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At least 649 records · Page 36Linked to original sources
PubMed1997

Differential effects of endothelin receptor activation on cyclic flow variations in rat mesenteric arteries.

BACKGROUND: Cyclic flow variations (CFVs) represent repetitive cycles of platelet adherence-aggregation and vasoconstriction, followed by dislodgment of platelet thrombi and restoration of blood flow at the site of vascular injury. Although activation of endothelin A (ETA) and endothelin B (ETB) receptors leads to vasoconstriction and nitric oxide release, respectively, the roles of endogenous endothelin-1 (ET-1) and its receptors in CFVs are unknown. METHODS AND RESULTS: A side branch of a mesenteric artery of male Wistar rats was cannulated and a short segment of the artery was mechanically injured to induce CFVs. After 20 minutes of saline infusion, either saline (negative control), BQ-123 (ETA receptor antagonist, 10 microg/min), BQ-788 (ETB receptor antagonist, 10 microg/min), or sarafotoxin S6c (ETB receptor agonist, 10 ng/min) was infused for 20 minutes from the side branch into the injured arterial segment. Percent (%) luminal stenosis as well as proximal and distal vessel diameters were observed and quantitatively measured every minute using intravital video microscopy and a micrometer-calibrated video screen. Both BQ-123 and sarafotoxin S6c significantly reduced CFVs represented by the mean luminal stenosis (BQ-123=29+/-13% and sarafotoxin S6c=27+/-11% reduction, respectively; P<.05 for both, compared with saline). In contrast, BQ-788 significantly increased CFVs (33+/-6% increase, P<.05 compared with saline). Moreover, the inhibitory effect of sarafotoxin S6c on CFVs was completely abolished in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME) (a nitric oxide synthase inhibitor, 10(-5) mol/L) in superfusate over the arteries (16.1+/-5% increase, P=NS compared with saline in the presence of L-NAME). In addition, BQ-123 caused a significant increase in the diameter of the vessel distal to the injured segment (12+/-4% increase, P<.05 compared with saline). CONCLUSIONS: Endogenous ET-1 release from sites of vascular injury contributes to CFVs and vasomotor tone in the rat mesenteric artery CFV model. ETA and ETB receptors have differential roles in CFVs: ETA receptor antagonism and ETB receptor stimulation reduce CFVs, the latter at least partially through increased nitric oxide formation.

Animals↗
PubMed1986

Velocity profiles of blood platelets and red blood cells flowing in arterioles of the rabbit mesentery.

Velocity profiles were determined in rabbit mesenteric arterioles (diameter 17-32 micron). A good spatial resolution was obtained by using the blood platelets as small and natural markers of flow, providing for the first time in vivo detailed, quantitative information about the shape of the velocity profiles in microvessels. In some experiments red blood cell velocity profiles were recorded as well. Easy detection of the cells of interest could be achieved by labelling them selectively with a fluorescent dye and visualizing them by intravital fluorescence video microscopy, using flashed illumination. Pairs of flashes were given with a short, preset time interval between both flashes, yielding in one TV picture two images of the same cell displaced over a certain distance for the given time interval. Velocity and mean radial position of cells, flowing within an optical section around the median plane of the vessel, were determined. The shape of the velocity profiles of platelets and red blood cells was similar. The profiles were flattened as compared to a parabola, both in systole and diastole. Vessel diameter did not change measurably during the cardiac cycle. As an index of the degree of blunting of the profiles, the ratio of the maximal and mean velocity of the profile was used, which is 2 for a parabola and 1 for complete plug flow. The index ranged from 1.39 to 1.54 (median 1.50), and increased with vessel diameter. Calculations showed that the blunting of the profiles cannot be explained by an influence of the finite depth of the optical section.

Animals↗
PubMed1991

A comparison of spreading and motility behaviour of 8701-BC breast carcinoma cells on type I, I-trimer and type V collagen substrata. Evidence for a permissive effect of type I-trimer collagen on cell locomotion.

