We tested the hypothesis that sympathoadrenal activity in humans is low during spaceflight and that this effect can be simulated by head-down bed rest (HDBR). Platelet norepinephrine and epinephrine were measured as indexes of long-term changes in sympathoadrenal activity. Ten normal healthy subjects were studied before and during HDBR of 2-wk duration, as well as during an ambulatory study period of a similar length. Platelet norepinephrine concentrations (half-life = 2 days) were studied in five cosmonauts, 2 wk before launch, within 12 h after landing after 11–12 days of flight, and at least 2 wk after return to Earth. Because of the long half-life of platelet norepinephrine, data obtained early after landing would still reflect the microgravity state. Platelet norepinephrine decreased markedly during HDBR (P < 0.001), whereas there were no significant changes when subjects were ambulatory. Platelet epinephrine did not change during HDBR. During microgravity, platelet norepinephrine and epinephrine increased in four of the five cosmonauts. Platelet norepinephrine concentrations expressed in percentage of preflight and pre-HDBR values, respectively, were significantly different during microgravity compared with HDBR [153 ± 28% (mean ± SE) vs. 60 ± 6%, P < 0.004]. Corresponding values for platelet epinephrine were also significant (293 ± 85 vs. 90 ± 12%, P < 0.01). The mechanism of the platelet norepinephrine and epinephrine response during spaceflight flight is most likely related to the concomitant decrease in plasma volume. HDBR cannot be applied to simulate changes in sympathoadrenal activity during microgravity.
sympathetic nervous activity in humans is closely related to the level of gravitational stress on the cardiovascular system. The baroreflexes adjust sympathetic activity to maintain a constant blood pressure despite variations in venous return of blood to the heart. Thus sympathetic activity is high when venous return is low such as in the upright position, whereas it is suppressed when venous return is increased such as during water immersion and antiorthostatic maneuvers.
Head-down bed rest (HDBR) has been applied to simulate cardiovascular changes during microgravity. In 1995 our laboratory reported, however, that plasma norepinephrine values were elevated during microgravity and above values obtained in the seated position on the ground (10). Blood samples were collected from an antecubital vein on the fifth to sixth day of flight. Later Ertl et al. (4) reported that baseline sympathetic activity as measured by microneurography was increased moderately by spaceflight. Furthermore, in the same study it was demonstrated that the norepinephrine spillover rate was significantly increased in space. These studies therefore suggested, contrary to expectations, that sympathetic activity is increased by spaceflight of 1- to 2-wk duration.
The aim of the present study was therefore to evaluate long-term changes in sympathoadrenal activity during HDBR and during microgravity by measuring platelet norepinephrine and epinephrine values. The HDBR study and ambulatory control study were done both with the subjects on a normocaloric diet and on a hypocaloric diet (−25%). The hypocaloric diet experiment was performed because cosmonauts tend to have a reduced calorie intake in space compared with subjects in ground-based observations, which might modulate sympathetic nervous activity.
Platelets circulate through all parts of the body and take up catecholamines from plasma. Platelet norepinephrine and epinephrine values are unaffected by acute changes in sympathoadrenal activity such as exercise and reflect chronic, more long-term changes (1, 2, 15). Furthermore, platelet epinephrine measurements may be a more reliable technique for detection of changes in epinephrine release than comparable measurements of epinephrine in forearm venous blood, because the platelet epinephrine concentration does not depend on the extraction ratio in forearm tissues. Platelets take up catecholamines from plasma in vivo, both norepinephrine and epinephrine. This is an active process. The final concentration of norepinephrine and epinephrine in the platelets is primarily dependent on the average plasma catecholamine concentrations. This is known especially from patients with pheochromocytoma, who have high values of platelet catecholamines in proportion to the plasma level. The concentration in the platelets decreases after the tumor as been removed although more slowly than in plasma (19).
Long-term changes in sympathoadrenal activity can also be recorded by multiple sampling procedures, but such procedures cannot easily be applied to spaceflight. We did not apply urine measurements of norepinephrine and epinephrine because renal function is influenced by microgravity and urinary excretion rate of water and sodium are decreased during spaceflight but not during HDBR (10, 11).
