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Am J Physiol Heart Circ Physiol 290: H941-H947, 2006. First published September 23, 2005; doi:10.1152/ajpheart.00645.2005
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Effect of erythrocyte aggregation at normal human levels on functional capillary density in rat spinotrapezius muscle

Sangho Kim,1 Aleksander S. Popel,2 Marcos Intaglietta,1 and Paul C. Johnson1

1Department of Bioengineering, University of California, San Diego, La Jolla, California; and 2Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland

Submitted 15 June 2005 ; accepted in final form 19 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previous studies have shown that functional capillary density (FCD) is substantially reduced by erythrocyte aggregation. However, only supranormal levels of aggregability were studied. To investigate the effect of erythrocyte aggregability at the level seen in healthy humans, the FCD of selected capillary fields in rat spinotrapezius muscle was determined with high-speed video microscopy under normal (nonaggregating) conditions and after induction of erythrocyte aggregation with Dextran 500 (200 mg/kg). To examine shear rate dependence, the effect was studied both at normal and reduced arterial pressures (50 and 25 mmHg), the latter achieved by short periods of hemorrhage. In a separate study, volume flow was determined in arterioles (52.1 ± 3.7 µm) under the same conditions. Before Dextran 500 infusion, FCD fell to 91% and 76% of control values, respectively, when arterial pressure was reduced to 50 and 25 mmHg. After Dextran 500 infusion, FCD was 96% at normal arterial pressure and fell to 79% and 37% of normal control values at 50 and 25 mmHg. All FCD values were significantly lower after dextran infusion. FCD reduction after lowering arterial pressure or dextran infusion appeared to be due to plasma skimming rather than capillary plugging. Reduction of FCD by dextran at reduced pressure was compensated by increased red blood cell flux in capillaries with red blood cell flow. We conclude that the level of aggregability seen in healthy humans is an important determinant of FCD only at reduced arterial pressure.

hemodynamics; red blood cell aggregability; in vivo blood rheology; in vivo microscopy; Dextran 500

FUNCTIONAL CAPILLARY DENSITY (FCD) is a measure of the number of capillaries with red blood cell flow in a defined tissue region. FCD is an important indicator of microcirculatory function as it reflects the diffusion distance for nutrients and waste products between flowing blood and the parenchymal cells. It also reflects the surface area available for exchange of fluid and solutes. FCD has been shown to decrease with reduction of arterial pressure (17) and with vasoconstriction (17, 30). However, whole organ studies have also shown that the surface area available for exchange of fluid and solutes (presumably the capillary bed primarily) can change independently of volume flow of blood (8). On this basis, it has been suggested that a mechanism separate from that for volume flow regulation exists for regulating the number of capillaries with flow.

The importance for normal physiological function of maintaining an adequate level of FCD is evident from the above considerations. Its importance is emphasized by studies (14) of severe hemorrhagic shock that have shown a high degree of correlation between FCD of the skeletal muscle layer of the hamster skinfold preparation and survival of the animal. In those studies, a decrease in FCD of 50% or more below control levels after transfusion of autologous blood indicated a poor prognosis for survival.

Red blood cell aggregation has been reported to have a significant effect on FCD. Mchedlishvili et al. (19) found that administration of Dextran 500 reduced the FCD in the rabbit cerebral cortex dramatically, from ~90% to ~10%. Vicaut et al. (32, 33) also found substantial reductions (>30%) in FCD in rat hearts and cremaster muscle after the infusion of Dextran 480. Both studies indicated that red blood cell aggregation can reduce FCD to a level that would increase the diffusion distance for nutrients and waste products and reduce available surface area for exchange of fluid and solutes. In addition, the reduction of FCD reported by Mchedlishvili et al. (19) would not have been compatible with survival in the shock study reported by Kerger et al. (14). However, the plasma concentrations of the dextrans employed in those studies were very high, two and a half to eight times greater than that required to elevate red blood cell aggregation in the rat to the level seen in healthy humans (4, 5).

