Exposure to a period of microgravity or bed rest produces several physiological adaptations. These changes, which include an increased incidence of orthostatic intolerance, have an impact when people return to a 1G environment or resume an upright posture. Compared with males, females appear more susceptible to orthostatic intolerance after exposure to real or simulated microgravity. Decreased arterial baroreflex compensation may contribute to orthostatic intolerance. We hypothesized that female rats would exhibit a greater reduction in arterial baroreflex function after hindlimb unloading (HU) compared with male rats. Mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were recorded in conscious animals after 13–15 days of HU. Baseline HR was elevated in female rats, and HU increased HR in both genders. Consistent with previous results in males, baroreflex-mediated activation of RSNA was blunted by HU in both genders. Maximum RSNA in response to decreases in MAP was reduced by HU (male control 513 ± 42%, n = 11; male HU 346 ± 38%, n = 13; female control 359 ± 44%, n = 10; female HU 260 ± 43%, n = 10). Maximum baroreflex increase in RSNA was lower in females compared with males in both control and HU rats. Both female gender and HU attenuated baroreflex-mediated increases in sympathetic activity. The combined effects of HU and gender resulted in reduced baroreflex sympathetic reserve in females compared with males and could contribute to the greater incidence of orthostatic intolerance in females after exposure to spaceflight or bed rest.
- simulated microgravity
- sympathetic nervous system
- bed rest
- hindlimb unweighting
prolonged exposure to microgravity or bed rest results in a series of adaptations and changes including a central shift in body fluids, followed by a reduction in total blood and plasma volume, and muscle atrophy (46). Upon return to a 1G environment or to normal activity, individuals display resting tachycardia, decreased exercise capacity, and increased incidence of orthostatic intolerance (6, 17, 19). The mechanisms that account for these changes are still unresolved, although cardiac atrophy (39), altered vascular reactivity (17, 32), hypovolemia (47), and altered baroreflex control of sympathetic nerve activity (11, 18, 48) have been suggested, and all potentially contribute. In addition, females appear to be less tolerant of the negative effects of returning from spaceflight or a period of bed rest, because females exhibit a greater incidence of orthostatic intolerance after spaceflight compared with men (23, 48). The mechanisms that account for the greater susceptibility of women to the effects of microgravity and bed rest are unknown.
The physiological response to an orthostatic challenge requires several compensatory mechanisms to maintain blood pressure and cerebral perfusion, including baroreflex-mediated increases in heart rate (HR) and vascular resistance (40). The arterial baroreceptor reflex is the primary controller of beat-to-beat arterial blood pressure and is critical in the cardiovascular response to an orthostatic stress (40). Previously, we demonstrated that male rats that have undergone hindlimb unloading (HU), a model for exposure to microgravity or bed rest, had attenuated baroreflex function. Male HU rats exhibit a blunted ability to reflexly increase sympathetic nerve activity compared with control rats (34). This effect is likely due to an adaptation within the central nervous system, because aortic baroreceptor afferent sensitivity is not altered by HU (36) and changes in neuronal activation at the rostral ventrolateral medulla are involved (24, 35). This alteration in baroreflex control of sympathetic nerve activity in male rats after HU affected both renal and lumbar sympathetic nerve activity (34), suggesting that it may be a generalized effect across sympathetic outputs to different vascular beds. These data are consistent with the hypothesis that exposure to microgravity or bed rest attenuates baroreflex control of sympathetic nerve activity contributing to orthostatic intolerance. It is unknown whether simulated microgravity affects baroreflex function similarly or to a greater extent in females compared with males. Less baroreflex-mediated sympathoexcitation after a period of spaceflight or bed rest in women compared with men could be a factor in the increased incidence of orthostatic intolerance upon return to earth or an upright posture.
This study was designed to test the hypothesis that exposure to a period of simulated microgravity attenuates baroreflex control of sympathetic nerve activity to a greater extent in females compared with males. This study utilized an established model for exposure to spaceflight or bed rest, the HU rat, which mimics the changes seen after spaceflight and bed rest (37). These changes include a central shift in body fluids while suspended (37), reductions in blood and plasma volume (5), atrophy of hindlimb postural muscles, and, when returned to a horizontal posture, a resting tachycardia (30, 34), evidence of decreased orthostatic tolerance (30), and reduced exercise capacity. To test our hypothesis, we evaluated arterial baroreflex control of renal sympathetic nerve activity (RSNA) and HR in both male and female conscious rats that underwent ∼14 days of either HU or control conditions.