SUBJECTS AND METHODS
All study protocols were reviewed and approved by Ethics Committees at the European Space Agency medical board and were in compliance with the Declaration of Helsinki II. All subjects provided informed consent to the procedures.
The HDBR Study
Nine normal subjects participated in all four study phases (Table 1). One subject participated only in phase 1 and another subject participated in the following three study periods. The mean age was 23.8 yr (range 21–29 yr). The mean body mass index was 23.0 kg/m2 (range 19.2–27.8 kg/m2). All subjects were healthy and had a normal heart rate and blood pressure.
Study phases 1 and 2 started with an adaptation period of 9 days (−9 to −1) when the subjects were ambulatory. This was followed by an intervention period of 14 days (+1 to +14), when the subjects at random were either ambulatory or subjected to −6° HDBR and vice versa.
Study phases 3 and 4 were performed in the same way except that all subjects were on a hypocaloric diet during the intervention period.
Phases 2 and 4 were the HDBR study, and phase 1 and 3 the ambulatory study.
The daily normocaloric diet consisted of protein (1 g per kg body wt per day), fat (30% of the energy; fatty acid composition was saturated and polyunsaturated fatty acids), and carbohydrate (remaining calories). In addition, the subjects received 50 ml of water per kg body wt, 2.5 mmol sodium/kg, 1,000 mg calcium, and 400 IU vitamin D per day.
The hypocaloric diet had an energy intake of 75% of the respective normocaloric ambulatory diet.
All subjects received the same amounts of water, protein, sodium, calcium, and vitamin D. Intake of alcohol and caffeine was not allowed. All other nutrients without experiment-specific requirements matched dietary recommended intake levels of the German Nutrition Society. In the adaptation period in the four phases all subjects received the normocaloric diet of identical nutrient composition.
Total energy expenditure was calculated as basal metabolic rate multiplied by the physical workload plus the calculated thermic effect of feeding.
The test subjects stayed in the laboratory all the time also during the ambulatory study phases. They were not allowed to do any exercise on a voluntary basis. However, in phases 1 and 3 they followed an exercise protocol that was two times 15 min of bicycle ergometry (∼125 W).
Blood samples for plasma and platelet catecholamines were collected from an antecubital vein. The samples were always collected in the morning at 7 AM with subjects in the supine position. The blood samples were immediately brought to the laboratory and prepared as described below. Blood samples were obtained on days −4 and −2 in the adaptation period and again on days +5, +9, and +14 during the intervention period. No samples for cateholamine analysis were obtained in the recovery period.
For all practical reasons, the preparation of blood samples differed in the HDBR study compared with the microgravity study. For this reason we have not compared absolute values in the two groups. Relative changes observed in relation to the corresponding basal values in the adaptation period and preflight may be compared.
Preparation of Blood Samples in the HDBR Study
Ten milliliters of blood were collected in polycarbonate tubes that contained 50 μl of 0.2 M NaEDTA solution per milliliter of blood. The EDTA blood was centrifuged without brake for 15 min at 20°C at 350 g. The upper two-thirds of the plasma, ∼3 ml, was transferred to a new tube and mixed gently. The number of platelets was counted in a Coulter counter and expressed as number × 106/ml. Samples of 108 platelets were collected and pipetted into Eppendorf tubes. These tubes were centrifuged for 15 min at 4°C at 1,800 g with brake. Supernatants were decanted and platelets frozen and stored at −20C° until analysis. The loss of platelets with the second centrifugation procedure and the decantation procedure was ∼4%.
It is important that no catecholamines are lost from the platelets during the preparation procedure. There was no agglutination of platelets before the final centrifugation and of course no coagulation (owing to the binding of Ca2+ to EDTA). In addition, we have analyzed platelet norepinephrine and epinephrine in 12 samples obtained from two subjects. Four samples were analyzed by the standard procedure; i.e., centrifugation was done without any delay. Four samples were allowed to stand for 25 min before centrifugation, and an additional four samples were centrifuged after a delay of 50 min. There was no decrease in platelet norepinephrine and epinephrine with time. The mean platelet norepinephrine concentration (±SE) was 62 ± 5, 62 ± 2, and 70 ± 2 pg/108 platelets with the standard procedure and with a delay of 25 and 50 min, respectively. The corresponding values for platelet epinephrine were 5 ± 1, 7 ± 2, and 8 ± 1 pg/108 platelets, respectively. None of these changes was significant.
