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1Swedish Defence Research Agency, Karolinska Institutet, Stockholm, Sweden; and 2Department of Automation Biocybernetics and Robotics, Jozef Stefan Institute, Ljubljana, Slovenia
Submitted 2 June 2008 ; accepted in final form 22 July 2008
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ABSTRACT |
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gravity; microgravity; vascular compliance; vascular deconditioning; vascular stiffness
Accordingly, we tested the hypothesis that arterial and arteriolar distensibility in limbs will increase in response to 5 wk of prolonged bedrest and that such increases will be especially pronounced in leg vessels.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Ten healthy male subjects took part in the study. Upon enrollment in the study, their average (range) age, body mass, and height were 23 (18–32) yr, 70.5 (57–86) kg, and 1.79 (1.66–1.90) m, respectively.
Potential subjects were recruited by advertisement. They were not included if they were taking any medication, had a family history of frequent deep vein thrombosis (DVT) or showed pathological results in a blood screening test for DVT susceptibility, had a history of vascular headaches (i.e., migraine or Hortons disease), or were unable to perform the prebedrest criterion tests (see Criterion Tests) and/or baseline experiments.
Subjects gave their informed consent to participate and were instructed that they were free to terminate any experiment and also to withdraw from the study at any time. The study protocol and experimental procedures were in accordance with the Declaration of Helsinki and were approved by the Committee for Medical Ethics of the Ministry of Health of the Republic of Slovenia.
General Study Protocol
The experiments and criterion tests reported herein were performed about 1 wk before bedrest (pre), within 12 h after the 35 days of bedrest (post), and again after 4 wk of recovery (recovery). For each subject, the order and schedule of the experiments and criterion tests were identical before and after bedrest.
Bedrest and Recovery Procedures
The bedrest procedure was conducted at the Valdoltra Orthopaedic Hospital in Ankaran, Slovenia. Subjects commenced and completed the 35-day bedrest period at predetermined intervals and in sequence over a 3-day period. During bedrest, subjects were accomodated in two rooms (with 5 individuals in each room). Each subject remained in the horizontal position at all times. He was allowed one pillow for the head and to occasionally lean on an elbow while eating or being transferred to a gurney. He was allowed to move his arms in and above the horizontal plane, whereas the legs had to be kept in the horizontal plane at all times. Muscular exercise (e.g., static contractions using the foot board of the bed as support) was prohibited. With the exception of alcohol-containing beverages, subjects were not restricted regarding food and drinks. Each subject was provided 3 meals/day; meals were designed by the hospital nutritionist. Hospital routines were adhered to at all times.
To ensure that subjects complied with the requirements of the bedrest protocol, and for subject safety, video cameras in the rooms provided continuous (24 h/day) surveillance of the subjects throughout the bedrest period. To minimize problems with bedrest-induced neck/back pain and stiffness of joints, each subject received physiotherapy twice a week and upon request. The therapy was performed with the subject in a horizontal position and consisted of massage as well as assisted (passive) stretching and assisted joint flexions. Each subject was screened for DVT twice every week throughout the bedrest period. Specifically, the popliteal veins were examined bilaterally using an ultrasound/Doppler system with a 6.0- to 11.0-MHz linear array transducer (Aspen, Acuson, Mountain View, CA).
During the 4-wk period between posttests and recovery tests, each subject resumed his normal ambulatory lifestyle and also participated in a supervised training regimen consisting of 11–12 1-h sessions of either cycle ergometry (n = 5) or lower body resistance training (n = 5).
Criterion Tests
Certain "criterion tests" were performed to evaluate subject compliance and hence to verify that the present bedrest procedure brought about the expected degrees of cardiovascular and musculoskeletal deconditioning. The tests included investigations of anthropometry, isometric muscle strength, aerobic work capacity, and orthostatic responses. Only the orthostatic response tests are reported in the present article; results from other criterion tests have been (cf. Ref. 1) or will be reported elsewhere.