Given the importance of FCD as an index of microvascular function, information on its status at normal levels of aggregation is needed. The current study was designed to determine whether red blood cell aggregation at levels normally seen in the human significantly affects FCD. Because the degree of red blood cell aggregation is inversely related to the shearing force, the study was carried out at normal and reduced arterial pressures. Red blood cell velocity and flux in capillaries were also determined at reduced arterial pressures. In a parallel study, we determined the effect of arterial pressure on flow in arterioles after the same protocol.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal preparation. Twenty-nine male Wistar-Furth rats (22 for FCD and 7 for volume flow measurements) weighing 140–350 g were used in this study. Animal handling and care were provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996). The study was approved by the University of California, San Diego, Animal Subjects Committee. The animal preparation used in the present study has been described previously (5, 15), and the reader is referred to those studies for a more complete description.

The rats were anesthetized with an intraperitoneal injection of 50 mg/kg pentobarbital sodium (Abbott). Additional anesthetic was administered intravenously throughout the experiment as needed. The animal was placed on a heating pad to maintain a body temperature of 37°C during surgery. A tracheal tube (PE-205) was inserted to provide a clear airway, the jugular vein was catheterized for administration of anesthetic or Dextran 500 during the course of the experiment, and the carotid artery was catheterized to measure arterial pressure and withdraw blood as needed for pressure reductions. All catheters were filled with a solution of heparinized saline (30 IU/ml) to prevent clotting. The rat spinotrapezius muscle was exteriorized and prepared for intravital microscopy as described in previous studies (5, 15). A temperature probe was placed beside the muscle to monitor temperature, which was maintained at 37°C during the experiment by a heating element attached to the animal platform.

Arterial pressure and blood rheological properties. As described in previous studies (5, 15), arterial pressure was continuously recorded with the use of a physiological data acquisition system (MP 100 System; Biopac Systems, Goleta, CA). The pressure data were transferred and stored in a microcomputer during the experiments. A blood sample of ~0.1 ml was withdrawn from the arterial catheter for measurement of hematocrit and red blood cell aggregation. Hematocrit was determined after centrifugation with a microhematocrit centrifuge (Readacrit, Clay Adams). The degree of red blood cell aggregation was assessed from duplicate measurements on a 0.035-ml blood sample with a photometric rheoscope (Myrenne Aggregometer; Myrenne, Roentgen, Germany). The aggregation index (M) obtained in the present study was based on the 10-s setting. All these measurements were taken during control as well as after infusion of Dextran 500.

Adjustment of red blood cell aggregability. Experiments were performed on animals under both normal (nonaggregating) conditions and after infusion of Dextran 500 (total 200 mg/kg body wt) to induce red blood cell aggregability in the range previously reported for healthy humans (M = 12–16) (18, 37). The dextran (average molecular mass 500 kDa; Pharmacia, Uppsala, Sweden) was dissolved in saline (6%) and infused in increments of 50 mg/kg over the course of 2–3 min. On the basis of a total blood volume of 5.5% of body weight (3), this represents a 6% increase in blood volume and a plasma dextran concentration of ~0.6% (wt/vol). Hematocrit and aggregation index values were determined ~10 min after Dextran 500 infusion. There was no discernable adverse reaction (e.g., visible swelling of the limbs) to the Dextran 500 infusion in the rats used in the study.

Experimental protocol. Two separate series of experiments on FCD and microcirculatory volume flow, respectively, were conducted. For the FCD measurements, an intravital microscope with a high-speed video camera (15) was used, whereas the volume flow measurements utilized a dual-slit technique (34) and an image shearing unit (3).

In both series of experiments, arterial pressure was reduced by removing blood via the carotid artery catheter into a heparinized syringe at the rate of ~2 ml/min. The amount of blood withdrawn to reach 50 mmHg was 1–3 ml and to reach 25 mmHg was 2–5 ml, depending on the body weight of the animal. Reduced arterial pressure was maintained within a range of ±10% of the desired value for ~10 min for image recordings or velocity and diameter measurements. The withdrawn blood was reinfused over a period of 0.5–2 min, and the arterial pressure was monitored until it returned to a steady-state value before the procedure was repeated.

