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1Department of Health and Kinesiology, Texas A&M University, and 3Department of Medical Physiology and 4Cardiovascular Research Institute, Texas A&M University Health Science Center, College Station; and 2Department of Anesthesiology, Baylor College of Medicine, Houston, Texas
Submitted 7 September 2004 ; accepted in final form 18 November 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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orthostatic intolerance; cerebral blood flow; hindlimb unloading; middle cerebral artery; vascular remodeling; nitric oxide synthase
To study the effects of chronic headward fluid shifts and elevations in arterial pressure on cerebrovascular control mechanisms, investigators have used head-down tail suspension (HDT) of rats as a model. Results from studies of this model indicate that 2-wk HDT induces hypertrophy of the medial layer of cerebral arteries (45) and enhances agonist-induced (48) and myogenic (18) vasoconstrictor responsiveness. In the case of the altered myogenic response, it was found that nonspecific inhibition of nitric oxide synthase (NOS) activity with NG-nitro-L-arginine methyl ester (L-NAME) abolished differences in cerebral artery myogenic vasoconstriction between groups, suggesting that HDT results in an attenuated basal release of NO or a diminished sensitivity of cerebral artery smooth muscle cells to NO (18).
The purpose of the present study was twofold: 1) to determine whether the enhanced vasoconstrictor responses of middle cerebral arteries (MCAs) from HDT rats result from diminished NO signaling through an endothelial NOS (eNOS), inducible NOS (iNOS), or neuronal NOS (nNOS) mechanism or, alternatively, occur as a result of reduced smooth muscle cell sensitivity to NO and 2) to investigate whether the enhancement in vasoconstrictor responsiveness of cerebral arteries in vitro have functional correlates in cerebral perfusion in vivo. To elucidate possible changes in autoregulatory control, regional CBF (rCBF) and CVR (rCVR) were determined in conscious control and HDT rats standing in a normal horizontal position and after 10 min of 75° head-up tilt (HUT). Given that HDT rats display alterations in arterial baroreflex control of the peripheral circulation (33), we hypothesized that perfusion of brain regions associated with neural control of peripheral vascular resistance would be compromised during a challenge of cerebral autoregulation with the HUT maneuver.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In Vitro Cerebral Artery Studies
After the 2-wk experimental period, the animals were anesthetized with pentobarbital sodium (35 mg/kg ip) and decapitated, and the brain was rapidly removed and placed in a 4°C physiological saline-albumin buffer solution (PSS). MCAs were isolated and mounted in vessel chambers containing PSS at 37°C as previously described (5, 6, 20, 39, 47). A micropipette filled with PSS was inserted into one end of the vessel and secured with 11-0 nylon ophthalmic suture. The other end of each vessel was cannulated with a second resistance-matched micropipette and secured with suture. After cannulation, each isolated vessel in the tissue chamber was transferred to the stage of an inverted microscope coupled to a video camera, a video micrometer, a videotape recorder, and a data acquisition system. To maintain a constant intraluminal pressure of 75 mmHg (16), the micropipettes cannulating the MCAs were connected to two independent hydrostatic fluid reservoirs; pressure was measured through sidearms of the reservoir lines with low-volume-displacement strain gauge transducers. Leaks were detected by pressurizing the vessel and then closing the valves to the reservoirs and verifying that intraluminal pressure remained constant. Vessels free of leaks equilibrated for at least 1 h at 37°C to develop basal tone; the bathing solution was replaced every 15 min during the equilibration period.
Experimental design.
