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International audience; A sedentary lifestyle has adverse effects on the cardiovascular system, including impaired endothelial functions. Subjecting healthy men to 7 days of dry immersion (DI) presented a unique opportunity to analyze the specific effects of enhanced inactivity on the endothelium. We investigated endothelial properties before, during, and after 7 days of DI involving eight subjects. Microcirculatory functions were assessed with laser Doppler in the skin of the calf. We studied basal blood flow and endothelium-dependent and -independent vasodilation. We also measured plasma levels of microparticles, a sign of cellular dysfunction, and soluble endothelial factors, reflecting the endothelial state. Basal flow and endothelium-dependent vasodilation were reduced by DI (22 + or - 4 vs. 15 + or - 2 arbitrary units and 29 + or - 6% vs. 12 + or - 6%, respectively, P < 0.05), and this was accompanied by an increase in circulating endothelial microparticles (EMPs), which was significant on day 3 (42 + or - 8 vs. 65 + or - 10 EMPs/microl, P < 0.05), whereas microparticles from other cell origins remained unchanged. Plasma soluble VEGF decreased significantly during DI, whereas VEGF receptor 1 and soluble CD62E were unchanged, indicating that the increase in EMPs was associated with a change in antiapoptotic tone rather than endothelial activation. Our study showed that extreme physical inactivity in humans induced by 7 days of DI causes microvascular impairment with a disturbance of endothelial functions, associated with a selective increase in EMPs. Microcirculatory endothelial dysfunction might contribute to cardiovascular deconditioning as well as to hypodynamia-associated pathologies. In conclusion, the endothelium should be the focus of special care in situations of acute limitation of physical activity.

International audience; Quantitative RT-PCR (qRT-PCR) and Western blotting studies on transporters at the blood-brain barrier (BBB) of isolated brain microvessels have produced conflicting data on their cellular distribution. A major problem is identifying cells expressing the genes of interest, since isolated brain microvessels are composed of several cell types and may be contaminated with mRNA or proteins from astrocytes and neurons. We isolated rat brain microvessels and examined microscopically samples at each step of isolation to evaluate microvessel purity. The expression of specific markers of endothelial cells (Glut-1, Flk-1), pericytes (Ng2), neurons (synaptophysin, Syn) and astrocytes (Gfap) was measured by qRT-PCR in order to select the protocol giving the least astrocyte and neuron mRNAs and the most endothelial mRNAs. We also evaluated the gene expression of drug transporters (Mdr1a, Mdr1b, Mrp1-5, Bcrp and Oatp-2) at each step to optimize their location in cells at the BBB. The Mdr1a, Mrp4, Bcrp and Oatp-2 gene profiles were similar to those of endothelium markers. The profiles of Mrp2 and Mrp3 closely resembled that of Ng2. Mrp5 and Mrp1 expression was not increased in the microvessel-enriched fraction, suggesting that they are ubiquitously expressed throughout the cortex parenchyma. We also evaluated by Western blotting the expression of P-gp, Mrp2, Gfap and Syn in the cortex and in the purest obtained microvessel fraction. Our results showed that P-gp expression strongly increased in microvessels whereas Mrp2 was not detected in any of the fraction. Surprisingly, Gfap expression increased in isolated microvessels whereas Syn was not detected. Our results showed that the strategy consisting of identifying gene expression at different steps of the protocol is useful to identify cells containing mRNA at the BBB and give overall similar results with protein expression.

Pulmonary hypertension (PH) is characterized by sustained vasoconstriction, with subsequent extracellular matrix (ECM) production and smooth muscle cell (SMC) proliferation. Changes in the ECM can modulate vasoreactivity and SMC contraction. Galectin-1 (Gal-1) is a hypoxia-inducible β-galactoside-binding lectin produced by vascular, interstitial, epithelial, and immune cells. Gal-1 regulates SMC differentiation, proliferation, and apoptosis via interactions with the ECM, as well as immune system function, and, therefore, likely plays a role in the pathogenesis of PH. We investigated the effects of Gal-1 during hypoxic PH by quantifying 1) Gal-1 expression in response to hypoxia in vitro and in vivo and 2) the effect of Gal-1 gene deletion on the magnitude of the PH response to chronic hypoxia in vivo. By constructing and screening a subtractive library, we found that acute hypoxia increases expression of Gal-1 mRNA in isolated pulmonary mesenchymal cells. In wild-type (WT) mice, Gal-1 immunoreactivity increased after 6 wk of hypoxia. Increased expression of Gal-1 protein was confirmed by quantitative Western analysis. Gal-1 knockout (Gal-1−/−) mice showed a decreased PH response, as measured by right ventricular pressure and the ratio of right ventricular to left ventricular + septum wet weight compared with their WT counterparts. However, the number and degree of muscularized vessels increased similarly in WT and Gal-1−/−mice. In response to chronic hypoxia, the decrease in factor 8-positive microvessel density was similar in both groups. Vasoreactivity of WT and Gal-1−/−mice was tested in vivo and with use of isolated perfused lungs exposed to acute hypoxia. Acute hypoxia caused a significant increase in RV pressure in wild-type and Gal-1−/−mice; however, the response of the Gal-1−/−mice was greater. These results suggest that Gal-1 influences the contractile response to hypoxia and subsequent remodeling during hypoxia-induced PH, which influences disease progression.

PURPOSE: To evaluate quantitative functional ultrasonography (US) in a murine gel model by using microbubble destruction kinetics to determine whether parametric indices provided with US could help assess angiogenesis. MATERIALS AND METHODS: Institutional Animal Subjects Committee approved experiments and procedures. In 36 normal mice, two 0.4-mL gel implants were placed subcutaneously on either side of spine. One implant contained 0.5, 1.0, or 1.5 microg human basic fibroblast growth factor (bFGF) per milliliter of gel. Functional US quantitative analysis of angiogenesis with microbubble contrast agent was performed on days 3, 6, 9, and 12; histologic data were collected. Time-intensity curve of implant was fitted to mathematic decay model to calculate fractional blood volume and fraction of blood replaced per unit of time. Microvascular density (MVD) and percentage of microvascular area (MVA) were measured after anti-CD31 staining. Spearman rank order correlation was used in analyses. RESULTS: bFGF-containing implants induced MVD of eight, 35, 42, and 42 vessels per square millimeter on days 3, 6, 9, and 12, respectively; in controls, MVD was four vessels/mm2 (P

International audience; Although Alzheimer s disease is classified as a neurodegenerative dementia, in Alzheimer s dementia there is increasing evidence of an association with vascular risk factors and vascular pathology. These observations are supported by epidemiological and pathological studies. Vascular factors may play a significant role in the pathogenesis of Alzheimer s dementia. Further research is needed to explore this relationship. Modification of vascular risk factors could make a significant impact towards prevention and treatment of Alzheimer s disease, which is a distressing and prevalent condition in the community. This article considers the relationship of these vascular risk factors and potential mechanisms associated with the aetiopathogenesis of Alzheimer s dementia.

