Axel R. Pries, Timothy W.

Secomb, Helfried Jacobs, Markus Sperandio, Kurt Osterloh and Peter Gaehtgens
Am J Physiol Heart Circ Physiol 273:2272-2279, 1997. You might find this additional information useful... This article cites 35 articles, 18 of which you can access free at: http://ajpheart.physiology.org/cgi/content/full/273/5/H2272#BIBL This article has been cited by 16 other HighWire hosted articles, the first 5 are: Bradykinin- and sodium nitroprusside-induced increases in capillary tube haematocrit in mouse cremaster muscle are associated with impaired glycocalyx barrier properties J. W. G. E. VanTeeffelen, A. A. Constantinescu, J. Brands, J. A. E. Spaan and H. Vink J. Physiol., July 1, 2008; 586 (13): 3207-3218. [Abstract] [Full Text] [PDF] J. Van Teeffelen, H. Vink, B. T. Ameredes, A. M. Jones, S. Egginton, L. F. Ferreira, G. W. Schmid-Schoenbein, W. L. Murfee, S. S. Segal, K. Tyml, P. McDonough, D. Keller, J. Buckwalter, B. Duling and L. B. Rowell J Appl Physiol, March 1, 2008; 104 (3): 895-899. [Full Text] [PDF] Fluorescence correlation spectroscopy can probe albumin dynamics inside lung endothelial glycocalyx A. P. Stevens, V. Hlady and R. O. Dull Am J Physiol Lung Cell Mol Physiol, August 1, 2007; 293 (2): L328-L335. [Abstract] [Full Text] [PDF] Cross-talk between cardiac muscle and coronary vasculature. N. Westerhof, C. Boer, R. R. Lamberts and P. Sipkema Physiol Rev, October 1, 2006; 86 (4): 1263-1308. [Abstract] [Full Text] [PDF] Hyaluronidase treatment of coronary glycocalyx increases reactive hyperemia but not adenosine hyperemia in dog hearts J. W. G. E. VanTeeffelen, S. Dekker, D. S. Fokkema, M. Siebes, H. Vink and J. A. E. Spaan Am J Physiol Heart Circ Physiol, December 1, 2005; 289 (6): H2508-H2513. [Abstract] [Full Text] [PDF] Medline items on this article's topics can be found at http://highwire.stanford.edu/lists/artbytopic.dtl on the following topics: Physiology .. Hemodynamics Cell Biology .. Glycocalyx Physiology .. Blood Circulation Physiology .. Microvasculature Physiology .. Absorption Medicine .. Etiology Updated information and services including high-resolution figures, can be found at: http://ajpheart.physiology.org/cgi/content/full/273/5/H2272 Additional material and information about AJP - Heart and Circulatory Physiology can be found at: http://www.the-aps.org/publications/ajpheart

Downloaded from ajpheart.physiology.org on March 15, 2010

This information is current as of March 15, 2010 .

AJP - Heart and Circulatory Physiology publishes original investigations on the physiology of the heart, blood vessels, and lymphatics, including experimental and theoretical studies of cardiovascular function at all levels of organization ranging from the intact animal to the cellular, subcellular, and molecular levels. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2005 by the American Physiological Society. ISSN: 0363-6135, ESSN: 1522-1539. Visit our website at http://www.the-aps.org/.

Microvascular blood ow resistance: role of endothelial surface layer

AXEL R. PRIES,1 TIMOTHY W. SECOMB,2 HELFRIED JACOBS,1 MARKUS SPERANDIO,1 KURT OSTERLOH,1 AND PETER GAEHTGENS1 1Department of Physiology, Freie Universitat Berlin, D-14195 Berlin, Germany; and 2Department of Physiology, University of Arizona, Tucson, Arizona 85724
Pries, Axel R., Timothy W. Secomb, Helfried Jacobs, Markus Sperandio, Kurt Osterloh, and Peter Gaehtgens. Microvascular blood ow resistance: role of endothelial surface layer. Am. J. Physiol. 273 (Heart Circ. Physiol. 42): H2272H2279, 1997.Observations of blood ow in microvascular networks have shown that the resistance to blood ow is about twice that expected from studies using narrow glass tubes. The goal of the present study was to test the hypothesis that a macromolecular layer (glycocalyx) lining the endothelial surface contributes to blood ow resistance. Changes in ow resistance in microvascular networks of the rat mesentery were observed with microinfusion of enzymes targeted at oligosaccharide side chains in the glycocalyx. Infusion of heparinase resulted in a sustained decrease in estimated ow resistance of 1421%, hydrodynamically equivalent to a uniform increase of vessel diameter by 1 m. Infusion of neuraminidase led to accumulation of platelets on the endothelium and doubled ow resistance. Additional experiments in untreated vascular networks in which microvascular blood ow was reduced by partial microocclusion of the feeding arteriole showed a substantial increase of ow resistance at low ow rates (average capillary ow velocities 100 diameters/s). These observations indicate that the glycocalyx has signicant hemodynamic relevance that may increase at low ow rates, possibly because of a shear-dependent variation in glycocalyx thickness. rheology; microvascular networks; shear rate; glycocalyx

ing diameters have been proposed: 1) irregularity of vessel lumen, 2) entrance effects at vascular branch points, 3) ow properties of white blood cells, and 4) restriction of ow by a macromolecular lining on the endothelial surface. Available studies (4, 20, 28, 34, 35) suggest that the rst three effects are not sufficient to quantitatively explain the observed high blood ow resistance in vivo. Therefore, the aim of the present study was to examine the effect of the glycocalyx on ow resistance by measuring changes in resistance after microinfusion of enzymes cleaving components of glycocalyx oligosaccharide side chains. Because changes of surface shear forces caused by blood ow might affect glycocalyx properties, the effect of blood ow rate on ow resistance was also examined. The rat mesentery preparation used is well suited for these studies, because its microvessels do not exhibit spontaneous changes of vascular tone that might inuence ow resistance during the experiments.
MATERIALS AND METHODS