2.5 ml of blood was collected in tubes as described above and centrifuged for 10 min at 1,800 g. The plasma was collected and stored at −20°C until analysis.
Blood samples for platelet measurements were collected from an antecubital vein in five male cosmonauts. The mean age was 41 yr (range 37–45 yr). They participated in three Soyuz missions to the International Space Station. Samples were collected ∼14 days before launch, after 11–12 days in flight within 12 h upon landing, and finally at least 14 days thereafter.
Samples for platelet norepinephrine and epinephrine measurements should preferably have been obtained inflight, but this was not possible because no centrifuge with an adjustable speed was available on the International Space Station. Because of the long half-life of platelet norepinephrine and epinephrine (see below), a sample taken after 11–12 days in flight and within 12 h after landing would still reflect the microgravity state. We tested the half-life of platelet norepinephrine in five normal subjects during the first 4 days of another HDBR study.
The mean half-life for platelet norepinephrine was 54 ± 12.5 h (±SE). There was a tendency for an inverse relationship between the half-life and the basal platelet norepinephrine values. The half time is of the same magnitude as reported by Chamberlain et al. (2) (44 h).
The platelets could not be counted at the sampling site, and therefore the preparation of the platelets had to be modified. After the initial centrifugation at 350 g, samples of 0.5 ml were added to Eppendorf tubes and centrifuged as described above (1,800 g with brake), decanted, and frozen at −20°C. In addition, at least two times 0.3 ml plasma samples were obtained and added to Eppendorf tubes. These samples were not centrifuged but were frozen and later applied for counting the number of platelets. A preliminary study indicated that the number of platelets remained the same before and after freezing. The mean platelet level in the cosmonauts preflight and within 12 h after landing averaged 243 ± 20 and 261 ± 56 × 109/l. These values were not significantly different and were well within normal range (140–340 × 109 platelets/l).
Plasma and platelet norepinephrine and epinephrine concentrations were quantified by a sensitive and precise radioenzymatic assay described earlier (8).
Results are presented as means ± SE. For the analysis of data obtained in the same group of subjects at different time intervals, we applied one-way repeated-measures ANOVA. Pairwise multiple comparison procedures were done with the Tukey's test. The paired t-test, the t-test, regression, and correlation analysis were applied in some tests. Statistical analysis was done with the SigmaStat version 2.0. A P value < 0.05 was considered significant.
Platelet norepinephrine decreased significantly during HDBR (phase 2; Fig. 1; P < 0.001).
The tendency for platelet norepinephrine to decrease during the normocaloric ambulatory study was not significant. The hypocaloric diet had no effect on platelet norepinephrine levels, which decreased during the HDBR (phase 4; P < 0.001) but remained unchanged during the ambulatory study period (Fig. 2).
The mean platelet norepinephrine level in the four experiments in the adaptation period before the intervention averaged 42.9 ± 9.8 (mean ± SE; phase 1), 41.2 ± 7.4 (phase 2), 34.4 ± 8.3 (phase 3), and 32.9 ± 8.3 (phase 4) pg/108 platelets. The values obtained in the adaptation period in phases 1 and 2 tended to be higher than in phases 3 and 4 (one-way repeated-measures ANOVA + Tukey's test; P < 0.05).
Platelet norepinephrine levels varied between individual subjects, but values in the same subject in the two samples obtained in the four adaptation periods were correlated [R values ranging from 0.98 to 0.60, P < 0.05 to 0.000 in all but two comparisons (n = 28 comparisons)]. There was also a strong positive correlation between platelet norepinephrine values in the adaptation period and during the intervention (phase 2, r = 0.92, P < 0.001; phase 4, r = 0.95, P < 0.001), indicating that the relative decrease in platelet norepinephrine was approximately the same in all subjects.
The corresponding values for platelet epinephrine in the adaptation period were 2.7 ± 0.7 (phase 1), 2.9 ± 0.9, 2.3 ± 0.8, and 2.6 ± 0.5 pg/108 platelets (not significant). Platelet epinephrine did not change significantly during HDBR and was not influenced by the hypocaloric diet.
