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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 microm) and density (0.045 vs. 0.57 microm(-2)) of internal elastic lamina fenestrae, smooth muscle cell density (3.50 vs. 1.53 microm(-3)), number of elastic laminae (3 vs. 2), and adventitial collagen structure (tortuous vs. straight). EG in elastic arteries was 4.5 microm 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., (Copyright 2007 S. Karger AG, Basel.)

There are basically two types of branching patterns in the terminal part of the arteriolar tree. On the one hand, in a number of tissues, including the developing chick embryo chorioallantoic membrane (CAM), the pattern is dichotomous, whereas in other tissues many arteriolar-arteriolar connections, arcades, are found. The structure of the branching pattern depends on the local physical and chemical environment. The goal of this study was to investigate whether substances with an effect on vascular growth influence the vascular branching pattern. We treated chick embryo CAMs daily from day 7 to day 14 postfertilization with 0.9% NaCl, angiotensin II (ANG-II), ANG-II in combination with different angiotensin receptor subtype antagonists, i.e., losartan and CGP 42112A, or the prostaglandin synthesis inhibitor acetylsalicylic acid (ASA). Arcade formation was quantified by counting the number of arcades per cm2 treated area, the branch-node ratio and mean surface area of arcade loops. ANG-II caused a 2-fold increase in the number of arcades versus 0.9% NaCl. Addition of ASA or losartan caused a further enhancement of arcade formation expressed in the number and branch-node ratio. CGP 42112A had no significant effect on arcade formation. From these data we hypothesize that ANG-II stimulates the process of capillary upgrading to arterioles by stimulation of arteriolar smooth muscle cell growth. Prostaglandins normally counteract this effect. After blockade of prostaglandin action, the ANG-II-induced arterialization is enhanced, resulting in pronounced arcade formation. The actions of losartan may be related to its inhibitory effects on prostaglandins rather than angiotensin receptor antagonism.

The observed positive correlation between cessation of red blood cell flow in capillaries at low perfusion pressures and the oxygen tension (PO2) in the superfusion solution may be due to oxygen-dependent arteriolar constriction. To test this hypothesis, we investigated capillary flow cessation during aortic occlusion and concomitant changes in diameters of terminal arterioles and capillaries in normal and vasodilated vascular beds of rabbit tenuissimus muscle (n = 15) by means of video intravital microscopy. In the vasodilated bed, arteriolar tone was eliminated by local application of 10(-4) M adenosine (ADO). The PO2 in the superfusate was varied locally, i.e., in the solution between objective lens and muscle surface. At a local PO2 of 40 mm Hg without ADO, flow ceased in about 50% of the capillaries during aortic occlusion while the arterioles dilated to 118% of control (median; p < 0.001). Addition of ADO led to an increase in arteriolar and capillary diameter to 220% (median; p < 0.001) and 121% (median; p < 0.05), respectively. Under ADO, the incidence of capillary flow cessation was reduced (p < 0.05) to about 20%. Elevation of the local PO2 from 40 to 100 mm Hg in the presence of ADO did not lead to a significant change in the incidence of flow cessation, nor to changes in arteriolar or capillary diameter. In the presence of ADO, median arteriolar and capillary diameters during aortic occlusion were 96% (p < 0.001) and 7% (p < 0.05) larger than their control diameters without ADO, respectively. In summary, it is suggested that the incidence of flow cessation may depend on both the arteriolar and the capillary diameter. Of these two factors, capillary diameter may be the most important one, because its changes affect the interaction between red blood cells and the vessel wall in the narrow capillaries, and, hence, the resistance to flow. In the presence of ADO, at elevated local PO2 levels flow cessation still occurs in about 20-30% of the capillaries, suggesting that arteriolar contraction is only in part responsible for the incidence of flow cessation.

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.

