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Departments of 1Aerospace Physiology and 2Aerospace Biodynamics, Fourth Military Medical University, Xi'an 710032, People's Republic of China
Submitted 21 October 2002 ; accepted in final form 26 February 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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rat; hindlimb unweighting; intermittent artificial gravity; countermeasure; myocardial contractility; skeletal muscle; atrophy; bone; osteopenia; centrifugation
In the past two decades, two ground-based human studies have provided promising data demonstrating the effects of daily +Gz gravitation in counteracting the deconditioning due to simulated microgravity. Shulzhenko and Vil-Viliams (40, 46) reported the beneficial effects of daily periodic exposures to +0.8 to +1.6 Gz during 3 or 28 days of dry immersion in alleviating the G tolerance reduction. Vernikos et al. (43, 44) reported that standing or walking for 2 or 4 h/day was effective in most cases to counteract the deconditioning effects of 4 days of -6° head-down bed rest. They concluded that various physiological systems benefit differently from daily short-duration +1 Gz gravitation; i.e., the preventive value appears to be system specific (43, 44).
The tail-suspended, hindlimb-unloaded (HU) rat model has also been used to assess the efficacy of daily gravitation alone or in combination with exercise in alleviating the adverse changes of muscles or bones due to simulated microgravity. Edgerton and colleagues (12, 15, 20, 22, 34, 36), D'Aunno and colleagues (10, 11), and Fitts and colleagues (13, 48, 49) provided convincing evidence that daily standing or exercise for short periods is effective in alleviating the effects of HU on hindlimb muscles of rats. For example, during 7 days of HU, periodic daily standing, very slow walking, or moderate running for a total of 40–60 min, or 3–4% of the total non-weight-bearing time, maintained a near-normal soleus (SOL) mass (12, 20, 22, 34). Centrifugation at 1.2 G (11) or 2.6 G (10) did not show additional benefit. Work on bones has been less relevant. Standing for 2 h/day was ineffective in preventing calcium loss and collagen-matrix reduction in the tibia of 15-day HU rats (23) and bone loss in the humerus of 35-day HU rats (38). It has been suggested that gravitational changes affect tibial growth plates according to Hert's curve; too much or too little loading results in lack of differentiation or closure of the epiphyseal plate (29, 30). However, in each of the aforementioned studies, only the changes in bone or muscle were studied. In addition, no information is available on how daily gravitation may affect changes in cardiac muscle and vessels due to simulated microgravity. Moreover, the experimental design of most of these animal studies did not permit dissociation of the effects of intermittent gravitation from the effects of exercise. Finally, the countermeasure effectiveness should be further evaluated over longer simulation periods, instead of over 7 days of HU (12, 13).
Therefore, we designed the present study to compare the effectiveness of daily gravitations, different in G vector (-Gx or +Gz), intensity, and duration, in preventing the deconditioning in a multitude of organ systems of the same animal during a midterm (4 wk) simulated microgravity. The research goal was to determine whether there are greater differences in physiological requirements of gravity among different organ systems. This study reports the effects of daily short-duration -Gx gravitation by standing (STD) at 1 G or centrifugation (CEN) at 1.5 or 2.6 G in preventing the adverse changes in hindlimb skeletal muscles, bone (femur), and cardiac muscle and discusses the problem of system specificity in G requirements.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Tail-suspended, HU rat model. The technique of tail suspension (31, 50), with modification from our laboratory, has been described in detail previously (8). Briefly, the tail was cleaned, air dried, and sprayed with a generous amount of benzoin and resin. A traction tape was preattached to a plastic bar, attached laterally along the proximal portion of the tail, and then secured by three tape strips. The tail was divided into four quadrants, and only an opposite pair of quadrants (lateral or dorsoventral) was used for application of the traction tape. Tape was replaced weekly using the alternate pair of quadrants. The rats were attached via the plastic bar in the tape to a swivel mounted at the top of the cage, allowing free 360° rotation. The rats were maintained in an about -30° head-down tilt position with their hindlimbs unloaded.