Ductal infiltrating carcinoma (d.i.c.) of human breast is a highly invasive neoplasm characterized by enhanced deposition of collagen. Paradoxically, enhanced collagen deposition is not correlated with inhibition of the migration of tumour cells into the host tissue. d.i.c. is characterized by the reappearance of 'embryonic' type I-trimer collagen and an increase in type V collagen content in the matrix. The effects of these two collagen types were compared with type I collagen as culture substrata on the spreading pattern, cytoskeletal organization and motile behaviour of 8701-BC breast carcinoma cells using rhodamine-phalloidin staining, a DNAase I-competition assay, scanning electron microscopy and time-lapse video-microscopy. Cells grown on type I collagen were stationary, showing a well-spread morphology and an extensive stress fibre pattern. Cells grown on type V collagen were also stationary, but displayed a poorly spread and elongated morphology. In contrast, cells grown on trimer collagen were motile and displayed a compact morphology and a reduced content of stress fibres. Both single-cell and group motility were detectable on trimer collagen substratum. These data are consistent with the existence of two opposite local signals, type I-trimer and type V collagens, which may confer a more or a less metastatic phenotype on breast carcinoma cells. Moreover, the synthesis of trimer collagen in d.i.c. is conceivably instrumental in providing new stromal pathways permitting tumour cells to infiltrate the host tissue.

Actins↗
PubMed1991

Microcirculatory pathways and blood flow in spleen: new insights from washout kinetics, corrosion casts, and quantitative intravital videomicroscopy.

The dynamics of splenic microcirculatory blood flow and the role of the spleen with respect to red blood cells (RBCs) have been elucidated by means of several different experimental approaches. First, the organ was treated as a 'black box' and input/output relationships were studied, such as the kinetics of RBC and plasma washout during Ringer perfusion. Second, microcorrosion casts were prepared from minimal amounts of injected material, such that 'fast' pathways for flow were selectively identified. Third, high-resolution video-microscopy was carried out in rat and mouse spleens transilluminated in vivo, with subsequent quantitative analysis of the data. New insights were obtained regarding fast and slow pathways for RBC flow; the origin of the high intrasplenic hematocrit; immature RBCs and the spleen; the pH, O2 tension and glucose concentration within the reticular meshwork; microcirculatory pathways bordering the white pulp; entry of blood into venous sinuses, both by open ends in the marginal sinus/zone and via interendothelial slits in sinus walls; spontaneous cyclic contractions of capillary walls in vivo, impeding RBC flow (i.e. endothelial contractility); and leukocyte interactions with walls of venous vessels. The different experimental approaches have provided complementary information and have clarified a number of important issues about which uncertainty existed in the literature. Exploiting the quantitative analysis of high-resolution intravital videomicroscopic recordings has an exciting potential for gaining new insights into the workings of this complex and neglected organ.

Animals↗
PubMed1987

Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae.

Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of 51Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cells (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of 51Cr release from radiolabeled monolayers. From these studies, we conclude that neutrophils are capable of killing Candida hyphae selectively within human vascular endothelial cell monolayers and may have protective rather than detrimental effects on endothelial cell integrity.

Candida albicans↗
PubMed1999

Dimensional and mechanical dynamics of active and stable edges in motile fibroblasts investigated by using atomic force microscopy.