Downloaded from ajpheart.physiology.org on March 15, 2010

SEVERAL physiologically important functions of microves-

sels are known to be affected by the surface characteristics of microvascular endothelium. These characteristics are determined by a macromolecular lining, referred to as the glycocalyx, that includes both molecules directly bound to the plasma membrane of the endothelial cells and components adsorbed from the blood plasma. The glycocalyx has been shown to inuence several aspects of vascular function, including the metabolic status of endothelial cells (31), endothelial permeability to water and solutes (32), leukocyte adhesion and emigration (11), and microvascular hematocrit (6). The effect of the glycocalyx on capillary hematocrit has been attributed to a restriction of the luminal volume available for ow of plasma and red blood cells (6, 7). Such a restriction could also alter vascular ow resistance. Observations of blood ow in microvascular networks and direct measurements of pressure drop and volume ow rates in vivo have shown that the resistance to blood ow is about twice as large as expected from tube ow studies (12, 13, 23). A number of mechanisms that could increase ow resistance in vivo compared with that of glass tubes with correspondH2272 0363-6135/97 $5.00 Copyright

Intravital microscopy. Experiments were conducted in the microvasculature of the rat mesentery with intravital microscopy after approval of the procedures used by university and governmental committees on animal care. Details of the animal preparation and the setup used for intravital microscopy have been described elsewhere (22). Male Wistar rats (body wt 300450 g) were prepared for intravital microscopy of the mesenteric microcirculation after premedication (atropine 0.1 mg/kg im and pentobarbital sodium 20 mg/kg im); anesthesia (ketamine 100 mg/kg im); cannulation of trachea, jugular vein, and carotid artery; and abdominal midline incision. During the experiments, which lasted for up to 2 h, the level of anesthesia and uid balance were maintained by infusion of physiological saline (24 ml kg 1 h 1 iv) containing 0.3 mg/ml pentobarbital sodium. Heart rate and arterial blood pressure (range 105140 mmHg) were continuously monitored via the catheter in the carotid artery. The animals were transferred to a special stage mounted on an intravital microscope. The small bowel was exteriorized, and fat-free portions of the mesentery were selected for investigation with a 25, NA 0.6 saltwater immersion objective (Leitz). In this preparation, vessels generally do not exhibit any spontaneous smooth muscle tone. As a precaution to prevent the development of tone and thus temporal variation of ow resistance during the measurement period, papaverine (10 4 M) was continuously superfused. Two series of experiments using different approaches were performed. Bifurcation experiments. In a series of 28 experiments, changes in the ows in the two daughter branches of a bifurcating feeding arteriole were used to analyze the effect of enzymes and of buffer solutions with altered osmolarity microinfused into one daughter vessel and its dependent vessel network (Fig. 1). Changes of systemic hemodynamic

1997 the American Physiological Society

BLOOD FLOW RESISTANCE AND ENDOTHELIAL SURFACE LAYER

H2273

Fig. 1. Schematic representation of experimental setup used for the 2 series. A: network approach. Arteriolovenular pressure difference is measured using a single micropipette, determining venular pressures at beginning and end of experiment and measuring arteriolar pressures in between. In addition, volume ow rate in feeding arteriole is derived from arteriolar vessel diameter and centerline ow velocity measured by spatial correlation to estimate network ow resistance. Microinjection of heparinase was used to modify endothelial glycocalyx in investigated vascular bed. B: bifurcation approach. Flow velocity is continuously measured in the 2 daughter branches of an arteriolar bifurcation using a dual-window technique. From changes of ow distribution between daughter branches, effects of enzymes infused into a smaller side branch on ow resistance in dependent vascular network are calculated.

Downloaded from ajpheart.physiology.org on March 15, 2010

conditions, including blood pressure, upstream arterial vessel tone, and changes of blood rheological properties, can be assumed to have equivalent effects on the ows in both treated and untreated daughter vessels. Therefore, observed changes in the ow velocity ratio between these vessels reect changes in the resistance of the treated vessel and its dependent network, independent of any systemic effects of the treatment. Flow velocities in the daughter branches of the feeding vessel bifurcation (diameter 30 m) were measured using a dual-window method. Two rotatable pairs of photodiodes were mounted on micromanipulators and positioned in front of a screen onto which the microscopic image was projected (18). The photodiodes were aligned with the centerlines of the two daughter vessels. Given the overall magnication of the microscope system, the equivalent distance between the two photodiodes along the vessel axis was 5.5 m in the mesenteric plane. The output signals of the photodiodes were amplied and sampled at a frequency of 10 kHz. Recordings of 5-s duration were taken at intervals of 0.52 min and stored in a personal computer. These recordings were crosscorrelated off-line using a Fourier transform algorithm to determine the temporal shift (delay) between the intensity patterns derived from each of the photodiode pairs. From the delay and the photodiode distance, the centerline ow velocity was determined. In independent calibration experiments using a rotating disk assembly, it was ascertained that this velocity measurement system yields reliable results up to 50 mm/s. This method provides a high time resolution (frequency of measurements 100 Hz) and is well suited for measurement of relative changes in velocity in the two vessels, in the presence of rapid pulsatile uctuations of velocity occurring simultaneously in both branches. The percent change of resistance ( R) in the treated network, fed by the microinfused daughter branch, was estimated from the changes in the ow velocity ratio between the two branches. Vessel diameters remained constant during the experiment as ascertained by occasional measurements. Flow velocity (V) in each branch is inversely related to the ow resistance (R) in that branch and its downstream

ramications. Therefore, the resistance of the network fed by the daughter branch to which the microinfusion was applied (RT ) is related, both before and after enzyme treatment, to the resistance of the untreated control network (RU ) by RT / RU VU/VT (1)

