After spaceflight, humans typically exhibit an impaired ability to regulate blood pressure while standing (orthostatic intolerance), which has been attributed primarily to an inability to effectively increase peripheral vascular resistance (1). Hindlimb suspension of rats, a treatment that models the effects of microgravity, also results in impaired blood pressure regulation (16,17) and altered control of peripheral vascular resistance and of regional blood flow (19,20). In particular, hindlimb-suspended rats exhibit attenuated sympathetic nerve activity during hypotension and attenuated vasoconstriction during exercise in the splanchnic and hindlimb muscle vascular beds (19,20,30,31).
Like astronauts, hindlimb-suspended rats also exhibit impaired ability to thermoregulate during exercise (3,32). Based on the reported attenuated hemodynamic responses to exercise and posture after hindlimb-suspension, the impaired thermoregulation may be due to the inability to adequately vasoconstrict the splanchnic vasculature. We, therefore, hypothesized that splanchnic vasoconstriction is attenuated during heat stress. Vasoconstriction of the splanchnic vascular bed is particularly important during heat stress in the rat to allow diversion of blood flow to the tail for heat dissipation without compromising mean arterial pressure (MAP) (14). To assess splanchnic vasoconstriction during heat stress, we specifically examined changes in blood-flow velocity of the superior mesenteric artery, a large artery supplying blood to a substantial portion of the intestines.
In light of the reported impairment in sympathetic nerve firing rate after hindlimb suspension (20), it is also possible that alterations in the sympathetic innervation of mesenteric arteries contribute to the impaired ability to vasoconstrict this vascular bed. For example, Zhang et al. (33) found that 4 wk of hindlimb suspension resulted in a significant decrease in the density of norepinephrine-containing nerves in the hindlimb muscle vasculature. We, therefore, hypothesized that hindlimb suspension results in decreased sympathetic innervation of mesenteric arteries. This hypothesis was tested by comparing the density of the perivascular sympathetic nerves in small mesenteric arteries from hindlimb-suspended and control rats by using immunostaining for tyrosine hydroxylase.
The experimental protocol was approved by the University of Iowa Animal Care and Use Committee and was in compliance with the ACSM animal care standards. Sixteen male Sprague-Dawley rats (mean ± SD: 285 ± 38 initial body weight; 329 ± 38 g final body weight) underwent the hindlimb-suspension procedure as described by Morey (22) for 28 d. In brief, the tail was bandaged along its length with a hook at the distal end. The hook was connected to a paper clip, which was attached to a swivel and crossbar spanning the top of the cage, giving the rat full mobility around the cage in a 360° arc. The floor of the cage had a plastic grate that allowed the rat to move itself around by grabbing the grate and also allowed waste to drop out of the cage. The rats were suspended at an angle of 40–45° as measured from the angle of a line drawn through the anus and shoulder. Thirteen control rats (284 ± 43 initial body weight; 387 ± 29 g final body weight) were housed in the same room in the same type of cage for 28 d. The rats were housed in temperature controlled quarters with a 12:12 h light/dark cycle and fed standard rat chow and water ad libitum. Ambient temperature was maintained at 26–27°C because it has been previously shown that hindlimb-suspended rats have a drop in body temperature when housed at the standard temperature of 21–23°C (27,29).
After the 28-d treatment, an individual rat was anesthetized with sodium pentobarbital (Nembutal, 50 mg·kg−1, i.p.). Hindlimb-suspended rats were removed from suspension immediately before anesthetizing. The femoral artery of one leg was catheterized to monitor MAP. Doppler flow probes were placed around the superior mesenteric artery and iliac artery of the other leg to measure blood-flow velocity. Colonic temperature was monitored with a thermistor probe.
After surgery, baseline MAP, blood-flow velocities, and colonic temperature were measured during the last 5 min of a 30-min period in an environmental chamber maintained at 25°C. The temperature of the chamber was then set to 42°C, which was achieved within 21 ± 7 min (mean ± SD). Physiological parameters were measured every minute until the endpoint, defined as a drop in MAP to below 80 mm Hg.
The MAP was determined by connecting the femoral artery catheter to a pressure transducer and electronically averaging the input using a Gould recorder (Gould Instrument Systems, Valley View, OH). Blood-flow velocities were measured using Doppler flow probes (Iowa Doppler Products, Iowa City, IA) embedded in an epoxy cuff that wrapped around the vessel. Doppler signals were measured using a pulsed Doppler flowmeter (model #545C-4, University of Iowa Bioengineering Resource Facility, Iowa City, IA). Blood-flow velocity (in Khz Doppler shift) has been shown to be directly proportional to absolute blood flow (8,9). Percent change in arterial resistance was calculated from the blood-flow velocity data using the following formula: % change in resistance = 100% × (Rt − Rc)/Rc, where Rc is MAP divided by the mean velocity signal in the control period and Rt equals MAP divided by the mean velocity signal in the test period (14). MAP, blood velocity, and temperature were recorded using the Datalog computer program (University of Iowa Bioengineering Resource Facility, Iowa City, IA).
