INTRODUCTION

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ORTHOSTATIC INTOLERANCE IS common in astronauts after prolonged spaceflight. This microgravity-induced orthostatic intolerance appears to be due, in part, to a greater postural decrease in cardiac output and stroke volume (3). Changes in cardiac output can occur for two reasons: via the Frank-Starling mechanism through changes in venous filling pressure or through altered contractility of the heart. The observed changes in cardiac output after exposure to microgravity cannot be fully explained by changes in contractility or heart rate alone because orthostatic heart rate tends to be higher postflight (3). Therefore, changes in filling pressure regulated by systemic venous capacitance function may be important determinants of cardiac output after exposure to microgravity.

In terms of capacitance, the largest vascular region is the splanchnic venous bed (14). Animal studies have shown that splanchnic veins are highly responsive to baroreceptor-mediated sympathetic stimulation, as would occur on assuming the upright posture (5, 6, 16). These studies have demonstrated that sympathetic stimulation of splanchnic veins and venules will cause them to constrict, leading to significant volume shifts out of this vascular bed. Consequently, changes in splanchnic venous capacitance can have large effects on venous filling pressure. Capacitance changes can occur through changes in both vessel compliance and unstressed vascular volume. One way to experimentally assess these changes is to examine changes in the pressure-diameter relationships of individual vessels, particularly in the splanchnic regions of the body.

Several studies have shown that simulated microgravity in rodents results in a decreased arterial contractile response to sympathomimetics such as norepinephrine (NE; Refs. 4, 12). One study has also demonstrated an attenuated contractile response of the vena cava to NE after exposure to simulated microgravity (15). Thus the response of veins to sympathomimetics may be altered by microgravity.

The purpose of this study was to determine the effects of simulated microgravity on the pressure-diameter relationships of rat mesenteric small veins. We used rat hindlimb unweighting (HLU) as a model for microgravity because it has been shown to mimic the effects of spaceflight on the cardiovascular system (10). We constructed pressure-diameter curves of isolated mesenteric small veins from control and HLU rats, both before and after the vessels were stimulated with NE. Changes in venous tone after HLU would be evident as changes in slope (compliance) and/or intercept (unstressed vascular volume) of the pressure-diameter curve. We hypothesized that the pressure-diameter curves of vessels from HLU rats would be shifted to higher intercepts and would have lower slopes than those of vessels from control rats. Additionally, we hypothesized that the vessels from HLU rats would be less reactive to NE than vessels from control rats.

	METHODS

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Animals. This protocol was approved by the Institutional Animal Care and Use Committee at Johns Hopkins University School of Medicine. Eight male Sprague-Dawley rats underwent HLU for 21 days. Rats were maintained at a suspension angle of ~30°. The animals were placed in a jacket that was connected to a piece of tubing. The tail was attached to the tubing with adhesive material. The tubing was then attached to a crossbar and swivel apparatus across the top of the cage. Rats had full mobility in the cage using their forelimbs with ad libitum access to rat chow and water. Ten control rats were maintained in a normal cage environment. After the 21-day treatment period, rats were anesthetized with 120 mg/kg ketamine and 2 mg/kg acepromazine, intramuscularly. Body temperature was maintained at 37°C by placing animals on a heated tray.

Vein isolation. A midline incision was made in the abdominal wall, and a small portion of the small intestine was exteriorized. At this point, all vessels and nerves remained intact. The tissues were constantly bathed in 37°C Krebs buffer equilibrated with 95% O₂-5% CO₂. A small mesenteric vein that branches off from the first-order venules was cannulated with a 30-gauge needle that had a hole cut in the middle of it (Fig. 1). The vessel was tied to the needle with two 5-0 silk sutures. Once the vessel was secured to the needle, the vessel was dissected free of the other vessels and the mesentery. It was then placed in a small chamber and suffused with 37°C Krebs buffer that was bubbled with 95% O₂-5% CO₂.

View larger version (89K): [in this window] [in a new window]   Fig. 1.   A: control vessel in vivo. B: small vessel from A after cannulation with 30-gauge needle.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Experimental set-up. Once the vessel is cannulated and dissected out of the mesentery, as shown in Fig. 1B, it is placed in a small chamber. Pumps are used to circulate warm buffer solution through that chamber. Each end of the needle is then connected to a small, fluid-filled pressure reservoir. The height of these pressure reservoirs is adjusted to change the pressure inside the vein. The resulting change in diameter is then measured.

Experimental protocol. The pressure-diameter relationships of 14 vessels from 10 control rats and 12 vessels from 8 HLU rats were obtained before and after the addition of two cumulative doses of NE (10⁶ M and 10⁴ M). The pressure in the vein was increased in 2-cmH₂O steps from 0 to 12 cmH₂O. The pressure was maintained at each step for 60 s or until the diameter of the vessel stabilized. After stabilization at 12 cmH₂O, pressure in the vessel was then decreased to 0 cmH₂O, and the vessel was allowed to return to its baseline diameter. NE was then added to the suffusate and allowed to circulate for ~5 min. The pressurization procedure was then repeated.