Orthostatic responses were determined during a stand test performed in a temperature-regulated environment (28°C). Following a 10-min period of motionless rest in the supine position, the subject was asked to rapidly stand up. He then stood with his heels and back against a wall for 10 min or until the appearance of signs/symptoms of imminent syncope. The subject was instructed to remain still and to avoid conducting voluntary muscle contractions during the test period. The heart rate (HR) was derived from ECG recordings with the electrodes positioned in a five-lead precordial arrangement using a cardiomonitor (Physiocontrol Lifepak 8, Physio-Control, Redmond, WA). Arterial pressures (APs) [systolic AP (SAP), diastolic AP (DAP), and mean arterial pressure (MAP)] were measured using a volume-clamp technique (Portapres, TNO, Amsterdam, The Netherlands) with the pressure cuff placed around the midphalanx of the third finger of the right hand and the reference pressure transducer taped to the skin at the level of the heart. The right arm was supported by a mitella, and the distal portions of the fingers were positioned at heart level.
The postbedrest orthostatic test coincided with the completion of the 35-day bedrest period.
Vascular Pressure-Distension Experiments
Methods and procedures. The experiments were carried out with the subject positioned in a pressure chamber with one arm (the "test arm") or the lower portion of a leg (the "test leg") extended through a hole in the chamber door. During the arm pressure provocation, the subject was seated, whereas during the leg pressure provocation, he was in the supine position. The test arm/leg was supported at the level of the heart with the use of a stand external to the chamber. The test limb was hermetically sealed to the door hole slightly distally of the axilla or proximally of the knee with use of a short self-sealing rubber sleeve. Special harnesses were used to stabilize the subject and to prevent involuntary muscle activity and movements in the arm and leg as the pressure in the chamber was elevated. Room temperature was maintained at 28°C (range: 25–30°C).
Diameters of the arteries in the test limb were measured using ultrasonography, with all measurements being performed by the same sonographer. Measurements were conducted in B-mode image during end diastole (determined from ECG recordings) as wall-to-wall distance in the sagittal section using a 6.0- to 11.0-MHz linear array transducer (Aspen, Acuson). The brachial artery diameter was measured 
5 cm proximal to the cubital fossa, and the tibial posterior artery diameter was measured 5 cm proximal to the ankle. To ensure that the same vessel segment was investigated in the three trials, intra- and extravascular "landmarks" were assigned and recorded during the sonography. Subsequently, off-line measurements of the intima media (IM) thickness were made for both arteries; measurements were conducted in the B-mode sagittal section images as the distance between the leading edge of the lumen-intima echo and the intima-adventitia echo in the far wall of the vessel.
Flow was estimated in the brachial and tibial arteries by simultaneous measurements of vessel diameter and mean flow velocity using an ultrasound/Doppler technique (6.0- to 11.0-MHz transducer, Aspen, Acuson). Assuming a circular cross section of the artery, flow was calculated by multiplying vessel cross-sectional area (CSA) by the time integral of the mean flow velocity.
HR was derived from ECG recordings using a bipolar precordial lead. SAP and DAP were measured in the midphalanx of the third finger inside the chamber using the volume-clamp technique. The level of the heart was used as the point of reference for AP measurements. Arterial distending pressure (DP) was calculated, and arteriolar DP was approximated by adding chamber pressure to DAP (cf. Refs. 12 and 13).
Changes in forearm and lower leg volumes were estimated from impedance plethysmographic recordings using a tetrapolar constant-current impedance system (Minnesota Impedance Cardiograph model 304A, Instrumentation For Medicine, Greenwhich, CT) with four pairs of standard disposable pregelled electrodes placed on the test limb.
Each subject rated his perceived pain in the test limb using a ratio scale (4) in which pain can be rated from 0 (no pain) to 10 (very, very strong pain; almost intolerable).