Perfusion of capillaries was investigated in the tenuissimus muscle of young anesthetized New Zealand White rabbits during control conditions, complete aorta occlusion, and reactive hyperemia at various local oxygen tensions. Capillaries were visualized with bright-field microscopy. The number of capillaries perfused under experimental conditions was compared with that during control conditions. Capillary diameter was measured to assess whether the interventions caused changes in luminal diameter. During control conditions at a local pO2 of about 20 mm Hg, capillary perfusion fluctuates; instantaneous capillary density is smaller than anatomical capillary density. When the aorta is (partially) occluded, capillary perfusion becomes continuous and instantaneous capillary density equals anatomical capillary density. The latter is also observed during the early phase of reactive hyperemia, prior to the reappearance of flowmotion. Capillary diameter is not invariant during these interventions, but decreases by 8% during occlusion and increases by 12% during reactive hyperemia. The concomitant change in perimeter and cross-sectional area should be factored in with functional capillary density, when tissue exchange surface area or volume flow are considered. When during control conditions, the muscle becomes locally (under the microscope lens) exposed to a higher oxygen tension, capillary diameter does not change. However, the relative number of capillaries perfused at complete aorta occlusion is unity at low local oxygen, and diminishes with increasing local oxygen to become 0 at an oxygen tension of about 70 mm Hg. In preparations in which capillary diameter is not invariant under the experimental conditions, functional capillary density can only be used to compare the number of perfused capillaries with the number of capillaries anatomically present. Capillary diameter has to be factored in when tissue perfusion or exchange surface area are considered.

TMo date, capillary microscopy, transcutaneous oximetry (tcpO2) and laser Doppler fluxmetry are frequently used in the investigation of skin microcirculation in patients with lower limb ischaemia. The concomitant microcirculatory disturbances may be useful in addition to macrocirculatory parameters to discriminate the different degrees of ischaemic severity. The best ways of application of these methods and the choice of the best parameters to assess ischaemia have been insufficiently investigated. Therefore, skin microcirculation was investigated with the use of these techniques in 130 patients with different stages of lower limb ischaemia, divided according to their ankle-to-brachial pressure index (ABI). Patients were investigated in the sitting and the supine position. Measurements were performed at rest and during reactive hyperaemia following arterial occlusion, and before and after local skin heating. The reactive hyperaemic response using laser Doppler fluxmetry differed in every patient group investigated. Capillary red blood cell velocity was markedly impaired in critically ischaemic patients (ABI < 25%). Transcutaneous oxygen pressure measurements at rest rendered the highest positive predictive value (PV; 87%) to classify patients as having clinically severe ischaemia (Fontaine 3 or 4). Ankle and toe pressure measurements provided a PV value of 78%. Microcirculatory parameters and techniques appear to be useful as an addition to standard macrocirculatory techniques to assess the severity of lower limb ischaemia. This is particularly of importance in patients in whom macrocirculatory parameters are unattainable.

Recombinant human erythropoietin therapy (ReHuEpo) at short term leads to an increase in systemic vascular resistance and to a decrease in cardiac index and skin microcirculatory flow. Long-term adaptive changes might occur. We studied the effects of ReHuEpo therapy on macrocirculation and skin microcirculation in 8 normotensive and normovolemic hemodialysis patients before and after, respectively, 4 and 14 months of treatment. The reported macrocirculatory changes at short term were at long term not significantly different as compared with the initial values. The mean arterial pressure remained unaltered, as might be explained by the slow correction of the hematocrit and the decrease in cardiac output in all initially long-lasting normotensive patients who were maintained normovolemic. Left ventricular end-diastolic dimensions decreased, and also the left ventricular muscle mass decreased, depending on the initial left ventricular muscle mass. The skin oxygenation improved, whereas the maximal capillary flow decreased both at short- and long-term ReHuEpo treatment. The number of capillaries in the nail fold remained unchanged. However, the percentage of tortuosity decreased significantly during ReHuEpo therapy.

The sensitivity of fluorescence intravital microscopy for the detection of immunologically elicited inflammatory reactions in the skin was compared to assessment by tissue swelling measurement. Rats were immunized to the contact-sensitizing agent toluene diisocyanate (TDI) by its application to the skin and challenged by the same procedure at a different site one week thereafter. At 2, 6, or 12 hrs after challenge individual rats received a bolus of fluorescently labeled albumin intravenously. Vascular integrity was determined by estimating the surface of areas with increased vascular permeability as observed by fluorescence intravital microscopy; contact dermatitis (CD) was assessed just prior to infusion of labeled albumin determination of tissue swelling by a semi-automatic micrometer. At 12 hrs after challenge, CD was detectable by tissue swelling. Intravital fluorescence microscopy revealed massive extravasation of the fluorescent dye at the site of challenge. At 6 hrs after challenge, CD was not yet discernible by toe thickness measurements. However, by fluorescence intravital microscopy significant leakage of labeled albumin was observed. Thus, at 6 hrs after challenge fluorescence intravital microscopy was more sensitive than determination of tissue thickness increment for detection of a contact hypersensitivity reaction. At 2 hrs after challenge, CD was neither demonstrable by tissue swelling measurements, nor by fluorescence intravital microscopy. Therefore, an early component of a delayed-type hypersensitivity (DTH) reaction to a contact allergen as shown to occur in mice, remains to be demonstrated in the rat. On the other hand, fluorescence intravital microscopy revealed non-immunological skin irritation by a 5% TDI solution within 2 hrs after its application to the skin of non-immunized rats.

