M. G. A. oude Egbrink

Two-photon microscopy of vital murine elastic and muscular arteries : Combined structural and functional imaging with subcellular resolution

Understanding vascular pathologies requires insight in the structure and function, and, hence, an imaging technique combining subcellular resolution, large penetration depth, and optical sectioning. We evaluated the applicability of two-photon laser-scanning microscopy (TPLSM) in large elastic and small muscular arteries under physiological conditions. Elastic (carotid) and muscular (uterine, mesenteric) arteries of C57BL/6 mice were mounted in a perfusion chamber. TPLSM was used to assess the viability of arteries and to visualize the structural components elastin, collagen, nuclei, and endothelial glycocalyx (EG). Functionality was determined using diameter changes in response to noradrenaline and acetylcholine. Viability and functionality were maintained up to 4 h, enabling the assessment of structure-function relationships. Structural vessel wall components differed between elastic and muscular arteries: size (1.3 vs. 2.1 µm) and density (0.045 vs. 0.57 µm–2) of internal elastic lamina fenestrae, smooth muscle cell density (3.50 vs. 1.53 µm–3), number of elastic laminae (3 vs. 2), and adventitial collagen structure (tortuous vs. straight). EG in elastic arteries was 4.5 µm thick, covering 66% of the endothelial surface. TPLSM enables visualization and quantification of subcellular structures in vital and functional elastic and muscular murine arteries, allowing unraveling of structure-function relationships in healthy and diseased arteries.

Complementary roles of platelets and coagulation in thrombus formation on plaques acutely ruptured by targeted ultrasound treatment: a novel intravital model

Summary.  Background: Atherothrombosis is a major cause of cardiovascular events. However, animal models to study this process are scarce. Objectives: We describe the first murine model of acute thrombus formation upon plaque rupture to study atherothrombosis by intravital fluorescence microscopy. Methods: Localized rupture of an atherosclerotic plaque in a carotid artery from Apoe−/− mice was induced in vivo using ultrasound. Rupture of the plaque and formation of localized thrombi were verified by two‐photon laser scanning microscopy (TPLSM) in isolated arteries, and by immunohistochemistry. The thrombotic reaction was quantified by intravital fluorescence microscopy. Results: Inspection of the ultrasound‐treated plaques by histochemistry and TPLSM demonstrated local damage, collagen exposure, luminal thrombus formation as well as intra‐plaque intrusion of erythrocytes and fibrin. Ultrasound treatment of healthy carotid arteries resulted in endothelial damage and limited platelet adhesion. Real‐time intravital fluorescence microscopy demonstrated rapid platelet deposition on plaques and formation of a single thrombus that remained subocclusive. The thrombotic process was antagonized by thrombin inhibition, or by blocking of collagen or adenosine diphosphate receptor pathways. Multiple thrombi were formed in 70% of mice lacking CD40L. Conclusions: Targeted rupture of murine plaques results in collagen exposure and non‐occlusive thrombus formation. The thrombotic process relies on platelet activation as well as on thrombin generation and coagulation, and is sensitive to established and novel antithrombotic medication. This model provides new possibilities to study atherothrombosis in vivo.

Endothelial glycocalyx thickness and platelet-vessel wall interactions during atherogenesis

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.

Different effects of anesthetics on spontaneous leukocyte rolling in rat skin.

In immunological reactions, leukocytes need to travel from the intravascular space through the vessel wall into the surrounding tissue. The first step in this process is leukocyte rolling, which has often been studied in anesthetized animals. In this study, we investigated the effect of pentobarbital, Hypnorm and both components of the latter, fentanyl and fluanisone, on this primary leukocyte-endothelial cell interaction. Using intravital brightfield video microscopy, observations were made in postcapillary venules in the intact skin of the nailfold of trained conscious Lewis rats. Subsequently, the animals were anesthetized and observations were made in vivo. Leukocyte rolling was significantly elevated after injection of Hypnorm or fentanyl, while pentobarbital and fluanisone had no effect. None of the anesthetics affected leukocyte rolling velocity. Blood flow was significantly increased only after injection of Hypnorm and fluanisone. No correlation existed between the relative changes in leukocyte rolling and concomitant changes in blood flow. The results show that the level of leukocyte rolling can be affected by anesthetics. These changes are probably not mediated by changes in local hemodynamics. Pentobarbital anesthesia does not influence leukocyte rolling. Therefore, pentobarbital is a suitable anesthetic for observation of leukocyte rolling in skin. Hypnorm significantly increases the level of rolling in skin venules. This effect seems to be caused mainly by fentanyl.

Leucocyte and platelet adhesion in different layers of the small bowel during experimental total warm ischaemia and reperfusion

Ischaemia and reperfusion (IR) of the small bowel is involved in many clinical conditions. A key component in IR‐induced tissue damage is microvascular dysfunction. The aim was to investigate the role of leucocytes and platelets in capillary flow impediment and tissue damage.

The role of mast cells and histamine in leukocyte-endothelium interactions in four rat strains.

Abstract 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.