A series of in vitro experiments were performed to determine the effects of HDT on MCA vasoconstrictor responsiveness. The first series determined vasoconstrictor responsiveness to mechanical perturbations (transmural pressure and shear stress) and K+ in the presence and the absence of 10–5 M L-NAME. To evaluate myogenic vasoconstriction, intraluminal pressure was increased from 0 to 135 mmHg in increments of 15 mmHg by raising both PSS-fluid reservoirs simultaneously so that all pressure changes occurred in the absence of intraluminal flow. A passive pressure-diameter relation was similarly determined after vessels were incubated 1 h in Ca2+-free PSS. To determine MCA vasoconstrictor responses to shear stress (5, 6), changes in luminal flow were initiated by altering the height of the two fluid reservoirs in equal and opposite directions to generate incremental pressure differences of 2, 4, 6, 10, and 20 cmH2O across the vessels without changing mean intraluminal pressure (27). Flow velocity through the resistance-matched cannulating pipettes was previously determined at each pressure difference by measuring the velocity of red blood cells suspended in PSS (Vrbc) with a velocimeter (Microcirculation Research Institute, Texas A&M University). Volumetric flow (
) was calculated from Vrbc and pipette diameter (d) according to the equation (10):
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Intravascular shear stress (
) for the experimental vessels was calculated from volumetric flow as:
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where 
is viscosity and r is vessel radius. Finally, agonist-induced vasoconstrictor responses were determined by isotonically increasing the concentration of K+ (10–100 mM) in the vessel bath (20).
Because L-NAME abolished differences in basal tone and vasoconstrictor responses between groups, separate series of studies were conducted to determine whether the L-NAME effect was associated with an endothelium-dependent, iNOS, or nNOS signaling mechanism. The endothelium was removed in three groups of arteries to determine whether differences in basal tone and myogenic and flow- and K+-induced vasoconstriction were the result of alterations in endothelium-mediated vascular tone. Air was passed through the lumen to remove endothelial cells as previously described (5, 6); vasodilation of <5% to intraluminally applied 10–6 M 2-methylthio-ATP, an endothelium-dependent vasodilator (39, 47), confirmed that the endothelium was successfully removed. In other sets of arteries, MCAs were incubated with the iNOS inhibitor aminoguanidine (AG; 10–4 M) (22, 32) or the nNOS inhibitor 1-[2-(trifluoromethyl)phenyl]imidazole (TRIM, 10–4 M) (8, 28) for 30 min; basal tone and myogenic or flow- or K+-induced vasoconstriction were then assessed.
To test for the possibility that smooth muscle responsiveness to NO release is altered with HDT, the vasodilator responses of endothelium-intact MCAs to the abluminal application of the NO donor sodium nitroprusside (SNP; 10–10-10–4 M) were determined in the presence and absence of 10–5 M L-NAME in a final set of vessels (40, 48). To avoid possible tachyphylaxis, only one concentration-response determination was made per vessel. At the conclusion of each experiment, arteries were incubated in Ca2+-free PSS for 1 h to determine maximal diameter at an intraluminal pressure of 75 mmHg.
Drugs and reagents. KCl, SNP, L-NAME, AG, and TRIM were purchased from Sigma. PSS buffer contained (in mM) 145 NaCl, 4.7 KCl, 1.2 NaH2PO4, 1.17 MgSO4, 2.0 CaCl2, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, and 3.0 MOPS (Sigma), plus bovine serum albumin (10 mg/ml; USB Chemicals), with a pH of 7.4. Ca2+-free PSS buffer contained 2 mM EDTA, and CaCl2 was replaced with 2.0 mM NaCl. K+ solutions were prepared similarly to PSS buffer, except with varying concentrations of NaCl and KCl to achieve desired increases in the molar concentration of K+ (10–100 mM) without corresponding increases in bath osmolality (20). Thus the following concentrations of NaCl and KCl were used to achieve each dose: 10 mM K+ dose: 135 mM NaCl and 14.7 mM KCl; 20 mM K+: 125 mM NaCl and 24.7 mM KCl; 30 mM K+: 115 mM NaCl and 34.7 mM KCl; 40 mM K+: 105 mM NaCl and 44.7 mM KCl; 50 mM K+: 95 mM NaCl and 54.7 mM KCl; 60 mM K+: 85 mM NaCl and 64.7 mM KCl; 80 mM K+: 65 mM NaCl and 84.7 mM KCl; and 100 mM K+: 45 mM NaCl and 104.7 mM KCl.