Background —The progression of hypertension during NO deficiency is associated with renal vascular fibrosis due to increased extracellular matrix (mainly collagen I) formation. The purpose of the present study was to investigate whether endothelin-1 (ET-1) is involved in this pathophysiological process. Methods and Results —Treatment of rats for 4 weeks with the NO synthase inhibitor N ω -nitro- l -arginine methyl ester (L-NAME) 50 mg · kg −1 · d −1 increased systolic blood pressure to 159±12 mm Hg. In animals treated with L-NAME, histological evaluation of renal sections revealed an increased formation of extracellular matrix (Masson’s trichrome), and specifically of collagens (Sirius red). A part of this fibrosis was attributed to abnormal collagen I presence, because mRNA expression of the collagen I α1 chain (reverse transcription–polymerase chain reaction) and procollagen I formation (radioimmunoassay) were increased 3- and 2.5-fold, respectively, in the renal resistance vessels of hypertensive animals. In subsequent experiments, we examined whether ET-1 was involved in activation of collagen I formation. mRNA expression (RNase protection assay) of preproET-1 and ET-1 content (radioimmunoassay) were 10-fold and 3-fold increased, respectively, in renal microvessels of rats treated with L-NAME. Interestingly, in these vessels, ET-1 (immunostaining) was colocalized with sudanophilic lesions. Bosentan, an ET receptor antagonist (20 mg · kg −1 · d −1 ), coadministered with L-NAME canceled the increased mRNA expression and synthesis of collagen I and attenuated the severity of renal vascular lesions without affecting L-NAME–induced high blood pressure. Conclusions —These data demonstrate that ET-1 synthesis is increased in renal microvessels when NO production is suppressed. In this model of hypertension, ET-1 is a major activator of collagen I formation in renal resistance vessels and participates in the development of renal fibrosis without affecting systolic blood pressure.

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International audience; OBJECTIVES: To investigate the effects of recombinant human erythropoietin on brain oxygenation in a model of diffuse traumatic brain injury. DESIGN: Adult male Wistar rats. SETTING: Neurosciences and physiology laboratories. INTERVENTIONS: Thirty minutes after diffuse traumatic brain injury (impact-acceleration model), rats were intravenously administered with either a saline solution or a recombinant human erythropoietin (5000 IU/kg). A third group received no traumatic brain injury insult (sham-operated). MEASUREMENTS AND MAIN RESULTS: Three series of experiments were conducted 2 hours after traumatic brain injury to investigate: 1) the effect of recombinant human erythropoietin on brain edema using diffusion-weighted magnetic resonance imaging and measurements of apparent diffusion coefficient (n = 11 rats per group); local brain oxygen saturation, mean transit time, and blood volume fraction were subsequently measured using a multiparametric magnetic resonance-based approach to estimate brain oxygenation and brain perfusion in the neocortex and caudoputamen; 2) the effect of recombinant human erythropoietin on brain tissue PO₂ in similar experiments (n = 5 rats per group); and 3) the cortical ultrastructural changes after treatment (n = 1 rat per group). Compared with the sham-operated group, traumatic brain injury saline rats showed a significant decrease in local brain oxygen saturation and in brain tissue PO₂ alongside brain edema formation and microvascular lumen collapse at H2. Treatment with recombinant human erythropoietin reversed all of these traumatic brain injury-induced changes. Brain perfusion (mean transit time and blood volume fraction) was comparable between the three groups of animals. CONCLUSION: Our findings indicate that brain hypoxia can be related to microcirculatory derangements and cell edema without evidence of brain ischemia. These changes were reversed with post-traumatic administration of recombinant human erythropoietin, thus offering new perspectives in the use of this drug in brain injury.

Cardiovascular diseases are often revealed during exercise and are associated with cutaneous blood flow (CBF) dysfunction. Studies of CBF during exercise are consequently of interest. Laser speckle contrast imaging (LSCI) allows for non-contact and real-time recording of CBF at rest. We tested whether LSCI could allow the study of CBF during a cycling exercise using a specific signal treatment procedure that removes movement-induced arte- facts from the LSCI raw signal. We recorded the baseline CBF and peak post-occlusive reactive hyperaemia (PORH) from the cutaneous forearm using LSCI and the mean blood pressure before and during cycling (80 Wat 70 rpm) in nine healthy subjects. We determined the cross-correlation coef- ficient r between LSCI traces obtained before and during cycling and before and after a specifically designed signal processing technique. The results are presented as the me- dian (25th-75th centile) and expressed as the cutaneous vascular conductance (laser speckle perfusion units (LSPU) per millimetre of mercury). Cross-correlation r in- creased from 0.226±0.140 before to 0.683±0.170 after post-processing. After signal processing, the peak PORH during exercise was reduced (0.38 (0.30-0.52) LSPU/mmHg) compared with the peak PORH during the non-exercise phase (0.69 (0.63-0.74) LSPU/mmHg, p

Nipah virus (NiV) is a highly pathogenic, negative-strand RNA paramyxovirus that has recently emerged from flying foxes to cause serious human disease. We have analyzed the role of the nonstructural NiV C protein in viral immunopathogenesis using recombinant virus lacking the expression of NiV C (NiVΔC). While wild-type NiV was highly pathogenic in the hamster animal model, NiVΔC was strongly attenuated. Replication of NiVΔC was followed by the production of NiV-specific antibodies and associated with higher recruitment of inflammatory cells and less intensive histopathological lesions in different organs than in wild-type-NiV-infected animals. To analyze the molecular basis of NiVΔC attenuation, we studied early changes in gene expression in infected primary human endothelial cells, a major cellular target of NiV infection. The transcriptomic approach revealed the striking difference between wild-type and mutant NiV in the expression of genes involved in immunity, with the particularly interesting differential patterns of proinflammatory cytokines. Compared to wild-type virus, NiVΔC induced increased expression of interleukin 1 beta (IL-1β), IL-8, CXCL2, CXCL3, CXCL6, CCL20, and beta interferon. Furthermore, the expression of NiV C in stably transfected cells decreased the production of the same panel of cytokines, revealing a role of the C protein in the regulation of cytokine balance. Together, these results suggest that NiV C regulates expression of proinflammatory cytokines, therefore providing a signal responsible for the coordination of leukocyte recruitment and the chemokine-induced immune response and controlling the lethal outcome of the infection.

