INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ON EARTH, BLOOD AND BODY FLUIDS are generally pulled down into less compliant vessels of the lower body. Once a person leaves the Earth's gravitational field, there is a marked redistribution of fluid and fluid pressures within the body (16, 40). The lack of significant gravity in space allows blood to shift toward the more-compliant arteries of the torso, neck, and head (41), forcing the cardiovascular system to adapt to a new hydrostatic pressure column. Part of this adaptation may include a remodeling of the cerebral vasculature to protect the brain from the sudden elevations in arterial perfusion pressure. However, a return to normal gravity then produces an orthostatic stress on the cardiovascular system and, in many cases, a compromised ability to maintain adequate blood flow to the brain (3, 4). Under these circumstances, it is possible that alterations in cerebral perfusion may be related to the remodeling of the cerebral vasculature that occurred in response to the headward fluid shifts during zero gravity.

Several studies have shown that increased transmural pressures can induce structural remodeling of arteries (25, 34). For example, hypertrophy of cerebral arteries is stimulated by hypertensive increases in transmural pressure (17, 30). Heistad and Kontos (18) identified hypertrophic structural alterations as the major factor that attenuates the pressure-induced increases in cerebral blood flow and fluid diffusion during hypertension. Thus it has been hypothesized that the increased cephalic transmural pressures induced by weightlessness could stimulate the cerebral vasculature to undergo similar structural changes (21, 41). To test this hypothesis, tail-suspended hindlimb-unloaded (HU) rats were used to simulate the cephalic fluid shift that occurs on exposure to microgravity. Hindlimb unloading of rats is a widely utilized model that reproduces many of the effects that microgravity exposure has on humans, including cephalic fluid shifts (15, 27), reduced blood volume (9, 10), resting and exercise tachycardia (28), orthostatic hypotension (26), and reductions in aerobic power (7, 33).

Therefore, the purpose of this study was to determine whether the cerebral basilar artery morphology is altered by 14 days of hindlimb unloading. Furthermore, to determine whether vascular adaptations are localized to regions undergoing increases in transmural pressure, resistance artery morphology was also investigated in the splanchnic region (splenic and mesenteric arteries) where little or no change in transmural pressure occurs with hindlimb unloading.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. All procedures performed in this study were approved by the Texas A&M University Institutional Animal Care and Use Committee and conformed to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals [DHEW Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20892].

Thirty male Sprague-Dawley rats weighing ~350 g were obtained (Charles River), housed individually in a room maintained at 23°C with a 12:12-h light-dark cycle, and given rat chow and water ad libitum. The animals were randomly assigned to either HU (n = 16) or cage control (C; n = 14) groups. The hindlimbs of the HU animals were elevated to an approximate spinal angle of 40-45° from horizontal. This was done with a harness attached to the tail by modification of a technique previously described (19). Briefly, a harness consisting of a curved, molded plastic cast (X-Lite splint material; AOA/Kirschner) was placed around the wrapped (Co-Flex bandage, Andover) proximal two-thirds of the animals' tail. Moleskin adhesive material was placed as contact points between the tail skin and the cast to allow for adequate blood flow. Two hooks attached to opposite ends of the cast were connected by a small chain to a swivel apparatus fixed at the top of the cage. The length of the chain was adjusted to prevent the hindlimbs of the animal from touching any supportive surfaces while the forelimbs maintained contact with the cage floor. This allowed the animal free range of movement about the cage while achieving the desired experimental results. C animals were maintained in a normal cage environment. Both groups were kept in their respective conditions for 14 days. This time period has been shown to be sufficient to generate cardiovascular alterations (5, 6, 9, 10, 13, 26, 28) and cephalic fluid shifts (27, 28, 36) in HU animals. Additionally, Nordborg and Johannson (30) showed that hypertension-induced alterations in vascular morphology occur within this time frame.

