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Daily short-period gravitation can prevent functional and structural changes in arteries of simulated microgravity rats

Departments of ¹Aerospace Physiology, ²Anatomy, and ³Pathology, Fourth Military Medical University, Xi'an 710032, People's Republic of China

Submitted 23 February 2004 ; accepted in final form 20 April 2004

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This study was designed to clarify whether simulated microgravity-induced differential adaptational changes in cerebral and hindlimb arteries could be prevented by daily short-period restoration of the normal distribution of transmural pressure across arterial vasculature by either dorsoventral or footward gravitational loading. Tail suspension (Sus) for 28 days was used to simulate cardiovascular deconditioning due to microgravity. Daily standing (STD) for 1, 2, or 4 h, or +45° head-up tilt (HUT) for 2 or 4 h was used to provide short-period dorsoventral or footward gravitational loading as countermeasure. Functional studies showed that Sus alone induced an enhancement and depression in vasoconstrictor responsiveness of basilar and femoral arterial rings, respectively, as previously reported. These differential functional alterations can be prevented by either of the two kinds of daily gravitational loading treatments. Surprisingly, daily STD for as short as 1 h was sufficient to prevent the differential functional changes that might occur due to Sus alone. In morphological studies, the effectiveness of daily 4-h HUT or 1-h STD in preventing the differential remodeling changes in the structure of basilar and anterior tibial arteries induced by Sus alone was examined by histomorphometry. The results showed that both the hypertrophic and atrophic changes that might occur, respectively, in cerebral and hindlimb arteries due to Sus alone were prevented not only by daily HUT for 4 h but also by daily STD even for 1 h. These data indicate that daily gravitational loading by STD for as short as 1 h is sufficient to prevent differential adaptational changes in function and structure of vessels in different anatomic regions induced by a medium-term simulated microgravity.

hindlimb unweighting; cardiovascular deconditioning; postflight orthostatic intolerance; arteries; vascular remodeling; vasoreactivity; countermeasure; intermittent artificial gravity

Before a short-arm centrifuge can be tested in space, research must first elucidate whether intermittent gravity exposure requirements vary greatly for different physiological systems. System specificity should be considered as one of the prerequesties in proposing IAG as a potential countermeasure. Vernikos et al. (35) reported that daily standing (STD) or walking for 2 or 4 h was effective in most cases to counteract the deconditioning effects of a 4-day, –6° head-down bedrest. They concluded that various physiological systems benefit differentially from daily short-period passive +1 vertical acceleration (G_z), or activity in +1 G_z, i.e., the preventive value appears to be system specific (35). Our laboratory's previous work (46) has indicated that the minimum gravity exposure requirements vary greatly among cardiac and skeletal muscles and bone in rats. However, no relevant information is yet available on vessels. We further demonstrated that increasing gravity intensity to 2.6 G by centrifugation in dorsoventral (–G_x) vector (46) or daily +45° head-up tilt (HUT) as a means to increase hindlimb venous pressure and postural load (34) showed less benefit or no additional benefit in attenuating muscle and bone atrophy.

