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Background: Vascular endothelium is covered by a glycocalyx. Damage to the glycocalyx after systemic inflammation or ischaemia/reperfusion contributes to increased vascular permeability and leucocyte adhesion. The underlying mechanisms leading to ischaemia/reperfusion-induced glycocalyx shedding are incompletely understood, in terms of lack of oxygen, absence of flow, or return of oxygen., Methods: Isolated guinea pig hearts perfused with Krebs-Henseleit buffer at 37°C underwent 20 min of either stopped-flow ischaemia or hypoxic perfusion with subsequent reperfusion/reoxygenation (n = 6 each). Hearts perfused with normoxic buffer served as time controls. Epicardial transudate was collected to assess coronary net fluid filtration, colloid extravasation, and histamine release by mast cells. Syndecan-1 and heparan sulphate were measured in coronary effluent, together with lactate, purines, and the release of mast-cell tryptase β. Additional hearts were perfusion-fixed to visualize the glycocalyx., Results: Both ischaemia and hypoxia with reperfusion/reoxygenation resulted in significant increases in net fluid filtration (P < 0.05) and release of syndecan-1 and heparan sulphate in coronary effluent. These effects were already seen with the onset of hypoxic perfusion. Histamine was released during hypoxia and reoxygenation and also reperfusion, as was tryptase β, and high concentrations of adenosine (>1 µmol litre⁻¹, hypoxia group) and inosine (> 7 µmol litre⁻¹, ischaemia group) were measured in effluent (P < 0.05). Damage to the coronary glycocalyx was evident upon electron microscopy., Conclusions: Both ischaemic and hypoxic hypoxia initiate glycocalyx degradation, promoting an increase in permeability. A contributing mechanism could be purine-mediated degranulation of resident mast cells, with liberated tryptase β acting as potential 'sheddase'.

Background: Heterogeneity of vascular permeability has been suggested for the coronary system. Whereas arteriolar and capillary segments are tight, plasma proteins pass readily into the interstitial space at venular sites. Fittingly, lymphatic fluid is able to coagulate. However, heart tissue contains high concentrations of tissue factor, presumably enabling bleeding to be stopped immediately in this vital organ. The distribution of pro- and anti-coagulatively active factors in human heart tissue has now been determined in relation to the types of microvessels., Methods and Results: Samples of healthy explanted hearts and dilated cardiomyopathic hearts were immunohistochemically stained. Albumin was found throughout the interstitial space. Tissue factor was packed tightly around arterioles and capillaries, whereas the tissue surrounding venules and small veins was practically free of this starter of coagulation. Thrombomodulin was present at the luminal surface of all vessel segments and especially at venular endothelial cell junctions. Its product, the anticoagulant protein C, appeared only at discrete extravascular sites, mainly next to capillaries. These distribution patterns were basically identical in the healthy and diseased hearts, suggesting a general principle., Conclusions: Venular extravasation of plasma proteins probably would not bring prothrombin into intimate contact with tissue factor, avoiding interstitial coagulation in the absence of injury. Generation of activated protein C via thrombomodulin is favored in the vicinity of venular gaps, should thrombin occur inside coronary vessels. This regionalization of distribution supports the proposed physiological heterogeneity of the vascular barrier and complies with the passage of plasma proteins into the lymphatic system of the heart., (Copyright © 2010 S. Karger AG, Basel.)