The atomic force microscope (AFM) was employed to investigate the extension and retraction dynamics of protruding and stable edges of motile 3T3 fibroblasts in culture. Such dynamics closely paralleled the results of earlier studies employing video microscopy that indicated that the AFM force-mapping technique does not appreciably perturb these dynamics. Force scans permitted height determinations of active and stable edges. Whereas the profiles of active edges are flat with average heights of 0.4-0.8 micrometer, stable edges smoothly ascend to 2-3 micrometers within about 6 micrometers of the edge. In the region of the leading edge, the height fluctuates up to 50% (SD) of the mean value, much more than the stable edge; this fluctuation presumably reflects differences in underlying cytoskeletal activity. In addition, force mapping yields an estimate of the local Young's modulus or modulus of elasticity (E, the cortical stiffness). This stiffness will be related to "cortical tension," can be accurately calculated for the stable edges, and is approximately 12 kPa in this case. The thinness of the leading edge precludes accurate estimation of the E values, but within 4 micrometers of the margin it is considerably smaller than that for stable edges, which have an upper limit of 3-5 kPa. Although blebbing cannot absolutely be ruled out as a mechanism of extension, the data are consistent with an actin polymerization and/or myosin motor mechanism in which the average material properties of the extending margin would be nearly constant to the edge. Because the leading edge is softer than the stable edge, these data also are consistent with the notion that extension preferentially occurs in regions of lower cortical tension.

3T3 Cells↗
PubMed2003

Microscopy and calorimetry as complementary techniques to analyze sugar crystallization from amorphous systems.

A comparison of microscopic and macroscopic techniques to evaluate sugar crystallization kinetics is presented using amorphous lactose and lactose-trehalose mixtures. Polarized light video microscopy (PLV) and differential scanning calorimetry (DSC) were applied to measure crystallization kinetics, induction times and time for complete sugar crystallization at different storage temperatures (60-95 degrees C). DSC was also employed to measure the glass transition temperature (T(ag)) of the systems. PLV permitted direct observation, in real time, of growth of individual crystals and morphological aspects at a scale not detected by DSC. Taking the average of several microscopic observations, the results for temperature dependence of crystallization rate and time to complete lactose crystallization were similar to those obtained by DSC. Both PLV and DSC techniques showed that the presence of trehalose delayed lactose crystallization, without affecting the T(ag) value. For the analysis of sugar crystallization in amorphous systems, PLV and DSC proved to be complementary techniques. Validation of results obtained by PLV with results from DSC opens a new area of microstructural analysis of crystallizing systems.

Calorimetry, Differential Scanning↗
PubMed2006

A novel approach to the study of human microcirculation: Reactivity to locally applied angiotensin II in the conjunctival microvascular bed.

OBJECTIVE: To develop a state-of-the-art, computer-assisted intravital microscopy protocol to evaluate directly the effects of topically applied drugs on conjunctival arteriolar and venular diameters. METHODS: Fifty-one normotensive volunteers were studied. Video-recordings of the bulbar conjunctival microcirculation were made before and following eye drops containing angiotensin II (AngII) (0.001% w/w, or 0.01%) or phenylephrine (0.25%). The computer-assisted analyses of arteriolar and venular diameters were performed off-line. In different protocols the microvascular reactivity to the different eye drops were compared. RESULTS: AngII (0.01%) eye drops, but not AngII (0.001%), induced significant constriction in both arterioles (median, 19%) and venules (13%). Phenylephrine eye drops (pharmacological control) induced similar arteriolar (18%) and venular (12%) constrictions. Repeated AngII challenges with a 30-min interval revealed reproducible vasoconstriction responses (median arteriolar constriction, 11 and 17%, respectively; NS). The vasoconstriction responses following AngII challenges on two consecutive days revealed reproducible responses (median arteriolar constriction, 13 and 11%, respectively; NS). CONCLUSIONS: The present results demonstrate that the proposed model for noninvasive intravital video-microscopy of the conjunctival microcirculation is sensitive for measuring direct arteriolar and venular reactivity following topically applied drugs. We consider this model a valuable tool for sophisticated research on in-vivo microvascular reactivity in humans.

Administration, Topical↗
PubMed1998

Comparison of mitosis in binucleated and mononucleated newt cardiac myocytes.

