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The endothelial glycocalyx (EG), the luminal cover of endothelial cells, is considered to be atheroprotective. During atherogenesis, platelets adhere to the vessel wall, possibly triggered by simultaneous EG modulation. It was the objective of this study to investigate both EG thickness and platelet-vessel wall interactions during atherogenesis in the same experimental model. Intravital fluorescence microscopy was used to study platelet-vessel wall interactions in vivo in common carotid arteries and bifurcations of C57bl6/J (B6) and apolipoprotein E knock-out (ApoE-/-) mice (age 7 - 31 weeks). At the same locations, EG thickness was determined ex vivo using two-photon laser scanning microscopy. In ApoE-/- bifurcations the overall median level of adhesion was 48 platelets/mm2 (interquartile range: 16 - 80), which was significantly higher than in B6 bifurcations (0 (0 - 16), p = 0.001). This difference appeared to result from a significant age-dependent increase in ApoE-/- mice, while no such change was observed in B6 mice. At the same time, the EG in ApoE-/- bifurcations was significantly thinner than in B6 bifurcations (2.2 vs. 2.5 μm, respectively; p < 0.05). This resulted from the fact that in B6 bifurcations EG thickness increased with age (from 2.4 μm in young mice to 3.0 µm in aged ones), while in bifurcations of ApoE-/- mice this growth appeared to be absent (2.2 μm at all ages). During atherogenesis, platelet adhesion to the wall of the carotid artery bifurcation increases significantly. At the same location, EG growth with age is hampered. Therefore, glycocalyx-reinforcing strategies could possibly ameliorate atherosclerosis.

Dextrans bind to the surface of platelets, red blood cells and endothelium. We investigated whether a low doses (30 mg/kg IV) of 40-kDa (Dx40), neutral, 500-kDa (Dx500) or sulphated, 500-kDa (Dx500S) dextrans influence platelet distribution in rabbit mesenteric arterioles and venules (diameter 17-33 microns). Intravital fluorescence videomicroscopy was used to visualize platelets labelled in vivo with acridine red. Their concentration distribution determined within a thin optical section about the median vessel plane was expressed relative to the mean concentration in that vessel. In arterioles, Dx500 and Dx500S increased the relative platelet concentration in the centre [radial position (R): 0.0-0.4 R] from 0.60 to 1.07 (P < 0.001) and 1.20 (P < 0.003), and reduced it near the wall (0.8-0.9 R) from 1.59 to 0.93 (P < 0.02) and 0.95 (P < 0.03) respectively. In venules a similar, but non-significant, effect was observed. Dx40 did not change platelet distribution in arterioles, but decreased their concentration in venules in the centre from 1.08 to 0.71 (P < 0.03) and increased it at the wall from 0.89 to 1.27 (P < 0.04). The deformability of red blood cells was unchanged, but their aggregation tendency increased approximately two-fold after Dx500 and Dx500S injection, while Dx40 had no influence. Leucocyte margination in venules did not affect platelet distribution. Dextran injection did not change microvascular flow velocity or plasma viscosity, suggesting that the observed changes in arteriolar platelet distribution were caused by binding of dextran to the surface of platelets and/or red blood cells.

Platelet distribution was investigated in 21 venules (V) and 10 arterioles (A) of the rabbit mesentery (vessel diam 15-33 microns). Circulating platelets were labeled in vivo with the dye acridine red and observed with fluorescence video microscopy. Only platelets flowing in a thin (5-7 microns) optical section located about the median plane of the vessel were used. The relative position of each platelet, i.e., the distance of its centroid to the left vessel wall divided by the local vessel diameter, was determined. In addition, in 10 venules leukocyte margination was inhibited by intravenous injection of dextran sulfate (500,000 mol wt; 30 mg/kg body wt). The number of platelets per unit volume (i.e., platelet density) relative to the mean density was significantly higher in the vessel center of V (1.04) than of A (0.55; P less than 0.005). In contrast, near the wall this density was significantly higher in A compared with V. Mean values were as follows: at radial position (R) = 0.9-1.0, 0.30 in A and 0.11 in V (P greater than 0.05); at R = 0.8-0.9, 1.63 in A and 0.84 in V (P less than 0.002); at R = 0.7-0.8, 1.60 in A and 1.36 in V (P greater than 0.05); at R = 0.6-0.7, 1.16 in A and 1.60 in V (P less than 0.02); and at R = 0.5-0.6, 0.92 in A and 1.36 in V (P less than 0.02). These differences in platelet distribution between arterioles and venules are not caused by the presence of leukocyte margination in venules.

The influence of platelet-vessel wall interactions on leukocyte rolling was investigated in rabbit mesenteric venules (diameter, 21-40 microns) using intravital videomicroscopy. Puncture of the wall with glass micropipettes (tip, 6-8 microns) evoked the formation of a thrombus in all venules. In most vessels, emboli were produced as well. The rolling of leukocytes (i.e., their movement along the vessel wall at a velocity clearly lower than that of the other blood cells) was quantitated simultaneously in vessel segments upstream and downstream from a thrombus up to 10 minutes after puncture. During embolization the number of rolling leukocytes decreased significantly from the upstream to the downstream vessel segment (median decrease, 45%; p less than or equal to 0.001). It was still decreased by approximately 50% after embolization had stopped, indicating that the decrease in leukocyte rolling was not caused by inclusion of leukocytes in the emboli. In venules without embolization, leukocyte rolling did not change systematically, indicating that fluid dynamic changes induced by the thrombus do not influence leukocyte rolling. Inhibition of prostaglandin formation with aspirin (100 mg/kg) almost completely abolished the influence of the thromboembolic reaction on leukocyte rolling, but blockade of thromboxane A2 receptors with sulotroban (30 mg/kg) had no effect. In conclusion, this is the first report on a functional interaction in vivo, at a site of vessel wall injury, between platelets, vascular cells, and leukocytes. The findings suggest that substances produced by activated platelets and/or damaged vascular cells diminish leukocyte rolling. The identity of these substances is not yet clear, but the present study indicates that prostaglandins other than thromboxane A2 are involved.