Physiological parameters were compared between groups at time points representing 10% intervals (up to 100%) of the time between the beginning of heating and the point at which MAP dropped below 80 mm Hg. This allowed normalization of responses over a relative time period, because there was variability in the time to this endpoint.
A typical indication of the efficacy of the hindlimb-suspension treatment is atrophy of the hindlimb muscles. Although we did not measure the muscle weights of these particular rats (because of the need to instrument both legs during the heating protocol), other rats undergoing the hindlimb-suspension treatment concurrently in our laboratory have all indicated significant atrophy in the gastrocnemius, soleus, and plantaris muscles (15).
Immunostaining of perivascular sympathetic nerves.
For examination of perivascular sympathetic nerve innervation, one to three second-order mesenteric arteries were dissected from the mesentery of hindlimb-suspended rats (N = 6) and control rats (N = 6) that did not undergo heat stress. These artery segments were fixed overnight in 4% paraformaldehyde in phosphate-buffered saline (PBS) at pH 7.4, and then stored in PBS. Artery segments were immunostained with an antibody to tyrosine hydroxylase (1° antibody), which is the rate limiting enzyme in catecholamine synthesis and a marker for adrenergic sympathetic nerves (7). The following procedure was used: 2 h incubation in 0.2% Triton X-100 in PBS, 30 min in 0.05% Pontamine Sky Blue (used to reduce auto-fluorescence) (Sigma, St. Louis, MS) in PBS, rinse three times with PBS (5 min each time), incubate 48 h in polyclonal tyrosine hydroxylase antibody (CA-101 bTHrab, Protos Biotech, New York, NY; diluted to 1:1000), rinse three times with PBS (5 min each time), incubate 2 h in 2° antibody (GAM-Alexa 488, Molecular Probes, Eugene, OR; diluted to 1:500), rinse three times with PBS (5 min each time), and mount on slide with fluorescent-preserving medium (Vectashield, Vector, Burlingame, CA). Negative controls were performed by: 1) eliminating incubation with 1° antibody (to test the specificity of the 2° antibody), 2) eliminating incubation with both 1° and 2° antibodies (to test for autofluorescence), and 3) preabsorbing (2 h) the 1% antibody with tyrosine hydroxylase antigen (THA001 rrTH, Protos Biotech) (to test the specificity of the 1° antibody).
Labeled tyrosine hydroxylase was visualized with a laser-scanning confocal microscope (Bio-Rad, MRC-1024, Hercules, CA), with the following camera settings for all samples: Power = 10, Iris = 2.5, Gain = 1326, Blev = 3. Three digital images were recorded from different regions of each segment and analyzed using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA). The percentage of pixels in the image above a threshold gray-scale value (threshold = 87), and the mean gray-scale value above this threshold was calculated for each of the three images per segment and averaged. Together, these measures estimate the area of the artery wall containing sympathetic nerves. These values were then averaged within their given group (control or hindlimb-suspended) to compare between groups.
MAP, colonic temperature, and superior mesenteric and iliac artery resistances were compared between groups by using two-way repeated measures ANOVA and Tukey’s post hoc analysis. Baseline Doppler flowmeter values between the groups were compared using two-tailed unpaired t-tests. In the micrographs, percent pixels above threshold and mean gray-scale values of pixels above threshold were compared between groups using two-tailed unpaired t-tests.
During heat stress, all rats exhibited a progressive rise in colonic temperature, indicating that the heat load was sufficient to overwhelm the ability to dissipate heat (Fig. 1). At colonic temperatures above approximately 40°C, MAP increased until approximately 43°C, after which it fell precipitously. Iliac resistance declined at lower colonic temperatures, approached baseline at higher temperatures, then decreased at the highest final temperature. Superior mesenteric artery resistance remained stable at temperatures up to about 40°C, then increased progressively at higher temperatures, and declined sharply at the highest final temperature. Colonic temperature, blood pressure, and superior mesenteric artery resistance significantly increased in both groups during the heating protocol. None of these physiological responses were significantly different between the control and hindlimb-suspended rats (see Fig. 1). Additionally, when superior mesenteric artery and iliac artery blood-flow velocity values were used to calculate blood-flow conductance (the inverse of resistance), the values were not statistically different between control and hindlimb-suspended rats (data not shown). Baseline Doppler flowmeter values are indicated in Table 1 and were not significantly different between the two groups.