The animals were further divided into two subsets. Basilar, splenic, and mesenteric resistance arteries were harvested from the first subset of rats (C, n = 9; HU, n = 9) for the morphometric studies. After 14 days, HU and C animals from this subset were weighed and injected with pentobarbital sodium (30 mg/kg ip) to induce deep anesthesia. The animals were then decapitated, the skull roof was removed, and the various nerve connections were cut, freeing the brain from the skull. The brain was then placed in 4°C filtered physiological saline buffer solution (PSS) (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; pH 7.4.

The second subset of animals (C, n = 5; HU, n = 7) was used to determine the effects of acute (10 min) and chronic (2 wk) hindlimb unloading on mean ascending aortic pressure. After 13 days of normal cage activity or hindlimb unloading, the rats in this subset were injected with pentobarbital sodium (30 mg/kg ip). A Silastic catheter (OD 0.6 mm, ID 1.0 mm) was advanced into the ascending aorta via the right carotid artery, secured, and externalized on the dorsal cervical region as previously described (28). After a 24-h recovery period (HU rats remained in a head-down tail-suspended position during this period), mean ascending aortic pressure was measured in C rats during normal standing and after 10 min of hindlimb unloading. Aortic pressure was measured in the HU rats while they remained tail suspended. These pressures were subsequently used to estimate basilar arterial pressure changes with hindlimb unloading.

Vessel preparation. With the use of a stereomicroscope (×20-40 magnification), the basilar artery (C, n = 7; HU, n = 6) was removed from the ventral side of the brain. The organs of the lower abdomen were revealed, and a feed artery leading to the spleen (C, n = 8; HU, n = 8) and the fourth branch of the mesenteric artery (C, n = 9; HU, n = 9) were isolated with the aid of a stereomicroscope. All three vessel types were cut into 1.0- to 1.5-mm sections and cannulated on glass pipettes attached to a fluid reservoir as previously described (5). Cannulations were performed within a Lucite microvessel chamber containing PSS. Luminal pressure was set and maintained at 60 cmH₂O by raising the fluid reservoirs 60 cm above the cannulated vessel. Dilation was then induced by the addition of 10⁴ M sodium nitroprusside. Sodium nitroprusside was used because it has been previously shown that the dilatory response elicited by this agent is unaltered in hypertrophied basilar arteries from hypertensive rats (22, 23, 38). After nitroprusside-induced dilation, each isolated vessel was fixed with paraformaldehyde, stained with eosin, and embedded in paraffin.

Histomorphometry. The paraffin-embedded vessels were cut into 5-µm-thick cross sections that were mounted on glass microscope slides and stained with hematoxylin and eosin. Vascular structure was evaluated by measuring vessel media layer cross-sectional area (CSA), media outer perimeter, media wall thickness, number of media nuclei, luminal perimeter, and intraluminal CSA. Areas were measured by tracing the applicable perimeters as they appeared on the computer monitor with a computer mouse, from which the interior area was calculated via a pixel count. For example, media layer CSA was calculated by tracing the media outer perimeter and the luminal perimeter, directing the program to calculate the respective areas (total CSA and intraluminal CSA), and subtracting the latter from the former. Media wall thickness was measured at four points separated by 90° angles and averaged. All measurements were determined by using an image-processing and shape-analysis system with BioQuant TrueColor Windows, version 2.0 (R&M Biometrics, Nashville, TN) software. The image-processing system consisted of an Olympus light microscope with an attached video camera coupled to an image processor, optical mouse, and IBM personal computer.

The circumferential stress () of the basilar artery was calculated from basilar artery pressure (BAP), inner diameter of the basilar artery (BAD_i), and wall thickness (WT), where
BAP was estimated to be 80% of aortic pressure (11) and was converted from mmHg to dyn/cm² (1 mmHg = 1.334 × 103 dyn/cm²). BAD_i was calculated from intraluminal CSA. BAD_i and WT of the basilar artery from control rats were used to calculate  during control standing and acute (10 min) hindlimb unloading.

