Intravital Videomicroscopy Research Articles

Synergistic effect of signaling from receptors of soluble platelet agonists and outside-in signaling in formation of a stable fibrinogen-integrin αIIbβ3-actin cytoskeleton complex

IntroductionThrombus formation in the injured vessel wall is a highly complex process involving various blood-born components that go through specific temporal and spatial changes as observed by intravital videomicroscopy. Platelets bind transiently to the developing thrombus and may either become stably incorporated into or disengage from the thrombus. The aim of the present study was to reveal the processes involved in the formation of a stable thrombus. MethodsPlatelet-rich plasma and washed platelets were studied by the aggregometer. The aggregate stability was challenged by eptifibatide. Platelet Triton-insoluble fraction was prepared and the actin and αIIb content in the cytoskeleton was analyzed by western blot. ResultsMaximal actin polymerization is achieved 1min after platelet activation while maximal αIIbβ3–actin cytoskeleton association requires 5 to 10min of activation and fibrinogen-mediated platelet-to-platelet bridging. Thus, actin polymerization is dependent on platelet activation and requires neither αIIbβ3 integrin occupation nor platelet aggregation. Formation of a stable aggregate requires platelet activation for more than 1min, complete increase in actin cytoskeleton fraction and partial association of αIIbβ3 with the actin cytoskeleton. However, direct αIIbβ3 activation is not sufficient for cytoskeleton complex formation. Thus, stable αIIbβ3–fibrinogen interaction, representing stable aggregate, is achieved after more than 1min agonist activation, involving inside-out and outside-in signaling but not after direct integrin activation, involving only outside-in signaling. ConclusionsFormation of a stable fibrinogen–αIIbβ3–actin cytoskeleton complex is the result of the combined effect of platelet stimulation by soluble agonists, activation of αIIbβ3, fibrinogen binding and platelet-to-platelet bridging.

Poly(ADP-ribose) polymerase-1 inhibition in brain endothelium protects the blood-brain barrier under physiologic and neuroinflammatory conditions.

Blood-brain barrier (BBB) dysfunction seen in neuroinflammation contributes to mortality and morbidity in multiple sclerosis, encephalitis, traumatic brain injury, and stroke. Identification of molecular targets maintaining barrier function is of clinical relevance. We used a novel in vivo model of localized aseptic meningitis where tumor necrosis factor alpha (TNFα) was introduced intracerebrally and surveyed cerebral vascular changes and leukocyte-endothelium interactions by intravital videomicroscopy. Poly(ADP-ribose) polymerase-1 (PARP) inhibition significantly reduced leukocyte adhesion to and migration across brain endothelium in cortical microvessels. PARP inactivation diminished BBB permeability in an in vivo model of systemic inflammation. PARP suppression in primary human brain microvascular endothelial cells (BMVEC), an in vitro model of BBB, enhanced barrier integrity and augmented expression of tight junction proteins. PARP inhibition in BMVEC diminished human monocyte adhesion to TNFα-activated BMVEC (up to 65%) and migration (80-100%) across BBB models. PARP suppression decreased expression of adhesion molecules and decreased activity of GTPases (controlling BBB integrity and monocyte migration across the BBB). PARP inhibitors down-regulated expression of inflammatory genes and dampened secretion of pro-inflammatory factors increased by TNFα in BMVEC. These results point to PARP suppression as a novel approach to BBB protection in the setting of endothelial dysfunction caused by inflammation.

Neutrophils mediate blood-spinal cord barrier disruption in demyelinating neuroinflammatory diseases.

Disruption of the blood-brain and blood-spinal cord barriers (BBB and BSCB, respectively) and immune cell infiltration are early pathophysiological hallmarks of multiple sclerosis (MS), its animal model experimental autoimmune encephalomyelitis (EAE), and neuromyelitis optica (NMO). However, their contribution to disease initiation and development remains unclear. In this study, we induced EAE in lys-eGFP-ki mice and performed single, nonterminal intravital imaging to investigate BSCB permeability simultaneously with the kinetics of GFP(+) myeloid cell infiltration. We observed a loss in BSCB integrity within a day of disease onset, which paralleled the infiltration of GFP(+) cells into the CNS and lasted for ∼4 d. Neutrophils accounted for a significant proportion of the circulating and CNS-infiltrating myeloid cells during the preclinical phase of EAE, and their depletion delayed the onset and reduced the severity of EAE while maintaining BSCB integrity. We also show that neutrophils collected from the blood or bone marrow of EAE mice transmigrate more efficiently than do neutrophils of naive animals in a BBB cell culture model. Moreover, using intravital videomicroscopy, we demonstrate that the IL-1R type 1 governs the firm adhesion of neutrophils to the inflamed spinal cord vasculature. Finally, immunostaining of postmortem CNS material obtained from an acutely ill multiple sclerosis patient and two neuromyelitis optica patients revealed instances of infiltrated neutrophils associated with regions of BBB or BSCB leakage. Taken together, our data provide evidence that neutrophils are involved in the initial events that take place during EAE and that they are intimately linked with the status of the BBB/BSCB.