Evaluation of eNOS Expression
RT-PCR. MCAs were snap frozen and stored at –80°C in 0.5-ml microcentrifuge tubes. Vessels were pulverized in lysate buffer, and RNA was extracted with the RNAqueous filter system (Ambion). Real-time PCR was performed with TaqMan probes designed with the use of Primer Express from the published sequence for rat eNOS (forward primer: GAA CCT ACA GAG CAG CAA ATC CA; reverse primer: CAG TCC CTC CTG GCT TCC TCC A) and a TaqMan oligonucleotide probe (probe: CGA GCC ACA ATC CTG GTC CGT CTT) labeled with a fluorescent reporter dye and a quencher dye (Applied Biosystems). Levels of the target sequence were quantified relative to the cycle number (cycle threshold) at which the target and coamplified 18S ribosomal RNA reach a fixed threshold as previously described (40).
Immunoblot analysis of MCA protein. Differences in eNOS protein expression in MCAs were assessed by immunoblot analysis as previously described (40). Briefly, MCA proteins (1 µg total protein per sample) were subjected to SDS-polyacrylamide gel electrophoresis (8–16% gradient gel; Novex) and transferred to nitrocellulose membrane. Membranes were blocked and incubated with primary antibody overnight at 4°C. Primary antibody dilutions were as follows: eNOS (Transduction Labs) 1:1,250 and cyclophilin 40 (Affinity BioReagents) 1:1,000 in blocking buffer. After being washed, membranes were incubated with the appropriate horseradish peroxidase-conjugated species-specific anti-IgG (1:50,000 to 1:100,000 depending on primary antibody) for 2 h at 25°C. Peroxidase activity was detected with West Dura Extended (Pierce). Normalization for loading differences and blot-to-blot variability in density was accomplished by using ratios of the densitometry signals for eNOS vs. cyclophilin 40 protein. Cyclophilin 40 was chosen as a normalization control after verifying empirically that no quantitative differences in expression were observed in vessels from control and HDT samples when equal protein amounts were loaded.
In Vivo CBF Study
Surgical procedures. One day before the end of the animals’ treatment period, control and HDT rats were anesthetized with pentobarbital sodium (30 mg/kg ip; Nembutal, Abbott Labs). The HDT animals were anesthetized while remaining in the head-down position. A catheter [Silastic, inner diameter (ID) 0.6 mm, outer diameter (OD) 1.0 mm; Dow Corning] filled with heparinized (200 U/ml) saline and connected to a pressure transducer and a chart recorder was advanced toward the right atria of the heart via the right jugular vein. This catheter was subsequently used for the infusion of the tracer n-isopropyl-p-iodoamphetamine[isopropylmethyl-1,3-14C] ([14C]IPIA, 22–50 mCi/mmol; American Radiolabeled Chemicals). A second polyurethane catheter (Microrenathane; ID 0.36 mm, OD 0.84 mm; Braintree Scientific), used for the withdrawal of a reference blood sample and measurement of heart rate and arterial blood pressure, was implanted in the caudal artery of the tail and filled with heparinized saline as previously described (12). Both catheters were externalized and secured on the dorsal cervical region.