Microcirculatory and haemorheological parameters were investigated before and after plasmapheresis in eighteen patients with secondary Raynaud's phenomenon based on progressive systemic sclerosis. After 4 plasmaphereses, once a week, all patients claimed explicit improvement of their complaints. Raynaud's phenomenon and especially the reaction upon cold provocation had disappeared and skin ulcers healed. Red blood cell (RBC) velocity increased significantly (p less than 0.001) after 4 weeks plasmapheresis. RBC aggregation and plasma viscosity were significantly lower (p less than 0.001) after the last plasmapheresis than before treatment. After 3 years four patients were still free of complaints, but in 14 patients the symptoms of Raynaud's phenomenon had reappeared after 6 to 9 months. The skin ulcers, however, did not return in these patients. RBC aggregation and plasma viscosity returned to the initial values after 9 months, while skin capillary blood flow remained significantly enhanced for 24 months. The finding that restoration of RBC aggregation and plasma viscosity to normal level is associated with enhanced skin capillary blood flow, indicates that disturbed haemorheology plays a role in the diminished skin blood flow, as observed in patients with secondary Raynaud's phenomenon. In these patients, plasmapheresis can be considered to treat severe ischemia of the digits.

In 9 chronic hemodialysis patients, treated with recombinant human erythropoietin (rHuEpo), longitudinal studies were performed to investigate possible changes in macro- and microcirculatory parameters during normovolemia, as assessed echographically by determining the inferior vena cava diameter and adjusting dialysis dry weight. Hematocrit increased from 19 +/- 4 to 33 +/- 5% (p less than 0.001). Systemic vascular resistance increased from 1,020 +/- 259 to 1,283 +/- 245 dyn/s/cm-5 (p less than 0.02), while mean arterial pressure remained unchanged. Cardiac index decreased (4.9 +/- 1.4 to 3.8 +/- 0.9 liters/min/m2; p less than 0.02), caused by a decrease in heart rate (87 +/- 21 to 75 +/- 16 beats/min; p less than 0.02) and stroke index (59.9 +/- 15.2 to 51.0 +/- 10.7 ml/m2; p less than 0.02). Red blood cell volume increased (468 +/- 105 to 858 +/- 203 ml/m2; p less than 0.001) and plasma volume decreased inversely ([125I]-albumin; 2,008 +/- 338 to 1,664 +/- 225 ml/m2; p less than 0.001), whereas total blood volume remained unaltered (2,476 +/- 397 to 2,518 +/- 352 ml/m2; n.s.). Total body weight increased (57.8 +/- 12 to 62.1 +/- 12 kg; p less than 0.02), indicative of an anabolic effect of rHuEpo therapy. Skin capillary circulation as measured by transcutaneous oxymetry at 37 degrees C skin temperature impaired, reflected by the increase of the time to peak after arterial occlusion (82 +/- 21 to 121 +/- 25 s; p less than 0.02). The reactive hyperemic response following the release of occlusion showed a significant increase at high hematocrit (10.7 +/- 4.2 to 16.6 +/- 5.3 mm Hg; p less than 0.02), whereas resting transcutaneous Po2 values showed a slight but not significant increase (2.3 +/- 1.3 to 4.7 +/- 3.3 mm Hg; n.s.). The high number of pathological capillaries in hemodialyzed patients might be an additional factor for the increase in systemic vascular resistance.

Recruitment of terminal arterioles and capillaries was studied in the tenuissimus muscle of 16 young urethane anesthetized rabbits. In one set of experiments (8 animals) intravital fluorescence microscopy was used to visualize transverse arterioles and to assess the number of their first order side branches (functionally the terminal arterioles) perfused with red blood cells before and after vasodilation (10(-4)M adenosine, applied topically). In another set of experiments (8 animals) the number of muscle capillaries perfused with red blood cells from the same parent arteriole was determined at rest and during vasodilation (reactive hyperemia), using bright field microscopy. All capillaries and first order side branches that were perfused during vasodilation were also perfused at rest. These findings indicate that in this skeletal muscle preparation no recruitment of arterioles or capillaries occurs in an anatomical sense of the word.

