In vivo interactions between neutrophils and endothelial cells (EC) follow a multistep process involving two distinct neutrophil adhesion receptors. L-selectin, constitutively functional on resting neutrophils, mediates an activation-independent primary interaction resulting in rolling along the venular wall. Subsequent activation of rolling neutrophils induces upregulation and functional activation of beta 2-integrins (CD11/CD18) leading to firm attachment. Based on previous findings we hypothesized that, under shear force, rolling may be essential for successful neutrophil-EC recognition. Here we report results of our studies of human neutrophil behavior in interleukin (IL)-1-activated rabbit mesentery venules, an interaction that requires both L-selectin and beta 2-integrins. Rolling of human neutrophils is L-selection mediated; it was strongly reduced by monoclonal antibody inhibition or enzymatic removal of L-selectin. Furthermore, activation induced L-selectin shedding and, in a dose- and time-dependent fashion, rendered neutrophils unable to recognize inflamed EC despite expression of active beta 2-integrins, which promoted adhesion in vitro. Neutrophils activated for 5 min or longer lost most of their ability to roll. However, 1-3 min after activation, rolling was reduced (not abolished), and cells that were still able to roll displayed a significant tendency for a CD18-dependent transition from rolling to sticking. The whole sequence of events, rolling, sticking, and transendothelial migration, could be observed if an extravascular chemotactic stimulus was applied by superfusing mesenteries with leukotriene B4. Under such conditions, sticking and emigration was blocked when rolling was inhibited by enzymatic removal of L-selectin. Our results indicate that primary neutrophil interaction with inflamed EC through the L-selectin is a prerequisite for neutrophil function at physiological shear rates in vivo.

Our findings support the concept of cardiovascular and microvascular stabilization by infused FFP, in which the increase in microvascular perfusion associated with restored EG is essential for an optimal resuscitation strategy.

A system is described for the in vivo noninvasive measurement of intravascular PO2 at the microscopic level. Under special circumstances the method can also be used to measure interstitial PO2. The PO2 determination is based on the O2-dependent quenching of phosphorescence of palladium-porphyrins bound to albumin. This compound was injected intravenously in the dosage of 30 mg/kg body wt and dissolved in saline to a concentration of 15 mg/ml. The phosphorescence emission was excited by epi-illumination with a strobe xenon arc and measured by a photomultiplier in a well-defined tissue area as small as 15 x 30 microns. A selected portion of the phosphorescence decay was fitted by a single exponential, and the Stern-Volmer equation was used to calculate PO2. Calibration was performed in vitro using saline and blood and was in agreement with previous reports. In vivo observations were made in normal tissue regions from the unanesthetized hamster transparent skin fold chamber preparation. The method allows PO2 determinations, in the range of 0-80 mmHg, in microvessels with diameters of 15-100 microns. Simultaneous transillumination of the tissue also allows measurement of vessel diameter and red blood cell velocity in the same vessels.

Simultaneous measurements of intravascular and interstitial oxygen partial pressure (Po2) in any tissue have not previously been reported, despite the importance of oxygen in health and in disease. This is due to the limitations of current techniques, both invasive and noninvasive. We have optically measured microscopic profiles of Po2 with high spatial resolution in subcutaneous tissue and transplanted tumors in mice by combining an oxygen-dependent phosphorescence quenching method and a transparent tissue preparation. The strengths of our approach include the ability to follow Po2 in the same location for several weeks and to relate these measurements to local blood flow and vascular architecture. Our results show that (i) Po2 values in blood vessels in well-vascularized regions of a human colon adenocarcinoma xenograft are comparable to those in surrounding arterioles and venules, (i) carbogen (95% 02/5% CO2) breathing increases microvascular P02 in tumors, and (iii) in unanesthetized and anesthetized mice P02 drops to hypoxic values at <200 Am from isolated vessels but drops by <5 mmHg (1 mmlg = 133 Pa) in highiy vascularized tumor regions. Our method should permit noninvasive evaluations of oxygen-modifying agents and offer further mechanistic information about tumor pathophysiology in tissue preparations where the surface of the tissue can be observed.