Experimental protocol. Each experiment commenced with a 10-min baseline period with normal atmospheric pressure in the chamber. Thereafter, chamber pressure was increased stepwise every other minute, resulting in 2.0-min plateaus of negative pressure surrounding the test limb relative to that surrounding the rest of the body. For the arm, chamber pressure plateaus were 15, 60, 90, 120, and 150 mmHg, and for the leg, chamber pressure plateaus were 15, 60, 90, 150, 180, 210, and 240 mmHg above atmospheric pressure. Upon completion of the pressure provocation protocol, chamber pressure was rapidly reduced, and a 2.5-min recovery period at atmospheric pressure ensued. Recordings of HR, SAP, DAP, vessel diameters, flow, forearm/lower leg impedance, and ratings of perceived pain in the test limb were obtained during the last 0.5 min at each pressure level.
In all subjects, pressure provocations were performed in the left arm and left leg. For the individual subject, the two trials (leg and arm) were separated by 
1 h. The order of the trials was alternated among subjects.
Analyses
The statistical significance of differences was evaluated by repeated-measures ANOVA followed by Tukey's honestly significant difference post hoc test (Statistica Statsoft, Tulsa, OK).
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Orthostatic Responses
Before bedrest, all subjects could endure the 10-min stand test without any signs or symptoms of imminent syncope. They all exhibited normal HR and AP responses (means ± SD, 
HR upon standing = 29 ± 8 beats/min, SAP = 13 ± 13 mmHg, and DAP = 14 ± 7 mmHg). In the posttrial, only three subjects completed the stand test. The remaining seven subjects had to discontinue the provocation prematurely (P < 0.05) due to signs/symptoms of imminent syncope; the endurance time in the stand test for these subjects was 3.6 ± 2.1 min. The HR response was markedly exaggerated and AP responses were attenuated (P < 0.001) in the posttrial (HR = 45 ± 14 beats/min, SAP = –2 ± 17 mmHg, and DAP = 7 ± 8 mmHg) compared with before bedrest. After recovery, all subjects completed the stand test without any unwarranted symptoms and with normal HR and AP responses (HR = 21 ± 9 beats/min, SAP = 6 ± 12 mmHg, and DAP = 3 ± 8 mmHg).
Vascular Pressure Distension
Exposure to high intravascular pressures induced pain in both the arm and leg. Pain increased with increasing pressure to reach median values of 8 at the highest chamber pressures that could be attained in the different conditions. Pain was always considerably stronger in the arm than in the leg at any given level of increased intravascular pressure, and the pain response was aggravated by bedrest. Since it can be assumed that such pressure-induced pain predominantly results from distension of veins (12, 13), details of the pain responses will be reported in a forthcoming publication concerning the effects of prolonged bedrest on the mechanical properties of limb veins.
Arterial diameters.
Before bedrest, tibial artery diameter remained unchanged up to a DP of 250 mmHg (range: 205 to 305 mmHg) but then increased (P < 0.05) by 8 ± 7% at the highest DP (Fig. 1A). Bedrest increased (P < 0.001) pressure distension in the tibial artery threefold to 24 ± 11% at the highest DP that could be attained in all three conditions (average peak DP = 276; range = 245–305 mmHg). After recovery, the tibial artery showed a similar pressure-distension response as in the prebedrest trial (8 ± 5%). The diameter of the brachial artery was also preserved at slight and moderate elevations of DP. However, in the brachial artery, distension occurred at considerably lower DP than in the tibial artery (P < 0.001); the pressure at which brachial artery distension was initially observed in the prebedrest trial ranged from 150 to 180 mmHg for the different individuals (Fig. 1B). Bedrest increased pressure distension in the brachial artery by a third (P < 0.05) from a prebedrest value of 15 ± 9% to 20 ± 10% postbedrest at the highest DP (206 ± 18 mmHg) that could be attained in all three conditions; after recovery, the pressure distension was 13 ± 7% at this DP.
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Arterial flow.
