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This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium- and plasma-derived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell-vessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro- and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging.

Accumulation of intracellular free calcium (Ca2+i) may play an essential role in the ischemia/reperfusion injury of skeletal muscle. Although it has been shown that Ca2+i levels significantly increase during ischemia/reperfusion, it is still a matter of debate whether Ca2+i increases during ischemia alone. It was the aim of this study to monitor the in vivo Ca2+i levels in the rat spinotrapezius muscle during ischemia of varying duration and reperfusion, using a ratiometric fluorescence technique, and to investigate the relationship between the postischemic flow patterns and Ca2+i, if any. The muscle was loaded with Indo-1/AM and imaged by a cooled digital camera. Pre- and postischemic tissue perfusion was assessed by means of an analogue camera. Our results show that short-term ischemia (5, 15 and 30 min) and subsequent reperfusion (60 min) does not alter Ca2+i homeostasis and that tissue perfusion promptly recovers after the insult. One or two hours of ischemia resulted in changes in Ca2+i levels, varying from preparation to preparation; increases in some and no changes in others. In these preparations three distinct flow patterns - normal, compromised and no-reflow - could be distinguished during the 60-min reperfusion. Our main conclusion is that in skeletal muscle Ca2+i levels may increase, the increase probably depending on the muscle fiber type exposed.

The understanding of the regulation of the free cytosolic [Ca2+] ([Ca2+]i) in skeletal muscle is hampered by the lack of techniques for quantifying free [Ca2+]i in muscle fibres in situ. We describe a model for studying the dynamics of free [Ca2+]i in the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus (SOL) muscles of the rat in vivo using caffeine superfusion to induce changes in free [Ca2+]i. We assumed that differences in sensitivity between the two muscle types for this substance reflect differences in intracellular Ca2+ handling in the fibres of which these muscles consist. The Indo-1 ratiometric method, using intravital microscopy with incident light, was adapted to measure free [Ca2+]i in vivo. Fluorescence images were collected by means of a digital camera. Caffeine superfusion at 37 degrees C for 2 min, at concentrations of 1, 2, 5, 10 or 20 mmol/l, induced a concentration-dependent increase in free [Ca2+]i and revealed differences in caffeine sensitivity between the muscle types, with the SOL being more sensitive. In a separate set of experiments the contracture threshold, as assessed by topical application of caffeine, was determined in both muscle types. EDL had a higher threshold for developing contracture than SOL. These finding are in agreement with previous in vitro studies. We may conclude that the dynamics of free [Ca2+]i can be assessed reliably in intact mammalian muscle in vivo.

The objective of the present study was to determine the role of mast cells and histamine in leukocyte-endothelium interactions in mesenteric venules of four rat strains: Brown Norway, Lewis, Sprague-Dawley and Wistar. Intravital microscopy showed that the mast cell stabilizer cromoglycate (5 mg/kg i.v. just before exteriorization of the mesentery) did not affect the baseline level and velocity of leukocyte rolling in any of the four strains. This finding is in agreement with the observation that cromoglycate pretreatment only slightly influenced mast cell degranulation in all strains except the Brown Norway. After mast cell stabilization, only in Sprague-Dawley did topical administration of histamine (10(-4) M) result in a significant increase in the level of leukocyte rolling and a decrease in the rolling velocity compared with the time control. Histamine induced leukocyte adhesion only in the Brown Norway strain. In conclusion, the hypothesis presented in other studies, that degranulation of mast cells, and more specifically the release of histamine, is of major importance for the induction of leukocyte-endothelium interactions in rat mesenteric venules is not generally applicable; the present study shows a clear strain dependency.

When perfusion pressure is reduced, red blood cell flow in the capillaries of skeletal muscle ceases at a positive pressure difference across the vascular bed, while arterioles dilate and venules are not constricted. This flow cessation (i.e., cessation of red blood cell flow) and luminal diameter changes in capillaries following femoral arterial pressure reduction were investigated in the rabbit tenuissimus muscle in situ (n = 42) using intravital video microscopy. Arterial pressure was reduced by occlusion of the aorta distal to the renal arteries. During the experiments, leg and muscle were placed in a sealed box. The muscle was exposed to low PO2 by leading a gas mixture deprived of O2 through the box. Locally at the muscle surface, i.e., under the microscope objective, PO2 was varied by varying the PO2 in the superfusion solution. In all experiments, the remainder of the muscle was kept at low (< 20 mm Hg) PO2. The incidence of flow cessation was virtually zero at low local (< 20 mm Hg) PO2 and became almost 100% at local values above 70 mm Hg. Initial equivalent capillary diameters were 3.1-5.8 microm (median 4.0 microm) and did not correlate with local O2 tension. During aorta occlusion, capillary diameters significantly (P < 0.0001) decreased by a median value of 8% at all local PO2 values; in 14 out of 54 capillaries local diameter became less than 2.8 microm. The extent of diameter reduction did not correlate with PO2. In the 14 capillaries in which the diameter became less than 2.8 microm flow cessation occurred in only four cases. The minimal diameter reached was always at the site of an endothelial nucleus. The capillary diameter reductions are probably due to passive recoil. In the 48 capillaries in which flow ceased, only in four cases did a red blood cell stop at the site of the nucleus. We conclude that capillary diameter reductions (local and generalized) lead to a considerable increase in capillary resistance which contributes to the occurrence of flow cessation but cannot solely explain it.