Data analysis. Plots of pressure vs. diameter squared were constructed. Changes in diameter squared were used because they more closely represent the volume of a fixed-length vessel (volume is proportional to length × diameter squared). The slope of the pressure-diameter squared relationship is a measure of the compliance of the vessel. A steeper slope implies a more compliant vessel. The y-intercept of the pressure-diameter squared relationship approximates the unstressed vascular volume of the vessel. A smaller intercept implies a decreased unstressed vascular volume. The data were fitted to a line using the method of least squares, and the slopes and intercepts were determined for each vessel at each concentration of NE. For all of the vessels, the pressure-diameter squared relationship was nonlinear below 4 cmH₂O. The data points at 0 cmH₂O and 2 cmH₂O were not used in fitting the data to a line. Thus the y-intercept of each relationship is an extrapolation of the linear portion of each pressure-diameter squared curve. In fact, the unstressed volume of a vessel is defined as "a virtual volume estimated by extrapolating a linear portion of the capacitance relation to zero transmural pressure" (13).

	RESULTS

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HLU rats lost ~9% of their body weight during the 21-day HLU procedure, whereas control rats gained ~13% of their body weight. Consequently, the HLU rats were significantly lighter than the control rats when they were weighed at the time of experimentation (387 ± 9 vs. 446 ± 11 g, P < 0.01). Qualitatively, HLU rats had less fat surrounding the mesenteric blood vessels than did control rats. Additionally, the diameters of the small mesenteric veins from HLU rats, measured at 0 cmH₂O after the vessel was cannulated, were significantly larger than in control rats (523 ± 83 vs. 374 ± 29 µm, P < 0.05).

Figure 3 shows examples of actual data collected from one control and one HLU vein. For control veins, exposure to NE shifted the pressure-diameter squared curves downward, such that, at any given pressure, the vein had a smaller diameter squared. In the control vein, 10⁶ M NE caused ~6% change in diameter squared, and 10⁴ M NE caused ~19% change in diameter squared over the entire range of pressures. Veins from HLU animals did not exhibit the same reactivity to NE. In these veins, 10⁶ M NE caused ~0.5% change in diameter squared and 10⁴ M NE caused ~3% change in diameter squared over the entire range of pressures. The differences in percent change between the two groups were significantly different at both concentrations of NE (P < 0.05).

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Representative pressure-diameter squared relationships from one control (A) and one hindlimb-unweighted (HLU; B) vein before and after exposure to norepinephrine (NE). Note that in the control vessel, NE causes a leftward shift in the pressure-diameter squared curve. NE has no effect on the curve from the HLU vessel.

The average pressure-diameter squared relationships for veins from both control and HLU animals are shown in Fig. 4. The slopes of the curves and the vein diameter squared at a pressure of 0 cmH₂O (D₀²) are presented in Fig. 5. In veins from control rats, 10⁴ M NE caused a significant decrease in D₀² (P < 0.05). 10⁶ M NE caused a small decrease in D₀² in control vessels, but this decrease was not significant. Neither concentration of NE had a significant effect on D₀² in small veins from HLU rats. Additionally, D₀² was higher in HLU veins than in control veins for all NE concentrations (P < 0.05). NE had no effect on the slopes of the pressure-diameter squared curves from control or HLU veins, and there were no differences between the two groups in any slope value.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   The average pressure-diameter squared relationships for control and HLU veins before and after exposure to NE. The gray portion of the line represents the extrapolated values below 4 cmH2O. In the control vessels, 104 M NE causes a significant decrease in vein diameter squared at pressure of 0 cmH2O (D02; P < 0.05). Curves from the HLU rats are shifted to larger D02 values (P < 0.05), and NE has no effect on the D02 of these vessels. R2 values for control curves are 0.989, 0.967, and 0.991 for no NE, 106 M NE, and 104 M NE, respectively. R2 values for HLU curves are 0.989, 0.989, and 0.947 for no NE, 106 M NE, and 104 M NE, respectively. The slope and D02 values are displayed in Fig. 5.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   D02 (A) and slopes (B) of pressure-diameter relationships from control and HLU small mesenteric veins. Control vessels exhibited a significant decrease in diameter after exposure to 104 M NE (#P < 0.05). HLU vessels had larger diameters than control vessels for all concentrations of NE (*P < 0.05). Neither HLU nor NE had any effect on slope. Values are means ± SE.