Before bedrest, flow in the tibial artery remained unchanged up to a DP of 264 mmHg (range: 238–298 mmHg) but then increased by 96 ± 114% (P < 0.05; Fig. 2A). At the highest DP that could be attained in all three conditions, the pressure-induced increase in tibial artery flow was more pronounced (P < 0.01) postbedrest (50 ± 39 ml/min) than prebedrest (13 ± 23 ml/min) and than after recovery (15 ± 12 ml/min). In the brachial artery, flow also remained unaltered at slight to moderate DP elevations and increased (P < 0.001) at the highest DP. However, the pressure-induced increase in flow occurred at substantially lower DP in the brachial than tibial artery (P < 0.001). The brachial artery flow response was unaffected by bedrest, with increments at the highest DP that could be attained in all three conditions of 205 ± 129 ml/min in the prebedrest, 194 ± 88 ml/min in the postbedrest, and 207 ± 103 ml/min in the recovery trials (Fig. 2B).
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20 beats/min but did not affect HR responses during the pressure provocations. Both SAP and DAP increased in response to the arm and leg pressure provocations: SAP by 15–25 mmHg and DAP by 10 mmHg. Bedrest did not affect either baseline values or pressure responses of SAP and DAP.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Arterial and Arteriolar Pressure Distension
The results demonstrate that, in both the brachial and tibial arteries, diameter as well as flow remained unaltered at slight to moderate elevations of DP. At high DP, vessel diameter and flow increased markedly; the DP at which these increments occurred was much higher for the tibial than brachial artery. Bedrest augmented the pressure-induced increments in tibial artery diameter and flow as well as the pressure-induced decrease in lower leg impedance.
That distension in arteries and arterioles occurred at lower DP in the arm than in the leg confirms previous results (13) and supports the notion that, in humans, regional arterial/arteriolar compliances are distributed in such a way that the hydrostatic forces acting upon them in erect posture are partially counteracted. Since AP remained relatively stable during the course of a pressure provocation, the two- and threefold increments in tibial and brachial artery flow, respectively, observed at high DP predominantly reflect precipitous decrements in local peripheral resistance. The notion that precapillary resistance vessels were forced open by high DP was supported by the observation that tissue impedance, and hence presumably tissue edema (19), in the test limb changed at a much faster rate at the highest levels of DP. Thus, pressure-induced decrements in test limb impedance can be assumed to result from a combination of tissue edema formation and filling/distension of veins (12, 13). Since the distensibility, and hence the rate of blood pooling, in leg and arm veins decreases with increasing DP (12, 13), the increased rate of impedance changes observed at high DP must result from increased edema formation. A progressively increasing rate of edema formation suggests a concomitant increase in capillary permeability or, more likely, capillary filtration pressure. The most plausible explanation for a sudden increase in capillary pressure is an increase in the diameter of precapillary resistance vessels.
That pressure provocations consistently induced minute increments in HR and minute to modest increments in AP are in keeping with previous results (12, 13). The mechanisms underlying such cardiovascular responses are not clear, but it appears reasonable to assume that they might fully or in part be secondary to pressure-induced limb pain.
The present results thus indicate that both peripheral arteries and arterioles distend at high intravascular pressures, which concurs with our previous findings (12, 13). It is not possible to conclude that the pressure-induced increases in arterial diameter represented solely true passive distension. To some extent, the arterial diameter changes might have been secondary to the flow increments, reflecting reduced vascular muscle tone elicited by increased shear stress on the arterial wall (for a review, see Ref. 21). However, it appears unlikely that shear stress-mediated dilatation played a key role in the present pressure-induced arterial diameter increments since we have found that such increments also remain when the arterial flow augmentation is abolished by blocking of flow in the downstream vasculature (R. Kölegård and O. Eiken, unpublished observations).