Statistical analysis. Student's t-tests were used to determine whether differences in the measured parameters were significant between C and HU rats for each vessel type. A one-way ANOVA was used to compare aortic pressure and basilar artery circumferential stress during standing and after 10 min and 14 days of hindlimb unloading. Student-Newman-Keuls method was used as a post hoc test to determine the significance of differences among means. All values are presented as means ± SE. A P < 0.05 was required for significance.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Body and soleus muscle mass. Body mass did not change over the 14-day period of tail suspension in the HU rats (pre-HU, 417 ± 4 g; post-HU, 409 ± 9 g). Body mass at the time of death was not different between HU and C rats (430 ± 7 g) (P = 0.0897). Hindlimb unloading reduced both soleus muscle mass (C, 230 ± 13 mg; HU, 145 ± 13 mg) and the soleus-to-body mass ratio (C, 0.529 ± 0.023 mg/g; HU, 0.348 ± 0.026 mg/g). Soleus muscle atrophy, which is characteristic of reduced skeletal muscle weight-bearing activity, confirms the effectiveness of the hindlimb-unloading intervention.

Ascending aortic pressure. Mean aortic pressure increased from 124 ± 7 mmHg during standing to 148 ± 5 mmHg after 10 min of hindlimb unloading in the C rats. Aortic pressure remained elevated after 14 days of hindlimb unloading (145 ± 2 mmHg).

Basilar artery. Media wall thickness was 52% greater in basilar artery from HU than from C animals (Figs. 1 and 2) (HU 33.9 ± 4.1 µm; C 22.3 ± 3.2 µm). Correspondingly, media CSA was 39% greater in HU than C rats (Fig. 3) (HU 17,893 ± 2,539 µm²; C 12,904 ± 1,433 µm²). Intraluminal CSA was 58% lower in HU than C animals (Fig. 4) (HU 7,816 ± 3,045 µm²; C 13,469 ± 5,500 µm²), whereas outer perimeter was not significantly different between groups (Fig. 5). There were no differences in the number of media nuclei between groups (Fig. 6). The circumferential stress  of the basilar artery was elevated with 10 min of hindlimb unloading but was diminished with 14 days of unloading;  at 14 days was not different from that during control standing (Fig. 7).

View larger version (89K): [in this window] [in a new window]   Fig. 1.   Typical cross-sectional view of basilar artery from control (C; A) and 14-day hindlimb-unloaded (HU; B) rats. Bar, 20 µm.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Media thickness of basilar (C, n = 7; HU, n = 6), mesenteric (C, n = 9; HU, n = 9), and splenic (C, n = 8; HU, n = 8) arteries for C and HU rats. Values are means ± SE. * Mean is different from C mean (P < 0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Media cross-sectional area (CSA) of basilar, mesenteric, and splenic arteries for C and HU rats. Values are means ± SE. * Mean is different from C mean (P < 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Intraluminal CSA of basilar, mesenteric, and splenic arteries for C and HU rats. Values are means ± SE. * Mean is different from C mean (P < 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Media outer perimeters of basilar, mesenteric, and splenic arteries for C and HU rats. Values are means ± SE.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   No. of media nuclei in basilar, mesenteric, and splenic arteries for C and HU rats. Values are means ± SE.

View larger version (33K): [in this window] [in a new window]   Fig. 7.   Circumferential wall stress in basilar artery during normal standing and after 10 min and 14 days of hindlimb unloading. Values are means ± SE. * Mean is different from C mean (P < 0.05).

Splenic and mesenteric feed arteries. There were no significant differences in media wall thickness (Fig. 2), media CSA (Fig. 3), intraluminal CSA (Fig. 4), outer perimeter (Fig. 5), and number of nuclei (Fig. 6) between groups for either the splenic or mesenteric arteries. However, there was a tendency for splenic media wall thickness to decrease with HU (P < 0.1).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The purpose of this study was to determine whether hindlimb unloading produces structural alterations in basilar, mesenteric, or splenic resistance arteries. Hindlimb unloading did not affect media wall thickness, media CSA, or intraluminal CSA of mesenteric or splenic arteries but did cause increases in basilar artery media wall thickness and media CSA (Fig. 1). Furthermore, the increase in media CSA did not alter the outer perimeter of the basilar artery but reduced the vessel intraluminal CSA. The increase in media CSA appears to be the result of smooth muscle hypertrophy rather than hyperplasia, since the number of media nuclei was not elevated.