Adaptation of vessels to microgravity exhibits obvious regional specificity. Simulated microgravity may differentially induce upward and downward regulations in the structure, function, and innervation state of the cerebral and hindquarter vessels in rats (9, 10, 15, 21–23, 30, 41, 45, 49). Findings from bedrest and spaceflight human studies have indicated that the inability to adequately elevate the peripheral resistance and the altered autoregulation of cerebral vasculature are important factors responsible for postflight orthostatic intolerance (1, 8, 14, 16, 39, 45, 47). Hence, it is of great interest to consider the effectiveness of IAG in alleviating/preventing differential adaptations of vessels to microgravity. The findings in human studies that daily short-period standing or walking (35) or centrifugation to +0.8–1.6 G_z (36) can prevent postbedrest orthostatic intolerance or post-dry-immersion reduction in +G_z tolerance have suggested such effectiveness of IAG on vascular changes. We thus hypothesized that daily short-period restoration of the distribution of transmural pressure across arterial vasculature and hence the physical stress acting on arterial wall to their normal levels by daily STD or HUT would be effective in preventing differential adaptational changes in vessels from different anatomic regions due to simulated microgravity in rats. In the present study, the basilar and femoral/anterior tibial arteries were chosen to verify the hypothesis. Therefore, the aims of the present study were 1) to examine whether differential vasoreactivity changes in cerebral and hindlimb arteries could be attenuated/prevented by daily short-period STD for 1, 2, or 4 h, or +45° HUT for 2 or 4 h during a 28-day simulated microgravity; and 2) to examine whether differential structural changes in basilar and anterior tibial arteries could also be prevented by daily 4-h HUT or 1-h STD during 28 days of simulated microgravity. A part of the preliminary results concerning structural changes have been published as a meeting proceeding (48).

	MATERIALS AND METHODS

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Animals and Experimental Design

The experimental protocols and procedures described below were approved by the Animal Care and Use Committee of the Fourth Military Medical University and conform to American Physiological Society guidelines on care and use of animals.

Three separate protocols were carried out in this study.

Protocol 1.   In protocol 1, the effectiveness in preventing differential changes in vasoconstrictor responsiveness of basilar and femoral arteries was evaluated over 28 days of simulated microgravity. This study was a part of a research project requiring that effectiveness in preventing the deconditioning in a multitude of organs be examined on the same animal during 4 wk of simulated microgravity. Findings concerning skeletal and cardiac muscles and bone have been reported previously (34, 46). Briefly, 49 male Sprague-Dawley rats were randomly assigned to seven experimental groups (n = 7 rats/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), suspension for 20 h/day and STD for 4 h/day (Sus + STD₄), suspension for 22 h/day and +45° HUT for 2 h/day (Sus + HUT₂), and suspension for 20 h/day and +45° HUT for 4 h/day (Sus + HUT₄). During the 28-day period, daily gravitational treatments were conducted between 0800 and 1200.

Protocol 2.   In protocol 2, the effectiveness of daily +45° HUT for 4 h/day in preventing simulated microgravity-induced structural changes in the wall of basilar and anterior tibial arteries was evaluated over 28 days of simulated microgravity. Eighteen male Sprague-Dawley rats were randomly assigned to three groups (n = 6): Con, SUS, and Sus + HUT₄. During the 28-day period, daily gravitational treatments were conducted between 0800 and 1200.

Protocol 3.   Protocol 3 incorporated two sets of experiments. In experiment 1, the effectiveness of daily STD for 1 h/day in preventing simulated microgravity-induced structural changes in the wall of basilar and anterior tibial arteries was evaluated over 28 days of simulated microgravity. Fifteen male Sprague-Dawley rats were randomly assigned to three groups (n = 5 rats/group): Con, Sus, and Sus + STD₁. During the 28-day period, daily gravitational treatments were conducted between 0900 and 1000. Experiment 2 was performed to examine whether daily STD alone would affect the structural parameters of the wall of both basilar and anterior tibial arteries. Twenty-eight male Sprague-Dawley rats were randomly assigned to four groups (n = 7 rats/group): ambulatory control, ambulatory for 22 h/day and STD for 2 h/day, Sus, and Sus + STD₂. During the 28-day period, daily gravitational treatments were conducted between 0900 and 1100.

Tail-suspended Hindlimb Unloading Rat Model

The technique of Sus (26, 42) with modification from our laboratory (4) was used to simulate the cardiovascular deconditioning effects of microgravity in rats. Briefly, the tail was cleaned and air dried and sprayed with a generous amount of benzoin and resin. A strip of adhesive tape looped over a plastic bar in the middle of the tape was then 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 fish swivel mounted at the top of the Plexiglas cage. The animals were maintained in –30° head-down-tilt position with their hindlimbs unloaded. All animals received standard rat chow and water ad libitum and were caged individually in a room maintained at 23°C on a 12:12-h light-dark cycle (lights on at 0600). The simulation period lasted 28 days.