BACKGROUND: The cultured adult newt ventricular myocyte has been shown to undergo mitosis and cytokinesis in a fully differentiated state. Insight into its proliferation and cellular changes during the repair process involves obtaining a better understanding of the nuclear pattern (mononucleated, binucleated, or multinucleated) resulting from mitotic events. Mitosis is easily observable in cultured newt cardiac myocytes using phase-contrast microscopy. METHODS: From days 8-19 in culture, the process of mitosis in mononucleated and binucleated newt ventricular myocytes was recorded and timed by using time-lapse video microscopy. Cultured cardiac myocytes were double-stained for myosin and F-actin by using fluorescein isothiocyanate (FITC)-labeled MF20 and rhodamine phalloidin. RESULTS: Mitotic, mononucleated myocytes produced mononucleated daughter cells in 80% of the cases, whereas 20% were single, binucleated myocytes, In binucleated myocytes, only 32% underwent complete cytokinesis to produce two binucleated daughter cells, whereas 68% resulted in variably nucleated myocytes. Mononucleated and binucleated myocytes undergoing mitosis had similar time intervals for the period from nuclear breakdown (prometaphase) to the start of anaphase (108.7 minutes and 94.5 minutes, respectively), but the period between anaphase and midbody formation was significantly shorter in binucleated than in mononucleated myocytes (43.5 minutes and 69.3 minutes, respectively). The myofibrillae were not as well organized in binucleated myocytes as those observed in mononucleated myocytes. CONCLUSIONS: Mitosis in vitro appears to proceed more rapidly in binucleated newt cardiac myocytes, which have more poorly organized myofibrillae than mononucleated myocytes. Mitosis of cultured binucleated myocytes commonly results in variably nucleated daughter cells, whereas mononucleated myocytes produce predominantly mononucleated daughter cells.

Actins↗
PubMed1995

[Corneal changes and corneal healing after keratomileusis in situ. In vivo studies using confocal slit-scanning microscopy].

So far, no investigations of micromorphological changes in the human cornea after in situ keratomileusis have been published. In 10 eyes of five patients who had undergone in situ keratomileusis, wound healing processes were followed up with a newly developed confocal slit-scanning video-microscope. Post-operatively, alterations in the corneal endothelium and in the corneal stroma were found. Wide streaky distortions were observed within the stroma above and below the lamellar incision during the early days immediately after surgery. These distortions were probably induced by shearing stresses of the oscillating blade of the microkeratome. Small scattering centers were seen at the level of the lamellar incision, which are thought to reflect a very slight roughness of the interface. In the immediate postoperative period the corneal endothelium showed reversible inhomogeneitis similar to blebs after short-term contact lens wearing, which are thought to be due to a hypoxia during the surgery. The endothelial alterations and the streaky distortions within the stroma disappeared within 9 days after the surgery whereas the scattering centers regressed much more slowly and could still be observed 60 days after the surgery. The total amount of scattered light of the cornea, however, was not increased except in one patient. The results show that outside the level of the lamellar incision, in situ keratomileusis leads to only short-term alterations of the corneal micromorphology, and that confocal in vivo slit-scanning video-microscopy allows thorough examination of these alterations.

Adolescent↗
PubMed2006

pH-induced release from P2VP-PEO block copolymer vesicles.

The pH-induced release of hydrophilic dyes from poly(2-vinylpyridine-b-ethylene oxide) (P2VP-PEO) block copolymer vesicles is investigated. The structure of the vesicles is characterized using small-angle neutron scattering (SANS) and cryo-electron microscopy (cryo-TEM). A decrease of the pH below 5 leads to protonation and dissolution of the poly-2-vinylpyridine blocks which induces rupture and dissolution of the vesicle membrane. Details of the rupture, dissolution, and release process are studied by fluorescence video microscopy, gel electrophoresis, and high-performance ultrafiltration.

Cryoelectron Microscopy↗
PubMed2004

Quantitative imaging of lymphocyte membrane protein reorganization and signaling.