Acute Chagas disease induces cerebral microvasculopathy in mice.

Cardiomyopathy is the main clinical form of Chagas disease (CD); however, cerebral manifestations, such as meningoencephalitis, ischemic stroke and cognitive impairment, can also occur. The aim of the present study was to investigate functional microvascular alterations and oxidative stress in the brain of mice in acute CD. Acute CD was induced in Swiss Webster mice (SWM) with the Y strain of Trypanosoma cruzi (T. cruzi). Cerebral functional capillary density (the number of spontaneously perfused capillaries), leukocyte rolling and adhesion and the microvascular endothelial-dependent response were analyzed over a period of fifteen days using intravital video-microscopy. We also evaluated cerebral oxidative stress with the thiobarbituric acid reactive species TBARS method. Compared with the non-infected group, acute CD significantly induced cerebral functional microvascular alterations, including (i) functional capillary rarefaction, (ii) increased leukocyte rolling and adhesion, (iii) the formation of microvascular platelet-leukocyte aggregates, and (iv) alteration of the endothelial response to acetylcholine. Moreover, cerebral oxidative stress increased in infected animals. We concluded that acute CD in mice induced cerebral microvasculopathy, characterized by a reduced incidence of perfused capillaries, a high number of microvascular platelet-leukocyte aggregates, a marked increase in leukocyte-endothelium interactions and brain arteriolar endothelial dysfunction associated with oxidative stress. These results suggest the involvement of cerebral microcirculation alterations in the neurological manifestations of CD.

Microcirculatory Effects of Selective Endothelin-A Receptor Antagonism in Testicular Torsion

Testicular torsion without timely intervention causes incurable damage to the testis. We examined the causative role of microcirculatory injury in torsion induced testicular damage with particular regard to endothelin-A receptor activation. The microcirculatory consequences of testicular torsion were assessed in the presence or absence of endothelin-A receptor antagonism in rats. Microcirculatory perfusion changes (red blood cell velocity and pulsatile flow pattern alterations) were examined by an orthogonal polarization spectral imaging technique. Microcirculatory inflammatory alterations were assessed byfluorescence intravital video microscopy after 60-minute torsion followed by 240-minute reperfusion. As a specific endothelin-A receptor inhibitor, the antisense homology box derived peptide ETR-p1/fl was applied 10 minutes before reperfusion. Tissue accumulation of leukocytes was estimated by myeloperoxidase activity in tissue biopsies taken at the end of the 4-hour reperfusion period. In further experiments testicular weight as a marker of permanent damage was evaluated 45 days after torsion. The physiological pulsatile flow pattern ceased in the initial phase of reperfusion while leukocyte-endothelial interactions increased throughout the examined reperfusion period. Endothelin-A receptor antagonism caused earlier return of pulsatile flow and recovery of red blood cell velocity, and alleviated microcirculatory inflammatory reactions and atrophy. Results suggest a pathophysiological role of endothelin-A receptor activation in the pathogenesis of testicular torsion. This effect is related to deterioration in testicular perfusion and activation of microcirculatory inflammatory reactions.

