INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

ASTRONAUTS USUALLY WORK WITH feeling much mental stress on launching, in flight, and on landing. They are required to complete their duties precisely and carefully and sometimes must deal with dangerous accidents calmly under severe environment. This mental stress may influence their physiological functions, which can cause serious medical problems. Epidemiological and pathophysiological evidence shows that intense psychological or physical stress can be a specific "triggers" precipitating myocardial infarction and sudden death (21, 24, 25, 34). However, physiological effects of mental stress on humans during and after exposure to microgravitational environment have seldom been examined.

In humans, mental tasks induce cardiovascular changes that are typically reported as increases in arterial blood pressure and heart rate, vasoconstriction in renal and splanchnic vascular beds, vasodilatation in skeletal muscles, and a decrease (or at least no increase) in systemic vascular resistance (3, 5). The autonomic nervous system is the primary contributor to the cardiovascular, in particular cardiac, responses to mental stress (3). Vasomotor sympathetic nerve activity to skeletal muscle typically increases in response to mental stress, but limb vascular resistance decreases; thus changes in vascular resistance during mental stress are likely not due to withdrawal of sympathetic activity. Circulating catecholamines play a role in peripheral vasodilatation during mental stress via -adrenergic receptor activation in humans (13). Nitric oxide also contributes to this vasodilatation (8).

It is unknown whether these cardiovascular responses to mental stress are effected by an exposure to microgravity. Spaceflight and its ground-based analog, 6° head-down bed rest (HDBR), commonly alter autonomic functions (9, 11, 27, 33) and regulation of peripheral vasculatures (4, 12, 29, 33). It was demonstrated that spaceflight may impair the abilities to increase total peripheral resistance and plasma norepinephrine concentrations in response to orthostasis, which may contribute to orthostatic intolerance after spaceflight (4, 12). A recent study has demonstrated a reduction in maximal vasodilatation during reactive hyperemia after 14 days of HDBR, suggesting that HDBR impaired multiple vasodilatory systems (29). In contrast, Convertino and colleagues (6) have recently reported that 14 days of HDBR led to a substantial increase in the vasodilatory response to a -adrenergic agonist, suggesting that ₂-adrenoreceptor responsiveness increased after HDBR. These findings do not have direct relation to responses to mental stress in humans. However, if spaceflight and HDBR modify cardiovascular responses to mental stress in humans, it may have some influences on physiological and medical conditions of astronauts. It is thus a matter worthy to be considered how and whether or not microgravity alters cardiovascular changes under mental stress.

We aimed to examine effects of HDBR on cardiovascular, in particular vasomotor sympathetic and peripheral vasodilator, responses to mental stress. Vasomotor sympathetic nerve activity was directly recorded from the tibial nerve as muscle sympathetic nerve activity (MSNA) by using a microneurographic technique. We performed 14 days of HDBR and 10 min of mental arithmetic (MA) tests before and after HDBR in 16 healthy male volunteers. We then compared the responses of MSNA, calf blood flow, and vascular resistance to MA after HDBR with those before HDBR.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

Subjects. Twenty-four healthy male volunteers with a mean age of 22 ± 1 (SE) yr (range 19-36 yr), mean height of 168.0 ± 1.0 cm (range 158.0-176.1 cm), and mean weight of 62.8 ± 1.7 kg (range 49.0-75.7 kg) were studied. All subjects were evaluated as having normal physical fitness by detailed medical history, physical examination, complete blood count, resting electrocardiogram, a panel of blood chemistry analyses, and psychological tests. No subject smoked, used recreational drugs, or had chronic medical problems. All subjects gave informed consent to participate in the study, which was approved by the Human Research Committee, Research Institute of Environmental Medicine, Nagoya University, and the Ethical Committee of the National Space Development Agency of Japan.

HDBR protocol. Sixteen subjects were exposed to 14 days of strict adherence to 6° HDBR. During HDBR, the subjects were continuously monitored by staff nurses to ensure that they remained head down without interruption and that no physical exercise was performed by the subjects. Dietary intake was 2,000-2,100 kcal/day (55% carbohydrate, 25% fat, 20% protein), and fluid intake from daily drinks was ad libitum; the average was 1,153 ± 45 ml/day. The photoperiod was 16 h of light and 8 h of dark with lights on at 0700. Smoking and drinking caffeinated beverages were strictly prohibited throughout the course of the experiment. Each subject underwent MA tests 7-12 days before the start of HDBR and immediately after 14 days of HDBR.