Because RU is assumed to be constant during the experimental procedure, RT is proportional to the ratio of velocity in the untreated network (VU ) to velocity in the treated network (VT ). This ratio is not affected by changes in hemodynamic conditions upstream or downstream of the observed microvascular networks or by systemic rheological changes. If SC represents the average value of VU/VT in the control state before treatment of one network and S represents a VU/VT value after treatment, R in the treated network can be expressed as RT 100 (S SC )/SC (2)

Once the infusion micropipette was impaled into a side branch of one daughter vessel, approximately ve velocity recordings of 5-s duration were made before microinfusion was started. One of the following substances was then infused for 7 min: vehicle solution [147 mM NaCl, 4 mM KCl, 3 mM CaCl2, 10 mM 3-(N-morpholino)propanesulfonic acid buffer, and 0.25 g/100 g bovine albumin (Sigma); n 4]; vehicle solution in which osmolarity was altered by adjustment of NaCl concentration [50 mosmol/l (n 3), 800 mosmol/l (n 5)]; vehicle containing heparitin-sulfate lyase (10 U/ml, EC 4.2.2.8, Sigma; n 4); vehicle containing heparinase (100 U/ml, EC 4.2.2.7, Sigma; n 6); or vehicle containing neuraminidase (n 2, 0.2 U/ml; n 4, 5 U/ml; EC 3.2.1.18, Sigma). To avoid possible effects of high transmural pressure in the microinfused vessels, the perfusion pressure in the pipette was adjusted so that a small amount of blood ( 10 30% of the total ow rate) was still seen to ow in the arteriole in addition to the enzyme solution. As evidenced by direct pressure measurements in the network experiments (see below), the intravascular pressure increase elicited by microinfusion was 10 mmHg. After the infusion period,

H2274

BLOOD FLOW RESISTANCE AND ENDOTHELIAL SURFACE LAYER

velocity was recorded for up to 60 min. Sham experiments without microinfusion demonstrated that resistance ratios remained constant with time for similar observation periods. Network experiments. To estimate resistance changes on heparinase treatment, 12 experiments were performed with this enzyme using a different technique. The ow resistance of microvascular networks in the rat mesentery was estimated by measuring arteriolovenular pressure drop and volume ow rate before and after local microinfusion of heparinase (Fig. 1). The networks investigated were fed by arterioles with inner diameters of 30 m and drained by venules of 45-m diameter. As in previous studies in the same preparation, such networks consist of 450 individual vessel segments (21). Intravascular pressures in the feeding arteriole and the draining venule were measured according to the servo-nulling technique using a micropipette with a tip diameter of 1 m connected to a micropressure measuring system (model 5, IPM; Ref. 9). After an initial measurement of venular pressure, the micropipette was impaled into the main feeding arteriole where it remained during the experimental period (up to 60 min). For each determination of network ow resistance, arteriolar pressure was monitored and the image of the vessel, obtained with a charge-coupled device camera (MX, AIS), was recorded on videotape for 30 s using asynchronous ash illumination (113601, Chadwick-Helmuth). The experiment was completed by a measurement of venular pressure to test the stability of arteriolovenular pressure gradient and a measurement of zero pressure (in the superfusing solution) to test the stability of micropressure recordings. The venular pressures at the beginning and the end of the experiment averaged 13.4 1.8 and 11.8 2.1 mmHg, respectively. A linear interpolation of the two venular pressures for each experiment was used in the calculations of ow resistance at a given time during the experiment. Because the micropipette remained in place before and after heparinase microinfusion, observed changes in resistance could not have resulted from perturbations of pressure caused by micropipette insertion. Furthermore, such perturbations were probably small, because the pipette tip was much smaller than the impaled vessels. The video recordings were analyzed off-line to determine arteriolar ow velocity and vessel diameter using a digital image analysis system (23). The spatial correlation analysis of recordings obtained with asynchronous illumination allowed measurements of blood ow velocities up to 40 mm/s. A previously established method (23) based on parametric descriptions of the Fahraeus effect and a spatial averaging model was used to convert the obtained centerline velocities into mean blood ow velocities. A digital image analysis system was used to measure vessel diameters interactively (16). The precision of this method as determined by repeated measurements ranges between 0.5 and 1 m (20). Because arteriolar diameters exhibited no detectable variations during the experiments or after enzyme treatment, relative changes of network ow resistance were calculated from relative changes in pressure drop and arteriolar ow velocity. In 9 of 12 experiments of this series, the network was perfused for 5 or 10 min with heparinase in the vehicle solution described above via a micropipette impaled into the feeding arteriole. Velocity data and arteriolar pressure readings were sampled several times in the control state, as well as repeatedly for 30 min after the end of the heparinase infusion, to allow estimations of network ow resistance. In three additional experiments of this series, the inuence of variation of ow rate on network ow resistance was analyzed without any previous enzyme treatment. A graded