	DISCUSSION

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The main finding of this study is that, after 21 days of HLU, rat small mesenteric veins are not as reactive to NE as veins from control rats. This decreased reactivity is evidenced by the lack of change in vessel diameter in response to NE. NE shifted the pressure-diameter curves of mesenteric veins from control rats toward smaller diameters (D₀²), but NE did not have a significant effect on the D₀² of vessels from HLU rats. Additionally, the D₀² of HLU vessels were larger than control vessels. There were no differences in slopes between the vessels or in response to NE.

A shift in D₀² indicates a change in unstressed vascular volume, which is a key factor, along with compliance, in regulating vascular capacitance. Changes in vascular capacitance can contribute to changes in venous filling pressure and, therefore, in cardiac output (17).

Our results could have profound implications for understanding the pathophysiology of microgravity-induced orthostatic intolerance. The upward shift of D₀² in the HLU vessels implies that the unstressed vascular volume is ~40% larger after HLU. Additionally, after HLU, the vessels no longer constricted, implying that the unstressed vascular volume and compliance were unchanged after exposure to NE. Both of these factors indicate that venous return, especially during a sympathetic stimulus (e.g., orthostasis), could be attenuated after exposure to microgravity, leading to the observed exaggerated fall in cardiac output and stroke volume.

Most previous studies investigating the mechanisms for microgravity-induced orthostatic intolerance have focused on decrements in the vasoconstrictor properties of arteries (4, 9, 12). We believe that changes in the venous circulation may be at least as important as changes in the arterial circulation. In fact Buckey et al. (3), in their study on 14 astronauts postflight, concluded that "the principal postflight change in hemodynamic response to orthostatic stress was a greater postural decrease in stroke volume, presumably related to reduced cardiac filling." Additionally, recent data from our laboratory (1) suggest that, despite a greater increase in total peripheral resistance, rats subjected to orthostatic stress after 10-14 days of HLU have an exaggerated decrease in stroke volume and cardiac output. This points to the importance of venous filling pressure in this response.

Purdy et al. (12) studied the contractility of the jugular and femoral veins from rats after 20 days of HLU and found no differences in the contractile responses to NE or KCl. This points to the importance of the specific vascular bed studied. We chose to study the splanchnic venous bed because it is the largest vascular bed in terms of capacitance (14). Also, many animal studies have shown that the splanchnic bed is very responsive to baroreceptor and sympathetic stimulation, pointing to this vascular bed as a primary source of blood volume changes (2, 6, 16). We studied small mesenteric veins because >50% of the intestinal blood volume is in the venules and small veins (5, 18).

Sayet et al. (15) studied the influence of HLU and spaceflight on the contractile responses of the rat vena cava. In this study, HLU and spaceflight decreased the sensitivity of rat vena cava to NE, which was related to a decreased affinity of NE for _1B-adrenoceptors. This points to a possible mechanism underlying the observed lack of reactivity of the small mesenteric veins to NE after HLU. Because _1B-adrenoceptors are the primary receptors mediating constriction of venules (7, 8), a desensitization or downregulation of these receptors could lead to a defect in the venoconstriction response. This defect in the _1B-adrenoceptor response was further clarified by a study investigating the effects of HLU on the norepinephrine-induced calcium signal (11). This study showed that HLU decreased the norepinephrine-induced release of calcium from intracellular stores.

In this study, we did not observe any changes in the slopes of the pressure-diameter relationships either after HLU or with exposure of the vessel to NE, indicating that compliance was not affected. Several previous animal studies have observed small but significant changes in the slopes of pressure-diameter curves from mesenteric venules with sympathetic and baroreceptor stimulation (6, 16). There are two potential explanations for this discrepancy. The first explanation is that the number of vessels tested in the previous studies was larger than in this study. In these previous studies, the changes in unstressed vascular volume were much larger than changes in compliance, and a large number of vessels needed to be tested before the compliance changes reached statistical significance. Indeed, in the current study, the slope of the control vessels was slightly lower after exposure to 10⁴ M NE (Fig. 5), but this result was not statistically significant. Additionally, the previous studies were conducted in vivo, and sympathetic activity was stimulated through the baroreflex. That type of protocol may have more of an effect on compliance than the in vitro protocol that we used in this study. Nevertheless, previous whole animal studies by Shoukas and Sagawa (17) demonstrated that changes in capacitance occur primarily through changes in unstressed volume.

In conclusion, this study shows that HLU has pronounced effects on the pressure-diameter relationships of rat mesenteric small veins. HLU shifts the pressure-diameter relationship to higher diameters and attenuates the ability of the veins to constrict in response to NE, implying changes in the capacitance characteristics of the mesenteric venous bed. Given the influence that mesenteric venous capacitance has on venous filling pressure, these changes can partially explain the exaggerated postural decrease in stroke volume and cardiac output observed after exposure to microgravity.