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At least 1,207 records · Page 67Linked to original sources
PubMed2005

Angiogenesis: the major abnormality of the keratin-14 IL-4 transgenic mouse model of atopic dermatitis.

OBJECTIVE: Angiogenesis plays an important role in psoriasis, but its role in atopic dermatitis is unknown. The authors examined the dermal microvasculature of an IL-4 transgenic mouse model of atopic dermatitis to determine whether angiogenesis was present. METHODS: Transmission and scanning electron microscopy and confocal microscopy studies were performed. RESULTS: Transmission electron microscopy showed sprouting, transcapillary pillars of intussusception, thickened endothelial cells with large nuclei, and increased interendothelial junctional cleft number and length. Compared to nontransgenic littermates, there was a significant increase in the lengths and numbers of the interendothelial junctional clefts, along with a decrease in the length ratios of tight junction to interendothelial junctional clefts in both the early and late disease stages. In the early and late skin lesions, scanning electron microscopy of vascular corrosion casts showed disorganization of the capillary network hierarchy with increased density of capillary sprouts. Confocal microscopy of the animals with early and late skin lesions showed significant reduction in tight junction protein claudin-5. CONCLUSIONS: Angiogenesis is the major pathologic feature in this model of atopic dermatitis. The chronic skin inflammation is intertwined with and may cause the angiogenesis, but the angiogenesis itself is likely to be important in this disease process.

Animals↗
PubMed2004

Ultrasensitive confocal fluorescence microscopy of C-reactive protein interacting with FcgammaRIIa.

BACKGROUND: C-Reactive protein (CRP) is an acute phase protein with a suggested pathogenic role in cardiovascular disease. Previous reports proposed that the low-affinity IgG receptor FcgammaRIIa is the major receptor for CRP. However, these reports were met with criticism because the use of anti-CRP antibodies in the detection of CRP binding to FcgammaRIIa may have caused false-positive results. METHODS AND RESULTS: To resolve this controversy, we used ultrasensitive fluorescence microscopy to study the association, dissociation, and equilibrium of CRP binding to FcgammaRIIa. CRP indeed binds to FcgammaRIIa, with low association rates and dissociation rates. Anti-CRP antibodies markedly enhance binding, as is evident from the decrease of the equilibrium dissociation coefficient by 2 orders of magnitude. CONCLUSIONS: Our study demonstrates the virtues of single fluorophore labeling and highlights the pitfalls of immunolabeling in investigating CRP/Fc receptor interactions. Importantly, this article provides the first quantitative characterization of CRP binding to FcgammaRIIa and explains and reconciles the diverse and conflicting data presented in the literature.

Animals↗
PubMed2000

Nonhomologous bioinjectable materials in urology: 'size matters'?

OBJECTIVE: To assess the factors that influence how particles might become fixed in tissues or migrate from them, by measuring the size of the injectable particles, their susceptibility to phagocytosis and their affinity for fibroblast attachment in culture. MATERIALS AND METHODS: The particle size of three types of particulate unphysiological bioinjectable material, i.e. Urocol (Genesis Medical, Ltd., London), Macroplastiquetrade mark (Uroplasty Ltd., Reading, UK) and Urethrin (Mentor Medical Systems, Wantage, UK) was analysed using phase-contrast light microscopy and confocal microscopy. Human monocytes from peripheral blood were incubated with the three materials in phagocytic studies, where ingestion was determined by confocal microscopy. A fibroblast cell line was used to ascertain the ability of the particles to act as a substrate for cell attachment in culture. RESULTS: The mean (SEM) maximum particle diameters of Macroplastique, Urethrin and Urocol were 209 (5.10) microm, 49 (1.52) microm and 14 (0.39) microm, respectively. Rat peritoneal macrophages and human peripheral blood monocytes commonly ingested Urocol particles; the phagocytosis of Urethrin was rare and that of Macroplastique was not detected. Fibroblasts adhered to Urocol paste and Urethrin particles, but not to Macroplastique. CONCLUSION: Published reports of particle size and phagocytosis are confusing, but a relationship clearly exists. Macroplastique is the largest particle and is least likely to be phagocytosed by human mononuclear phagocytes. Urocol paste is the slowest to dissipate in culture conditions; the flat surfaces of Urethrin, but not Macroplastique, can serve as a substrate for fibroblast anchorage.

Animals↗
PubMed1999

Distribution of actin bundles in Bowman's capsule of rat kidney.