The selective PAC1 receptor agonist maxadilan inhibits neurogenic vasodilation and edema formation in the mouse skin

We have earlier shown that PACAP-38 decreases neurogenic inflammation. However, there were no data on its receptorial mechanism and the involvement of its PAC1 and VPAC1/2 receptors (PAC1R, VPAC1/2R) in this inhibitory effect.Neurogenic inflammation in the mouse ear was induced by topical application of the Transient Receptor Potential Ankyrin 1 (TRPA1) receptor activator mustard oil (MO). Consequent neurogenic edema, vasodilation and plasma leakage were assessed by measuring ear thickness with engineer's micrometer, detecting tissue perfusion by laser Doppler scanning and Evans blue or indocyanine green extravasation by intravital videomicroscopy or fluorescence imaging, respectively. Myeloperoxidase activity, an indicator of neutrophil infiltration, was measured from the ear homogenates with spectrophotometry. The selective PAC1R agonist maxadilan, the VPAC1/2R agonist vasoactive intestinal polypeptide (VIP) or the vehicle were administered i.p. 15 min before MO. Substance P (SP) concentration of the ear was assessed by radioimmunoassay.Maxadilan significantly diminished MO-induced neurogenic edema, increase of vascular permeability and vasodilation. These inhibitory effects of maxadilan may be partially due to the decreased substance P (SP) levels. In contrast, inhibitory effect of VIP on ear swelling was moderate, without any effect on MO-induced plasma leakage or SP release, however, activation of VPAC1/2R inhibited the increased microcirculation caused by the early arteriolar vasodilation. Neither the PAC1R, nor the VPAC1/2R agonist influenced the MO-evoked increase in tissue myeloperoxidase activity.These results clearly show that PAC1R activation inhibits acute neurogenic arterial vasodilation and plasma protein leakage from the venules, while VPAC1/2R stimulation is only involved in the attenuation of vasodilation.

Contraction‐evoked vasodilation and functional hyperaemia are compromised in branching skeletal muscle arterioles of young pre‐diabetic mice

To investigate the effects of pre-diabetes on microvascular network function in contracting skeletal muscle. We hypothesized that pre-diabetes compromises contraction-evoked vasodilation of branching second-order (2A), third-order (3A) and fourth-order (4A) arterioles, where distal arterioles would be affected the greatest. Intravital video microscopy was used to measure arteriolar diameter (in 2A, 3A and 4A) and blood flow (in 2A and 3A) changes to electrical field stimulation of the gluteus maximus muscle in pre-diabetic (The Pound Mouse, PD) and control (c57bl6, CTRL) mice. Baseline diameter and blood flow were similar between groups (2A: ~20 μm, 3A: ~14 μm and 4A: ~8 μm; 2A: ~1 nL s(-1) and 3A: ~0.5 nL s(-1) ). Single tetanic contraction (100 Hz; 200, 400, 800 ms duration) evoked rapid-onset vasodilation (ROV) and blood flow responses that were blunted by ~50% and up to 81%, respectively, in PD vs. CTRL (P < 0.05). The magnitude of ROV was up to 2-fold greater at distal arterioles (3A and 4A) vs. proximal arterioles (2A) in CTRL; however, in PD, ROV of only 4A was greater than 2A (P < 0.05). Rhythmic contraction (2 and 8 Hz, 30 s) evoked vasodilatory and blood flow responses that were also attenuated by ~50% and up to 71%, respectively, in PD vs. CTRL (P < 0.05). The magnitude of vasodilatory responses to rhythmic contraction was also up to 2.5-fold greater at 4A vs. 2A in CTRL; however spatial differences in vasodilation across arteriolar branch orders was disrupted in PD. Arteriolar dysregulation in pre-diabetes causes deficits in contraction-evoked dilation and blood flow, where greatest deficits occur at distal arterioles.

Abstract 82: Angiogenesis Following Hind-Limb Ischemia in Mice Generates a Functionally Abnormal Microvasculature