Changes in membrane protein localization are critical to establishing cell polarity and regulating cell signaling. Fluorescence microscopy of labeled proteins allows visualization of these changes, but quantitative analysis is needed to study this aspect of cell signaling in full mechanistic detail. We have developed a novel approach for quantitative assessment of membrane protein redistribution based on four-dimensional video microscopy of fluorescently labeled proteins. Our analytic system provides robust automated methods for cell surface reconstruction, cell shape tracking, cell-surface distance measurement, and cluster formation analysis. These methods permit statistical analyses and testing of mechanistic hypotheses regarding cell signaling. We have used this approach to measure antigen-dependent clustering of signaling molecules in CD4+ T lymphocytes, obtaining clustering velocities consistent with single-particle tracking data. Our system captures quantitative differences in clustering between signaling proteins with distinct biological functions. Our methods can be generalized to a range of cell-signaling phenomena and enable novel applications not feasible with single-particle studies, such as analysis of subcellular protein localization in live organ culture.

Algorithms↗
PubMed1999

Sex differences in cell migration in the preoptic area/anterior hypothalamus of mice.

The preoptic area/anterior hypothalamus (POA/AH) sits as a boundary region rostral to the classical diencephalic hypothalamus and ventral to the telencephalic septal region. Numerous studies have pointed to the region's importance for sex-dependent functions. Previous studies suggested that migratory guidance cues within this region might be particularly unique in their diversity. To better understand the early development and differentiation of the POA/AH, cytoarchitectural, birthdate, immunocytochemical, and cell migration studies were conducted in vivo and in vitro using embryonic C57BL/6J mice. A medial preoptic nucleus became discernible using Nissl stain in males and females between embryonic days (E) E15 and E17. Cells containing immunoreactive estrogen receptor-alpha were detected in the POA/AH by E13, and increased in number with age in both sexes. From E15 to E17, examination of the radial glial fiber pattern by immunocytochemistry confirmed the presence of dual orientations for migratory guidance ventral to the anterior commissure (medial-lateral and dorsal-ventral) and uniform orientation more caudally (medial-lateral). Video microscopy studies followed the migration of DiI-labeled cells in coronal 250-microm brain slices from E15 mice maintained in serum-free media for 1-3 days. Analyses showed significant migration along a dorsal-ventral orientation in addition to medial-lateral. The video analyses showed significantly more medial-lateral migration in males than females in the caudal POA/AH. In vivo, changes in the distribution of cells labeled by the mitotic indicator bromodeoxyuridine (BrdU) suggested their progressive migration through the POA/AH. BrdU analyses also indicated significant movement from dorsal to ventral regions ventral to the anterior commissure. The significant dorsal-ventral migration of cells in the POA/AH provides additional support for the notion that the region integrates developmental information from both telencephalic and diencephalic compartments. The sex difference in the orientation of migration of cells in the caudal POA/AH suggests one locus for the influence of gonadal steroids in the embryonic mouse forebrain.

Animals↗
PubMed2003

Comparison of three computer methods of sperm head analysis.

OBJECTIVE: Analysis of sperm heads using three different computer morphometrical tools and experimental conditions to find a more reliable and secure strategy among them. DESIGN: Controlled experiments on sperm morphology analysis from volunteers. SETTING: Laboratory of microscopy and imaging processing. PATIENT(S): Ten human semen samples donated by different zoospermic men. INTERVENTION(S): Semen samples were collected by masturbation after > or =72 hours of abstinence. MAIN OUTCOME MEASURE(S): Spermatozoon head morphology was compared by the use of different video-microscopy systems, three computer programs, and various staining conditions and manipulation by different operators. Nonbiological material in the form of latex beads was also used. RESULT(S): The data obtained suggest that the semiautomatic computer program is the most reliable and secure method for performing sperm analysis, besides the fact that it is a fast process compared with manual methods. CONCLUSION(S): Computer systems of sperm analysis should incorporate a step of interactive object identification to work properly, allowing the operator to confirm or correct possible computer misidentification. The latex beads were used to confirm the capability of all three computer programs to correctly evaluate nonbiological material.

Automation↗
PubMed1998

Semi-automated measurement of motility of human subgingival microflora by image analysis.