In this study we define the distribution of actin bundle arrangement in Bowman's capsule of rat renal corpuscles. Parietal cells of Bowman's capsule were examined by conventional light microscopy, electron microscopy and confocal microscopy. Within each parietal cell individual actin bundles are arranged in a parallel fashion running the length of the cell. Computer reconstructions obtained using confocal microscopy clearly show the lengths of actin bundles to be arranged, on a capsule level, end-to-end, at angles and perpendicular to bundles in adjacent cells. The bundles stain positively for non-muscle myosin and vinculin. The presence and arrangement of actin bundles in parietal cells is consistent with a role in reinforcing capsule structure.

Actins↗
PubMed1993

Comparative localization of receptors for plasmin and for urokinase on MCF 7 cells.

The receptor for plasmin and the receptor for urokinase-type plasminogen activator were characterized on MCF 7 cells either independently or simultaneously, using fluorescence microscopy and confocal microscopy. The plasmin receptor was visualized, as previously described by Correc et al. (Int. J. Cancer 50, 767, 1992) using biotinylated plasminogen and fluoresceinated streptavidin. The urokinase receptor was revealed by both polyclonal and monoclonal antibodies reacting specifically with this receptor and by binding of urokinase aminoterminal fragment. On unfixed cells, these methods gave the same heterogeneous patterns of surface staining, consisting of contours and grains, localized mainly at the upper nonadherent face of the tumor cells by confocal microscopy. Only a part of the cells was stained. When both receptors were characterized together, their presence was found on the same cells and they gave almost superimposable patterns in many cases, as shown by confocal microscopy. In contrast, when MCF 7 cells were fixed or permeabilized before staining, quite different patterns were observed: almost all the cells were labeled. The staining was mainly cytoplasmic and localized preferentially in the center and close to the upper face of the cells. Similar results were found with antiserum against urokinase receptor and monoclonal antivinculin antibody. It is likely that the receptor for urokinase and the receptor for plasmin have similar localizations on MCF 7 cells, thus resulting in a functional cooperation.

Breast Neoplasms↗
PubMed2005

Morphologic and functional alterations induced by low doses of mercuric chloride in the kidney OK cell line: ultrastructural evidence for an apoptotic mechanism of damage.

Mercury produces acute renal failure in experimental animal models, but the mechanism of tubular injury has not completely been clarified. There is an increased interest in the role of apoptosis in the pathogenesis of renal diseases that result primarily from injury to renal tubular epithelial cells. However, detailed studies of morpho-functional alterations induced by mercuric chloride in kidney cell lines are scarce. This work characterizes these alterations in OK cell cultures. Morphological alterations were profiled using light microscopy, transmission electron microscopy, and confocal microscopy, as well as mitochondrial functional assays in the cells exposed to low concentrations of HgCl2. At concentrations of 1 and 10 microM of HgCl2 there were no morphological or ultrastructural alterations, but the mitochondrial function (MTT assay) and intracellular ATP content was increased, especially at longer incubation times (6 and 9 h). At 15 microM HgCl2, both the mitochondrial activity and the endogenous ATP decreased significantly. At this concentration the OK cells rounded up, had increased number of cytoplasmic vacuoles, and detached from the cell monolayer. At 15 microM HgCl2 ultrastructural changes were characterized by dispersion of the ribosomes, dilatation of the cisterns of the rough endoplasmic reticulum, increase of number of cytoplasmic vacuoles, chromatin condensation, invaginations of the nuclear envelope, presence of cytoplasmic inclusion bodies, and alterations in the size and morphology of mitochondria. At 15 microM HgCl2 apoptotic signs included membrane blebbing, chromatin condensation, mitochondrial alterations, apoptotic bodies, and nuclear envelope rupture. Using confocal microscopy and the mitochondrial specific dye MitoTracker Red, it was possible to establish qualitative changes induced by mercury on the mitochondrial membrane potential after incubation of the cells for 6 and 9h with 15 microM HgCl2. This effect was not observed at short times (1 and 3h) with this same concentration, neither with 1 and 10 microM HgCl2 in all the studied times. Taken together, these findings indicate that low concentrations of HgCl2 induce apoptosis by inhibiting mitochondrial function, and the OK cell line may be considered a useful tool for the study of programmed cell death involving mercurial species and other heavy metals.

Adenosine Triphosphate↗
PubMed1992

Confocal-line microscopy.