All procedures were approved by the Institutional Animal Care and Use Committee of the University of Missouri-Columbia and were conducted in accordance with the American Physiological Society's “Guiding Principles in the Care and Use of Animals.” Age-matched male (n = 24) and female (n = 20) Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were housed in temperature (21–24°C)- and light (12:12-h light-dark cycle)-controlled facilities. Animals consumed food and water ad libitum. In the female animals, the 4- to 5-day estrous cycle was monitored by analyzing daily vaginal cytology.
Rats were randomly assigned to HU or cage control (CC) groups. All rats were briefly anesthetized with isoflurane to implant the suspension apparatus and apply a small lightweight thoracic cast (∼10 g; Specialist plaster bandages; Johnson & Johnson). The suspension apparatus consisted of two stainless steel wires (0.029-mm diameter; Small Parts) passed through the ∼5th and ∼7th sacral intervertebral space and formed into a circle (∼4.5-cm diameter) with a small grommet (∼0.5-cm diameter). A week was allowed for the animals to recover before the HU animals were acclimated to HU. The acclimation consisted of hindlimb suspension for 1–3 h per day for 3 days. Care was taken to ensure the angle of suspension (∼30–35°) was adequate to eliminate weight bearing by the hindlimbs. After the acclimation period, HU animals were suspended for 13–15 days. CC animals were maintained in individual cages for a similar time period. Female HU rats maintained normal estrous cycles during the suspension period.
Surgery was performed 2 days before the experiment (∼day 12 of the HU period). Surgery was performed under isoflurane anesthesia using sterile technique. Arterial and venous catheters (PE-10 fused to PE-50) were implanted in the abdominal aorta and vena cava via the femoral artery and vein. Catheters were filled with heparinized saline (10 U/ml), closed with an airtight plug, and exteriorized in the dorsal cervical region. A left renal nerve was exposed by a retroperitoneal approach, dissected free of connective tissue, and placed on a recording electrode. The electrode consisted of two silver, Teflon-insulated wires (Medwire; 0.005-in. diameter) passed through Silastic tubing (0.025-in. diameter). The Teflon insulation was removed from the electrode tips, which were fashioned into hooks and placed in contact with the isolated renal nerve. The nerve and electrode were covered with a polyvinylsiloxane gel (Super Dent light; Darby Dental Lab Supply) that was allowed to harden before closure. A ground wire was implanted in the surrounding muscle. The electrode and ground wire were exteriorized with the catheters in the dorsal cervical region. The rats were treated postoperatively with 10 ml of subcutaneous saline. After recovery from anesthesia, the animals were returned to their cages or the suspension apparatus for 48 h before any experimental manipulations. In female rats, the estrous cycle was monitored, and surgery was performed on the second day of diestrus, which occurred between days 11 and 13 of HU. The experiment was then performed 2 days later (days 13–15 of HU). Because the estrus and early diestrus stages of the cycle are characterized by low and consistent estrogen and progesterone levels, this protocol was designed to maximize the number of female rats in the estrus or early diestrus stage of the cycle on the day of the experiment. Because of normal variability in estrous cycles and the possible effects of surgery, the stage of the cycle on the day of the experiment varied slightly (3 estrus, 13 diestrus, 4 proestrus). The duration of HU and CC period was varied similarly in the male animals (range 12–15 days). The average duration for all HU and control conditions in both male and female animals was 13.7 ± 0.2 days.
