Exercise plus volume loading prevents orthostatic intolerance but not reduction in cerebral blood flow velocity after bed rest

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1 Am J Physiol Heart Circ Physiol 302: H489 H497, First published November 11, 2011; doi: /ajpheart Exercise plus volume loading prevents orthostatic intolerance but not reduction in cerebral blood flow velocity after bed rest Sung-Moon Jeong, 1,2 Shigeki Shibata, 1 Benjamin D. Levine, 1 and Rong Zhang 1 1 Institute for Exercise and Environmental Medicine, Presbyterian Hospital of Dallas, and University of Texas Southwestern Medical Center at Dallas, Dallas, Texas; and 2 Department of Anesthesiology and Pain Medicine, College of Medicine, University of Ulsan, Asan Medical Center, Seoul, Korea Submitted 28 April 2011; accepted in final form 1 November 2011 Jeong SM, Shibata S, Levine BD, Zhang R. Exercise plus volume loading prevents orthostatic intolerance but not reduction in cerebral blood flow velocity after bed rest. Am J Physiol Heart Circ Physiol 302: H489 H497, First published November 11, 2011; doi: /ajpheart This study tested the hypothesis that reduction in cerebral blood flow (CBF) during orthostatic stress after bed rest can be ameliorated with volume loading, exercise, or both. Transcranial Doppler was used to measure changes in CBF velocity during lower body negative pressure (LBNP) before and after an 18-day bed rest in 33 healthy subjects. Subjects were assigned into four groups with similar age and sex: 1) supine cycling during bed rest (Exercise group; n 7), 2) volume loading with Dextran infusion after bed rest to restore reduced left ventricular filling pressure (Dextran group; n 7), 3) exercise combined with volume loading to prevent orthostatic intolerance (Ex-Dex group; n 7), and 4) a control group (n 12). LBNP tolerance was measured using a cumulative stress index (CSI). After bed rest, CBF velocity was reduced at a lower level of LBNP in the Control group, and the magnitude of reduction was greater in the Ex-Dex group. However, reduction in orthostatic tolerance was prevented in the Ex-Dex group. Notably, volume loading alone prevented greater reductions in CBF velocity after bed rest, but CSI was reduced still by 25%. Finally, decreases in CBF velocity during LBNP were correlated with reduction in cardiac output under all conditions (r ; P 0.001). Taken together, these findings demonstrate that volume loading alone can ameliorate reductions in CBF during LBNP. However, the lack of associations between changes in CBF velocity and orthostatic tolerance suggests that reductions in CBF during LBNP under steady-state conditions by itself are unlikely to be a primary factor leading to orthostatic intolerance. transcranial Doppler; spaceflight ORTHOSTATIC INTOLERANCE IS common after head-down-tilt (HDT) bed rest or spaceflight (5). The underlying mechanisms are likely to be multifactorial and are not completely understood (5, 7, 9, 20). In this regard, a substantial reduction in brain perfusion at the onset of cardiovascular collapse appears to be the final common pathway leading to orthostatic intolerance in most individuals (17, 19, 39). Notably, cerebral blood flow (CBF) is reduced during orthostatic stress even when mean arterial pressure is well maintained, that is, during the compensatory phases of cardiovascular response (3, 19). For example, reductions in CBF velocity during lower body negative pressure (LBNP), upright standing, or head-up tilt (HUT) may range from 10% to 30% under steadystate conditions (19, 34). Reductions in CBF velocity during Address for reprint requests and other correspondence: R. Zhang, Institute for Exercise and Environmental Medicine, Presbyterian Hospital of Dallas, 7232 Greenville Ave., Dallas, TX ( rongzhang@texashealth.org). LBNP were greater after HDT bed rest, but not after short-term space flight (15, 39). Whether reductions in CBF under steadystate conditions are related to orthostatic intolerance is not clear. We have proposed that reduction in CBF under steady-state conditions may reduce cerebrovascular reserve and contribute to orthostatic intolerance when systemic hemodynamic instability, as manifested by the onset of rapid reduction in arterial pressure and bradycardia, develops (19, 39). Central hypovolemia occurs after HDT bed rest or space flight as a result of cephalad fluid shift induced reduction in plasma volume (2). Our previous studies suggest that central hypovolemia induced by HDT bed rest contributes to