A simple incident illuminator (epiilluminator) for intravital microscopy using a mirror with 50% reflectance and 50% transmittance, placed at 45 degrees to the optical axis of the microscope, in combination with a polarizer and analyzer is described. Such a system can easily be mounted in a Ploemopak incident illuminator as used for fluorescence microscopy. Images of the red blood cells contained in the microvessels appear as dark on a light background. With the use of the system, microscopic images of good quality could be obtained from human skin capillaries, and skin, pial and intestinal microvessels in rats. In muscle, however, only the superficial capillaries could be adequately visualized.

Cessation of muscle capillary flow was studied in rabbit tenuissimus muscle, using intravital microscopy. Perfusion pressure was reduced by arterial occlusion or simultaneously elevated venous and tissue pressure. Muscle capillary flow ceased in the presence of arteriolar dilation at a median perfusion pressure of 17.5 mmHg during arterial occlusion and at 25.3 mmHg in case of simultaneously increased tissue and venous pressure. The pressure difference of 7.8 mmHg was confirmed in a series of experiments where arterial occlusion and increased tissue pressure were randomly alternated in the same animal. The mechanisms responsible for the difference in pressure level at which flow ceases in the capillaries under these circumstances are incompletely understood. Possible mechanisms are discussed.

The effect of simultaneously reduced perfusion and transmural pressure on muscle capillary flow was investigated in the tensor plagiopatagii muscle of unanesthetized bats. Perfusion and transmural pressure were simultaneously changed by suction applied to a sealed box containing the body of the bat, while the wing with the muscle under observation outside the box was exposed to atmospheric pressure. Flow ceased in almost all of the muscle capillaries at box pressures ranging from -46 to -96 mmHg (n = 40; median value: -66 mmHg). When flow in the muscle had ceased, flow was still present at a reduced level in capillaries on top of the muscle and in the web. No signs of obstruction to flow in the feeding or collecting vessels were found. The results are not in accordance with the phenomena causing cessation of flow as predicted by the theories of critical closure or interfacial tension. To restore flow in the muscle capillaries suction to the box had to be reduced by 14 mmHg (medium value; range 4 to 35 mmHg). The latter data conforms to the possible existence of yield stress in small vessels.

Conventional microscopes can be adapted for telescopic imaging by using a projection eyepiece which focuses the image formed by a standard microscope objective at such a large distance that light emanating from points on the object leaves the eyepiece as parallel bundles. A telescopic transfer lens forms the final image in its focal plane independent of the distance between the eyepiece and the transfer lens. In the system reported in this paper, the distance between the eyepiece and transfer lens can be varied from 0 to 280 mm without appreciable vignetting or image degradation for imaging on 35 mm film or smaller formats. This allows great flexibility in the vertical location of the plane of observation while permitting rigid fixation of the recording system-transfer lens combination. By use of a 45 degrees rotatable mirror between the transfer lens and the recording systems, two or more recording systems can be securely mounted and adjusted in advance and rapid selection among them can be made during the course of an experiment.

A non-invasive, non-traumatic model has been developed for long term study of the vascular anatomy, blood cell velocity and vascular reactivity of the cutaneous microcirculation of the rat. In the anesthetized rat, the hairless area of a toe close to the nail could be visualized by intravital video microscopy without manipulation of the skin. Good image quality of the vascular bed was obtained with the use of an incident illuminator equipped with a 50% mirror at 45 degrees to the optical axis and a polarizer-analyzer system in combination with a mercury lamp. This microscope system allows for visualization of the superficial vasculature of the skin, while microvessels can be repeatedly identified for several weeks. To test for vascular reactivity off-line analysis of blood cell velocity was performed in the control situation and during reactive hyperemia, using a video dual-window system and a cross-correlator. The mean control velocity in venules selected for testing of vascular reactivity was 70 micron/s. In these vessels a reproducible pattern of reactive hyperemia was seen with a mean peak velocity of 655 micron/s following arterial occlusion of 180 s. Possible applications of the model are discussed.