Modes of Daily Short-duration Gravitation Treatment

Two modes of gravitational loading, i.e., stationary ground support, or STD and +45° HUT were adopted to simulate the counteracting effects of IAG.

HUT.   For short-duration HUT exposure, the suspended rat was released from suspension and then placed into a 50-cm-long, tube-like metallic mesh cage, which was maintained at the +45° angle. Within the cage, the rat could move upward and downward in a head-up position, but it could not turn around. One component gravity vector was in +G_z (from head to tail) direction. Food and water were provided ad libitum at the front end of the cage.

STD.   For short-duration STD exposure, the suspended rat was released from suspension and then placed in a similar cage maintained in a horizontal position. The gravity vector was in the –G_x (from dorsal to ventral) direction.

Vessel Preparation and Vasoreactivity Measurement

At the end of the 28-day simulation period, rats from protocol 1 were anesthetized with pentobarbital sodium (40 mg/kg ip) and killed by exsanguination via the abdominal aorta. The basilar arteries were carefully isolated from the brain under a dissecting microscope. Simultaneously, the femoral arteries were collected. These vessel segments were freed of fat and connective tissues and then cut into rings (in length: basilar artery, 2 mm; femoral artery, 3 mm) with scissors.

For force measurement, two stainless steel wires (each 40 µm in diameter) were carefully passed through the lumen of a basilar arterial ring under a dissecting microscope. The two ends of each wire were fixed to a specially made plastic holder designed according to Mulvany and colleagues (27, 28) with modifications (49). Then each wire was stretched tightly by adjusting two fine screws on each side of the holder. One holder was fixed to a micrometer-controlled device to allow the vessel to be stretched by known increments. The other was tightly attached to a force-displacement transducer (TB-651T, Nihon Kohden, Tokyo, Japan), which was connected via an amplifier to a polygraph recorder (RM-6000, Nihon Kohden) for isometric force recording. Similarly, femoral arterial rings were mounted on two stainless steel wires (0.2 mm in diameter) and connected to a force-displacement transducer and micrometer-controlled device. Each vessel apparatus was mounted in an organ bath (20-ml volume for basilar arterial ring, 10-ml volume for femoral arterial ring) filled with Krebs solution. The Krebs solution was maintained at 37°C and was continuously bubbled with a gas mixture of 95% O₂-5% CO₂. After a 60-min equilibration at zero resting force, the vessel rings were individually stretched by 40-mg increments. At each increment of passive resting force, the contractile response to 100 mM KCl was determined until optimal resting force was identified for each individual vessel ring. Thus all subsequent pharmacological experiments were started after the rings were allowed to equilibrate at these initial resting forces for 60 min. Contractile responses to drugs are presented as absolute values in milligrams or grams of force.

Changes in vascular responsiveness of basilar and femoral arterial rings to 10–100 mM KCl from Con, Sus, and Sus plus daily gravitation groups from protocol 1 were examined and compared. Concentration-response relationships were determined by cumulative addition of KCl (10–100 mM) to rings with endothelium intact.