Aims: Antithrombin is an important inhibitor of the coagulation system, additionally exerting specific anti-inflammatory effects on endothelial cells. Healthy vascular endothelium is coated by the endothelial glycocalyx, diminution of which increases capillary permeability, e.g. after ischaemia. Antithrombin is known to infiltrate the glycocalyx, binding to glycosaminoglycans, and to preserve the glycocalyx after application tumour necrosis factor-alpha. We investigated the influence of antithrombin on glycocalyx subjected to ischaemia/reperfusion., Methods and Results: Isolated guinea pig hearts were perfused with Krebs-Henseleit buffer (KHB). Antithrombin was applied to achieve physiological levels (1 U/mL) before inducing 20 min of ischaemia (37 degrees C). Hearts were reperfused for 20 min at constant flow (baseline perfusion pressure 70 cmH(2)O) with KHB or KHB plus 2 g% hydroxyethyl starch (130 kDa). Coronary net fluid filtration was assessed directly by measuring transudate formation on the epicardial surface. Post-ischaemic coronary release of syndecan-1 and heparan sulfate was quantified by ELISA. Hearts were perfusion-fixed to visualize the glycocalyx by electron microscopy. Ischaemia/reperfusion caused degradation of the glycocalyx, enhanced coronary perfusion pressure, and increased vascular permeability. Antithrombin significantly reduced post-ischaemic glycocalyx shedding, coronary perfusion pressure, coronary leak, and tissue oedema formation compared to untreated hearts. Additional application of colloid augmented these actions of antithrombin. Electron microscopy revealed a mostly intact glycocalyx after antithrombin treatment., Conclusion: Antithrombin preserves the endothelial glycocalyx, sustaining the vascular barrier function and reducing interstitial oedema. The potentiated effect of colloid in these hearts suggests that the prevention of shedding should be of functional benefit also in vivo.

Background: The astonishing thickness of the endothelial glycocalyx, which rivals that of endothelial cells in the microvasculature, was disclosed in the last 15 years. As already demonstrated, this structure plays a key role in the regulation of inflammation and vascular permeability., Methods and Results: Two components of the glycocalyx, syndecan-1 and heparan sulfate, were measured in arterial blood of 18 patients undergoing surgery of the ascending aorta with cardiopulmonary bypass (n=12 with and n=6 without deep hypothermic circulatory arrest) and of 14 patients undergoing surgery for infrarenal aortic aneurysm. Basal values of syndecan-1 (1.2 microg/dL) and heparan sulfate (590 microg/dL) of patients were similar to those of control subjects. Anesthesia and initiation of surgery caused no changes. Global ischemia with circulatory arrest (n=12) was followed by transient 42- and 10-fold increases in syndecan-1 and heparan sulfate, respectively, during early reperfusion (0 to 15 minutes). After regional ischemia of heart and lungs (cardiopulmonary bypass; n=6), syndecan-1 increased 65-fold, and heparan sulfate increased 19-fold. Infrarenal ischemia was followed by 15- and 3-fold increases, respectively (n=14). The early postischemic rises were positively correlated (r=0.76, P<0.001). Plasma concentrations of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 did not change. Circulating polymorphonuclear granulocytes and the level of postischemic heparan sulfate corresponded negatively. Immunohistochemical imaging and immunoassay of isolated hearts (guinea pig) substantiated syndecan-1 and heparan sulfate as components of the endothelial glycocalyx released into the coronary venous effluent. Electron microscopy revealed shedding of the glycocalyx after ischemia/reperfusion., Conclusions: This study provides the first evidence in humans for shedding of the endothelial glycocalyx during ischemia/reperfusion procedures.

Background: Shear stress induces coronary dilatation via production of nitric oxide (NO). This should involve the endothelial glycocalyx (EG). A greater effect was expected of albumin versus hydroxyethyl starch (HES) perfusion, because albumin seals coronary leaks more effectively than HES in an EG-dependent way., Methods: Isolated hearts (guinea pigs) were perfused at constant pressure with Krebs-Henseleit buffer augmented with 1/3 volume 5% human albumin or 6% HES (200/0.5 or 450/0.7). Coronary flow was also determined after EG digestion (heparinase) and with nitro-L-arginine (NO-L-Ag)., Results: Coronary flow (9.50 +/- 1.09, 5.10 +/- 0.49, 4.87 +/- 1.19 and 4.15 +/- 0.09 ml/min/g for 'albumin', 'HES 200', 'HES 450' and 'control', respectively, n = 5-6) did not correlate with perfusate viscosity (0.83, 1.02, 1.24 and 0.77 cP, respectively). NO-L-Ag and heparinase diminished dilatation by albumin, but not additively. Alone NO-L-Ag suppressed coronary flow during infusion of HES 450. Electron microscopy revealed a coronary EG of 300 nm, reduced to 20 nm after heparinase. Cultured endothelial cells possessed an EG of 20 nm to begin with., Conclusions: Albumin induces greater endothelial shear stress than HES, despite lower viscosity, provided the EG contains negative groups. HES 450 causes some NO-mediated dilatation via even a rudimentary EG. Cultured endothelial cells express only a rudimentary glycocalyx, limiting their usefulness as a model system., (Copyright 2007 S. Karger AG, Basel.)