Table 2 shows plasma norepinephrine during the four phases. In the HDBR with normocaloric diet plasma norepinephrine decreased significantly, but the decrease occurred already between the first and second sample in the adaptation period. A similar response was seen in the adaptation period phase 4. During phase 3 (hypocaloric and ambulatory), plasma norepinephrine increased significantly at the end of the intervention period. Thus there was no change in plasma norepinephrine that could be related to HDBR. Plasma epinephrine values were low, with mean values ranging from 0.00 to 0.02 ng/ml. No significant differences were observed.
Figure 3 shows platelet norepinephrine values in the cosmonauts.
Preflight values were within normal range but in the lower end. Epinephrine values averaged preflight 1.5 ± 0.3, inflight 3.8 ± 1.1, and postflight 2.1 ± 0.2 pg/108 platelets. During microgravity, platelet norepinephrine and epinephrine increased in four of the five cosmonauts, but the change was not significant.
Platelets from subjects participating in the HDBR study and from the cosmonauts were processed and stored in different ways as described earlier, and a comparison of the absolute values may not be relevant. The relative changes may, however, be compared. Platelet norepinephrine during microgravity and during HDBR expressed in percentage of basal values (preflight or pre-HDBR values, respectively) were significantly different (152.6 ± 28 vs. 59.8 ± 5.7%, P < 0.004) for a difference between inflight and HDBR (Fig. 4).
Comparison of inflight values with values from the phase 4 study was also significant (152.6 ± 28 vs. 57 ± 6.6%, P < 001).
Comparing platelet epinephrine in the same way as norepinephrine indicated that platelet epinephrine was significantly different during microgravity compared with the HDBR experiment [293 ± 85 vs. 90 ± 12% (phase 2) and 89 ± 18% (phase 4), P < 0.01 and 0.02, respectively]. Thus there was a marked and highly significant difference in platelet norepinephrine and epinephrine responses during microgravity compared with HDBR. The lack of a decrease in platelet norepinephrine in cosmonauts compared with participants in the HDBR study cannot be explained by the relatively lower preflight values. In the phase 4 study four subjects had mean values in the adaptation period below 20 pg/108 platelets (range from 5 to 17.5), and all values decreased during HDBR.
The different sympathoadrenal response to HDBR compared with microgravity is most likely related to cardiovascular changes. Both microgravity and HDBR cause an increase in the end-diastolic volume at the beginning of exposure to these conditions. The underlying mechanism may, however, be different. During microgravity, the increase in central transmural venous pressure is due to an increase in venous return and to a more negative intrapleural pressure (17, 18), whereas the increase during HDBR is only due to increased venous return. Intravascular volumes decrease during both conditions and to approximately the same extent by 12% (9, 14). Later on there may also be remodeling of the heart muscle and some atrophy (13). Cardiac ouput is increased during weightlessness in parabolic flights and during the early phase of microgravity but gradually decreased during prolonged exposures (16; Norsk P, unpublished observations).
We did not observe any changes in plasma norepinephrine during HDBR, but it must be noted that all samples were obtained with the subjects in the supine position. Forearm venous plasma norepinephrine is correlated to muscle sympathetic nerve activity (3), which also has been found to be unchanged during short-term HDBR (7, 12). Goldstein et al. (5) observed that urinary excretion rates of norepinephrine decreased during HDBR compared with the supine position. In the present experiment, platelet norepinephrine, but not epinephrine, decreased markedly during HDBR. The decrease in platelet norepinephrine during HDBR is most likely due to the fact that the subjects were not allowed to sit up or stand up during HDBR and they were not allowed to perform exercise.
Several studies have now demonstrated, contrary to expectations, that sympathetic nervous activity is not decreased during microgravity, and it is most likely increased compared with ground-based values. In 1995 our laboratory reported that plasma norepinephrine values were elevated inflight and above values observed in the seated position in ground-based experiments (10). Ertl et al. (4) concluded that baseline sympathetic neural outflow was increased moderately inflight. Furthermore, in the same study it was demonstrated that the norepinephrine spillover rate was significantly increased in space. In this study, the steady-state concentration of the norepinephrine tracer was measured in venous blood and not in arterial blood, and the calculated clearance values are therefore too high and to some extent dependent on variations in the local uptake of the tracer in the forearm tissue. Results of the present study are in accordance with our previous study in which we showed that plasma norepinephrine concentrations were increased during microgravity. Thus results from all three studies, which applied different techniques to study sympathetic nervous activity, support the concept that sympathetic activity is moderately increased during microgravity.