The confocal-line (CL) technique combines some of the characteristics of confocal-scanning microscopy with those of conventional-imaging methods. It is based on the introduction of line-shaped illumination and linear image detection, as an alternative to the current confocal-point (CP) approach. Although confocal only in one dimension, the proposed solution offers performance and features adequate to a variety of biological and non-biological applications and is also adaptable to an increased number of microscopical observations and measurements. The absence of moving components in the optical path and the use of electronic linear imagers permits flexible and fast operation that appears particularly relevant in many fields of basic and applied research. For instance, transmission, reflection and emission images can simultaneously be collected from the same area of the specimen, with slight adjustments to the optical setup. Useful extensions of CL microscopy to the field of spectral imaging are obtained with the introduction of a slit, a polychromator and an area detector, substituting for the linear imager. Prototype instrumentation has been constructed working from the cited principles and some tests have been performed on selected applications.

Autoradiography↗
PubMed2001

Spatial distribution of perylenequinones in lichens and extended quinones in quincyte using confocal fluorescence microscopy.

The application of confocal fluorescence microscopy and microspectrofluorimetry to the characterization of the distribution of organic compounds in bulk lichens and mineral structures is demonstrated. Perylenequinones and extended quinones were chosen as both model compounds and as the naturally occurring fluorophores. These molecules occur, respectively, in corticolous microlichens and in a pink-colored mineral called quincyte. The structures of quincyte and of the lichens Cryptothelium rhodotitton and Graphis hematites are described, and the possibilities of energy dissipation and photoprotection mechanisms in these lichens are discussed. This study also illustrates how, for a wide range of specimens, naturally occurring quinone fluorophores in the specimen can be exploited directly to yield chemical and structural information without using fluorescent labelling. These intrinsic quinonoid compounds have molecular fluorescence yields and laser damage thresholds comparable or superior to common microscopy dyes, and can therefore be used to obtain high-contrast 3D fluorescence imaging without the complications introduced by dye labelling.

Lichens↗
PubMed1997

Distribution of sarcomere length and intracellular calcium in mouse skeletal muscle following stretch-induced injury.

1. The effect on sarcomere organization of stretching intact single skeletal muscle fibres by 50% of their optimum length (Lo) during ten consecutive short tetani was investigated. Stretch reduced tetanic force to 36 +/- 4% of the pre-stretch condition. Sarcomere organization was analysed using both electron and confocal microscopy. For confocal microscopy the striation pattern was examined by fluorescently staining F-actin with rhodamine-phalloidin. 2. Electron microscopy revealed that fibres which had been stretched during contraction contained areas of severe sarcomere disorganization, as well as adjacent sarcomeres of normal appearance. 3. Confocal images of stretched fibres, which had been fixed and stained with rhodamine-phalloidin, showed focal regions of overstretched sarcomeres and regions where sarcomeres of adjacent myofibrils were out of alignment with each other. Analysis of all sarcomeres along the length of fibres showed regions of sarcomere inhomogeneity were distributed throughout the fibre length and cross-section. 4. Fibres were microinjected with the fluorescent [Ca2+]i indicator fura-2 before being stretched. Conventional wide-field fluorescence imaging microscopy showed that the tetanic [Ca2+]i was reduced after stretching but remained uniformly distributed. 5. This study confirms the finding that stretch-induced muscle injury has components caused by disorganization of the myofibrillar array and by failure of tetanic Ca2+ release. The structural damage is spatially heterogeneous whereas the changes in Ca2+ release appear to be spatially homogeneous.

Animals↗
PubMed1989

Detection of diaminobenzidine reactions using scanning laser confocal reflectance microscopy.

We used scanning laser confocal microscopy to visualize sites of peroxidatic activity as detected by the diaminobenzidine (DAB) reaction. Imaging was achieved by employing the reflectance mode of this instrument. Intense reflectance was detected after DAB localization of endogenous granule-associated myeloperoxidase in neutrophils and of the exogenous tracer horseradish peroxidase in mouse oocytes. Detection of DAB reaction products with confocal reflectance microscopy will probably be an important addition to the utility of this cytochemical technique.

3,3'-Diaminobenzidine↗
PubMed1997

Study of bone formation around dense hydroxyapatite implants using light microscopy, image processing and confocal laser scanning microscopy.