reduction of blood ow in the microvessel networks was achieved by partial occlusion of the feeding arteriole with a blunted micropipette proximal to the point of pressure measurement. Graded ow reduction was repeated after varying the systemic hematocrit (HS ) to different levels between 0.52 and 0.07 by isovolemic or hypervolemic exchange of blood with homologous red blood cells, plasma, or hydroxyethyl starch solution (100 g/l, mol wt 200,000/0.5 in physiological saline where 0.5 indicates the degree of substitution in the starch molecule; Fresenius) according to a procedure described previously (23). Because reduction of ow rate in the feeding arteriole by partial occlusion may lead to upstream phase separation and thereby decrease hematocrit and ow resistance in the network studied, the hematocrit in the feeding arteriole was continuously measured by off-line videodensitometric analysis (17, 22). The image of the vessel was recorded on videotape using monochromatic illumination (wavelength 448 nm). An in vivo calibration for each vessel was established, assuming that the discharge hematocrit in the arteriole (H) in the unoccluded state was equal to HS. Experimental values of optical density (OD) measured in the unoccluded vessel during variation of HS were used to determine the parameters a and b of the linear regression OD a b (2HS H2 ) S (3)

Downloaded from ajpheart.physiology.org on March 15, 2010

With this calibration equation, H was calculated from the measurements of OD obtained during the occlusion procedures, when H was expected to be lower than HS. The effect of changes in H on network ow resistance R was determined using the equation R A B [(1 HS )C 1] (4)

The empirical parameters A, B, and C were determined by a tting procedure using the set of experimental data on network ow resistance R for different levels of HS in the unoccluded state. An equation of this form was used previously to adequately describe the relation between ow resistance and hematocrit in tube ow (19). The resistance measurements made during partial occlusion (R0 ) were then corrected (RC ) for the measured changes in hematocrit according to the equation A RC R0 A B [(1 B [(1 HS)C H)
C

1] 1] (5)

Statistics. Mean values were compared by a one-way analysis of variance procedure using Tukey-B correction for post hoc multiple comparisons. Signicance is indicated at the P 0.05 level.
RESULTS

Table 1 gives averaged morphological and hemodynamic values for arterioles of both bifurcation and network experiments. In the bifurcation experiments, vessel diameters were measured at the site of velocity measurement. In these experiments, no statistically signicant difference between the parameters for the two branches (infused and untreated) of the observed bifurcation was observed. Figure 2 shows results of heparinase infusion in a typical bifurcation experiment. Flow velocity in the untreated branch exhibited no substantial change during the experimental period, whereas ow velocity in the treated branch increased substantially after the

BLOOD FLOW RESISTANCE AND ENDOTHELIAL SURFACE LAYER

H2275

Table 1. Mean values and standard deviations of morphological and hemodynamic parameters for arterioles feeding networks studied
Diameter, m Pressure, mmHg P, mmHg Flow Velocity, mm/s Volume Flow, nl/min Shear Rate, diam/s

Bifurcation experiments (n Mean SD Mean SD 20.3 3.6 29.6 5.4 NM NM

28) 144 98 740 412 330 188 546 216

6.7 2.1 9) 16.2 7.2

Network experiments (n 64.1 15.3 52.2 15.5

P, pressure drop from feeding arteriole to draining venule; diam, vessel diameters; NM, not measured; n, no. of experiments.

enzyme infusion. This indicates a reduction of ow resistance in the microvascular network fed by the treated branch. Average changes of ow resistance with time after vehicle infusion and heparinase infusion in the bifurcation experiments are shown in Fig. 3. Postinfusion values of ow resistance change are slightly

Fig. 3. Changes in network ow resistance over time after 7-min microinfusion of vehicle (n 4 experiments) and heparinase (100 U/ml, n 6 experiments). Values from individual bifurcation experiments were averaged for successive 5-min time intervals and are shown with standard errors. For observation times exceeding 35 min, data were averaged over 10 and 15 min.

Downloaded from ajpheart.physiology.org on March 15, 2010

elevated after infusion of the vehicle (n 4 experiments), but this is statistically signicant only for the rst 5 min of the postinfusion period. In contrast, ow resistance in the heparinase infusion experiments (n 6) exhibited a signicant and sustained decrease during the observation period that ranged from 30 to 60 min in individual experiments. Microinfusion of neuraminidase into one daughter branch of arteriolar bifurcations led to a signicant elevation of ow resistance of the treated microvessel network already in the rst measurement after the end of the microinfusion and a further progressive increase with time (Fig. 4). Intravital microscopic inspection showed that this resistance increase was accompanied by adherence and rolling of large numbers of platelets on the endothelial surface in all vessel branches downstream of the infusion site.

Fig. 2. Representative bifurcation experiment. A: velocity recordings for both daughter branches in control period showing pulsatile velocity changes. B: velocity values averaged for 8-s recording intervals before and after heparinase microinfusion into 1 branch. After infusion, velocity in control branch has changed only slightly, whereas velocity in treated branch has increased substantially (SD not shown for clarity). C: resistance change (means SD) in treated branch calculated according to Eq. 2 from individual velocity measurements in both daughter branches.

Fig. 4. Changes in network ow resistance over time on infusion of neuraminidase (5 U/ml, n 4 experiments) in bifurcation experiments. Data representation as in Fig. 3.

