K. Ley

Red Cell Distribution at Microvascular Bifurcations

The distribution of red cell and blood volume flow was studied at 65 arteriolar bifurcations in the rat mesentery. Hematocrit and flow velocity were measured simultaneously in all three vessel segments constituting a bifurcation. Blood flow distribution was manipulated by irreversibly occluding downstream side branches of one of the daughter vessels. The dependence of fractional red cell volume flow on fractional blood flow was described using a three-parameter (X0, B, A) logit function. The critical volume flow fraction below which only plasma enters a downstream branch (X0), the nonlinearity of the relation between red cell and blood volume flow (B), and the asymmetry of that relation which is described by the parameter A decrease with increasing diameter of the vessel feeding the bifurcation. At diameters above 30 microns, phase separation is very limited. In addition, the nonlinearity parameter B decreases with decreasing hematocrit in the feeding vessel. The asymmetry parameter A strongly depends on the diameter ratio between the two daughter branches: For a given fractional blood flow, the smaller branch receives more red cells than the larger branch. Using a model for plasma skimming based on the assumption of a planar separating surface, the shape of the radial hematocrit profile in the feeding vessel has been calculated. The model predicts a decrease in local hematocrit from the vessel axis toward the wall with a distinct marginal zone free from cell centers. With increasing vessel diameter the hematocrit profile becomes more blunted while the width of the marginal zone increases.

Topological structure of rat mesenteric microvessel networks

Microvascular lengths, diameters, and flow directions were determined in all vessel segments (n = 1303) between bifurcations in three complete rat mesenteric microvessel networks (25 mm2 each) using intravital video- and photomicroscopy. The classification of vessel segments as arteriolar, venular, or av-segments (all segments connecting the arteriolar to the venular tree) was based on purely topological criteria. The topological structure of the networks was analyzed using the Horton-Strahler technique and a new generation scheme. Generation numbers were assigned to the vessel segments on the basis of the number of upstream (in the arteriolar tree) and downstream (in the venular tree) bifurcations. The mean generation number of the av-segments, a characteristic parameter of the generation scheme, reflects the topological structure of the network more accurately than Hortons branching ratio Rb. Both the arteriolar and venular tree of the mesenteric networks were found to be dichotomous branching structures which were neither strictly symmetric nor strictly asymmetric. The topological information obtained was compared to network models generated by different random branching algorithms. The result of this comparison suggests that the network structure changes at a certain generation level. Distal to this generation level, the mesenteric networks resemble a model network generated by random branching at any segment, while the proximal portion is similar to a model allowing random branching at terminal segments only.

Flow‐dependent regulation of arteriolar diameter in rat skeletal muscle in situ: role of endothelium‐derived relaxing factor and prostanoids.

1. Arteriolar diameter in the resting rat spinotrapezius muscle was studied by intravital video microscopy before and after blockade of the L‐arginine‐EDRF (NG‐nitro‐L‐arginine, L‐NNA) or the cyclo‐oxygenase‐prostacyclin (indomethacin) pathway. Blockade of either pathway leads to a decrease of arteriolar diameter of 25‐40%, while the combined blockade of both results in vasoconstriction of 50‐60%. 2. Alteration of blood flow velocity elicited by partial micropipette occlusion induces corresponding changes of vessel diameter. The flow‐dependent diameter response is reduced by about 80% by L‐NNA. By contrast, blockade of prostanoid production shows no significant influence on vessel response to blood flow alteration in the range tested. 3. Transient overshooting vasodilatation is seen for about 1 min following the sudden restoration of flow velocity subsequent to occlusion. In contrast to the initial phase of this response, the late phase is blocked by L‐NNA. 4. The findings suggest that basal release of endothelium‐derived relaxing factor (EDRF) and prostanoids leads to additive and independent dilator effects, and that flow‐dependent diameter changes are primarily mediated by EDRF. 5. If present data are compared with literature reports, it appears that arterial flow sensitivity is most pronounced in the smallest vessels. In such vessels, flow‐dependent dilatation will amplify even small changes of volume flow by more than four times.

Redistribution of red blood cell flow in microcirculatory networks by hemodilution.