All experiments began with a stabilization period of ∼30 min during which baseline hemodynamic parameters were recorded. At the end of this period, baroreflex curves were generated by ramp infusion of the α1-adrenergic agonist phenylephrine hydrochloride (PE; 1 mg/ml) and the vasodilator sodium nitroprusside (SNP; 1 mg/ml) over a 2- to 3-min period. Each ramp infusion produced an ∼50 mmHg change in MAP with a 1–2 mmHg/s rate of change. The rate of change in MAP was held constant by observing the pressure change and varying the rate of the infusion pump to produce a smooth ramp increase or decrease in MAP. Volume infused did not exceed 100 μl. To minimize any potential effects of released humoral agents (e.g., angiotensin II, vasopressin) on baroreflex function, baroreceptors were activated (PE infusion) before being unloaded (SNP infusion). MAP, HR, and RSNA were allowed to return to within 10% of baseline values before proceeding with the SNP infusion (∼20 min after end of PE infusion).
At the end of the experiments, the rats were euthanized with an overdose of pentobarbital administered through the venous catheter. The soleus and plantaris muscles and adrenal glands were removed, blotted dry, and weighed.
In each animal, background noise in the nerve recording was determined by elimination of RSNA by complete ganglionic blockade with hexamethonium bromide (30 mg/kg) and atropine methyl nitrate (0.05 mg/kg). Absolute RSNA was defined as the recorded nerve activity after subtraction of the background noise. In each animal, absolute RSNA at the start of the experiment was defined as 100%. Baroreflex-mediated changes in RSNA were calculated as a percentage of this baseline (%control). To evaluate baroreflex sympathetic reserve, we calculated the reflex change in RSNA as a percentage of the maximum baroreflex-mediated sympathoexcitation.
Arterial baroreflex-mediated changes in HR and RSNA for each rat were fit to a sigmoidal logistic function (28) by using a standard software package (SigmaPlot; SPSS, Chicago, IL). The following equation was used to relate RSNA or HR to MAP The sigmoidal curve is described by four parameters (P1–P4). P1 is the maximum level of RSNA or HR in response to a decrease in MAP or the upper plateau. P2 is a coefficient that is used to calculate the peak slope of the curve at the midpoint. P3, or midpoint, is the MAP at the point of greatest slope. P4 is the minimum level of RSNA or HR in response to an increase in MAP or the lower plateau. Figure 1 illustrates representative data of baroreflex-mediated changes in RSNA from an individual rat in each group. Raw data were fit to a sigmoid curve by using the above equation. The instantaneous slope or gain (GMAP) of arterial baroreflex control of RSNA or HR was calculated using the following equation The curve parameters (P1–P4) and peak gain were averaged within a group. Average baroreflex and instantaneous gain curves for each group were generated from the average parameters. Comparison of body weight before and after the HU or CC period within a group was analyzed using Student's t-test. All other data were analyzed using two-way analysis of variance (ANOVA) with the factors being gender (male or female) and treatment (CC or HU). When ANOVA indicated a significant interaction, differences between individual means were analyzed using a least significant difference test (44). A probability of P < 0.05 was considered statistically significant. All statistical analyses were performed using SigmaStat (SPSS). All data are expressed as means ± SE.
Body, soleus, plantaris, and adrenal gland weights are presented in Table 1. As expected, age-matched male rats weighed more than female rats both before and after the HU period. The female and male CC and female HU groups gained weight during the 14-day period. The male HU animals maintained their body weight but did not significantly gain or lose any weight during the HU period. Whether expressed as absolute values or normalized to body weight, atrophy of the soleus and plantaris muscles was evident in both male and female HU groups compared with CC animals. Neither gender nor HU affected adrenal gland weight.
Consistent with the literature (3, 15, 48), resting MAP was lower and resting HR was higher in females compared with males (Table 2). In addition, both HU groups exhibited a resting tachycardia compared with the CC animals. Resting MAP was higher in both HU groups. A resting tachycardia and hindlimb postural muscle atrophy are markers of cardiovascular deconditioning (46) and confirm that the HU protocol resulted in cardiovascular deconditioning.
Baroreflex control of HR.
In general, baroreflex control of HR was similar between groups (Fig. 2). All groups had characteristic sigmoidal baroreflex-mediated HR responses to changes in pressure. The instantaneous gain throughout the range of MAP (Fig. 2B) was similar between the groups. However, maximum baroreflex-induced tachycardia was greater in HU compared with CC rats (Table 3). Baroreflex curve midpoint, minimum HR, and maximum gain of arterial baroreflex control of HR were not altered by gender or HU (Table 3).