Role of newly formed platelets in thrombus formation in rat after clopidogrel treatment: comparison to the reversible binding P2Y12 antagonist ticagrelor

Platelet P2Y₁₂ receptors play an important role in arterial thrombosis by stimulating thrombus growth. Both irreversibly (clopidogrel) and reversibly binding (ticagrelor, AZD6140) P2Y₁₂ antagonists are clinically used for restricted periods, but possible differences in platelet function recovery after drug cessation have not been investigated. We treated WKY rats with a single, high dose of 200 mg/kg clopidogrel or 40 mg/kg ticagrelor. Blood was collected at different time points after treatment. Flow cytometry confirmed full platelet protection against ADP-induced αIIbβ₃ activation shortly after clopidogrel or ticagrelor treatment. At later time points after clopidogrel treatment, a subpopulation of juvenile platelets appeared that was fully responsive to ADP. Addition of ticagrelor to clopidogrel-treated blood reduced αIIbβ₃ activation of the unprotected platelets. In contrast, at later time points after ticagrelor treatment, all platelets gradually lost their protection against ADP activation. Perfusion experiments showed abolishment of thrombus formation shortly after clopidogrel or ticagrelor treatment. Thrombus formation on collagen was determined under high shear flow conditions. At later time points, large thrombi formed in the clopidogrel but not in the ticagrelor group, and unprotected, juvenile platelets preferentially incorporated into the formed thrombi. However, platelets from both groups were still similarly reduced in assays of whole blood aggregation. Conclusively, recovery of rat platelet function after ticagrelor differs mechanistically from that after clopidogrel. This difference is masked by conventional platelet aggregation methods, but is revealed by thrombus formation measurement under flow. Juvenile platelets formed at later time points after clopidogrel treatment promoted thrombus formation.

Local Application of Adenosine Induces an Increase of Capillary Diameter in Skeletal Muscle of Anesthetized Rabbits

The effects of locally applied adenosine (ADO) and/or femoral artery pressure reduction (induced by complete aorta occlusion) on capillary diameter were investigated in the tenuissimus muscle of anesthetized rabbits. Capillaries were visualized by means of intravital video microscopy. Diameters were measured using an image shearing device. During control femoral artery pressure (median: 83 mm Hg) and without ADO, median capillary diameter was 4.3 microns (range: 3.2-5.3 microns; 27 capillaries in 7 animals). Complete aorta occlusion (median femoral artery pressure: 18 mm Hg) resulted in a reduction of capillary diameter to 3.9 microns (2.7-4.7 microns; p < 0.0001). Subsequent reactive hyperemia resulted in an increase in diameter to 5.2 microns (3.7-6.0 microns; p < 0.0001). Locally applied ADO (10(-4) M) probably led to complete vasodilation of the arterioles, because their diameters did not further increase during reactive hyperemia after complete occlusion. ADO (10(-4) M) induced an increase of control capillary diameter to 5.5 microns (4.1-6.4 microns; median relative increase: 27%; p < 0.0001), resulting in a decrease of capillary resistance by 61%. In the presence of ADO, aorta occlusion resulted in a capillary diameter decrease to 4.7 microns (3.4-6.1 microns); p < 0.0001). Subsequent reactive hyperemia resulted in an increase to maximally 5.6 microns (4.3-6.4 microns; p < 0.0001). This diameter was approximately the same as the control diameter during ADO. During occlusion in the presence of ADO, capillary diameter was significantly larger (11%; p < 0.0001) than during control without ADO. The capillary diameter changes induced by the various interventions were mainly passive, i.e., proportional to capillary transmural pressure changes. However, capillary diameter was larger during aortal occlusion in the presence of ADO than during control femoral artery pressures without ADO, even though capillary pressure was probably higher in the latter case. It is proposed that the prolonged increase in transmural capillary pressure due to ADO may induce changes in capillary wall configuration, leading to larger diameters.

Two-photon lifetime imaging of blood and blood vessels

We investigated the potential of two-photon excitation microscopy for the imaging in large blood vessels. Experiments were carried out on isolated rat aorta, labeled with a DNA/RNA dye. Images of the vessel wall indicated that a penetration depth of more than 200 micrometers could be reached. Moreover, blood cells and platelets inside blood vessels could be imaged through the vessel wall. Fluorescence Lifetime Imaging (FLIM) was used as a contrast mechanism for discrimination of autofluorescence from fluorescence of labeled blood cells. We were able to observe labeled blood cells through the vessel wall and identify them by their morphology and characteristic fluorescent lifetimes.

Reinforcing pillars for quality culture development: a path analytic model

ABSTRACT To this date, research on the interplay between organisational structure/managerial and organisational value/psychological elements which impact on educational quality is scarce and fragmented. As a consequence of a lack of knowledge in this area, institutions often address these elements in isolation, moving past integral approaches, which reinforce the organisations’ quality culture. In order to examine interrelationships between context characteristics, work-related psychological attitudes of staff and enhancement practices, a path analysis was performed on data collected from academics with teaching coordination roles. The findings highlight the paramount importance of a ‘human relation’ value orientation; this orientation influences empowerment, commitment and communication satisfaction. Rational goal values and ownership are positively related to quality enhancement practices. It is advocated that institutional policies and strategies directed at educational quality enhancement should leave discretionary space for the availing of academics’ expertise. Nurturing collaborative teaching/learning communities with explicit concern for morale, involvement and development, deserves further cultivation.