Tissue Preparation and Histomorphometry by Light and Electron Microscopy

At the end of the 28-day simulation period, the rats in protocols 2 and 3 were anesthetized with pentobarbital sodium (40 mg/kg ip) and heparinized (1,000 U/kg ip). The chest was opened by a midthoracic incision to expose the heart and aortic arch. A perfusion cannula was inserted through the left ventricular wall and advanced into the root of the aorta, the ventricle was tightly ligated with a nylon suture to prevent back flow, and the right atrium was opened for effluent. Krebs solution (37°C), containing 10 µg/ml sodium nitroprusside to induce systemic vasodilation, was infused into the vascular system at 80 mmHg (MP-25S transducer, Nihon Kohden) for 15 min. Then the vasculature was perfused and fixed with 250 ml of 4% formaldehyde in PBS. Anatomically defined segments of basilar and anterior tibial arteries were immediately removed. The two landmarks for the two arterial segments were 1 mm distal to the convergence of the two vertebral arteries and 1 mm from popliteal arteries, respectively. Vessel samples were then postfixed in the same fixative solution overnight. Vessel segments from rats of protocol 2 and experiment 1 of protocol 3 were treated in 1% osmium tetroxide for 2 h, then dehydrated in a series of ethyl alcohol baths of progressively increasing concentration and 100% acetone, and cleared in several xylene washes before being embedded in Epon 812. Thick (5 µm) cross sections for light and thin (70–80 nm) longitudinal middlemost sections for electron microscopy were taken. The thick cross sections were mounted on glass slides and stained with toluidine blue, which deeply stains the media in contrast to the internal elastic lamina and adventitia. Vessel wall media cross-sectional area (CSA), media thickness (T), and luminal diameter (D) were measured under an optical microscope and analyzed by an image analysis system (Leica Q570C). T and D were measured at four points separated by 90° angles and averaged from at least five measurements on three sections from each vessel. The thin longitudinal sections were mounted on grids, stained with uranyl acetate and lead citrate, and examined with H-300 electron microscope (Hitachi). The thin sections provide cross-sectional views of the smooth muscle cells due to their circumferential arrangement. Thus mean cell CSAs (A_C) were measured and calculated. Then the mean number of smooth muscle cell layers (N_CL) in the media was calculated by dividing the mean T by the mean cell diameter, which was derived from the A_C (28, 38).

In experiment 2 of protocol 3, only thick cross sections (5 µm) were taken from the middlemost section of the basilar and anterior tibial arteries embedded in parafin. The 5-µm-thick sections were mounted on glass slides and stained by the van Gieson-Orcein method (23). Parameters T and D were measured under an optical microscope and analyzed by an image analysis system (Leica Q570C). Vessel wall media CSA was calculated by the equation CSA = [(r + T)² – r²], where r is the luminal radius (r = D/2).

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, and then a Student-Newman-Keuls post hoc test was used to determine the significance of group differences. Means are considered significantly different when the P value is <0.05.

	RESULTS

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Body weight data of rats for all experimental groups from protocol 1 have been summarized in Table 1 from data in our laboratory's previous papers (34, 46). In both protocol 2 and 3, the initial body weights were similar among different groups, and there were no significant differences in final body weight among Con, Sus, and the treatment groups (Table 2).

As shown in Figs. 1 and 2, cumulative application of KCl elicited concentration-dependent contractile responses in all ring preparations of the two kinds of arteries. After 28 days of Sus, the maximal isometric contractile responses of basilar and femoral arterial rings to KCl were significantly increased (P < 0.05) and decreased (P < 0.05), respectively, compared with those of respective arterial rings from Con rats. However, daily gravitational loading by HUT for 2 or 4 h, or STD for 1, 2, or 4 h, prevented the differential functional changes in different vessels due to suspension alone. The KCl-induced maximal contractile responses of both basilar and femoral arterial rings of the five daily gravitation groups showed no statistical differences compared with those of respective Con rats, whereas the differences with those of Sus rats were statistically significant (P < 0.05). Among different groups, the differences of the agonist concentrations that produced 50% of the maximal vasoconstrictor response values were statistically nonsignificant (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Concentration-response curves showing contractility to KCl for basilar (A) and femoral (B) arterial rings from control (Con), tail-suspension (Sus), and tail-suspension plus standing (Sus + STD) rats. Values are means ± SE; n = 7 per group. EC50, concentrations that produced 50% of the maximal vasoconstrictor response values. NS, non-significant. *Significant differences (P < 0.05) between Sus and either of the other 2 groups, i.e., Con and Sus + STD.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Concentration-response curves showing contractility to KCl for basilar (A) and femoral (B) arterial rings from Con, Sus, and Sus plus head-up tilt (Sus + HUT) rats. Values are means ± SE; n = 7 per group. *Significant differences (P < 0.05) between Sus and either of the other 2 groups, i.e., Con and Sus + HUT.