Extensive angiogenesis can occur in ischemic muscle. However, the extent to which newly formed microvessels regulate blood flow in ischemic and regenerating muscle remains unknown. To answer this, we developed an intravital video microscopy approach to red blood cell imaging in the extensor digitorum longus (EDL) muscle of mice following femoral artery excision. After surgery, blood flow ceased in the EDL microvasculature, followed by widespread muscle necrosis. However, 10 days later an extensive and highly branched network of flowing neovessels had regenerated. On day 14, branch pruning was evident, and vessel length density reached that of uninjured EDL. By day 28, the network grossly resembled that of the uninjured EDL, and complete arteriovenous units had developed. However, lumenal cross-sectional area of neocapillaries was 40% greater than uninjured capillaries (p&lt;0.05). In addition, 47% of neoarterioles flowed directly into venules, bypassing capillaries. Mean red blood cell flow in neocapillaries was only 59% of uninjured capillary flow (p&lt;0.05) and displayed reduced flow heterogeneity (p&lt;0.05). To evaluate the functional responsiveness of the regenerated network, the EDL was subjected to local hypoxia (2% O 2 ) via a polydimethylsiloxane (PDMS) membrane. Red blood cell flow in neocapillaries increased to only 66% of that in uninjured EDL capillaries (54.6±9.0 vs 82.6±7.4 μm/sec increase, p&lt;0.05). Furthermore, the ability to sustain a hypoxic flow response was profoundly reduced in neocapillaries (p&lt;0.05). Conclusions: Despite robust angiogenesis, the regenerated network in skeletal muscle is characterized by enlarged capillaries, slow red blood cell transit, non-functional flow units, and impaired flow responsiveness. These findings suggest that: 1) capillary density and bulk flow are inadequate indicators of functional angiogenesis; and 2) strategies beyond stimulating angiogenesis are needed to optimize flow to ischemic tissues.

Endothelial progenitor cells augment collateralization and hemodynamic rescue in a model of chronic cerebral ischemia.

Surgical flow augmentation for treatment of cerebral hemodynamic impairment remains controversial. Here, we investigated the benefit of endothelial progenitor cell (EPC) treatment in a rat model of chronic cerebral hypoperfusion. At repeated time points after 3-vessel occlusion (3-VO), animals were treated with 1 × 10(6) DiI-labeled (a) ex vivo-expanded embryonic-EPC (e-EPC), (b) cyclic AMP-differentiated embryonic-endothelial progenitor-derived cells (e-EPDC as biologic control) or, (c) saline. The cerebrovascular reserve capacity (CVRC) was assessed immediately before and on days 7 and 21 after 3-VO. Structural effects were assessed by latex perfusion, immunohistochemistry, and intravital fluorescence video microscopy on day 21. Three-vessel occlusion resulted in a significant impairment of the CVRC with better functional recovery after treatment with e-EPC (16.4±8%) compared with e-EPDC (3.7±8%) or saline (6.4±9%) by day 21 (P<0.05), which was paralleled by a significant increase in the vessel diameters of the anterior Circle of Willis, a significantly higher number of leptomeningeal anastomoses and higher parenchymal capillary density in e-EPC-treated animals. Interestingly, despite in vivo interaction of e-EPC with the cerebral endothelium, e-EPC incorporation into the cerebral vasculature was not observed. Our results suggest that EPC may serve as a novel therapeutic agent in clinical trials for nonsurgical treatment of chronic cerebral hemodynamic impairment.

Remote ischemic preconditioning differentially affects NADPH oxidase isoforms during hepatic ischemia–reperfusion

AimsWe investigated the function of major superoxide-generating enzymes after remote ischemic preconditioning (IPC) and hepatic ischemia–reperfusion (IR), with the specific aim of assessing the importance of the most relevant NADPH oxidase (NOX) isoforms, NOX2 and NOX4, in the mechanism of action. Main methods60-min partial liver ischemia was induced in Sprague–Dawley rats in the presence or absence of remote IPC (2×10-min limb IR), and hepatic microcirculatory variables were determined through intravital video microscopy and lightguide spectrophotometry during reperfusion. Inflammatory enzyme activities (myeloperoxidase (MPO) and xanthine oxidoreductase (XOR)), cytokine production (TNF-α and HMGB-1), liver necroenzyme levels (AST, ALT and LDH) and NOX2 and NOX4 protein expression changes (Western blot analysis) were assayed biochemically. Key findingsIn this setting, remote IPC significantly decreased the IR-induced hepatic NOX2 expression, but the NOX4 expression remained unchanged. The remote IPC provided significant, but incomplete protection against the leukocyte–endothelial cell interactions and flow deterioration. Hepatocellular damage (AST, ALT and LDH release), cytokine levels, and XOR and MPO activities also diminished. SignificanceRemote IPC limited the IR-induced microcirculatory dysfunction, but the protective effect did not affect all NOX homologs (at least not NOX4). The residual damage and inflammatory activation could well be linked to the unchanging NOX4 activity.

Alteration of the systemic and microcirculation by a single oral dose of flavan-3-ols.