International audience; OBJECTIVE: The microcirculation of septic patients has been characterized only semiquantitatively. Our goal was to characterize the sublingual microcirculation in healthy volunteers and patients with septic shock quantitatively. Our hypotheses were that 1) hyperdynamic blood flow is absent in septic shock; 2) nonsurvivors show more severe alterations than survivors; and 3) quantitative and semiquantitative microcirculatory parameters have a similar performance. DESIGN: Prospective, observational study. SETTING: Teaching intensive care unit in a university-affiliated hospital. SUBJECTS: Twenty-five normal volunteers and 25 patients with septic shock. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The sublingual microcirculation was evaluated by means of sidestream dark field imaging. Semiquantitative and quantitative microcirculatory parameters were determined through the use of applied software. Septic patients showed decreased perfused capillary density (13.2±4.4 mm/mm² vs. 16.6±1.6 mm/mm²), proportion of perfused capillaries (0.78±0.23 vs. 1.00±0.01), microvascular flow index (2.15±0.61 vs. 2.97±0.03), and red blood cell velocity (830±183 µm/sec vs. 1332±187 µm/sec) along with increased heterogeneity flow index (1.64±1.14 vs. 0.25±0.19) compared with controls. No differences were found in total capillary density (16.9±2.2 vs. 16.7±1.6). Only 4% of capillaries analyzed showed red blood cell velocities>75th percentile of the velocities of the normal volunteers. The nonsurvivors exhibited decreased perfused capillary density, proportion of perfused capillaries, and microvascular flow index along with increased heterogeneity flow index compared with the survivors. The correlations between microvascular flow index and proportion of perfused capillaries, total capillary density and number of grid-crossing capillaries, and red blood cell velocities and microvascular flow index gave high R values (0.92, 0.65, and 0.52, respectively; p

International audience; For more than two decades, methods for the non-invasive exploration of cutaneous microcirculation have been mainly based on optical microscopy and laser Doppler techniques. In this review, we discuss the advantages and drawbacks of these techniques. Although optical microscopy-derived techniques, such as nailfold videocapillaroscopy, have found clinical applications, they mainly provide morphological information about the microvessels. Laser Doppler techniques coupled with reactivity tests are widespread in the field of microvascular function research, but many technical issues need to be taken into account when performing these tests. Post-occlusive reactive hyperemia and local thermal hyperemia have been shown to be reliable tests, although their underlying mechanisms are not yet fully understood. Acetylcholine and sodium nitroprusside iontophoresis, despite their wide use as specific tests of endothelium-dependent and -independent function, respectively, show limitations. The influence of the skin site, recording conditions, and the way of expressing data are also reviewed. Finally, we focus on promising tools such as laser speckle contrast imaging.

International audience; For more than two decades, methods for the non-invasive exploration of cutaneous microcirculation have been mainly based on optical microscopy and laser Doppler techniques. In this review, we discuss the advantages and drawbacks of these techniques. Although optical microscopy-derived techniques, such as nailfold videocapillaroscopy, have found clinical applications, they mainly provide morphological information about the microvessels. Laser Doppler techniques coupled with reactivity tests are widespread in the field of microvascular function research, but many technical issues need to be taken into account when performing these tests. Post-occlusive reactive hyperemia and local thermal hyperemia have been shown to be reliable tests, although their underlying mechanisms are not yet fully understood. Acetylcholine and sodium nitroprusside iontophoresis, despite their wide use as specific tests of endothelium-dependent and -independent function, respectively, show limitations. The influence of the skin site, recording conditions, and the way of expressing data are also reviewed. Finally, we focus on promising tools such as laser speckle contrast imaging.

Retention of poorly deformable red blood cells (RBCs) by the human spleen has been recognized as a critical determinant of pathogenesis in hereditary spherocytosis, malaria, and other RBC disorders. Using an ex vivo perfusion system, we had previously shown that retention of Plasmodium falciparum–infected RBCs (Pf-RBCs) occur in the splenic red pulp, upstream from the sinus wall. To experimentally replicate the mechanical sensing of RBCs by the splenic microcirculation, we designed a sorting device where a mixture of 5- to 25-μm-diameter microbeads mimics the geometry of narrow and short interendothelial splenic slits. Heated RBCs, Pf-RBCs, and RBCs from patients with hereditary spherocytosis were retained in the microbead layer, without hemolysis. The retention rates of Pf-RBCs were similar in microbeads and in isolated perfused human spleens. These in vitro results directly confirm the importance of the mechanical sensing of RBCs by the human spleen. In addition, rigid and deformable RBC subpopulations could be separated and characterized at the molecular level, and the device was used to deplete a stored RBC population from its subpopulation of rigid RBCs. This experimental approach may contribute to a better understanding of the role of the spleen in the pathogenesis of inherited and acquired RBC disorders.

Growth hormone (GH) exerts its actions via coordinated pulsatile secretion from a GH cell network into the bloodstream. Practically nothing is known about how the network receives its inputs in vivo and releases hormones into pituitary capillaries to shape GH pulses. Here we have developed in vivo approaches to measure local blood flow, oxygen partial pressure, and cell activity at single-cell resolution in mouse pituitary glands in situ. When secretagogue (GHRH) distribution was modeled with fluorescent markers injected into either the bloodstream or the nearby intercapillary space, a restricted distribution gradient evolved within the pituitary parenchyma. Injection of GHRH led to stimulation of both GH cell network activities and GH secretion, which was temporally associated with increases in blood flow rates and oxygen supply by capillaries, as well as oxygen consumption. Moreover, we observed a time-limiting step for hormone output at the perivascular level; macromolecules injected into the extracellular parenchyma moved rapidly to the perivascular space, but were then cleared more slowly in a size-dependent manner into capillary blood. Our findings suggest that GH pulse generation is not simply a GH cell network response, but is shaped by a tissue microenvironment context involving a functional association between the GH cell network activity and fluid microcirculation.