The bone reaction to hydroxyapatite (HA) implants inserted transcortically and extending into the medullary canal of rat tibiae was quantitatively assessed using light microscopy, confocal laser scanning microscopy and an image processing system. Sixty-five male rats (6 weeks old) were divided into two groups, 60 for histological observation and image analysis and five for time-labelling. In the histological observation, control sections of 168 days showed a few bone trabeculae in the fatty bone marrow, and Ti implants had become gradually encapsulated with a thick bone tissue layer; however, HA implants became almost completely encapsulated with a thin bone tissue layer during the 168 day experimental period. Histometrical analysis of the percent bone contact revealed that Ti implants showed a continuous increasing curve, and HA implants showed rapid increase in the initial healing period up to 14 days, with 96% bone contact reaching a plateau at 84 days after operation. There was a significant difference in the percent of bone contact between Ti and HA implants throughout the experimental period. Confocal laser scanning microscopic observations revealed the presence of calcein at the 14th day and only slight alizarin colour layer in the bone tissue at the 28th day, both indicating bone formation. These findings suggest that the activity of bone formation was higher at the 14th day than at the 28th day. Also, the percentage of bone contact of HA is superior to titanium throughout the experimental period, and the ascending patterns of both implants are quite different to each other.

Animals↗
PubMed2000

Caveolar structure and protein sorting are maintained in NIH 3T3 cells independent of glycosphingolipid depletion.

Glycosphingolipids have been proposed to be critical components of clustered lipids within cell membranes that serve as rafts for the attachment and sorting of proteins to the cell membrane. Density gradient centrifugation was used to isolate and to ascertain the lipid composition of caveolin-enriched membranes. These membranes demonstrated a significant enrichment of sphingolipids and cholesterol containing up to 20 and 30%, respectively, of the cellular glucosylceramide and lactosylceramide. A specific inhibitor of glucosylceramide synthase, d-threo-1-phenyl-2-palmitoyl-3-pyrrolidino-propanol, was used to test the hypothesis that glycosphingolipids are required for the sorting of proteins to caveolae. When NIH 3T3 cells were depleted of their glucosylceramide based glycosphingolipid mass, the caveolar structure remained intact as determined by electron microscopy and confocal microscopy. The caveolar proteins caveolin and annexin II sorted normally to caveolae, as determined by immunoblotting and confocal microscopy. When the GPI-linked protein B61 was inducibly expressed in these cells, sorting to caveolar membranes occurred normally, even in the presence of glucosylceramide depletion. These observations suggest that protein sorting to caveolae in fibroblasts occurs independently of glycosphingolipid synthesis.

3T3 Cells↗
PubMed2000

Inputs from intrinsic primary afferent neurons to nitric oxide synthase-immunoreactive neurons in the myenteric plexus of guinea pig ileum.

Nitric oxide synthase (NOS) immunoreactivity occurs in two groups of neurons in the guinea pig small intestine: descending interneurons that are also immunoreactive for choline acetyltransferase (ChAT), and inhibitory motor neurons that lack ChAT immunoreactivity. Interneurons that are involved in local reflexes would be expected to have inputs from intrinsic primary afferent (sensory) neurons, most of which are calbindin-immunoreactive. We examined this possibility using triple staining for NOS, ChAT and calbindin immunoreactivity and investigated the relationships between calbindin-immunoreactive varicosities and the cell bodies of NOS-immunoreactive neurons, using high-resolution confocal microscopy and electron microscopy. By confocal microscopy, we found that the cell bodies of ChAT/NOS interneurons received 84 +/- 23 (mean +/- SD) direct appositions from calbindin-immunoreactive varicosities and that the cell bodies of NOS-inhibitory motor neurons received 82 +/- 20 appositions. Electron-microscopic examination of the relations of 265-calbindin-immunoreactive varicosities, at distances within the resolution of the confocal microscope (300 nm), to 30 NOS-immunoreactive nerve cells indicated that 84% formed close contacts or synapses and 16% were separated from neurons by thin glial cell processes. Thus, each NOS-immunoreactive nerve cell receives about 70 synaptic inputs or close contacts from the calbindin-immunoreactive varicosities of intrinsic primary afferent neurons. It is concluded that there are monosynaptic reflex connections in which intrinsic primary afferent neurons synapse directly with motor neurons and di- or poly-synaptic reflexes in which ChAT- and NOS-immunoreactive neurons are interneurons, interposed between intrinsic primary afferent neurons and NOS-inhibitory neurons.

Afferent Pathways↗
PubMed2000

Fluorescein-labeled naloxone binding to mu opioid receptors on live Chinese hamster ovary cells using confocal fluorescent microscopy.