The average time to the heat stress endpoint was 80 ± 10 min (mean ± SD) in control rats (N = 7) and 72 ± 8 min in hindlimb-suspended rats (N = 10) after the start of heating. This represented a change in colonic temperature over time of 0.074 ± 0.011°C·min−1 (mean ± SD) in controls rats and 0.089 ± 0.014°C·min−1 in hindlimb-suspended rats. These parameters were not significantly different between groups.
Figure 2 contains representative illustrations from each group of the tyrosine hydroxylase immunostaining of the artery wall, which indicate a network of sympathetic nerve varicosities. No significant staining was apparent in vessels when 1° antibody was eliminated, primary and secondary antibody was eliminated, or antigen was added (data not shown), which confirms the specificity of the 1° and 2° antibodies. When the tyrosine hydroxylase staining was analyzed, percent pixels above threshold (indicating a positive stain) were not different between the groups (Table 2). Mean gray-scale values of pixels above threshold (indicating the intensity of staining) were also not different between groups. The average standard deviation within the triplicate measurements of each vessel was relatively small (1.98 for percent pixels above threshold, 3.25 for mean gray-scale values of pixels above threshold) compared with the standard deviation of the group means (Table 2), indicating a relative homogeneity of sympathetic innervation along a given vessel. These results indicate that the sympathetic nerve innervation is quantitatively similar between the groups.
The purpose of this study was to characterize the cardiovascular responses to heat stress after hindlimb suspension. The results indicate that 28-d hindlimb suspension does not alter the peripheral vascular responses to heat stress or the rate of rise in core temperature in the anesthetized rat. Additionally, 28-d hindlimb suspension does not alter the sympathetic innervation of second-order mesenteric arteries.
The particular hemodynamic responses examined were changes in MAP, mesenteric artery resistance, and iliac artery resistance. The reason for evaluating responses of these particular parameters is because others have reported that after hindlimb suspension, these vascular beds exhibit impaired resistance responses during exercise (19,31) and impaired sympathetic nerve discharge during the baroreflex response to hypotension (20). Moreover, control of resistance in these vascular beds is important for a normal response to heat stress. Specifically, vasoconstriction of the mesenteric vascular bed during heat stress is necessary for cardiovascular stability (26). This vasoconstriction serves to maintain blood pressure as iliac and caudal resistances decrease in order to increase blood flow to the tail for heat dissipation (23,25). The results of this study indicate no impairment in the ability to increase resistance in the mesenteric vascular bed or to decrease resistance in the hindlimb vascular bed during heat stress.
It has been previously found that hindlimb-suspension impairs the baroreflex-mediated sympathetic output to the splanchnic and hindlimb vascular beds (20); therefore, one might expect that this impairment would inhibit the normal hemodynamic response to heat stress. However, it is not clear whether the baroreflex modulates the sympathetic response to heat stress in rats. In conscious rats, Kregel et al. (12) found that the baroreflex attenuated the hemodynamic responses to heat stress (i.e., arterial baroreceptor-denervated rats had an exaggerated increase in MAP, heart rate, and visceral vasoconstriction) and conferred significant thermal tolerance. In anesthetized rats, as used in this study, the baroreflex does not appear to modulate the hyperthermic control of vascular resistance (10). Because of the reported impairment in baroreflex response after hindlimb-suspension, it is possible that conscious exposure to heat stress may reveal altered vascular resistance responses. Anesthesia was used in the present study, however, to allow exposure of the animal to a high magnitude of heat stress in a humane manner. Moreover, the hemodynamic responses observed in this study were similar to those observed in passively heated unanesthetized rats (14).
In the present study, mesenteric resistance response was not impaired during heat stress after hindlimb suspension, which is in contrast to the reported attenuated increase in resistance to the viscera during exercise after hindlimb suspension (19,31). The impaired resistance increase during exercise could be due in part to altered baroreflex-mediated sympathetic nerve discharge, because hemodynamic responses are modified by baroreflex function during exercise (26). In the anesthetized rat, the increase in splanchnic resistance during heat stress is due primarily to sympathetic vasoconstriction discharge (4,10); however, the baroreflex does not appear to influence the hemodynamic responses, as mentioned above (10). Therefore, the impaired splanchnic vasoconstriction during exercise, but not during heat-stress (under anesthesia) in the hindlimb-suspended rat, may be due to the influence of the baroreflex.