Modes of daily short-duration -Gx gravitation. Two modes of -Gx vector gravitation were used to simulate the counteracting effect of IAG. One mode was stationary ground support, or standing (STD). For STD, the HU rat was removed from suspension and placed in an upright standing position. To restrict ambulatory activity, each rat was placed in a 50-cm-long, tubelike metallic mesh cage, which was maintained in a horizontal position. Food and water were provided ad libitum at the front end of the cage. The other mode of -Gx gravitation was centrifugation (CEN). The animal centrifuge radius is 2 m. The centrifugal acceleration reported is the gravito-inertial resultant. The animals were centrifuged at -Gx vector. For CEN, rats removed from suspension were placed in a 15 x 7 x 10-cm metallic multicompartment cage. During CEN, all animals were placed individually in each compartment, and no food or water was given.
Animals and Experimental Design
Animal care and use. All procedures were reviewed and approved by the Animal Care and Use Committee of the Fourth Military Medical University and conform to the American Physiological Society guidelines on care and use of animals. Male Sprague-Dawley rats were obtained from the Animal Center of the Fourth Military Medical University. The rats were allowed to acclimate to the animal room of our department for 7 days before the onset of each experiment. Rats of each group were housed individually in identical 30 x 26 x 26-cm Plexiglas cages in the same room. Food and water were provided ad libitum throughout the course of the experiment. The animal room was maintained at 23°C on a 12:12-h light-dark cycle (lights on at 0600).
Experimental design. Three separate protocols were carried out in this study.
In protocol 1, the counteracting effectiveness of daily STD of various durations in preventing/alleviating muscle and bone atrophy was evaluated over 28 days of HU. Thirty-five adult male Sprague-Dawley rats (250–260 g body wt) were randomly assigned to five groups (n = 7/group): control (CON), tail suspension (SUS), suspension for 23 h/day and STD for 1 h/day (SUS + STD1), suspension for 22 h/day and STD for 2 h/day (SUS + STD2), and suspension for 20 h/day and STD for 4 h/day (SUS + STD4). During the 28-day period, daily gravitation treatments were conducted between 0800 and 1200.
In protocol 2, the effectiveness of daily treatments at 1 G (STD), 1.5 G, or 2.6 G (CEN) for 1 h/day in preventing/alleviating muscle and bone atrophy was evaluated over 28 days of HU. Thirty male Sprague-Dawley rats (150–160 g body wt) were randomly assigned to five groups (n = 6/group): control (CON), tail suspension (SUS), suspension for 23 h/day and STD for 1 h/day (SUS + STD1), suspension for 23 h/day and centrifugation at 1.5 G for 1 h/day (SUS + CEN1.5G), and suspension for 23 h/day and centrifugation at 2.6 G for 1 h/day (SUS + CEN2.6G). During the 28-day period, treatments were conducted between 1500 and 1700.
In protocol 3, the effectiveness of daily STD for 1, 2, or 4 h/day in preventing contractility depression of cardiac muscle and SOL atrophy was determined over 28 days of HU. Fifty male Sprague-Dawley rats (140–150 g body wt) were randomly assigned to five groups (n = 10/group): control (CON), tail suspension (SUS), suspension for 23 h/day and STD for 1 h/day (SUS + STD1), suspension for 22 h/day and STD for 2 h/day (SUS + STD2), and suspension for 20 h/day and STD for 4 h/day (SUS + STD4). During the 28-day period, treatments were conducted between 0800 and 1200.
Qualitative and Quantitative Alterations in Skeletal Muscles
Tissue preparation and histochemistry. At the end of the 28-day suspension period, the rats from protocols 1 and 2 were anesthetized with pentobarbital sodium (40 mg/kg ip) and killed by exsanguination via the abdominal aorta. The left adrenal and testis were removed and weighed. Four muscles of the left hindlimb, SOL, medial gastrocnemius (MG), lateral gastrocnemius (LG), and extensor digitorum longus (EDL), were removed, trimmed of fat and connective tissue, and weighed. All four muscles were quick-frozen in isopentane, cooled by liquid nitrogen, and stored in a freezer at -70°C. This procedure was completed within 20 min after the muscles were removed from the rats. Within 2 wk of muscle excision, a 5-mm-thick cross section from the mid-belly of each muscle was mounted on a piece of cork with optimum cutting temperature compound. Serial 10-µm-thick cross sections of each muscle were cut in a cryostat at -20°C. The sections were then stained for myosin ATPase at an alkaline pH as described by Nwoye et al. (32).