The platelet measurements showed high epinephrine values during microgravity that were not observed in our previous study (10). The reason may be that in the first study samples were obtained from a forearm vein and epinephrine in arterial blood is extracted by forearm tissues (3). Platelets circulate through all parts of the body and take up catecholamines from plasma. Platelet epinephrine may therefore be a more reliable index of epinephrine release in the body than epinephrine in forearm venous blood.
The exact interrelationship during microgravity between the initial increase and gradual decrease thereafter in cardiac output and plasma volume and the increment in sympathetic nervous activity during spaceflight remains to be elucidated. Furthermore, the difference in the norepinephrine response to HDBR and microgravity should also be explained. Most likely the decrease in plasma volume inflight plays a major role for the increase in sympathetic nervous activity. There does not appear to be a pronounced early increase in urine output during weightlessness, but there may be a relative increase compared with the intake of fluid, because fluid and food intake decreases (14).
The reduction in plasma volume during HDBR has little influence on basal sympathetic nervous activity as long as the subjects are supine. After prolonged bed rest the subjects have a tendency to develop orthostatic hypotension, which largely can be corrected for by fluid intake (6).
The relationship between cardiac output and sympathetic nervous activity during spaceflight is more difficult to explain. The dilatation of the cardiopulmonary area during spaceflight should inhibit sympathetic nervous activity, but at the same time the distended central vasculature would induce a decrease in vascular compliance. This is probably also what occurs during the early period of spaceflight as observed during parabolic flights (17). During the subsequent decrease in cardiac output and stroke volume, arterial pulsation will decrease. This decrease in pulsation combined with a decrease in compliance of the central vascular wall may activate sympathetic nervous activity.
We cannot exclude the possibility that changes in sympathetic activity during microgravity are due to a decrease in the sensitivity to catecholamines, but this suggestion can hardly explain the difference between bed rest and microgravity. Furthermore, the sympathetic response during spaceflight is also unlikely to be an arousal reaction due to mental stress, because neither blood pressure nor heart rate increased inflight.
In conclusion, a relative high sympathoadrenal activity compared with preflight values seems to be an integrated part of the regulatory response to microgravity. Furthermore, HDBR cannot be applied to simulate changes in sympathoadrenal activity in humans during microgravity.
Platelet norepinephrine values were measured in blood samples obtained early after landing (<12 h). Because of the long half-life of norepinephrine in platelets of ∼2 days (Ref. 2 and the present study), values obtained early postlanding will still reflect the microgravity state. Although there is likely to be an increase in sympathetic activity during the return of the crew to Earth, increments in plasma norepinephrine are not likely to be very high owing to vasoconstriction, which will retain norepinephrine in the extracellular fluid. Furthermore, it has been shown in several studies that platelet norepinephrine and epinephrine are unaffected by acute short-term increments in sympathoadrenal activity such as exercise (1, 2), in which there is a marked increase in plasma norepinephrine and epinephrine. In further studies of sympathoadrenal activity during microgravity, platelet catecholamines should be measured in samples obtained in space. This can be done provided there is an access to a centrifuge with an adjustable speed on the international space station.
The preparation of platelet samples was different during HDBR compared with microgravity. The platelet norepinephrine levels in the preflight samples were also in the lower end of the normal range observed in the HDBR study. We cannot exclude that the loss of cateholamines from the samples may have been greater during the microgravity study compared with HDTB. The platelet number was, however, normal in the samples obtained from the cosmonauts and not different in the preflight and early postlanding sample. All samples in the microgravity experiments were prepared in the same way, and the relative changes in platelet catecholamines between the two groups can therefore be compared.
It is unclear whether the relatively low platelet catecholamine values found in the cosmonauts in preflight samples is a characteristic finding in this group of subjects. If this is the case it may indicate that cosmonauts have a relatively large plasma volume on Earth, because in normal subjects sympathetic nervous activity and plasma volume are inversely related (3).
This study was supported by the Danish Research Councils with grant no. 2006-01-0012.
Ulla Kjærulff-Hansen is thanked for excellent technical assistance.
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