Measurements. The MSNA was recorded by microneurography from the tibial nerve of unilateral leg. Calf blood flow was measured by using venous occlusion and mercury-in-Silastic strain gauge plethysmography (16) on the contralateral leg. Monitoring of the electrocardiogram from chest lead II, of blood pressure in peripheral artery with a pneumatic finger cuff (Portapres, TNO Institute of Applied Physics Biomedical Instrumentation TPD), and of respiration with a thermistor was performed. Blood pressure values were confirmed every minute by an automated upper-arm sphygmomanometer (BP203MII, Nippon Colin, Japan). The finger cuff of the Portapres was noninvasively attached to two digits of the left hand and inflated alternately to prevent pain due to continuous air pressure load. All variables except intermittent upper-arm pressure measurements were stored on a DAT recorder (PC-216Ax, Sony Magnescale) for further analysis.

Microneurography. A tungsten microelectrode with a shaft diameter of 120 µm and an electrode impedance of 2 to 5 M (model 26-05-1, Federick Haer, Bowdoinham, ME) was inserted percutaneously into the muscle nerve fascicles of the tibial nerve at the popliteal fossa without anesthesia. Nerve signals were fed into a high-input impedance preamplifier (Kohno Instruments, Nagoya, input impedance: 100 M; gain 40,000), with two active band-pass filters set between 500 and 5,000 Hz, and were monitored with a loudspeaker. MSNA was identified according to the following discharge characteristics: 1) pulse synchronous spontaneous efferent discharges, 2) afferent activity being evoked by tapping of the soleus muscle, but not in response to a gentle skin touch, and 3) enhancement during phase II of Valsalva maneuver.

Venous occlusion plethysmography. Calf blood flow was measured by using venous occlusion and mercury-in-Silastic strain gauge plethysmography (16). While the subject was supine, the calf was placed 10-15 cm above the right atrium to collapse the veins. Occlusion cuffs were placed around the thigh just above the knee and around the ankle. Care was taken to place the strain gauge in the same place in the pre- and post-HDBR trials. With the ankle cuff inflated to 250 mmHg to arrest the circulation to the foot, calf blood flow was measured by inflating the thigh cuff to 50 mmHg for 7 s each. Calf blood flow determinations were performed at 30-s intervals. Calf vascular resistance was calculated by dividing mean blood pressure by calf blood flow.

Mental stress test. The MA task consisted of subtraction of a two- or three-digit number from a three- or four-digit number. The investigator randomly sampled the MA task from preliminarily stored 800 questions, which were comparable among all trials (pre- and post-HDBR, control trials 1 and 2), depending on the subject's skill level. Each subject was presented with many MA questions in each trial by the investigator and could not memorize them. Each MA test was thus considered to be identical to the other tests. All subjects were urged to work more quickly throughout the task so that they remained actively engaged in the task. Both the task presentation and subject responses were primarily nonverbal to minimize the effect of investigator bias and speaking on MSNA and cardiovascular changes. The subjects were asked to assess their numerical rating of perceived stress on completion of the protocol, using a standard five-point scale of 0 (not stressful), 1 (somewhat stressful), 2 (stressful), 3 (very stressful), and 4 (very, very stressful) (22).

Experimental protocol. MA test was performed before and after HDBR. The subjects rested quietly in supine position for over 30 min. At least 10 min after a satisfactory recording site for MSNA was obtained, pre-MA baseline recordings of MSNA, finger blood pressure, upper-arm blood pressure, calf blood flow, heart rate, and respiration were performed for 10 min. MA test was then given for 10 min in supine position. All sympathetic and cardiovascular variables including MSNA were continuously recorded during 10 min of MA and 2 min of recovery.

Data analysis. MSNA was full-wave rectified and fed through a resistance-capacitance integrating circuit with a time constant of 0.1 s to obtain the integrated MSNA, which was displayed along with the electrocardiogram, blood pressure, and respiration on a pen recorder (RECTI-HORIZ, NEC San-Ei, Tokyo, Japan). MSNA was expressed as 1) MSNA burst rate, i.e., the mean number of sympathetic bursts per min, and 2) total MSNA, i.e., the sum of the MSNA burst amplitudes of all bursts during each analyzed period. For calculation of MSNA burst amplitude in the mean voltage neurogram, all amplitudes of bursts were measured using a digitizing tablet. As MSNA burst amplitude depended on electrode position, which varied from day to day, it is impossible to detect differences in absolute burst amplitudes. The mean value per minute of total MSNA (the sum of all MSNA burst amplitudes) during 10 min of pre-MA rest was given the arbitrary value of "100 arbitrary units/min," and all total MSNA during each minute of pre-MA, MA test, and recovery were expressed in relation to this value.