Image processing. Fibers stained lightly and darkly are referred to as types I and II, respectively. The cross-sectional areas (CSAs) of type I and II fibers and the proportion of type I fibers were determined using an image-processing system (model Q500MC, Leica). For each muscle, five cross sections were taken, and in each section, five microscopic fields were analyzed at x100 magnification. For each muscle, the CSAs of a population of 
500 fibers were determined.
Bone Sampling and Mechanical Testing
The right femurs were immediately removed from each animal and stripped of soft tissues, and their wet weight and middiaphysial diameter (mediolateral aspect) and length were measured using an electronic balance (precision = 1 x 10-4 g; model ACA-100, Denver Instrument) and hand-held precision calipers (±0.02 mm) successively. Their volume was determined by water displacement. Then their dry (118°C for 48 h) and ash (800°C for 24 h) weights were determined. The ratio of wet mass to volume produced an estimate of bone density. The left femurs were removed and kept at -70°C for mechanical testing within 3 days. Frozen specimens were thawed in saline solution at room temperature. Femurs were placed in a material testing machine (model 1195, Instron), supported by two steel rods separated by a distance of 16 mm, and loaded to failure in three-point bending at the middiaphysis with a cross-head speed of 2 mm/min. Data for elastic and maximum loads and coefficients for bending rigidity and bending toughness were obtained and calculated from stress-strain curves.
Contractile Performance of Papillary Muscle
Contractile performance of papillary muscle was determined according to Capasso et al. (6). At the end of the 28-day suspension period, the rats of protocol 3 were anesthetized with ether. The heart was rapidly excised and placed in oxygenated Krebs-Henseleit solution. The left soleus, adrenal, and testis were also removed and weighed. Left ventricular papillary muscle was removed and suspended horizontally in a continuous-perfusion myograph. The solution was maintained at 30°C, bubbled with 95% O2-5% CO2, and perfused at 10 ml/min. The nontendinous end of the papillary muscle was inserted into a spring-loaded clip, which was mounted on a micrometer assembly and used to adjust the muscle length. The tendinous end was tied to the stainless steel hook of a force transducer (model TB-651, Nihon Kohden). Papillary muscle was driven by square-wave impulses (10-ms duration, 130% threshold voltage, 0.2 Hz frequency) from an electronic stimulator (model SEN-3301, Nihon Kohden) through platinum wires placed on both sides of the muscle. The length-tension relation was obtained after an equilibration period of 90 min, during which the length associated with maximum developed force (Lmax) was determined. The length-tension curve was generated by reducing muscle length in 2% Lmax decrements to 88% of Lmax. A reproducible sequence of 10 contractions in each step was selected for analysis. At the end of each experiment, the muscle length at Lmax was measured, the muscle was weighed, and the CSA of the papillary muscle was calculated. The isometric parameters at Lmax, i.e., developed tension (DT), resting tension, peak rate of tension rise, peak rate of tension decline, time to peak rate of tension rise, time to peak tension (TPT), and time to half-relaxation, were measured and calculated.
Statistical Analysis
Values are means ± SE, except body weight data, which are means ± SD. A one-way ANOVA was used to determine the overall differences; then a Student-Newman-Keuls post hoc test was used to determine group differences. The 0.05 level of probability was chosen as significant for all analysis.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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The results from protocols 1 and 2 are shown in Tables 1 and 2, respectively. Initial body weights were similar among different groups. At the end of 28 days of HU, there were no significant differences in final body weight between CON and SUS rats, but the final body weight was significantly less in various treatment groups than in the respective CON group (P < 0.05 or P < 0.01).