The effect of isovolemic hemodilution on red blood cell flow distribution was studied in complete self-contained microvessel networks of the rat mesentery. Hematocrit, diameter, and length of all vessel segments as well as the topological structure were determined in control networks (systemic hematocrit, 0.54) and after hemodilution (systemic hematocrit, 0.30). Hemodilution was performed by exchanging blood with hydroxyethyl starch (MW 450,000; 6%) or homologous plasma. With hemodilution, the decrease of microvessel hematocrit exceeded that of systemic hematocrit. The average discharge hematocrit in capillaries was 79% of systemic hematocrit in the control group and 73% with hemodilution (p less than 0.001). The heterogeneity of capillary hematocrit within the network, expressed by the coefficient of variation, increased from 0.4 to 0.7. By using the morphological and topological data of four networks, the distribution of hematocrits was also calculated using a hydrodynamic flow model. The modeling results were found to be in close agreement with the experimental data. This indicates that the observed changes can be deduced from established rheological phenomena, most of all phase separation at arteriolar bifurcations. The changes in hematocrit distribution after hemodilution are accompanied by a redistribution of red blood cell flow within the network: relative to total red blood cell flow, red blood cell flow in the distal capillaries of the network increases by about 40% at the expense of the proximal capillaries that are close to the feeding arteriole and that exhibit the highest red blood cell flow under control conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Venulo-arteriolar communication and propagated response

The effect of microinjection of norepinephrine (10−5 M) into precapillary microvessels of the rat mesentery was studied using intravital microscopy. Upon application, in 29 out of 40 cases (73%) flow ceased at the site of drug application, although in most cases the precapillary microvessels themselves did not show a diameter change due to a lack of smooth muscle cells as confirmed by transmission electron microscopy. In 17 out of the 29 cases with flow cessation (59%), an intimate contact between the venule draining the site of application and the supplying arteriole was found. Initial constriction was seen at the site where the venule crossed the arteriole. Constriction propagated both up- and downstream along the arteriole, and also across arteriolo-arteriolar arcades. Arteriolar constriction could be abolished by intentionally occluding the venule draining the norepinephrine solution. It is proposed that venuloarteriolar contacts and propagated vasomotor response may contribute to local blood flow regulation by providing a feedback loop between tissue capillaries and resistance arterioles. In three complete mesenteric microvessel networks, the arterioles (n=34) supplying 273 out of 401 capillaries (68%) were in close proximity to venules draining these same capillaries. Each of these arterioles served, on average, 43 capillaries, showing a bimodal distribution with peaks at 4 to 16 and at 64 to 256 capillaries. On average, 62% of all capillaries drained by a given venule crossing an arteriole originated from this very arteriole, indicating a reasonably effective feedback.

Segmental differences of microvascular permeability for FITC-dextrans measured in the hamster cheek pouch

An intravital microscopic method for quantitative measurement of interstitial concentrations of fluorescent tracers has been applied to the investigation of microvascular permeability in the hamster cheek pouch. Some nanoliters of FITC-dextran mean mol wt (Mw) 20,000, 3000, or sodium fluorescein (Mw 376) were injected into an arteriole of the exposed cheek pouch via micropipet. The extravasation of fluorochromes was measured by a photodensitometric method including two sets of calibration procedures (in vitro and in vivo). Postcapillary and collecting venules exhibited the highest absolute increase of fluorochrome concentration in the tissue for all tracer molecules tested when compared to arterioles or capillaries. The permeability of the vascular wall was quantified, assuming that diffusion processes play the main role for the transport of the investigated molecules under the experimental conditions of a high concentration gradient across the membrane. Permeability coefficients P (cm/sec) and apparent diffusion coefficients D (cm2/sec) of the microvascular wall were calculated using a mathematical model for one-dimensional diffusion in composite media. The analysis is based on measured data of interstitial diffusion coefficients of the tracers used. For all tracer molecules tested, the wall of the capillaries and postcapillary venules was significantly more permeable than the arteriolar wall. For the largest test molecule (FITC-dextran Mw 20,000), the permeability coefficient of the vessel wall showed a maximum in the postcapillary venules. These findings support the concept of a gradient of permeability with a nonuniform distribution of exchange capacity only for the precapillary microvessels. A marked preponderance of venular over capillary permeability could, if at all, only be detected for FITC-dextran Mw 20,000. The present study characterizes the vessel wall by apparent diffusion coefficients which are, for FITC-dextran Mw 3000, and free fluorescein, roughly three orders of magnitude lower than the apparent diffusion coefficients in connective tissue.