International audience; Perfusion magnetic resonance imaging (MRI) offers relative safety (no ionizing radiation), high spatial/temporal resolution, multi-orientation imaging capability, and relative low cost when compared with sophisticated techniques, such as positron emission tomography (PET). Several methods have been recently proposed to image perfusion. Some techniques mimic conventional nuclear medicine principles, but use radioactively inert tracers. Other approaches directly use blood as an endogenous natural tracer. Such methods are totally noninvasive, and offer original insights, for instance by monitoring variations in blood oxygenation in human brain cortex during activation tasks. These methods are presented and compared. Emphasis is given on advantages and drawbacks, and potential clinical applications.

International audience; Recent developments in the use of magnetic resonance (MR) to measure and image diffusion and blood microcirculation ("perfusion") are summarized. After a brief description of the effects of diffusion and perfusion on the MR signal, the different methods (conventional spin-echo, stimulated-echo, gradient-echo, and echo-planar imaging) that have been proposed and used to image and measure diffusion and perfusion by gradient sensitization are presented, along with their advantages and limitations. The difficulties of diffusion/perfusion imaging related to both hardware and software are then discussed. Special attention is given to specific problems encountered with in vivo studies and data analysis. Finally, the potential biologic and clinical applications are outlined, and some examples are presented.

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The current paradigm in Plasmodium falciparum malaria pathogenesis states that young, ring-infected erythrocytes (rings) circulate in peripheral blood and that mature stages are sequestered in the vasculature, avoiding clearance by the spleen. Through ex vivo perfusion of human spleens, we examined the interaction of this unique blood-filtering organ with P falciparum–infected erythrocytes. As predicted, mature stages were retained. However, more than 50% of rings were also retained and accumulated upstream from endothelial sinus wall slits of the open, slow red pulp microcirculation. Ten percent of rings were retained at each spleen passage, a rate matching the proportion of blood flowing through the slow circulatory compartment established in parallel using spleen contrast-enhanced ultrasonography in healthy volunteers. Rings displayed a mildly but significantly reduced elongation index, consistent with a retention process, due to their altered mechanical properties. This raises the new paradigm of a heterogeneous ring population, the less deformable subset being retained in the spleen, thereby reducing the parasite biomass that will sequester in vital organs, influencing the risk of severe complications, such as cerebral malaria or severe anemia. Cryptic ring retention uncovers a new role for the spleen in the control of parasite density, opening novel intervention opportunities.

International audience; AIM: Exercise training is known to improve endothelium-dependent relaxation in the coronary and skeletal muscle arteries. However, the effects of exercise training on peripheral nonworking tissue, including microcirculation, are still unclear. Therefore, we investigated the effect of chronic and regular aerobic exercise on cutaneous microvascular endothelial function in rats. METHODS: We assessed the effect of physical training on skin microcirculation in 7 sedentary (SED) and 21 training rats (Wistar-Kyoto), submitted to a treadmill training protocol (15 m/min; 15% incline; 60 min/day; 8 weeks). Training rats were divided into 3 groups, exercising 1 day/week (Ex1), 3 days/week (Ex3) or 5 days/week (Ex5). Cutaneous blood flow was recorded before the beginning of the training protocol, after 4 weeks and at the end of the training program. Hyperemic response (RH) was the flow reaction obtained after sudden release of the garrot. For data analysis, cutaneous vascular conductance (CVC) was indexed as cutaneous blood flow divided by mean arterial blood pressure (in millimeters of mercury, mmHg) and normalized to baseline values. RESULTS: At baseline, CVC was not different among groups (SED or training) at 3 steps of experimental protocol. The hyperemic stimulus significantly increased normalized CVC only in group Ex3 after 4 weeks (P

International audience; Aminopeptidase A (APA) generated brain angiotensin III, one of the main effector peptides of the brain renin angiotensin system, exerting a tonic stimulatory effect on the control of blood pressure in hypertensive rats. The distribution of APA in human brain has not been yet studied. We first biochemically characterized human brain APA (apparent molecular mass of 165 and 130 kDa) and we showed that the human enzyme exhibited similar enzymatic characteristics to recombinant mouse APA. Both enzymes had similar sensitivity to Ca(2+). Kinetic studies showed that the K(m) (190 mumol/L) of the human enzyme for the synthetic substrate-l-glutamyl-beta-naphthylamide was close from that of the mouse enzyme (256 mumol/L). Moreover, various classes of inhibitors including the specific and selective APA inhibitor, (S)-3-amino-4-mercapto-butyl sulfonic acid, had similar inhibitory potencies toward both enzymes. Using (S)-3-amino-4-mercapto-butyl sulfonic acid, we then specifically measured the activity of APA in 40 microdissected areas of the adult human brain. Significant heterogeneity was found in the activity of APA in the various analyzed regions. The highest activity was measured in the choroids plexus and the pineal gland. High activity was also detected in the dorsomedial medulla oblongata, in the septum, the prefrontal cortex, the olfactory bulb, the nucleus accumbens, and the hypothalamus, especially in the paraventricular and supraoptic nuclei. Immunostaining of human brain sections at the level of the medulla oblongata strengthened these data, showing for the first time a high density of immunoreactive neuronal cell bodies and fibers in the motor hypoglossal nucleus, the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, the Roller nucleus, the ambiguus nucleus, the inferior olivary complex, and in the external cuneate nucleus. APA immunoreactivity was also visualized in vessels and capillaries in the dorsal motor nucleus of the vagus and the inferior olivary complex. The presence of APA in several human brain nuclei sensitive to angiotensins and involved in blood pressure regulation suggests that APA in humans is an integral component of the brain renin angiotensin system and strengthens the idea that APA inhibitors could be clinically tested as an additional therapy for the treatment of certain forms of hypertension.