orthostatic intolerance through its effects on baroreflex regulation of arterial pressure and/or exacerbating reductions in cardiac output during orthostatic stress (16, 31). Recent studies suggest that changes in cardiac output may influence CBF directly independent of changes in blood pressure (14, 21). Our previous study showed that central hypovolemia and reduction in cardiac output after HDT bed rest were not associated with reduction in CBF velocity under supine resting condition (39). However, the impact of hypovolemia and reduction in cardiac output on CBF during orthostatic stress has not been carefully studied. Countermeasures such as volume loading and exercise have been used to prevent orthostatic intolerance (31, 36). One of the potential mechanisms may be to ameliorate reductions in stroke volume or cardiac output during orthostatic stress (31). However, the effects of these countermeasures on brain perfusion remain unknown. The purpose of this study was test the hypothesis that reduction in CBF velocity during LBNP after bed rest would be ameliorated with volume loading, exercise, or both through their effects on cardiac output. In addition, we hypothesized that improvement in CBF during LBNP would lead to improvement in orthostatic tolerance. METHODS Subjects. Thirty-three healthy young subjects (29 men, 4 women) with a mean age of 31 9 years, height of cm, and weight of kg participated in this study. Two independent but related bed rest studies with the same HDT interventions and duration were conducted. The first bed rest study without countermeasures consisted of 12 subjects and served as a control group (24 5 years; 11 men, 1 woman). Part of the data from this study were published previously focusing on changes in CBF velocity during LBNP after bed rest (39). The second bed rest study with countermeasures was conducted after 5 years from the first bed rest study to determine the effects of exercise and volume loading on orthostatic tolerance (31). In the second bed rest study, 21 subjects were assigned randomly into three groups with sex as a stratification factor to balance the experiments: /12 Copyright 2012 the American Physiological Society H489

2 H490 1) exercise group (n 7; 38 3 years; 1 woman), 2) Dextran infusion group (Dextran group; n 7; 42 2 years; 1 woman), and 3) a combined exercise and Dextran infusion group (Ex-Dex group; n 7; 35 3 years; 1 woman) (31). All subjects had normal ECG and were normotensive. They also had normal echocardiography and were free of drugs and regular use of medication. All subjects gave their written informed consent, and the study protocol was approved by the Institutional Review Boards of the University of Texas Southwestern Medical Center at Dallas and Texas Health Presbyterian Hospital at Dallas. HDT bed rest. The detailed study protocol has been described previously (31, 39). Briefly, after baseline experiments, subjects were placed in 6 degree HDT bed rest for 18 days. Subjects were allowed to elevate on one elbow for meals but otherwise were restricted to the head-down position at all times. Subjects were housed in the General Clinical Research Center at the University of Texas Southwestern Medical Center/Parkland Hospital and given a standard diet. Fluids were allowed ad libitum, but all fluid intake and urine output were recorded. All experiments involving measurements of CBF velocity before and after bed rest and changes in CBF velocity during LBNP were repeated after 18 days of HDT bed rest. Dextran infusion after bed rest. The reduction in plasma volume after bed rest was estimated by the Evans Blue dye method (8). Changes in hematocrit were measured via microcentrifuge techniques. In the Dextran group or the Ex-Dex group, the amount of Dextran infusion was determined to restore the reduced left ventricular filling pressure to the pre-bed rest level (31). Briefly, a 6-F balloon-tipped, fluid-filled catheter (Swan-Ganz; Baxter) was placed through the antecubital vein into the pulmonary artery to measure pulmonary capillary wedge pressure (PCWP) (20). With continuous monitoring of PCWP, the amount of Dextran equivalent to the plasma loss estimated by the Evans Blue dye method was infused rapidly ( 1,000 ml/h) and allowed to equilibrate for 30 min followed by repeated assessment of PCWP. If the mean PCWP was still below the pre-bed rest level, additional Dextran was infused in 100- to 250-ml increments until the pre-bed rest level of PCWP was achieved. Of note, these procedures with invasive