The purpose of this investigation was to quantitatively estimate bacterial motility by image analysis, and to apply this method for the measurement of motility of human subgingival microflora. We developed a semi-automated method for the quantification of bacterial motility using video microscopy, digitization and image processing. Moving images of both authentic bacterial samples and clinical samples were recorded using a phase contrast microscope with a high speed (1/100 s) shutter camera. The motility was evaluated by measuring the total number of pixels remaining after the subtraction of 2 serial video images. The total number of pixels was significantly correlated with both the sum of the velocity of each bacterial cell and the number of motile bacteria on the same original images. Motility of subgingival microflora from 140 clinical samples tested was measured at 0 pixels to 3600 pixels, whereas the effect of Brownian movement was less than 150 pixels. The motility of subgingival microflora estimated with this image analysis system did not differ much from objective judgments by the naked eyes of experts. These results suggest that a semi-automated image analysis system may be useful in the evaluation of the motility of human subgingival microflora.

Adolescent↗
PubMedDate not supplied

[Development of instrumental and technical measurement aspects for clinical capillary microscopy].

Clinically oriented investigation in microcirculation was stimulated by the internist O. Müller. After the second World War new electronic methods were established and later converted into clinical techniques. Therefore it became possible to measure capillary blood cell velocity, dynamic capillary blood pressure and transcapillary diffusion of fluorescent tracers in human skin. For many years vital capillaroscopy has been successfully used to study the microcirculation of human skin capillaries. Capillary video-microscopy at the finger nailfold in connection with local cold exposure test are presented as a method with clinical applicability in vasopastic disease.

Capillary Permeability↗
PubMed2005

Kinetics and strain variation of phagosome proteins of Entamoeba histolytica by proteomic analysis.

The protozoan parasite Entamoeba histolytica ingests and feeds on microorganisms and mammalian cells. Phagocytosis is essential for cell growth and implicated in pathogenesis of E. histolytica. We report here the dynamic changes of phagosome proteins during phagosome maturation by proteomic analysis using reversed-phase capillary liquid chromatography and ion trap tandem mass spectrometry. Phagosomes were isolated at various intervals after internalization of latex beads. Immunoblot analysis and electron microscopy verified successful isolation of phagosomes. A total of 159 proteins were identified from the reference strain HM1 at different stages of phagosome maturation. Approximately 70% of them were detected in a time-dependent fashion, suggesting dynamism of phagosome biogenesis. The kinetics of representative proteins were verified by immunoblots and also by video microscopy of live transgenic amebae expressing green fluorescent protein-fused EhRab7A. Furthermore, we observed significant differences in phagosome profiles between HM1 and two recent clinical isolates. Approximately 60% of 229 proteins detected in at least one of these three strains were identified only in one strain, while approximately 20% of these proteins were detected in all three strains. These data should provide significant insights into molecular characterization of phagosome biogenesis, and help to elucidate the pathogenesis of this important infection.

Animals↗
PubMed1993

Translocation of cytoplasm and nucleus to fungal penetration sites is associated with depolymerization of microtubules and defence gene activation in infected, cultured parsley cells.

We describe a novel system of reduced complexity for analysing molecular plant-fungus interactions. The system consists of suspension-cultured parsley (Petroselinum crispum) cells infected with a phytopathogenic fungus (Phytophthora infestans) which adheres to a coated glass plate and thus immobilizes the plant cells for live microscopy. Conventional light and electron microscopy as well as time-lapse video microscopy confirmed the virtual identity of fungal infection structures and of several characteristic early plant defence reactions in the cultured cells and whole-plant tissue. Using this new system to approach previously unresolved questions, we made four major discoveries: (i) rapid translocation of plant cell cytoplasm and nucleus to the fungal penetration site was associated with local depolymerization of the microtubular network; (ii) the directed translocation was dependent on intact actin filaments; (iii) a typical plant defence-related gene was activated in the fungus-invaded cell; and (iv) simultaneous activation of this gene in adjacent, non-invaded cells did not require hypersensitive death of the directly affected cell.

Actins↗