance test, HG or SW treatment significantly shortens latency and prolongs total time compared to those of controls. In addition, TUNEL staining shows a few apoptotic cells in cortex and hippocampus in the HG, SW and HG+SW groups, and the number of TUNEL positive cells was found to be the most in the HG+SW group. Furthermore, rats with combined HG and SW treatment reveal a synergistic effect in both the Y-maze and the passive avoidance tests, as well as increased neuronal cell death. These findings suggest that simulated weightlessness may exacerbate hypergravity-induced impairment of learning and memory, likely caused by neuronal cell death in rats.

Daily 4-h head-up tilt is effective in preventing muscle but not bone atrophy due to simulated microgravity.

To assess the potential value of intermittent artificial gravity as an efficient countermeasure, our previous studies have showed that daily 4-h standing (STD) is sufficient in counteracting muscle atrophy but not bone atrophy induced by simulated microgravity. The aim of the present study was to determine whether intermittent gravitational loading by daily 2-h or 4-h, +45 degrees head-up tilt (HUT) is more effective than STD in counteracting muscle and, particularly, bone atrophy due to simulated microgravity. Sprague-Dawley male rats weighing 290-300 g were subjected to a 28-d tail-suspension to simulate microgravity deconditioning. Daily HUT for 2, or 4 h was used to provide intermittent gravitational loading in foot-ward and tail-ward directions. The results showed that 4 h/d HUT was sufficient, and 2 h/d was less effective, in preventing adverse changes in muscle weights, fiber types, and cross-sectional areas (CSA) of muscles due to a 28-d simulated microgravity. The % protections by 4 h/d HUT in maintaining the CSAs of type I fibers in soleus, medial and lateral gastrocnemius and extensor digitorum longus muscles were 103%, 82%, 102%, and 83%, respectively. However, according to changes in physical and mechanical properties of femur, daily 4-h HUT was ineffective in attenuating the adverse changes in bone due to a 28-d simulated microgravity. Reductions in wet, dry, and ash weights and decreases in mechanical strength of femur did not show significant improvement by daily 2-h or 4-h HUT. Taken together, the findings indicate that the countermeasure effectiveness of daily 2-h or 4-h HUT for muscles is comparable with that by daily STD with the same durations. Daily 4-h HUT, as 4-h STD, is also ineffective in attenuating adverse changes in bone mass, but seems partially effective in preventing declines in mechanical properties due to simulated microgravity.