Statistical analysis. Data are expressed as means ± SE. A two-way repeated-measures analysis of variance [condition (before vs. after HDBR, control trial 1 vs. 2) and time (pre-MA control, MA test, and recovery)] was performed (Figs. 14). Tests for simple effects were done with a Scheffé's F procedure when the main effect was found to be significant. A Wilcoxon signed-rank test was performed to compare baseline variables between before and after HDBR. Significance was set at P < 0.05. 

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Muscle sympathetic nerve activity (MSNA) burst rate (A) and its changes from pre-mental arithmetic (MA) baseline (B) and total MSNA (C) during 10 min of pre-MA baseline control, 10 min of MA, and 2 min of recovery before and after head-down bed rest (HDBR). Rec, recovery measurement after MA. Values are means ± SE. * P < 0.05 between before and after HDBR. # P < 0.05 vs. all corresponding pre-MA baseline control values.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Calf blood flow (A) and its changes from pre-MA baseline (B) and calf vascular resistance (C) and its changes from pre-MA baseline (D) during 10 min of pre-MA baseline control, 10 min of MA, and 2 min of recovery before and after HDBR. Values are means ± SE. * P < 0.05 between before and after HDBR. # P < 0.05 vs. all corresponding pre-MA baseline control values.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Heart rate (A) and its changes from pre-MA baseline (B) and mean blood pressure (C) and its changes from pre-MA baseline (D) during 10 min of pre-MA baseline control, 10 min of MA, and 2 min of recovery before and after HDBR. Values are means ± SE. * P < 0.05 between before and after HDBR. # P < 0.05 vs. all corresponding pre-MA baseline control values.

View larger version (57K): [in this window] [in a new window]   Fig. 4.   MSNA burst rate (A), total MSNA (B), calf blood flow (C), calf vascular resistance (D), heart rate (E), and mean blood pressure (F) during 10 min of pre-MA baseline control, 10 min of MA, and 2 min of recovery in control trials 1 and 2. There was no difference between control trials 1 and 2. Values are means ± SE. # P < 0.05 vs. all corresponding pre-MA baseline values.

	RESULTS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

Baseline measurements. Pre-MA baseline values for all variables before and after HDBR are presented in Table 1. Baseline MSNA burst rate, calf vascular resistance, and heart rate were higher, but baseline calf blood flow was lower after HDBR than before HDBR. Baseline mean blood pressure remained unchanged after HDBR.

MSNA. The perception of the severity of MA after HDBR (2.5 ± 0.5) was similar to that before HDBR (2.3 ± 0.6). MSNA responses to the experimental protocols are shown in Fig. 1. MSNA responses to MA test, expressed both as burst rate and total MSNA, were significantly changed by HDBR (time × condition interaction; P < 0.001 for both expressions of MSNA). MSNA burst rate did not increase during MA test before HDBR, but significantly increased after HDBR. Total MSNA significantly increased during MA test before HDBR due to an elevation in MSNA burst amplitude. The increase in total MSNA was significantly augmented after HDBR. The absolute level of MSNA was higher after HDBR than before HDBR (P < 0.001).

Calf blood flow and calf vascular resistance. The responses of blood flow and vascular resistance in the calf to the experimental protocols are shown in Fig. 2. These responses to MA test were significantly altered by HDBR (time × condition interaction; both, P < 0.01). Calf blood flow significantly increased, and calf vascular resistance significantly decreased from baseline levels before HDBR, whereas they did not change after HDBR. The absolute level of calf blood flow during MA was lower, whereas that of calf vascular resistance was higher, after HDBR compared with before (P < 0.001 for both).

Heart rate and mean blood pressure. The responses of heart rate and mean blood pressure to the experimental protocols are shown in Fig. 3. There was no difference in the responses of heart rate and mean blood pressure between before and after HDBR.