Background: Fluid extravasation may lead to myocardial edema and consequent reduction in ventricular function. Albumin is presumed to interact with the endothelial glycocalyx. The authors' objective was to compare the impact of different resuscitation fluids (human albumin, hydroxyethyl starch, saline) on vascular integrity., Methods: In an isolated perfused heart model (guinea pig), Krebs-Henseleit buffer was augmented with colloids (one third volume 5% albumin or 6% hydroxyethyl starch 130/0.4) or crystalloid (0.9% saline). Perfusion pressure and vascular fluid filtration (epicardial transudate formation) were assessed at different flow rates. After global, stopped-flow ischemia (37 degrees C, 20 min), hearts were reperfused with the same resuscitation fluid additives. In a second series, the authors applied the respective perfusates after enzymatic digestion of the endothelial glycocalyx (heparinase, 10 U over 15 min)., Results: Both 5% albumin and 6% hydroxyethyl starch decreased fluid extravasation versus saline (68.4 +/- 5.9, 134.8 +/- 20.5, and 436.8 +/- 14.7 microl/min, respectively, at 60 cm H(2)O perfusion pressure; P < 0.05), the corresponding colloid osmotic pressures being 2.95, 5.45, and 0.00 mmHg. Digestion of the endothelial glycocalyx decreased coronary integrity in both colloid groups. After ischemia, a transient increase in vascular leak occurred with Krebs-Henseleit buffer containing hydroxyethyl starch and saline, but not with albumin. The authors observed no difference between intravascular and bulk interstitial colloid concentration in the steady state. Notwithstanding, electron microscopy revealed an intact endothelial glycocalyx and no interstitial edema in the albumin group., Conclusion: Ex vivo, albumin more effectively prevented fluid extravasation in the heart than crystalloid or artificial colloid. This effect was partly independent of colloid osmotic pressure and may be attributable to an interaction of albumin with the endothelial glycocalyx.

Atrial natriuretic peptide (ANP) is reported to enhance vascular permeability in vivo. Our aim was to evaluate the impact of ANP on coronary extravasation of fluids and macromolecules and on the integrity of the endothelial glycocalyx. Isolated guinea pig hearts (n = 6/group) were perfused with Krebs-Henseleit buffer in a Langendorff mode. A 6% hydroxyethyl starch (HES) solution was infused into the coronary system for 20 min without (Control group) and simultaneously with (ANP group) ANP at 10(-9) M. In two further series, the glycocalyx was enzymatically degraded by means of heparinase (Hep) application (10 IU over 15 min), followed again by the infusion of HES in the absence (Hep group) and presence (ANP+Hep group) of ANP. Net fluid filtration, extravasation of HES, electron microscopic visualization of the glycocalyx, and quantification of shedding of syndecan-1, a component of the glycocalyx, were determined. An increase in fluid leak was observed in ANP, ANP+Hep, and Hep hearts [+29%, +31%, +14%, respectively; a decrease was observed in Control hearts (-13%)]. Similarly, an accelerated extravasation of colloid was observed in these three groups. Coronary release of syndecan-1 increased 9- to 18-fold during infusion of ANP. Electron microscopy revealed a dramatic degradation of the glycocalyx after ANP. These results indicate that the endothelial glycocalyx serves as a barrier to transmural exchange of fluid and colloid in the coronary vascular system. ANP causes rapid shedding of individual components of the glycocalyx and histologically detectable degradation. Thus the permeability-increasing effect of ANP may be at least partially related to changes in the integrity of the endothelial glycocalyx.