H2276

BLOOD FLOW RESISTANCE AND ENDOTHELIAL SURFACE LAYER

Fig. 5. Arteriolovenular ow resistance estimated in network experiments (n 9 experiments) in control situation and after microinfusion of heparinase for 5 or 10 min.

Figure 5 compares absolute values of ow resistance obtained in the network experiments for the untreated state with those seen after microinfusion of heparinase. Although in all but one experiment a signicant reduction of resistance was seen after 5 min of infusion, only a small additional effect was seen after 10 min. To summarize the results of both experimental designs, changes in ow resistance on treatment were averaged for each individual experiment over the postinfusion observation period. Mean values ( SD) of these averages are compared in Fig. 6 for the different treatment groups. The infusion of vehicle as well as of buffer solutions with altered osmolarity (bifurcation experiments) led to small but not signicant increases of ow resistance. No change in resistance was observed after injection of heparitin-sulfate lyase (bifurcation experiments). In contrast, heparinase treatment resulted in a substantial reduction of ow resistance in both the network experiments (mean reduction 20.8 8.5%, 10-min infusion; 16 13.4%, 5-min infusion)

Fig. 7. Flow resistance obtained in network experiments as a function of pseudo-shear rate (U, vessel diameters/s) in feeding vessel. Repetitive graded microocclusion was used in 3 microvascular networks to vary blood ow and hence shear rate level. Flow resistance for each occlusion procedure yielding 510 individual resistance measurements was normalized with respect to that determined at highest shear rates to allow superimposition of data for different networks and different systemic hematocrits. Shear rate in feed arteriole is given in vessel diameters per second. HS, systemic hematocrit.

Downloaded from ajpheart.physiology.org on March 15, 2010

and the bifurcation experiments ( 14.2 6.5%, 7-min infusion). These changes were statistically signicant. The average resistance increase for the rst 15 min after the end of the neuraminidase infusion was 98.5 25% for the higher enzyme concentration tested (bifurcation experiments). The effect of alterations in blood ow rate by partial microocclusion of the feed vessel on network ow resistance is shown in Fig. 7. Measured ow velocities in the feeding arteriole were converted into pseudoshear rates (U) by dividing by vessel diameter. If it is assumed that measured velocities represent means over vessel cross sections and that velocity proles are parabolic, pseudo-shear rates may be converted to wall shear rates by multiplying by 8. Because these assump-

Fig. 6. Changes of network ow resistance for treatment groups of both experimental approaches. Duration of microinfusion was 7 min unless indicated otherwise. For each experiment, data were averaged over respective observation periods (3060 min); values are means SD of these averages. Filled and hatched bars, results of bifurcation and network approaches, respectively. * Signicant changes of ow resistance (analysis of variance, P 0.05). n, No. of experiments.

BLOOD FLOW RESISTANCE AND ENDOTHELIAL SURFACE LAYER

H2277

tions may not be valid, the use of U values is preferable. At pseudo-shear rates in the feeding vessel below 100 diameters/s, ow resistance increased to about twice the value measured at high shear rates. This increase was seen both in the raw data (not shown) and in data corrected for the hematocrit reduction occurring during the occlusion procedure (Fig. 7). Data obtained over a large range of systemic hematocrit (0.070.52) exhibit very similar trends. This suggests that HS does not substantially inuence the increase in network ow resistance at low levels of U.
DISCUSSION

The key nding of the present study is that heparinase microinfusion led to a sustained 1421% decrease in ow resistance of microvascular networks downstream of the site of infusion. Such an effect was not found when enzyme-free vehicle or solutions of altered osmolarity were infused. If endothelial swelling or shrinking occurs during hypo- or hyperosmolar microperfusion, this effect is apparently rapidly reversed on reperfusion with normal blood and thus not detectable in the present experimental design. Heparitinsulfate lyase led to no measurable decrease of ow resistance. After neuraminidase, a doubling in ow resistance was observed, consistent with the results of Muhleisen et al. (14) for the isolated perfused ileum. In the present experiments, microscopic observation showed the presence of a layer adjacent to the endothelium composed mainly of adherent and rolling thrombocytes. Glycocalyx thickness. Changes in resistance imply changes either in the effective vessel diameter or the apparent blood viscosity. Because the amount of solution microinfused was very small (between 1 and 7 l), the systemic concentration of the enzymes applied is probably negligible, and it can be assumed that the rheological properties of blood entering the experimental site on terminating the microinfusion were unaffected by the preceding treatment. In addition, the results obtained in the bifurcation experiments are independent of changes of systemic hemodynamic conditions including blood viscosity. Therefore, the observed changes in ow resistance most likely reect changes in effective vessel diameter. The vessels investigated were fully dilated, and no pressure changes were seen after the end of microinfusion. Therefore, changes in morphological vessel diameter are unlikely. However, the decrease in network ow resistance after heparinase treatment could result from reduction of the thickness of the glycocalyx, leading to an increase of the effective vessel diameter available for red blood cell and plasma motion. The increase in effective vessel diameter that would be required to produce the observed decrease in resistance may be estimated using a theoretical simulation of ow in microvascular networks (22). This simulation uses geometries (diameters and lengths of the vessels, network topologies) determined previously (22) for networks in the rat mesentery of a size similar to the networks studied in the present study and assumes a