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At the end of the two experiments, the mean wet weights of the left testis of the SUS and the treatment groups were 44% and 34% of those of the respective CON group (P < 0.01), and the differences among the SUS and various treatment groups were not significant. There were no significant differences in left adrenal weight among CON, SUS, and treatment groups.
Muscle Weights
The data from protocol 1 are listed in Table 3. After 28 days of HU, the absolute wet weights of SOL, MG, LG, and EDL were 49%, 9%, 20%, and 11% less, respectively, than in CON rats (P < 0.05 or P < 0.01). Daily gravitation significantly attenuated atrophy of SOL: the wet weights of SOL were 38%, 28% (both P < 0.01), and 12% (not significant) less in SUS + STD1, SUS + STD2, and SUS + STD4 rats, respectively. However, for MG, LG, and EDL, the effects on reducing muscle atrophy were significant only when relative wet weights were considered (Table 3).
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The data from protocol 2 are listed in Table 4. After 28 days of HU, the absolute wet weights of SOL, MG, LG, and EDL were 60%, 22%, 26%, and 10% less, respectively, than in CON rats (P < 0.05 or P < 0.01). Although -Gx gravitation for 1 h/day at 1, 1.5, or 2.6 G attenuated the atrophy of SOL, increasing the G level was associated with a lessened counteracting effect. The wet weights of SOL were 33%, 41%, and 55% less in SUS + STD1, SUS + CEN1.5G, and SUS + CEN2.6G rats, respectively (P < 0.01). The effects in reducing muscle atrophy became more apparent when the changes were considered in terms of relative wet weights of muscles (Table 4).
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Muscle Fiber Types and Fiber CSA
The results from protocol 1 are depicted with the changes in relative wet weight of muscles in Fig. 1. After 28 days of HU, the mean CSAs of type I fibers of SOL, MG, LG, and EDL were 61%, 25%, 30%, and 35% less, respectively, than in CON rats (P < 0.05 or P < 0.01). The mean CSAs of type II fibers of SOL, MG, and LG were also reduced: 45%, 17%, and 19% less, respectively, than in CON rats (P < 0.05 or P < 0.01). HU for 28 days decreased the percentage of type I fibers in SOL and MG from 70% and 58% to 46% and 41%, respectively, whereas it had no significant effects on the percentage of type I fibers in LG and EDL (Fig. 1). Daily -Gx gravitation by STD resulted in a significant reduction in the shrinkage of type I and II fibers in SOL, MG, and LG and type I fiber in EDL. It also significantly reduced the decrease in the percentage of type I fibers in SOL and MG.
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Figure 2, in which results are presented from protocol 2, shows that after 28 days of HU the mean CSAs of type I fibers of SOL, MG, LG, and EDL were mostly affected, being 69%, 30%, 27%, and 25% less, respectively, than in CON rats (P < 0.05 or P < 0.01), and those of type II fibers were less affected, being 50%, 17%, and 20% less in SOL, MG, and LG, respectively, than in CON rats (P < 0.05 or P < 0.01). HU for 28 days also resulted in a significant decrease in the percentage of type I fibers in SOL and MG from 70% and 60% to 36% and 35%, respectively (P < 0.01). The efficacy of 1 h/day of periodic gravitation at 1 G (STD), 1.5 G (CEN), or 2.6 G (CEN) was related to the kind of muscle and the G level. For SOL and MG, the efficacy in maintaining the fiber size and fiber type composition was, in most cases, significantly decreased as the G level was increased from 1 to 2.6 G (Fig. 2, A–C). In EDL, CEN2.6G seemed to be more effective.
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Physical and Mechanical Properties of the Femur
Data from protocol 1 are listed in Table 5. After 28 days of HU, the wet, dry, and ash weights, diameter, and density of the femur were significantly decreased in SUS rats compared with CON rats (P < 0.01). Dry weight, ash weight, and diameter of the femur were also significantly lower in all the "SUS plus STD" groups than in the CON group (P < 0.01 or P < 0.05) but were significantly higher than in SUS rats in some cases (P < 0.01 or P < 0.05). The mechanical parameters, i.e., elastic load (Fe), maximum load (Fm), and bending rigidity coefficient (Cr), were significantly lower (P < 0.01) in SUS than in CON rats. Fe, Fm, and Cr were also significantly lower in SUS + STD than in CON rats (P < 0.05 or P < 0.01) but, in most cases, were significantly higher than in SUS rats (P < 0.01 or P < 0.05).