Experimental protocol. After 24 h of recovery from the catheter implantation surgery, control and HDT animals were randomly assigned to undergo one of two experimental conditions: 1) 15 min of standing in the horizontal plane and 2) 5 min of horizontal standing followed by 10 min of 75° HUT. To accomplish horizontal standing and HUT, the animals were placed in a Plexiglas hemicylinder (Rodent ECU; Braintree Scientific) fixed on a tilting support axis. The concept of challenging arterial pressure and cerebral autoregulation of perfusion was based on this experimental intervention described by Martel et al. (30). After placement into the Plexiglas hemicylinder, all catheters and instrumentation were immediately connected with all animals in the horizontal position. At the end of the 15-min experimental period, the reference blood withdrawal was initiated, and on the appearance of blood in the caudal catheter the [14C]IPIA (0.1 µCi/g body wt in 0.7 ml of physiological saline) was injected quickly into the right jugular vein. After 30 s, euthanasia solution (0.22 ml/kg, Euthanasia-5 solution; Henry Schein) was infused through the jugular catheter and blood withdrawal continued for 30 s. The animals were immediately removed from the Plexiglas hemicylinder and exsanguinated. The brain with the pituitary and pineal attached was quickly removed from the skull, frozen in isopentane chilled to –40°C, and stored at –80°C until sectioning. Each brain was cut into 20-µm-thick sections with a cryostat (–18°C). Representative sections were mounted onto glass slides and placed in contact with radiograph film (Biomax MR; Kodak) in light-tight cassettes. After a 12-day exposure, the film was developed, producing autoradiographic images.
Blood flow determination.
CBF was measured with quantitative autoradiography as previously described (3, 4, 19). Concentrations of the tracer (radioactivity/g brain tissue) were determined from the autoradiographs by comparing the optical densities of various brain regions with the optical densities produced with calibrated standards packed with tissue sections in the cassettes. The total radioactivity in the blood withdrawn (DPMB) was calculated according to the equation:
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where DPMRS is the mean radioactivity of sample aliquots taken from the total blood withdrawn, MassTB is the mass (g) of the total blood withdrawn, and MassRS is the mass of the aliquots. Blood flow for individual regions (rCBF) was calculated according to the equation:
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where rCBF is given in milliliters per 100 grams per minute and QRS is the rate of blood withdrawal from the caudal artery (0.4 ml/min). The rate of withdrawal was chosen to provide sufficient blood to measure radioactivity without altering arterial blood pressure. CBF was graphically displayed and calculated with the use of an image analysis system (MCID MI; Imaging Research).
Arterial pressure, heart rate, and vascular resistance determination. Pressure recordings were made with the pressure transducer (BP100; ADInstruments) at the level of the animal's head and recorded with the MacLab system and MacLab Chart software (ADInstruments) from the start of each experimental condition to just before start of blood withdrawal, because simultaneous pressure recordings and blood withdrawal were not possible. Mean arterial pressure was electronically averaged from pulsatile pressure measurements from the caudal catheter. Heart rate was estimated from pulsatile caudal pressure tracings. rCVR (mmHg·ml–1·min·100 g) was calculated by dividing mean arterial pressure (mmHg) by the tissue blood flow rate (ml·min–1·100 g–1).
Statistical Analysis
All values are presented as means ± SE. A value of P < 0.05 was required for significance. The development of basal tone was expressed as the percent constriction relative to maximal diameter and was calculated as:
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where IDmax is the maximal inner diameter recorded at a pressure of 75 mmHg and IDB is the starting baseline diameter. Recorded active myogenic responses, passive diameter measurements in response to pressure changes, active flow responses, and responses to changes in K+ concentration ([K+]) were normalized according to the formula:
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where IDS is the steady-state diameter measured subsequent to each incremental change in stimulus. The data are normalized to the maximal diameter to account for potential differences in vessel size between control and HDT groups (45).
Pressure-response, flow-response, and concentration-response curves were evaluated by using ANOVA with one within (intraluminal pressure, flow, or agonist concentration) and one between factor. Planned contrasts were conducted at each intraluminal pressure, flow, or concentration level to determine whether differences existed between groups. Student's unpaired t-tests were used to determine whether differences in developed spontaneous tone, maximal diameter, wall thickness at maximal diameter, body mass, soleus muscle mass, and the soleus muscle-to-body mass ratio were significant between groups.
In the blood flow studies, a one-way ANOVA was used to compare arterial pressure, heart rate, blood flow, vascular resistance, body mass, soleus muscle mass, and the soleus muscle-to-body mass ratio across treatment-condition groups, and an unpaired multiple-comparison procedure (Student-Newman-Keuls) was used as a post hoc test to determine the significance of differences among means. For all analyses, the 0.05 level was used to indicate statistical significance.