cardiac catheterization were conducted on day 15 of bed rests to measure changes in cardiac mechanics (31). After an additional 3 days of bed rest, the same amount of Dextran determined earlier was infused under supine resting conditions. Baseline hemodynamic measurements were repeated 30 min after completion of the Dextran infusion. Maximal LBNP was then applied to determine orthostatic tolerance. This study design would minimize the potential effects of invasive catheterization on LBNP tolerance (31). Exercise during HDT bed rest. Subjects in the exercise groups, including the exercise alone or combined exercise and Dextran infusion group, exercised on a supine ergometer three times a day, 30 min a session, at 75% of maximal heart rate determined from a pre-bed rest testing of maximal oxygen consumption (V O 2max) in the upright position (31). Polar heart rate monitor (Polar Electro, Finland) was used to monitor heart rate during exercise. Target heart rate was strictly controlled during each of the exercise sessions and reviewed carefully to ensure compliance. LBNP test. Orthostatic tolerance was measured using graded LBNP, beginning at 15 mmhg for 5 min and then increased to 30 and 40 mmhg for 5 min each, followed by an increase in LBNP by 10 mmhg every 3 min until signs or symptoms of presyncope or syncope were observed (39). LBNP was implemented with a Plexiglas tank sealed at the level of the iliac crests in the supine position. A cumulative stress index (CSI; CSI sum of the magnitude of LBNP at each level times duration) was used to assess orthostatic tolerance (39). In this study, changes in systemic and cerebral hemodynamics under steady-state conditions, that is, during the compensatory phases of LBNP, were presented from rest to 60 mmhg LBNP for statistical data analysis since most subjects tolerated this level of LBNP (pre-bed rest, n 28; post-bed rest, n 23). In addition, we measured arterial pressure and CBF velocity immediately before the onset of presyncopal symptoms to assess cerebrovascular responses during transient changes in arterial pressure. Instrumentation. ECG was obtained to monitor heart rate (HP B; Hewlett-Packard). Beat-by-beat arterial pressure was obtained using photoplethysmography (Finapres; Ohmeda). Intermittent blood pressure was measured in the arm by electrosphygmomanometry (Suntech Medical Instruments 4240). Cardiac output was measured using a rebreathing method (20). Cardiac output was measured at baseline under resting conditions and during the last minute of each level of LBNP ( 40 mmhg) and then every other level of LBNP ( 50 mmhg) (19, 39). Stroke volume was calculated as cardiac output divided by heart rate measured during rebreathing. Heart rate presented in this study was measured as time average of beat-to-beat changes not including the rebreathing period, since rebreathing itself may affect heart rate. Transcranial Doppler was used to measure CBF velocity in the middle cerebral artery (MCA) using a 2-MHz probe (Pioneer Nicolet, for the first bed rest study; Multiflow, DWL, Germany, for the second bed rest study). The probe was placed over the temporal window and was fixed at a constant angle and position using a custom-made probe holder to fit each subject s facial bone structure to allow for repeated studies (11). End-tidal CO 2 was monitored via a nasal cannula using a mass spectrometer (Marquette MGA 1100 Mass Spectrometer). For technical reasons, end-tidal CO 2 measurement was not available during the first bed rest study. Data acquisition and analysis. All experiments were performed in the morning in the supine position at least 2 h after a light breakfast. Subjects refrained from consuming caffeinated beverages or alcohol for at least 12 h before any testing. For pre-bed rest test, after the subjects rested in the supine position for at least 30 min, 6 min of heart rate, arterial pressure, and CBF velocity were recorded during spontaneous respiration as baseline data. LBNP was then performed. After bed rest, the same procedures for data collection were repeated. Of note, for the second bed rest study, an additional 6 min of data collection was repeated after Dextran infusion. The analog arterial pressure and spectral envelope of the CBF velocity Doppler signal were sampled at 100 Hz to obtain beat-to-beat mean arterial pressure and mean CBF velocity with each cardiac cycle (39). Beat-to-beat values of mean arterial pressure, CBF velocity, and heart rate were averaged again over 6 min at baseline and over the