Reproducibility of variables in responses to MA testing. To examine the reproducibility of MSNA, calf blood flow, and vascular resistance in response to MA testing, we performed repeated MA tests (control trials 1 and 2) without HDBR. The responses of variables in these control trials were summarized in Fig. 4. There was no difference in any baseline variables during pre-MA rest between the two trials. The perception of the severity of MA was similar in control trials 1 (2.4 ± 0.6) and 2 (2.5 ± 0.7). MSNA burst rate remained unchanged, but total MSNA significantly increased during MA tests due to an elevation in burst amplitude similarly in control trials 1 and 2. Calf blood flow increased, whereas calf vascular resistance decreased significantly, and heart rate and mean blood pressure increased significantly during MA testing in both trials, and their responses were similar in the two trials.

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

Mental stress is one of the important factors for maintenance of healthy condition of astronauts under very stressful working surroundings; however, very few investigators have examined physiological effects of mental stress on humans during and after spaceflight or simulated microgravity. This study is the first to examine how and whether or not HDBR affects influences of mental stress on cardiovascular, in particular vasomotor sympathetic and multiple vasodilator, responses. The major new findings of the present study were 1) the increase in MSNA in response to 10 min of MA was augmented, but 2) the increase in calf blood flow and decrease in calf vascular resistance in response to MA disappeared after 14 days of HDBR. In contrast, increases in heart rate and mean blood pressure during MA remained unchanged after HDBR, similar to previous study (30). These findings suggest that 14 days of HDBR might augment vasomotor sympathoexcitation but attenuate vasodilatation in the calf during mental stress in humans. The present study may raise a possibility that intense mental tension (i.e., on dangerous accident) can cause large vasomotor sympathoexcitation in astronauts.

Previous studies have reported variable MSNA responses to mental tasks (1, 2, 7, 14, 15, 32), possibly dependent on varied stress perceptions (5). In the present study, total MSNA significantly increased during MA before HDBR due to increase in burst amplitude, although increase in MSNA burst rate failed to be significant. We are confident of the reproducibility of MSNA response to MA employed here, because similar MSNA response patterns were observed with similar levels of stress perceptions in the control repeated MA tests on separated days and the pre-HDBR MA test.

Before HDBR, MA caused calf vasodilatation, which decreased vascular resistance and increased blood flow in the calf. The calf vasodilatation during MA before HDBR may be consistent with the previous vasodilator responses of forearm (8, 13) and calf (10, 15) during mental stress. Because MSNA did not decrease, but rather slightly increased during MA before HDBR, the calf vasodilatation observed before HDBR may not be due to a withdrawal of neural vasoconstrictor drive. This concept may be consistent with the previous study that color word conflict test increased MSNA and local norepinephrine release, but induced an increase in blood flow and decrease in vascular resistance in calf (10). Therefore, other mechanisms alone or in combination may contribute to the calf vasodilatation during MA before HDBR. Major possible mechanisms might be -adrenergic (13) or nitric oxide-mediated (8) vasodilatation, but the present study cannot provide a clear answer.

After HDBR, the increase in MSNA in response to MA was augmented. One possible mechanism of the augmented MSNA response seems to be enhanced stress perception of MA after HDBR. A subsequent study demonstrated that a stimulation of MSNA is governed primarily by perceived stress, which depends, in part, on the absolute difficulty of the task (5). In the present study, however, levels of numerical ratings of perceived stress of MA did not differ before and after HDBR. Therefore, the increase in stress perception of MA after HDBR cannot account for the augmented MSNA increase. The second possible explanation is that same perceived stress might cause larger increases in MSNA after HDBR (i.e., uncoupling of the perceived stress-MSNA enhancement relationship). Another possibility is impairment of arterial baroreflex-mediated inhibition of MSNA after HDBR. There may be an inhibitory effect of the arterial baroreflex on MSNA in response to blood pressure elevation during a mental task (1), because MSNA neurons receive strong inhibitory input from arterial baroreceptors, whereas the arterial baroreflex control of vasomotor sympathetic outflow may be impaired after real and simulated spaceflight (4, 9, 26, 33). Thus we suggest that the magnitude of arterial baroreflex-mediated inhibition of MSNA in response to blood pressure elevation during MA might be attenuated after HDBR, contributing partly to the augmentation of MSNA increase in response to MA after HDBR. However, this suggestion has a limitation. If impairment of the arterial baroreflex is responsible for the larger increase in MSNA and arterial baroreflex control of heart rate was also reduced after HDBR, similarly to previous studies (9, 11, 19, 33), increase in heart rate should have been augmented during MA after HDBR. In the present study, heart rate response was not altered after HDBR.