Several systematic reviews have reported that flow mediated dilatation (FMD) was significantly increased in subjects after ingestion of chocolate that contains flavan-3-ols; however, the mechanisms responsible for this effect are not clear. In this study, we evaluated the effects of a single oral dose of flavan-3-ols on the systemic circulation and microcirculation in the cremaster muscle using intravital video microscopy in vivo. The cremaster muscle in rats was spread over a plastic chamber and a gastric tube was placed into the stomach. Blood flow in the cremasteric artery was determined using a laser Doppler flowmeter, while blood pressure and heart rate were measured by the tail-cuff method. Red blood cell velocity in arterioles and blood flow in the artery were significantly increased 5 min after the administration of 10 mg/kg flavan-3-ols compared with distilled water treatment. The number of capillaries recruited in the cremaster muscle was also significantly increased 15 min after treatment. Microscopic observation confirmed that increased shear stress on endothelial cells was maintained during the measurement period. The mean arterial blood pressure and heart rate were also significantly elevated soon after administration and returned to baseline before the end of the observation period. Plasma nitrate and nitrite levels, and NO phosphorylation of aortic tissue were significantly increased at 60 min after administration of flavan-3-ols. According to these results, a single oral dose of flavan-3-ols elevates blood pressure and flow transiently, and these effects induce NO production through increased shear stress on endothelial cells.

Protective effects of antithrombin on free groin flaps after secondary venous stasis in the rat model

The anticoagulant activity of heparin is well established and led to its widespread clinical use for the prophylaxis and treatment of venous thrombosis in microsurgery. Heparin accelerates antithrombin (AT)-mediated inhibition of clotting and fibrinolytic proteinases. The aim of the study is to determine whether the focussed delivery of AT by rinsing of free adipocutaneous groin flaps shows protective effects on flap survival, following a fatal secondary venous stasis in the rat model. Further, intravital video microscopy (IVM) is used to detect substance-specific alterations in microvascular perfusion with special focus on regional differences between central and peripheral flap regions. Free microvascular groin flaps (n = 22) were transplanted to the neck in adult Sprague-Dawley rats. The flap pedicle was re-explored and the distal stump of the flap artery was catheterised 20 h later. Animals in group I (n = 11) were treated with 1 ml of Ringer's solution administered over 10 min via intraarterial infusion. Those in group II (n = 11) received 1 ml of AT (50 IU/kg). Afterwards, the flap vein was clamped for 35 min. The skin of the flaps and the native contralateral groin was examined by IVM using the plasma-marker fluorescein isothiocyanate (FITC)-dextran and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE)-labelled thrombocytes. After 14 days, the viability of the flaps was evaluated. The treatment with AT significantly increased the functional capillary density (FCD) of the flaps. After 14 days, flap necrosis occurred in nine animals of group I and three animals of group II, respectively. No partial flap loss was detected. The focussed delivery of AT resulted in significantly improved flap salvage. The results may reinforce the clinical custom of AT substitution in the setting of major surgical procedures such as elaborate microsurgical reconstructions, at least in cases with diminished AT levels.

Pulmonary Microvascular Albumin Leak Is Associated with Endothelial Cell Death in Murine Sepsis-Induced Lung Injury In Vivo

Sepsis is a systemic inflammatory response that targets multiple components of the cardiovascular system including the microvasculature. Microvascular endothelial cells (MVEC) are central to normal microvascular function, including maintenance of the microvascular permeability barrier. Microvascular/MVEC dysfunction during sepsis is associated with barrier dysfunction, resulting in the leak of protein-rich edema fluid into organs, especially the lung. The specific role of MVEC apoptosis in septic microvascular/MVEC dysfunction in vivo remains to be determined. To examine pulmonary MVEC death in vivo under septic conditions, we used a murine cecal ligation/perforation (CLP) model of sepsis and identified non-viable pulmonary cells with propidium iodide (PI) by intravital videomicroscopy (IVVM), and confirmed this by histology. Septic pulmonary microvascular Evans blue (EB)-labeled albumin leak was associated with an increased number of PI-positive cells, which were confirmed to be predominantly MVEC based on specific labeling with three markers, anti-CD31 (PECAM), anti-CD34, and lectin binding. Furthermore, this septic death of pulmonary MVEC was markedly attenuated by cyclophosphamide-mediated depletion of neutrophils (PMN) or use of an anti-CD18 antibody developed for immunohistochemistry but shown to block CD18-dependent signaling. Additionally, septic pulmonary MVEC death was iNOS-dependent as mice lacking iNOS had markedly fewer PI-positive MVEC. Septic PI-positive pulmonary cell death was confirmed to be due to apoptosis by three independent markers: caspase activation by FLIVO, translocation of phosphatidylserine to the cell surface by Annexin V binding, and DNA fragmentation by TUNEL. Collectively, these findings indicate that septic pulmonary MVEC death, putatively apoptosis, is a result of leukocyte activation and iNOS-dependent signaling, and in turn, may contribute to pulmonary microvascular barrier dysfunction and albumin hyper-permeability during sepsis.