last 2 to 3 min at each level of LBNP as the steady-state values. Arterial pressure and CBF velocity at the onset of presyncopal symptoms were obtained as mean values averaged over the last 15 s of data immediately before the release of LBNP. Cerebrovascular resistance index (CVRI) was calculated from mean arterial pressure divided by mean CBF velocity. Statistical analysis. Data are presented as means SD and were analyzed using SigmaStat software (SPSS, version 3.1). Changes in plasma volume, hematocrit, and CSI as well as body weight before and after bed rest were compared using paired t-tests within the groups. In the Dextran group and the Ex-Dex group, changes in systemic and cerebral hemodynamics before and after Dextran infusion also were compared using paired t-tests. Two-way repeated-measures ANOVA was used to detect the main and interactive effects of bed rest and LBNP on the systemic and cerebral hemodynamic variables within and between the groups. The Holm-Sidak method was used for post hoc comparisons. The relationship between changes in CBF velocity and cardiac output during LBNP was assessed using Pearson s linear correlation analysis. A P value of 0.05 was considered statistically significant. RESULTS Reduction in plasma volume and orthostatic tolerance after bed rest. All the subjects completed the experiments. Their demographic characteristics are given in Table 1. As expected, plasma volume was reduced after bed rest in both the control

3 Table 1. Demographic data and pre- and post-bed rest changes in plasma volume and orthostatic tolerance H491 Group and Dextran groups (P 0.01), but not in the exercise and Ex-Dex groups (Table 1). Thus the amount of Dextran infusion needed to restore the reduced left ventricular filling pressure to its pre-bed rest level tends to be larger in the Dextran group than that in the Ex-Dex group (Table 1). Hematocrit appears to be increased slightly with reduction in plasma volume. However, these changes were not statistically significant (Table 1). After bed rest, orthostatic tolerance (CSI) was reduced by 24 8% in the control group (Table 1). Despite restoration of plasma volume with Dextran infusion, CSI was reduced still by 25 7% in the Dextran group. In addition, a greater reduction in CSI by 43 5% was observed in the exercise group (Table 1). However, reduction in orthostatic tolerance was prevented completely in the Ex-Dex group (Table 1). Systemic and cerebral hemodynamics under resting conditions. Hemodynamic data under resting conditions before and after bed rest are shown in Fig. 1 and Table 2. Before bed rest, no significant differences in hemodynamic measurements were observed among the groups. After bed rest, resting cardiac output and stroke volume were decreased in the control and Dextran groups. Heart rate in the control group was increased to compensate for decreases in stroke volume (Table 2). No significant changes in these variables (except for a reduction in stroke volume in the Ex-Dex group) were observed in the Exercise and Ex-Dex groups (Table 2). Arterial blood pressure, CBF velocity, CVRI, and end-tidal CO 2 did not change in all the groups (Fig. 1 and Table 2). As expected, stroke volume and cardiac output were increased after volume loading in both the Dextran and Ex-Dex groups (Table 2). However, no significant increases in CBF velocity were observed associated with increases in cardiac output. Hemodynamics during LBNP. Before bed rest, systolic pressure was reduced at high levels of LBNP in the control group. Diastolic and mean blood pressure did not change in all the groups (Fig. 1 and Table 2). As expected, cardiac output and stroke volume were reduced associated with increases in heart rate in all the groups (Table 2). CBF velocity during LBNP was reduced gradually in all the groups and reached a significant level at 40 mmhg LBNP in the control and 50 and 60 Control Exercise Dextran Ex-Dex Sex, male/female 11/1 6/1 6/1 6/1 Age, years Height, cm Weight, kg Pre Post * * * * 0.92 Plasma volume, l Pre Post * * Hematocrit, % Pre Post Cumulative stress index, mmhg min Pre , Post * * * Dextran infusion, ml Values are means SD. Ex-Dex group, the combined exercise and Dextran infusion countermeasures; Pre, pre-bed rest; Post, post-bed rest. *P 0.05, comparisons between pre- and post-bed rest in the same group. P values of the last column are the main effects of ANOVA for groups under the same bed rest conditions. mmhg LBNP in the control and Ex-Dex groups (Fig. 1). End-tidal CO 2 was