relationship between apparent blood viscosity, vessel diameter, and hematocrit deduced from studies of blood ow in mesenteric networks (23). According to this simulation, a uniform increase of effective radius in all perfused microvessels of 0.35 m could account for the observed reduction of ow resistance in the bifurcation experiments on microinfusion of heparinase. For the network experiments, a value of 0.55 m is obtained. These values represent lower bounds on the thickness of the layer present on the endothelial surface in the untreated state. Equivalent reductions in resistance could also result from a nonuniformly distributed increase in diameter. In that case, the increase in effective radius would be larger than the above estimates in some parts of the networks and smaller in others. Because, for the purpose of this estimation, the glycocalyx is considered as a stiff, impermeable layer of uniform thickness lining all vessels in the network, the assumption of a permeable glycocalyx, permitting axial plasma ow (5), would increase these estimates. Also, the removal of the glycocalyx by the heparinase treatment was probably incomplete, because heparinase acts at specic sites within the oligosaccharide side chains of glycoproteins residing on the endothelial surface and does not cleave other (nonsulfated) sugar components or the protein backbone of these molecules. The above estimates of glycocalyx thickness are consistent with results of previous studies. On the basis of measurements of capillary hematocrit in resting striated muscle, Duling and Desjardins (7) suggested that plasma ow is retarded in a layer on the endothelial surface, the thickness of which was estimated between 0.8 and 1.8 m. Further studies (6) showed that treatment with heparinase caused a persistent increase in capillary hematocrit, indicating a reduction in the thickness of the layer. Using a different approach, Vink and Duling (33) observed capillaries in the hamster cremaster muscle and demonstrated a layer of 0.40.5 m adjacent to the endothelial cells that was inaccessible to owing red blood cells and plasma. In contrast, the apparent glycocalyx thickness seen in electron microscopic studies is 0.1 m (8, 15). However, electron microscopy requires xation and staining procedures that may cause collapse of the glycocalyx proteoglycans (6). Indirect methods have led to higher estimates of layer thickness. From measurements of the sialic acid concentration on the surface of vascular endothelium (2), Silberberg (29) estimated a thickness of the glycocalyx gel under native conditions of at least 0.5 m and noted that this gel would collapse to 0.05 m if dehydrated during preparation for electron microscopy. Possible contribution of plasma proteins to glycocalyx properties. Endothelial cells bind plasma proteins, including brinogen and albumin (26, 37). Observations of endothelium-coated beads sedimenting in different media (24) imply that plasma proteins affect uid ow past endothelial cells. Surprising reductions of falling velocities from expected values were found in plasma but not in other media. Based on Stokes law for the

Downloaded from ajpheart.physiology.org on March 15, 2010

H2278

BLOOD FLOW RESISTANCE AND ENDOTHELIAL SURFACE LAYER

drag on a sphere, the reduction in sedimentation velocity in plasma can be interpreted as an increase in the effective radius of the beads by 25 m. Formation of a layer of this thickness capable of impeding ow may depend on the low uid shear rate on the surface of the falling beads ( 3.5 s 1 ) compared with average wall shear rates in the microvessels in the mesentery ( 200 2,000 s 1; Ref. 21). These observations indicate that plasma protein levels may inuence the mechanical properties of the glycocalyx and suggest a possible origin for its stiffness. Adsorption of plasma proteins to other glycocalyx components may generate a colloid osmotic pressure exceeding that of free plasma (36) if the plasma protein molecules are loosely bound and retain signicant mobility. For mechanical equilibrium, such an increase of colloid osmotic pressure must be balanced by tension in the membrane-bound glycoproteins (30). To compress the glycocalyx, an applied force must overcome this pressure increment. The pressure exerted by the rim of a substantially deformed red blood cell is of the order /L, where is the shear elastic modulus of the membrane and L is a characteristic length scale. Taking 0.006 dyn/cm and L 1 m yields 60 dyn/cm2; a similar estimate is implied by more detailed calculations (27). An increase in colloid osmotic pressure within the glycocalyx by only 1% (i.e., 330 dyn/cm2, based on a colloid osmotic pressure of 25 mmHg) could thus generate sufficient force to resist penetration by red blood cells. The nding that short-term microinfusion of articial plasmas with altered protein content had little effect on capillary hematocrit (10) may indicate that equilibration of proteins between plasma and glycocalyx exhibits time constants above 10 min. Effect of ow rate on ow resistance. When blood ow rate was reduced by graded microocclusion, network ow resistance remained approximately constant at U levels down to 100 s 1 in the feeding arterioles (Fig. 7). However, ow resistance increased at ow rates below this level. The resistance increase at low shear rates was similar for all levels of systemic hematocrit (range 0.070.52) and seems therefore to be independent of red blood cell concentration. Several mechanisms for this behavior may be proposed. 1) In bulk shear ow, blood exhibits strong shear-thinning behavior. However, ow resistance in narrow glass tubes (diameter 30 m) perfused with red blood cells suspended in plasma shows little or no change of ow rate with U in the range from 1 to 100 s 1 (1, 25). Because average U values in venules with diameters 20 m are about one-eighth of those in the feeding arteriole (21), the threshold U level of 100 s 1 in the feeding arteriole corresponds to 12 s 1 in larger venules. Therefore, the observed increase in resistance is not accounted for by the rheological behavior of blood in narrow uniform tubes. 2) The observed increase of resistance on microocclusion could result from a passive diameter change caused by the reduction of capillary pressure. Bosman et al. (3) showed passive changes in capillary diameter by 6% and 12% during aortic occlusion causing a fall of capillary pressure and reac-