In protocol 2, the light micrographs of cross sections of basilar and anterior tibial arteries from Con, Sus, and Sus + HUT₄ rats show that the vessel walls are smooth without invaginations and that the thickness is consistent, verifying that the vessels were fully relaxed and rightly cross-sectionally cut. It allowed a reliable and accurate measurement of vessel wall dimensions. From electron micrographs of longitudinal sections of basilar and anterior tibial arteries from Con, Sus, and Sus + HUT₄ rats, outlines of smooth muscle cells can be distinguished and satisfactory for the measurement of CSA of each smooth muscle cell. The histomorphometric data are summarized in Table 3. Sus for 28 days resulted in differential structural changes in basilar and anterior tibial arteries. Compared with those of the Con group, T and media CSA of basilar artery from Sus increased by 53% (P < 0.01) and 17% (P < 0.01), respectively, whereas the T and CSA of anterior tibial artery from Sus decreased by 19% (P < 0.01) and 15% (P < 0.05), respectively (Table 3). Moreover, N_CL in the media of basilar artery of Sus rats was significantly greater than that of Con (P < 0.01), whereas the N_CL in the media of anterior tibial arteries of Sus group was significantly less than that of Con (P < 0.05). However, daily +G_z gravitation by 4 h/day HUT completely prevented these changes due to suspension alone. There were no significant differences in T, CSA, and N_CL between Sus + HUT₄ and Con groups (Fig. 3 and Table. 3). Both D and CSA of smooth muscle cell (A_C) of the two kinds of vessels showed no significant differences among Con, Sus, and Sus + HUT₄ groups (Table 3).

View larger version (32K): [in this window] [in a new window]   Fig. 3. Relative changes in media thickness (T), media cross-sectional area (CSA), and number of smooth muscle cell layers (NCL) in the media of basilar and anterior tibial arteries from rats of Con, 28-day Sus, and 2 gravitational treatment groups [Sus + HUT4 (A) and Sus + STD1 (B)]. Values are means ± SE; n = 6 or 5 per group. *P < 0.05, **P < 0.01 vs. Con; +P < 0.05, ++P < 0.01 vs. Sus.

View larger version (96K): [in this window] [in a new window]   Fig. 4. Electron micrographs of longitudinal sections of the wall of basilar (top) and anterior (bottom) tibial arteries from Con, a 28-day Sus, and Sus + STD1 rats. Vessel anatomy, with regions of internal elastic lamina (IEL), media (M), and adventitia (A), is clearly visible.

	DISCUSSION

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It has been shown that simulated microgravity may induce differential structural and functional changes in cerebral and hindquarter arteries due to changes in local prevailing hemodynamic conditions (45). Furthermore, it has also been shown for hindquarter arteries that these adaptational changes are more striking in distal arteries with relatively larger medial area (45). For example, for femoral artery, the medial CSA decreased by 4 and 7%, whereas for anterior tibial artery CSA decreased by 10 and 21%, respectively, after 2 and 4 wk of simulated microgravity (23, 45). To our knowledge, a comparison of vasoreactivity changes between femoral and anterior tibial arteries of the same animal during simulated microgravity has never been made. However, as inferred from the data from abdominal aorta and mesenteric and femoral anteries (22, 45), functional change of these two kinds of hindlimb vessels should be similar with that of anterior tibial artery being more striking. For methodological reasons, particularly for the convenience in comparing structural changes of the two kinds of arteries with comparable size, we chose femoral and anterior tibial arteries to represent hindlimb arteries, respectively, in functional and structural studies.