Torres Filho IP, Torres LN, Salgado C, Dubick MA. Plasma syndecan-1 and heparan sulfate correlate with microvascular glycocalyx degradation in hemorrhaged rats after different resuscitation fluids. Am J Physiol Heart Circ Physiol 310: H1468 -H1478, 2016. First published April 1, 2016; doi:10.1152/ajpheart.00006.2016.-The endothelial glycocalyx plays an essential role in many physiological functions and is damaged after hemorrhage. Fluid resuscitation may further change the glycocalyx after an initial hemorrhage-induced degradation. Plasma levels of syndecan-1 and heparan sulfate have been used as indirect markers for glycocalyx degradation, but the extent to which these measures are representative of the events in the microcirculation is unknown. Using hemorrhage and a wide range of resuscitation fluids, we studied quantitatively the relationship between plasma biomarkers and changes in microvascular parameters, including glycocalyx thickness. Rats were bled 40% of total blood volume and resuscitated with seven different fluids (fresh whole blood, blood products, and crystalloids). Intravital microscopy was used to estimate glycocalyx thickness in Ͼ270 postcapillary venules from 58 cremaster preparations in 9 animal groups; other microvascular parameters were measured using noninvasive techniques. Systemic physiological parameters and blood chemistry were simultaneously collected. Changes in glycocalyx thickness were negatively correlated with changes in plasma levels of syndecan-1 (r ϭ Ϫ0.937) and heparan sulfate (r ϭ Ϫ0.864). Changes in microvascular permeability were positively correlated with changes in both plasma biomarkers (r ϭ 0.8, P Ͻ 0.05). Syndecan-1 and heparan sulfate were also positively correlated (r ϭ 0.7, P Ͻ 0.05). Except for diameter and permeability, changes in local microcirculatory parameters (red blood cell velocity, blood flow, and wall shear rate) did not correlate with plasma biomarkers or glycocalyx thickness changes. This work provides a quantitative framework supporting plasma syndecan-1 and heparan sulfate as valuable clinical biomarkers of glycocalyx shedding that may be useful in guiding resuscitation strategies following hemorrhage.

Arteriolar and venular oxygen tension distribution was studied in the subcutaneous connective tissue of the chamber window preparation in conscious Syrian golden hamsters as a function of the systemic PO2, PCO2, pH, arterial pressure and hematocrit, microvascular red blood cell (RBC) velocity, vessel diameter, and blood flow in the same microvessels. PO2 was measured with the phosphorescence decay technique using Pd-meso-tetra(4-carboxyphenyl)porphyrin (30 mg/kg body wt iv). Systemic arterial and venous PO2s were 71.6 +/- 13.1 and 28.4 +/- 5.1 mmHg, while oxygen tension was 45.1 +/- 13.3 mmHg in arterioles and 30.1 +/- 10.7 mmHg in venules. The relatively low arteriolar PO2 and the small arteriolar-venular PO2 gradient indicate that some blood oxygen exits directly to the tissue or is shunted before reaching the capillaries. RBC velocity was the strongest correlate of microvascular PO2 (arterial correlation coefficient = 0.503 and venous correlation coefficient = 0.560, P < 0.001). Microvascular PO2 was also correlated with blood flow, vessel diameter, blood pH, and PCO2 but not with systemic PO2. Arterial oxygen tension was only significantly related to PCO2, pH, and hematocrit. These findings suggest that oxygen delivery to the tissue improves with increasing blood flow velocity and that microvascular PO2 is a locally regulated parameter in the absence of major systemic perturbations.

BackgroundRestoration of endothelial glycocalyx (EG) barrier may be an essential therapeutic target for successful resuscitation. The aim of this study was to compare in vivo the effects of resuscitation with normal saline (NS) to lactated Ringer’s solution (LR), 5% albumin and fresh frozen plasma (FFP) on their ability to maintain EG and barrier function integrity, mitigate endothelial injury and inflammation, and restore vascular homeostasis after hemorrhagic shock.MethodsAnesthetized rats (N = 36) were subjected to hemorrhagic shock (bled 40% of total blood volume), followed by resuscitation with 45 ml/kg NS or LR, or 15 ml/kg 5% albumin or FFP. Microhemodynamics, EG thickness, permeability, leukocyte rolling and adhesion were assessed in >180 vessels from cremaster muscle, as well as systemic measures.ResultsAfter hypotensive resuscitation, arterial pressure was 25% lower than baseline in all cohorts. Unlike FFP, resuscitation with crystalloids failed to restore EG thickness to baseline post shock and shedding of glycocalyx proteoglycan was significantly higher after NS. NS decreased blood flow and shear, and markedly increased permeability and leukocyte rolling/adhesion. In contrast, LR had lesser effects on increased permeability and leukocyte rolling. Albumin stabilized permeability and white blood cell (WBC) rolling/adhesion post shock, comparable to FFP.ConclusionsResuscitation with NS failed to inhibit syndecan-1 shedding and to repair the EG, which led to loss of endothelial barrier function (edema), decline in tissue perfusion and pronounced leukocyte rolling and adhesion. Detrimental effects of NS on endothelial and microvascular stabilization post shock may provide a pathophysiological basis to understand and prevent morbidity associated with iatrogenic resuscitation after hemorrhagic shock.

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