Baroreflex control of RSNA.
Baroreflex control of RSNA was affected by gender and by HU (Fig. 3). All groups displayed reflex decreases in RSNA during elevations in MAP and sympathoexcitation during decreases in MAP (Fig. 3A). However, both HU and female gender reduced the maximum RSNA in response to decreases in MAP (Figs. 3A and 4A). When combined, these effects led to the female HU group having the least sympathoexcitatory response to a decrease in MAP. The MAP at the curve midpoint was higher in both male and female HU compared with CC animals (Fig. 4B). In addition, there was a trend (P = 0.06) for the midpoint to be lower in females compared with males. These effects on midpoint are consistent with the higher resting MAP in the HU compared with control animals and in males compared with females (Table 2). The minimum RSNA in response to increases in MAP was not altered by gender or HU (Fig. 4C). Peak slope of the baroreflex curve was less in females and reduced by HU in both males and females (Fig. 4D). Although there was a trend for an interaction of gender and HU on peak slope, this was not statistically significant (P = 0.09).
We evaluated the effects of gender and HU on the ability to reflexly increase sympathetic nerve activity, or baroreflex sympathetic reserve. This was defined as the reflex increase in RSNA, calculated as a percentage of the peak baroreflex-mediated sympathoexcitation. Baroreflex sympathetic reserve was reduced in females of both groups compared with males (Fig. 5). In addition, compared with CC animals, HU in both genders decreased baroreflex sympathetic reserve. Therefore, in relation to maximum baroreflex sympathoexcitation, female HU animals had the least baroreflex sympathetic reserve or ability to increase RSNA in response to a hypotensive stimulus.
This study tested the hypothesis that a period of HU attenuates baroreflex control of sympathetic nerve activity to a greater extent in female compared with male rats. In both males and females, HU resulted in a resting tachycardia and hindlimb postural muscle atrophy, which are markers of deconditioning (46). Baroreflex control of HR was similar between males and females but was shifted upward by HU in both genders. The major finding of this study is that both female gender and HU result in blunted baroreflex-mediated sympathoexcitation. Thus HU appeared to produce similar reductions in baroreflex sympathoexcitation in both genders. When combined, the effects of female gender and HU resulted in female HU animals having the least ability to increase RSNA in response to a hypotensive challenge.
Baroreflex alterations after HU.
Similar to our previous results in males (34), HU blunted the ability to reflexly increase RSNA in response to decreases in MAP. In the current study, HU altered baroreflex control of RSNA in females to a similar extent as in the males. The mechanisms that account for the alteration in baroreflex function after HU are unknown but probably involve the central nervous system (24, 35, 36). These effects may be associated with changes in blood volume, cardiopulmonary reflex function, or neurohumoral homeostasis.
In the current study, the gain of baroreflex control of HR was minimally affected consistent with other studies using 7–14 days of HU (5, 16, 34, 38, 50). The variation between the effects of HU on baroreflex control of RSNA and HR may be due to the fact that HR represents an end-organ response and is controlled by both parasympathetic and sympathetic inputs. A point to consider is that nitric oxide has a positive chronotropic effect in human heart transplant patients (8). In addition, further work in healthy humans with intact autonomic innervation of the heart has demonstrated an effect of nitric oxide to potentiate the antiadrenergic component but not the direct action of muscarinic receptors on heart rate (9). Because we used a nitric oxide donor to lower blood pressure in this study, these effects of nitric oxide may have limited our ability to detect changes in baroreflex control of HR between groups. In addition, changes in responsiveness of the heart or vagal control of heart rate could offset potential changes in cardiac sympathetic control and result in unaltered baroreflex control of HR. Further study is required to investigate the independent contributions of the sympathetic and parasympathetic components of baroreflex control of HR after HU.
Baroreflex alterations after spaceflight or bed rest.