The results that simulated microgravity induced differential changes in vasoreactivity of basilar and femoral arterial rings are consistent with the results obtained in previous work on conduit and small arteries (9–11, 15, 21, 22, 45, 47, 49). Delp et al. (11) have shown for the first time with aortic ring and extended later by Ma et al. (22) with arterial rings from several kinds of hindquarter conduit arteries that vasoconstrictor responsiveness to both receptor-mediated and nonreceptor-mediated agonists are generally depressed in hindquarter arteries of rats subjected to tail suspension. Zhang et al. (49) have further showed that the vasoconstrictor responsiveness of basilar arterial ring is generally enhanced across many vasoconstrictors. Thus, in the present study, only the vasoconstrictor response to KCl was tested.

The morphometric data of the present work confirm and extend the main conclusions of the previous work (23, 41, 45, 47). Our morphometric data strongly suggest that differential changes in media CSA of basilar and anterior tibial arteries are due to an increase and decrease in N_CL, i.e., hyperplasia and aplasia, respectively. Simulated microgravity-induced medial hypertrophy in basilar arteries is quite similar to that observed in mesenteric arteries of spontaneously hypertensive rats (27, 28, 38). In both cases, T, CSA, and N_CL were significantly increased with the average CSA of vascular smooth muscle cells unchanged. However, Wilkerson et al. (41) reported that simulated microgravity-induced medial hypertrophy of cerebral artery appeared to be due to hypertrophy of individual vascular smooth muscle cells rather than hyperplasia. The reason for this discrepancy remains unclear. Nevertheless, the possibility of simultaneous hypertrophy in smooth muscle cells still cannot be excluded, because we only measured and calculated the CSA of smooth muscle cells in two dimensions rather than in three dimensions (27, 28). Recently, Dickhout and Lee (12) reported that increased smooth muscle cell length, and not hyperplasia, in prehypertensive spontaneously hypertensive rats is responsible for the observed increase in N_CL in the mesenteric arteries. Whether region-specific changes in smooth muscle cell length are involved in vascular adaptation to microgravity remains to be elucidated. In addition, the present study has further confirmed that the media CSA of the vessel wall is one of the most reliable parameters for comparative studies on vascular remodeling, because it is not affected by the in vivo morphological states of the vessels at the time of fixation (2, 19). It is intriguing that the relative changes in vascular media CSA due to simulated microgravity reported by Wilkerson et al. (41), Mao et al. (23), and the present work are consistent. However, the changes in D are quite inconsistent among the three studies. This is apparently due to different perfusion pressures used during in situ fixation. In the present study, the perfusion pressure was 80 mmHg, whereas those used in the two relevant studies were 26 mmHg (23) and 44 mmHg (41), respectively. According to Lee (19), to preserve the in vivo morphology, vessels should be perfusion fixed at a low flow rate and perfusion pressure (16–24 mmHg) in a maximally relaxed state, and the medial CSA parameter instead of wall thickness alone should be used. In the present study, to compare T and N_CL in the media among different groups, it was necessary to make the intraluminal CSA of fully relaxed vessels nearly in the same size and in situ fixed. Thus a higher perfusion pressure of 80 mmHg (5) was adopted to perfuse the sodium nitroprusside-relaxed vasculature. Under such a higher perfusion pressure, the intraluminal diameters for each kind of vessels studied were very close among different groups (see Tables 3–5).