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Data from protocol 2 are listed in Table 6. Two weight parameters and diameter and density of the femur were significantly lower in the SUS and the three countermeasure groups than in the CON group (P < 0.01); however, length of the femur was significantly greater in SUS and SUS + 1.5 G rats (P < 0.05). In most cases, there were no significant differences in these physical parameters between the SUS group and each of the three countermeasure groups. Fe, Fm, and Cr were significantly lower in the SUS group and the three countermeasure groups than in the CON group (P < 0.01), but, in most cases, these parameters were significantly higher in the countermeasure groups than in the SUS group (P < 0.01 or P < 0.05). However, there were no significant differences in physical and mechanical parameters among the three countermeasure groups of different G magnitude.
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Cardiac Muscle Contractility
With respect to the general conditions of the animals from protocol 3, there were no significant differences between the CON and any of the other three experimental groups, except for the final body weight of the SUS + STD4 group, which was significantly less (P < 0.01) than the CON group (Table 7). However, 28 days of HU resulted in a significant weight loss in SOL of SUS rats, and daily STD had an obvious ameliorating effect on weight loss. Table 8 shows isometric myocardial contraction data. No significant differences were noted in resting tension or CSA of papillary muscles among the five groups. SUS for 28 days resulted in significant reductions in DT, peak rate of tension rise, and peak rate of tension decline compared with the CON rats (P < 0.05). The timing parameters, i.e., time to peak rate of tension rise and TPT, were significantly increased in SUS rats (P < 0.05). Daily -Gx gravitation by STD for 1, 2, or 4 h showed a significant effect in preventing the adverse changes in mechanical and timing parameters due to SUS alone (P < 0.01 or P < 0.05).
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Passive and active length-tension relations among the five groups are depicted in Fig. 3. No significant differences in resting length-tension relations were noted among the five groups. However, the curve describing the active length-tension relation of the papillary muscle of the SUS rats shifted significantly downward (P < 0.01) compared with the curves of the other four groups (P < 0.01). Further analysis showed no significant differences in DT at any muscle length among the CON and the three SUS + STD groups.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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To titrate exclusively the effectiveness of periodic passive -Gx vector gravitations of different exposure durations and at different G levels, STD for 1, 2, or 4 h/day was used to provide periodic -1 Gx at 1 G and CEN at 1.5 or 2.6 G for 1 h/day was used to provide periodic -Gx hypergravitation at >1 G to SUS rats. In previous studies (11–13, 36, 41), STD ("stationary ground support" or "weight bearing") has been used because of its effectiveness in preventing muscle atrophy, but rarely osteopenia. CEN is the only choice allowing us to titrate the effectiveness of periodic -Gx hypergravitation at various G levels and has also been used in related studies (10, 11, 29, 30). During 28 days of HU, SUS alone did not affect the body weight gains of the rats, but SUS combined with various periodic gravitation treatments significantly decreased the final body weight gains in most cases. However, decreases in final body weight of treatment groups of protocol 1 were <10%. The reasons remain unclear. Restriction of movements in small tubelike cages during STD and reduced food consumption and more physical activity in treatment groups might be important factors. Although reduced final body weight gains in treatment groups suggest possible stressful influences of periodic gravitations, weights of the adrenals showed no significant differences among different experimental groups. Perhaps this kind of stress is insufficient to enlarge significantly the absolute adrenal mass (11) or induce only transient adrenal hypertrophy (30, 31, 42). The influence of daily short-duration hypergravitation on growth rate seems not to be a surprising result, because chronically centrifuged rats manifest signs and symptoms of stress, such as immobility, anorexia, and impaired thermoregulation, in the first few days and, finally, a decrease in growth rate and body mass (5, 11, 12, 30). Another disadvantage of using centrifugation to mimic IAG in animal studies is that the centrifuged rats usually assume a "rump-high crouching" posture (30), and the G forces imposed might not be transmitted via their limbs in a standing or walking posture (12). Another limitation of the present study is that measures were not taken to prevent cryptorchidism in the HU rats, which prevented collection of reliable data on the effectiveness of daily gravitation in preventing adverse changes in the testis (18). Despite these limitations, the main findings of the present study are most likely to be the consequence of intermittent -Gx gravity exposure on various physiological systems of HU rats.