Our findings that bedrest augmented the pressure-induced increments in tibial artery diameter and flow as well as the pressure-induced decrease in lower leg impedance suggest that removal of the hydrostatic pressure components that act along the blood vessels in erect posture during normal daily life activities markedly increases pressure distension in both arteries and precapillary resistance vessels. The possibility that the increased pressure distension in arteries/arterioles of the lower leg was attributable to removal of AP perturbations induced by muscular exercise, or to any other general/systemic effect associated with prolonged bedrest, rather than to the removal of the large hydrostatic pressure gradients acting along the vessels in upright posture seems unlikely. Physical training is believed to increase arterial distensibility, at least when distensibility is not determined by measuring the pressure-distension relationship but merely defined as the vessel diameter excursions during a normal pulse-pressure wave (28). In addition, the vasculature in the arm was not affected to the same extent as that in the lower leg by the present bedrest; a slight augmentation of brachial artery pressure distension was observed postbedrest, but, judging from the responses of brachial artery flow and forearm impedance, bedrest did not affect the distensibility of forearm arterioles. Even though during normal life activities local AP in the arm vessels commonly exceeds heart-level AP, the average decrease in local vascular pressure imposed by prolonged horizontal bedrest is much larger in the vascular circuits of the legs.
These observations confirm and extend previous findings indicating that in vivo wall stiffness in both arteries and arterioles may adapt to meet the demands imposed by the hydrostatic pressure gradients that act along the vessels. Previous experiments have shown that the distensibility of arteries/arterioles can be considerably reduced by pressure habituating the vessels. A 5-wk pressure-habituation regimen during which an intermittently applied vascular DP was gradually increased resulted in substantial attenuations of pressure-induced increments in brachial artery diameter and flow (11). The present results suggest that even after lifelong adaptation to intravascular pressure gradients associated with erect posture, arteries and arterioles need regular exposure to such pressure gradients to maintain their pressure resistance characteristics.
The question arises as to the mechanisms underlying the bedrest-induced increments in arterial and arteriolar distensibilitity. It is well documented that the wall structure of arteries may change in response to changes in the strain of the wall (for reviews, see Refs. 14 and 29). Indeed, it can be assumed that changes in the lumen-to-wall thickness ratio would affect the pressure-distension relationship of the vessels (14). Our findings that ultrasonographic determinations of the IM thickness and the lumen-to-IM thickness ratio in the tibial artery were unaffected by bedrest do not exclude significant atrophy of the vessel wall, since decrements in the IM thickness of <0.1 mm, corresponding to 20% of the present thicknesses, are not necessarily detected using this technique (24). Furthermore, the 10% reduction in postbedrest CSA of the tibial artery IM layers supports the notion of bedrest-induced atrophy of the walls of leg arteries. It has been suggested that changes in arterial wall thickness are more accurately determined by measuring CSA than by measuring wall thickness (29). Increasing the gravitational pull acting along longitudinally oriented vessels in rats, by suspending them from their tails or hindlimbs for prolonged periods of time, may increase the CSA in cranially located conduit and resistance arteries and arterioles and decrease the CSA of corresponding vessels located in the caudal portion of the body (6, 10).
It is also possible that bedrest-induced reductions in neurovascular input/sensitivity and/or in the local release of vasoconstrictive substances may have contributed to the increased distensibility of leg arteries/arterioles observed in the postbedrest pressure provocation. There are indications that prolonged bedrest reduces vasoconstrictor as well as flow-mediated and nitroglycerin-mediated conduit artery dilatatory responses in humans (2, 25). It has been shown that prolonged tail suspension in rats reduces maximal isometric contractile tension in the abdominal aorta, irrespective of whether contractions are evoked by receptor- or nonreceptor-mediated vasoconstrictors (20). It has been suggested that gravity-dependent changes in vascular responsiveness observed in tail-suspended rats might, in part, be induced by changed expressions of endothelial and neuronal nitrous oxide (NO) as well as of inducible NO synthase (17, 27). In contrast to the aforementioned studies, there is evidence suggesting that extended exposures to bedrest/microgravity does not affect vasoconstrictor capacity in humans, at least not in arm vessels (for a review, see Ref. 9). Thus, the mechanisms underlying bedrest-induced increments in vascular distensibility may be multifactorial and need to be further investigated.