The distribution of blood platelets flowing in arterioles (21-35 microns) of the mesentery of anesthetized rabbits was studied using intravital fluorescence microscopy. Sites were selected without upstream branch points within at least 10 vessel diameters. The distribution was determined by counting in flashed video frames the number of platelets present in each of six equal segments across the vessel. Only platelets were counted that could be localized objectively within a thin optical section around the median plane of the vessel. It could be shown that differences in counting volume between the six segments were negligible. Because of the use of flashed pictures (flash duration less than 0.1 ms; interval 180 ms), the method is independent of differences in velocity over the cross-sectional area of the vessel. In all measurements (15 sites in 13 vessels in 10 animals) the distribution was nonuniform, the wall segments containing the highest platelet numbers. The general distribution as calculated from all measurements (total platelet number 6,571) and expressed in percentages was found to be 23.0, 14.6, 12.5, 12.1, 13.6, and 24.2.

Velocity profiles were determined in rabbit mesenteric arterioles (diameter 17-32 micron). A good spatial resolution was obtained by using the blood platelets as small and natural markers of flow, providing for the first time in vivo detailed, quantitative information about the shape of the velocity profiles in microvessels. In some experiments red blood cell velocity profiles were recorded as well. Easy detection of the cells of interest could be achieved by labelling them selectively with a fluorescent dye and visualizing them by intravital fluorescence video microscopy, using flashed illumination. Pairs of flashes were given with a short, preset time interval between both flashes, yielding in one TV picture two images of the same cell displaced over a certain distance for the given time interval. Velocity and mean radial position of cells, flowing within an optical section around the median plane of the vessel, were determined. The shape of the velocity profiles of platelets and red blood cells was similar. The profiles were flattened as compared to a parabola, both in systole and diastole. Vessel diameter did not change measurably during the cardiac cycle. As an index of the degree of blunting of the profiles, the ratio of the maximal and mean velocity of the profile was used, which is 2 for a parabola and 1 for complete plug flow. The index ranged from 1.39 to 1.54 (median 1.50), and increased with vessel diameter. Calculations showed that the blunting of the profiles cannot be explained by an influence of the finite depth of the optical section.

Velocity profiles, as determined in vivo in rabbit mesenteric arterioles with fluorescently labeled platelets as natural flow markers, were used to calculate least estimates of the actual wall shear rate in these microvessels (17-32 micron diam). The fit of the velocity data points described the profile as close to the wall as 0.5 micron. To satisfy the no-slip condition, a thin layer of fluid with a steep velocity gradient near the wall was assumed. Least estimates of wall shear rate, as calculated from the fitted platelet-velocity profiles and using the mean velocity gradient in this layer of fluid, ranged from 472 to 4,712 s-1 with a median value of 1,700 s-1. Red blood cell center-line velocities varied between 1.3 and 14.4 mm/s (median 3.4). The wall shear rates were at least 1.46-3.94 (median 2.12) times higher than expected on the basis of a parabolic velocity distribution but with the same volume flow in the vessel. Considerable spatial differences in wall shear rate might exist even within a short segment of a vessel.

In rabbits, a clear microscopic image of individual, fluorescent blood platelets flowing in a mesenteric microvessel could be obtained over the whole cross-sectional area of the vessel, following an intravenous injection of a solution containing 5-15 mg of the fluorochrome Acridine Red. Most of the circulating platelets were labeled. Activation of the platelets by the injected dye was not seen. A fall in platelet count or hematocrit following injection did not occur. Electron microscopy revealed no gross ultrastructural changes. In vitro, the dye reduced platelet aggregation in a dose dependent way. In vivo, aggregation and adhesion of platelets as induced by laser injury or transection of an arteriole was observed in all cases, even following multiple injections of Acridine Red. Primary hemostatic plug formation times as measured in mesenteric arterioles were normal after the first and second injection of 5 mg Acridine Red, but prolonged after subsequent injections. Bleeding times as measured on the ear were prolonged following injection of 15 mg of the dye. It is concluded that this labeling procedure allows the study of the rheological behavior of the platelets in vivo. Whether this technique can also be used to study functional platelet behavior in vivo, needs further investigation.

Blood platelets labeled in vivo with the fluorochrome acridine red can be localized objectively within a thin optical section, using fluorescence microscopy. This allows the study of the rheological behavior of platelets in vivo as exemplified by the assessment of the concentration distribution and the orientation of platelets flowing in small arterioles of the rabbit mesentery.

Spatial orientation of blood platelets flowing in small arterioles of the rabbit mesentery was investigated using intravital fluorescence microscopy. Only platelets were studied that could be localized objectively within a thin optical section around the median plane of the vessel. The orientation of a platelet was assessed from its microscopic image and described by an angle pair, assuming the platelet to be an ellipsoidal body and using an empirical frequency distribution for its thickness to diameter ratio. The orientation of the platelets was not random; they tended to align themselves with their equatorial planes parallel to the wall. The degree of alignment increased from the center of the vessel toward the wall. As a corollary a frequency distribution of their diameter (3.15 +/- 0.72 micron (mean +/- sd) was obtained in vivo.