tive hyperemia causing an elevation of capillary pressure. Such diameter changes are too small to explain the resistance increase of up to 100% observed in the present experiments with decreasing shear rate. 3) The thickness of the glycocalyx may increase at very low ow rates (U 100 s 1 ). Desjardins and Duling (6) reported that capillary hematocrit in muscle capillaries increased with ow velocity from 0.18 to 0.27 mm/s. Capillary velocities in this range correspond to those at which ow-dependent resistance was evident in the present study. This last mechanism is therefore consistent with observed ow dependence of ow resistance observed here. Discrepancy between in vivo and in vitro estimates of network ow resistance. Measured values of ow resistance in microvascular networks have been found to be about twice as large as expected from measurements of ow resistance in glass tubes (23). Flow restriction by the glycocalyx is a possible reason for this difference. The relation between glycocalyx thickness and network ow resistance can be estimated using the theoretical simulation already described. According to this simulation, a uniform, impermeable glycocalyx of 1.5-m thickness would produce the difference in the ow resistance of microvascular networks of the rat mesentery compared with predictions based on glass tube rheology. In reality, however, other effects may also contribute to the increased ow resistance in vivo. The hydrodynamic resistance of a vessel with irregular luminal contour is larger than the resistance of a uniform tube with the same mean diameter (20) because contour irregularity leads to additional energy dissipation caused by transient deformation of red blood cells (28). Additional ow resistance may occur at vascular branch points (28). Available estimates of these effects suggest that they may cause a reduction of effective vessel radius by not more than 0.5 m. The effect of white blood cells on ow resistance of vascular beds has been debated, and reported resistance increases caused by white blood cells in skeletal muscle range from 1 to 20% (4, 34, 35). In the mesentery, capillaries are wider than in skeletal muscle and effects of white blood cells on resistance can be expected to be lower. Thus the observed difference between ow resistance in vivo and in vitro is only partly explained by effects of vascular irregularity, entrance phenomena, and white blood cells. The remaining discrepancy can, however, be accounted for by the presence of a stiff, impermeable layer with thickness of 0.5 m adjacent to the endothelium. In conclusion, the present ndings support the concept of a macromolecular layer (glycocalyx) at the luminal surface of microvascular endothelium that contributes signicantly to microvascular ow resistance. This layer may have signicant implications for the many physiological functions in which endothelial cells are involved. Such functions could therefore be affected by changes in plasma protein composition that modify the composition and thus relevant properties of the endothelial glycocalyx.

Downloaded from ajpheart.physiology.org on March 15, 2010

BLOOD FLOW RESISTANCE AND ENDOTHELIAL SURFACE LAYER The expert technical help of B. Giesicke and M. Ehrlich as well as the assistance of A. Scheuermann in preparing the manuscript are gratefully acknowledged. This study was supported by the Deutsche Forschungsgemeinschaft (Pr 271/11, 12, 51, and 52) and by National Heart, Lung, and Blood Institute Grant HL-34555. Address for reprint requests: A. R. Pries, Freie Universitat Berlin, Dept. of Physiology, Arnimallee 22, D-14195 Berlin, Germany. Received 20 February 1997; accepted in nal form 30 July 1997. REFERENCES 1. Alonso, C., A. R. Pries, O. Kiesslich, D. Lerche, and P. Gaehtgens. Transient rheological behavior of blood in low-shear tube ow: velocity proles and effective viscosity. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H25H32, 1995. 2. Born, G. V. R., and W. Palinski. Unusually high concentrations of sialic acids on the surface of aortic endothelium. Br. J. Exp. Pathol. 66: 543549, 1985. 3. Bosman, J., G.-J. Tangelder, M. G. A. Oude Egbrink, R. S. Reneman, and D. W. Slaaf. Capillary diameter changes during low perfusion pressure and reactive hyperemia in rabbit skeletal muscle. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H1048 H1055, 1995. 4. Braide, M., B. Amundson, S. Chien, and U. Bagge. Quantitative studies on the inuence of leukocytes on the vascular resistance in a skeletal muscle preparation. Microvasc. Res. 27: 331352, 1984. 5. Damiano, E. R., B. R. Duling, K. Ley, and T. C. Skalak. Axisymmetric pressure-driven ow of rigid pellets through a cylindrical tube lined with a deformable porous layer. J. Fluid Mech. 314: 163189, 1996. 6. Desjardins, C., and B. R. Duling. Heparinase treatment suggests a role for the endothelial cell glycocalyx in regulation of capillary hematocrit. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): H647H654, 1990. 7. Duling, B. R., and C. Desjardins. Capillary hematocritwhat does it mean? News Physiol. Sci. 2: 6669, 1987. 8. Haldenby, K. A., D. C. Chappell, C. P. Winlove, K. H. Parker, and J. A. Firth. Focal and regional variations in the composition of the glycocalyx of large vessel endothelium. J. Vasc. Res. 31: 29, 1994. 9. Intaglietta, M., and W. R. Tompkins. Micropressure measurements with 1 micron and smaller cannulae. Microvasc. Res. 3: 211214, 1971. 10. Keller, M. W., D. N. Damon, and B. R. Duling. Determination of capillary tube hematocrit during arteriolar microperfusion. Am. J. Physiol. 266 (Heart Circ. Physiol. 35): H2229H2238, 1994. 11. Ley, K., P. Gaehtgens, C. Fennie, M. S. Singer, L. A. Lasky, and S. D. Risen. Lectin-like cell adhesion molecule 1 mediates leukocyte rolling in mesenteric venules in vivo. Blood 77: 2553 2555, 1991. 12. Lipowsky, H. H., S. Kovalcheck, and B. W. Zweifach. The distribution of blood rheological parameters in the microcirculation of cat mesentery. Circ. Res. 43: 738749, 1978. 13. Lipowsky, H. H., S. Usami, and S. Chien. In vivo measurements of apparent viscosity and microvessel hematocrit in the mesentery of the cat. Microvasc. Res. 19: 297319, 1980. 14. Muhleisen, H., G. V. R. Born, and C. C. Michel. Effects of neuraminidase on vascular resistance under conditions of maximum vasodilatation in the isolated perfused rat ileum (Abstract). J. Physiol. (Lond.) 430: 38P, 1990. 15. Pino, R. M. The cell surface of a restrictive fenestrated endothelium. II. Dynamics of cationic ferritin binding and the identication of heparin and heparan sulfate domains on the choriocapillaris. Cell Tissue Res. 243: 157164, 1986. 16. Pries, A. R. A versatile video image analysis system for microcirculatory research. Int. J. Microcirc. Clin. Exp. 7: 327345, 1988.