reduced at high levels of LBNP in the Dextran and Ex-Dex groups, and a similar trend was observed in the exercise group, although not significant (Table 2). Finally, CBF velocity decreased substantially associated with reductions in arterial pressure at the onset of presyncopal symptoms in the control and Dextran groups and a similar reduction was observed in the Exercise and Ex-Dex groups even though no significant reductions in arterial pressure from the baseline were observed at maximal LBNP (Fig. 1). Of note, no significant differences in hemodynamic responses to LBNP were observed before bed rest among the groups. After bed rest, similar changes in systolic and diastolic pressure were observed as pre-bed rest in all the groups (Table 2). However, mean blood pressure was reduced at 50 and 60 mmhg LBNP in the Control group (Fig. 1). Relative to pre-bed rest, reductions in stroke volume and cardiac output and increases in heart rate during LBNP were greater in the control group (Table 2). CBF velocity during LBNP was reduced at a lower level of LBNP in the control group and to a greater magnitude in the Ex-Dex group (Fig. 1). Notably, restoring plasma volume with Dextran infusion normalized greater reductions in cardiac output and stroke volume and prevented exaggerated reduction in CBF velocity after bed rest in the Dextran group (Fig. 1 and Table 1). Reductions in end-tidal CO 2 during LBNP were similar between pre- and post-bed rest in the exercise, Dextran, and Ex-Dex groups (Table 2). Of note, reductions in CBF velocity during LBNP were correlated linearly with changes in cardiac output under all conditions (averaged r ; P 0.05; Fig. 2). Finally, similar to pre-bed rest, CBF velocity was reduced significantly associated with reduction in arterial pressure at the onset of presyncopal symptoms in the control and Dextran groups (Fig. 1). DISCUSSION This is the first study of effects of volume loading and exercise countermeasures on changes in CBF after HDT bed P

4 H492 Downloaded from Fig. 1. Pre- and post-bed rest changes in mean blood pressure (MBP), cerebral blood flow velocity (CBFV), and cerebrovascular resistance index (CVRI) during lower body negative pressure (LBNP). Ex-Dex group, the combined exercise and Dextran infusion countermeasures. BS, baseline; Dx, Dextran infusion; Max, maximal level of LBNP at the onset of presyncopal symptoms. Pre-bed rest maximal LBNP was 70 7 (range, ), ( ), ( 60 80), and mmhg ( 30 90) in the control, exercise, Dextran, and Ex-Dex groups, respectively. Post-bed rest maximal LBNP was (range, 40 90), ( 40 80), 59 9( 40 70), and mmhg ( 40 80) in the control, exercise, Dextran, and Ex-Dex groups, respectively. Solid lines and filled circles are pre-bed rest, and dashed lines and open circles are post-bed rest. The error bars are SD. *P 0.05 compared with baseline; P 0.05 comparison between pre- and post-bed rest at each level of LBNP. by on November 20, 2017 rest. The main findings are threefold. First, we found that volume loading alone prevented greater reduction in CBF velocity during LBNP after bed rest. However, contrary with our hypothesis, prevention of greater reduction in CBF velocity did not lead to an improvement in orthostatic intolerance. Second, we found that exercise combined with volume loading prevented orthostatic intolerance but did not prevent greater reduction in CBF velocity during LBNP. Third, we observed that reductions in CBF velocity during LBNP correlated linearly with changes in cardiac output under all conditions, suggesting an important role of cardiac output in regulation of brain perfusion during orthostatic stress. In the following sections, we will discuss the potential underlying mechanisms as well as the pathophysiological consequences of these findings. Central hypovolemia, cardiac output, and CBF during orthostatic stress. Previous studies of upright standing, HUT, or LBNP showed that CBF velocity was reduced during orthostatic stress even when mean arterial pressure was well maintained (3, 19). This reduction in CBF velocity, presumably