FCD determination. To determine FCD, the microscope objective was focused on a planar region of the capillary network, and the high-speed video camera was used to record the red blood cell movements in the capillaries for a period of at least 10 s. A field that had a large vessel or readily distinguishable vascular network geometry was chosen for the origin, enabling us to return to that field after recording other fields. During the control period, recordings were obtained from 10 to 12 microscopic fields adjacent to the original field. For each field, the objective focus level was adjusted to visualize as many capillaries as possible. A handdrawn map of all capillaries that could be observed by vertical focus adjustment was made for each field. A frame rate of 250–750 frames/s was used and was adjusted to maximize image clarity for plasma gaps and red blood cell movements in the capillaries. In general, with higher frame rates, it was easier to distinguish individual red blood cell movements in capillaries, whereas we could get information on larger numbers of capillaries with lower frame rates. FCD was estimated by counting the number of capillaries that contained moving red blood cells during a 10-s observation period as used in previous studies (10, 33). The FCD for each animal was measured at normal and reduced arterial pressures (50 and 25 mmHg) without dextran and then repeated after dextran infusion. During reduced arterial pressures, we recorded the microscopic fields starting from the origin for ~10 min, at which time arterial pressure began to rise. As a result, two to four fewer fields were scanned for reduced flow compared with the number scanned for the control period.

To estimate the total number of capillaries in the observed fields, a vasodilator (papaverine; Sigma, St. Louis, MO) was dissolved in saline (1 mg/ml), and 0.5 ml of the solution was applied topically over the muscle in three animals. FCD was determined before and after application of the papaverine.

Measurement of red blood cell velocity and flux in capillaries. Red blood cell velocity in functional capillaries was determined from video recordings with a video caliper (model 308, Colorado Video) and a video timer (model VTG, For-A) to record the time required for a red blood cell to travel between two caliper lines separated by a known distance. Capillaries where red blood cell images were well focused were selected for the measurements.

For red blood cell flux determination, capillaries that remained functional during the reduced flow situations were chosen. Red blood cell flux (cells/s) was determined by counting the number of red blood cells passing a caliper line during a fixed time period. The red blood cell flux was measured in the same capillary at the reduced flow situations before and after dextran infusion. Local hematocrit changes during the reduced flow conditions could be determined from the number of cells within the predetermined section.

In the red blood cell velocity and flux measurements, data were obtained from six randomly selected experiments (three each at 50 and 25 mmHg arterial pressures). The measurements were taken in at least 10 capillaries for each condition. Thus a total of 30–34 measurements were used to compare velocity and flux before and after dextran infusion at each level of reduced arterial pressure.

Volume flow determination. Velocity and diameter measurements were made in arterioles (30–70 µm ID). The mean velocity was estimated by using dual-slit velocity and a correction factor of 1.6 (1), whereas the vessel diameter was determined with an image shearing unit. The diameter and velocity for a vessel chosen for the study were measured for a period of 2–3 min at control, at 50- and 25-mmHg arterial pressures before and after dextran infusion as with the FCD determinations. A manual map of the microcirculatory bed guided the repeated measurements of selected vessels. Because we had a stable period of ~10 min at reduced arterial pressure, a maximum of two vessels could be studied for each animal with all six conditions. The order of measurements at 50 and 25 mmHg was varied randomly between experiments. A total of eight volume flow measurements for each condition was obtained. Pseudoshear rate ({gamma}) in the arterioles was calculated from the following equation: {gamma} = V/D [where V is mean velocity of red blood cells (in mm/s) and D is vessel diameter (in mm)].

Statistical analysis. A paired t-test was used to determine differences in experimental and physiological parameters between normal and dextran-treated animals. All of the data are reported as means ± SE. The statistical significance of differences among the three different arterial pressure groups for normal or dextran-treated animals was determined by using a one-way ANOVA test with Scheffé's, Bonferroni, and Tukey tests. All statistical tests were done by using commercially available software packages (SPSS 11.0 for Windows and Prism 4.0 for Windows). For all tests, P < 0.05 was considered statistically significant. All FCD data are expressed as a percentage of the capillaries with flow in the same fields at normal arterial pressure before administration of Dextran 500.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Systemic values. Control arterial pressure was 126 ± 3.7 mmHg and was not significantly different between FCD and volume flow studies or after dextran infusion. Systemic hematocrit (42.0 ± 0.37%) in normal rats was not significantly different from that (41.8 ± 0.34%) after dextran treatment. The index of aggregation for normal rats was negligibly small (0.1 ± 0.05), whereas in dextran-treated rats, it was 14.7 ± 1.02 and 13.5 ± 1.13 in the FCD and volume flow studies, respectively. The aggregation index was not significantly different between the two studies.