Exposure to microgravity undoubtedly removes all gravitational blood pressure gradients and leads to a redistribution of transmural pressure and blood flow across and within the arterial vasculature, which may well initiate differential adaptation of vessels in different anatomic regions (10, 16, 39, 45). It has also been speculated that, in addition to hypovolemia, the microgravity-induced autoregulations in the structure and function of vessels might be among the most important mechanisms responsible for postflight cardiovascular dysfunction and orthostatic intolerance (8, 10, 40, 45, 47). Hypothetically, when astronauts are exposed to microgravity, blood vessels in cerebral and dependent body regions are chronically exposed to higher and lower than normal upright, 1-G blood pressure, respectively. These local hemodynamic changes may induce differential autoregulations in resistance vessels in cerebral and dependent body regions. When astronauts return to the 1-G environment, the microgravity-induced down- and upregulations in resistance vasculature of dependent body regions and brain may act synergistically in the development of orthostatic hypotension with compromised cerebral blood flow. Findings from spaceflight human studies have indicated that the inability to adequately elevate the peripheral resistance (1) and enhanced cerebral vasoconstriction (14) might be important factors responsible for postflight orthostatic intolerance. The reason for the limited success of exercise-based countermeasures in preventing or alleviating postflight orthostatic intolerance might be that, in a microgravity environment, exercise alone cannot produce an acceleration field and hence cannot restore the vascular hydrostatic pressure gradients (16, 18, 39, 45, 47). Many investigators agree that artificial gravity may ultimately be required in future long-duration spaceflights. Recent studies have shown that continuous exposure to gravity does not seem necessary and that, instead, intermittent exposure may suffice (18, 32, 35–37, 43, 44). The fact that humans sleep horizontally each night with no ill effects implies that humans do not require constant exposure to gravity along the vertical axis of the body (+G_z). However, the minimum requirements of gravity exposure to maintain 1-G homeostasis remain unknown. Several ground-based human studies have provided promising data, suggesting potential benefits of IAG in preventing cardiovascular deconditioning due to microgravity exposure (32, 35–37). Vil-Viliams et al. (36, 37) reported the beneficial effects of daily short-period exposure to +0.8–1.6 G_z during a 3- or 28-day dry immersion in alleviating the gravity tolerance reduction. Sasaki et al. (32) reported the benefit of a daily +2 G_z treatment for 30 min twice per day in maintaining cardiovascular autonomic function during 4-day head-down bedrest. Vernikos et al. (35) reported that daily STD for 4 h completely prevented and STD for 2 h partially prevented decreases in post-HDBR orthostatic tolerance. They further indicated that various physiological systems benefit differentially from daily short-period +G_z gravitation. Therefore, it is very important to further elucidate the minimum requirement of intermittent gravitation to maintain 1-G homeostasis of vessels during simulated microgravity.

The Sus rat model has also been used to assess the efficacy of daily gravitation alone or in combination with exercise in alleviating/preventing the adverse changes of muscle or bones that might occur during simulated microgravity. Our laboratory's previous work (46) designed to compare the effectiveness of daily gravitation treatment in a multitude of organ system of the same animal has indicated that the minimum gravity exposure required to maintain 1-G homeostasis varies greatly among cardiac and skeletal muscles and bone. No relevant information is yet available on vessels. The present study reports for the first time that daily 2- or 4-h HUT, or 1-, 2-, or 4-h STD prevented the differential changes in function of cerebral and hindlimb arteries that might occur due to 28 days of simulated microgravity alone, and surprisingly, STD for 1 h/day is sufficient to prevent the adverse functional changes in arteries. The vascular rings examined were from the same animals of our laboratory's previous work (34, 46). We further showed that 4 h/day HUT or 1 h/day STD fully prevented differential changes in structure of vessels in different regions with separate groups of animals. The present work has further confirmed that the gravity requirement might vary greatly among different organ systems. The responsiveness of skeletal muscle to such gravitation treatment is moderate (4 h/day exposure was required to prevent soleus atrophy), whereas bone is most resistant (4 h/day exposure is ineffective in attenuating the adverse changes of femur during a 28 days of simulated microgravity) (34, 46). Thus further studies are needed to elucidate whether IAG is feasible as a gravity-based multisystem countermeasure.