A general method of confocal laser scanning microscopy was used to demonstrate specific binding of fluorescein-labeled naloxone (FNAL, 10-50 nM) to stably transfected mu opioid receptors on live Chinese hamster ovary cells. Nonspecific binding was visually indistinguishable from autofluorescence in cells with intact cell membranes. Fluorescent labeling of cell perimeters, not present in control nontransfected cells, reversed in transfected cells upon washout of FNAL or following the addition of either unlabeled naloxone (25 microM) or the mu specific antagonist CTOP (1 microM). The addition of the delta and kappa specific agonists DPDPE (1 microM) and U50488 (1 microM), respectively, failed to reverse the labeling. Further evidence of specific binding was obtained from kinetic experiments, where it was observed that only transfected cells showed a time-dependent exponential change in fluorescence that permitted estimation of association and dissociation binding rate constants of (5.8+/-0.5, mean+/-S.E.M.)x10(5) M(-1) s(-1) and (3.3+/-0.6)x10(-3) s(-1), respectively and a kinetically derived dissociation constant of 5.7+/-1.4 nM. These estimates were comparable to those obtained under similar conditions in radioligand binding experiments using [3H]-naloxone.

Animals↗
PubMed2004

Fibroblasts form a body-wide cellular network.

"Loose" connective tissue forms a network extending throughout the body including subcutaneous and interstitial connective tissues. The existence of a cellular network of fibroblasts within loose connective tissue may have considerable significance as it may support yet unknown body-wide cellular signaling systems. We used a combination of histochemistry, immunohistochemistry, confocal scanning laser microscopy (confocal microscopy), and electron microscopy to investigate the extent and nature of cell-to-cell connections within mouse subcutaneous connective tissue. We found that fibroblasts formed a reticular web throughout the tissue. With confocal microscopy, 30% of fibroblasts' processes could be followed continuously from one cell to another. Connexin 43 immunoreactivity was present at apparent points of cell-to-cell contact. Electron microscopy revealed that processes from adjacent cells were in close apposition to one another, but gap junctions were not observed. Our findings indicate that soft tissue fibroblasts form an extensively interconnected cellular network, suggesting they may have important and so far unsuspected integrative functions at the level of the whole body.

Animals↗
PubMed2005

Endoscopic confocal fluorescence microscopy of normal and tumor bearing rat bladder.

PURPOSE: We evaluated the possibility of performing endoscopic fiber-optic confocal microscopy in a rat bladder model and we distinguished different cell types. MATERIAL AND METHODS: Rhodamine 123 (Molecular Probes, Eugene, Oregon) (100 microM) was instilled for 30 minutes in 5 tumor bearing rat bladders (AY27). Five normal rats served as controls. A Cell-viziotrade mark confocal microscopy fiber was placed transurethrally in contact with normal or transformed bladder wall. Frozen sections were obtained from the same spots and subjected to conventional fluorescence microscopy and anatomical-pathological analysis. RESULTS: The different cells types present in rat epithelium (umbrella, intermediate and basal cells) could easily be identified with the Cell-viziotrade mark device due to their differences in morphology and fluorescence intensity. Individual AY-27 cells could not be demarcated due to the strong fluorescence signal but the entire tumor appeared as a brightly homogenous fluorescent blot surrounded by small inflammatory cells. CONCLUSIONS: We report the feasibility of endoscopic, in vivo, fiber-optic confocal microscopy in the rat bladder. We distinguished tumors from normal epithelium and visualized the different epithelial cell types in nontransformed rat bladder epithelium.

Animals↗
PubMed1991

Confocal fluorescence microscopy in modern cell biology.

Confocal fluorescence microscopy has become a major tool in modern cell biology. The paper explains the basic principles and especially the depth discrimination properties of confocal microscopy. An important application is described briefly and outlined with some figures. The paper concludes with remarks on features to be expected in the near future.

Animals↗
PubMed1998

Examination of blood-brain barrier transferrin receptor by confocal fluorescent microscopy of unfixed isolated rat brain capillaries.

Isolated rat brain capillaries were analyzed by confocal microscopy. Fluorescent immunoliposomes bearing the OX26 anti-transferrin receptor monoclonal antibody were synthesized and incubated with freshly isolated unfixed microvessels to visualize binding to luminal and abluminal membranes of the endothelium. Intactness of the endothelial structure was demonstrated by computer-aided reconstruction of a series of consecutive optical sections. These results indicate that analysis of unfixed brain capillaries by confocal microscopy offers the possibility of assigning the presence of membrane receptors to either the luminal or the basolateral plasma membrane domain.

Animals↗