Effectiveness of Daily Short-Duration -Gx Exposure by STD in Preventing Myocardial Contractility Depression Over 28 Days of HU
In the present study, as has been shown in our previous work (52, 55), 28 days of HU resulted in a significant reduction in DT and a significant prolongation in TPT of isolated papillary muscle. The present study showed for the first time that depression of myocardial contractility can be prevented by STD for 1 h/day over the 28 days of HU (Table 8). In the same study, 62%, 76%, and 89% of the left soleus wet weight was maintained by STD for 1, 2, and 4 h/day, respectively (Table 7).
Conflicting data have been reported for the effects of microgravity on cardiac mass and contractility. In most studies, decreased heart size during spaceflight observed by echocardiography was attributed primarily to reduced cardiac chamber volume and not loss of heart muscle. Also, indexes of cardiac contractility remain unchanged or increased slightly (for review see Refs. 7 and 47). However, evidence showing a loss of ventricular mass and a decrease in contractility of cardiac muscle in astronauts and bed-rest subjects has also been reported (2, 21, 28, 47). In D-2 astronauts and bed-rest subjects, marked reductions of 12% and 7.4%, respectively, in cardiac mass detected by magnetic resonance imaging (MRI) have been demonstrated (28). The atrophic change in cardiac muscle has been suggested as a nonneural mechanism (28, 54) accounting for postflight orthostatic intolerance. Conflicting data have also been reported in animal studies. Goldstein et al. (14) reported that the average cross-sectional area of fibers of left ventricular papillary muscle decreased significantly by 20% in rats flown on COSMOS 2044 for 14 days. Our previous work (52, 55) showed the nature and time course of depression in contractility of papillary muscle of rats subjected to 4 and 13 wk of tail suspension. Koenig et al. (25) demonstrated with invasively instrumented rhesus monkeys that, after 4 days of exposure to head-down tilt, the reduced orthostatic tolerance during graded lower body negative pressure was associated with a lowered cardiac contractility. Ray et al. (35) reported that 7 days of microgravity exposure and 7 or 28 days of HU did not reduce cardiac mass in rats. Additionally, simulated microgravity also had no effect on the peak rate of rise in left ventricular pressure. Reviewing the relevant work, they further indicated that reductions in cardiac mass might be related to relatively large reductions in body mass that occur in conjunction with the HU treatment. They further pointed out that when the reductions in body mass are relatively small (e.g., <10–12%), no change in cardiac mass occurs (35). Reductions in cardiac mass also paralleled the decrease in body mass due to chronic food restriction (17). In protocol 3 of the present study, the final body weights were comparable among CON, SUS, and the two treatment groups; the final body weight was 5.6% less in the SUS + STD4 group than in the CON group. Hence, the possible confounding influence of less gain in final body weight can be ruled out, although cardiac mass was not measured.
Despite the progress that has been made, whether hypovolemia and hypokinesia/hypodynamia during microgravity may lead to a reduction in cardiac mass and a decrement in performance (28, 52, 54) remains to be answered. To address these issues, methods with extremely good precision in measuring cardiac mass, such as MRI, should be considered in human studies (28). In animal studies, load-insensitive, inotropic state-sensitive index of cardiac contractility, such as Emax, the slope of the end-systolic pressure-volume relation (24, 37), and morphometric analysis (1) should be used to measure the cardiac performance and to quantitatively analyze the morphology of the myocardium, respectively. Reliable data and detailed information about central and ventricular hemodynamics during microgravity exposure also appear to be crucially important in understanding cardiac adaptation to microgravity (25, 28, 35, 47, 54). Nevertheless, the present study has provided data suggesting that intermittent gravitation might be a potential countermeasure to prevent myocardial changes if they occur due to microgravity.