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RESULTS |
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Body mass of control rats (428 ± 13 g) tended to be greater than that of HDT rats (396 ± 12 g) (P = 0.070). Hindlimb unloading reduced soleus muscle mass of HDT rats (143 ± 17 mg) relative to control soleus muscle mass (221 ± 12 mg). Similarly, the soleus-to-body mass ratio of HDT rats (0.351 ± 0.021 mg/g) was lower than that of control rats (0.486 ± 0.018 mg/g). Soleus muscle atrophy, which is characteristic of reduced skeletal muscle weight-bearing activity, confirms the effectiveness of the HDT intervention.
Vessel Characteristics
Maximal MCA intraluminal diameter was similar between groups (control 232 ± 4 µm, HDT 223 ± 3 µm), although MCA wall thickness was greater in HDT rats (control 11 ± 1 µm, HDT 16 ± 1 µm). The development of basal tone was greater in MCAs from HDT than from control rats (Fig. 1), and, correspondingly, baseline diameter was less in HDT animals (control 180 ± 7 µm, HDT 143 ± 7 µm). Both nonspecific NOS inhibition with L-NAME and endothelial cell removal abolished differences in basal tone and baseline diameter between groups, whereas iNOS and nNOS inhibition had no effect.
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MCAs from HDT rats vasoconstricted more to changes in transmural pressure than those from control rats (Fig. 2A). This group difference was abolished with L-NAME treatment (Fig. 2A) and endothelium removal (Fig. 2B). Neither iNOS nor nNOS inhibition eliminated differences in myogenic vasoconstriction between groups (Fig. 2B). Passive pressure-diameter relations were not different between groups (Fig. 2A).
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Intraluminal flow induced greater vasoconstriction (Fig. 3A) and shear stress (Fig. 3B) in MCAs from HDT rats. To determine whether the greater vasoconstriction was the result of higher shear forces, diameter was plotted as a function of shear stress (Fig. 3C). The data demonstrate that shear stress induces greater vasoconstrictor responses in arteries from HDT rats. L-NAME treatment and endothelium removal abolished these differences between groups, whereas group differences remained with iNOS and nNOS inhibition (data not shown).
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Although arteries from HDT rats initially dilated less to low [K+] and subsequently constricted more to high [K+] (Fig. 4), the shape of the response curves was similar between groups. Differences in K+-induced vasodilation and constriction reflected the initial difference in MCA basal tone. NOS inhibition with L-NAME and endothelial denudation eliminated differences in K+ responses between groups (Fig. 4), whereas iNOS and nNOS treatment had no effect on K+ responses (data not shown). The data also indicate that the procedure to remove the endothelium from the MCA did not adversely affect the smooth muscle cells from constricting to K+ (Fig. 4).
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SNP-induced vasodilation did not differ between groups in either the presence or the absence of L-NAME (data not shown).
eNOS Expression
HDT had no effect on MCA eNOS mRNA expression (Fig. 5A) but resulted in a reduction in eNOS protein content (Fig. 5B).
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Mean heart rate was not different during horizontal standing (0° tilt) and 75° HUT between control and HDT rats (Table 1). Mean arterial pressure was similar between groups during horizontal standing. During HUT arterial pressure remained unchanged in control rats but decreased in HDT rats to a level lower than that in control animals.