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Comparison of Generated Parallel Capillary Arrays to Three‐Dimensional Reconstructed Capillary Networks in Modeling Oxygen Transport in Discrete Microvascular Volumes

We compare RMN to PCA under several simulated physiological conditions to determine how the use of different vascular geometry affects oxygen transport solutions. Three discrete networks were reconstructed from intravital video microscopy of rat skeletal muscle (84 × 168 × 342 μm, 70 × 157 × 268 μm, and 65 × 240 × 571 μm), and hemodynamic measurements were made in individual capillaries. PCAs were created based on statistical measurements from RMNs. Blood flow and O₂ transport models were applied, and the resulting solutions for RMN and PCA models were compared under four conditions (rest, exercise, ischemia, and hypoxia). Predicted tissue PO₂ was consistently lower in all RMN simulations compared to the paired PCA. PO₂ for 3D reconstructions at rest were 28.2 ± 4.8, 28.1 ± 3.5, and 33.0 ± 4.5 mmHg for networks I, II, and III compared to the PCA mean values of 31.2 ± 4.5, 30.6 ± 3.4, and 33.8 ± 4.6 mmHg. Simulated exercise yielded mean tissue PO₂ in the RMN of 10.1 ± 5.4, 12.6 ± 5.7, and 19.7 ± 5.7 mmHg compared to 15.3 ± 7.3, 18.8 ± 5.3, and 21.7 ± 6.0 in PCA. These findings suggest that volume matched PCA yield different results compared to reconstructed microvascular geometries when applied to O₂ transport modeling; the predominant characteristic of this difference being an over estimate of mean tissue PO₂. Despite this limitation, PCA models remain important for theoretical studies as they produce PO₂ distributions with similar shape and parameter dependence as RMN.

The free groin flap in the rat: a model for improving microsurgical skills and for microvascular perfusion studies

The goal of this study was to evaluate the free groin flap in the rat transplanted to the neck as a tool for extending microsurgical skills and to assess its suitability as a model for microvascular perfusion studies following secondary venous ischaemia. An analysis of 60 consecutive groin flap transplantations was performed in male Sprague Dawley rats with special regard to anatomy and operation times (Part I, animals No. 1–60). Following flap transplantation, the animals No. 10–30 (n = 21) were used for the determination of the critical time period of a complete venous stasis of the free groin flap resulting in a total flap loss (Part II). The flaps of animals No. 31–41 (n = 11) were used for assessing the feasibility and reproducibility of intra-vital video microscopy (IVM) of the flaps (Part III). The mean total operation time decreased from 166 (± 26) minutes ins the first 10 animals to 126 (± 21) minutes and 130 (± 12) minutes in the latter two groups of 10 animals, respectively. After a critical period of 35 minutes of a complete artificial venous stasis a complete flap necrosis occurred. IVM detected a higher functional capillary density of the skin of the transplanted groin flaps in the animals in which the flaps were rinsed with 1 ml of Ringer's lactated solution prior to I/R. In conclusion, this model is simple and reliable. The model may be a useful tool for evaluating and comparing the effects of various anticoagulants or vasomotor drugss on microvascular perfusion in critically compromised free flaps.