Effectiveness of Daily Short-Duration -Gx Exposure by STD or CEN in Preventing Muscle Atrophy Over 28 Days of HU
In this study, as has been shown by others (16, 36), the relative effect of HU on muscle mass (based on wet weight) was as follows: slow extensors (SOL) > fast extensors (MG and LG) > fast flexors (EDL). The HU-induced changes in fiber CSAs of type I and II fibers and percentages of type I fibers in the four kinds of muscles showed a pattern similar, but not identical, to that of the absolute wet weight of respective muscles (Figs. 1 and 2), which corroborates previous studies on fiber-size and fiber-type changes in response to simulated microgravity in rats (12, 13, 36).
One important finding of the present study was that daily STD treatment significantly attenuated the atrophy of SOL, MG, LG, and EDL over 28 days of HU (Table 3, Fig. 1). According to the changes in most of the indexes of muscle atrophy, prolonging the exposure duration from 1 to 2 to 4 h/day showed a further atrophy-alleviating effect, with STD for 4 h/day being sufficient to maintain the muscle fiber CSAs, fiber type composition, and relative wet weight of all four muscles of suspended rats comparable to CON rats. However, with regard to the index of absolute wet weight, only SOL and MG showed complete prevention. Although the efficacy of STD had been reported by several groups (11–13, 36, 41), the present study titrated the efficacy of STD from 1 to 2 to 4 h/day, indicating clearly the duration dependency of the effectiveness of intermittent STD in attenuating HU-induced muscle atrophy. This duration dependency also supports the contention that the countermeasure effectiveness is due to the treatment. In similar work (15, 20, 22), despite a loss of initial body weight or no weight gain, the countermeasure effects were still manifest. It is apparent that this efficacy should also be related to the sustaining period of HU (12, 13). However, most of the pervious studies with a total of 40–60 min/day of STD as countermeasure were conducted over 7 days of HU (11, 15, 20, 22, 34). It has been shown that similar treatment was less effective over 14 days of HU than over 7 days of HU (48). In this context, Thomason et al. (41) showed that 2 or 4 h/day of stationary ground support for 4 wk significantly reduced the atrophic response of SOL, adductor longus, plantaris, and vastus lateralis, resulting in a sparing of myofibril protein and the relative and absolute slow myosin isoform contents of the SOL. The findings of the present study are consistent with those reported by Thomason et al. From the results of 7-day studies (11, 15, 20, 22, 34, 36), it could be speculated that much shorter periods of STD interspersed throughout the day would be more effective than one long daily period of STD of the same total duration. Another surprising finding is that a very short duration of STD with high-resistance intermittent exercise (high-load-bearing climbing) was more effective than STD alone in preventing the HU-induced atrophy of the SOL (22). In addition, the effectiveness of STD for 40 min/day alone (3, 48) or combined with resistance exercise (49) during 14 days of HU has also been examined and shown in single type I SOL fibers on the basis of their force-velocity-power and force-pCa relations.
Another important observation of this study was that as the magnitude of the daily 1-h gravitation increased from 1 G to 1.5 G to 2.6 G, the efficacy of alleviating muscle atrophy was markedly reduced in SOL and MG but increased in EDL (Table 4, Fig. 2). The design of protocol 2 was similar to that of D'Aunno et al. (10), except the latter was a 7-day study that was confined to SOL with muscle weight as the only index of atrophy. Their results are inconsistent with our results, in that they show no significant differences among 1 G (STD), 1.5 G, and 2.6 G (CEN) (10). This discrepancy might be related to the difference in the sustaining period of HU between the two studies.