FCD. A total of 1,450 functional capillaries (~60–100 capillaries/animal) were studied and 4–12 capillaries were observed in each field. Capillaries were considered functional when they contained moving red blood cells. We seldom saw capillaries with stationary red blood cells. All FCD data are expressed as a percentage of the capillaries with red blood cell flow in the control state. To determine whether there were, in addition, capillaries without red blood cell flow at this time, we applied the vasodilator (papaverine-saline solution) to the muscle surface. In these studies (n = 3), vasodilation increased the number of capillaries with red blood cell flow by 29 ± 1.76%, indicating that a substantial number of capillaries were not perfused with red blood cells in the control state.

Consistent with this observation, after reduction of arterial pressure or infusion of Dextran 500, red blood cell flow was occasionally observed in capillaries that had not been seen previously. These new, functional capillaries were counted in the study and comprised ~9% of the total. Figure 1 shows the effect of changes in arterial pressure and red blood cell aggregability on FCD. Arterial pressure reduction alone significantly reduced FCD. Before Dextran 500 infusion, FCD fell to 91.0 ± 0.97% and 76.1 ± 4.87% of control values, respectively, when arterial pressure was reduced from 126 ± 3.7 to 48 ± 1.4 and 25 ± 1.8 mmHg. Dextran 500 significantly reduced FCD to 95.9 ± 1.61% of normal control values at 124 ± 4.2 mmHg and to 79.4 ± 1.43% and 36.7 ± 6.47% of control levels at 47 ± 1.4 and 24 ± 2.4 mmHg. The reduction in FCD due to Dextran 500 was small but significant (P < 0.05) at normal arterial pressure, and the difference was more pronounced at arterial pressures of 50 (P < 0.005) and 25 mmHg (P < 0.0001).


Figure 1
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  		Fig. 1. Functional capillary density (FCD) with changes in arterial pressure before and after infusion of Dextran 500 to increase red blood cell (RBC) aggregability. Data are normalized to FCD at normal arterial pressure before dextran infusion. *P < 0.05, **P < 0.005, and {dagger}P < 0.0001; n = 7 for normal arterial pressure and n = 6 for reduced arterial pressure.

 
The spatial pattern of FCD reduction appeared either as an absence of red blood cell flow in an individual capillary or in a group of capillaries fed by a precapillary vessel. The prevalence of the second pattern was somewhat greater after dextran infusion. Analysis of results at 25 mmHg showed that only 11% of capillaries were part of the second pattern, but this rose to 45% after dextran. The difference was significant (P < 0.03).

Hemodynamics in capillaries. To determine whether the decrease in FCD after dextran infusion was compensated by an increase in flow, red blood cell velocity and flux were determined in functional capillaries at 50 and 25 mmHg. This analysis was not feasible at normal arterial pressure due to the very small change (4%) in FCD with dextran at that pressure. As shown in Fig. 2A, there was no significant effect on velocity at 50 mmHg, but a significant difference (P < 0.05, 40.5 ± 3.13 vs. 54.3 ± 4.46 µm/s) was seen at 25 mmHg. In Fig. 2B, comparison of red blood cell flux in the same capillaries revealed a highly significant increase at 25 mmHg (P < 0.001, 2.7 ± 0.31 vs. 4.8 ± 0.49 cells/s) after dextran infusion, whereas we found no significant difference at 50 mmHg.


Figure 2
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  		Fig. 2. A: RBC velocity in functional capillaries at reduced arterial pressure before and after Dextran 500 infusion. B: RBC flux in same capillaries as in A. *P < 0.05; **P < 0.001.