International audience; Staining and imaging glial cells in vivo while observing the microvasculature could help understand brain physiology, namely neuronal-glial-vascular communication. Two-photon excitation microscopy provides a means to monitor these interactions at the cellular level in living animals, but the cells of interest must be fluorescent. Injecting dyes intravenously is a rapid and quasi noninvasive method to stain cells in the brain. It necessitates that the dye is soluble in the blood plasma and crosses the blood brain barrier (BBB). We demonstrate here, using two-photon imaging, that sulforhodamine B (SRB) crosses the BBB and stains in vivo, specifically mouse astrocytes. This is confirmed by experiments on primary neurons and astrocytes cultures showing the preferential SRB staining of the latter. SRB is rapidly eliminated from the blood, which allows repeated injections in longitudinal studies.

International audience; Activin receptor-like kinase 1 (ALK1) is an endothelial-specific type I receptor of the TGFbeta receptor family that is implicated in angiogenesis and in the pathogenesis of the vascular disease, hereditary hemorrhagic telangiectasia (HHT). In the absence of a specific ligand, ALK1 cellular functions have been mainly studied through the use of a constitutively active form of this receptor (ALK1ca) and are still debated. We previously reported that ALK1ca inhibits proliferation and migration of human endothelial cells suggesting that ALK1 plays an important role in the maturation phase of angiogenesis (Lamouille et al., 2002, Blood 100: 4495-4501). In the present work, we further analyzed the role of ALK1 in the migration of human dermal microvascular endothelial cell (HMVEC-d) and observed that silencing endogenous ALK1 expression with siRNAs accelerates endothelial cell migration in the wound assay. Further, we demonstrate that ALK1-induced inhibition of migration is Smad-independent. Using a panel of kinase inhibitors, we found that HMVEC-d wound closure was completely inhibited by a JNK inhibitor and to a lower degree by an ERK kinase inhibitor. Further, HMVEC-d wounding induced activation of both JNK and ERK, and these were inhibited by ALK1ca expression. Taken together, these results support a significant role for ALK1 as a negative regulator of endothelial cell migration and suggest the implication of JNK and ERK as mediators of this effect.

Vascular endothelial growth factor (VEGF) is one of the most important regulators of physiological and pathological angiogenesis. Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway and overexpression of VEGF are common denominators of tumors from different origins. We have established a new link between these two fundamental observations converging on VEGF mRNA stability. In this complex phenomenon, tristetraprolin (TTP), an adenylate and uridylate-rich element-associated protein that binds to VEGF mRNA 3′-untranslated region, plays a key role by inducing VEGF mRNA degradation, thus maintaining basal VEGF mRNA amounts in normal cells. ERKs activation results in the accumulation of TTP mRNA. However, ERKs reduce the VEGF mRNA-destabilizing effect of TTP, leading to an increase in VEGF expression that favors the angiogenic switch. Moreover, TTP decreases RasVal12-dependent VEGF expression and development of vascularized tumors in nude mice. As a consequence, TTP might represent a novel antiangiogenic and antitumor agent acting through its destabilizing activity on VEGF mRNA. Determination of TTP and ERKs status would provide useful information for the evaluation of the angiogenic potential in human tumors.

International audience; Knowledge of the blood volume per unit volume of brain tissue is important for understanding brain function in health and disease. We describe a direct method using two-photon laser scanning microscopy to obtain in vivo the local capillary blood volume in the cortex of anesthetized mouse. We infused fluorescent dyes in the circulating blood and imaged the blood vessels, including the capillaries, to a depth of 600 microm below the dura at the brain surface. Capillary cortical blood volume (CCBV) was calculated without any form recognition and segmentation, by normalizing the total fluorescence measured at each depth and integrating the collected intensities all over the stack. Theoretical justifications are presented and numerical simulations were performed to validate this method which was weakly sensitive to background noise. Then, CCBV had been estimated on seven healthy mice between 2%+/-0.3% and 2.4%+/-0.4%. We showed that this measure of CCBV is reproductible and that this method is highly sensitive to the explored zones in the cortex (vessel density and size). This method, which dispenses with form recognition, is rapid and would allow to study in vivo temporal and highly resolute spatial variations of CCBV under different conditions or stimulations.

Received: 22 January 2007Accepted: 22 January 2007Published online: 14 February 2007© Springer-Verlag 2007This review intends tosummarize all articlespublished in Intensive Care Medicine in2006, grouped by specific topicsP. AndrewsWestern General Hospital, Intensive CareMedicine Unit,Edinburgh, UKE. AzoulaySaint Louis Hospital, Intensive CareMedicine Unit,Paris, FranceM. AntonelliUniversita Cattolica del Sacre Cuore,Department of Intensive Care andAnesthesiology,Rome, ItalyL. Brochard (✉)AP-HP, Hopital Henri Mondor, UniversiteParis 12, Reanimation Medicale, INSERMU 651,94000 Creteil, Francee-mail: laurent.brochard@hmn.aphp.frTel.: +33-1-49812545Fax: +33-1-42079943C. Brun-BuissonAP-HP, Hopital Henri Mondor, UniversiteParis 12, Reanimation Medicale,Creteil, FranceD. De BackerErasme Hospital, Service des SoinsIntensifs,Brussels, BelgiumG. DobbRoyal Perth Hospital, Intensive CareMedicine Unit,Perth, AustraliaJ.-Y. FagonEuropean Georges Pompidou Hospital,Intensive Care Medicine Unit,H. GerlachVivantes-Klinikum Neukoelln, Departmentof Anesthesiology,Berlin, GermanyJ. GroeneveldVUMC, Intensive Care Medicine Unit,Amsterdam, The NetherlandsD. MacraeRoyal Brompton Hospital, PediatricIntensive Care Unit,London, UKJ. ManceboHospital Sant Pau, Intensive Care MedicineUnit,Barcelona, SpainP. MetnitzUniversity Hospital of Vienna, Departmentof Anesthesia and General Intensive CareMedicine,Vienna, AustriaS. NavaFondazione S. Maugeri, Intensive CareMedicine Unit,Pavia, ItalyJ. PuginUniversity Hospital of Geneva, IntensiveCare Medicine Unit,Geneva, SwitzerlandM. PinskyUniversity of Pittsburgh Medical Center,Intensive Care Medicine Unit,Pittsburgh Pennsylvania, USAP. RadermacherUniversity Medical School of Ulm,Department of Anesthesia,Ulm, GermanyC. RichardUniversity Hospital of Le Kremlin-Bicetre,Intensive Care Unit,Le Kremlin-Bicetre, France