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Relative to that in the control rats, rCBF in HDT rats during horizontal standing was lower in 21 of 38 regions measured (Table 1, Fig. 6). During HUT, HDT rats had lower cerebral perfusion rates in all but three regions compared with the same condition in control rats. In the medulla, for example, HUT increased perfusion of all medullary regions in control animals and decreased medullary blood flow in HDT rats. rCVR during horizontal standing was higher in HDT than control rats in 19 of 38 regions (Table 2, Fig. 6). With HUT, rCVR in HDT animals was greater in 26 regions, including most areas in the basal ganglia, thalamus, hypothalamus, pons, cerebellum, and medulla (Table 2, Fig. 6).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Results from the present study demonstrate that the distinguishing characteristic of cerebral arteries from HDT rats is the greater level of basal tone (Fig. 1). This difference in tone is manifest throughout the physiological range of pressures (Fig. 2A), over a span of flow- or shear stress-mediated vasoconstrictions (Fig. 3), and throughout the range of K+-induced vasoconstrictions (Fig. 4). Nonspecific inhibition of NOS activity with L-NAME eliminated differences in basal tone between groups and, correspondingly, eliminated differences in myogenic and shear stress- and K+-induced vasoconstrictor responses. Removal of the endothelium likewise abolished differences in basal tone and vasoconstrictor responses. In contrast, inhibition of iNOS and nNOS activity, which has been suggested to contribute to the increased cerebral artery myogenic vasoconstriction in HDT rats (29), did not abolish differences in basal tone or vasoconstrictor responses between groups. These data indicate that the observed effects of L-NAME occur by abolishing differences in basal levels of endothelial NO release or bioavailability. This assertion that HDT diminishes basal levels of NO in cerebral arteries is further supported by the lower eNOS protein content in MCAs from HDT rats (Fig. 5B), indicating that HDT alters this and possibly other components of the NOS signaling pathway. Although the effects of L-NAME and endothelium removal to normalize vasoconstrictor responses between MCAs from control and HDT rats appear to result from a reduction in basal endothelial NO bioavailability with HDT, an alternative possibility is a diminished smooth muscle sensitivity to the relaxing effects of NO. Results with an NO donor demonstrate that MCA responsiveness to NO is not reduced by HDT, and thus differences in basal tone and vasoconstrictor responses appear to be the result of diminished basal levels of endothelium-derived NO.
The stimulus for the downregulation of eNOS protein is presently unknown. However, it has been widely reported that eNOS expression is sensitive to chronic changes in intravascular blood flow and shear stress (9, 34). We previously showed (44) that CBF during HDT is reduced by 
25%, presumably to protect the brain from elevations in fluid filtration and edema. Previous studies also showed that the unloading of the postural soleus muscle with HDT results in a chronic reduction of blood flow (31) and, like the cerebral arteries, lower eNOS protein expression in soleus muscle resistance arteries (46); vascular eNOS protein content remains unaltered in muscles that do not experience chronic reductions in flow with HDT (46). Regulation of eNOS expression by shear stress is predominantly at the transcriptional level (17), yet differences in mRNA expression levels did not occur at this time point (Fig. 5A). However, MCA levels of eNOS mRNA are diminished in rats exposed to HDT for shorter periods of time (unpublished observation). This indicates that there is a time-dependent decrease in eNOS mRNA that renormalizes to control levels. Alternatively, HDT may regulate eNOS protein stability or translation independent of transcription, as has been previously reported for other stimuli (37).
These vascular adaptations described in vitro appear to have functional consequences for cerebral perfusion and vascular resistance in vivo. In the conscious, horizontally standing rats there were no differences in arterial pressure between groups. Despite the groups having similar arterial pressures, mean CBF was 24% lower in the HDT rats, and in several regions CBF was <45% of that in control animals under normal standing conditions. This level of CBF has been proposed as the minimum perfusion required for maintaining normal energy metabolism in the rat brain (14, 15). In the present study we did not assess cerebral autoregulation by establishing the CBF-pressure relation across a range of arterial pressures in control and HDT animals, and therefore it is not possible to conclude whether HDT alters cerebral autoregulation and the autoregulatory range. However, previous work has shown that NOS inhibition in rats causes a downward vertical shift in the cerebral autoregulatory curve, i.e., autoregulation with a lower flow for a given arterial pressure (26). Therefore, the downregulation of the eNOS signaling mechanism in cerebral arteries may contribute to the lower cerebral perfusion in HDT rats during standing, although they had the same arterial pressure as control animals.