 
Volume flow. The diameter of arterioles selected in the present study (n = 8) was 52.1 ± 3.68 µm. Neither arterial pressure reduction nor Dextran 500 infusion caused a significant change in arteriolar diameter. In normal rats, control volume flow in arterioles was 28.9 ± 5.12 nl/s and, as shown in Fig. 3, fell to 23.1 ± 2.42% of control at 48 ± 2.9 mmHg and 9.4 ± 1.90% of control at 28 ± 2.2 mmHg. After dextran infusion, volume flow in arterioles was 88.0 ± 7.84% of control at 129 ± 4.1 mmHg and fell to 20.9 ± 2.34% at 52 ± 2.6 mmHg and 8.5 ± 0.81% at 27 ± 1.2 mmHg. While volume flow values at each pressure level were lower after Dextran 500 infusion, the differences were not statistically significant.


Figure 3
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  		Fig. 3. Volume flow in arterioles (n = 8) before and after dextran infusion with change in arterial pressure. Data normalized to values at normal arterial pressure before dextran infusion.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Principal findings. The main purpose of the present study was to determine the effect of red blood cell aggregation at the level seen in healthy humans on FCD. Because aggregation is dependent on shear rate, the study was performed at control and at reduced arterial pressures. The levels of aggregability induced in our FCD and volume flow studies, 14.7 ± 1.02 and 13.5 ± 1.13, respectively, are in the range reported for healthy humans, 12.4 (18) and 16 (37). In the latter study, only data in the hematocrit range of our animals were used. The principal finding of this study is that in the rat, inducing red blood cell aggregation to the degree seen in the human causes a small but significant decrease in FCD at normal arterial pressure and a much more pronounced effect as arterial pressure is reduced to 50 and 25 mmHg. This finding is consistent with our hypothesis. Arterial pressure reduction alone also reduced FCD significantly. Before examining these findings in detail, we will consider other known effects of red blood cell aggregation in the microcirculation.

Effects of red blood cell aggregation on microcirculatory function. The effects of red blood cell aggregation on total vascular resistance are complex and appear to depend on several factors that act in opposition to each other. As shown in small glass tubes, reducing flow rate increases aggregate formation, which increases effective viscosity but at the same time increases axial migration, which causes formation of a cell-poor, low-viscosity layer near the wall (26, 27). As a consequence, the net effect of aggregation is very dependent on local factors such as vascular network topology (6, 7).

Studies in the cat gastrocnemius muscle have shown that venous vascular resistance is inversely related to flow rate and that this effect is due to the shear rate dependence of red blood cell aggregation, which is a normal feature of cat blood (7) and blood of other athletic mammalian species but not sedentary species (25). This feature has been shown to be important in maintaining capillary hydrostatic pressure relatively constant during large changes in skeletal muscle blood flow (7). Studies on the spinotrapezius muscle of the rat with red blood cell aggregation induced by Dextran 500 have shown that this effect is due at least in part to a blunting of the velocity profile in venules at low shear rates (4). The formation of aggregates whose size is dependent on shear rate has also been demonstrated in venules of this vascular bed (5). Whereas aggregation normally leads to increased axial migration and formation of a cell-poor layer near the vessel wall as shown by in vitro studies (9, 24, 26, 27), this effect is mitigated in the venular network by frequent infusion of red blood cells from tributary vessels (6).

Red blood cell aggregation has been reported to have a very different effect in the arterial network, with the formation of a cell-poor layer at the vessel wall and reduction of wall shear stress being the greater effect (2, 36). As a consequence, increased aggregation over a period of days leads to reduced nitric oxide formation by the arterial vessels (2).

The existence of opposing effects of aggregation can lead to either a net increase or decrease in total vascular resistance (32). However, at high plasma concentrations, Dextran 500 has been shown to increase total vascular resistance very significantly. Mchedlishvili et al. (20) reported a flow reduction of ~50% in arterioles of rat mesentery together with a similar increase of arterial pressure after elevating Dextran 500 to 4.8% in plasma. Durussel et al. (11) reported a reduction of >50% in red blood cell velocity in venules as well as in arterioles in rat mesentery and cremaster with a Dextran 500 concentration of 4.1% in plasma. These dextran concentrations are seven to eight times greater than those used by Bishop et al. (4, 5, 6) and in the present study. Bishop and coworkers did not find a significant increase of arterial pressure in the rat with this dextran concentration, nor did we. In addition, we did not find a significant decrease in flow although a downward trend in our study (Fig. 3) was noted. Thus it appears that red blood cell aggregation at the level normally seen in humans has, at most, a slight effect on total vascular resistance in rat muscle.