Evidence of arterial remodeling induced by hypertension may provide insight into the morphological alterations of cerebral arteries that result from hindlimb unloading. For example, the resistance arteries leading to the brain of spontaneously hypertensive rats are subjected to constant higher pressures and adapt to this pressure elevation by increasing media layer thickness (17, 25, 30, 34). The remodeling caused by hypertension is thought to be largely due to the intrinsic myogenic response to loading (24, 25, 31), in which the distension of the artery produces a counterregulatory contraction of the vascular smooth muscle that leads to hypertrophy. An important functional consequence of the thickening of the cerebral vasculature leading to the brain is the prevention of edema formation within the brain. Brain tissue is extremely sensitive to vascular leakage, and increases in perfusion pressure without an appropriate vascular response to counterbalance the increased pressure would increase fluid diffusion into the brain. In fact, the thickening of the cerebral wall has been shown to aid in stroke prevention and maintain the blood-brain barrier in stroke-prone rats subjected to hypertension (37). The basilar artery is a major intracranial conduit to cerebral tissue arterioles and capillary beds through which most diffusion occurs. Thus a thickening of the basilar artery with hypertension is presumably an adaptive response to attenuate the elevated fluid pressure from being passed downstream to vessels within the cerebral tissue, thereby providing protection against cerebral edema (17, 25, 30, 34).

Hargens et al. (14) have reported similar arterial adaptations in the legs of giraffes. Because giraffes have such a large hydrostatic column, high arterial tone is necessary to counteract the intense fluid pressure in the lower extremities to control fluid diffusion and prevent edema. As juvenile giraffes grow to their full adult stature, the hydrostatic column expands, and the fluid pressure to the legs is elevated. Correspondingly, the media wall of the arterial vasculature in the lower extremities thickens. Thus, in this experiment of nature, the vascular smooth muscle cells in the legs hypertrophy in response to increases in transmural pressure and arterial distension (14).

In a comparable sequence of events, hindlimb unloading induces pressure changes indicative of an elevated hydrostatic fluid pressure to the head, including acute and chronic increases in aortic pressure (present study; Ref. 28) and acute elevations in intracranial pressure (15, 27). Because the basilar artery is a major site of resistance in the cerebral circulation (11), then it might be expected that hindlimb unloading-induced elevations in arterial pressure would increase the distension of the basilar artery and provide a stimulus for smooth muscle hypertrophy. To test this hypothesis, circumferential stress was calculated after acute (10 min) and chronic (14 day) hindlimb unloading. The results indicate that basilar artery circumferential stress is elevated acutely but that the structural remodeling that occurs with chronic unloading (i.e., increased wall thickness and decreased intraluminal CSA) serves to normalize this stress (Fig. 7). Therefore, these data are consistent with the notion that the smooth muscle hypertrophy that occurs in intracranial arteries serves to attenuate pressure elevations in cerebral arterioles and capillaries and, thus, diminish the risk of cerebral edema.

A functional consequence of the change in cerebral arterial structure may be an alteration in vascular responsiveness of cerebral vessels. For example, Geary et al. (13) have shown that cerebral arteries from HU rats have an increased myogenic responsiveness to changes in intraluminal pressure. Thus it appears that hindlimb unloading causes an elevation in cerebral artery circumferential stress, which increases the loading or stretch of the vascular smooth muscle and induces an autoregulatory myogenic contraction. Over time, the maintenance of an elevated tone purportedly produces arterial smooth muscle hypertrophy and correspondingly alters vascular responsiveness (e.g., enhanced myogenic autoregulation). A similar sequence of events has been proposed by Folkow (12). In examining spontaneously hypertensive rats, Folkow postulated that hypertrophied arterial smooth muscle increases myogenic and agonist-mediated vasoconstrictor responsiveness and, perhaps, decreases vasodilator responsiveness. The relationship between elevated transmural pressure, smooth muscle hypertrophy, and enhanced vasoconstrictor responsiveness has been experimentally substantiated in the hypertension literature (17, 18, 25, 30, 34). Thus the present findings of basilar artery hypertrophy and the report of hindlimb unloading-induced increases in myogenic autoregulation (13) are similar to the structural and functional arterial adaptations induced by hypertension.