Background: The impact on the endothelial glycocalyx for the extravasation of colloidal infusion solutions has not been investigated sufficiently., Methods: Isolated guinea pig hearts were perfused with Krebs-Henseleit buffer in a Langendorff mode. Solutions of 0.9% saline, 5% albumin (70 kd), or 6% hydroxyethyl starch (200 kd) were infused into the coronary system for 20 min at a rate of one third of the coronary flow, also during reperfusion after 15 min of ischemia, and after enzymatic digestion of the endothelial glycocalyx by heparinase. Net coronary fluid filtration was assessed directly by measuring the formation of transudate on the epicardial surface, and solute extravasation was assessed by measuring albumin and hydroxyethyl starch in the coronary effluent and transudate. Hearts were perfusion fixed to visualize the endothelial glycocalyx using transmission electron microscopy., Results: Only infusion of hydroxyethyl starch, not infusion of albumin, significantly decreased net coronary fluid filtration. Heparinase application without ischemia increased coronary leak by 25% but did not accelerate the passage of colloids. Ischemia alone did not alter permeability. However, there was a large (approximately +200%), transient (approximately 4 min) increase in permeability for water, albumin, and hydroxyethyl starch after ischemia with heparinase application. Also, histamine (10 m) only increased permeability after pretreatment of the hearts with heparinase. The thickness of the glycocalyx after colloid administration was 0.2-0.3 microm. No glycocalyx could be detected after application of heparinase., Conclusion: The endothelial glycocalyx acts as a competent barrier for water and colloids. Only after its destruction do changes in endothelial morphology (postischemic reperfusion or histamine application) become effective determinants of coronary extravasation.

Unlabelled: Polymorphonuclear neutrophils (PMN) play a crucial role in the initiation of reperfusion injury. In a previous study, we found that ketamine reduced the postischemic adherence of PMN to the intact coronary system of isolated guinea pig hearts. Because ketamine is a racemic mixture (1:1) of two optical enantiomers, we looked for possible differences in action between the stereoisomers. Seventy-six guinea pig hearts were perfused in the "Langendorff" mode under conditions of constant flow (5 mL/min) using modified Krebs-Henseleit buffer. After 15 min of global warm ischemia, freshly isolated human PMN (10(6)) were infused as a bolus into the coronary system during the second minute of reperfusion. PMN adhesion was expressed as the numeric difference between PMN recovered in the effluent and those applied. Series A hearts received 5 microM S(+), 5 microM R(-), or 10 microM racemic ketamine starting 20 min before ischemia and during reperfusion. In Series B hearts, 10 microM nitro-L-arginine, an inhibitor of NO synthase, was added to the perfusate. In Series C, PMN were preincubated for 15 min with 5 microM S(+)- or R(-)-ketamine. Coronary vascular leak was assessed by measuring the rate of formation of transudate on the epicardial surface. Ischemia/reperfusion without anesthetics increased coronary PMN adherence from 25.5% +/-2.3% (basal) to 35.3%+/-1.5% of the number applied. S(+)-ketamine reduced postischemic adherence in each series (A, 25.5%+/-5.1%; B, 22.5%+/-1.7%; C, 25.3%+/-7.7%), as did racemate (A, 26.4%+/-3.7%). Although 5 microM R(-)-ketamine had no effect on adhesion (A, 30.5%+/-6.7%; B, 34.3%+/-5.1%; C, 34.3%+/-4.3%), it significantly increased vascular leak in the presence of NOLAG. These findings indicate stereoselective differences in biological action between the two ketamine isomers: S(+)-ketamine inhibited PMN adherence, R(-)-ketamine worsened coronary vascular leak in reperfused isolated hearts., Implications: In this study, we demonstrated stereoselective differences in the biologic action of the two ketamine isomers in an animal model of myocardial ischemia. Polymorphonuclear neutrophil adherence to the coronary vasculature after ischemia was inhibited by S(+)-ketamine, whereas R(-)-ketamine increased coronary vascular fluid leak.