5 Table 2. Pre- and post-bed rest changes in systemic hemodynamics during lower body negative pressure H493 Baseline Dextran infusion Lower body negative pressure, mmhg Pre-bed rest Control group SBP, mmhg * * DBP, mmhg HR, beats/min * 97 15* * CO, l/min * * * * SV, ml * 51 15* 39 10* 32 11* EtCO 2, mmhg Exercise group SBP, mmhg DBP, mmhg HR, beats/min * CO, l/min * SV, ml * 43 15* EtCO 2, mmhg Dextran group SBP, mmhg DBP, mmhg HR, beats/min * CO, l/min * * * SV, ml * 53 14* 34 7* EtCO 2, mmhg * 32 4* 30 5* Ex-Dex group SBP, mmhg DBP, mmhg HR, beats/min * * CO, l/min * * SV, ml * 51 16* 34 16* EtCO 2, mmhg * Post-bed rest Control group SBP, mmhg * * DBP, mmhg HR, beats/min * * * * CO, l/min * * * SV, ml * 36 9* 31 7* 18 5* EtCO 2, mmhg Exercise group SBP, mmhg DBP, mmhg HR, beats/min * * * CO, l/min * * SV, ml * 38 17* 34 9* EtCO 2, mmhg * 25 7* Dextran group SBP, mmhg DBP, mmhg HR, beats/min * CO, l/min * * SV, ml * * 32 19* EtCO 2, mmhg * 32 3* 31 5* 27 5* Ex-Dex group SBP, mmhg DBP, mmhg HR, beats/min * * * CO, l/min * * SV, ml * * 28 5* EtCO 2, mmhg * Values are means SD. SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate; CO, cardiac output; SV, stroke volume; EtCO 2, end-tidal CO 2. CO was not measured at 50 mmhg LBNP. *P 0.05 compared with baseline; P 0.05 comparison between pre- and post-bed rest at each level of LBNP. blood flow, may reflect cerebral vasoconstriction during orthostatic stress and cannot be explained fully by reduction in arterial CO 2 or sympathetic activation (29, 37). Studies in rats have shown that endothelium-dependent vasodilation of the cerebral blood vessels was impaired under simulated microgravity conditions (22). However, the opposite was observed in the peripheral vascular beds in humans after HDT bed rest (1, 6). Notwithstanding these discrepancies, it is possible that

6 H494 Downloaded from Fig. 2. Linear relationship between changes in cardiac output (CO) and CBFV during LBNP. The regression lines were estimated from the group-averaged data at each level of LBNP. Solid lines and filled circles are pre-bed rest, and dashed lines and open circles are post-bed rest. The equations above the lines are pre-bed rest, and the equations below the lines are post-bed rest. either endothelial dysfunction and/or reductions in flow-mediated vasodilation associated with decreases in cardiac output or stroke volume (reduced shear stress) may contribute to cerebral vasoconstriction and the observed reduction in cerebral blood velocity during LBNP (37). Several studies support a role of cardiac output in regulation of brain perfusion (14, 21). For example, increases in CBF velocity have been observed associated with an increase in cardiac output after acute volume expansion even without changes in arterial pressure (21). In addition, increases in CBF velocity during dynamic exercise were attenuated in patients with heart failure or in healthy subjects with 1 -adrenergic blockade to reduce increase in cardiac output (14). Finally, increases in cardiac output with leg tensing during upright standing were correlated with increases in CBF velocity (33). However, it must be emphasized that the relationship between changes in cardiac output and CBF is not likely to be simple and can be influenced by many other factors, such as cerebral perfusion pressure, cerebrovascular tone, as well as the intrinsic and/or extrinsic neural activities (13). This study extended these previous findings by showing that acute volume loading ameliorated greater reduction in CBF velocity during LBNP after bed rest. Furthermore, we found that reductions in CBF velocity during LBNP were correlated linearly with changes in cardiac output regardless of the countermeasures used. These findings support our hypothesis that central hypovolemia after bed rest contributes to reductions in CBF during orthostatic stress through its effects on cardiac output. However, in contrast with previous studies, CBF velocity did not change associated with either reduction in cardiac output after bed rest or increases in cardiac output with Dextran infusion under resting conditions (21). A chronic cerebrovascular adaptation to HDT bed rest may explain the lack of association between reduction in cardiac output and CBF velocity observed under resting conditions given the fact that brain perfusion is determined mainly by its metabolic rate and perfusion pressure (13). However, the reason why CBF velocity did not increase with acute volume loading with Dextran infusion is not apparent. It is possible that effects of volume loading on CBF via normalization of central hypovolemia may be different from that of volume expansion under normovolemic conditions (21). In addition, CBF may be more sensitive to acute decreases in cardiac output during LBNP than increases in cardiac output with volume expansion. Notably, the slope of the cardiac output-cbf velocity relationship appears to be steeper after bed rest in the control, Dextran, and exercise groups (Fig. 2). In addition, there appears to be a downward shift of regression lines in the Ex-Dex group after bed rest (Fig. 2). These data, if confirmed by further by on November 20, 2017