It is possible that the above-described arterial remodeling in HU rats may also occur in humans when cerebral arterial pressure is elevated, such as during head-down-tilt bed rest and exposure to microgravity. For example, cerebral blood flow velocity (21), intracranial pressure (29), and extracranial pressure (35) increase during head-down tilt. Similarly, cerebral blood velocity temporarily increases in space (39) and parabolic flight (2), and indirect measurements made on a single cosmonaut (8) and monkeys (20) indicate that intracranial pressures are higher in microgravity. Thus simulated and real microgravity have been shown to produce cephalic fluid shifts (13, 16, 39-41) that appear to increase cerebral artery pressure.

Although direct experimental evidence is lacking in humans, indirect evidence suggests that the cephalic fluid shifts induced by head-down tilt and microgravity may also result in alterations of cerebral artery morphology and, correspondingly, vascular responsiveness. Zhang et al. (42) studied the effects of head-down-tilt bed rest on cerebral hemodynamics by using lower body negative pressure as a gravitational stress. When middle cerebral artery blood flow velocity was plotted against mean arterial pressure changes during progressive lower body negative pressure, blood flow velocity for a given arterial pressure was lower after bed rest. These investigators concluded that there was an impairment of cerebral autoregulation after bed rest, which may compromise cerebral blood flow during an orthostatic stress. These observations of a potential maladaptation in cerebral autoregulation are consistent with the findings of Buckey et al. (3), where astronauts returning from 9-14 days in space were asked to perform a 10-min stand test (3). Nine of the fourteen astronauts tested could not complete the stand test, and of these nine nonfinishers, two were normotensive. These two normotensive nonfinishers are consistent with the notion that a cephalic fluid shift may enhance cerebral arterial autoregulation and contribute to the etiology of orthostatic intolerance after spaceflight.

Unlike the basilar artery, alterations in the vascular smooth muscle layer of resistance arteries from the mesentery and spleen were not observed. The lack of remodeling in these vessels may reflect an absence of change in transmural pressure with hindlimb unloading in these vascular beds, since these tissues would be at or near the hydrostatic indifference point in the rat. However, even in the absence of a structural remodeling, previous work has demonstrated a compromised ability of these vascular beds to constrict. For example, during exercise, normal reductions in splenic and mesenteric blood flow are absent in 14-day HU rats (28). This apparent deficit in vasoconstriction could be due to decreases in sympathetic nerve activity or a diminished vasoconstrictor responsiveness of the arteries. In support of the latter possibility, Overton and Tipton (32) reported that elevations in mesenteric vascular resistance during infusions of phenylephrine and norepinephrine were attenuated in head-down and horizontally suspended rats. The decrease in vasoconstrictor response in both the head-down and horizontally suspended rats indicates that a change in fluid pressure is not the stimulus for adaptation in this tissue. In addition, the present study suggests that the decrease in vasoconstrictor responsiveness does not result from smooth muscle atrophy.

In conclusion, the findings of the present study demonstrate that media layer CSA and thickness are elevated as a result of smooth muscle hypertrophy in the basilar artery of 14-day HU rats. This adaptation is consistent with the hypothesis that a chronic elevation in cephalic arterial pressure increases the distension of the basilar artery, and the corresponding increase in circumferential stress serves as the mechanical stimulus for the smooth muscle hypertrophy. Furthermore, this hypertrophy may alter the autoregulatory responsiveness of cerebral arteries (13). If similar adaptations occur in the cerebral vasculature of humans undergoing prolonged bed rest or spaceflight, these arterial adaptations may compromise cerebral blood flow when fluid pressure is abruptly altered, such as during the assumption of the upright posture in a normal gravitational environment (1, 3, 42).