H2279

17. Pries, A. R., G. Kanzow, and P. Gaehtgens. Microphotometric determination of hematocrit in small vessels. Am. J. Physiol. 245 (Heart Circ. Physiol. 14): H167H177, 1983. 18. Pries, A. R., K. Ley, M. Claassen, and P. Gaehtgens. Red cell distribution at microvascular bifurcations. Microvasc. Res. 38: 81101, 1989. 19. Pries, A. R., D. Neuhaus, and P. Gaehtgens. Blood viscosity in tube ow: dependence on diameter and hematocrit. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1770H1778, 1992. 20. Pries, A. R., D. Schonfeld, P. Gaehtgens, M. F. Kiani, and G. R. Cokelet. Diameter variability and microvascular ow resistance. Am. J. Physiol. 272 (Heart Circ. Physiol. 41): H2716 H2725, 1997. 21. Pries, A. R., T. W. Secomb, and P. Gaehtgens. Structure and hemodynamics of microvascular networks: heterogeneity and correlations. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H1713H1722, 1995. 22. Pries, A. R., T. W. Secomb, P. Gaehtgens, and J. F. Gross. Blood ow in microvascular networks: experiments and simulation. Circ. Res. 67: 826834, 1990. 23. Pries, A. R., T. W. Secomb, T. Gessner, M. Sperandio, J. F. Gross, and P. Gaehtgens. Resistance to blood ow in microvessels in vivo. Circ. Res. 75: 904915, 1994. 24. Reinhart, W. H., C. M. Boulanger, T. F. Luscher, A. Haeberli, and P. W. Straub. Inuence of endothelial surface on ow velocity in vitro. Am. J. Physiol. 265 (Heart Circ. Physiol. 34): H523H529, 1993. 25. Reinke, W., P. Gaehtgens, and P. C. Johnson. Blood viscosity in small tubes: effect of shear rate, aggregation, and sedimentation. Am. J. Physiol. 253 (Heart Circ. Physiol. 22): H540H547, 1987. 26. Schneeberger, E. E., and M. Hamelin. Interaction of serum proteins with lung endothelial glycocalyx: its effect on endothelial permeability. Am. J. Physiol. 247 (Heart Circ. Physiol. 16): H206H217, 1984. 27. Secomb, T. W. Flow-dependent rheological properties of blood in capillaries. Microvasc. Res. 34: 4658, 1987. 28. Secomb, T. W., and R. Hsu. Motion of red blood cells in capillaries with variable cross-sections. J. Biomech. Eng. 118: 538544, 1996. 29. Silberberg, A. The physiological role of a thick gel-like layer over the endothelial surface (Abstract). Int. J. Microcirc. Clin. Exp. 9, Suppl. 1: 204, 1990. 30. Silberberg, A. Polyelectrolytes at the endothelial cell surface. Biophys. Chem. 41: 9, 1991. 31. Suarez, J., and R. Rubio. Regulation of glycolytic ux by coronary ow in guinea pig heart. Role of vascular endothelial cell glycocalyx. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H1994H2000, 1991. 32. Turner, M. R., G. Clough, and C. C. Michel. The effects of cationised ferritin and native ferritin upon the ltration coefficient of single frog capillaries. Evidence that proteins in the endothelial cell coat inuence permeability. Microvasc. Res. 25: 205222, 1983. 33. Vink, H., and B. R. Duling. Identication of distinct luminal domains for macromolecules, erythrocytes and leukocytes within mammalian capillaries. Circ. Res. 79: 581589, 1996. 34. Warnke, K. C., and T. C. Skalak. The effects of leukocytes on blood ow in a model skeletal muscle capillary network. Microvasc. Res. 40: 118136, 1990. 35. Warnke, K. C., and T. C. Skalak. Leukocyte plugging in vivo in skeletal muscle arteriolar trees. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H1149H1155, 1992. 36. Wiederhielm, C. A., and L. L. Black. Osmotic interaction of plasma proteins with interstitial macromolecules. Am. J. Physiol. 231: 638641, 1976. 37. Witte, S. The inuence of the brinolytic system on the affinity of brinogen for the endothelial-plasma interface. Thromb. Res. 52: 111117, 1988.

Downloaded from ajpheart.physiology.org on March 15, 2010