Effectiveness of Daily Short-Duration -Gx Exposure by STD or CEN in Alleviating Bone Atrophy Over 28 Days of HU
The data in Table 5 clearly indicate that HU in rats for 28 days resulted in adverse changes in physical and mechanical parameters of the femur. The results corroborate previous studies on adverse changes in physical and mechanical properties of bone due to real/simulated microgravity (33). Among the physical parameters, the decreases in wet weight, diameter, and density of the femur were prevented by daily short-duration STD over 28 days of HU, whereas the decreases in dry weight, ash weight, and Fe, Fm, and Cr were only partially alleviated in most cases by daily STD. Two recent reports also favor the limited effectiveness of STD in preventing bone atrophy due to non-weight bearing. Jain et al. (23) showed that STD for 2 h/day resulted in prevention of wet weight loss (water content reduction) of the tibia but no improvement in ash weight and tibia calcium content reductions over 15 days of HU. They showed further that STD for 4 h/day was only partially successful in preventing the bone demineralization (23). In the suspended rats, 50% forelimb weight bearing was associated with bone loss in the humerus, which was not ameliorated by 2 h of full weight bearing daily over 35 days of HU (38).
Data from protocol 2 showed that 1, 1.5, or 2.6 G for 1 h/day significantly improved physical and mechanical parameters of the femur relative to SUS alone; however, all the data were still significantly lower than for CON rats (Table 6). Although there were no significant differences in physical and mechanical parameters examined among the three treatment groups, our unpublished histochemical and immunohistochemical findings further demonstrated that 1 G (STD) or 1.5 G (CEN) for 1 h/day appeared to be more effective than 2.6 G. The latter corroborates the results reported by Montufar-Solis et al. (29, 30), obtained from histomorphometric analysis of tibial growth plates, suggesting the possibility of a range of G that will maintain the normal growth and homeostasis of the bone. Finally, rats used in protocols 1 and 2 were of growing age, and their body weights were <400 g in protocol 1.
Comparison of Countermeasure Effectiveness of Daily -Gx Exposure Among Different Physiological Systems
Although previous work supports IAG as a potential countermeasure (40, 43–46), comprehensive studies over longer simulation periods are needed to further examine the physiological requirements for gravity of various physiological systems. If the requirements varied to a greater extent, it would appear to be inappropriate to consider IAG as an important protocol for future gravity-based countermeasures.
The present findings suggest that the physiological requirements for gravity may vary greatly among different organ systems. It is surprising that STD for 1 h/day was sufficient to prevent myocardial contractility depression and differential vasoreactivity changes in vessels (58) over 28 days of HU. On the other hand, STD for 4 h/day was effective in preventing mass reduction and histomorphometric changes in the SOL. However, the bone mass and mechanics were more resistant to such treatments, being partially alleviated by STD for 4 h/day over 28 days of HU. Further study is needed to determine whether daily short-duration gravitation combines with supplemental countermeasures, such as exercise and dynamic loading (26, 38), would be sufficient to prevent bone atrophy.
In contrast to human studies, our findings on myocardial contractility and vasoreactivity support the results obtained in a 4-day head-down bed-rest study (43, 44) that STD for 2 h/day completely prevented orthostatic intolerance after head-down bed rest. Our results on bone atrophy also support the possibility that STD for 2 or 4 h was only partially effective in alleviating increased calcium excretion. However, in the same study, walking for 2 or 4 h was more effective, indicating the importance of muscle activity and impact loading in maintaining bone density. With respect to the G level appropriate for IAG, Vil-Viliams et al. (46) pointed out that it should be close to 1 G (0.8–1.6 G), which can be well tolerated by most subjects. The present study and related work (10, 11, 29, 30) with rats also showed no additional benefit of increasing G level in preventing muscle and bone atrophy.
In conclusion, we are just starting to understand the physiological requirements of intermittent exposure to gravity in preventing adverse changes during real/simulated microgravity. In the present study, we demonstrated that the responsiveness of cardiac muscle, hindlimb muscle, and bone of HU rats to daily short-duration -Gx gravitation varies greatly. Therefore, comprehensive ground-based human and animal studies involving a multitude of physiological systems should be conducted to further evaluate the countermeasure effectiveness of intermittent gravity exposure on various tissues/organs (particularly bone loss) over longer periods of simulated microgravity.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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This research was supported by National Natural Science Foundation of China Grant 30171032 and Defence Medical Fund Grants 98Z083 and 01Q114.
Preliminary results have been published as meeting proceedings (57).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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