Differential adhesion of granulocytes to five distinct phenotypes of cultured microvascular endothelial cells.

Adhesion of isolated human polymorphonuclear granulocytes (PMNs) to five different phenotypes of cultured microvascular endothelial cells derived from bovine corpora lutea was investigated by measuring the myeloperoxidase content of cell lysates. Untreated and interleukin 1 (IL-1) -pretreated confluent monolayers were overlaid with unstimulated and phorbol ester (PMA)-stimulated PMNs in the absence and presence of the monoclonal antibody IB4 recognizing and functionally blocking beta 2 (CD18) of the leukocyte integrins. Unstimulated PMN adhesion was highest on type 4, followed by type 3 and 5 endothelial cells. This adhesion was not inhibited by treatment with IB4. IL-1 pretreatment of endothelial cells resulted in a significant increase of PMN adhesion on types 1, 2, and 4, most of which was also beta 2 integrin-independent. PMA-stimulation of PMNs increased adhesion to maximal values on cell types 1 and 5, which was largely blocked by IB4. Type 2 endothelial cells supported significantly less PMA-stimulated PMN adhesion than all other types. In the presence of IB4, adhesion of PMNs to untreated and IL-1-pretreated type 3 and 4 endothelial cells was significantly reduced by PMA. This reduction of beta 2 integrin-independent adhesion by PMA stimulation is compatible with possible shedding of the lectin-like leukocyte adhesion molecule, L-selectin, from PMNs. Differential PMN adhesion may reflect distinctive expression of endothelial adhesion molecules in different phenotypes of microvascular endothelial cells. Endothelial specialization within the microcirculation may have important functional consequences for the inflammatory response in vivo.

Preferential distribution of leukocytes in rat mesentery microvessel networks

Distribution of leukocytes in rat mesenteric microvessel networks was studied using intravital fluorescence video microscopy. A digital image analysis system was used to measure vessel diameters, flow velocities and leukocyte fluxes in 306 capillaries of 8 networks. Capillaries were defined as vessel segments connecting divergent to convergent branch points. Their topological position within the network was quantified by a generation number defined as the number of bifurcations between the capillary and the arteriole feeding the network.Proximal capillaries (generation numbers 4 and 5) were slightly but significantly smaller in diameter (8.9±0.4) μm, mean ± SEM) than distal ones (generation numbers 20 and 21, 10.1±0.4 μm). Average capillary flow velocity decreased markedly from 2.0±1.0 mm·s−1 in proximal to 0.41 ±0.06 mm·s−1 in distal capillaries. Average leukocyte concentration was 3.4±0.5·109 l−1 and thus significantly below systemic values (6.0·109 l−1) in proximal capillaries, and above in distal ones (11.7±2.6·109l−1).The analysis of flow and leukocyte flux partition at 138 bifurcations showed preferential distribution of leukocytes to the daughter capillary with higher flow rate. This suggests a tentative explanation for the observed leukocyte accumulation along the microvascular tree: due to their low fractional flow, proximal capillaries draw relatively leukocytepoor blood from the arteriole feeding the network; this leads to an increased leukocyte concentration in distal capillaries. As a consequence of the concomitant increase of capillary diameter with increasing generation number, leukocytes are preferentially flowing through larger capillaries and are excluded from small ones.

Structural, hemodynamic and rheological characteristics of blood flow in the circulation

This chapter outlines some of the basic features of the circulation which appear to be of particular relevance for an understanding of hemorheological phenomena. The behavior of blood flowing through the various compartments of the cardiovascular system is determined by both its own rheological properties and the structural design and hemodynamic characteristics of the circulatory system. Thus, changes of blood rheology, which may occur in diseased states, cannot be interpreted in terms of their possible effects on blood flow in vivo, unless the hemodynamic state and possible alterations of vascular morphology are also appreciated. In fact, a multitude of pathophysiological situations or clinical disorders is characterized by an alteration, from the normal, of blood rheology and vascular hindrance; these may, in some cases, even have a common cause.