Background: Polymorphonuclear neutrophils (PMNs) contribute to postischemic reperfusion damage in many organs and tissues, a prerequisite being adhesion of PMNs to vascular endothelial cells. Because adhesion processes involve orderly interactions of membrane proteins, it appeared possible that "membrane effects" of volatile anesthetics could interfere. We investigated the effects of halothane, isoflurane, and sevoflurane on postischemic adhesion of human PMNs in the intact coronary system of isolated perfused guinea pig hearts., Methods: The hearts (n = 7-10 per group) were perfused in the "Langendorff" mode under conditions of constant flow (5 ml/min) using modified Krebs-Henseleit buffer equilibrated with 94.4% oxygen and 5.6% carbon dioxide. Global myocardial ischemia was induced by interrupting perfusion for 15 min. In the second minute of reperfusion (5 ml/min), a bolus dose of 6 x 10(5) PMNs was injected into the coronary system. The number of cells reemerging in the coronary effluent was expressed as a percentage of the total number of applied PMNs. Halothane, isoflurane, and sevoflurane, each at 1 and 2 minimal alveolar concentration (MAC), were vaporized in the gas mixture and applied from 14 min before ischemia until the end of the experiment., Results: Under nonischemic conditions, 24.7 +/- 1.3% of the injected neutrophils did not reemerge from the perfused coronary system. Subjecting the hearts to global ischemia augmented retention (36.4 +/- 2.8%, P < .05). Application of halothane reduced adhesion of neutrophils to 22.6 +/- 2.1% and 24.2 +/- 1.8% at 1 and 2 MAC, respectively (P < .05). Exposure to 1 and 2 MAC isoflurane was similarly effective, whereas basal adhesion was not significantly influenced. Sevoflurane-treated hearts (1 and 2 MAC) also showed decreased adhesion of PMNs (23 +/- 2.3% and 24.8 +/- 1.8%, respectively; P < .05) and an identical reduction resulted when sevoflurane (1 MAC) was applied only with the onset of reperfusion., Conclusions: Although the mechanism of action of volatile anesthetics remains unclear in these preliminary studies, their inhibitory effect on ischemia-induced adhesion of PMNs may be beneficial for the heart during general anesthesia.

Unlabelled: Eltanolone is a new steroid anaesthetic agent that is 5-beta reduced derivative of progesterone. In the present study we investigated the haemodynamic effects of eltanolone or thiopentone in patients scheduled for coronary artery bypass grafting., Methods: After obtaining approval of the institutional ethics committee and informed patient consent, 40 patients (age 45-70 years, ASA III and IV, ejection fraction > 50%, cardiac index > 2.5 l/min per m2) were randomly assigned to four groups, each containing 10 patients: After premedication with 2 mg flunitrazepam, anaesthesia was induced with 3 mg/kg thiopentone in group 1, 0.5 mg/kg eltanolone in group 2, 0.75 mg/kg eltanolone in group 3, 1.0 mg/kg eltanolone in group 4. Each patient additionally received 3 mirograms/kg fentanyl after induction and 0.1 mg/kg pancuronium. Heart rate, mean arterial pressure, pulmonary arterial pressure, central venous pressure, pulmonary artery occlusion pressure and cardiac output were recorded in the awake state, 2 min after induction of anaesthesia, and 1 and 5 min after intubation. Cardiac index and systemic vascular resistance were calculated., Results: Two minutes after induction, mean arterial pressure was significantly lower than the baseline (P < 0.05) in each group. Mean arterial pressure changes were more prominent in the case of eltanolone, but intergroup tests did not reveal significant differences between the four groups. There was a fall in cardiac index in all groups, and these changes reached the level of significance only in the thiopentone patients. The most obvious difference between eltanolone and thiopentone was systemic vascular resistance. It dropped significantly 2 min after induction with eltanolone at all dosages. In contrast, there was an increase in systemic vascular resistance following induction of anaesthesia with thiopentone. Intergroup tests also showed significantly (P < 0.05) lower systemic vascular resistance 1 and 5 min after intubation with eltanolone compared to thiopentone., Discussion: Mean arterial pressure reduction induced by eltanolone is most likely the result of the combination of a decrease in cardiac contractility and peripheral vasodilatation. In contrast, mean arterial pressure reduction in the case of thiopentone seems to be exclusively related to the negative inotropic properties of the drug. Results of a dosage finding study [5] with eltanolone revealed an AD50 of 0.33 mg/kg. In our study 0.5 mg/kg eltanolone brought all the patients to sleep within 2 minutes. The haemodynamic results do not show any significant difference up to twofold dosage. Therefore, the therapeutic margin seems to be large. Because of considerable interindividual variability additional studies in larger collectives are required for definitive evaluation of the drug.