Arterial baroreflex control of heart rate and muscle sympathetic nerve activity has been studied in humans after spaceflight or bed rest. After either bed rest or spaceflight, evaluation of baroreflex control of heart rate has focused on the relationship of R-R interval and a recorded blood pressure variable (either mean arterial, systolic, or carotid pressure, depending on the study) produced during spontaneous variations in blood pressure (12, 26), valsalva maneuver (19, 27), neck pressure (11, 19), or real or simulated head-up posture (13, 15). All of these studies except for one (13) reported a decreased HR baroreflex sensitivity or transfer function after exposure to real or simulated microgravity. By design, these studies predominantly evaluated the parasympathetic limb of the baroreflex, because immediate reflex changes in R-R interval were measured. It is interesting to point out that the one study in humans that demonstrated no change in the gain of the complete carotid sinus and aortic arch baroreflex arc utilized steady-state changes in arterial pressure, which would involve reflex-mediated changes in both parasympathetic and sympathetic inputs to the heart (13). In the current study, we used slow ramp changes in arterial pressure to elicit baroreflex responses, which likely also involve both branches of the autonomic nervous system. It is possible that baroreflex control of cardiac sympathetic activity and parasympathetic nerve activity are differentially affected by bed rest or spaceflight. In addition, changes in HR or R-R interval are end-organ responses. Besides changes in control of cardiac sympathetic or parasympathetic nerve activity, changes in transmitter release, receptors, or signal transduction to affect cardiac rate could play a role in the possible blunting of baroreflex control of the heart (R-R interval) after bed rest or spaceflight.
Reflex increases in sympathetic nerve activity during upright tilt have been reported to be unaltered (29, 39), enhanced (27) or blunted (43) after bed rest or a period of spaceflight in humans. However, in the studies that showed either enhanced or unaltered baroreflex function, either the subjects were not orthostatic intolerant (29) after exposure to real or simulated microgravity or they were not tested for orthostatic tolerance (27, 39). In the one study in humans in which orthostatic intolerance after exposure to a period of simulated microgravity was verified, baroreflex control of sympathetic nerve activity was blunted (43). In addition, individuals that are orthostatic intolerant after spaceflight or bed rest exhibit attenuated vasoconstriction and reduced norepinephrine release upon standing or head-up tilt, suggesting decreased activation of the sympathetic nervous system (6, 18, 32, 48).
Gender differences in baroreflex function.
In general, compared with men, women have higher resting HR, lower resting blood pressure, and lower basal sympathetic tone to the vasculature (10, 15, 42, 45, 48). In studies evaluating humans and animals, baroreflex control of HR has been reported to be blunted (1, 2, 4, 10, 41), similar (14, 15), or enhanced (7) in females compared with males. As discussed above, reflex control of HR is influenced by many factors. The method used to evaluate baroreflex function is an important consideration. For example, baroreflex control of HR was attenuated in females compared with males when abrupt changes (bolus method) but not when slower changes in MAP (similar to the current study) were utilized to evoke baroreflex changes in HR (2). Also, baroreflex control of sympathetic nerve activity varies with levels of gonadal hormones in women (33). Baroreflex control of lumbar sympathetic nerve activity in rats was previously reported to be similar between males and females, although the female rats were not studied at any particular stage of the estrous cycle (7). Hinojosa-Laborde et al. (25) reported that when stage of the estrous cycle is considered, baroreflex control of sympathetic nerve activity is enhanced in proestrus but attenuated in diestrus. However, when the females were analyzed as a group (disregarding estrous stage), baroreflex control of sympathetic nerve activity was similar between males and females (25). The current results showing that baroreflex control of sympathetic nerve activity is blunted in female rats in estrus/diestrus compared with male rats are consistent with these data. As mentioned earlier, women are less tolerant to an acute orthostatic stress (10, 21, 23, 42, 48). It seems likely that blunted baroreflex-mediated sympathoexcitation could contribute to the reduced ability of women to compensate during an orthostatic challenge.
Orthostatic intolerance after spaceflight or bed rest.