Although we observed no impairment of the ability to decrease resistance in the hindlimb during heat stress, there is a reported impairment of vasodilation to hindlimb working muscle during exercise after hindlimb suspension (19,31). These differences are likely due to the mechanisms of hindlimb vasodilation during the two different stimuli. Vasodilation during exercise may be primarily due to local factors (muscle metabolites and nitric oxide are leading candidates) (5,28). A study by McCurdy et al. (18) indicated that after hindlimb suspension there is decreased maximal response to adenosine and nitric oxide in gastrocnemius and soleus muscle arterioles, which may account for the decreased ability to vasodilate these muscles during exercise. During heat stress, however, vasodilation in the hindlimb may be primarily due to activation of sympathetic vasodilator nerves (13). Therefore, the present study may suggest that the sympathetic vasodilator mechanism is intact after hindlimb suspension.
Our finding that hindlimb suspension did not impair thermoregulation is in contrast to the results of a study by Woodman et al. (32), which found a greater increase in core temperature during exercise in hindlimb-suspended rats. The fact that the rats in their study were conscious and exercising may account for the observed differences between the two studies, because impaired baroreflex may have been involved. We can only conclude from the present study that passive heating in anesthetized rats does not impair thermoregulation.
As mentioned previously, baroreflex-mediated sympatho-excitation to the splanchnic region is impaired after hindlimb suspension (20). This impairment has been attributed to an alteration in central modulation of sympathetic outflow rather than an alteration in the afferent baroreceptor signal (21). Our results suggest that sympathetic output to the mesentery is not impaired during heat stress, because the resistance responses during heat stress are almost exclusively due to sympathetic output (11). Our results also indicate that the extent of the perivascular innervation of the arteries in the mesentery is not compromised. Moreover, a study by Overton and Tipton (24) showed that the resistance response to injected tyramine (which causes release of norepinephrine from sympathetic nerves) was not altered in the mesentery (conversely, resistance response to adrenergic agonists was reduced), which suggests that sympathetic neurotransmitter storage is not depressed, and may even be enhanced, after hindlimb suspension. Collectively, these results suggest that in the mesentery, the change in sympathetic control may only be at the level of the brain stem medulla and may be specific to baroreflex-mediated output.
The results of this study may not reflect the thermoregulatory ability in humans after real or simulated microgravity. Like rats, humans exhibit impaired thermoregulation during exercise after spaceflight or bedrest (3,6). This has been attributed at least in part to an attenuated increase in skin blood flow. This impaired forearm blood flow has also been observed during passive heating of humans after bedrest (2). Analogous to the increased skin blood flow in humans during heating, blood flow increases to the tail of rats during heating, and is a major thermoregulatory mechanism (23,25). Although we did not directly measure tail blood flow, our data suggest that tail blood flow was similar between hindlimb-suspended and control rats. Specifically, the identical changes in iliac artery blood flow suggest that tail blood flow was similar because this major artery supplies blood to the tail as well as the hindlimb. This preservation of tail vasodilation during heating after hindlimb suspension is, therefore not representative of the reported changes in skin blood flow in humans after bedrest.
In summary, this study demonstrated that hindlimb suspension did not impair hemodynamic responses to passive heat stress or sympathetic innervation of mesenteric small arteries. Together with other studies, these results suggest that after hindlimb suspension sympathetic vasoconstrictor nerve fibers innervating the mesentery and hindlimb, and possibly the sympathetic vasodilator nerve fibers, are fully functional. The impaired ability to redistribute blood flow during exercise after hindlimb suspension is, therefore, likely due to altered central control of the baroreflex and altered vascular response to local factors.
The authors would like to thank Ron Matthes for his expert surgical assistance, Christopher Wilson for assistance in caring for the rats, Kathy Walters for assistance in developing the immunostaining protocol, and Dr. Charles Tipton for training in the hindlimb-suspension procedure. This study was made possible by the generous support of the National Aeronautics and Space Administration (Graduate Student Researchers Program Fellowship (GRSP97–050)) and the American College of Sports Medicine (ACSM-NASA Space Physiology Research Grant).
Carl V. Gisolfi died June 3, 2000.
Address for correspondence: Robin Looft-Wilson, John B. Pierce Laboratory, 290 Congress Avenue, New Haven, CT 06519; E-mail: firstname.lastname@example.org.
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Keywords:©2002The American College of Sports Medicine
HEMODYNAMICS; THERMOREGULATION; ARTERIAL RESISTANCE; SIMULATED MICROGRAVITY