7 studies, would have significant implications. First, assuming that the cardiac output-cbf velocity relationship reflects a potentially important flow autoregulation of the cerebral circulation (in contrast with the traditional construct of pressure autoregulation), the increases in the slope of the cardiac output- CBF velocity relationship would suggest an impaired cerebral autoregulation, that is, for any given changes in cardiac output, there would be greater changes in CBF. Second, for a downward shift of this relationship in the Ex-Dex group after bed rest, CBF velocity would be smaller for any given cardiac output. These data then suggest that the proportion of cardiac output distributed to the brain during LBNP would be reduced. We speculate, but cannot prove, that exercise during bed rest may attenuate peripheral vasoconstriction, leading to a larger proportion of cardiac output to be distributed to the peripheral vascular beds during orthostatic stress (23). Although competitions for cardiac output between the systemic and cerebrovascular beds may be essential for maintaining arterial pressure under orthostatic stress, it may occur at the expense of reduction in CBF. The key question yet to be fully answered is if or how the changes in individual as well as interactions of systemic and cerebral regulation are related to orthostatic intolerance and control of brain perfusion after bed rest or space flight. Countermeasures, CBF, and orthostatic intolerance. Previous studies showed that orthostatic intolerance after bed rest was minimized by volume expansion with oral consumption of salt tablets and water (36). In addition, acute volume loading with Dextran infusion prevented heat stress-induced orthostatic intolerance in healthy subjects (18). In this study, orthostatic intolerance occurred even though reduction in plasma volume after bed rest was normalized by Dextran infusion. The underlying mechanisms leading to these discrepancies are not known. Differences in durations of bed rests and/or the extent of volume infusion used may influence a particular study outcome (31, 35, 36). Aerobic or resistance exercise training to maintain peak oxygen consumption or muscle mass at a pre-bed rest level failed to prevent orthostatic intolerance after bed rest (12). Moreover, aerobic exercise training with LBNP to simulate 1-g environment also failed to prevent orthostatic intolerance after bed rest (27). Consistent with these observations, we found that supine cycling exercise to maintain pre-bed rest ambulatory cardiac work, if anything, exaggerated rather than improved orthostatic intolerance after bed rest. Interestingly, exercise combined with volume loading prevented orthostatic intolerance, completely suggesting that maintaining cardiac mass and normalization of central hypovolemia both are necessary to prevent orthostatic intolerance after bed rest (31). The finding of orthostatic intolerance after bed rest despite prevention of a greater fall in CBF velocity with Dextran infusion was unexpected. These observations suggest that reduction in CBF during the compensatory phase of LBNP, that is, when mean arterial pressure is well maintained, is not likely to be a primary factor leading to orthostatic intolerance. This conclusion is consistent with our previous findings that superimposition of hyperventilation during LBNP resulted in further decreases in CBF velocity, but did not initiate hemodynamic instability or cause presyncopal symptoms (19). However, symptoms of presyncope or syncope eventually occur when CBF falls below a threshold that normal brain H495 metabolism cannot be maintained through a compensatory increase in brain tissue oxygen extraction (13). The substantial reduction in CBF velocity associated with a fall in arterial pressure in the control and Dextran groups suggests an impaired cerebral autoregulation under these conditions (Fig. 1) (38). In this regard, decreases in CBF under steady-state conditions of LBNP may reduce cerebrovascular reserve and hence contribute to orthostatic intolerance when the level of orthostatic stress continues to increase. Interestingly, even though no significant reductions in arterial pressure from the baseline were observed at the level of maximal LBNP, CBF velocity was reduced substantially in the exercise and