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Background and Purpose— Neonates that survive very preterm birth have a high prevalence of cognitive impairment in later life. A common factor detected in premature infants is their postnatal exposure to high oxygen tension relative to that in utero. Hyperoxia is known to elicit injury to premature lung and retina. Because data on the exposure of the brain to hyperoxia are limited, we studied the effects of high oxygen on this tissue. Methods— Rat pups were exposed from birth until day 6 to 21% or 80% O 2 . Cerebral vascular density was quantified by lectin immunohistochemistry. Immunoblots for several proteins were performed on brain extracts. We assessed cerebral functional deficits by visual evoked potentials. Results— Exposure of pups to hyperoxia leads to cerebral microvascular degeneration, diminished brain mass, and cerebral functional deficits. These effects are preceded by an upregulation of endothelial nitric oxide synthase (eNOS) in cerebral capillaries and a downregulation of Cu/Zn superoxide dismutase (SOD). The imbalance in nitric oxide (NO) production and antioxidant defenses favors the formation of nitrating agents in the microvessels revealed by increased nitrotyrosine (3-nt) immunoreactivity and decreased expression of NF-κB and the dependent vascular endothelial growth factor receptor 2. NOS inhibitors and eNOS deletion as well as an SOD mimetic (CuDIPS) restore vascular endothelial growth factor receptor-2 levels and nearly abolish the vasoobliteration. NOS inhibitors and SOD mimetic also prevent O 2 -induced diminished brain mass and functional deficit. Conclusions— Data identify NO and nitrating agents as major mediators of cerebral microvascular damage, ensuing impaired brain development and function in immature subjects exposed to hyperoxia.

International audience; Neisseria meningitidis is a commensal bacterium of the human nasopharynx. Occasionally, this bacterium reaches the bloodstream and causes meningitis after crossing the blood-brain barrier by an unknown mechanism. An immunohistological study of a meningococcal sepsis case revealed that neisserial adhesion was restricted to capillaries located in low blood flow regions in the infected organs. This study led to the hypothesis that drag forces encountered by the meningococcus in the bloodstream determine its attachment site in vessels. We therefore investigated the ability of N. meningitidis to bind to endothelial cells in the presence of liquid flow mimicking the bloodstream with a laminar flow chamber. Strikingly, average blood flows reported for various organs strongly inhibited initial adhesion. As cerebral microcirculation is known to be highly heterogeneous, cerebral blood velocity was investigated at the level of individual vessels using intravital imaging of rat brain. In agreement with the histological study, shear stress levels compatible with meningococcal adhesion were only observed in capillaries, which exhibited transient reductions in flow. The flow chamber assay revealed that, after initial attachment, bacteria resisted high blood velocities and even multiplied, forming microcolonies resembling those observed in the septicemia case. These results argue that the combined mechanical properties of neisserial adhesion and blood microcirculation target meningococci to transiently underperfused cerebral capillaries and thus determine disease development.

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International audience; PURPOSE: The purpose of this study was to assess the early effects of microbeam irradiation on the vascular permeability and volume in the parietal cortex of normal nude mice using two-photon microscopy and immunohistochemistry. METHODS AND MATERIALS: The upper part of the left hemisphere of 55 mice was irradiated anteroposteriorly using 18 vertically oriented beams (width 25 microm, interdistance 211 microm; peak entrance doses: 312 or 1000 Gy). At different times after microbeam exposure, the microvasculature in the cortex was analyzed using intravital two-photon microscopy after intravascular injection of fluorescein isothiocyanate (FITC)-dextrans and sulforhodamine B (SRB). Changes of the vascular volume were observed at the FITC wavelength over a maximum depth of 650 mum from the dura. The vascular permeability was detected as extravasations of SRB. RESULTS: For all times (12 h to 1 month) after microbeam irradiation and for both doses, the FITC-dextran remained in the vessels. No significant change in vascular volume was observed between 12 h and 3 months after irradiation. Diffusion of SRB was observed in microbeam irradiated regions from 12 h until 12 days only after a 1000 Gy exposure. CONCLUSION: No radiation damage to the microvasculature was detected in normal brain tissue after a 312 Gy microbeam irradiation. This dose would be more appropriate than 1000 Gy for the treatment of brain tumors using crossfired microbeams.

Background: The architectural frameworks of the skin microcirculation are rather complex and change continuously with aging. But these changes are yet poorly documented in vivo. Objectives: Using non-invasive methods belonging to the field of biometrology, the study aimed to investigate quantitatively the changes of the cutaneous microvasculature in different anatomic sites with age. Methods: Measurements were performed on crow’s feet, forehead, volar forearm and dorsum of hand in 50 women (aged 20–74 years who consisted of 10 probands in each live decades). The superficial vascular plexus was scanned by videocapillaroscopy and assessed with the software Capilab Toolbox®. The subpapillary vascular plexus was explored with laser Doppler flowmetry. The skin color a* was analyzed by chromametry. Results: A marked site and age effect on the skin microcirculation has been demonstrated. The density of capillary loops in the eldest group decreased by about 40–70% compared with the youngest group whereas the vascular length increased by 35–156%. The capillary density in the back of the hand was 4 times higher than in the crow’s feet. The vascular length in the crow’s feet was 3 times longer than in the back of the hand. Both blood flow and skin redness (a*) increased also with age. Conclusion: Both morphology and quantification of the cutaneous microvasculature showed changes with site and age. Videocapillaroscopy associated to an image processing and laser Doppler flowmetry revealed different vascular layers. So the combination of both instruments offers an easy way to observe the architectural frameworks in vivo.