One objective of this study was to investigate whether HDT affects the regional distribution of blood flow within the brain during postural stress. In humans regional CBF distribution has been shown to be altered during head-up as well as head-down tilt (36, 38, 42). Results from the present study demonstrate that regional variation in blood flow occurs in both control and HDT animals exposed to HUT. In HDT rats, HUT resulted in a 10% drop in arterial pressure, similar to that previously reported (30). The lower arterial pressure and higher CVR in HDT rats resulted in a 41% lower mean perfusion relative to that in control animals. The higher vascular resistance and lower cerebral perfusion in HDT rats during HUT occurred in several regions involved in cardiorespiratory control, such as insular cortex, hypothalamus, red nuclei, and the reticular formation and nuclei within midbrain, pons, and medulla, including the solitary tract nuclei and ventrolateral medulla (Fig. 6A). Other regions similarly affected include those involved in the maintenance of balance and equilibrium [vestibular nuclear area (Fig. 6B), caudate putamen, cerebellar vermis, and floccular lobe], hearing (inferior colliculus and cochlear nuclear area), vision (superior colliculus), and circadian rhythm regulation [suprachiasmatic nucleus (Fig. 6C) and pineal gland].
Blood flow to medullary areas during HUT was in sharp contrast between groups; perfusion was elevated in control animals with HUT, whereas flows were unchanged or reduced in the HDT animals (Fig. 6, A and B). Animal studies have shown that loss of vestibular input induces orthostatic hypotension (13, 25), and therefore reductions in flow in HDT rats to vestibular nuclei (Fig. 6B) and solitary tract and reticular nuclei, as well as cardiovascular control areas, could serve to disrupt the integrated postural/cardiorespiratory response to HUT. Although at present it is unclear whether regional CBF deficits have any direct systemic consequences, it is possible that an attenuated perfusion of regions involved in cardiovascular control could disrupt central integration of afferent input and contribute to the attenuated baroreflex control of sympathetic nerve activity reported in HDT rats (33). Such an effect could impair reflex control of arterial pressure during postural adjustments, such as that which occurred during HUT in the HDT rats of the present study. Indeed, reductions in flow through the MCA have been shown to precede alterations in arterial pressure with HUT (21, 35), supporting the notion that cerebral hypoperfusion may adversely affect neural control mechanisms and negatively impact tolerance of gravitational stress.
In conclusion, the present study demonstrates that prolonged cephalic fluid shifts and increases in arterial pressure enhance the basal tone of rat MCAs and, correspondingly, enhance vasoconstrictor responses to alterations in transmural pressure, luminal shear stress, and K+. When basal release of NO is blocked by nonspecific NOS inhibition, basal tone and vasoconstrictor responses do not differ between control and HDT rats. In contrast, neither specific iNOS nor nNOS inhibition has this effect. The effect of endothelium removal to return basal tone and vasoconstrictor responses to control levels and the lower MCA eNOS protein content in HDT rats indicate that the mechanism of enhanced basal tone and vasoconstriction is a downregulation of the eNOS signaling mechanism. The functional consequence of this and other vascular alterations (see, e.g., Ref. 45) with HDT appears to be greater CVR and lower cerebral perfusion. When applied to the human condition, these results suggest that alterations in cerebral autoregulation associated with microgravity or prolonged bed rest may be the result of diminished levels of cerebral artery endothelial NO. Although diminished NO bioavailability may be an appropriate adaptation of the cerebral vasculature to chronic elevations in arterial pressure, this adaptive response may limit the ability of cerebral vasculature to respond to abrupt alterations in pressure, such as that which occurs with orthostatic stress and acute hypotension (1, 2, 7, 24, 41, 49). Furthermore, spaceflight- and bed rest-induced deficits in locomotion, balance, and orthostatic tolerance may be related to regional reductions in perfusion of key regulatory areas within the brain.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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mean differs from control rat mean of the same condition (P < 0.05).