It also remains to be elucidated if, and to what extent, a bedrest-induced increase in pressure distension of the precapillary resistance vessels of the legs contributes to the orthostatic intolerance consistently observed after prolonged bedrest and spaceflights (15). Flow resistance in the leg vasculature constitutes a key determinant for orthostatic tolerance as well as for the capacity to withstand increased gravitoinertial load in the head-to-foot direction (3). However, it can be argued that the present findings suggest that, in erect posture at normal gravity, local AP in the lower portions of the legs is 200 mmHg, which might not be of sufficient magnitude to distend precapillary resistance vessels even after they have been deconditioned by 5 wk of horizontal bedrest. This does not exclude the possibility that an even longer period of bedrest or microgravity exposure might increase pressure distensibility in the precapillary vessels of the legs to the extent that significant distension of dependent arteries/arterioles would occur when assuming erect posture. There are indications that impaired arterial/arteriolar function contributes to the orthostatic intolerance induced by bedrest or microgravity (5, 7, 25). Furthermore, our results suggest that even slight increments of G load in the head-to-foot direction of <0.5 G will elevate pressure in the leg vessels to levels capable of substantially distending arteries/arterioles in the bedrest-deconditioned vasculature. Therefore, it must be considered that the well-documented reduction of G tolerance following bedrest or space missions (16) may, to a large extent, be attributable to increased pressure distension in the precapillary resistance vessels of the lower body.
Methodological Considerations
The present results of bedrest-induced effects on vascular distensibility should also be viewed in the context of subject compliance. Neither the video surveillance nor the criterion tests gave reason to doubt that all subjects complied with the requirements of the bedrest protocol. In general, long-duration bedrest induces signs of orthostatic intolerance in 50–60% of subjects (26). That the incidence of orthostatic intolerance varies considerably between studies is a consequence of different methods used to provoke orthostatic stress (i.e., stand tests, different tilt table, and lower body negative pressure protocols) and of the different indexes used to define orthostatic tolerance. The response is also dependent on the time elapsing between the termination of bedrest and the orthostatic provocation (cf. Ref. 15). In the present postbedrest stand test, all subjects exhibited exaggerated HR responses, and 70% of the subjects had to discontinue the orthostatic provocation because of signs/symptoms of imminent syncope. Thus, in the present study, bedrest induced the expected degrees of cardiovascular and, as reported previously (1), musculoskeletal deconditioning, confirming subjects' compliance with the protocol.
To increase the transmural pressure in the blood vessel of a subject's test limb, we used a method described in detail previously (12, 13). Briefly, the subject, except for his test limb, is enclosed in a pressure chamber. As chamber pressure is elevated, transmural pressures in the vasculature of the unexposed test limb are increased in direct proportion to the increase in chamber pressure.
Lumen diameters of the arteries were measured during end diastole with the use of ultrasonography. Arterial DP was calculated by adding applied chamber pressure to DAP, and changes in flow were also evaluated as functions of arterial DP. Since arterial flow in the forearm or lower leg is mainly controlled by local vascular resistance, it can be argued that flow should be treated as a function of arteriolar rather than arterial DP. The average arteriolar DP is however, difficult to determine, and hence the flow patterns in brachial and tibial arteries were analyzed as functions of peak arteriolar DP (i.e., DP at the upstream end of the arterioles), which, in turn, was assumed to correspond to arterial DP.
Even though blood volume was not measured in the present study, it is reasonable to assume that the 5-wk bedrest period reduced circulating blood volume by 500–600 ml (15). There is evidence suggesting that bedrest-induced reductions in circulating blood volume may increase baseline vascular resistance and thereby reduce the vasoconstrictor reserve in the forearm vasculature (for a review, see Ref. 9). However, judging from our observations that, under baseline conditions, AP as well as brachial and tibial artery flow, and hence flow resistance in the vasculatures of the forearm and lower leg, were unaffected by bedrest, it appears unlikely that a bedrest-induced blood volume reduction per se had any major impact on the pressure-distension determinations.
Conclusions
The present results indicate that removal of the hydrostatic pressure gradients that act along the blood vessels in erect posture markedly increases pressure distension in arteries and arterioles of the legs, suggesting that the increased stiffness observed in dependent arteries/arterioles is not inherent but rather represents an acquired feature that depends on regular exposures to such gradients.
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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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