Ex-Dex groups (Fig. 1). Whether these observations indicate a failure of cerebral autoregulation or the presence of other central or peripheral mechanisms leading to the reductions in CBF velocity under these conditions cannot be determined in this study. Study limitations. First, hemodilution with Dextran infusion may affect CBF. Normovolemic hemodilution with Dextran infusion or volume expansion with mannitol decreased blood viscosity, leading to increases in CBF (4, 32). We did not measure hematocrit and blood viscosity before and after Dextran infusion in this study. Therefore, decreases in hematocrit and blood viscosity with Dextran infusion may have attenuated reduction in CBF velocity during LBNP without fundamentally affecting brain perfusion. However, the amount of volume loading was relatively small consisting of only about 14% of total plasma volume in the Dextran group and 9% in the Ex-Dex group. Thus the effect of potential changes in blood viscosity associated with Dextran infusion on CBF velocity is likely to be trivial relative to the effects of LBNP. The fact that CBF velocity did not change significantly after Dextran infusion under resting conditions also supports this possibility. Second, transcranial Doppler was used for measuring changes in CBF velocity to reflect changes in blood flow. Changes in CBF velocity are proportional to changes in blood flow only if the diameter of the MCA remained unchanged. Several studies have demonstrated that during a variety of stimuli known to affect CBF, the diameter of the MCA remained relatively constant, including LBNP (10, 28, 30). Thus it is reasonable to assume that changes in CBF velocity were proportional to changes in CBF in the present study. Third, for the purpose of this study, a short period of 3 min of data was collected at each of high levels of LBNP (above 40 mmhg). These data were averaged to reflect changes in arterial pressure and CBF velocity under steady-state conditions. Our previous study suggests that dynamic autoregulation as quantified by a coherence function between beat-to-beat changes in arterial pressure and CBF velocity was impaired during LBNP after bed rest (39). A latter study used 6 min of data at each level of LBNP to assess dynamic cerebral autoregulation due to the consideration of lower statistical power for spectral estimation associated with short data segments (38). Thus further studies are needed to quantify dynamic cerebral autoregulation and its relation to orthostatic tolerance after HDT bed rest with and without countermeasures. In this regard, recent studies suggest that it is the magnitude of oscillations in CBF velocity rather than the steady-state mean values that was associated with orthostatic intolerance (24 26). Whether this is the case under the current study conditions needs to be determined.

8 H496 Finally, for technical reasons, end-tidal CO 2 was not measured in the control group during the first bed rest study. End-tidal CO 2 did not change before and after bed rest under resting conditions in the other three groups. In addition, the magnitude of reductions in end-tidal CO 2 during LBNP was similar before and after bed rest in the other three groups (Table 2). Thus the greater reductions in CBF velocity during LBNP in the control group after bed rest may not be induced by a greater reduction in arterial CO 2. In summary, we found that volume loading prevented greater reductions in CBF velocity during LBNP after bed rest. However, contrary with our hypothesis, prevention of greater reductions in CBF velocity under steady-state conditions did not lead to an improvement of orthostatic intolerance. Furthermore, exercise combined with volume loading prevented orthostatic intolerance, but did not prevent greater reduction in CBF velocity during LBNP. Taken together, these findings suggest that reductions in CBF under steady-state conditions, that is, during the compensatory phase of cardiovascular responses under orthostatic stress, are not likely to be a primary factor initiating or leading to orthostatic intolerance after bed rest. ACKNOWLEDGMENTS We thank the subjects for their willingness to participate in this study. GRANTS This study was supported in part by National Aeronautics and Space Administration Grant NNH047ZUU003N and a research fellowship award to Dr. S.-M. Jeong from the College of Medicine, University of Ulsan, Seoul, Korea. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). 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