International audience; A physiological pharmacokinetic (PBPK) model was used to estimate tumor microcirculation in nude mice with a grafted tumor. The kinetics of a rapid clearance blood pool agent, Vistarem, were investigated by dynamic MRI after bolus administration. Signal enhancements were recorded in arterial blood and in tumor tissue. To analyze these data, we developed a whole-body mathematical model of the agent's biodistribution using physiological parameters. The model included six compartments: arterial and venous plasma, tumor (split into capillaries and interstitium), and the rest of the body (also split into capillaries and interstitium). As an application, changes in tumor microcirculation parameters were evaluated in mice receiving either an antiangiogenic treatment (ZD4190) or a placebo. The analysis was performed in a Bayesian framework, and the model was fitted to experimental data using Markov Chain Monte Carlo techniques. Results showed a significant difference in tumor microcirculation between the two groups of mice when the microcirculation parameters are considered together. This whole-body physiological model enables to analyze jointly data in tumor tissue and in arterial blood. This leads to accurate estimates of microcirculation parameters and the evaluation of their uncertainty.

International audience; Abstract Skin topography and microvasculature undergo characteristic changes with age. Although several non-invasive bioengineering methods are currently available to measure them quantitatively, few publications have referred to their relationship with age in different anatomical sites. This study was carried out to observe the age-related changes of the skin topography and skin microcirculation. The microrelief was assessed with special processing software from scanning by interference fringe profilometry of silicone replicas performed on two sites (volar forearm and back of hand) on 50 female volunteers (aged 20–74 years who consisted of ten probands in each decade). The superficial vascular network of both sites was assessed by videocapillaroscopy, and the subpapillary vascular plexus was studied with laser Doppler flowmetry. Skin color, which is affected by blood flow, was observed by colorimeter. The skin roughness and the mean height between peak and valley increased with age. There were statistically significant differences between the evaluated sites. This study also shows that the capillary loops in the dermal papillae decrease but the subpapillary plexus increase with age. The interference fringe profilometry associated with videocapillaroscopy may be useful and accurate to measure the efficacy of medical or cosmetic products to delay skin aging.

E K-D and F S contributed equally to this work; Nitrative stress has an important role in microvascular degeneration leading to ischemia in conditions such as diabetic retinopathy and retinopathy of prematurity. Thus far, mediators of nitrative stress have been poorly characterized. We recently described that trans-arachidonic acids are major products of NO(2)(*)-mediated isomerization of arachidonic acid within the cell membrane, but their biological relevance is unknown. Here we show that trans-arachidonic acids are generated in a model of retinal microangiopathy in vivo in a NO(*)-dependent manner. They induce a selective time- and concentration-dependent apoptosis of microvascular endothelial cells in vitro, and result in retinal microvascular degeneration ex vivo and in vivo. These effects are mediated by an upregulation of the antiangiogenic factor thrombospondin-1, independently of classical arachidonic acid metabolism. Our findings provide new insight into the molecular mechanisms of nitrative stress in microvascular injury and suggest new therapeutic avenues in the management of disorders involving nitrative stress, such as ischemic retinopathies and encephalopathies.

International audience; We undertook this study to determine whether long-term high intensity exercise would modify cutaneous endothelial-dependent vasodilation. We compared a group of 9 highly trained windsurfers (mean age: 24.5 +/- 1.6 years) to a control group of 8 sedentary individuals (22.9 +/- 0.4 years, NS). Laser Doppler was used to measure cutaneous blood flow in the resting state (baseline), during post-occlusive hyperaemia (endothelium-dependent vasodilation), and local heating to 42 degrees C. Lipid profile was similar in both groups. Resting heart rate was significantly lower in windsurfers. Baseline cutaneous vascular conductance (CVC) values were similar in both groups (0.059 +/- 0.016 and 0.051 +/- 0.009). During reactive hyperaemia, normalized peak CVC value was significantly higher in the windsurfers group (1775.4 +/- 286.9 and 826.4 +/- 121.7 % baseline CVC; p = 0.01). Normalized peak CVC value in response to local heating (42 degrees C) was not significantly different between both groups (2359.4 +/- 346.1 and 1467.7 +/- 282.6 % baseline CVC). Endothelium-dependent vasodilation in cutaneous microcirculation is significantly enhanced in the forearm skin of highly trained windsurfers when compared to sedentary controls.

International audience; Hyperglycemia-induced oxidative stress is detrimental for endothelial cells, contributing to the vascular complications of diabetes. The mitochondrial permeability transition pore (PTP) is an oxidative stress-sensitive channel involved in cell death; therefore, we have examined its potential role in endothelial cells exposed to oxidative stress or high glucose level. Metformin, an antihyperglycemic agent used in type 2 diabetes, was also investigated because it inhibits PTP opening in transformed cell lines. Cyclosporin A (CsA), the reference PTP inhibitor, and a therapeutic dose of metformin (100 micromol/l) led to PTP inhibition in permeabilized human microvascular endothelial cells (HMEC-1). Furthermore, exposure of intact HMEC-1 or primary endothelial cells from either human umbilical vein or bovine aorta to the oxidizing agent tert-butylhydroperoxide or to 30 mmol/l glucose triggered PTP opening, cytochrome c decompartmentalization, and cell death. CsA or metformin prevented all of these effects. The antioxidant N-acetyl-l-cysteine also prevented hyperglycemia-induced apoptosis. We conclude that 1) elevated glucose concentration leads to an oxidative stress that favors PTP opening and subsequent cell death in several endothelial cell types and 2) metformin prevents this PTP opening-related cell death. We propose that metformin improves diabetes-associated vascular disease both by lowering blood glucose and by its effect on PTP regulation.

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Vessel size imaging (VSI) for brain tumor characterization was evaluated and the vessel size index measured by MRI (VSIMRI) was correlated with VSI obtained by histology (VSIhisto). Blood volume (BV) and VSI maps were obtained on 12 rats by simultaneous measurements of R2* and R2, before and after the injection of a macromolecular contrast agent, AMI-227. Immunostaining of collagen IV in vessels was performed. An expression was derived for evaluating VSI from stereologic measurements on histology data (VSIhisto). On BV and VSI images obtained from large-size tumors (n = 9), three regions could be distinguished and correlated well with histological sections: a high BV region surrounding the tumor, a necrotic area where BV is very low, and a viable tumor tissue region showing lower BV but higher VSI than the normal rat cortex, with the presence of larger vessels. The quantitative analysis showed a good correlation (Spearman rank's rho = 0.74) between VSIhisto and VSIMRI with a linear regression coefficient of 1.17. The good correlation coefficient supports VSI imaging as a quantitative method for tumor vasculature characterization.