We investigated the effects of adenosine and nitroglycerin (NTG) on coronary microvessel diameters (intravital fluorescence microscopy) and coronary perfusion (radioactive microspheres). Measurements were performed during baseline conditions (intravenous piritramid) and during controlled hypotension (mean arterial pressure approximately 60 mmHg) induced by halothane, adenosine, and NTG. Coronary vascular resistance (CVR) remained unchanged during halothane (-7%) but decreased during adenosine (-76%) and NTG (-29%). Coronary arteriolar diameters increased during all experimental steps. In the smallest vessels (20-40 microns), diameters increased by 14, 43, and 42% during halothane-, adenosine-, and NTG-induced hypotension, respectively. Diameter increases were less pronounced in larger vessels. The uniform action of adenosine and NTG in 20- to 500-microns arterial vessels is in contrast to the pronounced differences in reduction of CVR. Preferential dilation of arterioles < 20 microns or recruitment of coronary microvessels by adenosine might account for the more pronounced decrease of CVR during adenosine. Intracoronary application of adenosine (0.8 mg.kg-1.h-1) and NTG (1, 5, and 25 micrograms.kg-1.h-1) equally caused near-maximum dilation of coronary arterioles > 100 microns. However, NTG dilation of arterioles < 100 microns was dose dependent and exceeded large-vessel dilation only with the highest concentration of NTG.

An experimental model was established for fluorescence video microscopy of coronary microvessels. Nineteen dogs were anesthetized with a narcotic. Catheters were placed for hemodynamic monitoring and sampling of arterial and coronary venous blood. Myocardial perfusion was measured with radioactive microspheres. Following thoracotomy, movements of the myocardial surface area under investigation were restricted by a specially designed heart holder. Plasma was stained with FITC labelled dextran. Diameters were determined in arteriolar and venular microvessels greater than or equal to 20 microns. Measurements were performed during baseline conditions, i.e. only the basic anesthetic drug was applied, and during coronary vasodilation by continuous infusion of adenosine in a randomized sequence. Mean arterial pressure was reduced from 85 +/- 2 mmHg during baseline to 59 +/- 1 mmHg by infusion of 16.9 +/- 2.2 mg.kg-1.h-1 adenosine. Adenosine increased left ventricular blood flow by 253%, left ventricular oxygen demand remained unchanged. A total of 495 arteriolar and 170 venular diameters were measured during baseline condition and during adenosine infusion. Arteriolar diameters increased in all vessel segments between 20 and 600 microns, however, arterioles below a critical size of 100 microns had a greater dilating capacity than larger arterioles. Maximal decrease of segmental resistance occurred in 20-40 microns arterioles and amounted to 74%, which is less than the 82% decrease of total coronary resistance. Venular diameter changes, too, were more pronounced in smaller vessels.

The purpose of this study was to investigate the effects of the anesthetics enflurane and isoflurane and of the coronary vasodilator dipyridamole on myocardial oxygen balance and myocardial tissue oxygen tensions. The studies were performed in 24 open-chest dogs during basal anesthesia with a narcotic. Myocardial blood flow (MBF) was measured using radioactive microspheres, myocardial surface tissue PO2 by means of a platinum multiwire surface electrode. One control group and three experimental groups were studied: enflurane (1.1 vol%), isoflurane (0.7 vol%, both end-tidal concentrations), and dipyridamole (0.4 mg/kg). Mean arterial pressure significantly decreased to an average of 70 mm Hg in all three experimental groups. Although MBF was unchanged during enflurane (-18%) and isoflurane (+20%), it increased during dipyridamole (+304% p less than 0.05 vs baseline and control, enflurane, and isoflurane groups). Myocardial oxygen consumption decreased significantly during enflurane and isoflurane but remained unchanged during dipyridamole. Thus, the ratio between myocardial oxygen delivery and consumption increased 6% with enflurane (p less than 0.05 vs baseline), 47% with isoflurane (p less than 0.05 vs baseline and control group) and 280% with dipyridamole (p less than 0.05 vs baseline and control, enflurane, and isoflurane groups). Coronary venous PO2 remained unchanged during enflurane but increased significantly during isoflurane and dipyridamole. Left ventricular surface tissue PO2 was unchanged in enflurane and isoflurane animals and decreased slightly, yet significantly, during dipyridamole. All variables remained unchanged in the control group. Thus, isoflurane and dipyridamole interfered with MBF autoregulation and increased myocardial oxygen delivery out of proportion to myocardial demands.(ABSTRACT TRUNCATED AT 250 WORDS)