It is likely that a number of different mechanisms contribute to orthostatic intolerance. Increased incidence of orthostatic intolerance after real or simulated microgravity has been associated with hypovolemia (17, 47, 48), myocardial dysfunction (20, 39), changes in peripheral vascular responsiveness (17, 48), and alterations in neurohumoral regulation of cardiovascular function (11, 18, 48). Decreases in plasma volume and cardiac atrophy after exposure to microgravity or prolonged bed rest may contribute to orthostatic intolerance by reducing the ability to maintain stroke volume during an orthostatic challenge (20, 46, 47). Similarly, impaired peripheral vascular function could contribute to reduced orthostatic tolerance after real or simulated microgravity by limiting the ability to vasoconstrict and thus decreasing vascular resistance or venous return during an orthostatic challenge (46, 49). The current experiments along with other studies suggest that autonomic and neuroendocrine regulation of cardiovascular function is impaired after simulated microgravity or bed rest. It is reasonable to suggest that this may contribute to the regulation of arterial pressure and could be an important contributing factor to orthostatic intolerance (17, 18, 34, 46, 48).
Women are more susceptible to developing orthostatic intolerance than men either with or without exposure to real or simulated microgravity (10, 20, 22, 23, 48). It has been proposed that differences in center of gravity, blood volume, and autonomic responsiveness contribute to the gender difference in orthostatic tolerance (10, 42). Data from the current study suggest that female gender is associated with reduced baroreflex-mediated sympathetic reserve, and this reserve is reduced further by HU. Because reflex activation of sympathetic nerve activity is an important compensatory mechanism for an orthostatic challenge (40), these data are consistent with an increased incidence of orthostatic intolerance in both normal women and in women exposed to real or simulated microgravity.
In conclusion, in control animals, females compared with males had a decreased ability to increase sympathetic nerve activity during a hypotensive stimulus. HU in both genders produced attenuated baroreflex-mediated sympathoexcitation. The combined effects of female gender and HU resulted in the least sympathetic reserve occurring in females that had undergone simulated microgravity. The findings of this study are consistent with increased susceptibility of females to orthostatic stress observed after spaceflight or bed rest (10, 20, 23, 48). In addition to hypovolemia and changes in cardiac and vascular function, alterations in arterial baroreflex function after exposure to microgravity or prolonged bed rest likely contribute to the increased incidence of orthostatic intolerance, especially in females.
When comparing studies performed in humans after bed rest or spaceflight to studies in HU animals, several interesting differences need to be considered. The position of the subjects at the time of data collection varies among experiments. Studies in humans have been conducted while the subjects are head-down tilted or in microgravity, horizontal after a period of spaceflight or bed rest, or vertical, either upright or tilted. As expected, each of these positions evokes a slightly different cardiovascular response that must be considered when comparing among studies. Our studies in HU rats have been performed with the animals returned to a horizontal position after 14 days of HU. This is thought to mimic return to a 1G environment after spaceflight or resumption of an upright posture after bed rest. However, it is not known whether the freely moving post-HU rat best models humans that are horizontal or standing/head up after exposure to real or simulated microgravity. Also, in humans after bed rest or spaceflight, the incidence of orthostatic intolerance is increased, with females being affected to a greater extent than males (23, 48), and is more prevalent after long-duration spaceflight (31). In most human studies, not all subjects exposed to real or simulated microgravity become orthostatic intolerant. In addition, the cardiovascular response to an orthostatic test varies between the subjects that are tolerant or intolerant to the test (6, 32, 48). Because orthostatic tolerance is difficult to assess in rodents, it is unknown whether HU alters the ability to maintain pressure while undergoing head-up tilt in all, some, or none of the animals. The changes we observed in baroreflex control of sympathetic nerve activity are consistent with the increased incidence of orthostatic intolerance in females and after exposure to simulated or real microgravity. Although speculation, it is possible that the HU rat model relates specifically to the orthostatic intolerant subset of humans exposed to real or simulated microgravity.
This research was supported by National Aeronautics and Space Administration Grants NNA04CC62G (to C. M. Foley) and NAG21603 (to C. M. Heesch) and National Heart, Lung, and Blood Institute Grant HL-55306 (to E. M. Hasser). This investigation was conducted in a facility constructed with support from Research Facilities Improvement Program Grant C06 RR-16498 from the National Center for Research Resources.
We thank Sarah A. Friskey for excellent technical assistance in performing the surgical preparation and experimental procedures.
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