Daviet J-C, Preux P-M, Rebeyrotte I, Lacroix P, Dudognon P, Munoz M, Salle J-Y. Reliability of transcutaneous oxygen tension measurement on the back of the hand and complex regional pain syndrome after stroke. Arch Phys Med Rehabil 2004;85:1102–5. Objective To verify the reproducibility of measurement of transcutaneous oxygen tension (TcP o 2) on the back of the hand in control subjects and stroke patients in the assessment of the complex regional pain syndrome type I (CRPS I). Design Case series study. Setting Physical medicine and rehabilitation department at a university hospital. Participants Eighteen control subjects, 30 stroke patients without CRPS I, and 12 stroke patients with CRPS I. Interventions Not applicable. Main outcome measures TcP o 2 was measured on the back of hands on 2 consecutive days using a polarographic technique. The reproducibility was evaluated by using the intraclass correlation coefficient (ICC) and the coefficient of variation. Results In the controls, the values of TcP o 2 were not reproducible, with an ICC of .51 (95% confidence interval [CI], .23–.72). Similarly, in the hemiplegics with and without CRPS I, TcP o 2 was not reproducible, with an ICC of .43 (95% CI, −.15 to .74) and .69 (95% CI, .45–.84), respectively. The differences between the 2 upper limbs were even less reproducible in each population. Conclusions Measurement of TcP o 2 on the hand using our procedure did not seem to be sufficiently reproducible for application to a pathology such as CRPS I.

International audience; BACKGROUND: Despite aggressive resuscitation shock often results in multiple-organ failure characterized by increased energy demands of organs and decreased ability of effective energy production. The administration of ATP-MgCl(2) as a supportive measure has been investigated in various animal models of ischemia/reperfusion injury and hemorrhagic, endotoxic, and septic shock. INVESTIGATIONS: These studies showed improvement in organ blood flow, microcirculation, energy balance, cellular and mitochondrial, functions and restoration of immune competence, ultimately leading to increased survival. Originally these effects were attributed to direct energy provision by the ATP-Mg complex, but the minute amount of ATP infused compared to the body's ATP formation rate suggests that other mechanisms must be responsible for its beneficial properties such as stabilization of the cell membrane, phosphorylation of membrane proteins, decreased cell swelling, and improved microcirculatory perfusion. CONCLUSIONS: The experimental evidence currently available suggests the use of ATP-MgCl(2) as a therapeutic adjunct in patients with multiple-organ dysfunction. In addition, given the extremely short half-life which allows both rapid titration and control of the systemic hemodynamic response, for example, reduction in mean arterial pressure, ATP-MgCl(2) may be suitable as an alternative to other fast-acting vasodilators used for the management of acute pulmonary hypertensive crises and/or for the maintenance blood pressure during aortic cross-clamping.

International audience; Recent developments in the use of Magnetic Resonance Imaging (MRI) to measure and image molecular diffusion and blood microcirculation (perfusion) hold significant promise in the noninvasive evaluation of normal brain function and functional disorders. Molecular diffusion is the result of spontaneous random motion that involves all molecules and probes molecular motion at microscopic level. Using diffusion MRI, information on tissue geometry and compartmentation effects can be obtained. Diffusion MRI has been used to map myelin fiber orientation in brain with high accuracy. Diffusion MRI is also the only imaging modality which shows brain ischemia at a very early stage, even before T1w or T2w MR images become abnormal, offering great promises in the management of stroke patients. Also, diffusion imaging may be used to monitor tissue temperature changes noninvasively during hyperthermia or laser surgery. On the other hand, MRI can provide information on tissue perfusion. Several methods have been proposed, some of them including tracers or contrast agents. The most successful approach for brain function studies, however, is based on the sensitivity of MRI to magnetic effects induced by changes in the oxygenation status of hemoglobin (deoxyhemoglobin). These effects have already been used to characterize hematomas. These effects may also be exploited to detect small modulation in red blood cell oxygen content related to local variations in blood flow and oxygen consumption in tissues. In the brain cortex, such variations may be induced by external stimuli or internal cognitive processes. Capillary blood deoxyhemoglobin thus acts as a natural endogeneous contrast agent.(ABSTRACT TRUNCATED AT 250 WORDS)

International audience; The purpose of this paper is to review the basic principles of diffusion measurement with spin echoes. These principles can be combined with those of MR imaging to generate maps of diffusion coefficients. Diffusion imaging can be extended to imaging of other intravoxel incoherent motions (IVIM), such as blood microcirculation. Some of the technical problems encountered when implementing IVIM imaging are presented.

International audience; Recent developments have shown that diffusion and blood microcirculation (perfusion) could be imaged and measured noninvasively by MRI. The purpose of this presentation is to overview the different approaches that use B0 field gradients to monitor diffusion/perfusion. The principles of these different methods are discussed together with their limitations and their potential applications.

International audience; Recent developments have shown that diffusion and blood microcirculation (perfusion) could be imaged and measured noninvasively by MRI. The purpose of this presentation is to overview the different approaches that use B0 field gradients to monitor diffusion/perfusion. The principles of these different methods are discussed together with their limitations and their potential applications.

International audience; Recent developments have shown that diffusion and blood microcirculation (perfusion) could be imaged and measured noninvasively by MRI. The purpose of this presentation is to overview the different approaches that use B0 field gradients to monitor diffusion/perfusion. The principles of these different methods are discussed together with their limitations and their potential applications.

International audience; Intravoxel incoherent motion (IVIM) imaging is a method the authors developed to visualize microscopic motions of water. In biologic tissues, these motions include molecular diffusion and microcirculation of blood in the capillary network. IVIM images are quantified by an apparent diffusion coefficient (ADC), which integrates the effects of both diffusion and perfusion. The aim of this work was to demonstrate how much perfusion contributes to the ADC and to present a method for obtaining separate images of diffusion and perfusion. Images were obtained at 0.5 T with high-resolution multisection sequences and without the use of contrast material. Results in a phantom made of resin microspheres demonstrated the ability of the method to separately evaluate diffusion and perfusion. The method was then applied in patients with brain and bone tumors and brain ischemia. Clinical results showed significant promise of the method for tissue characterization by perfusion patterns and for functional studies in the evaluation of the microcirculation in physiologic and pathologic conditions, as, for instance, in brain ischemia.