Effect of arterial pressure reduction on FCD. On the basis of studies (11, 19, 20, 32, 33) in other laboratories, albeit at higher levels of aggregation, we hypothesized a reduction in FCD would occur at normal human levels of aggregation. As shown in Fig. 1, we demonstrated that arterial pressure reduction alone caused a significant reduction in FCD. This finding is consistent with previous findings in the hamster skinfold preparation during hemorrhagic hypotension (14). Acute local reduction of arterial pressure or vasoconstriction can produce the same effect (17, 30). In the absence of red blood cell aggregation, it would be expected that this is a reflection of the mechanical properties of an individual formed element passing through a narrow region in the capillary and that red blood cells would be present in the capillary lumen. However, in our study, stationary red blood cells were seldom observed in capillaries. Similarly, reduction of FCD during hemorrhage in the hamster skinfold preparation (14, 31) is seldom accompanied by stationary red blood cells in capillaries (A. G. Tsai, personal communication, March 30, 2005). These observations suggest that mechanical obstruction of red blood cells in the capillaries is not the principal mechanism by which FCD is reduced in rat muscle and hamster cheek pouch. In support of this possibility, a theoretical analysis based on the mechanical properties of the red blood cell indicates that localized reduction of luminal diameter from 7 to 4 µm would retard but not arrest movement of a human red blood cell (29). The limiting diameter for passage of a human red blood cell (diameter = 7.8 µm) is 2.8 µm (31) and would be less for the smaller rat erythrocyte (diameter = 6.8 µm). Considering the high elastic modulus of the capillary (22, 31), it seems unlikely that passive changes in luminal diameter are large enough to obstruct flow during reduction of arterial pressure or flow.

The most likely explanation for reduction of FCD in our preparation is plasma skimming at bifurcations in the capillary and precapillary network. This phenomenon is one of the more obvious features of microcirculatory networks. At a branch point where flow is unequal in the two daughter branches, extensive studies have shown that red blood cell flow into the branch with higher flow is favored (13, 16).

Groom et al. (12) have reported that spatial heterogeneity among capillaries substantially increases as flow falls. This observation is consistent with the concept of plasma skimming because small changes in the pressure differential between adjacent capillaries could lead to a shift between red blood cell flow and non-red blood cell flow in adjacent capillaries. In our study, the spatial pattern of decrease in FCD with arterial pressure reduction alone was typically absence of red blood cells from one capillary, while red blood cell flow continued in neighboring capillaries.

Effect of red blood cell aggregation on FCD. Current information on the effects of red blood cell aggregation on FCD is based on previous studies (11, 19, 20, 32, 33) where concentrations of Dextran 500, 480, or 70 in plasma were two and a half to eight times higher than in the present study. Whereas in those studies plasma viscosity would also have increased, the latter is apparently not a factor because Dextran 70 did not alter FCD when troxerutin was administered to prevent aggregation (33). A limitation of the previous studies is that such high dextran concentrations cause a degree of red blood cell aggregability beyond the levels seen in healthy humans and possibly beyond human disease states. In those studies (19, 32), at normal arterial pressure, FCD was reduced by 35% to 90%, but in our study, the corresponding change, although significant, was only 4%. Mchedlishvili et al. (19, 20) reported that capillaries without flow were filled with closely packed red blood cells. This phenomenon was not seen in the present study. The difference is probably a reflection of the difference in dextran concentration employed, which led, in turn, to a difference in the mechanism of FCD reduction.