Selective Activation of Cannabinoid Receptor 2 in Leukocytes Suppresses Their Engagement of the Brain Endothelium and Protects the Blood-Brain Barrier

Cannabinoid receptor 2 (CB2) is highly expressed in immune cells and stimulation decreases inflammatory responses. We tested the idea that selective CB2 activation in human monocytes suppresses their ability to engage the brain endothelium and migrate across the blood-brain barrier (BBB), preventing consequent injury. Intravital videomicroscopy was used to quantify adhesion of leukocytes to cortical vessels in lipopolysaccharide-induced neuroinflammation, after injection of ex vivo CB2-activated leukocytes into mice; CB2 agonists markedly decreased adhesion of ex vivo labeled cells in vivo. In an in vitro BBB model, CB2 activation in monocytes largely attenuated adhesion to and migration across monolayers of primary human brain microvascular endothelial cells and diminished BBB damage. CB2 stimulation in monocytes down-regulated active forms of integrins, lymphocyte function-associated antigen 1 (LFA-1), and very late antigen 4 (VLA-4). Cells treated with CB2 agonists exhibited increased phosphorylation levels of inhibitory sites of the actin-binding proteins cofilin and VASP, which are upstream regulators of conformational integrin changes. Up-regulated by relevant stimuli, Rac1 and RhoA were suppressed by CB2 agonists in monocytes. CB2 stimulation decreased formation of lamellipodia, which play a key role in monocyte migration. These results indicate that selective CB2 activation in leukocytes decreases key steps in monocyte-BBB engagement, thus suppressing inflammatory leukocyte responses and preventing neuroinflammation.

Levosimendan and its metabolite OR-1896 elicit KATP channel-dependent dilation in resistance arteries in vivo

BackgroundLevosimendan and its long-lived metabolite OR-1896 produce vasodilation in different types of vessels by activating ATP-sensitive (KATP) and other potassium channels. MethodsIn the present study we applied intravital videomicroscopy to investigate the in situ effects of levosimendan and OR-1896 on the diameters of real resistance arterioles (rat cremaster muscle arterioles with diameters of ~ 20μm). ResultsLevosimendan and OR-1896 induced concentration-dependent (1nM – 100μM) dilations to similar extents in these arterioles (maximal dilation from 23±2 to 33±2μm and from 22±1 to 32±1μm, respectively). The arteriolar dilations induced by the selective KATP channel opener pinacidil (1nM – 10μM) (maximal dilation from 22±4μm to 35±3μm) were diminished in the presence of the selective KATP channel blocker – glibenclamide (5μM) (maximal diameter attained: 22±1μm). Glibenclamide also counteracted the maximal dilations in response to levosimendan or OR-1896 (to 23±3μm or 22±5μm, respectively). ConclusionsIn conclusion, this is the first demonstration that levosimendan and OR-1896 elicit arteriolar dilation in vivo, via activation of KATP channels in real resistance vessels in the rat.

Vav Guanine Nucleotide Exchange Factors Regulate Atherosclerotic Lesion Development in Mice

Atherosclerosis requires migration of monocytes to the arterial intima, with subsequent differentiation into foam cells. We showed previously that the scavenger receptor CD36 contributes to the activation of Vav family guanine nucleotide exchange factors (Vavs) in aortae from hyperlipidemic apoE-null mice and that oxidatively modified low-density lipoprotein induced CD36-dependent activation of macrophage Vavs in vitro. We also discovered that CD36-dependent uptake of oxidized low-density lipoprotein and foam cell formation were reduced in Vav-deficient macrophages. We now tested the hypothesis that Vavs play a role in atherosclerotic lesion development. We showed that apoE/vav1 double-null mice fed a Western diet had significant reduction in total aortic lesion area (by en face analysis) compared with apoE-null mice, with no significant differences in body weight or plasma lipid profiles. Histological analysis of aortic sinus lesions showed fewer macrophages and foam cells in double-null mice compared with apoE-null mice, indicating impaired foam cell generation and homing of macrophages to atherosclerotic lesions. An intravital video microscopy-based adhesion assay with fluorescent (Qtracker655)-labeled monocytes showed reduced adhesion of vav1-null monocytes to hyperlipidemic carotid arteries compared with wild-type monocytes. Furthermore, fewer fluorescently labeled vav1-null monocytes accumulated in aortic sinus lesions in hyperlipidemic apoE-null mice. We also found that activation of RhoGTPase Rac and mitogen-activated protein kinase c-Jun N-terminal kinase-2 by CD36-specific oxidized phospholipids was dependent on Vavs. These results for the first time link Vavs to atherosclerotic lesion development and suggest that Vavs act as critical molecular links coupling hyperlipidemia with proatherogenic monocyte/macrophage responses.