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.

The influence of changes in systemic blood gas and pH values on the thromboembolic reaction following wall puncture was studied in rabbit mesenteric arterioles and venules (diameter 20-40 microns), using intravital video-microscopy. Under normal circumstances the number of emboli produced was higher in arterioles than in venules (6 and 1, respectively). The initial thrombus growth, the number of emboli produced per vessel and the total duration of the embolisation period were not significantly influenced by changes in blood gas and pH values in both arterioles and venules. Therefore, the observed difference in thromboembolic reaction between arterioles and venules cannot be explained by differences in blood gas and pH values in these microvessels. Since reduced velocity, as a measure of wall shear rate, did not correlate with the thromboembolic reaction in arterioles or venules, fluid dynamics can also not explain the difference, indicating that the thrombogenic or antithrombogenic activity of arteriolar and venular walls differs following injury. A combination of hypercapnia and hypoxia was found to result in a prolongation of the average time period needed to produce a new embolus in both vessel types. This prolongation in embolus production time was largely due to the occurrence of periods, in which the thrombus did not grow, reflecting hampering of the adhesion and aggregation of blood platelets to a growing thrombus under hypercapnic/hypoxic conditions.

The sympathetic nervous system is one of the primary factors controlling vascular resistance. Vascular smooth muscle contraction by sympathetic stimulation is mediated primarily by alpha-adrenoceptors. In this study, we investigated the effects of different forms of adrenergic stimulation on simultaneously measured macro- and microcirculatory dynamics in the rat mesenteric vascular bed. Macrocirculatory effects were measured by means of registration of changes in mean arterial pressure (MAP) and blood flow feeding this tissue via a miniaturized Doppler flow probe around the superior mesenteric artery. Microvascular changes were measured at the level of A2 arterioles by means of intravital microscopic registration of arteriolar diameters and red blood cell velocity (RBC vel). Topical or intra-arterial application of noradrenaline caused a dose-dependent decrease in arteriolar diameter and RBC vel at doses that did not cause significant systemic effects. On the other hand, during intravenous application of noradrenaline or other vasopressor substances an increase in MAP and decrease in organ (Doppler) blood flow was not parallelled by a decrease in arteriolar diameter or RBC vel. In contrast, RBC vel increased in a dose-related manner, whereas arteriolar diameter only secondarily decreased to a small degree. Similar results were obtained when the sympathetic nervous system was activated by means of electrical stimulation of the posterior hypothalamus. These data indicate that adrenergic stimulation can elicit different vascular responses. The ultimate microvascular effect depends upon the way of stimulation or route of drug administration.

Changes in vasomotion parameters and their consequences for local arteriolar resistance were studied in transverse arterioles and their first order side branches in the tenuissimus muscle of 10 young urethane anesthetized rabbits during stepwise reduction of arterial pressure, using intravital microscopy. To assess the influence of vasomotion on mean local arteriolar resistance, the effective vascular diameter, as a measure of mean flow carrying capacity, was calculated. The contribution of vasomotion to the mean local resistance is limited in transverse arterioles, but important in first order side branches, dominating the flow fluctuations in the downstream capillaries. During pressure reduction, an over-all increase in vasomotion cycle length and amplitude was found in both transverse arterioles and first order side branches, concomitant with an increase in effective arteriolar diameter and a decrease in local blood flow and reduced velocity, as a measure of wall shear rate. Flow autoregulation was observed in 70% of the arterioles. The changes in cycle length and amplitude showed only limited correlations with local blood flow, reduced velocity, arterial pressure and effective arteriolar diameter. This indicates that it is unlikely that only one of these variables is responsible for the changes in the vasomotion parameters.