To exclude the possibility that factors other than the intermittent change of gravity vector might be responsible for the beneficial effects on vessels; in protocol 3 of the present study, a second control group that received STD for 2 h/day alone was included (26). We have demonstrated that gravitational treatment alone does not influence the histomorphometric data of the vessels (see Table 5). It seems most likely that the counteracting effects are due to daily short-period –G_x/+G_z vector gravitation, which lower and elevate the blood pressure of the brain and hindlimb vessels, respectively (6, 24, 25, 31, 45). Because chronic 45° HUT resulted in a doubling of the hindlimb venous blood pressure, enhancement of venous myogenic response, sympathetic hyperinnervation of the hindlimb vessels (24, 25), and an increase in hindlimb postural load (17), we speculated that HUT (+G_z) may be more effective than STD (–G_x) in alleviating/preventing differential changes in the function of different arteries during simulated microgravity. However, the two kinds of intermittent gravitation treatment showed the same beneficial effects (see Figs. 1 and 2). This result is in accordance with our previous work on hindlimb muscle and bone (34). There are two possible explanations. One is that the minimum effective exposure time required for either –G_x or +G_z may be far <1 h/day; hence the differences cannot be revealed while the exposure time is 1 h/day. Another possibility is that the subtle differences in countermeasure effectiveness for vessels between intermitten –G_x and +G_z vector is hard to detect. Regardless of the mechanism, it is worth mentioning that daily –G_x gravitation for 60 min, or 4% of the total unloading time, is sufficient to maintain the function and structure of the vessels from different anatomic regions at their normal 1-G level in rats subjected to head-down suspension for 23 h/day during a 28-day period. The findings of the present work have provided further insight into the mechanisms involved in those reported in human studies (32, 35–37). The present study also supports the notion that restoration of Earth-like distribution of transmural is essential in counteracting cardiovascular deconditioning during real/simulated microgravity (16, 35, 39, 44, 45, 47). In addition, the enhanced orthostatic effects of acute maximal exercise and other additional proposals to apply missing gravitational stimuli during exercise (7, 8) might be due to some common primary mechanisms. It would be of great interest to elucidate the mechanisms by which daily 1-h gravitation is sufficient to prevent changes in vessels of rats subjected to simulated microgravity for 23 h/day during a 28-day simulation period. It appears that arterial wall tissue responds to sustained alterations in local stress due to removal of gravity, first by functional adjustment in myogenic tone and vasoreactivity, involving mechanisms like ion-channel remodeling in vascular smooth muscle cells (13), deficit in smooth muscle contractile apparatus (11, 29) in hindquarter arteries, and some other changes. Whereas the stress continues to act, structural autoregulation will occur, leading to a decrease and increase of myofibrillar protein content in hindquarter and cerebral arteries, respectively (45). Functional and structural changes are two interrelated and inseparable aspects of an integrated adaptation process of vessels. Nevertheless, it would be of great interest to further elucidate whether these interventions may be successful in preventing immediate or early functional changes of vessels due to acute or short-term (14 day) simulated microgravity. This would provide further insight into the understanding of the mechanisms underlying physiological and pathological vascular remodeling (20, 45).

In summary, the present study has demonstrated that daily gravitational loading by standing for as short as 1 h is sufficient to prevent differential adaptational changes in function and structure of cerebral and hindquarter vessels during 28 days of simulated microgravity. The present work has provided data to support that IAG may be efficacious in preventing vascular adaptational changes and hence cardiovascular deconditioning due to microgravity.

	GRANTS

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This study was supported by the National Natural Science Foundation of China (grant no. 30171032) and the Defence Medical Fund (grant nos. 98Z083 and 01Q114).

	ACKNOWLEDGMENTS

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The authors thank Deng Jing-Mao for technical assistance. We are particularly grateful to Dr. Robert M. K. W. Lee for helpful comments.

	FOOTNOTES

 

Address for reprint requests and other correspondence: L.F. Zhang, Dept. of Aerospace Physiology, The Fourth Military Medical Univ., Xi'an 710032, PR China (E-mail: zhanglf{at}fmmu.edu.cn).

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	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 

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