To maintain mass balance of red blood cells, reductions in FCD with elevated aggregability at each level of arterial pressure, as seen in our study, should be accompanied by proportionate increases in red blood cell flux. We considered the mass balance for red blood cells in capillary networks before and after Dextran infusion using volume flow, FCD, and red blood cell flux changes. The analysis was feasible only at reduced arterial pressures where larger changes were seen. After Dextran infusion, mean volume flow was reduced by 10% at both 50 and 25 mmHg. FCD fell by 13% and 52%, and red blood cell flux increased by 6% and 78% after dextran infusion at 50 and 25 mmHg. After the changes in FCD and red blood cell flux are combined, capillary flow fell by 8% and 14% after Dextran infusion at the two pressures. According to this calculation, there is only a minor discrepancy in mass balance for red blood cells of ~2% and 4%, respectively, at 50 and 25 mmHg compared with the measured values of volume flow. This calculation suggests that the reduction in FCD is effectively compensated by increased red blood cell flow in functional capillaries. We note, however, that the increase in red blood cell velocity shown in Fig. 2A accounts for only about one-third of the increase in red blood cell flux shown in Fig. 2B. From these data, it can be calculated that mean red blood cell concentration in the capillary flow stream increased from 6.67 to 8.84 cells/100 µm. This finding indicates that a significant fraction of plasma flow at this point may be through capillaries devoid of red blood cells and fits with the observation that stationary red blood cells were seldom seen in the capillary bed. Because application of a vasodilator increased the number of capillaries with red blood cell flow, plasma skimming may also be present under control conditions. Our estimate of the fraction of capillaries with flow under control conditions (78%) is identical to one previously obtained in this muscle by using an anatomical determination of the total number of capillaries (28).

FCD changes in relation to pseudoshear rate in arterioles are shown in Fig. 4. The mean pseudoshear rate in our study is ~20% lower than that calculated from a previous study (38) in this muscle for arterioles this size, but a wide variability was evident in both studies. This laboratory has previously reported a blunting of the velocity profile and formation of red blood cell aggregates in venules beginning at pseudoshear rates below 70 s–1 (4, 5). Although the pulsatile nature of flow in arterioles may retard aggregate formation, it is of note that the effect of Dextran 500 on FCD also became more apparent at these pseudoshear rates. We also noted in the present study that after dextran infusion, the pattern of decrease in FCD shifted with a greater tendency for two to five adjacent capillaries to be without red blood cell flow. It appears from this observation that an important effect of red blood cell aggregation at normal human levels of aggregability is to cause increased plasma skimming in the precapillary vessels. We speculate that red blood cell aggregates may be present in such vessels and that skimming may be secondary to axial migration of the red blood cell aggregates. With aggregation and reduced pressure in combination, we see the summated result of reduced pressure causing plasma skimming among individual capillaries and aggregation increasing the tendency for plasma skimming in precapillary vessels.


Figure 4
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  		Fig. 4. Normalized FCD values vs. pseudoshear rate in arterioles before and after infusion of Dextran 500. Solid line (R2 = 0.9278) and dotted line (R2 = 0.9487) represent nonlinear fits to data points.

 
The physiological importance of our finding is that normal human levels of red blood cell aggregability, through the plasma skimming phenomenon, introduce greater spatial heterogeneity in delivery of red blood cells to the capillary network. This in turn would increase heterogeneity in oxygen delivery to the tissues. Because the effect of aggregation on FCD was small at normal arterial pressure, the present finding suggests that it only becomes important under conditions of reduced flow. However, during hemorrhagic shock not treated by fluid replacement or in disease states such as occlusive vascular disease where blood flow is reduced to marginal levels, normal human levels of red blood cell aggregability would increase spatial heterogeneity and further compromise oxygen delivery to the tissues. With higher levels of aggregability, as seen in disease states (21, 23, 35), axial migration and plasma skimming may become more pronounced.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-52684, HL-62354, and HL-64395.


   ACKNOWLEDGMENTS
 
We thank Kristina Flores, Scott Dunning, and Jong Hoon Park for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. C. Johnson, Dept. of Bioengineering, Univ. of California, San Diego, La Jolla, CA 92093-0412 (e-mail: pjohnson{at}bioeng.ucsd.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Baker M and Wayland H. On-line volume flow rate and velocity profile measurement for blood in microvessels. Microcirc Res 7: 131–143, 1974.
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