MSNA has a close correlation to cardiac sympathetic activation (estimated by cardiac norepinephrine spillover) (31). In addition, MSNA increases circulating levels of plasma norepinephrine (14), partly contributing to cardiac adrenergic activation. Thus it could be argued that the higher absolute levels as well as greater responses of MSNA after HDBR may have important implications for the large cardiac adrenergic activation under mental stress, which could be a potent risk for the atrophic heart after microgravity (22). However, this speculation was tentative, because heart rate response was unchanged rather than augmented after HDBR.

After HDBR, the increase in calf blood flow and decrease in calf vascular resistance in response to MA were attenuated. These findings suggest that HDBR might change and impair the calf vasodilator response to mental stress. This concept may be consistent with the recent study by Shoemaker et al. (29). We cannot determine the precise mechanism(s) for the attenuation of calf vasodilatation during MA after HDBR, but there are several possibilities. First, the increase in MSNA in response to MA was augmented after HDBR. This augmented MSNA activation could counteract vasodilatation induced by other multiple vasodilatory systems during MA after HDBR. However, the first possibility alone cannot account for all the attenuation of vasodilatation during MA after HDBR, because the increase in MSNA remained unchanged, but the increase in calf blood flow and decrease in calf vascular resistance were reduced during the initial 4 min of MA after HDBR compared with the pre-HDBR trial.

The second possibility is impairment of -adrenergic vasodilatation during MA. However, it was recently reported that 14 days of HDBR led to a substantial increase in the vasodilator response to a -adrenergic agonist (6), suggesting that HDBR may cause selective increases in ₂-adrenoreceptor responsiveness. Thus the second possibility seems unlikely, although it remains unknown whether secretion of adrenaline from adrenal glands in response to mental stress was altered after HDBR.

The third possibility is impairment of nitric oxide-mediated vasodilatation during mental stress. The mechanisms for release of nitric oxide remain controversial, but some vascular formations of nitric oxide are directly facilitated by shear stress (18). Circulatory blood volume loss (9) and reduced absolute levels of calf blood flow after HDBR could decrease shear stress in vessels, thereby suppressing shear stress-induced nitric oxide release. However, this possibility remains tentative, because neither measurement of nitric oxide formation levels nor administration of nitric oxide synthase blocker was performed during MA in the present study.

We observed that resting supine MSNA increased after 14 days of HDBR. This finding is consistent with our previous studies demonstrating increases in resting MSNA after short [3 (17) and 6 (20) days] and prolonged [120 days (19)] duration of simulated microgravity. One possible mechanism for the increase in resting sympathetic tone after HDBR may be HDBR-induced circulatory blood volume loss, which might stimulate baroreflex functions to elevate sympathetic nerve outflow. However, our finding is inconsistent with a previous work by Shoemaker et al. (28) showing decreases in MSNA after 14 days of HDBR. One possible explanation is the difference in methodological designs, particularly in daily fluid consumption, which was smaller in the present study (1,153 ± 45 ml/day) than in the previous study (2,000 ml). However, further studies are necessary to address these issues.

A major postspaceflight problem is orthostatic intolerance. But, it may be difficult to consider the putative role of augmented sympathoexcitation and reduced vasodilatation during mental stress after HDBR in the pathophysiology of this orthostatic intorelance, because mental stress and orthostatic stress likely induce very different physiological responses in humans. However, when a subject who is exposed to spaceflight and HDBR reenters a gravitational environment, he may feel tension for presyncopal symptoms, because he must know the risk of orthostatic hypotension after microgravity. The present study tentatively suggests that the mental tension of orthostatic hypotension might partly contribute to increase sympathetic vasoconstrictor outflow and arterial tone and might not relate to impaired vasoconstrictor response during orthostatic hypotension after HDBR (4, 12).

In conclusion, 14 days of HDBR augmented vasomotor sympathoexcitation but attenuated vasodilatation in the calf muscle in response to mental stress. These findings suggest that total responses of peripheral circulation to mental stress might be altered after HDBR. The mechanism(s) responsible for these alterations are unknown.